LINC01018介导E2F1-CDK6通路调控结肠癌发病的机制

Involvlment of LINC01018 in the pathogenesis of colon cancer by mediating E2F1-CDK6 pathway

目的研究LINC01018参与结肠癌发病的具体机制。方法实时荧光定量PCR(qRT-PCR)法检测结肠癌组织和细胞以及癌旁组织和细胞中LINC01018的表达差异情况。建立稳定过表达LINC01018的HT-29细胞株。利用RNA结合蛋白免疫沉淀(RIP)实验检测LINC01018与E2F1蛋白之间是否存在相互作用。利用双荧光素酶实验检测E2F1对CDK6启动子的调控作用。在过表达LINC01018的HT-29细胞中同时上调E2F1或CDK6表达,再通过CCK-8实验、Transwell实验、Western blot方法检测HT-29细胞的增殖、侵袭、迁移以及细胞中CDK6及MMP-2蛋白表达情况。结果LINC01018在结肠癌组织及细胞中异常低表达。RIP实验结果显示,LINC01018与E2F1蛋白存在相互作用。双荧光素酶实验结果表明,E2F1蛋白能够增强CDK6启动子工作效率,E2F1对CDK6存在正调控作用;而LINC01018的过表达能够减弱E2F1对CDK6的正调控作用。回复实验显示,上调E2F1或CDK6表达能够减弱LINC01018过表达对HT-29细胞增殖、侵袭、迁移及CDK6、MMP-2蛋白表达的影响。结论LINC01018在结肠癌组织及细胞中异常低表达。LINC01018可能通过E2F1/CDK6/MMP-2轴调控HT-29细胞增殖、侵袭及迁移,并参与结肠癌的发病。

Objective To study the specific mechanism of LINC01018 involved in the pathogenesis of colon cancer.Methods The expression of LINC01018 in colon cancer tissues and cells and normal colon tissues and cells were detected by real time fluorescence quantitative polymerase chain reaction(qRT-PCR).HT-29 cell line which overexpresses LINC01018 stably was established.RNA binding protein immunoprecipitation(RIP)assay was used to detect the interaction between LINC01018 and E2F1 protein.Dual luciferase assay was used to detect the regulatory effect of E2F1 on CDK6 promoter.The expression of E2F1 or CDK6 was up-regulated in HT-29 cell line which overexpresses LINC01018,then the proliferation,invasion and migration of HT-29 cells and the expression of CDK6 and matrix metalloproteinase-2(MMP-2)in HT-29 cells were detected by cell counting method(CCK-8)assay,Transwell assay and Western blot.Results The expression of LINC01018 was abnormally low in colon cancer tissues and cells.The result of RIP assay showed that LINC01018 interacted with E2F1 protein.The result of dual luciferase assay showed that E2F1 protein could enhance the efficiency of CDK6 promoter,and E2F1 had a positive regulatory effect on CDK6.Overexpression of LINC01018 could attenuate the positive regulatory effect of E2F1 on CDK6.Up-regulation of E2F1 or CDK6 expression could attenuate the effects of LINC01018 overexpression on the proliferation,invasion,migration and expression of CDK6 and MMP-2 in HT-29 cells.Conclusions The expression of LINC01018 was abnormally low in colon cancer tissues and cells.LINC01018 may regulate the proliferation,invasion and migration of HT-29 cells through E2F1/CDK6/MMP-2 axis,and participate in the pathogenesis of colon cancer.