摘要
[目的]探究miR-19b-3p调控SMAD4通路对膀胱癌进展的影响。[方法]将T24细胞分为3组(5×10^(5)个/孔):空白对照组(不转染)、miR-19b-3p inhibitor组(转染miR-19b-3p特异性抑制慢病毒10μL)、miR-19b-3p阴性对照组(转染阴性对照慢病毒10μL)。qRT-PCR分析各组细胞中miR-19b-3p的表达;通过细胞划痕、平板克隆、Transwell实验分析miR-19b-3p对各组细胞迁移、增殖和侵袭能力的影响,miR-19b-3p与SMAD4之间的靶向相互作用通过双荧光素酶报告基因实验来验证。Western blot检测各组细胞SMAD4、TGF-β、Vimentin、E-cadherin和MMP9的蛋白表达量。[结果]与空白对照组、miR-19b-3p阴性对照组相比,miR-19b-3p inhibitor组的T24细胞的克隆形成数量、迁移率和侵袭细胞个数均降低(P<0.05)。双荧光素酶报告结果显示,miR-19b-3p能够靶向作用SMAD4(P<0.05)。与空白对照组、miR-19b-3p阴性对照组相比,miR-19b-3p inhibitor组的SMAD4表达升高,Vimentin蛋白表达下调,E-cadherin蛋白表达上调,MMP9表达下调(P<0.05)。[结论]miR-19b-3p能够靶向抑制SMAD4的表达。下调miR-19b-3p导致SMAD4的表达升高进而抑制膀胱癌细胞的增殖、迁移及侵袭。
[Objective]To investigate the effect of miR-19b-3p on the progression of bladder cancer by regulating TGF-βpathway.[Method]T24 cells were divided into 3 groups(5×10^(5) cells/well):blank control group(no transfected),miR-19b-3p inhibitor group(transfected with 10μL of miR-19b-3p-specific inhibitory lentivirus)and miR-19b-3p negative control group(transfected with 10μL of non-targeting negative control lentivirus).The expression of miR-19b-3p in each group of cells was analyzed by qRT-PCR.The effects of miR-19b-3p on cell migration,proliferation and invasion were an⁃alyzed by cell scratch assay,plate cloning assay and Transwell assay.The interaction targeted by miR-19b-3p onto SMAD4 was assessed utilizing a dual luciferase reporter gene system.Western blot was used to detect the protein expressions of SMAD4,TGF-β,Vimentin,E-cadherin and MMP9 in each group.[Result]Compared with the blank control group and miR-19b-3p negative control group,the colony formation,migration rate and invasive cell number of T24 cells in the miR-19b-3p in⁃hibitor group were decreased(P<0.05).Dual-luciferase reporter assay showed that miR-19b-3p could target SMAD4(P<0.05).Compared with the blank control group and miR-19b-3p negative control group,the miR-19b-3p inhibitor group had a significantly increased expression of SMAD4,a significantly decreased expression of Vimentin,a significantly in⁃creased expression of E-cadherin,and a significantly decreased expression of MMP9(P<0.05).[Conclusion]miR-19b-3p can target and inhibit the expression of SMAD4.Downregulation of miR-19b-3p can lead to an increase in SMAD4 ex⁃pression,thereby inhibitting the proliferation,migration and invasion of bladder cancer cells.
作者
吕建敏
张智源
左庆军
徐峰
LYU Jianmin;ZHANG Zhiyuan;ZUO Qingjun;XU Feng(Department of Urology,Shanghai Seventh People′s Hospital,Shanghai 200137,China)
出处
《生物技术》
2025年第6期704-709,715,共7页
Biotechnology
