Detection and Quantification of Immunoregulatory miRNAs in Human Milk and Infant Milk Formula

Abstract

Mammary gland secretory cells produce miRNA-rich milk. In humans, these miRNAs reach infant/neonate bloodstream, playing diverse roles, like neural system development, metabolism, and immune system maturation. Notwithstanding, still few works explore human milk miRNA content, and there are no reports at the population level. Our hypothesis was that miR-146b-5p, miR148a-3p, miR155-5p, mir181a-5p, and mir200a-3p immunoregulatory miRNAs are expressed in human colostrum/milk at a higher level than infant milk formulae. The aim of this work was to evaluate the expression of the five immunoregulatory miRNAs in human milk and compare it with their expression in infant milk formula. For this purpose, miRNA relative expression was measured by qPCR in cDNA prepared from total RNA extracted from sixty human colostrum/milk samples and six different formulae. The comparative Cт method 2−ΔCт using exogenous cel-miR-39 as internal control was employed, followed by statistical analysis. We found the relative expression levels of miRNAs are comparable among colostrum/milk samples, and these miRNAs are present in infant milk formulae but at very low concentrations. We conclude that the relative expression of the immunomodulatory miRNAs is comparable in all the human colostrum/milk samples and is higher than the expression in formulae.

Full text

Citation: Vélez-Ixta, J.M.; Benítez-
Guerrero, T.; Aguilera-Hernández, A.;
Martínez-Corona, H.; Corona-
Cervantes, K.; Juárez-Castelán, C.J.;
Rangel-Calvillo, M.N.; García-Mena,
J. Detection and Quantification of
Immunoregulatory miRNAs in
Human Milk and Infant Milk
Formula. BioTech 2022,11, 11.
https://doi.org/10.3390/biotech
11020011
Academic Editor: Maria Teresa Di
Martino
Received: 16 December 2021
Accepted: 18 March 2022
Published: 20 April 2022
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4.0/).
Article
Detection and Quantification of Immunoregulatory miRNAs in
Human Milk and Infant Milk Formula
Juan Manuel Vélez-Ixta 1, Tizziani Benítez-Guerrero 1, Arlene Aguilera-Hernández 1, Helga Martínez-Corona 1,
Karina Corona-Cervantes 1, Carmen Josefina Juárez-Castelán1, Martín NoéRangel-Calvillo 2
and Jaime García-Mena 1, *
1Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del
Instituto Politécnico Nacional (Cinvestav), Av. Instituto Politécnico Nacional 2508,
Ciudad de México 07360, Mexico;
juan.velez@cinvestav.mx (J.M.V.-I.); tizziani.benitez@cinvestav.mx (T.B.-G.);
arlene.aguilera@cinvestav.mx (A.A.-H.); helga.martinez@cinvestav.mx (H.M.-C.);
karina.corona@cinvestav.mx (K.C.-C.); carmen.juarez@cinvestav.mx (C.J.J.-C.)
2Hospital General “Dr. JoséMaría Rodríguez”, Instituto de Salud del Estado de Mexico,
Ecatepec de Morelos 55200, Estado de México, Mexico; drrangelcalvillo@gmail.com
*Correspondence: jgmena@cinvestav.mx; Tel.: +52-5747-3800 (ext. 5328)
Abstract:
Mammary gland secretory cells produce miRNA-rich milk. In humans, these miRNAs reach
infant/neonate bloodstream, playing diverse roles, like neural system development, metabolism,
and immune system maturation. Notwithstanding, still few works explore human milk miRNA
content, and there are no reports at the population level. Our hypothesis was that miR-146b-5p,
miR148a-3p, miR155-5p, mir181a-5p, and mir200a-3p immunoregulatory miRNAs are expressed
in human colostrum/milk at a higher level than infant milk formulae. The aim of this work was
to evaluate the expression of the five immunoregulatory miRNAs in human milk and compare it
with their expression in infant milk formula. For this purpose, miRNA relative expression was
measured by qPCR in cDNA prepared from total RNA extracted from sixty human colostrum/milk
samples and six different formulae. The comparative C
т
method 2
−∆Cт
using exogenous cel-miR-39
as internal control was employed, followed by statistical analysis. We found the relative expression
levels of miRNAs are comparable among colostrum/milk samples, and these miRNAs are present in
infant milk formulae but at very low concentrations. We conclude that the relative expression of the
immunomodulatory miRNAs is comparable in all the human colostrum/milk samples and is higher
than the expression in formulae.
Keywords: human milk; qPCR; microRNA; RNA; formula milk; breastfeeding
1. Introduction
According to the Mexican national survey ENSANUT [
1
], the panorama of breast-
feeding in Mexican women 12–49 years old with children up to 24 months of age is not
favorable since only 28.3% of children under six months are exclusively breastfed. It is
common that children under 12 months of age are fed infant formula, and just a small
portion of children corresponding to 29% of the studied population was breastfed up to
two years of age. Therefore, these data allow us to conclude that there is still a deficiency in
breastfeeding practices in Mexico that must be tackled and managed from a collaborative
perspective [2].
MicroRNAs (miRNAs) are short 19–24 nucleotides non-coding RNAs that derive from
characteristic hairpin precursors. They constitute one of the more abundant classes of
gene-regulatory molecules, suppressing the expression profiles of protein-coding genes
at the post-transcriptional level [
3
,
4
]. miRNAs have been described in human milk (HM)
as unique bioactive components. During lactogenesis, milk particles are produced by
secretory epithelial cells located in the alveoli of the mammary gland and are secreted in
BioTech 2022,11, 11. https://doi.org/10.3390/biotech11020011 https://www.mdpi.com/journal/biotech

BioTech 2022,11, 11 2 of 9
vesicles into the lumen [
5
]. The vesicles carrying miRNAs are taken up by the infant during
feeding. Later, they are transported and assimilated by the cells to perform their function.
Colostrum has an important immune function due to its high immunoglobulins and
bioactive components content [
6
]. In the human body, miRNAs are present in extracellular
fluids like saliva, urine, plasma, and milk [
7
]. HM contains macronutrients, micronutrients,
bioactive compounds, growth factors, and immunological factors [8].
In recent years, the study of bioactive components, for example, miRNAs, immunoglob-
ulins, maternal cells, among others, has been of great interest. There is solid evidence of the
presence of miRNAs in HM, in addition, they have been found expressed in milk of other
species like goat, cow, and pig [
9
]. In HM, miRNAs remain stable since they are associated
with extracellular vesicles, fat globules, and cellular components. There is evidence that
miRNAs play an important role in establishing the neonate’s immune system and have
other beneficial effects on infant health [9].
Exosome-derived miRNAs, such as miR-148a, induce cell proliferation and protein
expression in normal colon epithelial cells [
10
]. Moreover, miR-148a promotes CD70 and
LFA-1 gene overexpression through hypomethylation. These genes are associated with cell
survival and T-cell activation, respectively [
11
]. In a study done using six-month mature
HM, high content of miR-181 and miR-155 was found. These miRNAs are involved in
the differentiation of B cells [
12
]. Examples of other highly enriched miRNAs in HM are
miR-17 and miR-92, which have functions, such as regulation of monocyte development,
differentiation of B cells and T cells [
13
]. In another report, the presence of miRNAs with
important immunological functions, for example, miR-148a-3p, miR-30b-5p, miR-182-5p,
and miR-200a-3p, was demonstrated in exosomes derived from HM [
14
]. Another group
showed the presence of miR-21, miR-16, and miR-146b-5p in pre-term HM [15].
The presence of miRNAs has been established in cow’s milk based infant milk formula
(IMF) by next-generation sequencing and quantitative real-time PCR. In these products,
the miRNA content is generally lower than HM [
7
]. Since cow’s milk is the most frequently
used component of infant formulas, the same group reported that around 90% of miRNAs
found in human milk are also found in cow and goat’s milk. On the other hand, previous
studies did not detect abundant miRNA content in IMFs [
16
]. The aim of this work was to
explore the expression of five different immunoregulatory miRNAs in Mexican HM and
compare it with the content in IMF.
2. Materials and Methods
2.1. Type of Study
Descriptive cross-sectional study of five selected miRNA expression in human milk
and infant milk formula (IMF).
2.2. Sample
Human colostrum/milk (HCM) samples were selected from a collection of samples
of a previous study made in the public hospital “Dr. JoséMaría Rodríguez” located in
Ecatepec de Morelos, State of Mexico (19_36 03500N, 99_ 303600W), and kept at
−
70
◦
C
until used. The protocol was approved by the Ethics Committee of the General Hospital
(Project identification code: 217B560002018006) [
17
]. The inclusion criteria were: Healthy
women between 0 to 7 postpartum days, of Mexican origin for at least two generations, who
gave birth by vaginal delivery or non-elective C-section with 37–41 weeks of gestational age.
The exclusion criteria were: Smoking, probiotic or alcohol consumption, being affected by
diabetes, overweight, or obesity, and using antibiotics during the last trimester of pregnancy.
Infant milk formulae (IMF) were selected among the most consumed products in Mexico
City (Table S1). IMF were reconstituted with nuclease-free water (Sigma-Aldrich
®
, St. Louis,
MO, USA, Cat. W4502-1L) following the manufacturer’s instruction.

BioTech 2022,11, 11 3 of 9
2.3. Oligonucleotide Selection and Design
For this research, miR-146b-5p, miR148a-3p, miR155-5p, mir181a-5p, and mir200a-3p
were selected based on literature search in PubMed portal (https://pubmed.ncbi.nlm.nih.
gov/, accessed on 15 August 2020), including original and review articles from years 2000 to
2020, searching for the terms “miRNAs, human milk, milk formula and immunoregulatory
miRNAs”. Raw sequences were obtained from miRBase (www.mirbase.org/, accessed
on 15 August 2020). Stem-loop primers for cDNA synthesis and primers for qPCR were
designed using CLC Workbench v.8.1.3 software (https://www.qiagen.com/, accessed on
15 August 2020) based on a previous report [18] (Table S2).
2.4. RNA Extraction
RNA was extracted from 1 mL HCM or IMF, respectively. Samples were centrifugated
at max speed, for 10 min, at 4
◦
C in Neofuge 13R Centrifuge (HealForce
®
, Shanghai, China).
Cellular pellet and lipid phase were washed with cold PBS pH 7.4. Subsequently, total RNA
was extracted with mirVana
™
miRNA Isolation Kit (Invitrogen
™
, Cat. AM1560, Carlsbad,
CA, USA), following manufacturer instructions. 3
µ
L of exogenous miRNA (33 fmol)
cel-miR-39 (microRNA (cel-miR-39) Spike-In Kit, Cat. 59000, Thorold ON, Canada) were
added to each sample before lysis buffer. RNA was eluted in 100
µ
L volume and stored
at
−
20
◦
C. RNA concentration and purity were assessed with NanoDrop
™
2000 (Thermo
Fisher Scientific, Cat. ND-2000, Waltham, MA, USA).
2.5. cDNA Synthesis
cDNA synthesis was made using 5–50 ng extracted RNA in 15
µ
L reaction volume,
using MicroRNA Reverse Transcription Kit (Applied Biosystems
™
, Cat. 4366596, Waltham,
MA, USA), following manufacturer’s instructions. Individual cDNA reactions were pre-
pared for each miRNA evaluated for each sample. Reactions were placed in Thermocycler
2720 (Applied Biosystems, Waltham, MA, USA) for 30 min at 16
◦
C; 30 min, 42
◦
C; 5 min,
85 ◦C; and 10 min, 4 ◦C.
2.6. miRNA Quantification by qPCR
qPCR was made using 1
µ
L of cDNA and Maxima SYBR Green/ROX qPCR Master
Mix (Thermo Scientific
™
, Cat. K0222, Waltham, MA, USA) reagents in a final volume of
25
µ
L, as described by the manufacturer. Reactions were placed on PCR plates (Applied
Biosystems
™
, Cat. 4375816, Waltham, MA, USA) and qPCR run in StepOne
™
Real-Time
PCR System Thermocycler (Applied Biosystems
™
, Cat. 4376357, Waltham, MA, USA)
according to the two-step cycling protocol: Denaturation of 95
◦
C for 10 min; 40 cycles of
95
◦
C for 15 s, 60
◦
C for 60 s and finally, a melt curve of 95
◦
C for 15 s, 60
◦
C for 60 s, and
95 ◦C for 15 s. Quantitation was made in triplicate.
2.7. Bioinformatics and Statistical Analyses
Data obtained from this work were analyzed using R environment [
19
] and Rstu-
dio 1.4.1717 software [
20
]. The R-libraries employed were “readr” [
21
], “dplyr” [
22
],
“scales” [
23
], and “tidyr” [
24
]. Cтwere calculated by Step One Software v2.2.2 (Applied
Biosystems
™
, Waltham, Massachusetts, USA) and exported to comma separated values
(.csv) format. Afterwards, the comparative Cтmethod 2
−∆Cт
[
25
] was used for statistical
analysis. Internal control for this study was cel-miR-39. One way Analysis of Variance
(ANOVA) was performed to seek differences in relative miRNA levels in HCM. Kruskal–
Wallis one-way analysis of variance was carried out to seek for differences in miRNA levels
at different postpartum days. Further, Mann–Whitney U test (Wilcoxon rank-sum test)
was used to seek differences in relative miRNA levels between both groups (HCM and
IMF). Tables were elaborated with R and exported to html for inclusion in this work using
‘sjPlot’ library [
26
]. Plots were elaborated with ‘ggplot2’ library [
27
] and exported to Joint
Photographic Experts Group format (.jpg) to 600 dpi/ppi resolution.

BioTech 2022,11, 11 4 of 9
3. Results
3.1. miRNA Relative Expression Is Comparable among the Studied Human Milk Samples
We studied the expression of five different immuno-related miRNAs in human colostrum/
milk samples collected from 60 women in a period of 0 to 7 postpartum days. Women
were healthy, normal weight, around 23 years-old, mostly with vaginal delivery (Table 1).
Samples were collected in a period of three months from 16 October 2017 to 29 January
2018 (Figure S1). Total RNA was extracted, and miR-146b-5p, miR148a-3p, miR155-5p,
mir181a-5p, mir200a-3p were quantified as described in Materials and Methods. Results for
mir181a-5p were discarded due to technical problems with the primer FmiR181 (Table S2),
which amplified a spurious product in the qPCR reaction for the non-template control. The
results for miR-146b-5p, miR148a-3p, miR155-5p, and mir200a-3p indicated that the relative
expression for all of them was comparable, as shown by the one-way ANOVA statistical
test of the relative expression units (2
−∆Cт
) (Pr(>F) = 0.7136) (Figure 1). There is a tendency
of higher miRNAs relative expression at postpartum day-3 (Figure S2). We also evaluated
whether there was an association between miRNA relative expression and mother age but
found no association in our data (Figure S3).
Table 1. Data for 60 participant mothers in the study.
Anthropometric Data Mean SD
Age (years) 22.93 ±4.95
Height (m) α1.57 ±0.07
Weight (Kg) β56.48 ±10.01
BMI γ23.22 ±3.74
Type of delivery n %
Elective caesarean 4 6.67
Non elective caesarean 13 21.67
Vaginal 43 71.66
Parity n %
Primiparous δ16 27.12
Multiparous δ43 72.88
Milk extraction n %
Manual ε34 82.93
Pump ε7 17.07
Socioeconomic data n %
Residence Mexico City (19◦2501000 N 99◦080440 0 O) 15 25.00
Estado de México (19◦210150 0 N 99◦370510 0 O) 36 60.00
Guerrero State (17◦3604700 N 99◦570000 0 O) 2 3.33
Hidalgo State (20◦280420 0 N 98◦510490 0 O) 1 1.67
Oaxaca State (16◦530530 0 N 96◦240510 0 O) 3 5.00
Puebla State (19◦000130 0 N 97◦530180 0 O) 2 3.33
Veracruz State (19◦2600500 N 96◦220590 0 O) 1 1.67
Activity Housewife δ52 88.14
Student δ2 3.39
General employee δ5 8.47
Education Primary school (6 years) 20 33.33%
Secondary school (3 years) 36 60.00%
High school (3 years) 1 1.67%
University school (4–5 years) 3 5.00%
α
55 mothers;
β
50 mothers;
γ
48 mothers;
δ
59 mothers;
ε
41 mothers. BMI = weight (kg)/[height (m)]
2
, n,
indicates number of data for the category. Anthropometric data correspond to the start of pregnancy.

BioTech 2022,11, 11 5 of 9
BioTech 2021, 10, x FOR PEER REVIEW 5 of 10
Education Primary school (6 years) 20 33.33%
Secondary school (3 years) 36 60.00%
High school (3 years) 1 1.67%
University school (4–5 years) 3 5.00%
α 55 mothers; β 50 mothers; γ 48 mothers; δ 59 mothers; ε 41 mothers. BMI = weight (Kg)/[height
(m)]2, n, indicates number of data for the category. Anthropometric data correspond to the start of
pregnancy.
Figure 1. Expression of selected miRNAs in human colostrum/milk. hsa-miR146 (miR-146b-5p); hsa-
miR-148 (miR148a-3p), hsa-miR-155 (miR155-5p), hsa-miR-200 (mir200a-3p). The Y-axis shows the
relative expression of each miRNA with respect to the internal control cel-miR-39, and the X-axis
shows the corresponding miRNA. Each dot in the plot represents a sample. The double horizontal
lines indicate the standard error of the mean. One-way ANOVA statistical test shows no significant
differences in miRNA levels, Pr(>F) = 0.7136.
3.2. Selected miRNAs Were Identified in Infant Milk Formulae
Next, we studied the relative expression of miR-146b-5p, miR-148a-3p, miR-155-5p,
mir-181a-5p, mir-200a-3p in six different infant milk formulae (IMF) available in the local
market (Table S1). Total RNA was extracted after reconstituting the powder, as instructed
by the manufacturer in triplicate. miR-146b-5p, miR-148a-3p, miR-155-5p, mir-181a-5p,
and miR-200a-3p miRNAs were quantified as described in Materials and Methods. As
mentioned before, the results for miR-181a-5p were discarded due to technical problems
with the primer FmiR181 (Table S2). The results for miR-146b-5p, miR-148a-3p, miR-155-
5p, and mir-200a-3p indicated that the relative expression of miR-146b-5p and miR-155-
5p was higher in IMF than the expression of hsa-miR-148 and hsa-miR-200. In addition,
the Mann-Whitney U (Wilcoxon rank-sum) test indicated that the relative expression of
miRNAs was higher in the human milk samples in comparison to the IMF (p = 9.0 × 10−7
for miR-146; p = 1.1 × 10−7 for miR-148; p = 9.3 × 10−3 for miR-155, and p = 8.0 × 10−6 for mir-
200) (Figure 2).
Figure 1.
Expression of selected miRNAs in human colostrum/milk. hsa-miR146 (miR-146b-5p);
hsa-miR-148 (miR148a-3p), hsa-miR-155 (miR155-5p), hsa-miR-200 (mir200a-3p). The Y-axis shows
the relative expression of each miRNA with respect to the internal control cel-miR-39, and the X-axis
shows the corresponding miRNA. Each dot in the plot represents a sample. The double horizontal
lines indicate the standard error of the mean. One-way ANOVA statistical test shows no significant
differences in miRNA levels, Pr(>F) = 0.7136.
3.2. Selected miRNAs Were Identified in Infant Milk Formulae
Next, we studied the relative expression of miR-146b-5p, miR-148a-3p, miR-155-5p,
mir-181a-5p, mir-200a-3p in six different infant milk formulae (IMF) available in the local
market (Table S1). Total RNA was extracted after reconstituting the powder, as instructed
by the manufacturer in triplicate. miR-146b-5p, miR-148a-3p, miR-155-5p, mir-181a-5p,
and miR-200a-3p miRNAs were quantified as described in Materials and Methods. As
mentioned before, the results for miR-181a-5p were discarded due to technical problems
with the primer FmiR181 (Table S2). The results for miR-146b-5p, miR-148a-3p, miR-155-5p,
and mir-200a-3p indicated that the relative expression of miR-146b-5p and miR-155-5p was
higher in IMF than the expression of hsa-miR-148 and hsa-miR-200. In addition, the Mann-
Whitney U (Wilcoxon rank-sum) test indicated that the relative expression of miRNAs was
higher in the human milk samples in comparison to the IMF (p= 9.0
×
10
−7
for miR-146;
p= 1.1
×
10
−7
for miR-148; p= 9.3
×
10
−3
for miR-155, and p= 8.0
×
10
−6
for mir-200)
(Figure 2).

BioTech 2022,11, 11 6 of 9
BioTech 2021, 10, x FOR PEER REVIEW 6 of 10
Figure 2. Comparative expression of selected hsa-miRs in colostrum/milk (n = 60) with respect to
milk formula (n = 18). The graphics show the relative expression of each miRNA with respect to the
internal control cel-miR-39. (A) hsa-miR-146, (B) hsa-miR-148, (C) hsa-miR-155, (D) hsa-miR-200.
The Y-axis shows the relative expression of each miRNA, and the X-axis shows the type of sample.
Each dot in the plot represents a sample. The double horizontal lines indicate the standard error of
the mean. Mann-Whitney U (Wilcoxon rank-sum) test was made. ** indicates p < 0.01 and ***
indicates p < 0.001.
4. Discussion
miRNAs play an important role in the regulation of gene expression and contribute
to the pathogenesis of complex diseases, such as IBD [28]. Several studies have revealed
that levels of certain miRNAs are altered in IBD patients in comparison to healthy
individuals, such as miR-124, miR-320, miR-21, miR-31, and miR-141 [29]. Further, work
done on Crohn’s disease suggests that dysregulation of miRNAs at the level of the
intestinal mucosa may play an important role in the early stages of the disease [30].
Studies in human milk (HM) have shown evidence of the presence of miRNAs, where
they remain stable, even under very low pH conditions. This supports the notion that
miRNAs can remain stable in the acidic conditions of the gastrointestinal tract and
consequently, are able to be absorbed in the gut. Many miRNAs present in HM have been
described, some with unknown function, but others with immunoregulatory function.
Among the miRNAs found in a study of HM using massive sequencing, miR-146b-5p,
miR-200a-3p, and miR-148-3p which were measured in our work, were found among the
10 most abundant [31]. A similar expression of miR-148a-3p, miR-146b-5p, and miR-
200a/c-3p has been reported in different studies discussed in a recent systematic review,
indistinctly of factors like the fraction of HM, lactational age, and health status of the
mother and her offspring [32].
The miRNA expression after birth has been monitored. The expression of miRNA-
146b, analyzed in the lipid and skimmed fraction of HM, was reported at relatively stable
Figure 2.
Comparative expression of selected hsa-miRs in colostrum/milk (n = 60) with respect to
milk formula (n = 18). The graphics show the relative expression of each miRNA with respect to the
internal control cel-miR-39. (
A
) hsa-miR-146, (
B
) hsa-miR-148, (
C
) hsa-miR-155, (
D
) hsa-miR-200. The
Y-axis shows the relative expression of each miRNA, and the X-axis shows the type of sample. Each
dot in the plot represents a sample. The double horizontal lines indicate the standard error of the
mean. Mann-Whitney U (Wilcoxon rank-sum) test was made. ** indicates p< 0.01 and *** indicates
p< 0.001.
4. Discussion
miRNAs play an important role in the regulation of gene expression and contribute to
the pathogenesis of complex diseases, such as IBD [
28
]. Several studies have revealed that
levels of certain miRNAs are altered in IBD patients in comparison to healthy individuals,
such as miR-124, miR-320, miR-21, miR-31, and miR-141 [
29
]. Further, work done on
Crohn’s disease suggests that dysregulation of miRNAs at the level of the intestinal mucosa
may play an important role in the early stages of the disease [30].
Studies in human milk (HM) have shown evidence of the presence of miRNAs, where
they remain stable, even under very low pH conditions. This supports the notion that
miRNAs can remain stable in the acidic conditions of the gastrointestinal tract and con-
sequently, are able to be absorbed in the gut. Many miRNAs present in HM have been
described, some with unknown function, but others with immunoregulatory function.
Among the miRNAs found in a study of HM using massive sequencing, miR-146b-5p,
miR-200a-3p, and miR-148-3p which were measured in our work, were found among the 10
most abundant [
31
]. A similar expression of miR-148a-3p, miR-146b-5p, and miR-200a/c-3p
has been reported in different studies discussed in a recent systematic review, indistinctly
of factors like the fraction of HM, lactational age, and health status of the mother and her
offspring [32].
The miRNA expression after birth has been monitored. The expression of miRNA-146b,
analyzed in the lipid and skimmed fraction of HM, was reported at relatively stable levels
from the first to the second month after delivery [
15
]. In another work evaluating a different
group of miRNAs, the expression of miR-146b-5p is reported stable in samples obtained

BioTech 2022,11, 11 7 of 9
during two, four, and six months after birth [
33
]. On the other hand, miR-148a suffered
a slight decline in its expression in late lactation. Additionally, a differential expression
was found between women with normal weight and women who are overweight or
obese. A decrease of 30% was registered in obesity with respect to the healthy group
during the first month of lactation. This decrease was not observed two months later,
where significant differences in the expression of this miRNA among the groups were not
found [
34
]. However, in another report, differences in the expression of miR-148 in skim
milk (0 to 1 month postpartum) were not observed [
35
]. In another study, the presence of
miR-200a-3p was reported consistently and highly expressed in all human milk samples
evaluated [
36
]. Regarding variations in miRNA expression during the day, miR-146b has
shown little variation in its expression [15].
It is noteworthy that studied miRNAs could be found in IMF despite the processing
involved in their production, however, similar results, yielding lower miRNA in IMF with
respect to HCM have been previously reported [
7
,
37
], despite the fact that miRNAs are
known to be stable and resistant to different adverse conditions, like abrupt temperature
and pH changes and endonuclease activity [
31
,
38
]. It is also important to remark that HCM
samples used in this study were collected in late 2017 and early 2018, so they had been stored
for at least three years at
−
70
◦
C, while the IMF were prepared and processed immediately
after purchase. To this point, no reports have been found discussing the stability of miRNAs
over prolonged periods of time, and although the long storage time of the samples could
be a limitation of this work, it is interesting to note that there is a higher expression of
the miRNAs studied in HCM, despite the age of the samples. Moreover, consistent with
these results, another study focused on miRNA presence in infant gastric content and
found that miRNA profile of gastric content of formula-fed infants was lower than that of
breastfed infants, nevertheless, their presence indicates miRNA stability and transfer [
39
].
The limitations of this work are the small number of different immunoregulatory miRNAs
and IMF that were monitored.
5. Conclusions
The relative expression of the four immunoregulatory miRNAs analyzed in this study
is comparable in all the human colostrum/milk samples analyzed. The same miRNAs
are not abundantly expressed in the infant milk formulae studied in this work. The
expression of the immunomodulatory miRNAs is higher in human colostrum/milk than
the expression in infant milk formulae. This observation supports the importance of
the human breastfeeding practice. For instance, further studies analyzing human milk
miRNA profile characterization by high-throughput sequencing could be useful. Human
milk miRNA expression could be related with different factors, like breastfeeding period,
mother’s age and weight, for this reason, appropriate studies are needed. Moreover, it is
an important matter to compare human miRNA profile with different health conditions
present in pregnancy, e.g., obesity, metabolic syndrome, and gestational diabetes.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/
10.3390/biotech11020011/s1, Figure S1. Time frame graphic for sample procurement in the study,
Figure S2. Expression of selected hsa-miRs in colostrum/milk at different time, Figure S3. Expression
of miRNAs in colostrum/milk v.s. the age of mothers. Table S1. Nutrimental information of Infant
Milk Formulae, and Table S2. Primer sequences used in this study.
Author Contributions:
Conceptualization, J.M.V.-I., M.N.R.-C. and J.G.-M.; methodology, J.M.V.-I.,
T.B.-G. and A.A.-H.; validation, J.M.V.-I., T.B.-G. and A.A.-H.; formal analysis, J.M.V.-I.; investigation,
J.M.V.-I., T.B.-G., A.A.-H., H.M.-C., K.C.-C., C.J.J.-C., M.N.R.-C. and J.G.-M.; resources, M.N.R.-C. and
J.G.-M.; data curation, J.M.V.-I., T.B.-G., A.A.-H. and K.C.-C.; writing—original draft preparation,
J.M.V.-I., T.B.-G. and J.G.-M.; writing—review and editing, J.M.V.-I., T.B.-G., A.A.-H., H.M.-C., K.C.-C.,
C.J.J.-C., M.N.R.-C., J.G.-M.; visualization, J.M.V.-I.; supervision, C.J.J.-C., M.N.R.-C. and J.G.-M.;
project administration, M.N.R.-C. and J.G.-M.; funding acquisition, M.N.R.-C. and J.G.-M. All authors
have read and agreed to the published version of the manuscript.

BioTech 2022,11, 11 8 of 9
Funding:
This research was funded by Fondo SEP-Cinvestav-2018-174; and CONACyT FORDECYT-
PRONACES/6669/2020.
Institutional Review Board Statement:
The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the Ethics Committee of the General Hospital (Project
identification code: 217B560002018006).
Informed Consent Statement:
Informed consent was obtained from all subjects involved in the
study.
Data Availability Statement: Not applicable.
Acknowledgments:
We express our gratitude to Alberto Piña-Escobedo, Carolina Miranda-Brito,
Rodrigo García-Gutiérrez for support in the laboratory work; to Viridiana Rosas-Ocegueda and
Alma Lemus-Hernández for administrative assistance. We thank the CONACyT M. Sc. Fellowship
for J.M.V.-I. (997152), A.A.-H. (911623), H.M.-C. (1078225); Ph. D. Fellowship for T.B.-G. (635676),
K.C.-C. (777953), and Post-Doctoral Fellowship for C.J.J.-C. (321600). J.G.-M (19815) is Fellow from
the Sistema Nacional de Investigadores (SNI), Mexico.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
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