Dedifferentiation Of Epidermal Cells Into Epidermal Like Stem Cells And Differential Expression Of MicroRNAs Induced By Protein Kinase C Inhibitor

Objective To investigate the feasibility of dedifferentiation of epidermal cells into epidermal like stem cells and analysis the differential expression of microRNAs induced by protein kinase C inhibitor GF109203 X.Methods(1) Human primary epidermal cells were obtained with enzyme digestion method.(2) The cells were randomly divided into 2 groups as follows, Experimental group: primary epidermal cells were cultured for 2 days before treated with GF109203 X,of which final concentration is 10 μM.Controlled group:the same volume of DMSO was substitute for GF109203 X as in experimental group. Monoclonal antibody of integrin β1,keratin 19(CK19),CK14,and CK10 were detected by immunocytochemical staining after 2 days induced.(3) Total RNA was isolated using TRIzol and purified with RNeasy mini kit according to manufacturer’s instructions. RNA quality and quantity was measured by using NanoDrop ND-1000 spectrophotometer and RNA Integrity was determined by gel electrophoresis. The miRCURYTM Array Power Labeling kit was used according to the manufacturer’s guideline for miRNA labeling,then the labeled samples were hybridized on the miRCURYTM LNA Array(v.18.0). Following hybridization, washed several times using Wash buffer kit and then the slides were scanned using the Axon GenePix 4000 B microarray scanner. Scanned images were then imported into GenePix Pro 6.0 software for grid alignment and data extraction, then normalized expressed data and analysis the differentially expressed miRNAs,Real-Time reverse transcriptase polymerase chain reaction(RT-PCR) was performed to confirm the array results.(4)Enrichment analysis was conducted for partially of the targets of differentially expressed microRNAs.Results(1) Cells of experimental group formed distinct clones after 4 days of culture: expressions of integrin β1、 CK19 and CK14 were positive but CK10 was negative. Cells of controlled group formed few clones after 4 days of culture: expression of CK10 was positive but expressions of integrin β1、 CK19 and CK14 were negative.(2)In the experimental group, 45 microRNAs were up-regulated, and 34 down-regulated. The most significantly up-regulated microRNAs were hsa-miR-1-3p, hsa-miR-374a-5p, hsa-miR-144-5p,etc; while hsa-miR-31-5p,hsa-let-7c-3p,hsa-miR-514a-3p and so on were significantly down-regulated.(3) The findings of RT-PCR on hsa-mi R-1-3p of up regulation and hsa-miR-31-5p of down regulation were in high concordance with microarray results.(4)Enrichment analysis shows that some target genes of differentially expressed micro RNAs were proved to play an important role in cell proliferation and differentiation,cell cycle progressment, apoptosis.Conclusion Protein kinase C inhibitor GF109203 X may induce the epidermal cells to convert into epidermal like stem cells, and give rise to differentially expressed microRNAs of epidermal cells.