TIPE3干扰质粒对SW480结肠癌细胞生长的作用及相关机制探讨

摘要

Objective:To study the effect of interference TIPE3 on the colon cancer cell growth by transfecting SW480 colon cancer cells with the TIPE3 interference plasmid were detected.Methods:Transfecting the constructed TIPE3-shRNA-pSIREN-RetroQ plasmid to SW480 cells.To determine the highest interference efficiency plasmid ,the mRNA and protein levels of recombined plasmid were detected by RT-PCR and Western blot separately and tested the cell proliferation with CCK 8.Meanwhile,apoptosis rate of SW480 cells was determined by flow cytometry assay with AnnexinV-FITC/PI.To further determined the effects of recombined plasmid on cell development ,the level of protein involved in proliferation and apoptosis were detected by Western blot .Results:The most effecient in-terference plasmids were successfully constructed.We found that the cell survival rate decreased when interference TIPE 3 gene express-ing in colorectal cancer cells .Flow cytometry indicated that interefering the expression of TIPE 3 would increase the sensitivity of SW 480 cell to apoptosis induced by aDR5ScFv.The results of Western blot showed that low expression of TIPE 3 would activate caspase3 and downregulate the expression of p-AKT,p-PDK1 and PCNA.Conclusion:Interference TIPE3 could promote apoptosis and inhibit prolif-eration in SW480 colon cancer cells .%目的:通过TIPE3干扰质粒转染SW480结肠癌细胞,验证干扰TIPE3表达对SW480结肠癌细胞生长的影响并探讨相关机制.方法:构建TIPE3-shRNA-pSIREN-RetroQ干扰质粒,通过脂质体转染法成功将干扰质粒导入SW480细胞,通过RT-PCR、Western blot检测重组质粒的干扰效率.应用CCK-8方法检测SW480细胞生存率.AnnexinV-FITC/PI流式细胞法检测细胞凋亡.使用Western blot检测细胞增殖、凋亡相关分子的表达情况.结果:成功设计、构建和筛选具有生物活性且干扰效率最佳的TIPE3-shRNA-pSIREN-RetroQ干扰质粒.CCK-8检测证实干扰SW480结肠癌细胞TIPE3表达可以抑制细胞生长.流式结果显示,TIPE3-shRNA3干扰组的凋亡率为(27.99±1.087)%,显著高于正常细胞组(12.10±2.213)%及转染空载质粒组(11.44±0.2770)%.证实了降低TIPE3表达可以增加SW480对aDR5ScFv所诱导的细胞凋亡敏感性.Western blot结果显示干扰TIPE3表达可以活化caspase3蛋白,降低p-AKT、p-PDK1、PCNA等分子的表达.结论:干扰TIPE3的表达对SW480结肠癌细胞具有促进凋亡,抑制增殖的作用.