MicroRNA-mediated Regulation Of MSCs Differentiation To Sweat Gland-like Cells: Targeting NF-κB

Objective:In previous study in our laboratory, we reported that, when BrdU-labeled adult bone-marrow-derived mesenchymal stem cells (MSCs) were co-cultured with heat-shocked confluent sweat gland cells (SGCs) in vitro, a subset of MSCs differentiated into sweat gland-like cells (SGLCs). However, the underlying molecular mechanism of the phenomenon has not been further investigated. Thus, we designed a systemic scheme of experiments to investigate the microRNA-mediated regulating mechanisms in MSCs differentiation to SGLCs.Methods:It has been illustrated that Ectodysplasin-A1 (EDA-A1) could stimulate ectodermal organ development in normal mice, and was sufficient to rescue the hair growth and sweat gland in tabby mice. It also has been reported that epidermal growth factor (EGF) could enhanced the percentage of MSCs differentiation into SGCs. In this article,1μg/ml EDA-A1,50 ng/ml EGF and the injured microenviroment from heat-shocked SGCs was applied to induce MSCs differentiated into SGCs. Then the profiles of microRNA (microRNA) differential expression in MSCs before and after induced differentiation and matured SGCs were arrayed by microRNA microarray. Subsequently, the significantly changed microRNAs which targeted the genes related to SGCs development and MSCs differentiation by bioinformatics prediction were screened out for further validation, which performed by transfecting MSCs with synthetic has-miR-138-5p mimics (inhibitor)-cy3, synthetic has-miR-146a-5p mimics (inhibitor)-cy3 and synthetic microRNA mimics (inhibitor)-cy3 negative control, and Lipofectamine 2000, respectively. After transfection, the expression of target genes was determined by RT-PCR and the phenotype was assayed by immunohistochemisty. Finally, the critical targets and key signaling pathways involved in the microRNAs regulation during the phenotype conversion were discussed in this article.Results:After 10d of induction by indirect co-culture with heat-shocked hSGCs and inducing factor as EDA-A1 and EGF, part of MSCs at the upper layer, similar as "cobblestone" morphology. Determined by imunofluorescence, some induced MSCs exhibited positive expression of SGC markers:CEA, CK19, CK7, CK8 and CK14.The detection results of microRNA expression profiles showed, SGLCs VS MSCs,19 microRNAs were up-regulated significantly, while 49 microRNAs were down-regulated; SGCs VS MSCs,120 microRNAs up-regulated and 59 microRNAs down-regulated. In order to screen microRNAs to be validated in the future, we picked out the 37 microRNAs which occurred simultaneously in the result of significantly changed microRNAs in SGLCs VS MSCs and SGCs VS MSCs. By bioinformatics prediction, we analyzed the target genes of these 37 differentially expressed microRNAs and found a series of them correlated with the regulation of SGCs development and MSCs differentiation, by targeting cytokine CEA, KRT, and signaling pathway EDA/EDAR, NF-κB, Wnt, ERK et al. Especially, has-miR-138-5p and has-miR-146a-5p targeted several critical genes in the NF-κB signaling pathway simultaneously.48 h after MSCs transfected with microRNAs, a predominance of cytoplasm full of red fluorescence was observed under fluorescence microscope. Flow cytometric analysis indicated 98.56% transfection efficiency with Lipofectamine 2000. The result of RT-PCR showed that, after 48 h transfection with has-miR-138-5p inhibitor, the expression of h-KRT19, h-NFKB1, h-RELA and h-RELB was significantly increased in MSCs, but h-KRT16 has no expression before or after transfection. Oppositely, the expression of h-NFKB1, h-RELA and h-RELB was decreased after transfection with has-miR-138-5p mimics. Moreover, the expression of h-IRAK1, h-TRAF2 and h-TRAF6 was significantly increased in MSCs after 48h transfection with has-miR-146a-5p inhibitor, while significantly decreased after transfection with has-miR-146a-5p mimics.It also has been demonstrated that miR-146 was an NF-kB negative regulator. The phosphorylation of IκBα on serine 32, which is essential for its degradation, was decreased to～40% of control level in cells expressing miR-146a. We inferred that, caused by has-miR-138-5p reduction, the expression of h-NFKB1, h-RELA and h-RELB was markedly increased, thus NF-κB signaling pathway was strongly activated, then, as a kind of negative feedback adjustment, has-miR-146a-5p increased, which leaded to the reduction of h-IRAK1, h-TRAF2 and h-TRAF6, thereby inhibiting the activation of NF-κB pathway at upper signal adaptins.More evidences showed, a few of MSCs expressed sweat gland-specific makers after transfected with has-miR-138-5p inhibitor, which meant that inhibiting has-miR-138-5p might promote phenotype conversion, but conversion efficiency was limit. More work need to be carried out to understand the underlying mechanisms in MSCs differentiation to SGLCs.Conelusions:The evidence that NF-κB was activated by down-regulated has-miR-138-5p and inhibited by up-regulated has-miR-146a-5p might indicate the synergism of these microRNAs on regulating NF-κB pathway, and NF-κB might be the key signaling pathway that microRNAs mediated in regulation of MSCs differentiation to SGLCs.