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Abstract

Mesenchymal stem cells (MSCs), isolated from different adult sources, have great appeal for therapeutic applications due to their simple isolation, extensive expansion potential, and high differentiative potential.

In our previous studies we isolated MSCs form amniotic fluid (AF-MSCs) and skin (S-MSCs) and characterized them according to their phenotype, pluripotency, and mRNA/microRNAs (miRNAs) profiling using Card A from Life Technologies.

Here, we enlarge the profiling of AF-MCSs and S-MSCs to the more recently discovered miRNAs (Card B by Life Technologies) to identify the miRNAs putative target genes and the relative signaling pathways. Card B, in fact, contains miRNAs whose role and target are not yet elucidated.

The expression of the analyzed miRNAs is changing between S-MSCs and AF-MSCs, indicating that these two types of MSCs show differences potentially related to their source. Interestingly, the pathways targeted by the miRNAS deriving from Card B are the same found during the analysis of miRNAs from Card A.

This result confirms the key role played by WNT and TGF-β pathways in stem cell fate, underlining as other miRNAs partially ignored up to now deserve to be reconsidered. In addition, this analysis allows including Adherens junction pathways among the mechanisms finely regulated in stem cell behavior.

Keywords

amniotic fluid miRNAs MSCs profiling skin

Introduction

Human mesenchymal stem cells (MSCs) are the ideal candidates for different cellular therapies due to their simple isolation from different sources, extensive proliferation rate, and high differentiative potential.^1–7

In human bone marrow-derived MSCs, the silencing of Dicer or Drosha, two key components in the biogenesis of canonical microRNAs (miRNAs), blocks both osteogenic and adipogenic differentiation, establishing that miRNAs are critical regulators of differentiation.⁸

miRNAs are a class of 18–22 nucleotides length small RNAs which can attenuate gene expression by inhibiting translation in the cytoplasm or promoting mRNA degradation in the nucleus.⁹

Current literature data show that miRNAs play a crucial role in stem cell fate specification, including self-renewal, differentiation, and somatic cell reprogramming.^10,11

We have previously isolated and characterized MSCs from skin (S-MSCs) and amniotic fluid (AF-MSCs)^12–16 and also analyzed the expression level of 377 more highly considered miRNAs (TaqMan® Array Human MicroRNA Card A Set v2.0, Life Technologies).¹⁷

Even if both kinds of isolated MSCs satisfy the minimal criteria for MSCs definition,¹² they showed a different expression of miRNAs belonging to the WNT signaling, MAPK signaling, and TGF-β signaling pathways.¹⁷

These differences underline the importance of miRNAs in the fine regulation of the stem cell fate through the above-mentioned pathways that are involved in the maintenance, self-renewal, and differentiation of human MSCs.^18,19 In order to improve the actual knowledge about the involvement of miRNAs and relative targets on MSCs differentiation, we enlarge the profiling of AF- and S-MSCs to the TaqMan® Array Human MicroRNA Card B Set v2.0 (Life Technologies) that collects the more recently discovered miRNAs whose targets are still unclear.

Materials and methods

Isolation, culture, and characterization of AF-MSCs and S-MSCs

Amniotic fluids (n = 3) and skin biopsies (n = 3) were collected during routine amniocenteses for prenatal diagnosis and from mammary gland of healthy adult patients undergoing cosmetic plastic surgery, respectively, in accordance with the Politechnic Marche University Ethical Committee. AF-MSCs and S-MSCs were isolated, cultured, and characterized as previously described.^20–22

RNA extraction

To isolate small RNA (<200 nucleotides), approximately 1×10⁶ cells from each sample were processed using Total RNA Purification Kit (Norgen Biotek Corporation, Thorold, ON, Canada) according to the manufacturer’s protocol.

TaqMan array cards

Reverse transcription (RT) and pre-amplification reactions (Megaplex Primer Pools, Card B v2.0, Life Technologies) were performed in multiplex modality starting from 300 ng of total RNA for each sample according to the manufacturer’s protocol.

Simultaneous synthesis of cDNA for mature miRNAs (384) was performed using Megaplex Reverse Transcription Human Pool B (Life Technologies), which is a set of pre-defined pools of 380 stem-looped reverse transcription primers. RT-qPCR was performed using the Applied Biosystems 7900HT Fast Real-Time PCR System, and default thermal-cycling conditions. Controls are included in each card.

Analysis of miRNAs microarray data

MiRNAs expressed at a detectable level in more than 85% of samples were compared based on their relative expression to the overall miRNAs expression on each array, using the median and mean of four small nucleolar RNAs normalization analysis. The ΔΔCt for each miRNAs was defined as the difference of expression between S-MSCs and AF-MSCs. It was calculated with the following equation: ([Ct S-MSCs miR-median or small nucleolar RNAs mean Ct values obtained in the profiling of S-MSCs] – [Ct AF-MSCs miR-median or small nucleolar RNAs mean Ct values obtained in the profiling of AF-MSCs]). A two-fold or greater difference considered a significant finding. The mean values compared by two-tailed t test and Kruskal–Wallis test.

Computational prediction of miRNAs target genes

For prediction of potential miRNA-mRNA interactions and the relative signaling pathways, DIANA-mirPath (DIANA-microT v.3.0.)²³ and miRDB were used. In the DIANA-mirPath output page, all pathways are sorted according to a descending enrichment statistical score (−lnP) along with the number and name of each miR’s target gene involved in each KEGG (Kyoto Encyclopedia of Gene and Genomes) pathway. The input dataset enrichment in each KEGG pathway is represented by the negative natural logarithm of the P value (−lnP). KEGG was also used to find the signaling pathways related to the putative target genes identified with miRDB.^24–26

Results

Similar and different miRNAs expression in S-MSCs compared to AF-MSCs

Comparing the profiling of miRNAs spotted in the card B in S-MSCs (n = 3) with AF-MSCs (n = 3), most of them showed different expression patterns.

We identified and analyzed a mean of 160 miRNAs expressed at a detectable level in more than 85% of samples; no significant differences were noted among individual donors of each cellular type.

As before,¹⁷ we divided expressed miRNAs into three groups: group A, including miRNAs similarly expressed in S-MSCs and AF-MSCs (n = 34; fold-change = 1); group B, including miRNAs upregulated (n = 30; fold change ⩾2; P <0.05); and group C, including miRNAs downregulated (n = 89; fold-change ⩽–2; P <0.05) in S-MSCs versus AF-MSCs (Table 1).

Table 1.

Differentially expressed miRNAs from Card B by Life Technologies in S-MSCs compared to AF-MSCs.

Group A: miRNAs similarly expressed in S-MSCs vs. AF-MSCs	hsa-miR-378, hsa-miR-30d, hsa-miR-34b, hsa-miR-500, hsa-miR-520h, hsa-miR-549, hsa-miR-564, hsa-miR-567, hsa-miR-630, hsa-miR-634, hsa-miR-635, hsa-miR-640, hsa-miR-645, hsa-miR-767-5p, hsa-miR-767-3p, hsa-miR-31-3p, hsa-miR-302b-5p, hsa-miR-376a-5p, hsa-miR-93-3p, hsa-miR-135b-3p, hsa-miR-15b-3p, hsa-miR-933, hsa-miR-942, hsa-miR-124-5p, hsa-let-7a-3p, hsa-miR-431-3p, hsa-miR-7-2-3p, hsa-miR-10b-3p, hsa-miR-708-3p, hsa-miR-16-1-3p, hsa-miR-1292, hsa-miR-1301, hsa-miR-128, hsa-miR-1825
Group B: miRNAs upregulated in S-MSCs vs. AF-MSCs	hsa-let-7c-3p, hsa-let-7g-3p, hsa-miR-1183, hsa-miR-1300, hsa-miR-148a-5p, hsa-miR-185-3p, hsa-miR-223-5p, hsa-miR-24-2-5p, hsa-miR-29b-1-5p, hsa-miR-302d-5p, hsa-miR-30a-3p, hsa-miR-30d-3p, hsa-miR-363-5p, hsa-miR-425-3p, hsa-miR-519e5p, hsa-miR-548K, hsa-miR-580, hsa-miR-585, hsa-miR-592, hsa-miR-600, hsa-miR-601, hsa-miR-606, hsa-miR-608, hsa-miR-631, hsa-miR-641, hsa-miR-665, hsa-miR-888-3p, hsa-miR-892b, hsa-miR-92a-1-5p, hsa-miR-96-3p
Group C: miRNAs downregulated in S-MSCs vs. AF-MSCs.	hsa-miR-101-5p, hsa-miR-105-3p, hsa-miR-106b-3p, hsa-miR-1203-, hsa-miR-122-3p, hsa-miR-1224-3p, hsa-miR-1238, hsa-miR-1251, hsa-miR-125b-2-3p, hsa-miR-1262, hsa-miR-1263, hsa-miR-1272, hsa-miR-1276, hsa-miR-1282, hsa-miR-1284, hsa-miR-1285, hsa-miR-129-3p, hsa-miR-1296, hsa-miR-130a-3p, hsa-miR-132-5p, hsa-miR-138-2-3p, hsa-miR-144-5p, hsa-miR-17-3p, hsa-miR-181a-2-3p, hsa-miR-182-3p, hsa-miR-192-3p, hsa-miR-193b-5p, hsa-miR-195-3p, hsa-miR-19b-1-5p, hsa-miR-200c-5p, hsa-miR-206, hsa-miR-20a-5p, hsa-miR-20b-3p, hsa-miR-218-1-3p, hsa-miR-22-5p, hsa-miR-221-5p, hsa-miR-24-1-5p, hsa-miR-26a-1-3p, hsa-miR-302d, hsa-miR-30a-5p, hsa-miR-30b-3p, hsa-miR-337-3p, hsa-miR-33a, hsa-miR-34a-3p, hsa-miR-367-5p, hsa-miR-374a-3p, hsa-miR-452-3p, hsa-miR-454-5p, hsa-miR-497-3p, hsa-miR-513C, hsa-miR-516-3p, hsa-miR-518c-5p, hsa-miR-518e-5p, hsa-miR-518f-5p, hsa-miR-524, hsa-miR-543, hsa-miR-548I, hsa-miR-550, hsa-miR-554, hsa-miR-557, hsa-miR-566, hsa-miR-571, hsa-miR-578, hsa-miR-581, hsa-miR-593, hsa-miR-595, hsa-miR-603, hsa-miR-605, hsa-miR-609, hsa-miR-613, hsa-miR-614, hsa-miR-622, hsa-miR-623, hsa-miR-625-3p, hsa-miR-628-3p, hsa-miR-633, hsa-miR-647, hsa-miR-650, hsa-miR-744-3p, hsa-miR-769-3p, hsa-miR-769-5p, hsa-miR-921, hsa-miR-924, hsa-miR-92b-5p, hsa-miR-936, hsa-miR-943, hsa-miR-944, hsa-miR-99a-3p, hsa-miR-99b-3p

Identification of signaling pathways regulated by miRNAs

Using DIANA MiRpath prediction analysis software we identified the pathways targeted by miRNAs equally or deregulated in S-MSCs compared with AF-MSCs.

We did not find in DIANA all the miRNAs reported in Figure 1 but only 17 of the equally expressed (Group A), 11 of the upregulated (Group B), and 33 of the downregulated (Group C) (Table 2).

Figure 1.

KEGG (Kyoto Encyclopedia of Gene and Genomes) Adherens Junction pathway. The arrows identified the connection point between this pathway and the WNT/TGF-β signaling pathways.

Table 2.

Pathways targeted by miRNAs belonging to groups A, B, and C.

		DIANA	miRDB
Group A (miRNAs similarly expressed in S-MSCs and AF-MSCs)
Ubiquitin mediated proteolysis	Genes (n)	53	37
Ubiquitin mediated proteolysis	miRNAs (n)	13	9
Adherens Junction	Genes (n)	34	17
Adherens Junction	miRNAs (n)	15	11
Group B (miRNAs upregulated in S-MSCs compared to AF-MSCs)
Adherens Junction	Genes (n)	23	37
Adherens Junction	miRNAs (n)	8	15
Axon guidance	Genes (n)	34	12
Axon guidance	miRNAs (n)	15	11
Group C (miRNAs downregulated in S-MSCs compared to AF-MSCs)
WNT	Genes (n)	80	73
WNT	miRNAs (n)	28	32
TGF-β	Genes (n)	52	85
TGF-β	miRNAs (n)	27	24

For each group (A, B, and C), two pathways were found with a –ln(p) value higher than 10 and targeting over 30% of genes belonging to the same pathway.

The relative KEGG (Kyoto Encyclopedia of Gene and Genomes) pathways are: ubiquitin mediated proteolysis and Adherens junction for group A; Adherens junction and axon guidance for group B; and WNT and TGF-β for Group C (Table 2).

Subsequently, miRDB software was used to complete the identification of targets of miRNAs belonging to Groups A, B, and C. This analysis confirmed the previous results: the KEGG pathways were the same identified by DIANA (Table 2).

Adherens Junction, WNT, and TGF-β signaling pathways are key players in the maintenance, self-renewal, and differentiation of MSCs, and are related one to each other (Figure 1).

Discussion

Human MSCs are the most promising candidates for therapeutic applications. Therefore, the definition of markers suitable for the identification of the level of stemness, growth, and differentiation potential is necessary to improve the molecular characterization of MSCs and drive their future differentiation for regenerative medicine.²⁷ It is now well accepted that miRNAs play a key role in their maintenance and differentiation. After profiling miRNAs in skin-derived MSCs (S-MSCs) and amniotic fluid-derived MSCs (AF-MSCs) by the use of the TaqMan® Array Human MicroRNA Card A, we enlarged our analysis to less known miRNAs spotted in card B. Card B collects the miRNAs whose targets and roles are not yet validated. Interestingly, the miRNAs downregulated in S-MSCs compared to AF-MSCs target the WNT and TGF-β signaling pathways; these pathways were also targeted by miRNAs spotted in Card A,¹⁷ underlining their importance in the regulation of the stem cells fate.

The equally expressed and the upregulated miRNAs in S-MSCs compared to AF-MSCs matched the Adherens Junction signaling pathway. This pathway is strictly related to the dual process of Epithelial to Mesenchymal Transition (EMT) and the reverse Mesenchymal to Epithelial Transition (MET) that are in turn highly connected to many other cellular functions and fundamentally important in defining stem cells character and stem cell transitions.²⁸ It is important to note as the regulation of the Adherens Junction pathway passes also through the WNT and TGF-β pathways (Figure 1). In conclusion, this deeper analysis in S- and AF-MSCs of the less known miRNAs (by the use of card B by Life Technologies) highlights how the stem cell fate is a finely regulated mechanism, whose behavior is affected by more miRNAs than previously estimated. In this scenario, the role played by miRNAs less investigated up to now deserves to be reconsidered; moreover, their changing expression between MSCs derived from two different sources (skin and amniotic fluid) confirms that the so-called MSCs are really a heterogeneous reservoir, depending on their derivation. This conclusion has to be kept in mind for the future uses of stem cells for therapeutic approaches.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This work was supported by grant FIRB-RBAP10MLK7_003 and FIRB-RBAP1153LS_004 from Ministero dell’Istruzione, dell’Università e della Ricerca, Rome, Italy.

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New miRNAs network in human mesenchymal stem cells derived from skin and amniotic fluid

Abstract

Keywords

Introduction

Materials and methods

Isolation, culture, and characterization of AF-MSCs and S-MSCs

RNA extraction

TaqMan array cards

Analysis of miRNAs microarray data

Computational prediction of miRNAs target genes

Results

Similar and different miRNAs expression in S-MSCs compared to AF-MSCs

Identification of signaling pathways regulated by miRNAs

Discussion

Footnotes

Declaration of conflicting interests

Funding

References