lncRNA ARHGAP5-AS1靶向调控miR-155-5p抑制肾癌细胞增殖和侵袭

lncRNA ARHGAP5-AS1 inhibits the proliferation and invasion of renal cancer cells by targeting and regulating miR-155-5p

目的探讨lncRNA ARHGAP5-AS1在肾癌组织和细胞株中的表达及其对肾癌细胞株增殖和侵袭的影响及其分子机制。方法采用GEPIA数据库分析肾癌组织中ARHGAP5-AS1的表达,并分析其与肾癌患者临床分期、总生存期和无病生存期的关系。采用实时荧光定量聚合酶链反应(RT-qPCR)法检测肾癌细胞(786-O、Caki-1、OS-RC-2、ACHN、A-498)中ARHGAP5-AS1的表达水平。向肾癌OS-RC-2细胞中转染pcDNA3.1-ARHGAP5-AS1质粒或pcDNA3.1质粒,记为ARHGAP5-AS1组和对照组。采用集落形成实验和Transwell实验检测OS-RC-2细胞增殖和侵袭能力的改变。采用双荧光素酶报告基因实验验证ARHGAP5-AS1和miR-155-5p的靶向关系。采用Starbase v3.0在线数据库分析ARHGAP5-AS1和miR-155-5p在肾癌组织中表达的相关性。采用RT-qPCR法检测miR-155-5p表达水平的变化。Western blotting法检测Raf/MEK/ERK分子通路蛋白p-Raf、p-MEK、p-ERK、p-FBW7、c-MYC的表达变化。计量资料以均数±标准差(±s)表示,两组间比较采用独立样本t检验,多组间比较采用单因素方差分析。结果ARHGAP5-AS1在肾癌组织中呈低表达(P<0.01),其表达水平与肾癌患者临床分期、总生存期和无病生存期均有关(P<0.01)。ARHGAP5-AS1在肾癌细胞株(786-O、Caki-1、OS-RC-2、ACHN、A-498)中均呈低表达(P<0.01)。与对照组相比,ARHGAP5-AS1组OS-RC-2细胞的增殖能力和侵袭能力均显著降低(P<0.01)。双荧光素酶报告基因实验证实ARHGAP5-AS1靶向结合miR-155-5p(P<0.01)。ARHGAP5-AS1与miR-155-5p在肾癌组织中的表达呈负相关(P<0.01)。与对照组相比,ARHGAP5-AS1组OS-RC-2细胞中miR-155-5p表达显著降低(P<0.01)。与对照组相比,ARHGAP5-AS1组OS-RC-2细胞中Raf/MEK/ERK分子通路蛋白p-Raf、p-MEK、p-ERK、p-FBW7、c-MYC表达水平降低。结论lncRNA ARHGAP5-AS1在肾癌组织中低表达并与肾癌患者临床分期和生存期相关,ARHGAP5-AS1靶向调控miR-155-5p表达抑制肾癌细胞的增殖和侵袭。

ObjectiveTo explore the expression of lncRNA ARHGAP5-AS1 in renal cancer tissues and cell lines,and the effect of ARHGAP5-AS1 on the proliferation and invasion of renal cancer cell lines and its molecular mechanism.MethodsThe GEPIA database was used to analyze the expression of ARHGAP5-AS1 in renal cancer tissues,and its relationship with clinical stage,overall survival and disease-free survival of renal cancer patients was analyzed.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression level of ARHGAP5-AS1 in renal cancer cells(786-O,Caki-1,OS-RC-2,ACHN,A-498).Renal carcinoma OS-RC-2 cells were transfected with pcDNA3.1-ARHGAP5-AS1 plasmid or pcDNA3.1 plasmid,denoted as ARHGAP5-AS1 group and control group.Colony formation assay and Transwell assay were used to detect changes in the proliferation and invasion ability of OS-RC-2 cells.Dual-luciferase reporter gene experiment was used to verify the targeting relationship between ARHGAP5-AS1 and miR-155-5p.The Starbase v3.0 online database was used to analyze the correlation between the expression of ARHGAP5-AS1 and miR-155-5p in renal cancer tissues.RT-qPCR was used to detect the expression level changes of miR-155-5p.Western blotting was used to detect the expression changes of Raf/MEK/ERK molecular pathway proteins p-Raf,p-MEK,p-ERK,p-FBW7,and c-MYC.The measurement data were expressed as mean±standard deviation(±s),the independent sample t-test was used for comparison between two groups,and one-way analysis of variance was used for comparison between multiple groups.ResultsARHGAP5-AS1 was lowly expressed in renal cancer tissues(P<0.01),and its expression level was related to the clinical stage,overall survival and disease-free survival of patients with renal cancer(P<0.01).ARHGAP5-AS1 showed low expression in renal cancer cell lines(786-O,Caki-1,OS-RC-2,ACHN,A-498)(P<0.01).Compared with the control group,the proliferation and invasion abilities of OS-RC-2 cells in ARHGAP5-AS1 group were significantly reduced(P<0.01).Dual-luciferase reporter gene experiment confirmed that ARHGAP5-AS1 targets and binds to miR-155-5p(P<0.01).The expression of ARHGAP5-AS1 and miR-155-5p in renal cancer tissues was negatively correlated(P<0.01).Compared with the control group,the expression of miR-155-5p in OS-RC-2 cells in the ARHGAP5-AS1 group was significantly reduced(P<0.01).Compared with the control group,the expression levels of Raf/MEK/ERK molecular pathway proteins p-Raf,p-MEK,p-ERK,p-FBW7,and c-MYC in OS-RC-2 cells in the ARHGAP5-AS1 group were reduced.ConclusionslncRNA ARHGAP5-AS1 is lowly expressed in renal cancer tissues and is related to the clinical stage and survival of renal cancer patients.ARHGAP5-AS1 inhibits the proliferation and invasion of renal cancer cells by targeting the expression of miR-155-5p.