TIPE3干扰质粒对SW480结肠癌细胞生长的作用及相关机制探讨

Effect and mechanism of TIPE3 interference plasmid on SW480 colorectal cancer growth

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-p SIREN-Retro Q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。Annexin V-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-p SIREN-Retro Q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±0.277 0)%。证实了降低TIPE3表达可以增加SW480对a DR5Sc Fv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。

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-p SIREN-Retro Q 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 CCK8. Meanwhile,apoptosis rate of SW480 cells was determined by flow cytometry assay with Annexin V-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 interference plasmids were successfully constructed. We found that the cell survival rate decreased when interference TIPE3 gene expressing in colorectal cancer cells. Flow cytometry indicated that interefering the expression of TIPE3 would increase the sensitivity of SW480 cell to apoptosis induced by a DR5 Sc Fv. The results of Western blot showed that low expression of TIPE3 would activate caspase3 and downregulate the expression of p-AKT,p-PDK1 and PCNA. Conclusion： Interference TIPE3 could promote apoptosis and inhibit proliferation in SW480 colon cancer cells.