The Effect And Mechanism Of Mi R-155 On The Apoptosis Of TNF-?-induced Human Umbilical Vascular Endothelial Cells

Objective: Detecting the expression and effects of miR-155 on TNF-?-induced HUVECs apoptosis, and exploring the mechanisms of mi R-155 regulates TNF-?-induced HUVECs apoptosis.Methods:(1) First, depending on the processing concentration of TNF-?, HUVECs were divided into six groups, namely 0 ng/mL group, 1ng/mL group, 10 ng/mL group, 20 ng/mL group, 40 ng/mL group and 100ng/mL group; then various concentrations groups depending on the processing time period were divided into different groups, 0 h group, 12 h group, 24 h group and 48 h group. MTT assay was used to detect the cell survival rate and Heochst33342 staining was used to detect the extent of cell apoptosis. Thus we know the optimal concentration and time of TNF-? induced HUVECs apoptosis.(2) Real-time Quantitative PCR Detecting System(qPCR) was used to detect the expression level of mi R-155 of TNF-? induced HUVECs apoptosis.(3) By transfection of mi R-155 mimics and anti-miR-155 to make mi R-155 overexpression or inhibit miR-155 expression in HUVECs. MTT assay to detect the effects of mi R-155 on HUVECs apoptosis induced by TNF-?. Hoechst 33342 staining and Annexin V-FITC/PI double staining to detect the extent of apoptosis.(4) MiR-155 potential target genes were forecasted by bioinformatics software and study reports and confirmed by Western blot.Results:(1) With the extension of TNF-? treatment time andconcentration increased, the survival rate of HUVECs gradually reduced,but apoptosis rate increased. The optimum processing conditions are 10ng/ml and 24 h.(2) qPCR results showed that the expression level of mi R-155 was increased significantly in TNF-? group(P<0.01).(3) MTT assay showed that the cell survival rate was improved significantly by transfection of miR-155 mimics in TNF-?-induced HUVECs(P<0.01).On the contrary, the cell survival rate was significantly decreased by transfection of anti-mi R-155 in TNF-?-induced HUVECs(P<0.01). The cell apoptosis rate was decreased significantly by transfection of miR-155 mimics detected by Hoechst 33342 staining and Annexin V-FITC/PI double staining(P<0.01). But the cell apoptosis rate was significantly increased by transfection of anti-miR-155 mimics(P<0.01).(4) mi R-155 directly inhibited FADD ? Caspase-3 and Active-caspase-3 protein expression through targeting their 3'UTR tested by bioinformatics analyses and western blot.Conclusions: miR-155 inhibits HUVECs apoptosis induced by TNF-? through down-regulating FADD and Caspase-3 protein expression.