lncRNA KCNQ1OT1在结肠癌细胞中的促癌功能和下游调控通路研究

Cancer-promoting function of lncRNA KCNQ1OT1 in colon cancer cells and its downstream regulatory pathways

目的明确lncRNA KCNQ1OT1在结肠癌HCT-116和HT-29细胞中的功能,探讨其作用的分子机制。方法在结肠癌HCT-116和HT-29细胞系中转染KCNQ1OT1的过表达质粒及空白对照,siRNA干扰KCNQ1OT1及空白对照;分别在两个细胞系中进行CCK8、划痕实验、Transwell、细胞侵袭实验以验证KCNQ1OT1对细胞功能的影响;免疫共沉淀(RNA immune precipitation,RIP)和双荧光素酶标记实验分别验证KCNQ1OT1与miR-125a-5p的特异结合以及miR-125a-5p和SMURF1蛋白的特异结合。过表达和敲低KCNQ1OT1、miR-125a-5p后,Western hlot验证SMURF1的表达;完成过表达KCNQ1OT1同时过表达miR-125a-5p、敲低KCNQ1OT1同时敲低miR-125a-5p后检测SMURF1表达情况的Resecue实验,验证KCNQ1OT1与miR-125a-5p的调控关系。结果在结肠癌HCT-116和HT-29细胞中,过表达的KCNQ1OT1能促进结肠癌细胞的增殖、迁移和侵袭;KCNQ1OT1敲低后,结肠癌细胞的增殖、迁移和侵袭受抑制;miR-125a-5p与KCNQ1OT1及SMURF1均能特异性结合;过表达KCNQ1OT1促进SMURF1的表达,敲低KCNQ1OT1抑制SMURF1的表达;过表达miR-125a-5p抑制SMURF1的表达,敲低miR-125a-5p促进SMURF1的表达;过表达KCNQ1OT1的基础上同时表达miR-125a-5p能显著抑制SMURF1的表达,敲低KCNQ1OT1的基础上同时敲低miR-125a-5p能促进SMURF1的表达。结论在结肠癌细胞中,lncRNA KCNQ1OT1通过KCNQ1OT1-miR-125a-5p-SMURF1调控轴,促进结肠癌细胞的增殖、迁移和侵袭。

Objective To clarify the function of IncRNA KCNQ1 OT1 in HCT-116 and HT-29 colon cancer cell lines,and explore the molecular mechanism of its role in colon cancer cells.Methods HCT-116 and HT-29 cells transfected by LncRNA KCNQ10 T1 overexpression plasmid and blank control served as pcDNA3.1-KCNQ1 OT1 group and pcDNA3.1 group,and the two cell lines cells knocked down by siRNA and blank control served as si-KCNQ1 OT1 group and NC group.CCK8,scratch assay,Trans well and cell invasion assay were conducted to verify the effect of lncRNA KCNQ1 OT1 on cell function.RNA immune Precipitation(RIP)assay and double luciferase labeling assay were applied to verify the binding interaction between KCNQ1 OT1 and miR-125 a-5 p and the binding interaction between miR-125 a-5 p and SMURF1.After overexpression and knockdown of KCNQ1 OT1 and miR-125 a-5 p,the expression of SMURF 1 was verified by western blot.Rescue experiment was performed to detect the expression of SMURF1 after co-transfection and co-knockdown of KCNQ1 OT1 and miR-125 a-5 p.Results In HCT-116 and HT-29 cells,overexpressed KCNQ1 OT1 could promote the proliferation,migration and invasion of colon cancer cells.After knockdown of KCNQ1 OT1,the proliferation,migration and invasion of colon cancer cells were inhibited.miR-125 a-5 p could specifically bind to both KCNQ1 OT1 and SMURF1.Overexpression of KCNQ1 OT1 promoted the expression of SMURP1,while knockdown of KCNQ1 OT1 inhibited the expression of SMURF1.Overexpression of miR-125 a-5 p inhibited the expression of SMURF1,and knockdown of miR-125 a-5 p promoted the expression of SMURF 1.On the basis of overexpression of KCNQ1 OT1,expression of miR-125 a-5 p could significantly inhibit the expression of SMURF1,while knockdown of miR-125 a-5 p and KCNQ1 OT1 could promote the expression of SMURF1.Conclusion In colon cancer cells,lncRNA KCNQ1 OT1 promotes the proliferation,migration and invasion of colon cancer cells by regulating the KCNQ10 T1-miR-125 a-5 p-SMURFl axis.