Effects of AT1R gene silencing in apoptosis induced by STZ in NIT-1 cells

MO Li-wen; LIANG Xing-huan; KUANG Xiao-cong; LI Hai-bin; LI Li; CAI Jin-wei; LU De-cheng

Abstract：Objective To investigate the effects of silencing angiotensin Ⅱtype 1 receptor ( AT1R) in apoptosis induced by STZ in mice islet cells NIT -1.Methods Three pairs of siRNA targeting AT1R were designed and synthe-sized.They were transfected into NIT -1 by liposome transfection .Fluorescence microscopy was used to examine the effect of different siRNA concentration on the transfection efficiency .AT1R mRNA expression was detected with real -time PCR to compare silencing effects among the three siRNA sequence and different durations of interference .NIT-1 cells were al-located into 4 groups.The control group did not receive intervention .The STZ group was treated with 5 mmol/L STZ for 30 min.The si-AT1R group was transfected with 40 nmol/L si-AT1R and detected 24 h later.The si-AT1R+STZ group was transfected with 40 nmol/L si-AT1R and 24 h later treated with 5 mmol/L STZ for 30 min.Caspase-3 mR-NA expression was detected by Real -time PCR.Apoptosis rate was measured by Hoechst 33342 staining microscopy and Annexin V-FITC/PI-labeled flow cytomety .Results Transfection with 40 nmol/L siRNA had the greatest fluores-cence.The maximum inhibitory effects of AT1R siRNA transfection on AT1R expression was 40 nmol/L at 24 h with si-AT1R-2.The expression of AT1R mRNA was markedly down regulated by 92.20% compared with that in the control group (P<0.05).The expression of caspase -3 mRNA in the STZ group increased by 2.37 times compared with the control group (P<0.05).The expression of caspase -3 mRNA in the si-AT1R+STZ group decreased by 11.28%compared with the STZ group , but the difference was not statistically significant .Apoptosis rate in the STZ group increased by 3.27 times compared with the control group ( P<0.05) .The cells treated with both si -AT1R and STZ had a signifi-cantly decreased apoptosis rate ( by 26.82%) compared with the STZ group ( P<0.05 ) .Conclusion This study suc-cessfully silenced AT1R in NIT-1 cells and obtained the best efficiency of silence with 40 nmol/L si-AT1R-2 for 24 h.RNA interference of AT1R gene can reduce the expression of Caspase -3 gene and apoptosis induced by STZ , sugges-ting that AT1R mediates the process of apoptosis induced by STZ in NIT -1 cells.