长链非编码RNA FMO4-AS5调控miRNA-620表达对肾癌细胞增殖能力、细胞周期和免疫逃逸的影响  

Effects of long non-coding RNA FMO4-AS5 regulating miRNA-620 expression on the proliferation ability,cell cycle and immune escape of renal cancer cells

作　　者：余登祥 李贤龙 柳永 陈小刚 刘飞[3] 许浩 Yu Dengxiang;Li Xianlong;Liu Yong;Chen Xiaogang;Liu Fei;Xu Hao(Department of Urology,Huangshi Central Hospital,Affiliated Hospital of Hubei Polytechnic University,Huangshi 435000,China;Key Laboratory of Kidney Disease Occurrence and Intervention of Hubei Province,Huangshi 435000,China;Department of Urology,The Second Affiliated Hospital of Nanchang University,Nanchang 330008,China)

机构地区：[1]黄石市中心医院(湖北理工学院附属医院)泌尿外科,黄石435000 [2]肾脏疾病发生与干预湖北省重点实验室,黄石435000 [3]南昌大学第二附属医院泌尿外科,南昌330008

出　　处：《肿瘤研究与临床》2024年第4期262-267,共6页Cancer Research and Clinic

基　　金：国家自然科学基金(81760459)。

摘　　要：目的探讨长链非编码RNA FMO4-AS5调控miRNA-620(miR-620)表达对肾癌细胞增殖能力、细胞周期和免疫逃逸的影响。方法通过cBioPortal在线数据库(数据更新至2022年1月)分析FMO4-AS5在肾癌组织(323例)和正常肾组织(153例)中的表达水平。采用LncBook预测软件分析FMO4-AS5直接靶向结合的微小RNA(miRNA)。实时荧光定量聚合酶链反应(qRT-PCR)检测正常肾小管上皮细胞HK-2和肾癌细胞株ACHN、CAKI1、786-P、A498中FMO4-AS5的相对表达水平,选择FMO4-AS5相对表达水平最高的786-P细胞进行后续实验。将786-P细胞分为对照组和si-FMO4-AS5组,分别转染0.4μg pcDNA-si-NC质粒和0.4μg pcDNA-si-FMO4-AS5质粒。采用平板克隆实验检测两组786-P细胞的增殖能力,流式细胞术检测786-P细胞周期,酶联免疫吸附试验(ELISA)检测白细胞介素1β(IL-1β)、干扰素γ(IFN-γ)、单核细胞趋化蛋白1(MCP-1)水平。采用双荧光素酶报告基因实验验证FMO4-AS5和miR-620的靶向关系。蛋白质印迹法检测786-P细胞中JAK1-STAT3信号通路相关蛋白和细胞周期蛋白CDK9、cyclin T1的表达。结果cBioPortal在线数据库中,肾癌组织中FMO4-AS5相对表达水平高于正常肾脏组织(P<0.01)。肾癌细胞株ACHN、CAKI1、786-P、A498中FMO4-AS5相对表达水平分别为2.79±0.30、4.47±0.41、7.26±0.52、3.65±0.74,均较肾小管上皮细胞HK-2细胞高(1.03±0.22)(均P<0.01)。对照组和si-FMO4-AS5组细胞克隆形成数分别为(226±17)个和(54±16)个,si-FMO4-AS5组细胞克隆形成数低于对照组(t=7.15,P<0.01)。与对照组比较,si-FMO4-AS5组G0/G1期细胞比例增高(t=5.25,P<0.01),G2/M期、S期细胞比例均降低(t=3.32,P<0.05;t=5.35,P<0.01)。对照组和si-FMO4-AS5组MCP-1分别为(2.01±0.17)pg/ml和(5.54±0.52)pg/ml,IFN-γ分别为(1.51±0.49)pg/ml和(3.71±0.29)pg/ml,IL-1β分别为(1.28±0.19)pg/ml和(3.21±0.38)pg/ml,si-FMO4-AS5组MCP-1、IFN-γ、IL-1β水平均较对照组增高(均P<0.01)。双荧光素酶报告基因实验�Objective To explore the effect of long non-coding RNA FMO4-AS5 regulating the expression of miRNA-620(miR-620)on the proliferation ability,cell cycle and immune escape of renal cancer cells.Methods The expression levels of FMO4-AS5 in renal cancer tissues(323 cases)and normal renal tissues(153 cases)were analyzed by cBioPortal online database(data was updated to January 2022).LncBook prediction software was used to analyze the microRNA(miRNA)directly targeted by FMO4-AS5.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect the relative expression levels of FMO4-AS5 in normal renal tubular epithelial cells HK-2 and renal cancer cell lines ACHN,CAKI1,786-P,and A498.The 786-P cells with the highest relative expression level of FMO4-AS5 were selected for subsequent experiments.The 786-P cells were divided into the control group and si-FMO4-AS5 group,which were transfected with 0.4μg pcDNA-si-NC plasmid and 0.4μg pcDNA-si-FMO4-AS5 plasmid,respectively.Plate cloning assay was used to detect the proliferation ability of 786-P cells in the two groups,flow cytometry was used to detect the cell cycle of 786-P cells,enzyme-linked immunosorbent assay(ELISA)was used to detect interleukin 1β(IL-1β),interferonγ(IFN-γ),and monocyte chemotaxis protein 1(MCP-1)level.The dual luciferase reporter gene assay was used to verify the targeting relationship between FMO4-AS5 and miR-620.The expressions of JAK1-STAT3 signaling pathway-related proteins and cell cycle proteins CDK9 and cyclin T1 in 786-P cells were detected by using Western blotting.Results In the cBioPortal online database,the relative expression level of FMO4-AS5 in renal cancer tissues was higher than that in normal renal tissues(P<0.01).The relative expression levels of FMO4-AS5 in renal cancer cell lines ACHN,CAKI1,786-P,and A498 were 2.79±0.30,4.47±0.41,7.26±0.52,and 3.65±0.74,respectively,which were all higher than renal tubular epithelial cells HK-2 cells(1.03±0.22)(all P<0.01).The number of cell clone in the control gr

关 键 词：肾肿瘤 微RNAS 长链非编码RNA 细胞增殖 免疫逃逸 

分 类 号：R737.11[医药卫生—肿瘤]