miR-891a-5p通过CPEB1调控结肠癌细胞生物过程的机制研究

The mechanism of miR-891a-5p regulating the biological process of colorectal cancer cells by targeting CPEB1

目的 探讨miR-891a-5p通过细胞质多聚腺苷酸化元件结合蛋白1(CPEB1)调控结肠癌细胞增殖、凋亡、迁移、侵袭的机制。方法 运用实时荧光定量逆转录聚合酶链反应(qPCR)检测结肠癌组织、癌旁组织、人正常结肠上皮细胞和人结肠癌细胞中miR-891a-5p的表达;将LOVO细胞分为A组(不做任何处理)、B组(转染anti-miRcon)、C组(转染anti-miR-891a-5p)、D组(共转染anti-miR-891a-5p和si-con)、E组(共转染anti-miR-891a-5p和siCPEB1)、F组(转染miR-con)、G组(转染miR-891a-5p);CCK-8法检测细胞增殖率、Annexin V-FITC/PI法检测细胞凋亡率;蛋白免疫印迹实验检测细胞周期蛋白D1(Cyclin D1)、胱天蛋白酶-3酶原(Pro-caspase-3)、裂解的胱天蛋白酶-3(C-caspase-3)、N-钙黏蛋白(N-cadherin)、上皮细胞钙黏蛋白(E-cadherin)和波形蛋白(Vimentin)的蛋白表达;Transwell实验检测细胞迁移、侵袭;双荧光素酶报告基因检测实验检测细胞的荧光活性。结果 与癌旁组织相比,结肠癌组织miR-891a-5p表达升高,与正常结肠上皮细胞相比,结肠癌细胞中miR-891a-5p的表达均显著升高(P<0.05)。与B组相比,C组miR-891a-5p、Cyclin D1、N-cadherin、Vimentin蛋白表达下降,细胞增殖率、迁移、侵袭数量降低,C-caspase-3、CPEB1、E-cadherin蛋白表达和凋亡率升高(P<0.05);与D组相比,E组miR-891a-5p、Cyclin D1、N-cadherin、Vimentin蛋白表达水平升高,细胞增殖率、迁移、侵袭数量升高,C-caspase-3、CPEB1、E-cadherin蛋白表达和凋亡率降低(P<0.05)。miR-891a-5p与CPEB1的3′-UTR存在连续的6个互补结合位点。与F组相比,G组WT-CPEB1荧光活性和CPEB1蛋白表达降低;与B组相比,C组WT-CPEB1荧光活性升高(P<0.05)。结论 miR-891a-5p可促进结肠癌细胞增殖、迁移、侵袭,抑制凋亡,其作用机制可能与靶向抑制CPEB1有关。

Objective To explore the mechanism of miR-891a-5p regulating the proliferation,apoptosis,migration and invasion of colon cancer cells through cytoplasmic polyadenylation element binding protein 1(CPEB1).Methods Real-time fluorescence quantitative reverse transcription polymerase chain reaction(qPCR) was used to detect the expression of miR-891a-5p in colon cancer tissue,paracancer tissue,human normal colon epithelial cells and human colon cancer cells.LOVO cells were divided into the group A(without any treatment),the group B(transfected with anti-miRcon),the group C(transfected with anti-miR-891a-5p),the group D(co-transfected with anti-miR-891a-5p and si-con),group E(co-transfected with anti-miR-891a-5p and si-CPEB1),the group F(transfected with miR-con) and group G(transfected with miR-891a-5p).Cell proliferation rate was detected by CCK-8 assay,and apoptosis rate was detected by Annexin V-FITC/PI assay.Cyclin D1,Pro-caspase-3,C-caspase-3,N-cadherin,E-cadherin and Vimentin were detected by Western blot assay.Transwell assay was used to detect cell migration and invasion.Double luciferase reporter gene assay was used to detect the fluorescence activity of cells.Results The expression of miR-891a-5p in colon cancer tissue was significantly increased compared with that in normal colon epithelial cells(P <0.05).Compared with the group B,protein expressions of miR-891a-5p,Cyclin D1,N-cadherin and Vimentin were decreased in the group C,and cell proliferation rate,migration and invasion number and protein expressions of C-caspase-3,CPEB1 and E-cadherin and apoptosis rate were increased(P <0.05).Compared with the group D,the protein expressions of miR-891a-5p,Cyclin D1,N-cadherin and Vimentin,proliferation rate,migration and invasion number of cells were increased in the group E,while the protein expressions of C-caspase-3,CPEB1 and E-cadherin and apoptosis rate were decreased(P<0.05).There were six consecutive complementary binding sites between miR-891a-5p and CPEB1 at the 3 ’UTR.Compared with the group F,the fluorescence activity and CPEB1 protein expression of WT-CPEB1 were decreased in the group G.Compared with the group B,the fluorescence activity of WT-CPEB1 was increased in the group C(P<0.05).Conclusion miR-891a-5p can promote the proliferation,migration and invasion of colon cancer cells and inhibit apoptosis,which may be related to the targeted inhibition of CPEB1.