Effects Of SiRNA Inhibit Class I PI3K On Humangastric Cancer SGC7901 Cells

Objective: To amplificate recombinant adenovirus PI3K(â… )-RNAi-AD containing small interfering RNA (siRNA) structure which targeting to the Classâ… PI3K signaling pathway and investigate the effects of siRNA-mediated inhibition of classâ… PI3K signal transduction on the proliferation ,apoptosis and autophagy of gastric cancer SGC7901 cells.Methods:â‘ Amplify recombinant adenovirus PI3K(â… )-RNAi-AD and contral adenovirus NC-RNAi-GFP-AD, the titre of them was detected,meanwhile the most effective multiple of infection(MOI) of PI3K(â… )-RNAi-AD to SGC7901 cells was detected.â‘¡Human gastric cancer SGC7901 cells were transfected with recombinant adenovirus PI3K(â… )-RNAi-AD for 24h, 48h, 72h as the experimental group, the human gastric cancer SGC7901 cells were transfected with control adenovirus NC-RNAi-GFP- AD for 72h as the control group(cells were transfected with NC-RNAi-GFP-AD for 24h, 48h and 72h as the control group in MTT).â‘¢SGC7901 cells were transfected with the recombinant adenovirus, the siRNA structure which carried by the AD would then block Classâ… PI3K signaling pathway. The MTT assay was used to determine the inhibition effects on cell proliferation. Mitochondrial membrane potential was measured using the fluorescent probe JC-1. The expression of autophagy was monitored with monodansylcadaverin (MDC) staining and transmission electron microscopy(TEM). The specific protein of autouphagy was detected through western blot, and the ratio of LC3-â…¡/LC3-â… was calculated for indicating the level of autophagy.Results:â‘ The recombinant adenovirus PI3K(â… )-RNAi-AD and contral adenovirus NC-RNAi-GFP-AD have been amplified successfully. The titer of PI3K(â… )-RNAi-AD was 1.0×10¹¹ pfu/L, The titer of NC-RNAi-GFP-AD was 0.9×10¹¹ pfu/L. The efficiency of transfection is best when the (MOI) of adenovirus was 50.â‘¡After the siRNA targeting to Classâ… PI3K blocked Classâ… PI3K signal pathway, the inhibition ratio of 24h,48h,72h were 27.48%±2.7%,41.92%±2.02%,50.85%±0.92%; the percents of the cells with green fluorescence were 18.5%±1.64%(24h),39.5%±1.70%(48h),55.8%±1.86%(72h) compared with the control group 12.6%±1.11%(P<0.05); The monodansylcadaverin (MDC) staining and TEM observation displayed that the autophagic vacuoles of 24h,48h,72h encreased gradually and there were less autophagic vacuoles in the control group than that in the experiment groups; The respective ratio of LC3-â…¡/LC3-â… to 24h,48h,72h were 0.293±1.481%,0.488±1.199%,0.889±1.190% compared with the control group the ratio of LC3II/LC3I was 0.123±1.225% (P<0.05).Conclusion:â‘ The titer of the recombinant adenovirus PI3K(â… )-RNAi-AD was 1.0×10¹¹ pfu/L,the efficiency of transfection is best when the MOI of adenovirus was 50.â‘¡After Classâ… PI3K signaling pathway has been blocked by specific siRNA, the proliferation of SGC7901 cells was inhibited; the apoptosis of SGC7901 cells was enhanced; the level of autophagy was up-regulated.