摘要
目的探讨长链非编码RNA(lncRNA)FGD5反义RNA 1(FGD5-AS1)、miR-512-3p和Ras相关蛋白31(RAB31)在膀胱癌进展中的作用和调控机制。方法收集2019年1月至2021年12月于承德医学院附属医院行手术治疗的60例膀胱癌患者肿瘤组织及癌旁组织,并体外培养膀胱癌细胞系(5637、KU-19-19、T24、UM-UC-3)和正常尿路上皮细胞系(SV-HUC-1)。采用实时定量PCR检测肿瘤组织和癌旁组织以及膀胱癌细胞中FGD5-AS1、miR-512-3p和RAB31 mRNA表达,Pearson相关分析确定膀胱癌患者癌组织中miR-512-3p与FGD5-AS1、RAB31 mRNA表达之间的相关性。将sh-NC、sh-FGD5-AS1、sh-FGD5-AS1和NC抑制剂、sh-FGD5-AS1和miR-512-3p抑制剂转染至T24细胞中,分别记为阴性对照组、FGD5-AS1沉默组、抑制剂对照组、联合组;另设置正常组(不转染)。用CCK-8法检测细胞活力;Transwell小室测定细胞迁移和侵袭能力;Western blotting检测RAB31和E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(vimentin)表达;双萤光素酶报告基因和RNA Pull down实验检测miR-512-3p与FGD5-AS1和RAB31的靶向关系。结果膀胱癌组织与细胞中FGD5-AS1、RAB31 mRNA呈高表达,miR-512-3p呈低表达(P<0.05),且膀胱癌患者癌组织中FGD5-AS1、RAB31 m RNA的表达与mi R-512-3p表达呈负相关,FGD5-AS1与RAB31 mRNA表达呈正相关(r=-0.779、-0.649、0.652,均P<0.001)。沉默FGD5-AS1可上调miR-512-3p表达,下调RAB31 mRNA和蛋白表达,降低细胞活力、迁移和侵袭数以及N-cadherin、vimentin水平,升高E-cadherin水平(P<0.05);敲低miR-512-3p表达可明显减弱沉默FGD5-AS1对膀胱癌T24细胞增殖、迁移和侵袭以及上皮-间质转化(EMT)进程的抑制作用(P<0.05);FGD5-AS1可以海绵化miR-512-3p,而RAB31是miR-512-3p的靶标。结论沉默FGD5-AS1可能通过上调miR-512-3p、下调RAB31表达抑制膀胱癌细胞的增殖、迁移、侵袭和EMT进程。
Objective To investigate the function and regulatory mechanism of the long non-coding RNA(lncRNA)FGD5 antisense RNA 1(FGD5-AS1),miR-512-3p and Ras-related protein 31(RAB31)in the progression of bladder cancer.Methods Sixty pairs of tumor and adjacent tissues were collected from bladder cancer patients who underwent surgical treatment at the Affiliated Hospital of Chengde Medical University between January 2019 and December 2021.Bladder cancer cell lines(5637,KU-19-19,T24,and UM-UC-3)and a normal urothelial cell line(SV-HUC-1)were cultured in vitro.The expression of FGD5-AS1,miR-512-3p,and RAB31 mRNA in tumor tissues,adjacent tissues,and bladder cancer cells was detected using real-time quantitative PCR.Pearson correlation analysis was used to determine the correlation between miR-512-3p and FGD5-AS1,RAB31 mRNA levels in the cancer tissues of patients with bladder cancer.T24 cells were transfected with sh-NC,sh-FGD5-AS1,sh-FGD5-AS1 and NC inhibitors,or sh-FGD5-AS1 and miR-512-3p inhibitors,and labeled as the negative control group,FGD5-AS1 silencing group,inhibitor control group,and combination group,respectively.Another group,which was not transfected was set as normal control.CCK-8 was used to quantify cell viability.A transwell chamber was used to measure cell migration and invasion abilities.Western blotting was performed to detect RAB31,E-cadherin,N-cadherin,and vimentin.Dual luciferase reporter gene and RNA pull-down assays were used to detect the relationship between miR-512-3p,FGD5-AS1,and RAB31.Results FGD5-AS1 and RAB31 mRNA levels were high in bladder cancer tissues and cells,whereas miR-512-3p expression was low(P<0.05).FGD5-AS1 and RAB31 mRNA levels in bladder cancer tissues were negatively correlated with miR-512-3p,and the FGD5-AS1 and RAB31 mRNA levels were positively correlated(r=-0.779,-0.649,and 0.652,respectively;all P<0.001).Silencing FGD5-AS1 could upregulate miR-512-3p,downregulate RAB31 mRNA and protein levels,reduce cell viability,migration,and invasion,decrease N-cadherin and vimentin protein levels,and increase E-cadherin protein(P<0.05).Knocking of miR-512-3p significantly reduced the inhibitory effects of silencing FGD5-AS1 on the proliferation,migration,invasion,and epithelial-mesenchymal transition of T24 cells of bladder cancer(P<0.05).FGD5-AS1 could sponge miR-512-3p,and RAB31 was identified as a target of miR-512-3p.Conclusion FGD5-AS1 silencing inhibits the proliferation,migration,invasion,and epithelial-mesen chymal transition of bladder cancer cells by upregulating miR-512-3p and inhibiting RAB31 expression.
作者
李富博
李爱科
饶井芬
刘宝兴
石方玉
李文鑫
杨春丽
林萍萍
LI Fubo;LI Aike;RAO Jingfen;LIU Baoxing;SHI Fangyu;LI Wenxin;YANG Chunli;LIN Pingping(Department of Oncology,Affiliated Hospital of Chengde Medical College,Chengde 067020,China;Department of General Surgery,Xinglong country People’s Hospital,Chengde 067000,China)
出处
《中国医科大学学报》
北大核心
2026年第1期33-40,共8页
Journal of China Medical University
基金
河北省医学科学课题研究计划(20231360)。
