体外siRNA靶向抑制PI3Kp85α基因对人胃癌AGS细胞株的作用

目的 探讨小干扰RNA（siRNA）技术沉默PI3Kp85α基因表达对人胃癌细胞株AGS增殖、迁移及侵袭能力的影响。方法 设计3条靶向抑制PI3Kp85α基因的siRNA片段（siRNA-1、siRNA-2、siRNA-3），分别转染人胃癌AGS细胞，并设阴性对照组及空白组。Western blotting检测siRNA序列对PI3Kp85α蛋白表达的影响。利用MTT实验、划痕迁移实验和Transwell侵袭实验检测下调PI3Kp85α水平对AGS细胞增殖、迁移和侵袭能力的影响。结果 转染siRNA-1、siRNA-2和siRNA-3的AGS细胞中PI3Kp85α蛋白水平均低于阴性对照组及空白组（P＜0.05）。转染siRNA-3的AGS细胞PI3Kp85α蛋白表达降低最明显，抑制率达80％，故选择siRNA-3进行后续实验。转染24h后，PI3Kp85α siRNA转染组细胞增殖能力明显低于对照组细胞（P＜0.05）。24h体外划痕实验显示，PI3Kp85α siRNA转染组的划痕愈合能力明显低于对照组（P＜0.05）。Transwell实验显示，PI3Kp85α siRNA转染组细胞的细胞穿膜数明显低于对照组（P＜0.05）。结论 通过siRNA能明显下调PI3Kp85α蛋白在胃癌细胞AGS中的表达，并抑制肿瘤细胞的增殖、迁移和侵袭。

Role of siRNA of PI3Kp85a gene in gastric carcinoma AGS cells

Objective To investigate the influence of small interfering RNA （siRNA） targeting PI3Kp85αon proliferation, migration and invasion of gastric carcinoma cell line AGS. Methods Three siRNA with different sequences （ siRNA- 1, siRNA-2, siR- NA-3） were desinged and transfeeted into AGS cells. The AGS cells were divided into siRNA groups, universal scrambled negative siRNA groups and blank groups. After transfection, the protein expression of PI3Kp85α was detected by western blotting. MTT assay, wound healing and Transwell assays were performed to detect the effect of PI3Kp85α siRNA on AGS cell proliferation, migration and invasion. Results The expressions of PI3Kp85α protein significantly decreased in the siRNA-2 and siRNA-3 transfected cells. Western blotting analysis showed that cells transfected with siRNA-3 had the strongest inhibition of PI3Kp85α protein, with the inhibi- tion rate being 80%, and siRNA-3 was chosen in the subsequence experiments for gene knockdown. MTTassay and wound healing as- say showed that the proliferative and healing ability of PI3Kp85α-knockdown cells was significantly lower than that of the negative control cells since 24 hours after transfection, respectively （P〈0.05）. Transwell assay showed that the cells passing the polycarbonate membranes was significantly less in PI3Kp85α-knockdown cells than in the negative control ceils （P〈0.05 ）. Conclusion siRNA down-regnlating PI3Kp85α gene in human gastric carcinoma cell line AGS can inhibit cell proliferation, migration and invasion.