The Mechanism Of MiRNA199B-5P Regulating TGF-β2in Human Trabecular Meshwork Cells Under Oxidative Stress

Objective Increased intraocular pressure of glaucoma associated with trabecular meshwork oxidation denaturation and increased outflow resistance. Extracellular Matrix(ECM) abnormal deposition in primary open angle glaucoma(POAG) patients can lead to the trabecular meshwork outflow resistance increase. The experiment mainly study the expression of ECM in Human Trabecular Meshwork Cells (HTMC) under oxidative stress,and the influence of synthesis and secretionin of Transforming Growth Factor-β2(TGF-β2) by miRNA-199b-5p.Methods The different concentration of hydrogen peroxide (H2O2) stimulation HTMC, make use of the cell growth activity determined by MTT test in the HTMC.300uM H2O2treated HTMC1.5h, the expression of TGF-β2and Fibronectin (FN) were assayed by real-time PCR and immunohistochemical staining. Gene microarray detection the expression of miRNA-199b-5p in oxidative stress,and validates the the expression of miRNA-199b-5p by real-time PCR. Consults with bioinformatics, we found that miRNA-199b-5p binding TGF-β2.Detection the function of HTMC andTGF-β2content by overexpression miRNA-199b-5p.Results H2O2can inhibit the growth of cells activity and promote the expression of TGF-β2and FN in HTMC:After treated with H2O2the expression of TGF-beta2was increased at1h,2h,2h,4h,8h,12h,24h, relative expression were1.04±0.33,5.04±1.25,8.54±2.48,10.88±2.25,11.83±2.15,18.72±3.09,25.38±5.18, with significant difference statistically (P<0.01); the expression of FN was increased at1h,2h,2h,4h,8h,12h,24h, relative expression werel.00±0.172.82±0.30,4.56±0.35,5.22±1.86,6.65±2.18,11.06±7.57,17.10±4.12,with significant difference statistically (P<0.01).MicroRNA array and real-time PCR detect down-regulation of miRNA-199b-5p under oxidative stress. After treated with H2O2the expression of miRNA-199b-5p was decreased at1h,2h,2h,4h,8h,12h,24h, relative expression were1.00±0.16,0.50±0.17,0.57±0.39,0.41±0.14,0.45±0.09,0.38±0.12,0.17±0.04, with significant difference statistically (P <0.01).Overexpression miRNA-199b-5p can inhibition cell migration and lead to down-regulation of TGF-β2and FN in THMC. After transfected with miRNA199b-5P mimics24h, the expression of TGF-β2in the control group was2.54±0.38, the treatment group was1.18±0.29, with difference statistically significant (P<0.05). the expression of FN in the control group was2.54±0.29, the treatment group was1.04±0.08, with difference statistically significant (P<0.05).Conclusions In oxidative stress state the expression of TGF-β2is increased,while the miRNA-199b-5p expression decreased in HTMC. miRNA-199b-5p can negative regulation effect on the synthesis and secretion of TGF-β2though targeting3’-UTR of TGF-β2,we can infer that oxidative stress promote the TGF-β2expression increases to a certain extent with the miRNA-199b-5p downgrade.At the same time, the secretion of FN increased in HTMC under oxidative,ss may with the miRNA-199b-5p downgrade have certain relations.