摘要
目的探讨长链非编码RNA(long-chain non-coding RNA,lncRNA)AP000892.6对膀胱癌细胞迁移、侵袭和上皮间充质转化进程的影响及相关分子机制。方法应用GEPIA数据集分析AP000892.6在膀胱癌和癌旁组织中的表达。应用实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测膀胱癌细胞株647V、T24、UMUC3、5637、RT4和正常膀胱上皮细胞SV-HUC-1中AP000892.6的表达水平。将AP000892.6过表达质粒或阴性对照质粒转染到T24细胞,实验分为AP000892.6组和NC组,RT-qPCR法验证质粒转染效率。细胞划痕法和Transwell实验分别检测转染T24细胞的迁移和侵袭能力。应用生物信息学方法预测以及双荧光素酶报告基因实验验证AP000892.6的下游靶基因。RT-qPCR法检测AP000892.6下游靶基因的表达。Western blot法检测上皮表型蛋白E-cadherin、ZO-1和间充质表型蛋白Vimentin、N-cadherin、Twist的表达。结果与癌旁组织比较,AP000892.6在膀胱癌组织中表达降低(P<0.01)。与SV-HUC-1细胞比较,AP000892.6在膀胱癌细胞株中表达降低(P<0.05),T24细胞中AP000892.6的相对表达水平最低(P<0.01)。与NC组比较,过表达AP000892.6抑制膀胱癌T24细胞的迁移(P<0.01)和侵袭能力(P<0.01)。双荧光素酶报告基因分析证明miR-616-5p是AP000892.6的靶基因(P<0.01)。与NC组比较,AP000892.6对miR-616-5p表达具有负调控作用(P<0.01)。与NC组比较,AP000892.6组T24细胞中上皮表型蛋白E-cadherin、ZO-1表达上调,间充质表型蛋白Vimentin、N-cadherin、Twist表达下调。结论AP000892.6通过靶向miR-616-5p抑制膀胱癌T24细胞的迁移、侵袭和上皮间充质转化进程。
Objective To investigate the effect of long-chain non-coding RNA(lncRNA)AP000892.6 on the migration,invasion and epithelial-mesenchymal transition of bladder cancer cells and the related molecular mechanism.Methods The expression of AP000892.6 in bladder cancer and adjacent tissues was analyzed using GEPIA dataset.The expression levels of AP000892.6 in bladder cancer cell lines 647V,T24,UMUC3,5637,RT4 and normal bladder epithelial cells SV-HUC-1 were detected by real-time quantitative polymerase chain reaction(RT-qPCR).The AP000892.6 overexpression plasmid or negative control plasmid was transfected into T24 cells,and the experiment was divided into AP000892.6 group and NC group.The plasmid transfection efficiency was verified by RT-qPCR.The migration and invasion abilities of transfected T24 cells were detected by cell scratch method and Transwell assay,respectively.The downstream target gene of AP000892.6 was predicted and validated using bioinformatics method and dual-luciferase reporter gene experiment.The expression of AP000892.6downstream target gene was detected by RT-qPCR.Western blot was used to detect the expression of epithelial phenotype proteins E-cadherin,ZO-1 and mesenchymal phenotype proteins Vimentin,N-cadherin and Twist.Results Compared with adjacent tissues,the expression of AP000892.6 was decreased in bladder cancer tissues(P<0.01).Compared with SV-HUC-1 cells,the expression of AP000892.6 was decreased in bladder cancer cell lines(P<0.05),and the relative expression level of AP000892.6 in T24 cells was the lowest(P<0.01).Compared with the NC group,overexpression of AP000892.6 inhibited the migration(P<0.01)and invasive ability(P<0.01)of bladder cancer T24 cells.Dual-luciferase reporter gene analysis proved that miR-616-5p was the target gene of AP000892.6(P<0.01).Compared with the NC group,AP000892.6 negatively regulated the expression of miR-616-5p(P<0.01).Compared with the NC group,the expression of epithelial phenotype proteins E-cadherin and ZO-1 were up-regulated in T24 cells of AP000892.
作者
杨金辉
孙建涛
李小辉
盖强强
郝彤彤
YANG Jinhui;SUN Jiantao;LI Xiaohui(Department of Urology,Luoyang Central Hospital Affiliated to Zhengzhou University,Henan 471000,China)
出处
《医学研究杂志》
2023年第12期26-31,共6页
Journal of Medical Research
基金
国家自然科学基金资助项目(82103284)。
