摘要
目的探讨miR-338-5p通过靶向TSHZ3调控膀胱癌细胞增殖、迁移和侵袭的机制。方法实时荧光定量PCR(qRT-PCR)检测miR-338-5p在33例膀胱癌组织和癌旁组织中的表达;在膀胱癌T24和UM-UC-3细胞中分别转染mimics-NC、miR-338-5p mimics、inhibitor-NC和miR-338-5p inhibitor,qRT-PCR检测转染效率;采用CCK-8实验检测过表达或者敲低miR-338-5p对膀胱癌细胞增殖能力的影响;Transwell实验检测miR-338-5p对膀胱癌细胞迁移和侵袭能力的影响;TargetScan、miRDB和Targetminer数据库预测miR-338-5p潜在的靶基因,选定靶基因TSHZ3;双萤光素酶报告基因实验和Western blot验证miR-338-5p和TSHZ3的靶向关系;在膀胱癌细胞中共转染miR-338-5p mimics和TSHZ3过表达质粒,通过CCK-8和Transwell实验检测细胞的增殖、迁移和侵袭能力;Western blot检测过表达miR-338-5p或者TSHZ3对Wnt/β-catenin信号通路的影响。结果miR-338-5p在膀胱癌中表达上调(P<0.05);过表达miR-338-5p提高了膀胱癌细胞的增殖、迁移和侵袭能力(P<0.05),转染miR-338-5p inhibitor则降低了膀胱癌细胞的增殖、迁移和侵袭能力(P<0.05);生物信息学分析和双萤光素酶报告基因实验表明,miR-338-5p和TSHZ3存在靶向关系;挽救实验表明,同时过表达miR-338-5p和TSHZ3部分消除了过表达miR-338-5p对膀胱癌细胞增殖、迁移和侵袭能力的促进作用(P<0.05);Western blot结果显示,与mimics-NC组比较,过表达miR-338-5p降低了TSHZ3蛋白表达水平,提高了Wnt3a和β-catenin蛋白表达水平(P<0.05);过表达TSHZ3降低了Wnt3a和β-catenin蛋白表达水平(P<0.05)。结论miR-338-5p在膀胱癌中表达上调,通过靶向抑制TSHZ3的表达促进膀胱癌的增殖、迁移和侵袭,该机制可能与激活Wnt/β-catenin信号通路有关。
Objective To investigate the mechanism of miR-338-5p regulating the proliferation,migration and invasion of bladder cancer cells by targeting TSHZ3.Methods The expression of miR-338-5p in 33 samples of bladder cancer tissue and paracancerous tissue was detected by qRT-PCR.Bladder cancer T24 and UM-UC-3 cells were transfected with mimics-NC,miR-338-5p mimics,inhibitor-NC and miR-338-5p inhibitor,and the transfection efficiency was detected by qRT-PCR.CCK-8 assay was used to detect the effect of overexpression or knockdown of miR-338-5p on the proliferation of bladder cancer cells.Transwell assay was used to detect the effect of miR-338-5p on migration and invasion of bladder cancer cells.The potential target genes of miR-338-5p were predicted by TargetScan,miRDB and Targetminer databases,and TSHZ3 was chosen for the target gene.The targeting relationship between miR-338-5p and TSHZ3 was verified by dual-luciferase reporter gene assay and Western blot assay.miR-338-5p mimics and TSHZ3 overexpression plasmids were co-transfected in bladder cancer cells,and the proliferation,migration and invasion of cells were detected by CCK-8 and Transwell assays.Western blot assay was used to detect the overexpression of miR-338-5p or TSHZ3 against Wnt/β-catenin signaling pathway.Results miR-338-5p was significantly upregulated in bladder cancer(P<0.05).Overexpression of miR-338-5p increased the proliferation,migration and invasion of bladder cancer cells(P<0.05),while transfection of miR-338-5p inhibitor decreased the proliferation,migration and invasion of bladder cancer cells(P<0.05).Bioinformatics analysis and the dual-luciferase reporter gene assay showed that there was a targeting relationship between miR-338-5p and TSHZ3.The rescue experiment showed that co-transfection of miR-338-5p mimics and TSHZ3 overexpression plasmid partially eliminated the promoting effect of overexpression of miR-338-5p on the proliferation,migration and invasion of bladder cancer cells(P<0.05).Western blot results showed that overexpression of miR-338-5p GP-significantly decreased the expression level of TSHZ3 protein and increased Wnt3a andβ-catenin protein expression levels(P<0.05).In addition,overexpression of TSHZ3 reduced Wnt3a andβ-catenin protein expression levels(P<0.05).Conclusion miR-338-5p is upregulated in bladder cancer,and promotes the proliferation,migration and invasion of bladder cancer by targeting TSHZ3 expression.The mechanism may be related to the activation of Wnt/β-catenin signaling pathway.
作者
刘宏伟
朱奕
向玲宝
熊洪
陈瑞琦
LIU Hongwei;ZHU Yi;XIANG Lingbao;XIONG Hong;CHEN Ruiqi(Laboratory of Urology,Affiliated Hospital of Guangdong Medical University,Zhanjiang 524001,China)
出处
《天津医药》
CAS
北大核心
2023年第10期1025-1032,共8页
Tianjin Medical Journal
基金
广东省自然科学基金资助项目(2022A1515012195)
湛江市科技计划项目(2019A01028,2020A01022)。
