长链非编码RNA MINCR靶向微小RNA-876-5p调控膀胱癌细胞生物学行为的分子机制研究  

Molecular mechanism of long non-coding RNA MINCR regulating bladder cancer cell biological behavior by targeting microRNA-876-5p

作　　者：徐林飞[1] 张海涛[1] 刘晟[1] 蔡海荣[1] 胡恩平[1] Xu Linfei;Zhang Haitao;Liu Sheng;Cai Hairong;Hu Enping(Department of Urology Surgery,Taizhou Municipal Hospital,Zhejiang Province,Taizhou 318099,China)

机构地区：[1]浙江省台州市立医院泌尿外科,318099

出　　处：《中国医药》2021年第4期613-617,共5页China Medicine

基　　金：浙江省自然科学基金(LY12H05009);浙江省台州市科学技术局项目(20ywa33)。

摘　　要：目的探讨长链非编码RNA(LncRNA)MINCR靶向微小RNA(miR)-876-5p调控膀胱癌细胞增殖、迁移、侵袭及凋亡的分子机制。方法体外培养人正常膀胱上皮细胞SV-HUC-1与膀胱癌细胞系T24、RT4、UM-UC-3,采用反转录与荧光定量聚合酶链反应法检测LncRNA MINCR和miR-876-5p的表达量;将si-NC、si-MINCR、miR-NC、miR-876-5p mimics、anti-miR-NC、anti-miR-876-5p、si-MINCR+anti-miR-NC、si-MINCR+anti-miR-876-5p转染入T24细胞,分别记为si-NC组、si-MINCR组、miR-NC组、miR-876-5p组、anti-miR-NC组、anti-miR-876-5p组、si-MINCR+anti-miR-NC组、si-MINCR+anti-miR-876-5p组;采用噻唑蓝法检测细胞活力;采用Transwell小室实验检测细胞迁移及侵袭能力;采用流式细胞术检测细胞凋亡率;双荧光素酶报告基因实验检测LncRNA MINCR与miR-876-5p的靶向关系;蛋白质印迹法检测细胞周期蛋白D1(Cyclin D1)、基质金属蛋白酶(MMP)2、MMP-9、天冬氨酸蛋白水解酶3(Cleaved-caspase-3)蛋白表达量。结果与SV-HUC-1细胞比较,膀胱癌细胞系T24、RT4、UM-UC-3中LncRNA MINCR的表达水平升高[(2.25±0.20)、(1.84±0.18)、(1.61±0.12)比(1.00±0.10)],miR-876-5p的表达水平降低[(0.38±0.03)、(0.46±0.04)、(0.51±0.05)比(1.02±0.12)](均P<0.05)。与miR-NC组比较,miR-876-5p组细胞活力降低,迁移及侵袭细胞数减少,凋亡率升高,Cyclin D1、MMP-2、MMP-9蛋白水平降低,Cleaved-caspase-3蛋白水平升高(均P<0.05)。双荧光素酶报告实验证实LncRNA MINCR能够靶向结合miR-876-5p。与si-MINCR+anti-miR-NC组比较,si-MINCR+anti-miR-876-5p组细胞活力升高,迁移及侵袭细胞数增多,凋亡率降低,Cyclin D1、MMP-2、MMP-9蛋白水平升高,Cleaved-caspase-3蛋白水平降低(均P<0.05)。结论抑制LncRNA MINCR表达可减弱膀胱癌细胞增殖、迁移及侵袭能力,并诱导细胞凋亡,其作用机制与靶向调控miR-876-5p的表达有关。Objective To explore the molecular mechanism of long non-coding RNA(LncRNA)MINCR targeting microRNA(miR)-876-5p to regulate the proliferation,migration,invasion and apoptosis of bladder cancer cells.Methods Human normal bladder epithelial cells SV-HUC-1 and bladder cancer cell lines T24,RT4,UM-UC-3 were cultured in vitro.The reverse transcription and quantitative real-time polymerase chain reaction method was used to detect the expression of LncRNA MINCR and miR-876-5p.si-NC,si-MINCR,miR-NC,miR-876-5p mimics,anti-miR-NC,anti-miR-876-5p,si-MINCR+anti-miR-NC,si-MINCR+anti-miR-876-5p were transfected into T24 cells,and they were divided into si-NC group,si-MINCR group,miR-NC group,miR-876-5p group,anti-miR-NC group,anti-miR-876-5p group,si-MINCR+anti-miR-NC group,si-MINCR+anti-miR-876-5p group.The methyl thiazolyl tetrazolium method was used to detect cells viability.Transwell chamber experiment was used to detect cells migration and invasion ability.Flow cytometry was used to detect the apoptosis rate.The dual luciferase reporter experiment was used to detect the targeting relationship between LncRNA MINCR and miR-876-5p.Western blotting was used to detect the expression of Cyclin D1,matrix metalloproteinase(MMP)-2,MMP-9,and Cleaved-caspase-3 protein.Results Compared with SV-HUC-1 cells,the expression level of LncRNA MINCR in bladder cancer cell lines T24,RT4,UM-UC-3 increased[(2.25±0.20),(1.84±0.18),(1.61±0.12)vs(1.00±0.10)],and the expression level of miR-876-5p decreased[(0.38±0.03),(0.46±0.04),(0.51±0.05)vs(1.02±0.12)](all P<0.05).Compared with miR-NC group,the cells viability in miR-876-5p group decreased,the number of migration and invasion cells decreased,the apoptosis rate increased,the protein levels of CyclinD 1,MMP-2,and MMP-9 decreased,and the protein level of Cleavedcaspase-3 increased(all P<0.05).The dual luciferase report experiment confirmed that LncRNA MINCR could target miR-876-5p.Compared with si-MINCR+anti-miR-NC group,the cell viability in si-MINCR+anti-miR-876-5p group increased,the nu

关 键 词：膀胱癌 长链非编码RNA MINCR 微小RNA-876-5p 增殖 迁移 侵袭 凋亡 

分 类 号：R737.14[医药卫生—肿瘤]