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-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West; ern; 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-Ret; roQ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. 991. 087) %,显著高于正常细胞组(12. 102. 213) %及转染空载质粒组(11. 440. 277 0); %。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。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 CCK8. 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 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 aDR5ScFv. 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.国家自然科学基金; 福建省属公益类科研院所基本科研专项项