MicroRNAs as Peripheral Biomarkers in Aging and Age-Related Diseases

Abstract

MicroRNAs (miRNAs) are found in the circulatory biofluids considering the important molecules for biomarker study in aging and age-related diseases. Blood or blood components (serum/plasma) are primary sources of circulatory miRNAs and can release these in cell-free form either bound with some protein components or encapsulated with microvesicle particles, called exosomes. miRNAs are quite stable in the peripheral circulation and can be detected by high-throughput techniques like qRT-PCR, microarray, and sequencing. Intracellular miRNAs could modulate mRNA activity through target-specific binding and play a crucial role in intercellular communications. At a pathological level, changes in cellular homeostasis lead to the modulation of molecular function of cells; as a result, miRNA expression is deregulated. Deregulated miRNAs came out from cells and frequently circulate in extracellular body fluids as part of various human diseases. Most common aging-associated diseases are cardiovascular disease, cancer, arthritis, dementia, cataract, osteoporosis, diabetes, hypertension, and neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Variation in the miRNA signature in a diseased peripheral circulatory system opens up a new avenue in the field of biomarker discovery. Here, we measure the biomarker potential of circulatory miRNAs in aging and various aging-related pathologies. However, further more confirmatory researches are needed to elaborate these findings at the translation level.

Full text

This chapter was originally published in the book Progress in Molecular Biology and Translational
Science, Vol. 146 published by Elsevier, and the attached copy is provided by Elsevier for the author's
benefit and for the benefit of the author's institution, for non-commercial research and educational use
including without limitation use in instruction at your institution, sending it to specific colleagues who
know you, and providing a copy to your institution’s administrator.
All other uses, reproduction and distribution, including without limitation commercial reprints, selling or
licensing copies or access, or posting on open internet sites, your personal or institution’s website or
repository, are prohibited. For exceptions, permission may be sought for such use through Elsevier's
permissions site at:
http://www.elsevier.com/locate/permissionusematerial
From S. Kumar, M. Vijayan, J.S. Bhatti and P.H. Reddy, MicroRNAs as Peripheral Biomarkers in Aging
and Age-Related Diseases. In: P.Hemachandra Reddy, editor, Progress in Molecular Biology and
Translational Science, Vol. 146, Burlington: Academic Press, 2017, pp. 47-94.
ISBN: 978-0-12-811532-9
© Copyright 2017 Elsevier Inc.
Academic Press
Provided for non
-
commercial research and educatio
nal use only.
Not for reproduction, distribution or commercial use.

CHAPTER THREE
MicroRNAs as Peripheral
Biomarkers in Aging and
Age-Related Diseases
S. Kumar*
,1
, M. Vijayan*, J.S. Bhatti*
,†
, P.H. Reddy*
,{
*Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, TX, United States
†
Department of Biotechnology, Sri Guru Gobind Singh College, Chandigarh, India
{
Texas Tech University Health Sciences Center, Lubbock, TX, United States
1
Corresponding author: e-mail address: subodh.kumar@ttuhsc.edu
Contents
1. Introduction 49
2. Circulatory miRNAs 51
3. miRNAs Secretion in Circulatory Biofluids 51
4. Circulatory miRNAs as Biomarkers in Aging and Age-Associated Diseases 53
4.1 Aging 53
4.2 Cardiovascular Disease 54
4.3 Cancer 63
4.4 Arthritis 70
4.5 Cataract 72
4.6 Osteoporosis 73
4.7 Diabetes/Obesity 74
4.8 Hypertension 78
4.9 Neurodegenerative Diseases 80
5. Concluding Remarks 88
Acknowledgments 89
References 89
Abstract
MicroRNAs (miRNAs) are found in the circulatory biofluids considering the important
molecules for biomarker study in aging and age-related diseases. Blood or blood com-
ponents (serum/plasma) are primary sources of circulatory miRNAs and can release
these in cell-free form either bound with some protein components or encapsulated
with microvesicle particles, called exosomes. miRNAs are quite stable in the peripheral
circulation and can be detected by high-throughput techniques like qRT-PCR, microar-
ray, and sequencing. Intracellular miRNAs could modulate mRNA activity through
target-specific binding and play a crucial role in intercellular communications. At a path-
ological level, changes in cellular homeostasis lead to the modulation of molecular func-
tion of cells; as a result, miRNA expression is deregulated. Deregulated miRNAs came out
Progress in Molecular Biology and Translational Science, Volume 146 #2017 Elsevier Inc.
ISSN 1877-1173 All rights reserved.
http://dx.doi.org/10.1016/bs.pmbts.2016.12.013
47
Author's personal copy

from cells and frequently circulate in extracellular body fluids as part of various human
diseases. Most common aging-associated diseases are cardiovascular disease, cancer,
arthritis, dementia, cataract, osteoporosis, diabetes, hypertension, and neurodegenera-
tive diseases such as Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and
amyotrophic lateral sclerosis. Variation in the miRNA signature in a diseased peripheral
circulatory system opens up a new avenue in the field of biomarker discovery. Here, we
measure the biomarker potential of circulatory miRNAs in aging and various
aging-related pathologies. However, further more confirmatory researches are needed
to elaborate these findings at the translation level.
ABBREVIATIONS
AD Alzheimer’s disease
AFP alpha-fetoprotein
AGO Argonaut2
ALS amyotrophic lateral sclerosis
AUC area under curve
BMCs blood mononuclear cells
BMD bone mineral density
BPH benign prostatic hyperplasia
BC breast cancer
CA 19–9carbohydrate antigen 19-9
CAD coronary artery disease
CBL casitas B-lineage lymphoma protooncogene
CD226 cluster of differentiation 226
CEA carcinoembryonic antigen
cfPWV carotid–femoral pulse wave velocity
CgA chromogranin A
COPD chronic obstructive pulmonary disease
CRC colorectal carcinoma
CRP C-reactive protein
CSF cerebrospinal fluid
CTO chronic total occlusion
CVD cardio-cerebrovascular disease
DM diabetes mellitus
ERA early rheumatoid arthritis
HD Huntington’s disease
HDL high-density lipoproteins
HF heart failure
HTT Huntingtin protein
iCIMT increased CIMT
IFG impaired fasting glucose
IGF1 insulin-like growth factor 1
IGT impaired glucose tolerance
INDCs inflammatory neurological disease controls
IPAH idiopathic pulmonary hypertension
48 S. Kumar et al.
Author's personal copy

LuCa lung cancer
MCI mild cognitive impairment
miRNA microRNA
mRNA messenger RNA
MVs microvesicles
nCIMT normal carotid intima-media thickness
NDs neurodegenerative diseases
NINDCs noninflammatory neurological disease controls
NSCLC nonsmall cell lung cancer
PAG1 phosphoprotein-associated with glycosphingolipid microdomains 1
PBMCs peripheral blood mononuclear cells
PCa prostate cancer
PD Parkinson’s disease
PP pulse pressure
pre-miRNA precursor miRNA
pri-miRNA primary miRNA
qRT-PCR quantitative real-time polymerase chain reaction
RA rheumatoid arthritis
RF rheumatoid factor
RISC RNA-induced silencing complex
ROC receiver operating characteristic
rRNA ribosomal RNA
sALS sporadic amyotrophic lateral sclerosis
T1DM type 1DM
T2DM type 2DM
TOB2 transducer of ERBB2, 2
VHR very high risk
1. INTRODUCTION
The first microRNAs (miRNAs) were discovered in Caenorhabditis
elegans in 1993 by Lee and colleagues, and at present, more than 1881 precursor
and 2588 mature miRNAs have been identified as updated by miRbase-21
database released in June 2014 (http://www.mirbase.org/). miRNAs are iden-
tified in various human diseases such as cancer, viral infection, diabetes,
immune-related diseases, aging, and neurodegenerative disorders; their role
has been established in different aspects of disease such as diagnosis, pathogen-
esis, and therapeutics.
1–10
miRNA synthesis processing starts in the nucleus
with the formation of primary miRNA (pri-miRNA), then precursor miRNA
(pre-miRNA), and finally mature miRNA generated in the cell cytoplasm.
11
Approximately 2000 human genes encode different miRNAs, annealing at
49miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

3ʹUTR of nearly 60% of human genes and modulating their activity at the
transcription level.
1,11–13
Besides working as a gene modulator, in several cir-
cumstances, miRNAs are also secreted in the extracellular biofluids such as
blood, serum, plasma, saliva, and urine.
7,14
Circulatory miRNAs have also
been found to be quite stable in extracellular circulation and could be a good
bioindicator for aging and age-related disease assessment.
6,8,14–17
Aging is a multifactorial process characterized by a progressive loss of
physiological integrity, leading to impaired function and increased vulner-
ability to death.
18
Within the aging process, cellular and molecular changes
and damage lead to increased disease susceptibility and mortality.
7
A key fac-
tor for the aging process is cellular senescence. The most common cellular
processes that induce senescence in normal aging are epigenetic stress, prot-
eotoxic stress, oxidative stress, telomere damage, and DNA damage, while
disease-related senescence is accompanied by smoking, telomere damage,
and DNA damage.
19
Well-known aging-associated diseases are cardiovascu-
lar disease, cancer, arthritis, dementia, cataract, osteoporosis, diabetes, and
hypertension and neurodegenerative diseases (NDs) such as Alzheimer’s dis-
ease (AD), Huntington’s disease (HD), Parkinson’s disease (PD), and
amyotrophic lateral sclerosis (ALS) (Fig. 1). A less invasive method to
Fig. 1 Most common aging and age-related human diseases.
50 S. Kumar et al.
Author's personal copy

diagnose aging-associated pathologies and other NDs much earlier than cur-
rent methods is needed, and one encouraging alternative is through the use
of biomarkers. One such promising biomarker for aging and aging-related
diseases is circulatory miRNAs. The purpose of this chapter is to discuss the
latest developments in circulating miRNAs and their possible role in early,
noninvasive identification and assessment of aging-associated diseases.
2. CIRCULATORY miRNAs
In 2010, Weber and colleagues reported that miRNAs are present in
various biofluids such as blood, saliva, tears, urine, amniotic fluid, colostrum,
breast milk, bronchial secretions, cerebrospinal fluid (CSF), peritoneal fluid,
pleural fluid, and seminal fluid.
20
The stability and abundance of circulatory
miRNAs in biofluids, such as serum and plasma, are main factors that con-
tribute to their use as potential diagnostic and progression biomarkers in a
clinical context.
21
Starting in 2007, initial observations were made that
mature miRNAs are released from the cellular cytoplasm within extracellu-
lar vesicles.
22
At the time of writing, the presence of “circulatory miRNAs”
has been verified in 12 different biofluids.
20
3. miRNAs SECRETION IN CIRCULATORY BIOFLUIDS
Accumulating evidence shows that miRNAs are secreted in extra-
cellular spaces either in the microvesicles (MVs)-encapsulated form or
released in the vesicle-free form bound with some proteins or other com-
pounds.
16,17,23
Kumar and Reddy have mentioned five different ways of
miRNA transportation into extracellular circulation: (1) bound with
high-density lipoproteins (HDL) particle in nonvesicle form; (2) complex
form with Ago2 protein; (3) packaged within exosomes; (4) encapsulated
within MVs; and (5) accumulated in apoptotic bodies.
10
Arroyo et al. dem-
onstrated that the majority (90%) of circulatory miRNAs were circulated in
MV-free form bound with Ago2 family proteins.
24
Ago2 protein association
provides ample resistance to miRNAs from a nuclease-rich extracellular
environment, because of the high stability of Ago2 protein in biofluids.
23
In some instances, particular cells showed a specific miRNA transport mech-
anism for certain miRNAs such as miR-122, a liver-enriched candidate
exclusively detected with Ago2 complexes.
24
The HDL particle size of
8–12 nm is mainly constituted of phosphatidylcholine and apolipoprotein
A-I, which forms a stable ternary structure with extracellular plasma
miRNAs by divalent cation bridging.
17
HDL particles participate only in
51miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

a minor fraction of transportation of endogenous miRNAs in peripheral cir-
culation. miR-223, a well-known miRNA, circulates in plasma associated
with HDL in the case of atherosclerosis.
25
Another transportation mode is
via miRNA encapsulation in microparticles including MVs, exosomes, and
apoptotic bodies (Fig. 2). The chief functions of MVs and exosomes are
intercellular communication and transportation of various bioactive compo-
nents (DNA, RNA, miRNA, cytokines, and other proteins).
Exosomes are cell-derived vesicles, found in most biological fluids
including cultured cells media.
26
These are round natural nanovesicles or
multivasicular bodies, 50–100 nm diameter, originated from endosomes
and secreted from plasma membrane.
17
Exosomes play a precise role in sea-
rch of miRNAs as diagnostic molecules due to their unique properties: (1)
exosomes contain the disease-specific or deregulated miRNAs expressed
during pathogenic states and nonexosomal miRNAs can be easily removed
from healthy cells; (2) exosomes have the potential to cross blood barrier via
transcytosis, thus easily clearing endothelial cellular layers; and (3) miRNAs
within exosomes are quite a protective form of cellular RNase present in the
circulatory system.
27
Sometime, it appears that miRNAs released from
exosomes may be the remnants of dead cells. Cheng et al.’s study showed
that exosomes offer protective and more enriched sources of miRNAs
Fig. 2 Modes of circulatory miRNA secretion from cells.
52 S. Kumar et al.
Author's personal copy

compared to other sources of biomarkers.
27
MVs are slightly larger vesicles
(100–4000 μm diameter) generated from the cells via outward budding and
blebbing of the plasma membrane. MVs are secreted by different cell types
such as neurons, muscle cells, inflammatory cells, and tumor cells. However,
platelets are considered to be major sources of MV production and secre-
tion.
28
miR-150 is a well-studied miRNA that is secreted from human
blood cells and monocytes via MVs.
29
Finally, apoptotic bodies are the larg-
est MVs among all that are shed from cells during apoptosis and aid in miR-
126 transportation.
30,31
miRNAs found in extracellular circulation are well
packaged in macrovesicles, hence they are resistant to cellular RNase.
32
In
addition to macrovesicles, Ago2 protein also formed Ago2–miRNA com-
plex during miRNA processing into the cytoplasm, which also enhances
miRNA stability and participates in miRNA binding to target sites.
4. CIRCULATORY miRNAs AS BIOMARKERS IN AGING
AND AGE-ASSOCIATED DISEASES
This particular chapter introduces the concept of circulatory miRNAs
as minimal invasive biomarkers for aging and age-related diseases and dis-
cusses the opportunities and challenges associated with circulatory miRNAs
as biomarkers. We summarize the recently published literature on circula-
tory miRNAs in aging and age-related diseases, and provide an overview
of the future steps necessary to develop miRNA-based biomarkers for use
in clinical routine.
4.1 Aging
Aging is a complex biological process, highly regulated by multiple evolu-
tionary conserved mechanisms.
33
Cell senescence, the key process of aging,
is basically linked to complex cellular and molecular changes that occur in
the cells over time. Such major biological phenomena are telomere erosion,
changes in protein processing, lifestyle/epigenetics factors, and alteration in
gene expression.
33
Recent studies identified miRNAs as the regulator of
several pathways that are involved in aging and cellular senescence.
7,33–36
However, very few studies are available that show the significant alterations
of circulatory miRNA levels during aging in the human population.
16
Hooten and colleagues quantified the miRNA expression in peripheral
blood mononuclear cells (PBMCs) of young (mean age 30 years) and old
(mean age 64 years) individuals. They identified three miRNAs, miR-
151a-5p, miR-181a-5p, and miR-1248, that were found to be significantly
53miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

downregulated in older individuals.
7
Similarly, their level also lowered in
the serum samples of elderly rhesus monkeys (Table 1).
7
Further study on human serum samples from seven elderly males
(69.861.77 years old) and females (72.431.49 years old), and six young
males (26.170.83 years old) and females (23.171.52 years old), identified
miR-20a levels that were significantly lower in elderly male subjects when
compared to young male subjects.
34
A study on serum samples from healthy
subjects of different age groups, involving 21 subjects (age 22 years),
10 subjects (age 40 years), 10 subjects (age 59 years), and 9 subjects
(age 70 years), identified downregulation of 5 miRNAs (miR-29b,
miR-106b, miR-130b, miR-142-5p, and miR-340) and upregulation of 3
miRNAs (miR-92a, miR-222, and miR-375) in an age-dependent
manner.
37
Even in C57BL/6 mice, miR-34a levels were also increased in
the cochlea, auditory cortex, and plasma samples during the aging process.
36
4.2 Cardiovascular Disease
Cardio-cerebrovascular disease (CVD) is an important age-related disease
and the most common cause of mortality and morbidity worldwide.
CVD is accompanied by obesity, diabetes, hyperlipidemia, and hyperten-
sion, while atherosclerosis is the most prominent indication of CVD among
all complications.
38
miRNA analysis in the serum samples of atherosclerosis
and preatherosclerosis (patients with hyperlipidemia, hypertension, and dia-
betes) patients indicated a decreased expression of miR-92a, miR-126,
miR-130a, miR-222, and miR-370 levels in preatherosclerosis patients.
However, in the case of atherosclerosis, miR-21, miR-122, miR-130a,
and miR-211 were significantly increased, whereas miR-92a, miR-126,
and miR-222 were markedly decreased compared to healthy controls
(Table 1).
38
In coronary artery disease (CAD) distinct miRNA expression profiles
were observed in patients with typical unstable angina (UA) and angiograph-
ically documented CAD (n¼13), and in individuals with noncardiac chest
pain (control, n¼13). Microarray analysis of plasma samples revealed a sig-
nificant elevation of miR-106b/25 cluster, miR-17/92a cluster, miR-
21/590-5p family, miR-126*, and miR-451 expression in UA patients
compared to controls, as shown in Table 1.
39
In heart failure (HF) patients (n¼44), four miRNAs, miR-103, miR-
142-3p, miR-342-3p, and miR-30b, were significantly (P¼0.002–0.030)
downregulated compared to controls (n¼15), chronic obstructive pulmonary
54 S. Kumar et al.
Author's personal copy

Table 1 miRNAs Expression Changes in Human and Mice Samples in Aging and Age-Related Diseases
Sample Source Species miRNAs Status in Disease References
Aging
PBMC Human #miR-151a-5p, miR-181a-5p, miR-1248 Hooten et al.
7
Serum Monkey #miR-151a-5p, miR-181a-5p, miR-1248
Serum Human #miR-20a Sawada et al.
34
Serum Human "miR-92a, miR-222, miR-375
#miR-29b, miR-106b, miR-130b, miR-142-5p, miR-340
Zhang et al.
37
Plasma C57BL/6
mice
"miR-34a Pang et al.
36
Cardiovascular diseases
Serum Human "miR-21, miR-122, miR-130a, miR-211
#miR-92a, miR-126, miR-222
Jiang et al.
38
Plasma Human "miR-106b/25, miR-17/92a, miR-21/590-5p family,
miR-126*, miR-451
Ren et al.
39
Plasma Human #miR-103, miR-142-3p, miR-342-3p, miR-30b Ellis et al.
40
Plasma Human "miR-423-5p, miR-10b, miR-30d, miR-126 Hakimzadeh et al.
41
Cancer
Serum Human "200c, miR-605, miR-135a*
#miR-433, miR-106a
Alhasan et al.
42
Serum Human "miR-21-5p, miR-375, miR-205-5p, miR-194-5p
#miR-382-5p, miR-376c-3p, miR-411-5p
Huo et al.
43
Continued
Author's personal copy

Table 1 miRNAs Expression Changes in Human and Mice Samples in Aging and Age-Related Diseases—cont’d
Sample Source Species miRNAs Status in Disease References
Serum Human "miR-182, miR-183, miR-210
#miR-126
Zhu et al.
44
Serum Human "miR-429, miR-205, miR-200b, miR-203, miR-125b, miR-34b Halvorsen et al.
45
Serum Human "miR-23a-3p, miR-27a-3p, miR-142-5p, miR-376c-3p Vychytilova-Faltejskova
et al.
46
Serum and plasma Human "miR-4270, miR-1225-5p, miR-188-5p, miR-1202, miR-4281,
miR-1207-5p, miR-642b-3p, miR-1290, miR-3141
Hamam et al.
47
Urine Human "miR-375
#miR-148
Stuopelyte et al.
48
Plasma Human "miR-21, miR-375 Gao et al.
49
Plasma Human "miR-21, miR-152 Chen et al.
50
Plasma Human #miR-195 Su et al.
51
Plasma Human "miR-410-5p Wang et al.
52
Plasma Human "miR-148a
#miR-15b
Fre
`res et al.
53
Plasma Human "miR-34a
#miR-150
Aherne et al.
54
Exosomes Human "miR-141 Li et al.
55
Author's personal copy

Exosomes Human "let-7a, miR-1229, miR-1246, miR-150, miR-21,
miR-223, miR-23a
Ogata-Kawata et al.
56
Neutrophils Human "miRs-423-3p, 148a-3p, 18a-3p, 574-3p
#miR-26a-2-3p
Ma et al.
57
Blood human "miR-200c, miR-141 Antolı
´net al.
58
Blood
Tissue
Human "miR-193a-3p, miR-23a, miR-338-5p Yong et al.
59
Arthritis
Serum Human #miR-146a, miR-155, miR-16 Filkova
´et al.
60
Serum
Blood
Human "miR-125b Duroux-Richard et al.
61
Plasma Human "miR-4634, miR-181d, miR-4764-5p
#miR-342-3p, miR-3926, miR-3925-3p, miR-122-3p,
miR-9-5p, miR-219-2-3p
Wang et al.
62
PBMCs
Plasma
Human #miR-125b Hruskova et al.
63
Serum Human "miR-16-5p, miR-23-3p, miR-125b-5p, miR-126-3p,
miR-146a-5p, miR-223-3p
Castro-Villegas et al.
64
Plasma Human "miR-23, miR-223
Continued
Author's personal copy

Table 1 miRNAs Expression Changes in Human and Mice Samples in Aging and Age-Related Diseases—cont’d
Sample Source Species miRNAs Status in Disease References
Cataract
Lens
Epithelial cells
Human "miR-34a Chien et al.
65
"miR-15a-5p, miR-15a-3p, miR-16-1-5p
#miR-16-1-3p
Li et al.
55
Aqueous humor Human "miR-4484, miR-6515-3p, miR-3663-3p, miR-4433-3p,
miR-6717-5p, miR-4725-3p, miR-1202, miR-3197
#miR-4507, miR-3620-5p, miR-5001-5p, miR-6132, miR-4467,
miR-187-5p, miR-6722-3p, miR-4749-5p, miR-1260b, miR-4634
Tanaka et al.
66
Aqueous humor Human "miR-451a, miR-21, miR-16 Wecker et al.
67
Blood "miR-184, miR-4448, miR-30a, miR-29a, miR-29c,
miR-19a, miR-30d, miR-205, miR-24, miR-22, miR-3074
Osteoporosis
Circulating
monocytes
Human "miR-422a Cao et al.
68
Circulating
monocytes
Human "miR-133a Wang et al.
69
Serum Human "miR-152-5p, miR-335-5p, miR-320a
#miR-30e-5p, miR-140-5p, miR-324-3p, miR-19b-3p, miR-19a-3p,
miR-550a-3p, miR-186-5p, miR-532-5p, miR-93-5p, miR-378a-5p,
miR-16-5p, miR-215-5p, let-7b-5p, miR-29b-3p, miR-7-5p,
miR-365a-3p
Kocijan et al.
70
Author's personal copy

Serum Human "miR-122-5p, miR-125b-5p, miR-21-5p Panach et al.
71
Serum Human "miR-21, miR-23a, miR-24, miR-93, miR-100, miR-122a,
miR-124a, miR-125b, miR-148a
Seeliger et al.
72
Blood Human "miR-194-5p Meng et al.
73
Diabetes
Plasma Human #miR-126 Zhang et al.
74
Plasma Human "miR-101, miR-200a, miR-148b, miR-210, miR-155,
miR-320, miR-103, miR-145, miR-21*, miR-126, miR-148a
#miR-93, miR-146a
Assmann et al.
75
Plasma Human "miR-126-3p Olivieri et al.
76
Plasma Mice "miR-375 Latreille et al.
77
Blood Human "miR-142-3p
#miR-126a
Zhu et al.
78
Blood Human #miR-15a Al-Kafaji et al.
79
Serum Human "miR-9, miR-29a, miR-30d, miR34a, miR-124a,
miR-146a, miR-375
Kong et al.
80
Serum Human "miR-661, miR-571, miR-770-5p, miR-892b, miR-1303 Wang et al.
81
Serum Human "miR-25 Nielsen et al.
82
Serum Human #miR-223 Wen et al.
83
Continued
Author's personal copy

Table 1 miRNAs Expression Changes in Human and Mice Samples in Aging and Age-Related Diseases—cont’d
Sample Source Species miRNAs Status in Disease References
Hypertension
Serum Human "miR-1-2, miR-1957, miR-20a, miR-145, miR-27a, miR-23a,
miR-23b, miR-191, miR-130
#miR-30c-2, miR-99a, miR-328, miR-199a, miR-330, miR-204
Sarrion et al.
84
Plasma Human "miR-92a Huang et al.
85
Blood Human "miR-1, miR-133a, miR-26b, miR-208b, miR-499, miR-21 Parthenakis et al.
86
PBMCs Human #miR-9 and miR-126 Kontaraki et al.
87
PBMCs Human "miR-1, miR-208b, miR-499, miR-21
#miR-133a, miR-26b
Kontaraki et al.
88
Dementia
Plasma Human "miR-128, miR-132, mir-874, miR-134, miR-323-3p, miR-382 Sheinerman et al.
89
Alzheimer’s disease
PBMCs Human "miR-34a, miR-181b Schipper et al.
90
Blood Human "miR-26b-3p, miR-28-3p, miR-30c-5p, miR-30d-5p, miR-148b-
5p, miR-151a-3p, miR-186-5p, miR-425-5p, miR-550a-5p, miR-
1468,
miR-4781-3p, miR-5001-3p, miR-6513-3p
#let-7a-5p, let-7e-5p, let-7f-5p, let-7g-5p, miR-15a-5p, miR-17-3p,
miR-29b-3p, miR-98-5p, miR-144-5p, miR-148a-3p, miR-502-3p,
miR-660-5p, miR-1294, miR-3200-3p is
Satoh et al.
91
Author's personal copy

CSF Human "miR-146a, miR-100, miR-505, miR-4467, miR-766,
miR-3622b-3p, miR-296
#miR-449, miR-1274a, miR-4674, miR-335, miR-375, miR-708,
miR-219, miR-103
Denk et al.
2
CSF/ECF Human "miR-9, miR-125b, miR-146a, miR-155 Alexandrov et al.
92
Serum/CSF Human #miR-125b, miR-23a, miR-26b Galimberti et al.
93
Serum Human #miR-137, miR-181c, miR-9, miR-29a, miR-29b Geekiyanage et al.
94
Serum Human "miR-9
#miR-125b, miR-181c
Tan et al.
95
Serum Human "miR-3158-3p, miR-27a-3p, miR-26b-3p, miR-151b
#miR-36, miR-98-5p, miR-885-5p, miR-485-5p, miR-483-3p,
miR-342-3p, miR-30e-5p, miR-191-5p, let-7g-5p, let-7d-5p
Tan et al.
96
Serum Human #miR-31, miR-93, miR-143, miR-146a Dong et al.
97
Serum exosomes Human "miR-361-5p, miR-30e-5p, miR-93-5p, miR-15a-5p, miR-143-3p,
miR-335-5p, miR-106b-5p, miR-101-3p, miR-425-5p,
miR-106a-5p, miR-18b-5p, miR-3065-5p, miR-20a-5p, miR-582-
5p
#miR-1306-5p, miR-342-3p, miR-15b-3p
Cheng et al.
27
Plasma Human "miR-323b-5p, miR-545-3p, miR-563, miR-600,
miR-1274a, miR-1975
#let-7d-5p, let-7 g-5p,miR-15b-5p, miR-142-3p, miR-191-5p,
miR-301a-3p, miR-545-3p,
Kumar et al.
98
Continued
Author's personal copy

Table 1 miRNAs Expression Changes in Human and Mice Samples in Aging and Age-Related Diseases—cont’d
Sample Source Species miRNAs Status in Disease References
Plasma exosomes Human "miR-548at-5p, miR-138-5p, miR-5001-3p, miR-659-5p
#miR-185-5p, miR-342-3p, miR-141-3p, miR-342-5p,
miR-23b-3p, miR-338-3p, miR-3613-3p
Lugli et al.
99
Huntington’s disease
Plasma Human "miR-877-5p, miR-223-3p, miR-223-5p, miR-30d-5p, miR-128,
miR-22-5p, miR-222-3p, miR-338-3p, miR-130b-3p, miR-425-5p,
miR-628-3p, miR-361-5p, miR-942
Dı
´ez-Planelles et al.
100
Parkinson’s disease
Serum Human #miR-141, miR-214, miR-146b-5p, and miR-193a-3p Dong et al.
101
Serum Human "miR-223 ∗, miR-324-3p, mir-24
#miR-339-5p
Vallelunga et al.
102
Amyotrophic lateral sclerosis
Plasma Human "miR-4258, miR-663b, miR-4649-5p
#miR-26b-5p, miR-4299, let-7f-5p, miR-4419a,
miR-3187-5p, miR-4496
Takahashi et al.
103
Plasma
Muscle
Serum
Mice
Human
"miR-126 Toivonen et al.
104
Author's personal copy

disease (COPD) (n¼32), and other breathless patients (n¼59).
40
In the case
of CAD, patients with insufficient collateral network development showed
a significant elevation in miR-423-5p (P<0.05), miR-10b (P<0.05),
miR-30d (P<0.05), and miR-126 (P<0.001) levels in the aortic plasma
compared to controls. Similarly, chronic total occlusion patients miRNA
analysis also indicate significantly greater expression of miR-30d (P<0.05)
and miR-126 (P<0.001) relative to healthy controls (Table 1).
41
Current findings suggested the importance of blood-based miRNAs as
biomarkers that can be monitored easily. Therefore, miRNAs potentially
represent a convenient and minimally invasive tool for the diagnosis of
CVD and patient stratification.
4.3 Cancer
Cancer is the leading cause of death worldwide with 8.2 million
people dying from it each year (http://www.who.int/cancer/en/).
105
Uncontrolled/unwanted differentiation and proliferation of cells, local-
ized at a particular site or invading at different places in the body, lead
to cancer formation. In men, the most common types of cancers include
lung, prostate, colorectal, stomach, and liver, whereas in women, common
forms are breast, colorectal, lung, uterine cervix, and stomach cancer
(http://www.who.int/cancer/en/). Currently, cancer detection is based
on the body-specific protein biomarkers that are circulated in human
blood such as alpha-fetoprotein for liver cancer, chromogranin A for
neuroendocrine tumors and especially carcinoid tumors, nuclear matrix
protein 22 for bladder cancer and carbohydrate antigen 125 for ovarian
cancer, etc.
106
However, due to late-stage disease manifestation, patients’
mortality rate is higher in a disease such as cancer. Detection of miRNAs
in biological sources (such as blood, saliva, plasma, serum, and other bio-
fluids) and their remarkable stability against RNase enzyme provide a hope
for the development of a potential next-generation biomarker for cancer
screening. The molecular interaction between miRNA and their target
mRNA is well understood, and expression of most miRNAs is strongly
deregulated in all human malignancies.
107
Recent studies have explored
the role of circulatory miRNAs as biomarkers in various cancers.
6,105–107
4.3.1 Aging and Cancer
The proportion of cancer incidence in elderly persons (65 years or older) has
increased in most countries during the last few decades. According to Inter-
national Agency for Research on Cancer and International Association of
Cancer Registries, the proportions of all cancers among elderly men and
63miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

women were 61% and 56%, respectively.
108
All cancers combined (except
nonmelanoma skin cancer) were almost seven times more frequent among
elderly men (2158 per 100,000 person-years), and around four times more
frequent among elderly women (1192 per 100,000 person-years) than
among younger persons (30–64 years old), based on the standardized
rates.
108
Among elderly men, prostate cancer (451 per 100,000), lung can-
cer (449 per 100,000), and colon cancer (176 per 100,000) make up around
half of all diagnosed cancers. Prostate cancer itself occurred around 22 times
more frequently among elderly men than among younger men. The most
frequent cancers among elderly women are breast cancer (248 per
100,000), colon cancer (133 per 100,000), lung cancer (118 per 100,000),
and stomach cancer (75 per 100,000), with these making up 48% of all
malignant cancers.
108
For most cancers, significant geographical variations
in incidence rates are found among elderly individuals, reflecting socioeco-
nomic status, differing particularly between developing and developed
countries. In contrast with other major causes of death among the elderly,
cancer incidence and mortality have not declined in general, indicating that
primary prevention (especially cessation of tobacco smoking) remains a most
valuable approach to decrease mortality; for most major cancers (prostate,
colon, and breast), the causes remain almost unknown. Therefore, it is
important to emphasize the increasing need for research into the prevention
of cancer and the planning of treatment and care in the elderly.
Stable blood-based circulatory miRNA species have allowed for the dif-
ferentiation of patients with various types of human cancers. Studies found
that miR-21 has been identified as an “oncomir” in various tumors while
miR-152 is a tumor suppressor.
50
Expression of both miR-21 and miR-
152 were analyzed in patients with lung cancer (LuCa), colorectal carcinoma
(CRC), breast cancer (BC), and prostate cancer (PCa). Quantitative
real-time polymerase chain reaction (qRT-PCR) analysis of plasma samples
from a total of 204 cancer patients, 159 various benign lesions, and 228 nor-
mal subjects revealed a significant elevation of miR-21 and miR-152
expression in LuCa, CRC, and BC when compared with normal controls.
Upregulation of miR-21 and miR-152 levels was also observed in the
plasma samples of patients with benign lesions of lung and breast, as com-
pared to normal controls, respectively. However, no significant expression
variation of these two miRNAs was observed in PCa or prostatic benign
lesions as compared to controls. Receiver operating characteristic (ROC)
curve analyses revealed that miR-21 and/or miR-152 can discriminate
LuCa, CRC, and BC from normal controls.
50
64 S. Kumar et al.
Author's personal copy

4.3.2 Prostate Cancer
PCa is the primary noncutaneous malignancy among men in the Uni-
ted States, and is the second most common cause of cancer mortality.
42
Scano-miR profiling of very high-risk (VHR) PCa patients (n¼19)
showed the deregulation of five miRNAs: miR-200c, miR-605, miR-
135a*, miR-433, and miR-106a in serum samples. Among these, miR-
200c had most significant elevated serum levels in all patients with VHR
PCa, whereas circulating miR-605 and miR-135a*expression was reduced
and miR-433 and miR-106a expression was increased in patients with
VHR vs healthy volunteers (Table 1).
42
Further, biological pathway anal-
ysis showed the potential capability of these miRNAs as biomarkers for
VHR aggressive PCa.
Plasma circulating miRNAs were analyzed in 149 PCa patients,
57 healthy controls, and 121 noncancer patients (with benign prostatic
hyperplasia (BPH) and other urinary diseases). qRT-PCR results showed
a significant elevation of circulating miR-410-5p level in the PCa patients
compared to healthy controls or noncancer patients (Table 1). ROC curve
analysis showed the diagnostic properties of plasma miR-410-5p with an
area under curve (AUC) value of 0.8097 (P<0.001) in PCa patients.
52
Further study on plasma samples from 57 PCa patients and 28 BPH
patients showed upregulation of miR-21 and miR-375 by Taqman-based
qRT-PCR assay. However, in the BPH group, the median relative expres-
sion levels of miR-21 and miR-375 were 0.07 and 0.55, respectively,
whereas in the PCa group, the median values of miR-21 and miR-375 were
1.32 and 1.74, respectively (Table 1). ROC analysis illustrated that the AUC
values for miR-21 and miR-375 were 0.799 and 0.757, respectively
(P¼0.000126). These results showed the discriminating power of circula-
ting miR-21 and miR-375 in PCa patients from BPH controls at early
stages.
49
Analysis on 56 prostate cancerous and 16 noncancerous tissues detec-
ted differentially expressed 754 miRNAs by TaqMan Low Density Array.
Highly abundant miRNAs were selected and were analyzed in urine speci-
mens of 215 patients with BPH and 62 asymptomatic controls by
qRT-PCR. Validation analysis identified most significant miR-148a
and miR-375 in urine specimens (Table 1). Both miRNAs strongly
improved the diagnostic power of the prostate-specific antigen test with
AUC values of 0.79 and 0.84 for miR-148a and miR-375, respectively.
48
Thus, urine-circulating miRNAs may be a noninvasive tool for sensitive
and specific detection of PCa.
65miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

Besides serum, plasma, and urine, exosome miRNAs are considered
important sources as peripheral circulatory miRNAs. Exosomes isolated
from the serum of PCa patients, patients with BPH, and healthy controls
were analyzed for miR-141 expression. Exosomes contain a higher expre-
ssion of miR-141 compared to whole serum samples in healthy controls.
The level of serum exosomal miR-141 was found to be significantly
higher in the PCa patients compared to the patients with BPH and the
healthy controls (3.85-fold, P¼0.0007 and 4.06-fold, P¼0.0005, respec-
tively) (Table 1). Additionally, expression of miR-141 was significantly
higher in metastatic PCa compared with localized PCa (P<0.0001).
ROC curve analysis revealed that serum exosomal miR-141 yielded an
AUC value of 0.86, with 80% sensitivity and 87.1% specificity in dis-
criminating patients with metastatic PCa from the patients with localized
PCa.
55
A study suggested that the serum exosomes may serve as a more
suitable material compared with the whole serum for measuring circula-
ting miRNAs in patients.
Further, in prostate cancer patients, the concentration of miR-21-5p,
miR-141-3p, miR-100-5p, and miR-375 was found to be increased in
the serum samples while the expression of miR-141-3p was increased in
both serum and plasma samples.
105
Beside their diagnostic value, plasma
miRNAs, miR-20a-5b, miR-21-5b, and miR-145-5p, were also useful
for the prediction of recurrence of PCa followed by localized treatment
of cancer.
109
4.3.3 Lung Cancer
LuCa is a leading cause of cancer-related deaths in the world, and up to 85%
of lung cancer is classified as nonsmall cell lung cancer (NSCLC).
44
Circu-
lating miRNAs have been reported to be stably expressed and detected in
plasma/serum and to function as potent biomarkers in NSCLC. The diag-
nostic and prognostic value of miR-195 was evaluated in the plasma samples
of patients with NSCLC. qRT-PCR analysis of 100 NSCLC patients and
100 healthy volunteers showed significant downregulation miR-195 in
NSCLC patients compared with healthy controls (P<0.001). Decreased
plasma miR-195 level was significantly associated with lymph node metas-
tasis and advanced clinical stage (Table 1). Further, multivariate Cox regres-
sion analysis confirmed low plasma miR-195 expression as an independent
unfavorable prognostic factor for NSCLC patients.
51
Findings indicated that
plasma miR-195 might serve as a promising biomarker for the early detec-
tion and prognosis evaluation of NSCLC.
66 S. Kumar et al.
Author's personal copy

Serum-circulating miRNAs could be used as a biomarker to detect early
lung cancer. The levels of miRNAs were analyzed in serum samples from
112 NSCLC patients and 104 controls (20 current smokers without lung
cancer, 23 pneumonia patients, 21 gastric cancer patients, and 40 healthy
controls) by Taqman probe-based qRT-PCR. Data showed significantly
upregulation in serum levels of miR-182, miR-183 and miR-210, and
downregulation of miR-126 level in NSCLC patients compared with
healthy controls (Table 1). Further, ROC curve analysis revealed that the
miR-182, miR-183, miR-210, or miR-126 level could serve as a diagnostic
biomarker for NSCLC early detection, with a high sensitivity and specific-
ity. Combination of these four miRNAs with carcinoembryonic antigen
(CEA) further increased the diagnostic value, with an AUC value of
0.965 (sensitivity, 81.3%; specificity, 100.0%; and accuracy, 90.8%).
44
In
addition, serum levels of miR-182, miR-183, miR-210, and miR-126
could be used to distinguish NSCLC or early-stage NSCLC from current
tobacco smokers without lung cancer and pneumonia or gastric cancer
patients with a high sensitivity and specificity.
Halvorsen and colleagues tested the diagnostic potential of serum
miRNAs on 38 NSCLC patients, 16 patients suffering from COPD, and
16 healthy volunteers. TaqMan Low Density Arrays analysis showed dereg-
ulation of 754 unique miRNAs. Further, validation study by qRT-PCR
revealed six ideal miRNAs, miR-429, miR-205, miR-200b, miR-203,
miR-125b, and miR-34b, with a significantly higher abundance in NSCLC
patients serum compared to controls. Further, these miRNAs revealed a sig-
nificant AUC value 0.89 for stages I–IV and 0.88 for stage I/II after ROC
curve analysis.
45
Thus, the accessibility and stability of these circulating
miRNAs make them promising biomarkers as a supplement in future scre-
ening studies.
Peripheral neutrophils, an important component of innate and adaptive
immune systems, also showed aberrant miRNA expression in NSCLC
patients. miRNAs were examined in neutrophils of 15 patients with stage
I NSCLC and 15 smokers without cancer, and showed deregulation of five
important miRNAs: miR-423-3p, miR-148a-3p, miR-18a-3p, miR-574-
3p, and miR-26a-2-3p. Further, a validation study on 82 patients with lung
cancer and 73 controls showed a high level of miR-423-3p, miR-148a-3p,
miR-18a-3p, and miR-574-3p, and low expression of miR-26a-2-3p in
neutrophils of patients with NSCLC vs controls. However, a set of only
two miRNAs, miR-26a-2-3p and miR-574-3p, were developed to pro-
duce 77.8% sensitivity and 78.1% specificity for NSCLC detection.
57
67miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

4.3.4 Breast Cancer
BC is the most common type cancer and is the second cause of
cancer-related mortality among women. Annually, the number of women
who are newly diagnosed with breast cancer is currently exceeds 235,000
and there are around 40,000 deaths as a result of breast cancer in the United
States.
47
Circulating miRNAs can be used to predict BC recurrence.
miRNA profiling of serum samples from 48 BC patients identified signifi-
cantly deregulated miRNAs by Exiqon miRCURY qRT-PCR panels.
A further validation study on an independent set of sera from 20 patients
with BC recurrences and 22 patients without recurrences identified four
upregulated miRNAs (miR-21-5p, miR-375, miR-205-5p, and miR-
194-5p), and three downregulated miRNAs (miR-382-5p, miR-376c-
3p, and miR-411-5p) in recurrent patients (Table 1).
43
A novel approach was used to isolate circulating miRNAs through an
enrichment step using speed-vacuum concentration, which resulted in a
fivefold increase in miRNA abundance. Microarray expression profiling
on samples from 23 BC and 9 normal controls identified significantly
upregulated 18 miRNAs in BC patients (P<0.05). Expression of nine
miRNAs, miR-4270, miR-1225-5p, miR-188-5p, miR-1202, miR-4281,
miR-1207-5p, miR-642b-3p, miR-1290, and miR-3141, was subsequently
validated using qRT-PCR in a cohort of 46 BC and 14 controls (Table 1).
Expression of these miRNAs was found to be higher in patients with stages
I, II, and III, compared to stage IV, suggesting a potential utilization for early
detection.
47
A plasma miRNA profile was determined by qRT-PCR in a cohort of
378 women. Eighty-four miRNAs were found to be significantly deregu-
lated in patients with metastatic BC compared to controls. The eight most
significant miRNAs were measured in the first profiling cohort composed of
41 primary BC and 45 controls. A further validated study in diverse cohorts
of 108 primary BC, 88 controls, 35 BC in remission, 31 metastatic BC, and
30 gynecologic tumors found miR-148a to be the most significantly
upregulated miRNA and the most significantly downregulated one was
miR-15b.
53
Expression of miR-200c and miR-141 was examined in blood samples
of 57, stages I–IV, BC patients and 20 age-matched controls by qRT-PCR.
miR-200c expression was significantly downregulated (P<0.0001) in BC
patients compared to controls and yielded an area under the ROC curve
of 0.79 (90% sensitivity and 70.2% specificity). However, the miR-141 level
was significantly higher in the blood of patients with stages I–III, lymph
68 S. Kumar et al.
Author's personal copy

node metastasis, and HER2 negative tumors.
58
Thus, miR-200c and miR-
141 were independent prognostic factors and associated with distinct out-
comes of BC patients.
4.3.5 Colorectal Cancer
Over the past decade, research has shown that altered miRNA expression
found in CRC and circulating miRNAs are involved in CRC detection,
progression, and outcome.
110
Recently, Vychytilova-Faltejskova and col-
leagues did a three-phase biomarker study on 427 colon cancer patients
and 276 healthy donors. Serum samples were screened for miRNA expres-
sion by using Illumina small RNA sequencing and validated by qRT-PCR.
Analysis showed 54 significantly deregulated miRNAs in sera of CRC
patients compared to healthy donors (P<0.01). However, in a final vali-
dation study, the most significant upregulation of only four miRNAs,
miR-23a-3p, miR-27a-3p, miR-142-5p, and miR-376c-3p, was observed
(Table 1). Diagnostic accuracy of these miRNAs was also established with
AUC value ¼0.917, distinguishing patients from controls with sensitivity of
89% and specificity of 81% (AUC ¼0.922).
46
Cell-free circulatory nature of miRNAs as biomarkers in CRC was
assessed by Aherne et al. on 48 plasma samples comprising normal, polyp,
adenoma, early, and advanced cancer samples. Results showed 667 der-
egulated miRNAs. Three miRNAs (miR-34a, miR-150, and miR-923)
were selected for further validation in a cohort of 97 subjects divided into
the same five groups, and in an independent public dataset of 40 CRC sam-
ples and paired normal tissues. High levels of miR-34a and low miR-150
levels distinguished groups of patients with polyps from those with advanced
cancer (AUC ¼0.904), and low circulatory miR-150 levels separated
patients with adenomas from those with advanced cancer (AUC ¼0.875).
In addition, altered expression of miR-34a and miR-150 can distinguish
an independent public dataset of 40 CRC samples and paired normal
tissues.
54
Microarray analyses were performed on 88 primary CRC patients and 11
healthy controls in exosome-enriched fractions of serum samples for circu-
latory miRNA detection. Serum exosomal level of seven miRNAs, let-7a,
miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a, were
found to be significantly higher in primary CRC patients compared
to healthy controls (Table 1). Interestingly, their levels were signifi-
cantly downregulated after surgical resection of CRC tumors. Further,
69miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

high sensitivities of the seven selected miRNAs showed significant AUC
values by ROC curve analysis with tumor markers (CA 19-9 and
CEA).
56
CRC tissues and blood samples were analyzed for biomarker validation
by stem-loop qRT-PCR. Seven miRNAs, miR-150, miR-193a-3p, miR-
23a, miR-23b, miR-338-5p, miR-342-3p, and miR-483-3p, were found
to be differentially expressed in both tissue and blood samples. However,
significant positive correlations were observed in the tissue and blood levels
only with three miRNAs (miR-193a-3p, miR-23a, and miR-338-5p). Fur-
ther, ROC curve analysis of these miRNAs yielding an AUC value of 0.887
(80.0% sensitivity, 84.4% specificity, and 83.3% accuracy) confirmed them as
a classifier for CRC detection.
59
The discovery of circulatory miRNAs as biomarkers brought forward a
new understanding of the basic mechanisms of carcinogenesis, and opened
up exciting prospects for diagnostics and prognostics. Although still an
emerging field, needing much exploration, the hope is to apply circulating
miRNAs to cancer diagnosis and treatment.
4.4 Arthritis
Rheumatoid arthritis (RA) is another age-related chronic inflammatory
autoimmune disease, which affects approximately 1% of the world’s pop-
ulation.
62,63
RA is associated with persistent synovitis, leading to severe
joint destruction, development of joint deformities, and increased risk of
cardiovascular diseases.
63
Establishment of early-stage detection para-
meters and treatment response would be beneficial for patients with early
rheumatoid arthritis (ERA) to prevent ongoing joint damage. Circula-
ting miRNA expression was analyzed in the serum samples of ERA
patients (n¼34) and patients with established RA (n¼26). Levels of
three miRNAs, miR-146a, miR-155, and miR-16, were decreased in
ERA patients in comparison with established RA (Table 1).
60
Analysis
revealed that miR-223 may serve as a marker of disease activity, and
miR-16andmiR-223maybepossiblepredictorsfordiseaseoutcome
in ERA.
60
miRNA array analysis of plasma samples from RA patients (n¼75,
containing 44 active RA and 31 nonactive RA) unveiled differential
expression of nine miRNAs as compared to controls subjects (n¼70).
miR-4634, miR-181d, and miR-4764-5p levels were increased, whereas
miR-342-3p, miR-3926, miR-3925-3p, miR-122-3p, miR-9-5p, and
70 S. Kumar et al.
Author's personal copy

miR-219-2-3p expression levels were decreased in RA patients. The
AUC values for nine individual miRNAs ranged from 0.6254 to 0.818;
however, the best miRNA candidates that showed significant differences
in their expression between RA and other control groups were miR-122-
3p, miR-3925-3p, miR-342-3p, and miR-4764-5p.
62
miR-125b level was found to be significantly elevated in the total blood
and serum samples of RA patients compared to osteoarthritic and healthy
donors. However, miR-125b upregulation was also found in patients with
other forms of chronic inflammatory arthritis. However, higher serum levels
of miR-125b at disease flare were associated with good clinical response to
treatment with rituximab 3 months later (P¼0.002); hence it could be a
potential predictive biomarker in response to rituximab treatment.
61
In con-
trast, the treatment-naı
¨ve early phase of RA patients showed significantly
lowered expression of miR-125b in the PBMCs and plasma samples than
healthy controls. While after 3 months of treatment, it was significantly
(P¼0.042) increased particularly in responders, ROC analysis indicated a
significant (P¼0.048) AUC value 0.652 (95% CI 0.510–0.793) in the
patients after 3 months of therapy. miR-125b in PBMCs of treatment-naı
¨ve
patients may present a novel biomarker for monitoring the treatment out-
come during the early phase of RA.
63
Further, miRNA expression levels in RA patients (n¼95) before and
after anti-TNFα/DMARDs combination therapy may also be potential
novel biomarkers for predicting and monitoring therapy. Serum levels of
six miRNAs, miR-16-5p, miR-23-3p, miR125b-5p, miR-126-3p,
miR-146a-5p, and miR-223-3p, were found significantly upregulated in
RA patients by anti-TNFα/DMARD combination therapy.
64
However,
those miRNAs were only increased in responder patients after therapy,
and paralleled with the reduction of TNFα, interleukin (IL)-6, IL-17, rheu-
matoid factor, and C-reactive protein. In plasma samples of RA patients,
miR-23 and miR-223 may serve as both predictors and biomarkers of
response to anti-TNFα/DMARDs combination therapy.
64
Churov and
colleagues discussed around 10 reports and found more than 20 miRNAs
that were found to be deregulated in the blood components such as
serum/plasma, PBMCs, neutrophils, and PB T cells and PBCD4
+
cells of
RA patients. The most significant circulatory miRNAs were miR-16,
miR-21, miR-24, miR-26a, miR-125a-5p, miR-125b, miR-126-3p,
miR-223, and miR-451.
111
These were elevated in the plasma or serum
and are considered to be the most promising noninvasive biomarkers for
the detection of RA.
71miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

4.5 Cataract
Cataracts, the most common cause of blindness worldwide, are significantly
related to the aging process.
65
Role of miR-34a has been identified in the lens
senescence in age-related cataract patients. Study on the lens epithelium sam-
ples of 110 patients with four age groups: between 55 and 64 years (n¼25;
22.7%), between 65 and 74 years (n¼35; 31.8%), between 75 and 84 years
(n¼28; 25.5%), and older than 85 years (n¼22; 20%) revealed that miR-34a
expression levels were significantly different between each age group and it is
found to be greater in patients with older age.
65
A further study was also conducted on the lens epithelial cells by Li and
colleagues, on 60 age-related cataract patients (including 20 with cortical
cataracts, 20 with nuclear cataracts, and 20 with posterior subcapsular cata-
racts) and 20 normal patients. Expression of miR-15a-5p, miR-15a-3p, and
miR-16-1-5p was decreased in normal lens epithelial cells but was higher at
significant levels in corresponding cells of patients with cortical, nuclear, or
posterior subcapsular cataracts (P<0.01) (Table 1), whereas miR-16-1-3p
expression was relatively high in normal lens epithelial cells, but significantly
decreased in cells of patients from each cataract group (P<0.01).
55
Next-generation sequencing (NGS) techniques of human aqueous
humor samples identified 158 miRNAs in four samples; an additional
59 miRNAs were present in at least three samples. The aqueous humor
miRNA profile shows some overlap with published NGS-derived inven-
tories of circulating miRNAs in blood plasma with high prevalence of
human miR-451a, miR-21, and miR-16. In contrast to blood, miR-184,
miR-4448, miR-30a, miR-29a, miR-29c, miR-19a, miR-30d, miR-205,
miR-24, miR-22, and miR-3074 were detected among the 20 most preva-
lent miRNAs in aqueous humor.
67
Tanaka and colleagues conducted a microarray analysis of aqueous humor
samples from glaucoma (n¼10), cataract (n¼5), and epiretinal membrane
patients (n¼5), revealing the disease-related extracellular miRNAs pro-
files.
66
Eight miRNAs were found to be significantly upregulated in glau-
coma patients compared to controls, as follows: miR-4484, miR-6515-3p,
miR-3663-3p, miR-4433-3p, miR-6717-5p, miR-4725-3p, miR-1202,
and miR-3197, whereas 10 downregulated miRNAs were miR-4507,
miR-3620-5p, miR-5001-5p, miR-6132, miR-4467, miR-187-5p, miR-
6722-3p, miR-4749-5p, miR-1260b, and miR-4634 (Table 1). The two
miRNAs miR-3620-5p and miR-6717-5p showed the maximum AUC
value (0.88) to distinguish the patient and controls.
66
72 S. Kumar et al.
Author's personal copy

4.6 Osteoporosis
Osteoporosis is a systemic skeletal disorder characterized by increased risk of
bone fracture (BF) due to fragility and reduction in bone mass. BFs, partic-
ularly hip fracture, are a major concern in health care because of the asso-
ciated morbidity and mortality, mainly in the elderly.
71,72
It has also been
postulated that miRNAs might play important roles in age-related bone loss
disorders, bone remodeling, postmenopausal osteoporosis, and osteoporotic
fracture patients.
112
Wang and colleagues identified miR-133a as a promising molecule
where expression level varies between patients with low bone mineral den-
sity (BMD) (n¼10) compared with the high BMD (n¼10) groups during
postmenopausal in Caucasian women. Microarray analysis of circulating
monocytes revealed the significant (P¼0.007) higher expression of miR-
133a in patients with low BMD. Further, bioinformatic target gene analysis
showed three potential osteoclast-related target genes, CXCL11, CXCR3,
and SLC39A1 of miR-133a.
69
In women with postmenopausal osteoporosis, significant miRNA sig-
nature was identified as biomarkers. miRNAs array analysis of circulating
monocytes (osteoclast precursors) from 10 high BMD and 10 low BMD
postmenopausal Caucasian women identified upregulation of miR-422a
at the marginal significant level (P¼0.065) in the low BMD compared with
the high BMD group. A more significant upregulation of miR-422a was
identified in the low BMD group by qRT-PCR analysis (P¼0.029)
(Table 1). Additionally, qRT-PCR analyses of miR-422a target genes
showed the negative correlation with these five gene (CBL, CD226,
IGF1, PAG1, and TOB2) expressions, suggesting miR-422a as the potential
miRNA biomarker underlying postmenopausal osteoporosis.
68
Though
postmenopausal osteoporosis is the most common cause of low-traumatic
fractures, bone loss and low-traumatic fractures also occur in premenopausal
state in women and in young males. In men, late-stage bone loss is described
as “male idiopathic osteoporosis.”
113
Circulating serum miRNAs showed
the differential expression pattern in patients with premenopausal, post-
menopausal, and male idiopathic osteoporosis. Three miRNAs were com-
monly upregulated, miR-152-5p, miR-335-5p, and miR-320a, while
16 were downregulated: miR-30e-5p, miR-140-5p, miR-324-3p, miR-
19b-3p, miR-19a-3p, miR-550a-3p, miR-186-5p, miR-532-5p, miR-
93-5p, miR-378a-5p, miR-16-5p, miR-215-5p, let-7b-5p, miR-29b-3p,
miR-7-5p, and miR-365a-3p by qRT-PCR analysis among patients with
73miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

prevalent low-traumatic fractures and control subjects (Table 1). Interest-
ingly, eight miRNAs had AUC values >0.9 for the classification of fracture
patients.
70
In 2015, Meng and colleagues identified miR-194-5p as a potential bio-
marker for postmenopausal osteoporosis. Microarray analysis on the blood
samples from osteopenia (n¼23) and osteoporosis patients (n¼25) identi-
fied five miRNAs (miR-130b-3p, miR-151a-3p, miR-151b, miR-194-5p,
and miR-590-5p); however, only miR-194-5p expression was increased
and found to be enriched in multiple osteoporosis-related pathways.
73
Fur-
ther study by Panach and colleagues identified three valuable miRNAs,
miR-122-5p, miR-125b-5p, and miR-21-5p, which were upregulated in
serum samples of brain fracture patients with respect to controls. Remark-
ably, the miR-21-5p level was correlated with CTx (r¼0.76;P<0.00001),
a marker of bone resorption, confirming its diagnostic potential.
71
miRNAs analysis in the serum of 30 osteoporotic and 30 nonosteoporotic
patients revealed the significant upregulation of 9 miRNA candidates,
namely miR-21, miR-23a, miR-24, miR-93, miR-100, miR-122a,
miR-124a, miR-125b, and miR-148a in osteoporotic patients (Table 1),
whereas onthe bone tissue of 20 osteoporotic and 20 nonosteoporotic patients
only six miRNAs (miR-21, miR-23a, miR-24, miR-25, miR-100, and
miR-125b) displayed a significantly higher expression.
72
However, a total
of five miRNAs display a common upregulation in both serum and bone
tissue.
These studies reveal an important role for several miRNAs in osteopo-
rotic state and in postmenopausal osteoporosis patients, and suggests that
they may be used as biomarkers for diagnostic purposes and may be a target
for treating bone loss and optimizing fracture healing in osteoporotic
patients.
4.7 Diabetes/Obesity
Diabetes mellitus (DM) is an age-related metabolic disorder characterized by
insulin secretion from pancreatic βcells that is insufficient to maintain blood
glucose homeostasis. DM has a global public health issue, estimated to aff-
ect 450 million people, and the economic cost is projected to be $490 bil-
lion/year by 2030.
114,115
It is a disease resulting from insufficient production
of the insulin hormone pancreatic cells (type 1 DM, T1DM) or from inef-
fective insulin action (type 2 DM, T2DM).
114
For both types, T2DM is
more common in humans, comprising 85%–90% of total DM cases,
74 S. Kumar et al.
Author's personal copy

and it has been considered a progressive metabolic disorder. The role of
miRNAs as biomarkers has been explored in both types of diabetes; how-
ever, very few studies are available in the case of T1DM.
67
T1D is usually diagnosed when >80% of the pancreatic beta-cells are
destroyed by the immune system. Plasma miRNA expression was analyzed
in 25 T1D patients and 20 age- and gender-matched nondiabetic controls
by using Stem-loop RT-Pre-Amp Real-time PCR. Results showed a
significant two- to fivefold downregulation of miR-93*and miR-146a
and 2–40fold upregulation of miR-101, miR-200a, miR-148b, miR-
210, miR-155, miR-320, miR-103, miR-145, miR-21*, miR-126, and
miR-148a in T1D patients (Table 1).
75
A study by Zhang and colleagues on plasma samples identified miR-126
as a potential biomarker for early prediction of T2DM in susceptible indi-
viduals. The study included 30 subjects in each three groups: normal (fasting
glucose), T2DM-susceptible, and T2DM individuals. Five miRNAs, miR-
29b, miR-28-3p, miR-15a, miR-223, and miR-126, were selected for the
qRT-PCR analysis. However, only miR-126 showed significantly reduced
expression in susceptible individuals and T2DM patients compared to nor-
mal individuals.
74
A comprehensive characterization of the serum miRNA profile in pati-
ents with T2DM-associated microvascular disease (T2DMC) demonstrated
deregulated miRNAs expression. Serum samples obtained from 184 T2DM
patients (92 with microvascular complications and 92 free of complications)
and 92 age/gender-matched controls were analyzed by using a TaqMan
Low Density Array. Initially, the levels of 754 miRNAs were markedly
upregulated in the patients’ groups; however, subsequently validated analysis
by qRT-PCR identified only five ideal miRNAs (miR-661, miR-571,
miR-770-5p, miR-892b, and miR-1303) that were significantly upre-
gulated in T2DM patients (P<0.05) (Table 1).
81
Thus, circulating miRNAs
are an emerging class of biomarkers for T2DM.
Even in children with newly diagnosed T1D levels of sera, miRNAs
were changed when compared with age-matched healthy controls and gly-
cemic controls. Global miRNA sequencing analyses on the pooled sera sam-
ples were performed on two groups: T1D cohorts (n¼275 and 129,
respectively), and one control group (n¼151). Twelve miRNAs were iden-
tified as upregulated in T1D patients (miR-152, miR-30a-5p, miR-181a,
miR-24, miR-148a, miR-210, miR-27a, miR-29a, miR-26a, miR-27b,
miR-25, and miR-200a) (Table 1). Several of these miRNAs were linked
with important molecular pathways such as apoptosis and beta-cell
75miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

networks. However, further analysis identified miR-25 as negatively asso-
ciated with residual beta-cell function (est.: 0.12, P¼0.0037), and posi-
tively associated with glycemic control (HbA1c) (est.: 0.11, P¼0.0035)
at 3 months after onset of the disease.
82
Thus, the study demonstrates that
miR-25 might be a “tissue-specific” miRNA for diagnosis in new onset
T1D children and may be a predictive circulating miRNA biomarker.
Another important circulatory miRNA is miR-126-3p, whose levels
were found to be deregulated in T2DM patients. Plasma samples from
193 patients with T2DM aged 40–80 years, and 136 healthy subjects aged
20–90 years were used to explore the combined effect of age and glycemic
state on miR-126-3p expression. Expression of miR-126-3p was signifi-
cantly higher in the oldest individuals compared with the youngest controls
(<45 vs >75 years) with relative expression level: 0.27 0.29 vs 0.48 0.39
(P¼0.047). However, age-based comparison between controls and T2DM
demonstrated significantly different miR-126-3p levels only in the oldest
(0.48 0.39 vs 0.22 0.23, P<0.005). Furthermore, miR-126-3p levels
were seen to be lower in patients with poor glycemic control, compared
with age-matched controls. The age-related increase in plasma miR-
126-3p found in controls was paralleled by a five- or sixfold increase in
intra/extracellular miR-126-3p in in vitro-cultured HUVECs undergoing
senescence.
76
Moreover, miR-126-3p expression was downregulated in
intermediate-age HUVECs grown in a high-glucose medium until
senescence.
Kong and colleagues identified seven diabetes-related serum miRNAs,
miR-9, miR-29a, miR-30d, miR34a, miR-124a, miR-146a, and miR-
375, having clinical significance during pathogenesis of type 2 diabetes
(T2D).
80
Serum sample analysis of 56 subjects including 18 cases of newly
diagnosed T2D (n-T2D) patients, 19 cases of prediabetes individuals
(impaired glucose tolerance and/or impaired fasting glucose), and 19 cases
of T2D-susceptible individuals with normal glucose tolerance (s-NGT)
showed upregulation of these miRNAs by qRT-PCR. Furthermore, differ-
ent statistical analyses showed that miR-34a was the most significant
miRNA that was able to discriminate patients and controls.
80
A study on the peripheral whole blood samples from patients with T2D
(n¼24), prediabetes individuals exhibiting impaired fasting glucose (IFG)
and impaired glucose tolerance (IGT) (n¼22), as well as healthy control
subjects (n¼24) investigated the expression miR-15a.
79
qRT-PCR analysis
indicated a significant downregulation of miR-15a in patients with T2D and
IFG/IGT individuals, compared with healthy control subjects (P<0.05).
76 S. Kumar et al.
Author's personal copy

Multivariate logistic regression analysis showed a significant association of
lower miR-15a expression with T2D and prediabetes (P<0.05). Further-
more, ROC curve analysis revealed that blood miR-15a was able to distin-
guish patients with T2D and IFG/IGT individuals from healthy controls
(AUC; 0.864).
79
Thus, the miR-15a level in peripheral whole blood may
serve as a potential biomarker for T2D and prediabetes.
A study on the mice model (375KO) identified miR-375 as an important
regulator of β-cell mass and function. Mice overexpressing miR-375 exhibit
normal β-cell mass and function.
77
Analysis of plasma samples from 375KO
indicated an elevation of the miR-375 level after acute and profound β-cell
destruction. Furthermore, these findings are supported by higher expression
of miR-375 levels in the circulation of T1D subjects, but not mature onset
diabetes of the young and T2D patients.
77
Altogether, the study suggests an
essential role for miR-375 in the maintenance of β-cell mass, and total
plasma miR-375 levels make this miRNA an unlikely biomarker for β-cell
function, but suggest a utility for the detection of acute β-cell death for auto-
immune diabetes.
An interesting meta-analysis on 38 miRNA expression profiling studies
selected some potent miRNAs as a biomarker for T2DM.
78
The top upre-
gulated miRNA in T2DM patients was miR-142-3p and the top down-
regulated miRNA was miR-126a. The dysregulation of miR-199a-3p
and miR-223 was highly pancreas-specific and liver-specific. miR-30e
was downregulated in patients with T2DM as well, while miR-92a was
downregulated in animal models of diabetes. Meta-analysis confirmed that
miR-29a, miR-34a, miR-375, miR-103, miR-107, miR-132, miR-142-
3p, and miR-144 are potential circulating biomarkers of type 2 diabetes. In
addition, miR-199a-3p and miR-223 are potential tissue biomarkers of
T2DM.
78
Obesity is also an age-related health complication and a serious risk factor
for many metabolic disorders, especially diabetes. Over the past decade, the
prevalence of obesity has increased dramatically across the world, especially
in developed countries.
83
Technically, obesity results from a chronic imbal-
ance between energy intake and energy expenditure. Recent studies have
proposed that miRNA expression is deregulated in obese patients, and
miRNAs are the potent regulator of many diseases related to obesity.
83
A study on 13 patients with type 2 diabetes, 20 obese patients, 16 obese
patients with type 2 diabetes, and 20 healthy controls detected three serum
miRNAs, miR-138, miR-15b, and miR-376a, that were found to have
potential as predictive biomarkers in obesity. miR-138 and miR-376a are
77miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

potential predictive tools for distinguishing obese patients from normal
healthy controls, diabetic patients, and obese diabetic patients. In addition,
the combination of miR-503 and miR-138 can be used to distinguish dia-
betic from obese diabetic patients.
116
Wen and colleagues identified miR-223 as a potent regulator of obesity.
A study on 121 subjects, including 41 normal weight, 40 overweight, and
40 obesity subjects quantified the miR-223 expression in the serum samples
by real-time PCR. The miR-223 expression was lower in both overweight
and obesity subjects compared with normal-weight control (1.06 vs 7.54,
P<0.001; 4.56 vs 7.54, P<0.001, respectively). However, after 3 months,
lifestyle intervention circulating miR-223 level was increased significantly in
both overweight and obese groups.
83
Taken together, we have demonstrated a group of diabetes-related cir-
culatory miRNAs with biomarker properties; however, several technical
and scientific obstacles need to be overcome for miRNAs to become a part
of the diagnostic arsenal to identify individuals with diabetes mellitus and its
devastating complications.
4.8 Hypertension
Hypertension is a leading cause of cardiovascular disease, including CAD,
HF, chronic kidney disease, peripheral vascular disease, and stroke.
117
Idi-
opathic pulmonary hypertension (IPAH) is a rare disease characterized by
a progressive increase in pulmonary vascular resistance leading to HF.
Serum microarray expression profiling of circulating miRNAs in
12 well-characterized IPAH patients and 10 healthy volunteer showed sig-
nificant changes in 61 miRNAs. Nine miRNAs (miR-1-2, miR-1957,
miR-20a, miR-145, miR-27a, miR-23a, miR-23b, miR-191, and miR-
130) were upregulated, whereas six miRNAs (miR-30c-2, miR-99a,
miR-328, miR-199a, miR-330, and miR-204) were downregulated
(Table 1). However, the important one was miR-23a because it was corre-
lated with the patients’ pulmonary function as well as controlling the expres-
sion of 17% of the significantly changed mRNAs including PGC1α, which
was recently associated with the progression of IPAH. Furthermore, the
silencing of miR-23a leads to an increase of PGC1αexpression.
84
Parthenakis and colleagues evaluated the overexpression of six miRNAs,
miR-1, miR-133a, miR-26b, miR-208b, miR-499, and miR-21, in periph-
eral blood of patients with well-controlled essential hypertension in relation
to arterial stiffness.
86
However, after 1 year of effective antihypertensive
78 S. Kumar et al.
Author's personal copy

therapy, only the miR-21 level showed a significant decrease in patients, and
it was correlated with changes in both carotid femoral pulse wave velocity
(cfPWV) and carotid radial pulse wave velocity (crPWV) independent of
blood pressure levels (r¼0.56 and r¼0.46, respectively; P<0.001 for
both).
86
Furthermore, low levels of miR-21 are strongly associated with
an improvement in arterial stiffness in patients with well-controlled essential
hypertension, independent of their blood pressure levels.
Kontaraki and colleagues evaluated the expression of miR-9 and miR-
126 in 60 patients with untreated essential hypertension and in 29 healthy
individuals. qRT-PCR analysis of PBMCs RNA showed significantly
lower miR-9 and miR-126 (P<0.001) expression levels in hypertensive
patients compared with healthy controls (Table 1). Interestingly, miR-9
levels showed a significant positive correlation with the left ventricular mass
index. Furthermore, both miR-9 and miR-126 expression levels showed
significant positive correlations with the 24-h mean pulse pressure (PP) in
hypertensive patients.
87
A further study on 102 patients with essential hypertension and
30 healthy individuals showed the deregulation of six miRNAs’ expression
in PBMCs by qRT-PCR. Hypertensive patients showed significantly lower
level of miR-133a and miR-26b, and higher expression of miR-1, miR-
208b, miR-499, and miR-21 compared with healthy controls. Essentially,
significant negative correlations in miR-1 and miR-133a were observed
with the left ventricular mass index, while miR-208b, miR-26b, miR-
499, and miR-21 expression showed a positive correlation with this index.
88
Plasma miR-92a expression was analyzed in 240 participants, including
60 healthy volunteers with normal carotid intima-media thickness
(nCIMT), 60 healthy volunteers with increased CIMT (iCIMT), 60 hyper-
tensive patients with nCIMT, and 60 hypertensive patients with iCIMT by
qRT-PCR.
85
miR-92a expression was significantly lowered (24.59 1.30
vs 27.76 2.13 vs 29.29 1.89 vs 33.76 2.08; P<0.001) in healthy con-
trols with nCIMT, followed by healthy controls with iCIMT, then hyper-
tensive patients with nCIMT and the highest expression in hypertensive
patients with iCIMT (Table 1). miR-92a levels also showed a significant
positive correlation with 24-h mean systolic BP, 24-h mean diastolic BP,
24-h mean PP, 24-h daytime PP, 24-h nighttime PP, CIMT, and cfPWV.
85
This evidence suggests that possibilities of circulating miR-92a represent a
potential noninvasive atherosclerosis marker in essential hypertensive
patients. Thus, results point to the utility of circulating miRNA expression
as a biomarker of disease progression.
79miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

4.9 Neurodegenerative Diseases
The most common NDs include AD, mild cognitive impairment (MCI),
PD, ALS, and HD. Besides AD, very few studies have demonstrated the
potential role of miRNAs as a noninvasive biomarker in other NDs.
miRNA profiling of whole blood samples of PD patients showed the down-
regulation of miR-1, miR-22p, and miR-29a in the patients compared to
controls, and differential expression of miRNAs signatures such as miR-16-
2-3p, miR-26a-2-3p, and miR-30a were used to differentiate between
treated and untreated patients (Table 1).
118
Furthermore, miRNA analysis
of plasma samples indicated upregulation of miR-181c, miR-331-5p,
miR-193a-5p, miR-196b, miR-454, miR-125a-3p, and miR-137 in PD
patients.
119
In 2014, a study conducted by Batta-Orfila and colleagues indi-
cated significant suppression of miR-19b, miR-29a, and miR-29c in the
serum samples of PD patients.
120
ALS is also a fatal neurodegenerative dis-
ease that progressively weakens neuronal cells that leads to degeneration of
upper and lower motor neurons.
104
A study on the SOD1-G93A mice, an
ALS mouse model, showed upregulation of miR-206 in skeletal muscle and
plasma through microarrays analysis. Even human ALS patients’ serum sam-
ples also revealed upregulation of miR-206.
104
A recent study by Takahashi
and colleagues covered the plasma samples of two cohort of ALS patients: (1)
ALS patients (n¼16) and healthy controls (n¼10); and (2) 48 ALS patients
(n¼48), healthy controls (n¼47), and disease controls (n¼30), the discov-
ery through microarray analysis and validation by qRT-PCR found the
upregulation of miR-4649-5, and downregulation of miR-4299 in patients
compared to controls (Table 1).
103
4.9.1 Dementia
MCI is a syndrome characteristic of early stages of many NDs. Recently, we
have identified two sets of circulating brain-enriched miRNAs: the miR-
132 family (miR-128, miR-132, and miR-874) normalized per miR-
491-5p and the miR-134 family (miR-134, miR-323-3p, and miR-382)
normalized per miR-370, capable of differentiating MCI from age-matched
control with high accuracy (Table 1). Here, we report a biomarker valida-
tion study of the identified miRNA pairs using larger independent sets of
age- and gender-matched plasma samples. Biomarker pairs detected MCI
with sensitivity, specificity, and overall accuracy similar to those obtained
in the first study. The miR-132 family biomarkers differentiated MCI from
AMC with 84%–94% sensitivity and 96%–98% specificity, and the miR-134
80 S. Kumar et al.
Author's personal copy

family biomarkers demonstrated 74%–88% sensitivity and 80%–92% speci-
ficity. When miRNAs of the same family were combined, miR-132 and
miR-134 family biomarkers demonstrated 96% and 87% overall accuracy,
respectively.
89
4.9.2 Alzheimer’s Disease
AD is the most important age-related neurological disorder and occurs in
elderly individuals. AD pathogenesis is associated with gradual loss of neu-
rons, synapses and synaptic function, abnormalities in mitochondrial func-
tion, and inflammatory responses.
121
In order to search for the miRNAs as a
promising biomarker to monitor the AD pathogenesis particularly in its
presymptomatic state, very few reports are available on human biofluid sam-
ples such as serum, plasma, CSF, and exosomes derived from serum and
plasma as well (Table 1). Most of the studies were conducted on human sub-
jects having MCI and AD dementia, and an almost equal number of healthy
controls were also included.
4.9.2.1 Circulatory miRNAs in Whole Blood
Human blood is the most vital and widely used specimen for human disease
assessments, and its testing is also minimally invasive. Reanalysis of a publi-
cally available small RNA-Seq data set identified differential expression of
27 miRNAs in 48 AD patients and 22 normal subjects.
91
Whole-blood spec-
imens were analyzed for miRNAs expression by single-end sequencing
on Hiseq 2000 (Illumina). Thirteen miRNAs (miR-26b-3p, miR-28-3p,
miR-30c-5p, miR-30d-5p, miR-148b-5p, miR-151a-3p, miR-186-5p,
miR-425-5p, miR-550a-5p, miR-1468, miR-4781-3p, miR-5001-3p, and
miR-6513-3p) were upregulated and 14 miRNAs (let-7a-5p, let-7e-5p,
let-7f-5p, let-7g-5p, miR-15a-5p, miR-17-3p, miR-29b-3p, miR-98–5p,
miR-144-5p, miR-148a-3p, miR-502-3p, miR-660-5p, miR-1294, and
miR-3200-3p) were found to be downregulated in AD patients compared
to controls (Table 1). Further, ROC curve analysis revealed a significant
discrimination potential of these 27 miRNAs for AD and controls.
91
However, the potential role of these miRNAs needs to be established in a large
population group.
4.9.2.2 Blood Mononuclear Cells as a Source of miRNAs
Early studies on blood mononuclear cells (BMCs) as a source of circulatory
miRNAs were conducted by Schipper and colleagues on 16 AD cases and
16 negative controls. Expression profiling of RNA samples showed
81miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

significant upregulation of miR-34a and miR-181b through microarray
and qRT-PCR analysis.
90
However, the study did not reveal these miRNAs
as a biomarker for AD, because the BMCs could not be good sources for
cell-free miRNAs. However, this study generated information about the
augmented level of miRNA expression in BMC and identification of
putative gene targets of these miRNAs and their probable role in AD
pathogenesis.
4.9.2.3 Serum as Sources of Circulatory miRNAs
Circulatory miRNAs as a biomarker for AD were mostly studied on the
patients’ sera samples (Table 1), as serum is considered to be the most suitable
and gentle circulatory biofluid and an appropriate source for cell-free
miRNAs. A study on seven AD patients and seven healthy controls sera
showed downregulation of five miRNA candidates, miR-137, miR-
181c, miR-9, miR-19a, miR-29b, by qRT-PCR when compared to neg-
ative controls.
94
Opposite to upregulation of miR-181b in BMCs, the sera
level of miR-181c was downregulated in AD patients. In the same direction,
Galimberti and colleagues also investigated serum samples from a cohort
consisting of 7 AD and 6 noninflammatory neurological disease control
(NINDC) subjects. The eighty-four most abundantly expressed miRNAs
were analyzed by a miRNA PCR array. The results showed a significant
downregulation of miR-125b, miR-223, miR-23a, and miR-26b in AD
compared to negative controls (Table 1).
93
This was further validated in a
large cohort of 15 AD, 12 NINDCs, 8 inflammatory neurological disease
controls (INDCs), and 10 frontotemporal dementia, demonstrating signifi-
cant downregulation only in miR-125b, miR-23a, and miR-26b in AD
patients. Additionally, expression analysis of these miRNAs in CSF of
AD and NINDCs showed a low level of only miR-125b and miR-26b
in AD patients. Interestingly, miR-26b also showed a negative correlation
with tau and Ptau protein level in AD patients. Even ROC curve analysis
showed a significant AUC value (0.77) for miR-26b; however, the miR-
125b AUC value was more accurate (0.82). This observation further sub-
stantiates the diagnostic potential of miR-26b and miR-125b to distinguish
AD from NINDCs.
93
A different study on the serum samples also indicated downregulation of
miR-125b in AD patients compared to healthy controls with more signif-
icant AUC values (0.85, P0.0001).
95
Downregulation of miR-181c and
upregulation of miR-9 were also observed in AD patients by qRT-PCR
analysis. Significant AUC values of miR-181c and miR-9 (0.74 and 0.62,
82 S. Kumar et al.
Author's personal copy

respectively) also revealed their importance as biomarkers for AD.
95
How-
ever, the main drawback of such studies was the small population cohort and
these were very preliminary data; hence, these observations need to be
replicated in a larger population.
Tan and colleagues conducted an important genome-wide expression
profiling of serum miRNAs on AD patients.
96
Their study included primary
screening of cohort 1: AD (n¼50) and negative control (n¼50), through
high-throughput sequencing of miRNAs.
96
Unique miRNAs were identi-
fied and examined against the miRbase database Release 19, which detected
only 90 miRNAs that were found to be significantly modulated in probable
AD patients. Out of only 96, miR-36 is identified as novel miRNA as it
was not listed on miRbase-19. The authors chose another 14 ideal miRNAs
that were expressed differentially (twofold) in AD patients and controls.
Among them four miRNAs (miR-3158-3p, miR-27a-3p, miR-26b-3p,
and miR-151b) were upregulated, whereas 10 miRNAs (miR-36, miR-
98-5p, miR-885-5p, miR-485-5p, miR-483-3p, miR-342-3p, miR-
30e-5p, miR-191-5p, let-7g-5p, and let-7d-5p) were downregulated in
AD patients compared to controls.
96
Further validation by qRT-PCR on
a large cohort (2) AD (n¼158) and negative control (n¼155) revealed
downregulation of only six miRNAs (miR-483-3p, miR-342-3p, miR-
98-5p, miR-191-5p, miR-885-5p, and let-7d-5p) in the probable AD
patients (Table 1). Additionally, ROC curve analysis of these miRNAs indi-
cated the highest diagnostic accuracy of miR-342-3p with an AUC value of
0.84, and a cut-off value of 0.93.
96
Such studies are recommended to expand
on a large cohort in a different ethnic population for better investigation of
the disease.
Dong and colleagues examined the AD patients, individuals with MCI,
and vascular dementia (VD) along with nondemented controls for the
expression profiling of serum miRNAs by Solexa sequencing and
qRT-PCR analysis.
97
Four miRNAs (miR-31, miR-93, miR-143, and
miR-146a) were downregulated in the AD patients compared to controls
in both the discovery and the validation set (Table 1). AUC values (0.72,
0.69, 0.70, and 0.70, respectively) through ROC curve analysis also showed
their significant discriminating power to AD patients from controls. How-
ever, expression of these miRNAs was not consistent in MCI and VD indi-
viduals where miR-93 and miR-146a were significantly elevated in MCI
cases, whereas miR-143 expression was decreased and miR-31 showed
no change compared to controls. In VD cases, miR-143 expression was
decreased, and miR-31, miR-93, and miR-146a levels were significantly
83miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

higher compared to controls.
97
Hence, differential expression of these
miRNAs can discriminate AD cases, but their ambiguous expression pattern
in MCI and VD cases could not explain the AD progression.
4.9.2.4 Serum Exosomal miRNAs
Analysis of serum exosomes for circulatory miRNA detection is supposed to
be more feasible and more fertile than whole serum. Exosomes, the cargo,
may offer an enriched population of miRNAs that were found to be free
from endogenous RNA contaminants, e.g., ribosomal RNA. Hence, exo-
somes may be considered a prominent house of disease-specific miRNA sig-
natures.
27
Exosomes were prepared from serum samples of AD patients,
MCI individuals, and healthy controls using specific kits, and were processed
for sequencing analysis of miRNAs differentially expressed in three groups.
An initial screening of first cohort indicated a significant upregulation of 14
miRNAs (miR-361-5p, miR-30e-5p, miR-93-5p, miR-15a-5p, miR-
143-3p, miR-335-5p, miR-106b-5p, miR-101-3p, miR-425-5p, miR-
106a-5p, miR-18b-5p, miR-3065-5p, miR-20a-5p, and miR-582-5p)
and downregulation of three miRNAs (miR-1306-5p, miR-342-3p, and
miR-15b-3p) (Table 1). Validation analysis through qRT-PCR of the sec-
ond cohort also confirmed the above observations, though the study lacked a
ROC curve analysis of deregulated miRNAs for diagnostic accuracy. Anal-
ysis of exosomal miRNA profiling is also a good approach in order to look
for disease-specific miRNA signatures for AD.
4.9.2.5 Plasma as Sources of Circulatory miRNAs
Blood-based plasma samples are another important sources of circulatory
miRNAs. Plasma is the largest component of human blood, making up
about 55% of its overall content. Important constituents of blood plasma
are immunoglobulins (antibodies), clotting factors, proteins albumin and
fibrinogen, enzymes, and water. The main function of plasma is the trans-
portation of cellular nutrients, hormones, and proteins to the different parts
of the body. Kumar and colleagues did a plasma miRNAs analysis in AD,
MCI, and healthy control patients. Primary testing by nCounter miRNA
assay (Nanostring Technology, Seattle, WA, USA) of cohort 1 revealed
upregulation of six miRNAs, miR-323b-5p, miR-545-3p, miR-563,
miR-600, miR-1274a, and miR-1975, and downregulation of seven
miRNAs, let-7d-5p, let-7g-5p, miR-15b-5p, miR-142-3p, miR-191-
5p, miR-301a-3p, and miR-545-3p, was also observed in AD and MCI
cases compared to healthy controls (Table 1). Validation studies on cohort
84 S. Kumar et al.
Author's personal copy

2 confirm the downregulation of these miRNAs expression in AD patients
by qRT-PCR. However, none of the miRNA candidate showed
upregulation by qRT-PCR analysis.
98
ROC curve analysis showed that
the best miRNA signatures were miR-142-3p and miR-301a-3p, having
100% specificity. However, miR-142-3p has better sensitivity (0.65) than
miR-301a-3p (0.25). The combination of two different miRNAs (miR-
545-3p and miR-15b) also displayed very significant diagnostic accuracy
with AUC value ¼0.96, sensitivity ¼0.9, and specificity ¼0.94. However,
based on AUC value, sensitivity, and specificity, the best-characterized indi-
vidual miRNAs were miR-191-5p, miR-15b-5p, and let-7d-5p.
98
Further-
more, to diagnose the AD at a preclinical early stage, the next step would be
needed to analyze the longitudinal plasma samples from a large cohort having
the MCI.
4.9.2.6 Plasma Exosomal miRNAs
Like serum, plasma exosomes also transport the disease-associated miRNAs.
A recent study by Lugli and colleagues identified differential expression of
plasma exosomal miRNAs in AD patients and controls upon Illumina deep
sequencing.
99
A total of 20 miRNAs were found to be deregulated, where
four (miR-548at-5p, miR-138-5p, miR-5001-3p, and miR-659-5p) were
upregulated and seven (miR-185-5p, miR-342-3p, miR-141-3p, miR-
342-5p, miR-23b-3p, miR-338-3p, and miR-3613-3p) were significantly
downregulated in AD patients compared to controls (Table 1). Among
those, miR-242-3p was more interesting because its brain-enriched nature
and its expression was reduced at a more significant level, as confirmed
by a t-test.
99
4.9.2.7 CSF and Extracellular Fluid Circulatory miRNAs
CSF is a clear biofluid, secreted by the choroid plexus and circulates into the
brain ventricles across the blood–brain barrier. CSF plays important role in
intercerebral transportation.
27
CSF is collected from the brain by a sophis-
ticated lumber puncture procedure. Studies showed that CSF is also a source
of circulatory miRNAs for assessment of neurological and neurodegene-
rative disorders. Microarray analysis of both CSF and extracellular fluid
(ECF) samples indicated significant overexpression of miR-9, miR-125b,
miR-146a, and miR-155 in AD cases compared to healthy controls
(Table 1).
92
A recent study on CSF samples from a large cohort of AD
(n¼22) and healthy controls (n¼28) identified 1178 miRNAs by Open
Array qRT-PCR.
2
Analysis showed upregulation of seven miRNAs
85miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

(miR-146a, miR-100, miR-505, miR-4467, miR-766, miR-3622b-3p,
miR-296) and downregulation of eight miRNAs (miR-449, miR-1274a,
miR-4674, miR-335, miR-375, miR-708, miR-219, and miR-103) in
AD patients compared to controls (Table 1). The diagnostic accuracy of
these miRNAs was measured by ROC curve analysis, but only miR-146a,
miR-375, miR-103, and miR-100 showed significant AUC values (0.97,
0.99, 0.87, and 0.72, respectively).
2
Expression analysis of miRNAs in
CSF and ECF also provides the informative biomarkers that can be used
to compare and detect AD from heterogeneous controls.
As discussed earlier, multiple serum/plasma/exosomal miRNAs have
been identified, and these miRNA may be useful in determining circulatory
biomarkers for AD. Furthermore, screening of large population with differ-
ent stages of disease progression is needed with a universally standardized
procedure.
4.9.3 Huntington’s Disease
Huntington’s disease (HD) is an inherited neurodegenerative disorder
which is caused by an unstable CAG triplet expansion in the HD gene,
encoding for a polyglutamine tract in the huntingtin protein (HTT).
100
Cir-
culatory miRNA profile was analyzed in the plasma samples from 15 symp-
tomatic patients, with 40–45 CAG repeats in the HTT gene, and 7 healthy
matched controls. A total of 752 human mature miRNAs had sequences
against human miRNome panels. Further analysis showed alteration of
168 plasma miRNAs in symptomatic patients. However, statistical analysis
indicated significant upregulation of 13 miRNAs (miR-877-5p, miR-223-
3p, miR-223-5p, miR-30d-5p, miR-128, miR-22-5p, miR-222-3p,
miR-338-3p, miR-130b-3p, miR-425-5p, miR-628-3p, miR-361-5p,
and miR-942) in HD patients as compared with controls (Table 1).
100
4.9.4 Parkinson’s Disease
PD is the second most common neurodegenerative disorder in the United
States, affecting approximately 1 million Americans and 5 million people
worldwide.
122
Very few studies are available that identify several dysregu-
lated circulating miRNAs in PD patients. Recently, Dong and colleagues
identified novel circulating miRNAs by screening of 169 PD patients and
180 healthy controls by Solexa sequencing technology and qRT-PCR.
Analysis showed a significant decreased in four serum miRNAs (miR-
141, miR-214, miR-146b-5p, and miR-193a-3p) in PD patients compared
with controls (Table 1). This 4-miRNA panel could be used to differentiate
86 S. Kumar et al.
Author's personal copy

HY stage 1 and 2 PD patients from controls and thus may be novel bio-
markers for the early detection of PD.
101
The miRNA expression also varies in PD and similar atypical conditions
such as multiple system atrophy (MSA), and circulating miRNAs could be
used to distinguish PD patients from MSA and healthy individuals. Serum sam-
ples were processed by TaqMan Low Density Array technology, and 754
miRNAs were analyzed. The nine most significant circulatory miRNAs were
identified that expressed differentially in 25 PD and 25 MSA patients as com-
pared to 25 controls. However, a validation study found four more specific
miRNAs: three were upregulated miR-223∗, miR-324-3p, and miR-24,
whereas miR-339-5p was downregulated in both diseases (Table 1). Specifi-
cally, miR-30c and miR-148b were downregulated in PD and miR-148b was
upregulated in MSA. However, comparison of MSA and PD showed three
upregulated miRNAs (miR-24, miR-34b, and miR-148b) in MSA serum.
102
4.9.5 Amyotrophic Lateral Sclerosis
Amyotrophic Lateral Sclerosis (ALS) is a lethal motor neuron disease that
progressively debilitates neuronal cells that control voluntary muscle activ-
ity.
104
miRNAs from the plasma of sporadic amyotrophic lateral sclerosis
(sALS) patients and healthy controls were analyzed using two cohorts: a
discovery cohort analyzed with microarray (16 sALS patients and
10 healthy controls) and a validation cohort confirmed with qPCR (48
sALS patients, 47 healthy controls, and 30 disease controls). Three
miRNAs were upregulated: miR-4258, miR-663b, and miR-4649-5p,
whereas six were downregulated significantly: miR-26b-5p, miR-4299,
let-7f-5p, miR-4419a, miR-3187-5p, and miR-4496 in the discovery
cohort (Table 1). Interestingly, upregulation of miR-4649-5p and down-
regulation of miR-4299 was not influenced by clinical characteristics,
hence they have the potential to be ALS diagnosis biomarkers.
103
To find biomarkers for ALS, miRNA alterations were studied in skeletal
muscle and plasma of mutated human superoxide dismutase 1 (SOD1-G93A)
mice, and subsequently miRNAs levels were tested in the serum from human
ALS patients. Muscles tissues from symptomatic SOD1-G93A mice (age
90 days) and their control littermates were first studied using miRNA micro-
arrays, and then evaluated with quantitative PCR from five age groups from
neonatal to the terminal disease stage (10–120 days). The only miR-206 was
found to be consistently altered in relative to various age/gender/muscle
groups and during the course of the disease pathology.
104
87miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

5. CONCLUDING REMARKS
To date, accumulating evidence has shown that changes in serum/
plasma/CSF/ECF/urine and other biofluids’ miRNA levels are correla-
ted with certain biological conditions such as aging and aging-related dis-
eases including CVD, cancer, arthritis, dementia, cataract, osteoporosis,
diabetes, hypertension, and NDs. Specific cellular and molecular changes
in miRNA transcription levels or at miRNA secretory levels have been
linked to the development and progression of human diseases. Experi-
mental observations indicate their novel informative biomarkers nat-
ure and/or therapeutic targets with higher sensitivity and specificity for
such diseases. Nevertheless, potential biomarker applications will require
a more refined understanding of the mechanisms regarding how circula-
tory miRNAs are changing with disease development and progression.
Additionally, the analysis of circulatory miRNAs as a biomarker has seve-
ral preanalytical as well as analytical challenges during application. There-
fore, some strengths and weaknesses still exist in the path of miRNAs
as a futuristic biomarker (Fig. 3). To overcome these challenges, more
population-based studies with constant analytical standardization is fur-
ther recommended to decide the clinical utility of miRNAs in the man-
agement of aging diseases.
Fig. 3 Summary of strengths and weaknesses of circulatory miRNAs as biomarkers in
human diseases.
88 S. Kumar et al.
Author's personal copy

ACKNOWLEDGMENTS
P.H.R. is supported by NIH Grants AG042178, AG047812, and the Garrison Family
Foundation.
REFERENCES
1. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell.
2004;116(2):281–297.
2. Denk J, Boelmans K, Siegismund C, Lassner D, Arlt S, Jahn H. MicroRNA profiling of
CSF reveals potential biomarkers to detect Alzheimer‘s disease. PLoS One. 2015;10(5):
e0126423.
3. Kumar S, Chawla YK, Ghosh S, Chakraborti A. Severity of hepatitis C virus (geno-
type-3) infection positively correlates with circulating microRNA-122 in patients sera.
Dis Markers. 2014;2014:435476.
4. Li Y, Kowdley KV. MicroRNAs in common human diseases. Genomics Proteomics
Bioinformatics.2012;10(5):246–253.
5. ZhangL,XuY,JinX,etal.Acirculating miRNA signature as a diagnostic bio-
marker for non-invasive early detection of breast cancer. Breast Cancer Res Treat.
2015;154(2):423–434.
6. Schwarzenbach H, Nishida N, Calin GA, Pantel K. Clinical relevance of circulating
cell-free microRNAs in cancer. Nat Rev Clin Oncol. 2014;11(3):145–156.
7. Noren Hooten N, Fitzpatrick M, Wood 3rd WH, et al. Age-related changes in micro-
RNA levels in serum. Aging (Albany, NY). 2013;5(10):725–740.
8. Vijayan M, Reddy PH. Peripheral biomarkers of stroke: focus on circulatory micro-
RNAs. Biochim Biophys Acta. 2016;1862(10):1984–1993.
9. Reddy PH, Tonk S, Kumar S, et al. A critical evaluation of neuroprotective and
neurodegenerative microRNAs in Alzheimer’s disease. Biochem Biophys Res Commun.
2016 [Epub ahead of print].
10. Kumar S, Reddy PH. Are circulating microRNAs peripheral biomarkers for
Alzheimer’s disease? Biochim Biophys Acta. 2016;1862(9):1617–1627.
11. Femminella GD, Ferrara N, Rengo G. The emerging role of microRNAs in
Alzheimer’s disease. Front Physiol. 2015;6:40.
12. Harries LW. MicroRNAs as mediators of the ageing process. Genes (Basel).
2014;5(3):656–670.
13. Kawahara Y. Human diseases caused by germline and somatic abnormalities in micro-
RNA and microRNA-related genes. Congenit Anom (Kyoto). 2014;54(1):12–21.
14. Machida T, Tomofuji T, Ekuni D, et al. MicroRNAs in salivary exosome as potential
biomarkers of aging. Int J Mol Sci. 2015;16(9):21294–21309.
15. Grasso M, Piscopo P, Confaloni A, Denti MA. Circulating miRNAs as biomarkers for
neurodegenerative disorders. Molecules. 2014;19(5):6891–6910.
16. Jung HJ, Suh Y. Circulating miRNAs in ageing and ageing-related diseases. JGenet
Genomics.2014;41(9):465–472.
17. Creemers EE, Tijsen AJ, Pinto YM. Circulating microRNAs: novel biomarkers and
extracellular communicators in cardiovascular disease? Circ Res. 2012;110(3):483–495.
18. Bao Q, Pan J, Qi H, et al. Aging and age-related diseases—from endocrine therapy to
target therapy. Mol Cell Endocrinol. 2014;394(1–2):115–118.
19. Childs BG, Durik M, Baker DJ, van Deursen JM. Cellular senescence in aging and
age-related disease: from mechanisms to therapy. Nat Med. 2015;21(12):1424–1435.
20. Weber JA, Baxter DH, Zhang S, et al. The microRNA spectrum in 12 body fluids. Clin
Chem.2010;56(11):1733–1741.
89miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

21. Ardila-Molano J, Vizcaı
´no M, Serrano ML. Circulating microRNAs as potential
cancer biomarkers. Rev Colomb Cancerol. 2015;19(4):229–238.
22. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated
transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange
between cells. Nat Cell Biol. 2007;9(6):654–659.
23. Turchinovich A, Samatov TR, Tonevitsky AG, Burwinkel B. Circulating miRNAs:
cell-cell communication function? Front Genet. 2013;4:119.
24. Arroyo JD, Chevillet JR, Kroh EM, et al. Argonaute2 complexes carry a population of
circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci
USA. 2011;108(12):5003–5008.
25. Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT. MicroRNAs
are transported in plasma and delivered to recipient cells by high-density lipoproteins.
Nat Cell Biol. 2011;13(4):423–433.
26. Keller S, Sanderson MP, Stoeck A, Altevogt P. Exosomes: from biogenesis and secre-
tion to biological function. Immunol Lett. 2006;107(2):102–108.
27. Cheng L, Doecke JD, Sharples RA, et al. Prognostic serum miRNA biomarkers
associated with Alzheimer’s disease shows concordance with neuropsychological and
neuroimaging assessment. Mol Psychiatry. 2015;20(10):1188–1196.
28. Boon RA, Vickers KC. Intercellular transport of microRNAs. Arterioscler Thromb Vasc
Biol. 2013;33(2):186–192.
29. Zhang Y, Liu D, Chen X, et al. Secreted monocytic miR-150 enhances targeted endo-
thelial cell migration. Mol Cell. 2010;39(1):133–144.
30. Zernecke A, Bidzhekov K, Noels H, et al. Delivery of microRNA-126 by apoptotic
bodies induces CXCL12-dependent vascular protection. Sci Signal. 2009;2(100):ra81.
31. Kosaka N, Iguchi H, Yoshioka Y, Takeshita F, Matsuki Y, Ochiya T. Secretory
mechanisms and intercellular transfer of microRNAs in living cells. JBiolChem.
2010;285(23):17442–17452.
32. Smith-Vikos T, Slack FJ. MicroRNAs circulate around Alzheimer’s disease. Genome
Biol. 2013;14(7):125.
33. Dimmeler S, Nicotera P. MicroRNAs in age-related diseases. EMBO Mol Med.
2013;5(2):180–190.
34. Sawada S, Akimoto T, Takahashi M, et al. Effect of aging and sex on circulating micro-
RNAs in humans. Adv Aging Res. 2014;3(2):152–159.
35. Ameling S, Kacprowski T, Chilukoti RK, et al. Associations of circulating plasma
microRNAs with age, body mass index and sex in a population-based study. BMC
Med Genomics. 2015;8:61.
36. Pang J, Xiong H, Yang H, et al. Circulating miR-34a levels correlate with age-related
hearing loss in mice and humans. Exp Gerontol. 2016;76:58–67.
37. Zhang H, Yang H, Zhang C, et al. Investigation of microRNA expression in human
serum during the aging process. J Gerontol A Biol Sci Med Sci. 2015;70(1):102–109.
38. Jiang Y, Wang HY, Li Y, Guo SH, Zhang L, Cai JH. Peripheral blood miRNAs as a
biomarker for chronic cardiovascular diseases. Sci Rep. 2014;4:5026.
39. Ren J, Zhang J, Xu N, et al. Signature of circulating microRNAs as potential
biomarkers in vulnerable coronary artery disease. PLoS One. 2013;8(12):e80738.
40. Ellis KL, Cameron VA, Troughton RW, Frampton CM, Ellmers LJ, Richards AM.
Circulating microRNAs as candidate markers to distinguish heart failure in breathless
patients. Eur J Heart Fail. 2013;15(10):1138–1147.
41. Hakimzadeh N, Nossent AY, van der Laan AM, et al. Circulating microRNAs char-
acterizing patients with insufficient coronary collateral artery function. PLoS One.
2015;10(9):e0137035.
42. Alhasan AH, Scott AW, Wu JJ, et al. Circulating microRNA signature for the diagnosis
of very high-risk prostate cancer. Proc Natl Acad Sci USA. 2016;113(38):10655–10660.
90 S. Kumar et al.
Author's personal copy

43. Huo D, Clayton WM, Yoshimatsu TF, Chen J, Olopade OI. Identification of a circu-
lating microRNA signature to distinguish recurrence in breast cancer patients.
Oncotarget. 2016;7(34):55231–55248.
44. Zhu W, Zhou K, Zha Y, et al. Diagnostic value of serum miR-182, miR-183, miR-
210, and miR-126 levels in patients with early-stage non-small cell lung cancer. PLoS
One. 2016;11(4):e0153046.
45. Halvorsen AR, Bjaanaes M, LeBlanc M, et al. A unique set of 6 circulating micro-
RNAs for early detection of non-small cell lung cancer. Oncotarget. 2016;7(24):
37250–37259.
46. Vychytilova-Faltejskova P, Radova L, Sachlova M, et al. Serum-based microRNA sig-
natures in early diagnosis and prognosis prediction of colon cancer. Carcinogenesis.
2016;37:941–950.
47. Hamam R, Ali AM, Alsaleh KA, et al. MicroRNA expression profiling on individual
breast cancer patients identifies novel panel of circulating microRNA for early detec-
tion. Sci Rep. 2016;6:25997.
48. Stuopelyte K, Daniunaite K, Bakavicius A, Lazutka JR, Jankevicius F, Jarmalaite S. The
utility of urine-circulating miRNAs for detection of prostate cancer. Br J Cancer.
2016;115(6):707–715.
49. Gao Y, Guo Y, Wang Z, et al. Analysis of circulating miRNAs 21 and 375 as potential
biomarkers for early diagnosis of prostate cancer. Neoplasma. 2016;63(4):623–628.
50. Chen H, Liu H, Zou H, et al. Evaluation of plasma miR-21 and miR-152 as diagnostic
biomarkers for common types of human cancers. J Cancer. 2016;7(5):490–499.
51. Su K, Zhang T, Wang Y, Hao G. Diagnostic and prognostic value of plasma micro-
RNA-195 in patients with non-small cell lung cancer. World J Surg Oncol.
2016;14(1):224.
52. Wang J, Ye H, Zhang D, et al. MicroRNA-410-5p as a potential serum biomarker for
the diagnosis of prostate cancer. Cancer Cell Int. 2016;16:12.
53. Freres P, Wenric S, Boukerroucha M, et al. Circulating microRNA-based screening
tool for breast cancer. Oncotarget. 2016;7(5):5416–5428.
54. Aherne ST, Madden SF, Hughes DJ, et al. Circulating miRNAs miR-34a
and miR-150 associated with colorectal cancer progression. BMC Cancer.
2015;15:329.
55. Li Y, Liu S, Zhang F, Jiang P, Wu X, Liang Y. Expression of the microRNAs hsa-miR-
15a and hsa-miR-16-1 in lens epithelial cells of patients with age-related cataract. Int
J Clin Exp Med. 2015;8(2):2405–2410.
56. Ogata-Kawata H, Izumiya M, Kurioka D, et al. Circulating exosomal microRNAs as
biomarkers of colon cancer. PLoS One. 2014;9(4):e92921.
57. Ma J, Li N, Lin Y, Gupta C, Jiang F. Circulating neutrophil microRNAs as biomarkers
for the detection of lung cancer. Biomark Cancer. 2016;8:1–7.
58. Antolin S, Calvo L, Blanco-Calvo M, et al. Circulating miR-200c and miR-141 and
outcomes in patients with breast cancer. BMC Cancer. 2015;15:297.
59. Yong FL, Law CW, Wang CW. Potentiality of a triple microRNA classifier:
miR-193a-3p, miR-23a and miR-338-5p for early detection of colorectal cancer.
BMC Cancer. 2013;13:280.
60. Filkova M, Aradi B, Senolt L, et al. Association of circulating miR-223 and miR-16
with disease activity in patients with early rheumatoid arthritis. Ann Rheum Dis.
2014;73(10):1898–1904.
61. Duroux-Richard I, Pers YM, Fabre S, et al. Circulating miRNA-125b is a potential
biomarker predicting response to rituximab in rheumatoid arthritis. Mediators Inflamm.
2014;2014:342524.
62. Wang W, Zhang Y, Zhu B, et al. Plasma microRNA expression profiles in Chinese
patients with rheumatoid arthritis. Oncotarget. 2015;6(40):42557–42568.
91miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

63. Hruskova V, Jandova R, Vernerova L, et al. MicroRNA-125b: association with disease
activity and the treatment response of patients with early rheumatoid arthritis. Arthritis
Res Ther. 2016;18(1):124.
64. Castro-Villegas C, Perez-Sanchez C, Escudero A, et al. Circulating miRNAs as poten-
tial biomarkers of therapy effectiveness in rheumatoid arthritis patients treated with
anti-TNFalpha. Arthritis Res Ther. 2015;17:49.
65. Chien KH, Chen SJ, Liu JH, et al. Correlation between microRNA-34a levels and lens
opacity severity in age-related cataracts. Eye (Lond). 2013;27(7):883–888.
66. Tanaka Y, Tsuda S, Kunikata H, et al. Profiles of extracellular miRNAs in the aqueous
humor of glaucoma patients assessed with a microarray system. Sci Rep. 2014;4:5089.
67. Wecker T, Hoffmeier K, Plotner A, et al. MicroRNA profiling in aqueous humor
of individual human eyes by next-generation sequencing. Invest Ophthalmol Vis Sci.
2016;57(4):1706–1713.
68. Cao Z, Moore BT, Wang Y, et al. MiR-422a as a potential cellular microRNA
biomarker for postmenopausal osteoporosis. PLoS One. 2014;9(5):e97098.
69. Wang Y, Li L, Moore BT, et al. MiR-133a in human circulating monocytes: a potential
biomarker associated with postmenopausal osteoporosis. PLoS One. 2012;7(4):e34641.
70. Kocijan R, Muschitz C, Geiger E, et al. Circulating microRNA signatures in patients
with idiopathic and postmenopausal osteoporosis and fragility fractures. J Clin
Endocrinol Metab. 2016;101(11):4125–4134.
71. Panach L, Mifsut D, Tarin JJ, Cano A, Garcia-Perez MA. Serum circulating micro-
RNAs as biomarkers of osteoporotic fracture. Calcif Tissue Int. 2015;97(5):495–505.
72. Seeliger C, Karpinski K, Haug AT, et al. Five freely circulating miRNAs and bone
tissue miRNAs are associated with osteoporotic fractures. JBoneMinerRes.
2014;29(8):1718–1728.
73. Meng J, Zhang D, Pan N, et al. Identification of miR-194-5p as a potential biomarker
for postmenopausal osteoporosis. PeerJ. 2015;3:e971.
74. Zhang T, Lv C, Li L, et al. Plasma miR-126 is a potential biomarker for early prediction
of type 2 diabetes mellitus in susceptible individuals. Biomed Res Int. 2013;2013:761617.
75. Assmann T, Coutinho M, Tschiedel B, Canani L, Crispim D. Circulating microRNAs
as biomarkers for type 1 diabetes mellitus. Diabetol Metab Syndr. 2015;7(suppl 1):A206.
76. Olivieri F, Bonafe M, Spazzafumo L, et al. Age- and glycemia-related miR-126-3p
levels in plasma and endothelial cells. Aging (Albany, NY). 2014;6(9):771–787.
77. Latreille M, Herrmanns K, Renwick N, et al. miR-375 gene dosage in pancreatic
beta-cells: implications for regulation of beta-cell mass and biomarker development.
J Mol Med. 2015;93(10):1159–1169.
78. Zhu H, Leung SW. Identification of microRNA biomarkers in type 2 diabetes: a
meta-analysis of controlled profiling studies. Diabetologia. 2015;58(5):900–911.
79. Al-Kafaji G, Al-Mahroos G, Alsayed NA, Hasan ZA, Nawaz S, Bakhiet M. Peripheral
blood microRNA-15a is a potential biomarker for type 2 diabetes mellitus and
pre-diabetes. Mol Med Rep. 2015;12(5):7485–7490.
80. Kong L, Zhu J, Han W, et al. Significance of serum microRNAs in pre-diabetes and
newly diagnosed type 2 diabetes: a clinical study. Acta Diabetol. 2011;48(1):61–69.
81. Wang C, Wan S, Yang T, et al. Increased serum microRNAs are closely associated with
the presence of microvascular complications in type 2 diabetes mellitus. Sci Rep.
2016;6:20032.
82. Nielsen LB, Wang C, Sorensen K, et al. Circulating levels of microRNA from children
with newly diagnosed type 1 diabetes and healthy controls: evidence that miR-25 asso-
ciates to residual beta-cell function and glycaemic control during disease progression.
Exp Diabetes Res. 2012;2012:896362.
83. Wen D, Qiao P, Wang L. Circulating microRNA-223 as a potential biomarker for
obesity. Obes Res Clin Pract. 2015;9(4):398–404.
92 S. Kumar et al.
Author's personal copy

84. Sarrion I, Milian L, Juan G, et al. Role of circulating miRNAs as biomarkers in idio-
pathic pulmonary arterial hypertension: possible relevance of miR-23a. Oxid Med Cell
Longev. 2015;2015:792846.
85. Huang Y, Tang S, Ji-Yan C, et al. Circulating miR-92a expression level in patients
with essential hypertension: a potential marker of atherosclerosis. J Hum Hypertens.
2016. [Epub ahead of print].
86. Parthenakis F, Marketou M, Kontaraki J, et al. Low levels of microRNA-21 are a
marker of reduced arterial stiffness in well-controlled hypertension. J Clin Hypertens
(Greenwich). 2016. [Epub ahead of print].
87. Kontaraki JE, Marketou ME, Zacharis EA, Parthenakis FI, Vardas PE. MicroRNA-9
and microRNA-126 expression levels in patients with essential hypertension: potential
markers of target-organ damage. J Am Soc Hypertens. 2014;8(6):368–375.
88. Kontaraki JE, Marketou ME, Parthenakis FI, et al. Hypertrophic and antihypertrophic
microRNA levels in peripheral blood mononuclear cells and their relationship to left
ventricular hypertrophy in patients with essential hypertension. J Am Soc Hypertens.
2015;9(10):802–810.
89. Sheinerman KS, Tsivinsky VG, Abdullah L, Crawford F, Umansky SR. Plasma micro-
RNA biomarkers for detection of mild cognitive impairment: biomarker validation
study. Aging (Albany, NY). 2013;5(12):925–938.
90. Schipper HM, Maes OC, Chertkow HM, Wang E. MicroRNA expression in
Alzheimer blood mononuclear cells. Gene Regul Syst Bio. 2007;1:263–274.
91. Satoh J, Kino Y, Niida S. MicroRNA-seq data analysis pipeline to identify blood bio-
markers for Alzheimer’s disease from public data. Biomark Insights. 2015;10:21–31.
92. Alexandrov PN, Dua P, Hill JM, Bhattacharjee S, Zhao Y, Lukiw WJ. MicroRNA
(miRNA) speciation in Alzheimer’s disease (AD) cerebrospinal fluid (CSF) and extra-
cellular fluid (ECF). Int J Biochem Mol Biol. 2012;3(4):365–373.
93. Galimberti D, Villa C, Fenoglio C, et al. Circulating miRNAs as potential biomarkers
in Alzheimer’s disease. J Alzheimers Dis. 2014;42(4):1261–1267.
94. Geekiyanage H, Jicha GA, Nelson PT, Chan C. Blood serum miRNA: non-invasive
biomarkers for Alzheimer’s disease. Exp Neurol. 2012;235(2):491–496.
95. Tan L, Yu JT, Liu QY, et al. Circulating miR-125b as a biomarker of Alzheimer’s
disease. J Neurol Sci. 2014;336(1–2):52–56.
96. Tan L, Yu JT, Tan MS, et al. Genome-wide serum microRNA expression profiling
identifies serum biomarkers for Alzheimer’s disease. J Alzheimers Dis. 2014;40(4):
1017–1027.
97. Dong H, Li J, Huang L, et al. Serum microRNA profiles serve as novel biomarkers for
the diagnosis of Alzheimer’s disease. Dis Markers. 2015;2015:625659.
98. Kumar P, Dezso Z, MacKenzie C, et al. Circulating miRNA biomarkers for
Alzheimer’s disease. PLoS One. 2013;8(7):e69807.
99. Lugli G, Cohen AM, Bennett DA, et al. Plasma exosomal miRNAs in persons with and
without Alzheimer disease: altered expression and prospects for biomarkers. PLoS One.
2015;10(10):e0139233.
100. Diez-Planelles C, Sanchez-Lozano P, Crespo MC, et al. Circulating microRNAs in
Huntington’s disease: emerging mediators in metabolic impairment. Pharmacol Res.
2016;108:102–110.
101. Dong H, Wang C, Lu S, et al. A panel of four decreased serum microRNAs as a novel
biomarker for early Parkinson’s disease. Biomarkers. 2016;21(2):129–137.
102. Vallelunga A, Ragusa M, Di Mauro S, et al. Identification of circulating microRNAs
for the differential diagnosis of Parkinson’s disease and multiple system atrophy. Front
Cell Neurosci. 2014;8:156.
103. Takahashi I, Hama Y, Matsushima M, et al. Identification of plasma microRNAs as a
biomarker of sporadic amyotrophic lateral sclerosis. Mol Brain. 2015;8(1):67.
93miRNAs as Potential Biomarkers for Human Diseases
Author's personal copy

104. Toivonen JM, Manzano R, Olivan S, Zaragoza P, Garcia-Redondo A, Osta R. Micro-
RNA-206: a potential circulating biomarker candidate for amyotrophic lateral sclerosis.
PLoS One. 2014;9(2):e89065.
105. He Y, Lin J, Kong D, et al. Current state of circulating microRNAs as cancer
biomarkers. Clin Chem. 2015;61(9):1138–1155.
106. Ma R, Jiang T, Kang X. Circulating microRNAs in cancer: origin, function and appli-
cation. J Exp Clin Cancer Res. 2012;31:38.
107. Khoury S, Tran N. Circulating microRNAs: potential biomarkers for common malig-
nancies. Biomark Med.2015;9(2):131–151.
108. Hansen J. Common cancers in the elderly. Drugs Aging.1998;13(6):467–478.
109. Shen J, Hruby GW, McKiernan JM, et al. Dysregulation of circulating microRNAs and
prediction of aggressive prostate cancer. Prostate. 2012;72(13):1469–1477.
110. Hollis M, Nair K, Vyas A, Chaturvedi LS, Gambhir S, Vyas D. MicroRNAs potential
utility in colon cancer: early detection, prognosis, and chemosensitivity. World
J Gastroenterol. 2015;21(27):8284–8292.
111. Churov AV, Oleinik EK, Knip M. MicroRNAs in rheumatoid arthritis: altered expres-
sion and diagnostic potential. Autoimmun Rev. 2015;14(11):1029–1037.
112. Sun M, Zhou X, Chen L, et al. The regulatory roles of microRNAs in bone remodeling
and perspectives as biomarkers in osteoporosis. Biomed Res Int. 2016;2016:1652417.
113. Gennari L, Bilezikian JP. Idiopathic osteoporosis in men. Curr Osteoporos Rep.
2013;11(4):286–298.
114. Chien HY, Lee TP, Chen CY, et al. Circulating microRNA as a diagnostic marker in
populations with type 2 diabetes mellitus and diabetic complications. J Chin Med Assoc.
2015;78(4):204–211.
115. Guay C, Regazzi R. Circulating microRNAs as novel biomarkers for diabetes mellitus.
Nat Rev Endocrinol. 2013;9(9):513–521.
116. Pescador N, Perez-Barba M, Ibarra JM, Corbaton A, Martinez-Larrad MT,
Serrano-Rios M. Serum circulating microRNA profiling for identification of potential
type 2 diabetes and obesity biomarkers. PLoS One. 2013;8(10):e77251.
117. Romaine SP, Charchar FJ, Samani NJ, Tomaszewski M. Circulating microRNAs and
hypertension—from new insights into blood pressure regulation to biomarkers of
cardiovascular risk. Curr Opin Pharmacol. 2016;27:1–7.
118. Margis R, Margis R, Rieder CR. Identification of blood microRNAs associated to
Parkinsonis disease. JBiotechnol. 2011;152(3):96–101.
119. Cardo LF, Coto E, de Mena L, et al. Profile of microRNAs in the plasma of Parkinson’s
disease patients and healthy controls. J Neurol. 2013;260(5):1420–1422.
120. Botta-Orfila T, Morato X, Compta Y, et al. Identification of blood serum
micro-RNAs associated with idiopathic and LRRK2 Parkinson’s disease. J Neurosci
Res. 2014;92(8):1071–1077.
121. Reddy PH. Abnormal tau, mitochondrial dysfunction, impaired axonal transport of
mitochondria, and synaptic deprivation in Alzheimer’s disease. Brain Res.
2011;1415:136–148.
122. Khoo SK, Petillo D, Kang UJ, et al. Plasma-based circulating microRNA biomarkers
for Parkinson’s disease. J Parkinsons Dis. 2012;2(4):321–331.
94 S. Kumar et al.
Author's personal copy