RNA结合蛋白TTP对肺腺癌细胞增殖影响的实验研究

RNA binding protein tristetraprolin inhibits proliferation of lung adenocarcinoma cells in vitro

目的探讨TTP(tritetraprolin)过表达对肺腺癌细胞增殖影响及其可能的作用机制。方法构建带有四环素可调控(tetracycline-on,Tet-on)的TTP基因表达系统,利用慢病毒感染的方法转染肺腺癌H1975及1299细胞,并在强力霉素(doxycycline,Dox)的诱导下稳定表达TTP。用实时荧光定量PCR和Western blot鉴定TTP受Dox调控表达的效果。分别采用CCK-8法、Transwell、平板克隆形成实验、流式细胞术检测TTP过表达后对细胞增殖、迁移、集落形成能力、凋亡和细胞周期的影响。Western blot法检测TTP过表达对凋亡基因在蛋白水平表达的影响。结果成功构建受Dox诱导调控的TTP过表达肺腺癌细胞模型。与TTP低表达组比较,Dox诱导TTP过表达组明显抑制H1975、H1299细胞增殖、迁移能力(H1975:P<0.01,H1299:P<0.001)及集落形成能力(P值均<0.0001),且使H1975细胞周期被阻滞在S期(P<0.01),但TTP过表达不影响肺腺癌细胞凋亡。结论 TTP过表达显著抑制肺腺癌细胞增殖、迁移、集落形成能力,使细胞周期被阻滞在S期,但TTP过表达不通过细胞凋亡途径影响肺腺癌细胞增殖和生长。

Objective To investigate the effect of tristetraprolin(TTP)on the proliferation of lung cancer cells and to explore the possible mechanism.Methods The human lung adenocarcinoma cells with tetracycline-inducible(Tet-on)expression regulatory system were established in H1975 and 1299 cells with the aid of lentivirus infection.Western blotting and q-PCR were used to confirm the overexpression of TTP in the cells after the doxycycline(Dox)treatment.Effect of TTP on the proliferation,migration and colony-forming capacity were detected by CCK-8 assay,Transwell test and colony forming unit test respectively.The cell apoptosis and cell cycle were measured by flow cytometry.The expression of cellular apoptosis-related proteins were determined by Western blotting.Results Lung adenocarcinoma cells with Dox-induced TTP overexpression were successfully established.Compared with the cells with low TTP expression,those with Dox-induced TTP overexpression siginicantly inhibited proliferation,migration(H1975:P<0.01,H1299:P<0.001)and colony-formation(all P<0.0001)in lung adenocarcinoma cells H1975 and H1299.What's more,H1975 cells were arrested at S stage(P<0.01).However,TTP overexpression exerted no effect on cell apoptosis.Conclusion TTP overexpression obviously inhibits proliferation,migration and colony-forming capacity,and arrests cell cycle at S stage in lung adenocarcinoma cells.But the mechanism is not due to the regulation of cell apoptosis.