Effects Of ShRNA Inhibit Class Ⅲ PI3K On Human Gastric Cancer SGC7901 Cells

Objective:①T o construct the plasmid containing Short Hairpin RNA (shRNA) structure which targeting to the ClassⅢPI3K signaling pathway of human gastric cancer SGC7901 cell, observe the inhibitory effect to the PI3KC3 mRNA expression and screen out the most effective shRNA sequence.②I nvestigate the effect of shRNA-mediated inhibition of classⅢPI3K signal transduction on the apoptosis and autophagy of gastric cancer SGC7901 cells.Methods:①Specific shRNA interference sequence for gene PI3KC3 mRNA of human ClassⅢPI3K signal transduction pathway was selected and four sites in different of PI3KC3 mRNA targeted to interfere with the recombinant plasmid shRNA. Gene sequencing was utilized to ensure that the shRNA sequence had been properly inserted into the vector.②Stomach adenocarcinoma SGC7901 cells were transfected with the recombinant plasmid, the shRNA structure which carried would then block ClassⅢPI3K signaling pathway. Morphological changes of the cells were observed 24 h, 48 h and 72 h after transfection with a microscope.③Total RNA of the transfected cells was collected 48 h after transfection. Real-time fluorescence quantitative PCR was applied to detect PI3KC3 expression, according to which the shRNA sequence with the best interference effect was decided.④The alteration of LC3, p53 and PUMA mRNA expression were examined by Real-time fluorescence quantitative PCR, respectively.Results:①Four shRNA sequence targeting ClassⅢPI3K signaling pathway were successfully constructed. Gene sequencing proved the shRNA interfering chains had been inserted into the plasmid vector properly.②2 4 h after transfected with recombinant plasmid, the volume of SGC7901 cells shrank, cell gap widened. Part of the cells fell off after 48 h. Cell loss was even more severe after 72h.③Real-time fluorescence quantitative PCR showed that PI3KC3 mRNA expression were reduced in all groups. Samples No.712 had down-regulated PI3KC3 mRNA expression to 15.17%±2.21% of that of the control group(p<0.05).④Real-time fluorescence quantitative PCR showed the expression of LC3 mRNA was down-regulated to 28.02%±2.65% of that of the control group's, but the expression of p53 and PUMA mRNA was up-regulated to 475.62%±8.36% and 180.24%±4.17% of that of the control group, respectively (P<0.05).Conclusion:①T he shRNA plasmid which targeting to the ClassⅢPI3K signaling pathway of human gastric cancer SGC7901 cell has been constructed successfully. It can effectively inhibit the target gene PI3KC3 mRNA expression. The interference sequence "5'-TCCGCTTAGACCTGTCGGATGAAGAGGCT-3'" which designed based on the PI3KC3 mRNA is the most effective shRNA sequence targeting to the ClassⅢPI3K signaling pathway of human gastric cancer SGC7901 cell.②The shRNA targeting to classⅢPI3K has down -regulated autophagy specific gene LC3 mRNA expressions.③The shRNA targeting to classⅢPI3K has up-regulated apoptosis related gene p53 and PUMA mRNA expressions.④After classⅢPI3K has been inhibited by specific shRNA, down-regulated autophagy may intruduce apoptosis in gastric cancer SGC7901 cell line.