长链非编码RNA CCAT1对膀胱癌细胞增殖、迁移和耐药的影响及其机制

Effects of long non-coding RNA CCAT1 on proliferation,migration and drug resistance of bladder cancer cells and its molecular mechanism

目的探讨长链非编码RNA(lncRNA) CCAT1对膀胱癌细胞增殖、迁移和耐药的影响及其机制。方法选取2019年6月到2021年6月新乡市中心医院收治的71例膀胱癌标本作为研究对象, 采用荧光定量聚合酶链反应(PCR)分析肿瘤组织和癌旁组织lncRNA CCAT1表达水平。采用浓度梯度递增法建立顺铂耐药细胞株T24/顺铂(DDP), 分别采用对照lncRNA和lncRNA CCAT1敲降慢病毒感染T24和T24/DDP细胞系, 分别为T24/对照组, T24/CCAT1 KD组, T24/DDP对照组和T24/DDP CCAT1 KD组。采用细胞计数试剂盒(CCK-8)分析细胞的增殖能力;采用划痕实验和Transwell实验分析细胞的迁移能力;采用流式细胞术分析细胞的耐药能力。采用生物信息学和双荧光素酶报告基因分析lncRNA CCAT1靶基因, 采用荧光定量PCR分析靶基因表达水平, 组间比较采用t检验。结果癌旁组织中lncRNA CCAT1表达水平(1.03±0.18)明显低于膀胱癌组织(2.78±0.48), 差异有统计学意义(t=28.341, P<0.05)。T24-对照组细胞24、48、72 h吸光度值(0.92±0.03、1.49±0.06、2.00±0.03)明显高于T24-CCAT1 KD组(0.70±0.06、1.17±0.04、1.56±0.10), 差异均有统计学意义(F=3.108, P<0.05)。T24-对照组细胞划痕愈合率[(87.75±3.50)%]明显高于T24-CCAT1 KD组[(1.03±0.18)%], 差异均有统计学意义(t=8.555, P<0.05)。T24-对照组细胞迁移数量[(123.25±17.08)个]明显高于T24-CCAT1 KD组[(90.75±11.32)个], 差异均有统计学意义(t=3.172, P<0.05)。T24/DDP对照组经顺铂处理后24、48、72 h吸光度值(0.73±0.09、1.27±0.10、1.84±0.12)明显高于T24/DDP CCAT1 KD组细胞(0.52±0.03、0.91±0.03、1.36±0.04), 差异均有统计学意义(F=4.094, P<0.05)。双荧光素酶报告基因显示lncRNA CCAT1是微小RNA(miR)-490-3p的海绵。T24/对照组细胞miR-490-3p表达水平(1.21±0.10)明显低于T24/CCAT1 KD组细胞(2.30±0.20), 差异有统计学意义(t=9.699, P<0.05)。结论 lncRNA CCAT1在膀胱癌中呈高表达, 通过miR-490-3p调节着膀胱癌细胞的增殖、迁移和耐药等过程。

Objective To investigate the effect and molecular mechanism of long non-coding RNA(lncRNA)CCAT1 on proliferation,migration and drug resistance of bladder cancer cells.Methods A total of 71 cases of bladder cancer in Xinxiang Central Hospital from June 2019 to June 2021 were selected as the research objects.The expression level of lncRNA CCAT1 in tumor tissues and adjacent tissues was analyzed by fluorescence quantitative polymerase chain reaction(PCR).Cisplatin resistant cell line T24/cisplatin(DDP)was established by concentration gradient method.ControlⅠlncRNA and lncRNA CCAT1 knockdown lentiviruses were used to infect T24 and T24/DDP cell lines,which served as T24/control group,T24/CCAT1 KD group,T24/DDP control group and T24/DDP CCAT1 KD group,respectively.The proliferation ability of cells was analyzed by cell counting kit-8(CCK-8)assay.The migration ability of cells was analyzed by scratch test and Transwell test.The drug resistance of cells was analyzed by flow cytometry.The target gene of lncRNA CCAT1 was analyzed by bioinformatics and double luciferase reporter gene,and the expression level of target gene was analyzed by fluorescence quantitative PCR.The measurement data were compared by t-test,Results The expression level of lncRNA CCAT1 in adjacent tissues(1.03±0.18)was significantly lower than that in bladder cancer tissues(2.78±0.48,t=28.341,P<0.05).The absorbance values at 24,48 and 72 h in T24/control group(0.92±0.03,1.49±0.06,2.00±0.03)were significantly higher than those in T24/CCAT1 KD group(0.70±0.06,1.17±0.04,1.56±0.10,F=3.108,P<0.05).The cell scratch healing rate in T24/control group[(87.75±3.50)%]was significantly higher than that in T24/CCAT1 KD group[(1.03±0.18)%,t=8.555,P<0.05].The number of migrating cells in T24/control group(123.25±17.08)was significantly greater than that in T24/CCAT1 KD group(90.75±11.32,t=3.172,P<0.05).The absorbance values in T24/DDP control group at 24,48 and 72 h after cisplatin treatment(0.73±0.09,1.27±0.10,1.84±0.12)were significantly higher than those in T24/DDP CCAT1 KD group(0.52±0.03,0.91±0.03,1.36±0.04,F=4.094,P<0.05).The double luciferase reporter gene showed that lncRNA CCAT1 was a sponge of microRNA(miR)-490-3p.The expression level of miR-490-3p in T24/control group(1.21±0.10)was significantly lower than that in T24/CCAT1 KD group(2.30±0.20,t=9.699,P<0.05).Conclusion LncRNA CCAT1 is highly expressed in bladder cancer.MiR-490-3p regulates the proliferation,migration and drug resistance of bladder cancer cells.