摘要
目的 探讨miR-4258在膀胱癌T24细胞分化和侵袭过程中的作用机制。方法 通过TargetScan和荧光素酶报告基因验证miR-4248靶基因。将T24细胞分为正常对照NG组、阴性对照miR-ctrl组、miR-4258inhibitor组和miR-4258mimics组,采用脂质体(Lipo 2000)细胞转染法转染;采用实时荧光定量聚合酶链反应(real-time qPCR)检测组织和细胞的miR-4258表达水平;运用免疫印迹(Western blot)检测Cadherin-19(CDH19)、E-cadherin、α-SMA的表达水平。通过Transwell检测侵袭能力的变化,明胶酶谱法测定基质金属蛋白酶2(MMP2)的活性。结果 miR-4258在膀胱上皮癌组织中的表达水平明显高于其癌旁组织,差异有统计学意义(t=3.711,P<0.01);生物信息学预测和荧光素酶报告基因验证miR-4258作用靶点为CDH19 3′UTR。miR-4258mimics转染T24细胞后,CDH19和上皮细胞钙黏蛋白(E-cadherin)的蛋白表达水平较miR-ctrl组显著下调(P<0.05),而α-SMA的表达水平较miR-ctrl组显著提高(P<0.01);上调miR-4258表达MMP2的活性较miR-ctrl组显著提高(P<0.01),同时T24细胞的侵袭能力也得以明显增强。结论 miR-4258调控靶点为CDH19,可能通过下调CDH19的表达水平,促进了T24细胞分化,增强了T24的迁移和侵袭能力。
Objective To investigate the mechanism of miR-4258 in the differentiation and invasion of bladder cancer T24 cells.Methods The miR-4248 target gene was verified with TargetScan and luciferase reporter.T24 cells were divided into normal control group,negative control group,miR-4258 inhibitor group,and miR-4258 mimics group.Cells were tranfected with Lipo 2000.The expression of miR-4258 was detected in tissues and cells with real-time qPCR.The expressions of cadherin-19(CDH19),E-cadherin andα-SMA were detected with Western blotting.The changes of invasion and the activity of matrix metalloproteinase 2(MMP2)were determined with Transwell assay and gelatin zymography,respectively.Results The expression of miR-4258 was significantly higher in cancer tissues than in adjacent tissues(t=3.711,P<0.01).Bioinformatic analysis and luciferase reporter confirmed that the target gene of miR-4258 was CDH193′UTR.Compared with the negative control group,the miR-4258 mimics group had significantly down-regulated protein expression of CDH19 and E-cadherin(P<0.05),significantly increased expression ofα-SMA(P<0.01),significantly higher activity of MMP2(P<0.01),and significantly enhanced invasion of T24 cells.Conclusion The target of miR-4258 is CDH19,which may promote the differentiation,migration and invasion of T24 cells by down-regulating the expression of CDH19.
作者
曹罗元
杨菁
富显果
董文婿
林应华
黄宝英
CAO Luoyuan;YANG Jing;FU Xianguo;DONG Wenxu;LIN Yinghua;HUANG Baoying(Department of Central Laboratory,Ningde Municipal Hospital,Affiliated Hospital of Ningde Normal University,Ningde 352100,China)
出处
《现代泌尿外科杂志》
CAS
2021年第3期244-248,273,共6页
Journal of Modern Urology
基金
福建省自然科学基金面上项目(No.2017J01166)
福建医科大学启航基金项目(No.2016QH098)。
