Maximally Expressed miRNAs of Milk in Cells, Plasma and Lipid Fraction of Human Milk and Antibodies-Abzymes Catalyzing Their Hydrolysis

: Human milk provides neonates with various components that ensure newborns’ growth, including protection from bacterial and viral infections. In neonates, the biological functions of many breast milk components can be very different compared with their functions in the body fluids of healthy adults. Catalytic antibodies-abzymes hydrolyzing peptides, proteins, DNAs, RNAs, and oligosaccharides were detected not only in the blood sera of autoimmune patients but also in human milk. Non-coding microRNAs (18–25 nucleotides) are intra- and extra-cellular molecules of different human fluids. MiRNAs possess many different biological functions, including regulating several hundred genes. Five of them: miR-148a-3p, miR-200c-3p, miR-378a-3p, miR-146b-5p and let-7f-5p were previously found in milk in increased concentrations. Here, we determined number of copies of these miRNAs in 1 mg of analyzed cells, lipid fractions, and plasmas of human milk samples. The relative amount of microRNA decreases in the following order: cells  lipid fraction > plasma. IgGs and sIgAs were isolated from milk plasma, and their activity in the hydrolysis of five microRNAs was compared. In general, sIgAs demonstrated higher miRNA-hydrolyzing activity than IgGs antibodies. The hydrolysis of five microRNAs by sIgAs and IgGs was site-specific. The relative activity of each microRNA hydrolysis was very dependent on the milk preparation. The correlation coefficients between the content of five RNAs in milk plasma and the relative activity of sIgAs than IgGs in their hydrolysis strongly depended on individual microRNA and changed from −0.01 to 0.80. Thus, it was shown that milk contains specific antibodies-abzymes hydrolyzing microRNAs specific for human milk.


Introduction
Short non-coding microRNAs (18-25 nucleotides) are intra-and extra-cellular molecules present in various human and animal fluids [1,2]. MicroRNAs have many different biological functions, including the regulation of up to several hundred genes [3,4]. Different changes of microRNAs (microRNA-regulated gene networks) can result in the realignment in the expression of a lot of genes in different cells. It was shown that human milk could contain a few dozens to thousands of various microRNAs [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. MicroRNAs of a mother's milk have important functions for lactating breasts and infants [5]. The known data strongly support that the milk microRNAs enter the systemic circulation of infants and have tissue-specific developmental and immunoprotective functions [5]. According to the literature data, human milk contains about 1400 mature miRNAs [5][6][7][8][9]. Based on the estimation of the value of the number of real-time polymerase chain reaction (PCR) cycles at which fluorescence exceeds the threshold value, it was concluded that miR-148a-3p, miR-200c-3p, miR-378a-3p, miR-146b-5p and let-7f-5p are contained in human milk in increased concentrations [5][6][7][8][9]. It was interesting to quantify and compare the relative content of these five microRNAs.
Because of the absence of apparent immunization, the existence of any ABZs in people without any immune diseases was considered not possible. For example, auto-abzymes were not detected in healthy people and patients demonstrating no very severe infringements of the immune status [11][12][13][14][15][16][17].
A particular group of healthy people are pregnant and lactating females. Women's milk contains different Abs (IgGs, IgAs, sIgAs and IgMs), and sIgAs are the major component (85-90%) [34,35]. The origin of milk IgGs is still debated; they could be partially produced locally by mammary gland specific cells or partially moved from the circulation system of female blood [34]. IgAs are synthesized by women's mammary gland B-lymphocytes [35]. IgA antibodies are produced by plasma cells in the mammary stroma, and then they are assembled to dimeric sIgAs on the basolateral surface of the epithelium [36]. During lactation, B cells stimulated by antigen in Peyer's patches switch from IgM production to dimeric IgA and migrate to the mammary gland [37,38].
The immune system of neonates during the first 4-6 months of life is immature: new-borns' mucous surfaces and respiratory and gastroenterological tracts are still poorly filled with antibodies [39]. Infants begin to produce antibodies in the intestine in the first 3-5 months of life. However, neonates are well protected by antibodies of their mother's milk (passive immunity), which covers mucous membranes with Abs against bacterial, viral, and other components [39]. Breast milk sIgAs are present in high concentration and active during at least 7-8 months after birth and play an important role in maintaining the passive humoral response [40].
Women during pregnancy and after the beginning of the lactation very often demonstrate a sharp exacerbation of AI reactions similar to those in typical autoimmune pathologies, including anti-phospholipid syndrome, systemic lupus erythematosus (SLE), multiple sclerosis (MS), thyroiditis, renal insufficiency, etc. [41][42][43][44][45]. Pregnancy realizes a range of specific changes in the immune system, leading to an increased risk of several diseases exacerbation and adverse maternal and fetal outcomes, including preeclampsia, fetal loss, and preterm birth [46,47]. SLE is often a disease during pregnancy [48,49] and sometimes leads to harmful situations for the mother and fetus [50]. The incidence of SLE exacerbation usually occurs during pregnancy and within three months after delivery [48,49,51].
There is sometimes a remission of multiple sclerosis in the third trimester of pregnancy, but the disease worsens in the first postpartum period [52]. Autoimmune thyroid reactions are found in approximately 18% of pregnant women [53]. It is important to emphasize that many different ADs could be "triggered" or "activated" in healthy females during their pregnancy and especially after childbirth [25,[41][42][43][44][45][54][55][56]. The pregnancy and the onset of lactation are special periods associated with the changes in the immune system of women [12][13][14][15][16][17]54,55]. These changes lead to the synthesis of various autoantibodies and abzymes in the blood and milk of women. The existence of abzymes in the blood and milk indicates the presence of AI reactions in women.
It was proposed earlier that different ADs could be originated from specific defects of hematopoietic stem cells [57]. It was later shown that various ADs development occurs due to specific changes in the differentiation profiles of bone marrow stem cells (HSCs) [58][59][60][61][62].
Spontaneous and accelerated by antigens development of SLE in SLE-prone MRL-lpr/lpr mice [58][59][60] and multiple sclerosis in EAE-prone C57BL/6 mice [61,62] leads to very similar specific reorganizations of their immune systems, which is bound with a production of abzymes hydrolyzing DNA and proteins.
Very similar changes (as in mice with deep SLE) in the differentiation profiles of HSCs were revealed in lactating mice [58][59][60]. Such changes in lactating mice are usually temporary and return to normal after 1-3 months. In contrast, there are further changes in the differentiation profiles in mice diseased with SLE during the deepening of the pathology [58][59][60]. As in mice with deep SLE pathology, the changes in the differentiation profiles in lactating mice lead to the production of abzymes with high catalytic activity.
Taking this into account, in this work, for the first time, a quantitative analysis of these microRNAs and an assessment of the relative activities of sIgAs and IgGs of human milk in the hydrolysis of these five microRNAs were carried out.
The statistical difference in the content of almost all microRNAs in the cell fraction was significant (p = 0.001-0.003), except for 148a-3p-278a-3p and 148a-3p-278a-3p (p = 0.79). A similar situation was observed for the lipid fraction (p = 0.001-0.04) except for two pairs of parameters: 148a-3p-278a-3p and 146b-5p-let-7f-5p (p = 0.1-0.96). The statistical significance of differences in the content of most microRNAs in milk plasma was also high (p = 0.001-0.01), except for 146b-5p-200c-3p and 148-3p-378-3p (p = 0.43-0.87). Thus, a quantitative analysis of the content of five microRNAs in plasmas, cells, and lipid fractions of human milk was carried out. At the same time, no uniformity in the content of all five miRNAs in each of the three milk fractions (plasmas, cells and lipid fractions) was found. The content of each microRNA in each of the fractions turned out to be specific.

Hydrolysis of microRNAs
The relative activity in the hydrolysis of five microRNAs was analyzed using seven IgG and sIgA preparations isolated from milk plasma as described in [73,74]. Typical patterns of miR-146b-5p and miR-148a-3p splitting by seven milk sIgAs and IgGs are given in Figure 1. Three out of seven IgG preparations are weakly hydrolyzed miR-146b-5p ( Figure 1A). The major cleavage sites of miR-146b-5p in the case of IgGs with numbers 3 and 4 are 10C-11U, 9C-10C and 6A-7U, while other IgGs weaker cleave this miRNA in these sites ( Figure 1A). Interestingly, 4 out of seven IgG preparations (especially with number 7) effectively hydrolyze miR-146b-5p at 18A-19A and 16U-17C sites. Exactly these 18A-19A and 16U-17C sites of the hydrolysis are the most typical and common for all seven sIgA preparations ( Figure 1A). The characteristic sites for IgGs in the case of sIgAs should be attributed to moderate hydrolysis sites.
There are much more major sites for hydrolysis of miR-148a-3p by IgGs and sIgAs than for miR-146b-5p. For most IgGs and sIgAs, the following sites can be classified as major: 12A-13U, 11C-12A, 9G-10A, 6C-7A, 5U-6C and 3U-4U ( Figure 1B). At the same time, for some IgGs and sIgAs, individual major hydrolysis sites were observed, which should be classified as average or minor in the case of other antibody preparations. This is pronounced especially in the case of sIgAs with numbers 4 and 5 that very effectively hydrolyze miR-148a-3p at 14C-15A site ( Figure 1B).
Only one common major site of miR-200c-3p hydrolysis for IgGs and sIgAs antibodies is 5A-6G site (Figure 2A). Four additional major sites are observed for three sIgA preparations with numbers 1, 4, and 5: 11G-12G, 10U-11G, 9A-10U, and 6G-7U. For sI-gA7, IgG1, IgG3, and IgG5, there is a pronounced average hydrolysis site-14C-15C. Interestingly, sIgA7 and IgG2-IgG6, in addition to efficient hydrolysis miR-200c-3p at 5A-6G site, split this microRNA at many other sites with approximately comparable efficiency ( Figure 2A). For all IgG preparations, the main major site of let-7f-5p hydrolysis is 7U-8G ( Figure 2B). In addition, all IgG preparations effectively hydrolyze this microRNA at two sites: 11A-12G and 10U-11A. Other sites of let-7f-5p hydrolysis in the case of some IgGs should be classified as moderate or minor. For seven sIgA preparations, no pronounced common sites of let-7f-5p hydrolysis were observed ( Figure 2B). Three IgGs (with numbers 1, 4 and 5) very effectively cleave this RNA at 5U-6A site, while in the case of sIgAs, this site is a minor one. 7U-8G site is major for four sIgA preparations with numbers 2, 3, 6 and 7 ( Figure 2B). Figure 3 demonstrates the patterns of the hydrolysis of miR-378a-3p by IgG and sIgA antibodies.
There are four moderate or minor sites of miR-378a-3p splitting by IgGs and sIgAs: 20U-21C, 16C-17A, 15U-16C and 11A-12G. At the same time, 7A-8C, 6G-7A, 5A-6G and 4A-5A sites are major in the case of two sIgAs, but they are average or minor for all other antibodies ( Figure 3). Table 3 shows the relative activities of seven individual IgG and sIgA preparations and the average values for these preparations in the hydrolysis of five microRNAs. Several of the antibody sets did not meet the Gaussian normal distribution. Considering this, for all sets of parameters, we calculated the median (M) and interquartile ranges (IQR) ( Table 3).  Coef. Correl   Interestingly, in the hydrolysis of miR-148a-3p and miR-200c-3p by sIgAs, on average, was 1.7-1.9 times more active than IgGs ( Table 3).
The opposite situation was observed for miR-146b-5p, which was hydrolyzed by IgGs approximately 1.8 times more efficiently than by sIgAs. The average activities of sIgAs and IgGs in the hydrolysis of miR-378a-3p and let-7f-5p were comparable (Table 3). The most average increased hydrolysis by sIgAs and IgGs was observed for let-7f-5p, and the lowest hydrolysis rate was found for miR-378a-3p (Table 3).
All antibodies were isolated from milk plasma. Therefore, we compared CCs between concentrations of various microRNAs in plasma (Table 4). All CCs were positive, but very different and varied from 0.007 for miR-200c-3p and let-7f-5p to 0.99 in the case of miR-148a-3p and miR-378a-3p (Table 4). It could be expected that the relative activity of antibodies in the hydrolysis of each of the five RNAs would correlate with the relative concentration of these microRNAs in milk plasma. However, this turned out to be far from the case. The CCs of the concentrations of miR-148a-3p (−0.01) and miR-200c-3p (−0.05) in plasmas with the efficiency of their hydrolysis by IgGs turned out to be weakly negative. The rest of the CCs were positive: 0.41-0.79 (Table 5). In the case of sIgAs, all CCs were positive and varied from 0.03 to 0.8. Table 5. Values of coefficient correlations between plasma microRNAs concentrations and relative activities of seven IgGs and sIgAs in the hydrolysis of five microRNAs *. miR-148a-3p miR-200c-3p miR-378a-3p let-7f-5p miR-146b-

Spatial Structures of Five miRNAs
Spatial structures of five miRNAs having minimal free energy were calculated. The relative amount (%) of every product of each microRNA hydrolysis by individual IgGs and sIgAs was calculated. Then, using the data of three independent experiments for each IgG and sIgA samples, the average percentage of every product corresponding to seven milk plasma IgG and IgA preparations was calculated. Figures 4-6 show the location of hydrolysis sites in the spatial structures of five microRNAs in the case of IgG and sIgA antibodies. As indicated above and can be seen in Figures 1-3, antibodies from milk plasma of various donors hydrolyze five microRNAs with different efficiencies and, in some cases, at different sites. Taking this into account, Figures 4-6 show averaged data on the efficiency of five microRNAs hydrolysis by seven IgG and sIgA preparations at each of the sites. Pronounced major hydrolysis sites of each microRNA in the case of only some antibody preparations are indicated in brackets. The main sites for more efficient cleavage of miR-148a-3p by IgG and sIgA antibodies are located in the specific loop of this microRNA ( Figure 4A,B). In the case of seven sIgAs, the hydrolysis at five sites of the loop is very different. Some sIgA preparations hydrolyze this microRNA at these sites 1.3-3.0 times more efficiently in comparison with the average values for all seven sIgAs (indicated in brackets) ( Figure 4A). The hydrolysis efficiency of the miR-148a-3p by seven IgG preparations was more comparable ( Figure 4B).
Four of the six hydrolysis sites of miR-200c-3p are also located in the loop of this RNA, but they can be classified as moderate cleavage sites in the case of IgGs ( Figure 4D). Several sIgA preparations more efficiently hydrolyze miR-200c-3p at two sites in this loop ( Figure 4C). The most efficient hydrolysis of this miR-200c-3p by sIgAs and IgGs occurs at the 5A-6D site outlying from the loop ( Figure 4C,D).
The double-stranded loop fragment of miR-378a-3p includes 16 of its 22 nucleotides ( Figure 5A,B). In addion, in this case, 8 of 11 hydrolysis sites are located in this specific loop. The relative average percentage of hydrolysis of miR-378a-3p by sIgAs (1.7-5.2%) and IgGs (1.6-4.4%) is relatively low. However, there are three hydrolysis sites of this microRNA in the region from 3G to 6G. In the case of several sIgA preparations, hydrolysis at the 4A-5A site proceeds more efficiently (12%).
Four of the eight hydrolysis sites of let-7f-5p are disposed in its loop, having no double-stranded regions ( Figure 5C,D). Of the six sites of pronounced hydrolysis, three are also located in a specific loop of miR-146b-5p. Some sIgA preparations hydrolyze these microRNAs at two sites (7U-8G and 10U-11A) more efficiently than other ones ( Figure 5D). However, one sIgA preparation hydrolyzes this microRNA most efficiently at 5U-6A site (24.4%). At the same time, the overall sites of maximum hydrolysis of let-7f-5p by sIgAs and IgGs are entirely different: 5U-6A and 7U-8G ( Figure 5C,D).
sIgAs and IgGs hydrolyze miR-146b-5p at six sites, three of which are located in the loop. Hydrolysis at these three loop sites is moderate ( Figure 6). The site of maximum hydrolysis of this microRNA by sIgAs and IgGs is 9C-10C6 adjacent to the loop ( Figure 6). Unlike sIgAs ( Figure 6A), several IgG more efficiently hydrolyze this microRNA at two sites: 5G-6A and 6A-7U ( Figure 6B). Regardless of the absence or presence of double-stranded regions in specific loops, all five microRNAs are mainly hydrolyzed by sIgAs and IgGs at the sites of their particular loops. Hydrolysis at some sites of all five microRNAs in the case of sIgAs and IgGs is comparable, however, in the case of some sites, significant differences are observed. The most striking differences in the hydrolysis of miRNAs by sIgAs and IgGs are observed in the case of a specific hairpin fragment of miR-200c-3p ( Figure 4C,D), 4A-5A site of miR-378a-3p ( Figure 5A,B), 5U-6A site of let-7f-5p ( Figure 5C,D), and 5G-6A and 6A-7U sites of miR-146b-5p.

Discussion
MicroRNAs regulate the expression of many genes through association with Argonaute [75][76][77][78][79][80][81]. At the same time, miRNAs are susceptible to degradation. Although association with Argonaute protects miRNAs from nucleases, extensive pairing with some unusual RNA targets can trigger miRNA hydrolysis [75,80]. It is believed that the degree of complementarity and the miRNA/target ratio is critical for efficient miRNA hydrolysis [75,77]. MiRNA and target form a duplex with an unpaired flexible linker, which leads to duplex bending and opening of the 3′ end of miRNA for enzymatic attack [77]. According to [76], endogenous RNA (Serpine1) controls the hydrolysis of two miRNAs (miR-30b-5p and miR-30c-5p) in mouse fibroblasts. Targeted miRNA degradation requires ZSWIM8 Cullin-RING E3 ubiquitin ligase [80]. Some studies have found other proteins involved in miRNA degradation, and it is also noted that the unprotected 3′-ends of miRNAs can become available for enzymatic attack by 3′-5′-exonucleases or as yet unidentified other cellular enzymes [75,78]. Thus, destruction occurs in a complex with Argonaute with miRNA at the 3′-end of the molecule.
The statistical difference in almost all microRNAs content in cells, plasmas, and lipid fractions was mainly significant (p = 0.001-0.04), except for some of them (p > 0.05). Positive CCs between the five miRNAs in most preparations of milk plasmas (0.14 to 0.92), cells (0.04 to 0.99), and lipid fractions varied over a wide range with several exceptions when negative correlations were observed from −0.005 to −0.37 (Table 1).
IgGs and sIgAs were isolated from milk plasmas, and their relative catalytic activity in the hydrolysis of five microRNAs was estimated (Table 3). It should be noted that the relative activity of IgGs and sIgAs in the hydrolysis of five microRNAs depended very strongly on the milk preparation. For example, the rate of hydrolysis of miR-200c-3p by IgG7 was 21.3 times higher than that for IgG2, while sIgA2 hydrolyzed this microRNA 20.2 times slower than sIgA1. Overall, the efficiency of some microRNAs hydrolysis by seven IgGs and sIgAs did not correspond to the normal Gaussian distribution. CCs of the hydrolysis of five RNAs by the seven IgGs ranged from 0.17 to 0.82, while for 7 sIgAs from −0.08 to 0.98 (Table 3). The CCs between concentrations of five microRNAs in plasmas were also very different and varied from 0.07 to 0.99 (Table 3). CCs between microRNAs concentrations in individual plasmas and RAs of Abs corresponding to these plasmas in the hydrolysis of five microRNAs for IgGs varied from −0.01 to 0.79 and for sIgAs from 0.03 to 0.80 (Table 3). Milk was collected from seven women at about the same time after lactation began. However, in the case of three RNAs, the CCs between the RAs of IgGs and sIgAs were negative (−0.07-−0.41), and for two RNAs, they were highly positive (0.87) ( Table 3).
The sites of five microRNAs hydrolysis by sIgAs and IgGs are indicated on RNA's spatial structures (Figures 4-6). Substantially, sIgAs and IgGs hydrolyze five microRNAs at the same sites, mainly located in the loop structures of the substrates (Figures 4-6). The hydrolysis efficiency of five RNAs by sIgAs and IgGs is predominantly comparable. At the same time, several particular of seven sIgA and IgG preparations hydrolyze these microRNAs much more efficiently than others. Hydrolysis of microRNAs occurs after their various bases: A, G, U and C (Figures 4-6). No pronounced specificity of microRNA hydrolysis after a specific base is observed. It is possible that, in principle, the spatial structures of different microRNAs are, to some extent, a more critical factor in determining cleavage sites.
As mentioned above, destruction of microRNAs occurs in a complex with Argonaute at the 3′-end of RNAs. It should be assumed that some part of microRNAs in milk can exist in a free form. And these microRNAs can be site-specific hydrolyzed by IgGs not at their 3-terminus.

Chemicals and Donors
Most chemicals of high quality used for this study were obtained from Sigma (St. Louis, MO, USA). Protein A-Sepharose, Superdex 200 HR 10/30 column, and Protein G-Sepharose were provided by GE Healthcare (GE Healthcare, New York, NY, USA). FastAP thermosensitive alkaline phosphatase and RNase A were from Fisher Scientific (Pittsburgh, PA, USA). Fluorescein isothiocyanate (FITC) was from Thermo Fisher (Thermo Fisher; MA, New York, USA). FITC-conjugates of oligonucleotides (ONs) were synthesized using the solid phase phosphoramidite method [82]. According to their analysis, all ribo-ONs were homogeneous according to reversed-phase chromatography and electrophoresis in 20% polyacrylamide gel.
The milk sampling protocol was confirmed by the Human ethics committee of Novosibirsk State Medical University (Novosibirsk, Russia; number 105-HIV; 07. 2010). The ethics committee supported this study in appliance with Helsinki ethics committee guidelines. All mothers gave written agreement to donate their milk for scientific studies. The mothers have no history of gastrointestinal, respiratory, autoimmune, rheumatologic, cardiovascular, or other system pathologies.

Purification and Analysis of RNAs
RNA preparations were isolated from different fractions of human milk: cell sediments, lipid fractions, and milk plasmas. A combination of special Lira reagents and buffers and columns (BiolabMix) was used for isolation. 1 mL of a special solution of Lira reagents was added to 100 μL of the sample and incubated for 10 min at room temperature with constant stirring. Then, 200 μL of chloroform was added, and the mixture was incubated for 10 min at room temperature. The resulting mixture was centrifuged for 12 min at 10,000× g at 4 °C. The aqueous upper phase was used for the isolation of RNAs. To RNA-containing aqueous phase, an equal volume of 96% ethanol was added, and this mixture was applied to the silicon membrane of a particular column (BiolabMix). After the column centrifugation in a special tube for 30 s at 10,000× g, at 4 °C, the filtrate realized from the column was removed. Then, to 150 μL of special Lira concentrated buffer WB1, 350 μL of ethanol was added, and 500 μL of the mixture was applied to the column. After centrifugation of the column for 30 s at 10,000× g, at 4 °C, the filtrate was removed. Then, this operation was repeated one more. For complete removal of the buffer, the column was centrifuged again for 3 min at 10,000× g. To remove from the column RNAs, 60 μL of special buffer for RNA elution was applied on the column, which was incubated at room temperature for 5 min, centrifuged for 1 min at 10,000× g, at 4 °C. To remove co-extracted DNA, RNA preparations obtained were incubated for 20 min at 37 °C with DNase I, and for inactivation of DNase I, the solution was heated to 70 °C for 5 min.

RNA Amplification
RNA amplification was performed according to a standard protocol using special primers on a Bio-Rad CFX Connect device (Hercules, CA, New York, USA).
In the first stage, microRNA reverse transcription was performed using specific Stem-loop primers and a reverse transcription kit including M-MuLV-RH reverse transcriptase (BiolabMix). The reaction mixture containing 2 μL RNA, 1 μL Stem-loop reverse transcription (RT)-primer, 12 μL H2O was heated to 65 °C for 5 min, and then cooled on ice for 2 min. The resulting mixture was centrifuged, 1 μL of M-Mul V and 4 μL of M-MuI V buffer were added. The mixture was incubated in a Bio-Rad CFX Connect cycler: 30 min at 16 °C followed at 30 °C for 30 s for 60 cycles, at 42 °C for 30 s, at 50 °C for 1 s and finally at 85 °C for 5 min. Then, reverse transcription products were analyzed by real-time polymerase chain reaction using a fluorescent probe. The reaction mixture (20 μL) contained 10 μL BioMaster HS-qPCR (2x) (BiolabMix), 5.6 μL H2O, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 1.0 μL fluorescent probe (2.5 μM), and 2 μL reverse transcription product. The reaction mixture was incubated in a Bio-Rad CFX Connect system at 95 °C for 5 min, followed for 45 cycles at 95 °C for 5 s, at 60 °C for 10 s, and 72 °C for 1 min. For reverse transcription of the mRNA of the reference genes, the oligo (dT) primer and the SYBR intercalating dye were used instead of the Stemloop primer.
Primers: Forward: GTCATCCCTGAGCTGAACGG Reverse: TTGAGGGCAATGCCAGCC The first step is reverse transcription of microRNAs was performed using oligo(dT) primers and reverse transcription kit containing reverse transcriptase M-MuLV-RH (Bi-olabMix). The reaction mixture containing 2 μL RNA, 1 μL oligo(dT)-primer, 12 μL H2O was heated to 65 °C for 5 min, and then cooled on ice for 2 min. After mixture centrifugation, 1 μL of M-Mul V and 4 μL of M-MuI V buffer were added. The mixture was incubated in a Bio-Rad CFX Connect cycler for 30 min at 16 °C, and then for 60 cycles at 30 °C for 30 s, 42 °C for 30 s and at 50 °C for 1 s and finally at 85 °C for 5 min. The reverse transcription products were analyzed by real-time PCR with the SYBR intercalating dye detection. The 20 μL reaction mixture contained: 10 μL HS-qPCR SYBR Blue (2×) (BiolabMix), 6 μL H2O, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), and 2 μL reverse transcription product. The reaction mixture was incubated in a Bio-Rad CFX Connect cycler at 95 °C for 5 min, followed for 45 cycles at 95 °C during 5 s, at 60 °C for 10 s, and at 72 °C for 1 min.
The calibration curves were obtained based on the amplification data of synthetic microRNA used in a concentration from 10 −3 to 10 −7 ng/μL). Using applicator software and calibration curves, the concentrations of each studied microRNA (ng/μL) in different human milk fractions were calculated and recalculated to the number of microRNA copies in 1 mg of the investigated fractions of human milk plasmas, cells, and lipid fractions under study.

Purification and Analysis of Antibodies
The milk of 7 healthy females residing in the Russia Novosibirsk region (120 mL at one time; 20-35 years old) was collected at 1.0-1.5 weeks after the onset of the lactation. Milk samples were collected using the standard sterile appliances intended for the collection of excess of mother's milk. During 1-3 h after collection, all samples were cooled to 4 °C, and centrifuged for 20 min at 14 thousand rpm using the Eppendorf centrifuge; cells, lipid phases, and milk plasma were obtained. Immunoglobulins were purified from each milk sample similar to [63][64][65][66][67][68]. There were no substantial variations in any analyzed parameters of antibodies and abzymes (relative content of Abs and their catalytic activities) within the sampling period of 1-4 weeks after the beginning of lactation [73,74].
To obtain IgGs, the milk plasma was delivered on a column with Protein G-Sepharose equilibrated with buffer A (20 mM Tris-HCl (pH 7.5), 0.15 M NaCl) as in [73,74]. The flow-through fraction containing sIgA antibodies was applied on a column with Protein A-Sepharose equilibrated with buffer A. All nonspecifically adsorbed proteins were eluted from Protein G-Sepharose and Protein A-Sepharose first using buffer A up to zero optical density (A280), then with buffer A supplemented with 0.3 M NaCl and 1.0% Triton X-100, and again with buffer A. IgGs and sIgAs were specifically eluted from each of sorbents with 0.1 M glycine-HCl (pH 2.6), immediately neutralized using 1.0 M Tris-HCl (pH 8.5), and then dialyzed against 20 mM Tris-HCl (pH 7.5) [73,74].
These microRNAs were chosen since they were found in human milk in increased concentrations [5,9].
The reaction mixture (15 μL) contained 50 mM Tris-HCl buffer (pH 7.5); 0.01 mg/mL one of labeled microRNA and 40 μg/mL IgG or sIgA antibodies similar to [73,74]. To get markers of ON length, limited statistical hydrolysis of microRNAs with unspecific alkaline RNase (splitting RNAs with comparable efficiency at all internucleoside bonds) and specific RNase T1 hydrolysis was used. The products after alkaline hydrolysis contained cyclic 3′-monophosphate that possess lower electrophoretic mobility; they give additional bands. Therefore, they were treated with FastAP thermally sensitive alkaline phosphatase. All gels were analyzed using Typhoon FLA 9500 laser scanner (GE Healthcare, New York, NY, USA). The results are reported as a mean ± standard deviation of at least three independent experiments.

Spatial Model of microRNAs
The spatial models of four microRNAs were generated by Predict a Secondary Structure server, which uses a combination of four algorithms for predicting the secondary structure of RNA similar to [69][70][71]: calculating a partition function, denoting the structure with minimal energy by http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html (accessed 14 February 2018).

Statistical Analysis
The relative activities of the IgG and sIgA preparations were estimated from a decrease in the fluorescence intensity of the initial microRNA in comparison with that of the control experiment corresponding to incubation of five microRNAs without Abs. The results were given as the mean and the standard deviation of at least two-three independent experiments for each IgG and sIgA preparation. Most of the sample sets did not meet the normal Gaussian distribution. To check for a normality of the values distribution law the criterion of Shapiro-Wilk's W Test was used. The correlation coefficients between different parameters were calculated using the criterion of Shapiro-Wilk's W Test. The differences between different groups of microRNAs of various groups of IgGs and sIgAs sets were estimated by the Mann-Whitney test (Statistica 10; Statistical Package, StatSoft. Inc., USA; http://www.statsoft.com/Products/STATISTICA-Features; StatSoft. Inc., New York, accessed 20 January 2010), the value p < 0.05 was considered statistically significant. The median (M) and interquartile ranges (IQR) are estimated.

Conclusions
In summary, we have quantified the relative concentrations of five microRNAs (miR-148a-3p, miR-200c-3p, miR-378a-3p, miR-146b-5p and let-7f-5p) which are found in a mother's milk in increased concentration. It was shown that human milk polyclonal sIgAs and IgGs possess site-specific microRNAs-hydrolyzing activities. It is shown that each milk preparation is characterized by a specific content of five microRNAs, IgG and sIgA antibodies-abzymes that hydrolyze these RNAs. The correlation coefficients of the content of five microRNAs in three fractions of milk: cells, plasmas, and lipid fractions and the content of microRNAs in plasmas with relative activity of sIgAs and IgGs in the hydrolysis of these five RNAs vary over a wide range from negative to positive.  The ethics committee supported this study in appliance with Helsinki ethics committee guidelines. All mothers gave written agreement to donate their milk for scientific studies. The mothers have no history of gastrointestinal, respiratory, autoimmune, rheumatologic, cardiovascular, or other system pathologies.

Data Availability Statement:
The data supporting our study results are included in the article.