The Effects Of MicroRNAs On HBM-MSCs Differentiation Into Sweat Gland Like-cells

Objectives1. Purifying the primary cultured sweat gland cells with Trypsase-EDTA solution, getting ready for the next experiments.2. Screening the differently expressed microRNAs between human bone marrow mesenchymal stem cells and sweat gland-like cells, and predicting target genes of the differently expressed microRNAs by bioinformatics technology. Searching crucial microRNAs in the process of human bone marrow mesenchymal stem cells differentiate into sweat gland-like cells and predicting the functions of these crucial microRNAs by bioinformatics technology.3. Exploring the functions of microRNAs in the process of human bone marrow mesenchymal stem cells differentiate into sweat gland-like cells. Methods1. The collagenase-II with concentration of 2% digest the particle skin in order to separate and extract sweat glands.2. The primary cultured sweat gland cells were separated and purified by Trypsase-EDTA solution, and identified by immunocytochemistry staining. The natural sweat glands morphological structure were observed in normal skin by hematoxylin-eosin staining.3. To demonstrate the multipotential differentiation of hBM-MSCs, the hBM-MSCs were separately induced to osteogenic differentiation and adipogenic differentiation by applying osteogenic differentiation basal medium and adipogenic differentiation basal medium, and to identify the results, respectively, by oil red O staining or alizarin red staining. The hBM-MSCs and heat-shocked sweat gland cells were separately cultured by the same one Transwell plate which established a co-culture system between heat-shocked sweat gland cells and hBM-MSCs. The hBM-MSCs were induced in 7 days and sweat gland cells surface markers were detected by immunofluorescence staining, RT-PCR and Western Blot.4. The total RNAs of hBM-MSCs and sweat gland-like cells were respectively extracted by TRIzol reagent. Next, the total RNAs were purified and hybridized to the microRNAs microarray. The target genes of these differential expression microRNAs were predicted and analyzed by bioinformatics technology. The real-time fluorescent quantitative PCR was applied to verify the results of the micro RNAs microarray test.5. The specific chemical synthesis microRNAs mimics, microRNAs inhibitor or microRNA Negative Control were separately transfected into hBM-MSCs of heat-shocked co-culture system. The transfected hBM-MSCs were continuously cultured in the co-culture system 7 days for inducing hBM-MSCs to differentiate into sweat gland-like cells. Then the sweat gland cells surface markers were detected by immunofluorescence staining, RT-PCR and Western Blot. Results1. More than 95% fibroblasts around primary cultured sweat gland cells were removed by Trypsase-EDTA solution, that obtains highly purified sweat gland cells.2. The hBM-MSCs, which were induced by Transwell plate co-culture system, expressed markers of sweat gland cells such as CEA, CK8 and CK19.3. Analysed the microRNAs expression profiles, we found 68 microRNAs which had a greater than 2-fold change in differential expression between hBM-MSCs and sweat gland-like cells. The 68 microRNAs include 19 up-regulated microRNAs and 49 down-regulated microRNAs. Among the up-regulated microRNAs, microRNA-132-3p, microRNA-4467, microRNA-4484, microRNA-146a-5p and microRNA-6126 were up-regulated over five times, and among the down-regulated microRNAs, microRNA-708-5p, microRNA-138-5p, microRNA-6812-5p, microRNA-138-1-3p, microRNA-1281, microRNA-3157-3p, microRNA-4298 and microRNA-4459 were down-regulated over five times.4. The results of real-time fluorescent quantitative PCR were consistent with microRNAs microarray results.5. Among the 68 microRNAs, which were predicted target genes by bioinformatics technology, 18 microRNAs involve in sweat gland development and formation signaling pathway such as NF-κB signaling pathway, MAPK/Erk signaling pathway, Wnt/β-Catenin signaling pathway and EDA/EDAR signaling pathway. Particularly the microRNA-138-1-3p not only changed obviously but also targeted to EDA gene of EDA/EDAR signaling pathway.6. The EDA gene expression level, CEA-positive cells and CK19-positive cells in microRNA-138-1-3p mimics transfection group are lower than the microRNA Negative Control transfection group. But the EDA gene expression level, CEA-positive cells and CK19-positive cells in microRNA-138-1-3p inhibitor transfection group are higher than the microRNA Negative Control transfection group. Conclusions1. The primary cultured sweat gland cells can be highly purified by Trypsase-EDTA solution with differential digesting time.2. The hBM-MSCs can be induced into sweat gland-like cells by Transwell plate indirectly co-culture system.3. There are 68 microRNAs differences at expression level between hBM-MSCs and sweat gland-like cells, these micro RNAs might play essential roles in hBM-MSCs induced to differentiate into sweat gland-like cells.4. microRNA-138-1-3p regulates hBM-MSCs to differentiate into sweat gland-like cells by regulating EDA gene expression in EDA/EDAR signaling pathway.