The Differential Expression Of MicroRNA Between Human Hypertrophic Scar And Normal Skin And Intervention Of Tetrandrine

Objective： To observe the microRNA differential expression profile betweenhyperplastic scar and normal skin and the effects of tetrandrine on microRNAexpression in human hypertrophic scar fibroblasts, and explore the mechanism ofhyperplastic scar and the significance of tetrandrine in the prevention and treatmentof hyperplastic scar.Methods：（1）The total RNA was extracted by Trizol from hyperplastic scarand normal skin tissues marked as experimental group T1and control group C1, andpurified by mirVana miRNA Isolation Kit, and then labeled and hybridized bymiRNA Complete Labeling and Hyb Kit. The images of hybridization wereanalyzed by Feature Extraction (v10.7) software. Data normalization and varianceanalysis were made by software of GeneSpring(GX10.0). The microarray resultswere confirmed by real-time reverse transcription-polymerase chain reaction（RT-PCR）. The targets of differentially expressed microRNAs were predicted bybioinformatics approaches.（2）Hypertrophic scar fibroblasts cultured in vitro fromhuman hypertrophic scar were randomly divided into two groups,one with theintervention of tetrandrine (5μg/ml)as the test group T2, the other without drug as acontrol group C2. The experimental and control groups were collected and analysedby microarray after48h culture. Real-Time reverse transcriptase polymerase chainreaction (RT-PCR) was performed to confirm the array results. The targets ofdifferentially expressed microRNAs validated by RT-PCR were functionallyannotated by bioinformatics approaches.Results：（1） In the hyperplastic scar,92microRNAs were up-regulated, and13down-regulated. The most significant up-regulated microRNAs werehsa-miR-564, hsa-miR-936, etc; while hsa-miR-451, hsa-miR-223, hsa-miR-363,hsa-miR-29b-1*and so on were significantly down-regulated. The findings ofRT-PCR on hsa-miR-21of up regulation and hsa-miR-451of down regulation werein high concordance with microarray results. Some target genes of microRNAs were proved to play an important role in fibroblast growth, collagen formation andcell adhesion, proliferation, apoptosis and differentiation.（2）Microarray analysisidentified a total of186microRNAs which exhibited lower expression inexperimental group than in control group, while9microRNAs demonstrated higherexpression. The significantly down-regulated microRNAs were hsa-miR-1246,hsa-miR-10b, hsa-miR-760, etc; and hsa-miR-27b, hsa-miR-29b-1*,hsa-miR-193a-3p and so on had the higher expression level. The findings ofRT-PCR on hsa-miR-27b of up regulation and hsa-miR-125b of down regulationwere in high concordance with microarray results. The target genes of them wereconfirmed to be related with several signaling pathways of cell proliferation,differentiation and apoptosis as well as the formation of collagen.Conclusion：There is a clear difference of microRNA expression betweenhuman hyperplastic scar and normal skin, which may be closely related with theformation, development and evolution of hyperplastic scar. The differentialmicroRNA expression in human hypertrophic scar fibroblasts regulated bytetrandrine in vitro supplies experimental basis for further understanding of theanti-fibrosis effect of tetrandrine.