过表达OTUD1对结肠癌细胞HCT116增殖和侵袭的影响及机制研究

Overexpression of OTUD1 in HCT116 cells inhibits cell growth,migration and invasion

目的:探讨卵巢癌结构域蛋白酶-1(ovarian tumor domain-containing protease-1,OTUD1)基因对人结肠癌细胞HCT116增殖和侵袭的作用及机制。方法:选取低表达OTUD1的人结肠癌细胞株,利用慢病毒包装技术过表达OTUD1基因,空载质粒作对照,嘌呤霉素筛选后构建稳定转染的结肠癌细胞株。RT-PCR(reverse transcription PCR)和Western blot技术检测转染效率。细胞迁移和侵袭实验检测2组细胞迁移和侵袭能力的改变。细胞增殖实验和流式细胞术检测2组细胞增殖能力的变化。克隆形成实验检测2组细胞的克隆形成能力。Western blot技术分析相关蛋白分子水平的变化,探讨其可能的机制。结果:6株结肠癌细胞株中HCT116细胞的OTUD1蛋白表达水平最低,并成功构建过表达OTUD1基因的结肠癌细胞株HCT116-OTUD1。与对照组HCT116-Control细胞相比,HCT116-OTUD1细胞株迁移(P=0.000)和侵袭能力减弱(P=0.000),CCK-8增殖实验显示实验组细胞增殖水平下降(P=0.004),EDU流式检测结果显示细胞增殖水平下降(P=0.000),克隆形成能力下降(P=0.017)。上皮间质转化(epithelial mesenchymal transition,EMT)相关分子E-cadherin表达水平上调,Vimentin表达水平下调,增殖相关信号通路分子p-AKT、p-ERK1/2表达水平下调。结论:过表达OTUD1可以通过减弱细胞的EMT作用来影响其迁移和侵袭能力,并抑制细胞的增殖能力,这种变化可能与MAPK/Erk和PI3K/Akt信号通路的激活受到抑制有关。

Objective：To investigate the effects and mechanism of OTUD1 gene on proliferation and invasion of human colon cancer line HCT116. Methods：Human colon cancer cell lines with lower expression of OTUD1 were selected. The OTUD1 gene in HCT116 cells was over expressed by lentivirus infection technique,and empty vector was used as control. After screening by puromycin,HCT116 cells with stably expressed of OTUD1 was constructed. RT-PCR and Western blot were used to detect the transfection efficiency. The abilities of migration and invasion in vitro were observed by Transwell assay. The CCK-8 cell proliferation assay was used to detect the proliferation ability. The colony formation ability was measured by colony forming assay. The changes of associated molecular level was detected by Western blot to predict its possible mechanism. Results：The expression of OTUD1 in HCT116 cells was the lowest in the 6 human colon cancer cell lines. HCT116-OTUD1 cells with stably-expressed OTUD1 were successfully established. Compared with that of control group,the migration and invasion ability of HCT116-OTUD1 cells was decreased（P=0.000）,the proliferation ability was inhibited（P =0.004）,the clone formation ability was decreased（P =0.017）,EDU level performed by flow cytometry was down-regulated（P=0.000）. Furthermore,the EMT-associated molecule E-cadherin was up-regulated,while Vimentin was down-regulated,and the proliferation associated molecule p-AKT and p-ERK were down-regulated. Conclusion：OTUD1 might promote HCT116 cells proliferation,migration and invasion.