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Current Cancer Drug Targets, 2018, 18, 000-000 1
RESEARCH ARTICLE
1568-0096/18 $58.00+.00 © 2018 Bentham Science Publishers
Interactions of Vascular Endothelial Growth Factor and p53 with miR-195
in Thyroid Carcinoma: Possible Therapeutic Targets in Aggressive Thy-
roid Cancers
Hamidreza Maroofa, Soussan Irania,b, Armin Ariannaa, Jelena Viderc, Vinod Gopalana,c and
Alfred King-yin Lama,*
aCancer Molecular Pathology, School of Medicine, Griffith University, Gold Coast, Queensland, Australia; bAssociate
Professor, Dental Research Centre, Research Centre for Molecular Medicine, Oral Pathology Department, Dental Fac-
ulty, Hamadan University of Medical Sciences, Hamadan, Iran; cSchool of Medical Science, Griffith University, Gold
Coast, Queensland, Australia
A R T I C L E H I S T O R Y
Received: December 16, 2017
Revised: May 27 , 2018
Accepted: June 01, 2018
DOI:
10.2174/1568009618666180628154727
Abstract: Background: The clinical pathological features, as well as the cellular mechanisms of
miR-195, have not been investigated in thyroid carcinoma.
Objective: The aim of this study is to identify the interactions of vascular endothelial growth factor
(VEGF), p53 and miR-195 in thyroid carcinoma. The clinical and pathological features of miR-195
were also investigated.
Method: The expression levels of miR-195 were identified in 123 primary thyroid carcinomas, 40
lymph nodes with metastatic papillary thyroid carcinomas and seven non-neoplastic thyroid tissues
(controls) as well as two thyroid carcinoma cell lines, B-CPAP (from metastasizing human papillary
thyroid carcinoma) and MB-1 (from anaplastic thyroid carcinoma), by the real-time polymerase
chain reaction. Using Western blot and immunofluorescence, the effects of exogenous miR-195 on
VEGF-A and p53 protein expression levels were examined. Then, cell cycle and apoptosis assays
were performed to evaluate the roles of miR-195 in cell cycle progression and apoptosis.
Results: The expression of miR-195 was downregulated in majority of the papillary thyroid carci-
noma tissue as well as in cells. Introduction of exogenous miR-195 resulted in downregulation of
VEGF-A and upregulation of p53 protein expressions. Upregulation of miR-195 in thyroid carci-
noma cells resulted in cell cycle arrest. Moreover, we demonstrated that miR-195 inhibits cell cycle
progression by induction of apoptosis in the thyroid carcinoma cells.
Conclusion: Our findings showed for the first time that miR-195 acts as a tumour suppressor and
regulates cell cycle progression and apoptosis by targeting VEGF-A and p53 in thyroid carcinoma.
The current study exhibited that miR-195 might represent a poten tial therapeutic target for patients
with thyroid carcinomas having aggressive clinical behaviour.
Keywords: VEGF-A, p53, micro RNA, miR-195, angiogenesis, thyroid carcinoma.
1. INTRODUCTION
Papillary thyroid carcinoma (PTC) is the most commonly
diagnosed thyroid cancer accounting for approximately 80%
of all thyroid tumours [1]. Previous studies have shown that
vascular endothelial growth factor (VEGF) exerts a pivotal
role in the control of angiogenesis and biological aggressive-
ness in thyroid carcinomas [2-6]. It is worth stating that, in
sustained hypoxic conditions in vitro/in vivo, p53 downregu-
lates VEGF expression through the retinoblastoma (Rb)
pathway in a p21-dependent manner [7, 8].
In thyroid and other carcinomas, miRNAs regulate cell
growth, differentiation, proliferation, cell metabolism and
cancer metastasis by directly or by regulating its downstream
*Address correspondence to this author at the Head of Pathology, Griffith
Medical School, Gold Coast Campus, Gold Coast QLD 42 22, Australia;
Tel: +61 7 56780718; Fax: +61 7 56780303; E-mail: a.lam@griffith.edu.au
target proteins [9-19]. miR-195, a member of the miR-15/16
family, has reported to play a pivotal role as a tumour sup-
pressor in many human can cers. It could promote apoptosis,
mainly through targeting p53 expression [20, 21]. miR-195 is
located at chromosome 17p13.1, the same genomic locus as
p53. P53 is the most frequently mutated gene in cancers and
its mutated form is involved in some thyroid carcinomas [22-
26]. Recently, the regu latory role of miR-195 in angiogenesis
and specifically its direct effect on VEGF-A has been
described in hepatocellular carcinomas [27, 28]. Taken to-
gether, it can be hypothesised that miR-195 acts as a key
regulator for the VEGF-A/p53 crosstalk in thyroid carcinoma
cells.
The underlying cellular mechanisms responsible for the
increased expression of p53 following miR-195 activation in
thyroid cancers are still unknown. In addition, the clinical
and pathological implications of miR-195 have not been in-
2 Current Cancer Drug Targets, 2018, Vol. 18, No. 1 Maroof et al.
vestigated in patients with thyroid carcinomas. In this study,
we aimed to identify the cellular implications of miR-195 as
well as the regulatory effects of miR-195 on p53, VEGF-A
and clinicopathological correlations in thyroid carcinomas.
2. MATERIALS AND METHODS
2.1. Patients and Tissue Samples
Patients with papillary thyroid carcinomas were recruited
from different collaborating hospitals in Australia. A
pathologist (AKL) reviewed the histological sections from
these carcinomas to confirm the diagnosis and classify the
carcinoma. The presence of co-existing lymphocytic
thyroiditis, psammoma bodies, calcifition or ossfication in
the tumour stroma was also noted. The clinical and patho-
logical features were recorded in database. The thyroid car-
cinomas were classified concerning the criteria defined by
the World Health Organization classification of endocrine
tumours [29, 30]. Only conventional and follicular variant of
papillary thyroid carcinoma are included in this study [31-
34]. The eighth edition of the American Joint Committee on
Cancer (AJCC) tumor-node-metastasis (TNM) staging
system was used to stage the thyroid tumours [35].
In total, 123 primary thyroid papillary carcinomas (in-
cluding 79 conventional papillary thyroid carcinomas and 44
follicular variants of papillary thyroid carcinoma), 40 lymph
nodes with metastatic papillary thyroid carcinomas and
seven non-neoplastic thyroid tissues were selected from ar-
chival formalin-fixed and paraffin embedded tissue. For the
use of tissue samples in this study, ethical approval was
obtained from Griffith University (MED/19/08/HREC). His-
tologic sections (4 µm thick) were cut from the selected tis-
sue blocks. The sections were stained with haematoxylin &
eosin. The author (AKL), who is a pathologist, reviewed the
histology of the thyroid carcinomas. All blocks containing
over 90% of cancer (with < 10% stromal tissue contamina-
tion) were selected.
2.2. Cell Culture
The thyroid cancer cell lines used in this study, B-CPAP
(from metastasizing human papillary thyroid carcinoma) and
MB-1 (from anaplastic thyroid carcinoma) were purchased
from Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmBH-German Collection of microorganisms
and cell cultures (DSMZ, Braunschweig, Germany).
Anaplastic thyroid carcinoma is the most clinical and bio-
logical aggressive form of thyroid carcinoma. Many of the
anaplastic thyroid carcinomas had co-existing papillary thy-
roid carcinoma implying that the anaplastic carcinoma dedif-
ferentiated from papillary thyroid carcinoma [36]. A non-
neoplastic thyroid follicular cell line (Nthy-ori 3-1) was
obtained from the European Collection of Cell Cultures
(ECACC) as a control. B-CPAP and Nthy-ori3-1 cell lines
were cultured in Roswell Park Memorial Institute medium
(RPMI 1640) (Invitrogen Carlsbad, CA, USA), 2 mM l-
glutamine (Invitrogen) supplemented with 10% heat & inac-
tivated fetal bovine serum (Invitrogen). MB-1 cells were
cultured in 80% RPMI 1640 (Invitrogen), 10% heat & inac-
tivated fetal bovine serum (Invitrogen) and 2 mM l-
glutamine (Invitrogen). The cell lines were authenticated in
the standard protocol (using multiplex polymerase chain
reaction of mini-satellite markers for DNA fingerprinting
and identification of short tandem repeats of cell lines and
cytogenetics). The passage numbers of these cell lines were
less than eight.
2.3. Isolation of miRNA
Total miRNA was extracted from formalin fixed paraffin
embedded tissue samples using miRNeasy extraction kits
(Qiagen Pty. Ltd., Hilden, NRW, Germany). In brief, the sec-
tions were deparaffinised by suspending in 1ml xylene and
then centrifuged. After adding one ml of ethanol (96–100%),
240µl proteinase K digestion buffer was added to the sections.
Then, 10µl proteinase K was added. After incubation at 56°C
for 15 minutes and then at 80°C for 15 minutes, 25µl DNase
booster buffer and 10 µl DNaseI were added. In the next step,
1200 µl ethanol (100%) was applied to the sample. Then,
500µl buffer RPE (Qiagen) was added. Finally, 30µl RNase-
free water was added and centrifuged to elute the RNA. Ex-
traction of total miRNA from the cells was performed using
NucleoSpin® miRNA Kit (MACHEREYNAGEL GmbH &
Co. KG, Düren, Germany). cDNA synthesis was done using
the miScript reverse transcription kit (Qiagen) according to the
manufacturer’s protocol.
2.4. Quantification of miR-195 Expression
The miR-195 expression level was quantified by real-
time quantitative PCR (qRT-PCR) using Hs_miR-195 miS-
cript Primer Assay (Qiagen) following the suggested proto-
col. A total volume of 20µl reaction mixture containing 10 µl
QuantiTect SYBR Green, 2 µl of each primer ((miScript
Universal Primer (Qiagen) and miR-195-5p 5' UAGCAG-
CACAGAAAUAUUGGC 3') , 2 µl of RNase-free water and
4 µl of template cDNA at 1.5 ng/µl were obtained. Samples
were normalised using the housekeeping gene RNU6B (Hs_
RNU6B_2 miScript Primer Assay, Qiagen). All qRT-PCR
reactions were carried out in triplicates with non-template
controls as previously published protocol [6]. The ΔCt
method was used to calculate miRNA expression levels. The
relative expression level of miR-195 was calculated and
quantified with the 2-ΔΔCt method after normalisation with
reference to expression of RNU6B. Normalised final data
was analysed using one-way (ANOVA) to determine if there
were significant differences in miRNA-195 expression be-
tween thyroid tissue in primary and metastatic sites. Addi-
tional co mparisons were to determine whether there were
significant differences in other thyroid cancer subgroups,
staging, patients’ gender and other clinicopathological char-
acteristics in the expression of miRNA-195.
2.5. Transient Transfection with miR-195 Mimic
The miR-195 mimic sequence (guide strand) 5'-
UAGCAGCACAGAAAUAUUGGC-3′ and HiPerFect trans-
fection reagent were purchased from Qiagen. The cell lines
were transiently transfected with miR-195 mimics (+ miR-
195), a non-targeting control (positive control) (+ miR-1)
and AllStars negative control siRNA (scramble control)
(Qiagen) immediately after being seeded at a density of
20×10 4 cells / well in 6 well plate, using the HiperFect
transfection reagent (Qiagen). Based on the Qiagen’s proto-
col and time-course experiment, a final concentration of 5nM
VEGF, p53 and miR-195 in Thyroid Carcinoma Current Cancer Drug Targets, 2018, Vol. 18 , No. 1 3
and a 48-hours transfection time was chosen for all
transfections. miR-195 mimic was added in 200 µl of serum-
free, antibiotic-free medium, supplemented with 5µl of
HiperFect. The mixture was allowed to stand for 15 minutes
at room temperature. The resulting 200 µl of transfection
reagent was added dropwise to each well containing 2 ml of
medium. Cells were maintained at 37 ºC and 5% CO2 and
monitored for 48 hours afterwards. The same protocol was
performed for miR-1 and scramble transfections.
2.6. Immunofluorescence
In the immunofluorescence analysis, cells were cultured
on a glass culture slide and transiently transfected for 48
hours. Then, cells were fixed in 4% cold paraformalde-
hyde/phosphate buffered saline (PBS) for 30 minutes. After
permeabilization with 0.4% Triton X-100 for 10 minutes,
cells were blocked with 5% normal goat serum/PBS (Sigma–
Aldrich St. Louis, MO, USA) for 45 minutes. Then, they
were incubated with antibodies against p53 (Pab 1801,
1:100; Santa Cruz Biotechnology, Dallas, TX, USA), VEGF-
A (A-20, 1:200 dilution; Santa Cruz Biotechnology)
overnight at 4 ºC. Thereafter, the cells were incubated with
Texas Red-labelled secondary antibody (1:3000 dilution;
Life technologies, St. Louis, MO, USA) for 2 hours at room
temperature. As negative control for each stain, the staining
was performed without the primary antibody. After being
counterstained with 4ˈ,6-diamidino-2-phenylindole (DAPI)
(Sigma–Aldrich), confocal laser scanning microscopy
images were performed with an Eclipse Ti-E microscope
(Nikon Pty. Ltd., Melville, NY, USA) using a plan
apochromat 60×/1.40 objective and NIS-Elements imaging
software platform (Nikon) with the following setting: image
Size 2,048 x 2,048 and 16 bit; Pixel/dwell of 25.2 µs; Pixel
Size 0.31 µm; laser power 10%; Master gain 600–1,000.
After the images were captured, the original image files were
converted into tagged image file format (TIFF) files.
2.7. Western Blot Analysis
After 48 hours of transient transfection, the cells were
lysed in Cell Lysis Buffer NP40 (50 mM Tris, pH 7 .4, 250
mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 1%
Nonidet P40, 0.02% NaN3) (Invitrogen) supplemented with
protease inhibitor cocktail (Sigma-Aldrich), phenylmethane-
sulfonyl fluoride solution (PMSF) (Sigma-Aldrich) and
phosphatase inhibitor cocktail (Cell Signaling, Danvers, MA,
USA). Then, whole protein lysates were quantified using the
Macherey-Nagel protein assay kit (MACHEREY-NAGEL).
Equal quantities of 30µg protein samples were run on a 4–
15% precast polyacrylamide gel (Mini-PROTEAN® TGX TM
Precast Gel, BIO-RAD, Hercules, CA, USA). Blocking was
performed with 5% non-fat milk in TBST (Tris-buffered
saline-Tween 20: 120 mmol/l Tris–HCl, pH 7.4, 150 mmol/l
sodium chloride, and 0.05% Tween 20) for 2 hours at room
temperature. The membrane was incubated with anti-p53,
1:100 dilution; anti-VEGF-A, 1:300 dilution and anti-ß-
actin, 1 :5000 dilution; Santa Cruz Biotechnology) overnight
at 4ºC. According to the manufacturer’s protocol, blots w ere
washed three times with TBST. Then, the blots were incu-
bated for 2 hours with horseradish peroxidase (HRP)-
conjugated secondary antibody (1:5000 dilution; Santa Cruz
Biotechnology) for 1.5 hours at room temperature. Bolts
were developed using Clarity™ Western ECL Blotting Sub-
strate kit (BIO-RAD). They were then visualised by using
VersaDoc-MP Imaging System (BIO-RAD) and analysed
with ImageJ software (National Institutes of Health, Be-
thesda, MD, USA).
2.8. Cell Cycle Analysis
For cell cycle analysis by flow cytometry, cells were cul-
tured in 6-well plate. After 48 hours of transient transfection,
the cancer cells were trypsinized and washed with ice-cold
PBS. They were then fixed in 70% ice-cold ethanol at -20ºC
for one hour. After centrifugation, the cells were washed
twice with PBS, stained with propidium iodide (PI) (50
mg/ml in PBS), RNase (50 mg/ml) and Triton X-100 (0.1%).
They were incubated for 40 minutes at 37 ºC and analysed
using a FACS Calibur flow cytometer (BD Biosciences, San
Jose, CA, USA). Each histogram was constructed with the
data from at least 10,000 events in triplicate. Data were
analysed to calculate the percentage of the cell population in
each phase using the FlowJo single-cell analysis software
(FLOWJO, LLC, Ashland, OR, USA).
2.9. Apoptosis Assay
Apoptosis assay was performed to measure the percent-
age of apoptotic cells using a Membrane Permeability/Dead
Cell Apoptosis Kit (Invitrogen). After 48 hours of transient
transfection, cells were harvested and washed twice with ice-
cold PBS. They were resuspended at the 25×10 4 cells/ml in
PBS. For staining, 1 µl of YO-PRO ®-1 and 1µl of PI were
added and kept in the dark for 20 minutes at room tempera-
ture. Cells were analysed for the number of apopto tic cells
using a FACS Calibur flow cytometer (BD Biosciences) and
calculated with FlowJo single-cell analysis software
(FLOWJO, LLC, Ashland, OR, USA).
2.10. Statistical Analysis
All experiments were performed at least three times. All
the clinical information, pathological data and miRNA ex-
pression changes were computerised. Statistical analysis was
performed using the Statistical Package for Social Sciences
for Windows (version 25.0; IBM SPSS Inc., Armonk, NY,
USA). Final normalised data were analysed as comparisons
of group means using Student’s t-test and ANOVA (using
Bonferroni and LSD correction) for continuous variables and
chi-square or likelihood ratio for categorical variables. Ex-
perimental results were expressed as means ± SD (standard
deviation). In addition, Pearson correlation (2-tailed) test
used for correlation analysis. A p value of < 0.05 was con-
sidered statistically significant and individual p-value was
shown in the figures. GraphPad Prism (Prism 7.0; Graph Pad
Software, San Diego, California, USA) was used to show the
charts and graph.
3. RESULTS
3.1. Expression Profiles of miR-195 in Primary Thyroid
Carcinomas and Clinicopathological Parameters
Downregulation of miR-195 expression was noted to be
predominant in papillary thyroid carcinoma as ~ 70% (n=86)
4 Current Cancer Drug Targets, 2018, Vol. 18, No. 1 Maroof et al.
Table 1. The relationship of miR-195 expression levels and clinicopathological characteristics of 123 papillary thyroid carcinomas.
miR-195 Expression
Clinical & Pathological Data
Total
Number
High
Low
Normal
P-value
Gender
Male
41
7 (17.1%)
32 (78%)
2 (4.9%)
0.002 *
Female
82
16 (19.5%)
54 (65.9%)
12 (14.6%)
Age
< 45
67
11 (16.4%)
49 (73.1%)
7 (10.4%)
0.691
≥ 45
56
12 (21.4%)
37 (66.1%)
7 (12.5%)
Tumour size (mm)
≤ 40 mm
110
13 (13%)
73 (73%)
14 (14%)
0.001*
> 40mm
13
10 (43.5%)
13 (56.5%)
0 (0.0%)
T staging
T1 or T2
78
9 (11.5%)
58 (74.4%)
11(14.1%)
0.020*
T3
45
14 (31.1%)
28 (62.2%)
3 (6.7%)
Lymph node metastasis
Positive
40
7 (17.5%)
33 (82.5%)
0 (0.0%)
0.017*
Negative
83
16 (19.3%)
53 (63.9%)
14 (16.9%)
TNM staging
Stages I or II
87
13 (14.9%)
62 (71.3%)
12 (13.8%)
0.147
Stage III
36
10 (27.8%)
24 (66.7%)
2 (5.6%)
Pathological v ariant
Conven tional
79
16 (20.3%)
52 (65.8%)
11 (13.9%)
0.355
Follicular
44
7 (15.9%)
34 (77.3%)
3 (6.8%)
Psammoma body
Present
52
9 (17.3%)
38 (73.1%)
5 (9.6%)
0.791
Absent
71
14 (19.7%)
48 (67.6%)
9 (12.7%)
Calcification in stroma
Present
62
14 (22.6%)
42 (67.7%)
6 (9.7%)
0.494
Absent
61
9 (14.8%)
44 (72.1%)
8 (13.1%)
Osseous metaplasia in stroma
Present
6
1 (16.7%)
5 (83.3%)
0 (0%)
0.639
Absent
117
22 (18.8%)
81 (69.2%)
14 (12%)
Lymphocytic thyroiditis
Present
40
9 (22.5%)
27 (67.5%)
4 (10%)
0.738
Absent
83
14 (16.9%)
59 (71.1%)
10 (12%)
* Statistically significant; High expression: fold change greater than 2; Low expression: fold change = less than 0.5; Normal expression = fold changes between 0.5 and 2
of the thyroid carcinomas exhibited low expression of miR-
195. Only 19% (n=23) of the papillary thyroid carcinoma
had miR-195 restoration. The other 11% (n=14) had a similar
expression as non-neoplastic thyroid tissue.
Table 1 showed the correlation between miR-195 expres-
sions and various clinicopathological features of papillary
thyroid carcinomas. A significant difference between miR-195
expression level and gender of the patient was detected
(p=0.002). High prevalence of miR-195 downregulation was
noted in male patients with papillary thyroid carcinomas (78%
versus 6 5.9%). Additionally, the miR-195 downregulation
was highly prevalent in patients with small thyroid carcinomas
(maximum dimension <40mm) when compared to those of
other larger carcinomas (73% versus 56.5%, p=0.001). Simi-
larly, patients with early T stages (T1or T2) cancers showed a
slight high prevalence of high expression of miR-195 high
expression when compared to patients with advanced T stage
(T3) cancers (74.4% versus 62.2%, p=0.020).
VEGF, p53 and miR-195 in Thyroid Carcinoma Current Cancer Drug Targets, 2018, Vol. 18 , No. 1 5
Of the 123 patients, 33% (n=40) had lymph node metasta-
ses. miR-195 expression was often down-regulated in papillary
thyroid carcinomas with lymph node metastases when com-
pared to those without lymph node metastases (82.5% versus
63.9%, p=0.017). In these 40 patients with lymph node metas-
tases, we compared the expression of miR-195 in primary can-
cer and metastatic cancer in the lymph node. There was also a
significant difference in miR-195 expression between them. In
primary carcinoma, 82.5% (33 of 40) showed low expression
of miR-195 whereas, in metastatic carcinoma in lymph node,
45% (18 of 40) revealed low expression of miR-195
(p=0.0001). Thus, lower expression levels were more
commonly detected in primary cancers when compared with
metastic cancers.
miR-195 expression level was down-regulated in thyroid
carcinoma cells (B-CPAP and MB-1). These findings are
compatible with miR-195 expression levels in tissues exam-
ined, as miR-195 was more often down-regulated in those
thyroid carcinomas w ith lymph node metastases when com-
pared to thyroid carcinomas without lymph node.
3.2. miR-195 Expression and its Target Effects on VEGF-
A and p53 Proteins
miR-195 was significantly downregulated in B-CPAP
(Metastasizing human papillary thyroid carcinoma) and MB-
1(human anaplastic thyroid carcinoma), when compared to
the non-cancer, immortalised thyroid cell line (Nthy-ori-3-
1cell line). The relative expression (ratio of expression) of
miR-195 in Nthy-ori-3-1cells was equal to “1” whereas the
ratios of expression were 0.44 ± 0.05, 0.19 ± 0.01, in B-
CPAP and MB-1 cell lines respectively (Fig. 1) (p< 0.05).
To investigate the biological effects of miR-195 restoration
on thyroid cancer cells, B-CPAP and MB-1 cell lines were
transfected with an exogenous miR-195 (mimics) . Cells
treated with the miR-195 showed significant restoration of
miR-195 levels (shown as a ratio of expression; 2.79 ± 0.01,
1.72 ± 0.33) in B-CPAP and MB-1 cells respectively when
compared to the Nthy-ori-3-1cells (Fig. 1) (P<0.05). miR-
195 was significantly overexpressed to investigate its spe-
cific regulatory function on VEGF-A and p53 expression
levels.
Western blot (Fig. 2) and immunofluorescence analysis
(Fig. 3) showed that miR-195 over-expression results in the
downregulation of VEGF-A protein expression. In addition,
thyroid cancer cells with high miR-195 lead to upregulation
of p53 protein expression. Control cell groups did not show
any significan t changes in VEGF and p53 protein expres-
sions after miR-195 transfection.
4. DISCUSSION
In the present study, we assessed the miR-195 expression
in a large cohort of patients with papillary thyroid carcinoma
and correlated its expression with v arious clinicopathological
features. The miR-195 was downregulated in ~ 70% of can-
cer tissues indicating its tumour suppressor role in the patho-
genesis of thyroid carcinomas. In thyroid cancers, the size of
the tumour and extent of local invasion of cancer cells (T
stage) plays a key role in predicting the biological aggressive
of cancer [36]. We believe that our results showed for the
first time that miR-195 is overexpressed in 19% of papillary
thyroid carcinomas. This means that miR-195 expression
pathway is complex. For instance, a study conducted by
Wang et al, displayed that miR-195 applies its function
through modulation of Raf1 gene, which is upregulated in
papillary thyroid carcinoma, and dysfunction of this gene
results in upregulation of miR-195 in papillary thyroid carci-
noma [37].
Fig. (1). miR-195 expression was downregulated in thyroid car-
cinoma cells. miR-195 expression significantly decreased in metas-
tasizing human papillary thyroid carcinoma (B-CPAP) and human
anaplastic thyroid carcinoma (MB-1) when compared to non-cancer
immortalised thyroid cells (Nthy-ori3-1). miR-195 transfection led
to restoration of m iR-195 in thyroid carcinoma cells shown as B-
CPAP (miR-195) and MB-1 (miR-195) when compared to Nthy-
ori3-1 cell line. Relative miR-195 expression after transfection with
miR-195, detected by qRT-PCR. RNU6B was used to normalize the
mRNA level. Each bar represents the mean of three independent
experiments. An asterisk (*) indicates p < 0.05, when compared to
Nthy-ori-3-1. The quantitative values were expressed as means ±
SD of triplicate measurements. They are representative of three
separate experiments.
In this study, low expression of miR-195 expression was
predominantly noted in thyroid carcinomas of smaller size
and lower T stages (Table 1). Therefore, miR-195 downregu-
lation enhances cancer progression or establishment of the
early growth of tumour masses. These clinical correlations of
miR-195 were not previously reported in thyroid carcinomas.
However, the association of miR-195 downregulation with
increased cancer progression is demonstrated in several can-
cers [38, 39]
In some other cancers, similar findings were often noted
in the study of miR-195. High circulating levels of miR-195
correlated with early pathological stages (T1 and T2) in
breast cancer [40]. Downregulation of miR-195 was also
reported in colorectal cancer [41]. However, in a study on
tongue squamous cell carcinoma tissues, downregulation of
miR-195 was significantly associated with larger and pathol-
ogically advanced tumours (T3, T4) [42]. Thus, the
expression level of miR-195 in cancer could be cancer-
specific. Further studies matching the expression profiling
between carcinoma tissue and blood samples could be done
to confirm these findings in thyroid carcinomas.
Lymph node metastasis is an important predictor of sur-
vival of patients with cancer. It is present in the advanced
stages of patients with cancer. In the current study, a signifi-
cant sub-population of metastatic thyroid cancers showed
decreased miR-195 expression level. Thus, miR-195 could be
a potential diagnostic and prognostic marker for thyroid
6 Current Cancer Drug Targets, 2018, Vol. 18, No. 1 Maroof et al.
Fig. (2). Restoration of miR-195 regulates VEGF-A and p53 expressions in carcinoma cells. miR-195 restoration down-regulates target
protein VEGF-A and p53 in B-CPAP (metastasizing human papillary thyroid carcinoma) and in MB-1 (human anaplastic thyroid carcinoma)
cells when compared to miR-1 transfected and scramble group. B-CPAP and MB-1 were transfected for 48 hours. The effect of this restora-
tion was examin ed in thyroid cancer cells using Western blotting. In both carcinomas, expression of VEGF-A was decreased in miR-195
mimic transfected group when compared to miR-1 transfected and scramble group. Expression of p53 was increased in miR-195 mimic trans-
fected group when compared to miR-1 transfected and scramble group. Sample loading control was β-actin; y-axis on Western blot compari-
son diagrams show VEGF-A and p53 protein expressions based on signal absorption. Results were representative of three independent ex-
periments. They were shown as mean ± SD; and (*) implies as probability value p < 0.05, when compared to controls.
Fig. (3). Confirmation of miR-195 mediated alteration of targets proteins in B-CPAP and MB-1 cells via immunofluorescence micros-
copy. VEGF-A and p53 protein expressions in B-CPAP and MB-1 cells at 48 hours after the transfection by immunofluorescence. Similar to
Western blot analysis, miR-195 restoration significantly reduced the expression level of VEGF-A protein in B-CPAP and MB-1 cells when
miR-195 mimic transfected group, miR-1 transfected (positive control) and scramble group (negative group) were compared. The expression
level of p53 w as increased in transfected cells when compared to miR-1 transfected and scramble group. Immunofluorescence images were
captured by a Nikon A1R+ confocal microscope using 60× objective with immersion oil. VEGF-A and p53 are stained red, and nuclei are
blue; Scale in the immunofluorescence images shows 5 and 10 µm. B-CPAP (metastasizing human papillary thyroid carcinoma) and in MB-
1(human anaplastic thyroid carcinoma).
cancer. In addition, a study on oesophageal squamous cell
carcinoma using microarray reported that down-regulated
miRNAs including miR-195 were significantly associated
with cancer cell invasion and lymph node metastasis [43].
These results further confirm the tumour suppressor role for
miR-195 in the progression of papillary thyroid carcinoma,
which is consistent with previous findings of miR-195 in
different cancers [44-48].
To investigate the interactive factors of miR-195 in me-
tastatic thyroid cancer carcinoma and tumorig enesis, we
have analysed its modulatory effects on VEGF-A and p53
protein expressions in thyroid carcinoma cells. VEGF-A is a
key proangiogenic factor secreted by carcinoma cells while
p53 act as a potent apoptotic and tumour suppressor factor in
human cancer [28, 49]. This study noted a significant down-
regulation of VEGF-A protein and upregulation of p53
VEGF, p53 and miR-195 in Thyroid Carcinoma Current Cancer Drug Targets, 2018, Vol. 18 , No. 1 7
Fig. (4). Effect of miR-195 on cell cycle distribution of thyroid carcinoma. Metastasizing human papillary thyroid carcinoma cells (B-
CPAP) (A) and human anaplastic thyroid carcinoma cells (MB-1) (B) were transfected for 48 hours. Propidium iodide (PI) solution was used
to stain nuclei so that they can be analy sed for DNA content by flow cytometry . Results showed that the cell number increased in G0-G1
phase and decreased in S and G2-M phases in the miR-195 transfected group when compared to miR-1 transfected and scramble group. An
asterisk (*) indicate statistically significant differences (p < 0.05, Student’s t-test) when compared to control cells. Data shown as
mean ± SD of three independent experiments. They represent percentage cells in different phases of the cell cycle w ith miR-195 related to
controls.
Fig. (5). Induction of apoptotic by re-expression of miR-195 in thyroid carcinoma. Following transfection of metastasizing human papil-
lary thyroid carcinoma cells (B-CPAP) (A) and human anaplastic thyroid carcinoma cells (MB-1) (B) for 48 hours, cells were subjected to
Annexin V/propidium iodide (PI) staining and flow cytometry analysis. Data shown as mean ± SD of three independent experiments. They
represent percentage of Annexin V-positive cells with miR-195 related to controls. The p ercentage of dead cells (Q1; upper left quadrant), late
apoptosis cells (Q2; PI+/Annexin V+; upper right quadrant), early apoptosis cells (Q3; PI-/Annexin V+; lower right quadrant) and live cells
(Q4; lower left quadrant), are indicated. Asterisks (*) indicate statistically significant differences (p < 0.05, Student’s t-test) when compared
to control cells.
8 Current Cancer Drug Targets, 2018, Vol. 18, No. 1 Maroof et al.
protein following miR-195 restoration. This finding was in
agreement with previous data indicated that suppression o f
VEGF-A and restoration of p53 following application of
miR-195 in hepatocellular carcinoma and cervical carci-
noma, respectively [28, 41, 50]. It has also been demon-
strated that p53 could identify the miR-195 promoter region
and overexpressed miR-195 [20], resulting in enhancement
of the miR-195 effects [48]. Therefore, miR-195 could act as
a potential candidate in controlling of the cell proliferation
and play a vital role in tumour development by regulating
p53 and VEGF protein expressions [51]. Downregulation of
miR-195 might contribute to the development of papillary
thyroid carcinomas by activating angiogenesis and by evad-
ing apoptosis [52].
In glioblastoma (brain tumour), miR-195 suppresses cell
proliferation by induction of cell cycle arrest at G0-G1 phase
[53]. This study has reported for the first time that the miR-
195 induced the cell cycle and apoptotic changes in thyroid
carcinoma cells. This effect on cell cycle is similar to results
for restorations of p53 functions reported in some cancers
[54-56]. Blocking of the thyroid carcinoma cell’s entry into
the ‘S’ transitional phase following miR-195 restoration
could be attributed to upregulation of p53 gene, and to down-
regulation of VEGF-A by miR-195 restoration.
In this study, we noted that miR-195 has dual functions in
thyroid can cer cells, operating as a tumour suppressor by di-
rectly inhibiting both cell proliferation and invasion , and as an
angiogenic inhibitor by targeting VEGF-A and p53 [53]. Resto-
ration of miR-195 could rebuild the suppressor function of p53
tumour suppressor function and modulates VEGF-A expression.
In tumour microenvironment, the lack of oxygen concentration
(hypoxia) because of tumour growth is demonstrated to regulate
VEGF-A expression. This hypoxic condition also stimulates the
binding of hypoxia-inducible factor (HIF) to the VEGF pro-
moter, promoting VEGF gene transcription [57]. Therefore, it is
reasonable to assume that miR-195 also, directly and indirectly,
regulates genes, which are involved in tumour microenviron-
ment, invasion and metastasis
In summary, our results demonstrated for the first time
that miR-195 plays a key role in thyroid carcinogenesis by
exhibiting its tumour suppressor properties in vitro. Multiple
novel clinicopathological associations noted in this study
suggests the potential role of miR-195 as a marker in predict-
ing thyroid cancer aggressiveness. Furthermore, modulatory
effects of miR-195 on VEGF-A and p53 proteins and its sub-
sequent effects on cell cycle events and apoptosis confirm its
significance in targeting molecular pathogenesis in thyroid
carcinomas. In addition, we have recently reported that miR-
34b-5p and miR-205 suppressed cancer growth in thyroid
cancer by supressing angiogenesis and apoptosis process [12,
58]. Thus, suppression of multiple miRNAs may have syner-
gic roles that could have the potential for targeting therapy in
thyroid carcinoma with aggressive clinical behaviours.
ETHICS APPROVAL AND CONSENT TO PARTICI-
PATE
Not applicable.
HUMAN AND ANIMAL RIGHTS
No Animals/Humans were used for studies that are the
basis of this research.
CONSENT FOR PUBLICATION
Not applicable.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
The authors would like to thank the funding support of
student scholarships from Griffith University and the project
grants of the Menzies Health Institute of Queensland from
Griffith University. In addition, we would like to thank the
staff of Menzies Health Institute of Queensland and Pathol-
ogy Queensland for their help in the laboratory work.
FUNDING
The authors would like to thank the funding support of
student scholarships from Griffith University, grant from
Queensland Cancer Council and the project grants of the
Menzies Health Institute of Queensland, Griffith University.
AUTHOR CONTRIBUTIONS
Hamidreza Maroof: cellar experimental works and manu-
script writing
Soussan Irani: works on the histology experiment
Armin Ariana: supervise histology experiment
Jelena Vider: help with flow cytometry
Vinod Gopalan: supervise the cellular experiment and
edit of manuscript
Alfred King-yin Lam: supervise the works and manu-
script editing
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