The Effect And Mechanism Of TGF-β Mediated Signal Pathway Modulated By MiR-21in Hypertrophic Scars

Objective Hypertrophic scar caused by trauma was one of the mosttroublesome critical problems in clinical practice, which was one type of fibroticdisease with unknown mechanism. According to the research of all kinds of fibroticdisease, miR-21could promote fibrosis positively via modulating the expression ofTGF-β and Smad7in post-transcriptional level. In our previous study, we found thatthe expression of miR-21was significantly higher in the HS tissue, and TGF-β couldpromote its expression. Therefore, we speculated that the mechanism of hypertrophicscars was a positive feed back between TGF-β and miR-21. In detail, the promotedexpression of miR-21after skin healed from trauma could accelerate expression ofTGF-β. Correspondingly，higher expression of TGF-β further reinforced the effect ofmiR-21in scar formation. Dermal Fibroblasts (normal skin and HS tissue) and scarmodel are to be adopted in combination with relative techniques such as real-time PCR,transfection, anti-miR, immunohistochemistry to confirm the hypothesis either in cell(in vitro) or in tissue (in vivo). The study would probably further clarify both the effectand mechanism in HS formation, which meight provide novel target in biologicalcontrol of HS.Methods(1) Collecting specimens of HS patients and normal skin; tissue embedded inparaffin, sliced for H&E staining and Masson’s trichrome and immunohistochemistry;Collagen quantitative detection; real-time PCR for detecting the different expressionlevel of miR-21mRNA in normal skin tissue and HS tissue;(2) Normal skin fibroblasts and HS fibroblasts culture and cell transfection;construction of miR-21overexpression and antisense RNA interference vector, q-PCR,WB for detecting the expression of Col I,Col III, Fibronectin and α-SMA, in order to observe the effect of miR-21on the biological characteristics of fibroblasts.(3) Cells transfected with mimic-miR-21or anti-miR-21, while the addition ofTGF-β1or TGF-β1receptor inhibitor, quantitative expression of PCR, ELISAdetection of TGF-β1and scar related protein; so as to clear the miR-21effect of scarformation is related with TGF-β pathway;(4) With a number of bioinformatics website and software (Targetscan, PicTar,miRanda and MicroInspector), prediction of the target genes of miR-21, screeningspecific target genes related to scar formation and muscle fibroblasts differentiation;the use of miR-21over expression and antisense expression vector, were transfectedinto human normal skin fibroblasts and HS fibroblasts, the regulatory effect ofqRT-PCR, Western-Blot observation of miR-21on the expression of specific targetgenes.ResultThe content of collagen in hypertrophic scar was higher than that of the normalskin; the expression of extracellular matrix such as COLI, COL III, Fibronectin (FN)and α-SMA in scar tissue significantly increased; miR-21expression in HS tissuesignificantly increased, scar fibroblasts transfected with anti-miR-21can reduce theexpression of scar related gene (FN, collagen, α-SMA); high expression of TGF-β1inHS tissue can promote the proliferation of fibroblasts, and promote the synthesis ofcollagen and other ECM; TGF-β enhanced the expression of miR-21in fibroblasts ofHS tissue, while inhibiting miR-21leaded to decreasing of profibrogenic activity ofTGF-β; miR-21inhibited expression of the target gene Smad7Conclusion1. The expression of miR-21increased significantly in hypertrophic scarcompared with normal skin.2. MiR-21promote the occurrence of the HS by affecting the expression ofTGF-β gene and regulating the specific target gene activities through thepost-transcription level.