MiR-31 Negatively Regulates FIH To Promote Hypertrophic Scar Fibrosis Through Downstream HIF-1α-TIMP1-MMP1 Pathway

Objective Hypertrophic scar(HS)formation associate with excessive extracellular matrix synthesis and deposition, but the formation biological mechanism remain to be unclear. Micro RNAs(mi RNA) are 19 to 24 nucleotides noncoding RNA which work by binding to complementary target sites in an m RNA, then preventing translation of the transcript or accelerating its decay. Our study analyzed the mi RNA expression difference by gene array in tissue isolated from normal skin(NS) and hypertrophic scar(HS),and found the mi R-31 was the most positive one, it may be a potential novel molecular target for the treatment of hypertrophic scarring. Hypoxia inducible factor1 α(HIF1α) in recent years have also been found to play an important role of fibrosis. Previous studies have shown that during wound repairing, the hypoxia-inducible factor-1a(HIF-1α) was up-regulation, but the molecular mechanisms responsible for the increase of HIF-1α is little known. In this paper, we will explore how mi R-31 and HIF-1α to promote the proliferation of scar, whether the effects exist between them, and to validate the effectiveness of the mi R-31 function by in vivo experiment. This study is expected to elucidate the the role of mi R-31 in HS, and provides theory support and the new target for the control of HS.Methods 1. Collecting specimens of hypertrophic scar patients and normal skin; tissue soaked with Faure Marin and embedded in paraffin for fixing, sliced for H&E staining and Masson’s trichrome and immunohistochemistry; RT-PCR for detecting the different expression level of mi R-31 m RNA in normal skin tissue and hypertrophic scar tissue; WB(Western blot) detect the protein level of HIF1α in NS and hypertrophic scar tissue.2. Hypertrophic scar fibroblasts and normal skin fibroblasts culture, detect the expression of mi R-31; Fn; Col1a1; Col3a1; TGF- β in different passages, verify the availability of cell model construction. Transfect mi R-31 mimic into NSFBs, and mi R-31 inhibitor into HSFBs. Detect the expression of mi R-31; Fn; Col1a1; Col3a1 by RT-PCR and WB after transfecting. So as to observe the effect of mi R-31 on the biological characteristics of fibroblasts. 3. Using the bioinformatics software and database,like:Targetscan, Pic Tar and mi Randa, by computing the nucleotide untranslated region combination to predict the target gene of mi R-31.Screening for specific target gene FIH which relative to hypoxia. Luciferase reporter and RT-PCR, WB detect the expression of FIH after transfecting mi R-31 mimic or inhibitor, to verify that mi R-31 targeting FIH. 4. Detect the expression of HIF1α; TIMP-1; MMP1 by RT-PCR and WB after transfecting, to observe the effect of mi R-31 on HIF1α and its downstream protein. 5. HSFBs were cultured in hypoxia environment, and detect the protein level of Fn; Col1a1; Col3a1; TIMP-1; MMP1 at the three time nodes of 24h; 48h; 72 h. Thus elucidating the reason of HIF1α promotes the formation of HS. 6. Establishment of HS model, observe the expression of Fn; Col1a1; of Col3a1 after local intervention of mi R-31 inhibitor in vitro.Result The expression of mi R-31 and HIF1α increased significantly in HS compared with NS, Fn; Col1a1 and Col3a1 three fibrosis index was positively correlated with mi R-31 in vitro experiments, these prompt that mi R-31 promotes the formation of HS. What’s more, HIF-1α has the similarity function with HIF-1α in fibroblasts. Fibroblast gradient hypoxia experiment proved that TIMP-1 is the downstream effects of HIF1 a gene, and it’s positively correlated with HIF-1α. MMP1 has been verified to promote the degradation of Fn Col1a1 Col3a1, and it is inhibited by TIMP-1. So, with hypoxia deepened, fibrosis overexpression is increased by inhibition of TIMP-1 on MMP1.FIH is the factor inhibiting HIF, it can inhibit of HIF1α transcription of downstream genes. Luciferase reporter, RT-PCR and WB experiment showed that FIH is the target gene of mi R-31, and mi R-31 can enhance the effect of HIF1α to promote fibrosis. In conclusion,mi R-31 enhance the pathway of HIF1α-TIMP-1-MMP1 to promote HS formaton. In order to further verify the role of mi R-31 in of HS, we designed animal experiments. After mi R-31 inhibitor intervention Murine Model of Hypertrophic Scar, HE, MASSON and immunohistochemical staining showed that Fn; Col1a1 and Col3a1 was decreased.Conclusion 1. In hypertrophic scar, the relative expression of mi R-31 is increased significantly. 2. Mi R-31 promotes fibrosis in HS formation by inhibiting FIH and inducing HIF-1α activity. 3. Mi R-31 inhibitor has inhibitory effect on nude mice HS model.