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Effects of siRNA Inhibit Class I PI3K on Humangastric Cancer SGC7901 Cells

Author: YuLiYan
Tutor: XingChunGen
School: Suzhou University
Course: Surgery
Keywords: ClassⅠPI3K SiRNA Proliferation Apoptosis Autophagy
CLC: R735.2
Type: Master's thesis
Year: 2011
Downloads: 32
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Abstract


Objective: To amplificate recombinant adenovirus PI3K（Ⅰ）-RNAi-AD containing small interfering RNA （siRNA） structure which targeting to the ClassⅠPI3K signaling pathway and investigate the effects of siRNA-mediated inhibition of classⅠPI3K signal transduction on the proliferation ,apoptosis and autophagy of gastric cancer SGC7901 cells.Methods:①Amplify recombinant adenovirus PI3K（Ⅰ）-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（Ⅰ）-RNAi-AD to SGC7901 cells was detected.②Human gastric cancer SGC7901 cells were transfected with recombinant adenovirus PI3K（Ⅰ）-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）.③SGC7901 cells were transfected with the recombinant adenovirus, the siRNA structure which carried by the AD would then block ClassⅠPI3K 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-Ⅱ/LC3-Ⅰwas calculated for indicating the level of autophagy.Results:①The recombinant adenovirus PI3K（Ⅰ）-RNAi-AD and contral adenovirus NC-RNAi-GFP-AD have been amplified successfully. The titer of PI3K（Ⅰ）-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 50.②After the siRNA targeting to ClassⅠPI3K blocked ClassⅠPI3K 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-Ⅱ/LC3-Ⅰto 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:①The titer of the recombinant adenovirus PI3K（Ⅰ）-RNAi-AD was 1.0×1011 pfu/L,the efficiency of transfection is best when the MOI of adenovirus was 50.②After ClassⅠPI3K 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.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Gastric neoplasms
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