Effects Of SiRNA Inhibit Class â…¢ PI3K On Human Gastric Cancer SGC7901 Cells

Objective:①To construct the plasmid containing small interfering RNA (siRNA) structure which targeting to the Class III PI3K signaling pathway, produce recombinant adenovirus PI3K(III)-RNAi-AD.②Investigate the effects of siRNA-mediated inhibition of class III PI3K signal transduction on the apoptosis and autophagy of gastric cancer SGC7901 cells.Methods:①Specific siRNA interference sequence for gene PI3KC3 mRNA of human Class III PI3K signal transduction pathway was selected and constructed the recombinant plasmid siRNA. The gene sequencing was utilized to ensure that the siRNA sequence had been properly inserted into the vector. The HEK293 cells was cotransfected by the shuttle plasmids and the genomic plasmid of the adenovirus.The recombinant adenovirus PI3K(III)-RNAi-AD was generated through AdMax? system.②Stomach adenocarcinoma SGC7901 cells were transfected with the recombinant adenovirus, the siRNA structure which carried would then block Class III PI3K signaling pathway. The MTT assay was used to determine the inhibition effects on growth. Morphological changes of the cells were observed 24 h, 48 h and 72 h after transfection with a microscope. Mitochondrial membrane potential was measured using the fluorescent probe JC-1. The expression of autophagy was monitored with monodansylcadaverin (MDC) staining. The specific protein of autouphagy was detected through western blot, and calculated the ratio LC3II/LC3I for indicating the level of autophagy.Results::①The siRNA plasmid which targeting to the Class III PI3K signal pathway and the recombinant adenovirus PI3K(III)-RNAi-AD has been constructed successfully. The tite of PI3K(III)-RNAi-AD is 2.0×1011 pfu/L, The tite of NC-RNAi-GFP-AD is 1.8×1011 pfu/L. The efficiency of transfection is best when the multiple of infection (MOI) of adenovirus is 30.②After the siRNA targeting to class III PI3K blocked Class III PI3K signal pathway, the absorbance of 24h,48h,72h were 0.30±2%,0.33±1.77%,0.42±2.36% respectively in the experiment groups compared with that the absorbance of24h,48h,72h were 0.40±4.25%,0.58±3.29%,0.85±3.25% respectively in the control groups (p<0.05), the inhibition ratio of 24h,48h,72h were 25.31%±7.3%,43.71%±4.35%,50.89%±2.84%; the percents of the cells with green fluorescence were 19.6%±2.65%(24 h),35.7%±6.62% (48 h),50.4%±7.39%(72h) compared with the control group the percents of the cells with green fluorescence was 12.3%±2.17% (p<0.05),that the percents of the cells with green fluorescence in total cells became larger indicaied that the mitochondrion membrane potential decreased more; The monodansylcadaverin (MDC) staining displayed that the autophagic vacuoles of 6h,12h,24h decreased gradually and there were more autophagic vacuoles in the control group of 24h than that in the experiment groups; The respective ratio LC3II/LC3I of 6h,12h,24h were 0.876±7.51%,0.356±2.99%,0.145±1.98% compared with the control group the ratio LC3II/LC3I of 72h was 1.267±4.93% (p<0.05).Conclusion:①The siRNA plasmid which targeting to the Class III PI3K signal pathway and the recombinant adenovirus PI3K(III)-RNAi-AD has been constructed successfully.②After Class III PI3K has been blocked by specific siRNA, the proliferation of cells was inhibited; the level of autophagy was down-regulated; the apoptosis of gastric cancer SGC7901 cells was enhanced.