CircRHOT1通过调控miR-29b/KDM2A对膀胱癌细胞增殖和迁移的影响  

Effects of circRHOT1 on bladder cancer cell proliferation and migration by regulating miR-29b/KDM2A

作　　者：王栋[1] 刘伟霞[2] 张玉瑞[1] 康郑军[1] 何笑凯[1] 何江[1] 朱海鹏[1] WANG Dong;LIU Wei-xia;ZHANG Yu-rui;KANG Zheng-jun;HE Xiao-kai;HE Jiang;ZHU Hai-peng(Department of Urology,the Fifth Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,Henan,China)

机构地区：[1]郑州大学第五附属医院泌尿外科,河南郑州450052 [2]郑州大学第五附属医院肾病内科,河南郑州450052

出　　处：《广东医学》2023年第5期543-549,共7页Guangdong Medical Journal

基　　金：河南省高等学校重点科研项目(21A320037)。

摘　　要：目的 探讨环状RNA RHOT1(circRHOT1)对微小RNA-29b-3p(miR-29b-3p)/赖氨酸去甲基化酶2A(KDM2A)轴的调控作用及对膀胱癌(BCa)细胞增殖和迁移的影响。方法 取人输尿管上皮永生化细胞(SV-HUC-1)和BCa细胞(5637、TCCSUP、T24和UM-UC-3),实时荧光定量PCR(RT-qPCR)检测细胞中circRHOT1、miR-29b-3p和KDM2A mRNA表达。取T24细胞,分为对照组、si-NC组、si-circRHOT1组、si-circRHOT1+anti-NC组、si-circRHOT1+anti-miR-29b-3p组。采用RT-qPCR检测各组细胞中circRHOT1、miR-29b-3p和KDM2A mRNA表达水平;CCK-8法检测各组细胞的增殖活性;Transwell小室法检测细胞迁移和侵袭能力;Western blot检测KDM2A和上皮-间质转化(EMT)相关蛋白E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)和N-钙黏蛋白(N-cadherin)表达;双荧光素酶报告基因和RNA结合蛋白免疫沉淀(RIP)验证T24细胞中circRHOT1、miR-29b-3p和KDM2A的靶向关系。结果 与SV-HUC-1细胞相比,BCa细胞(T24、5637、TCCSUP和UM-UC-3)中circRHOT1、KDM2A mRNA水平显著升高,miR-29b-3p水平显著降低(P<0.05)。敲低circRHOT1后,与对照组、si-NC组相比,si-circRHOT1组KDM2A的mRNA和蛋白水平、细胞活力、迁移和侵袭能力、N-cadherin、Vimentin水平均降低,miR-29b-3p、E-cadherin水平升高(P<0.05);抑制miR-29b-3p表达可升高KDM2A表达,并减弱敲低circRHOT1对增殖、迁移和侵袭的抑制作用(P<0.05)。双荧光素酶报告基因和RIP实验证实circRHOT1可靶向负调控miR-29b-3p表达,KDM2A是miR-29b-3p的靶标。结论 敲低circRHOT1可能通过靶向上调miR-29b-3p来抑制KDM2A表达,进而抑制BCa细胞增殖、迁移和侵袭。Objective To investigate the regulatory effect of circular RNA RHOTI(circRHOTI)on microRNA-29b-3p(miR-29b-3p)/lysine demethylase 2A(KDM2A)axis,and its impacts on proliferation and migration of bladder cancer(BCa)cells.Methods Human ureteral epithelial immortalized cells(SV-HUC-1)and BCa cells(5637,TCCSUP,T24 and UM-UC-3)were collected.Real-time quantitative PCR(RT-qPCR)was used to assess the expressions of circRHOT1,miR-29b-3p and KDM2A mRNA in the cells.T24 cells were collected and grouped into control group,si-NC group,si-circRHOT1 group,si-circRHOTI+anti-NC group,and si-circRHOT1+anti-miR-29b-3p group.RT-qPCR was applied to assess the expression levels of circRHOT1,miR-29b-3p and KDM2A mRNA.CCK-8 method was applied to detect the proliferation activity of cells in each group.Trans-well chamber method was applied to detect cell migration and invasion abilities.Western blot was applied to detect the expression of KDM2A and epithelial-mesenchymal transition(EMT)-related proteins E-cadherin,Vimentin and N-cadherin.Dual-luciferase reporter gene and RNA-binding protein immunoprecipitation(RIP)were applied to verify the targeting relationship of circRHOT1,miR-29b-3p and KDM2A in T24 cells.ResultssCompared with SV-HUC-1 cells,the mRNA levels of circRHOT1 and KDM2A in BCa cells(T24,5637,TCCSUP and UM-UC-3)were significantly increased,and the level of miR-29b-3p was significantly reduced(P<0.05).After knockdown of circRHOT1,compared with the control group and si-NC group,the mRNA and protein levels of KDM2A,cell viability,migration and invasion abilities,the levels of N-cadherin and Vimentin were decreased;and the levels of miR-29b-3p and E-cadherin were increased in the si-circRHOTI group(P<0.05).Inhibition of miR-29b-3p expression could increased KDM2A expression,and attenuated the inhibitory effects of knockdown of circRHOT1 on proliferation,migration and invasion(P<0.05).Dual-luciferase reporter gene and RIP experiments confirmed that circRHOT1 could target and negatively regulate the expression of miR-29b-3p,and KDM

关 键 词：环状RNA RHOT1 膀胱癌 微小RNA-29b-3p 赖氨酸去甲基化酶2A 

分 类 号：R737.14[医药卫生—肿瘤] R699.5[医药卫生—临床医学]