依维莫司负调控mTOR信号通路对膀胱癌BTT739细胞增殖和迁移的抑制作用及机制研究

To study the inhibitory effect and mechanism of the mTOR inhibitor everolimus on proliferation and migration in mouse bladder cancer BTT739 cells by inhibiting the mTOR signaling pathway

目的:探索mTOR抑制剂依维莫司(RAD001)对膀胱癌BTT739细胞在体外和小鼠体内增殖和迁移的抑制作用及其作用机制,为依维莫司在临床上治疗膀胱癌提供理论依据。方法:体外培养小鼠膀胱癌BTT739细胞,待其生长至对数期后用梯度浓度的RAD001(0 nmol/L、5 nmol/L、10 nmol/L和20 nmol/L)干预24 h,用MTT法检测细胞增殖的变化;Tranwell实验检测RAD001对BTT739细胞迁移的影响;ELISA试剂盒检测细胞分泌蛋白E-钙黏蛋白表达的变化。Western blot检测Akt、mTOR、4EBP和PTEN蛋白的表达,RTPCR检测mTOR mRNA的表达。将BTT739细胞接种于昆明鼠皮下,待成瘤后,用依维莫司干预14天,取小鼠肿瘤组织检测E-钙黏蛋白及AKT、mTOR、4EBP和PTEN蛋白表达的情况。结果:体外实验显示RAD001可以抑制BTT739细胞的增殖和迁移并与剂量有关(P<0.05)。还可以增加E-钙黏蛋白的含量,抑制癌细胞的转移。实验证明RAD001在体外抑制Akt、mTOR、4 EBP的表达,促进肿瘤抑制因子PTEN的表达(P<0.05)。将BTT739细胞接种于小鼠皮下后发现,RAD001在小鼠体内亦可以抑制病变情况,促进PTEN和E-钙黏蛋白的表达,抑制Akt、mTOR和4EBP的表达。结论:mTOR抑制剂依维莫司通过抑制mTOR信号通路抑制膀胱癌细胞BTT739的增殖、迁移和侵袭。

Objective：To explore the inhibitory effect and mechanismof everolimus（RAD001）,the inhibitor of mTOR,on proliferation and migration of mouse bladder cancer cell BTT739 in vitro and vivo,and also provide a theoretical basis for clinical treatment of bladder cancer with everolimus.Methods：The mouse bladder cancer BTT739 cells were cultured in vitro.The cells were treated with RAD001 （0 nmol/L,5 nmol/L,10 nnmol/L and 20 nnmol/L） for 24 h after the cells were grown to logarithmic phase.The proliferation of BTT739was detected by MTT assay.The effect of RAD001 on the migration of BTT739 cells was detected by Tranwell experiment.The expression of E-cadherin was detected by ELISA kit.Western blot was used to detect the protein expression of Akt,mTOR,4EBP and PTEN,and mTOR mRNA expression was detected by RT-PCR.BTT739 cells were inoculated subcutaneously in Kunming mice.After the tumor was established,the mice were treatmented with everolimus for 14 days.After that,we detect the expression of E-cadherin,AKT,mTOR,4EBP and PTEN protein in tumor tissues.Results：The vitro experiments showed that RAD001 could inhibit the proliferation and migration of BTT739 cells in dose-dependent manner （P＜0.05）.It also can increase the E-cadherin content and inhibit metastasis of cancer cell.Experiments show that RAD001 can inhibit the expression of Akt,mTOR and 4EBP in vitro and promote the expression of tumor suppressor PTEN （P＜0.05）.When inoculatedthe BTT739 cells in mice subcutaneously,we found that RAD001 can also inhibit the lesion in vivo,RAD001 promotes the expression of PTEN and E-cadherin,and inhibit the expression of Akt,mTOR and 4EBP.Conclusion：The mTOR inhibitor everolimus inhibits the proliferation,migration and invasion of bladder cancer cell BTT739 by inhibiting the mTOR signaling pathway.