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

Objective: To construct the plasmid containing shRNA structure which targeting to the ClassⅠPI3K signaling pathway of human gastric cancer SGC7901 cell, observe the inhibitory effect to the PI3KCA mRNA expression and screen out the most effective shRNA sequence.Methods: Select specific RNA interference sequence for ClassⅠPI3K signal transduction pathway of gene PI3KCA mRNA sequence, construct four sites in different of PI3KCA mRNA targeted to interfere with the recombinant plasmid shRNA. Using gene sequencing to ensure that the interference had been right sequence inserted into the vector;use plasmid transfection stomach adenocarcinoma SGC7901 cell.Using shRNA structure to block ClassⅠPI3K signaling pathway,autophagy-induced cell death occurred. Transfection at the first 24 h,48 h,72 h were observed morphological changes of gastric cancer cells,transfected cells were collected total RNA after 48 h,using real-time fluorescence quantitative PCR to detect target gene PI3KCA,autophagy-related gene MAP-LC3 mRNA expression changes;the total protein of transfected cells were collected after 72 h,using Western Blot to detect PI3KCA protein p110 catalytic subunit protein expression changes;through the above-mentioned test results,select the best sequence of shRNA interference.Results: Successful constructed four with ClassⅠPI3K signaling pathway interference targeting recombinant plasmids,gene sequencing has been proved the shRNA interfere chains have been inserted in the plasmid vector rightly;gastric cancer SGC7901 cells transfected with plasmid after 24 h, under the microscope,cells volume were shrinkage,cells gap were widened;after 48 h the cells fall off;after 72 h, more cell loss significantly;48 h later by real-time quantitative PCR,fluorescence detection PI3KCA each group,as well as the MAP-LC3 mRNA expression,4 group cells PI3KCA1 and PI3KCA2 mRNA expression were reduced,one of the Samples No.705 interference plasmid's PI3KCA1 mRNA expression down to the control group 2.4%±1.3% (p<0.05),PI3KCA2 mRNA expression down to control group's 0.4%±0.4% (p<0.05),4 groups of cell lines interference shRNA autophagy-related genes MAP-LC3 mRNA up-regulation expression,which interfere with plasmid Samples No.705's MAP-LC3 mRNA expression up to the control group's 5198.39±0.04% (p<0.05);72 h after ,the Western Blot detecting protein expression of p110 protein, the ClassⅠPI3K regulatory subunit, 4 group showed reduced protein expression,which interfere with plasmid Samples No.705's p110 protein expression down to the control group's 49.20%±14.11% (p <0.05).Conclusion:The shRNA plasmid which targeting to the ClassⅠPI3K signaling pathway of human gastric cancer SGC7901 cell has been constructed successfully. It can effectively inhibit the target gene PI3KCA mRNA and the corresponding protein expression and up-regulate autophagy-related genes MAP-LC3's mRNA expression. The interference sequence "5'-AGAGGTTTCAGGAGATGTGTTACAAGGCT-3'" which designed based on the PI3KCA mRNA's base site 3090-3118 is the most effective shRNA sequence targeting to the ClassⅠPI3K signaling pathway of human gastric cancer SGC7901 cell .