长链非编码RNA IRAIN对肾癌细胞增殖、迁移和侵袭的影响及其机制

Effect of long-chain non-coding RNA IRAIN on proliferation,migration and invasion of renal cell carcinoma cells and its molecular mechanism

目的:探讨长链非编码RNA(lncRNA)IRAIN对肾癌细胞增殖、迁移和侵袭的影响及其机制。方法:选取2017年7月至2019年7月河南大学淮河医院收治的肾癌组织和癌旁组织作为标本,采用荧光定量聚合酶链反应(RT-PCR)分析肿瘤组织和癌旁组织中lncRNA IRAIN表达水平;构建lncRNA IRAIN过表达慢病毒载体,感染人肾癌细胞株A498细胞为实验组,感染空白质粒为对照组。采用细胞计数试剂盒(CCK-8)、划痕实验和Transwell实验分析两组细胞增殖、迁移和侵袭的影响。采用RNA结合蛋白免疫沉淀实验和蛋白质印迹法(Western blot)分析与lncRNA IRAIN相互作用的蛋白及其表达水平。组间数据比较采用t检验。结果:癌旁组织lncRNA IRAIN表达水平(1.37±0.26)明显高于肾癌组织(0.51±0.17),差异有统计学意义(t=5.907,P<0.05)。对照组细胞吸光度(A)值(2.18±0.34)明显高于实验组(1.29±0.21),差异有统计学意义(t=4.017,P<0.05)。对照组细胞划痕愈合率[(89.38±7.82)%]明显高于实验组[(51.38±6.95)%],差异有统计学意义(t=5.115,P<0.05)。对照组细胞迁移数量(109.37±9.37)明显高于实验组(60.18±5.89),差异有统计学意义(t=4.202,P<0.05)。lncRNA IRAIN与Zeste增强子同源物2(EZH2)蛋白可能存在相互作用。对照组细胞p15和p21蛋白相对表达水平(1.15±0.18、1.30±0.20)明显高于实验组(0.52±0.15、0.67±0.21),差异有统计学意义(t=3.901、3.729,P<0.05)。结论:lncRNA IRAIN在肾癌组织中呈低表达,高表达lncRNA IRAIN可通过结合EZH2显著抑制肾癌细胞的增殖、迁移和侵袭等恶性细胞行为。

Objective To investigate the effect of long-chain non-coding RNA(lncRNA)IRAIN on proliferation,migration and invasion of renal cell carcinoma(RCC)cells and its molecular mechanism.Methods RCC tissues and adjacent tissues in Huaihe Hospital of Henan University and Clinical Medical College of Henan University from July 2017 to July 2019 were selected as samples,and the expression level of lncRNA IRAIN in tumor tissues and adjacent tissues was analyzed by fluorescence quantitative polymerase chain reaction(RT-PCR).The lentiviral vector of lncRNA IRAIN overexpression was constructed.Human RCC cell line A498 was infected as experimental group,and blank plasmid was infected as control group.Cell proliferation,migration and invasion were analyzed by cell counting kit-8(CCK-8)assay,scratch test and Transwell test in the control group and experimental group.The protein interacting with lncRNA IRAIN and its target protein expression levels were analyzed by RNA binding protein immunoprecipitation assay and Western blotting.The data were compared between groups by t-test.Results The expression level of lncRNA IRAIN in the RCC tissues(1.37±0.26)was significantly higher than that in adjacent tissues(0.51±0.17,t=5.907,P<0.05).The absorbance(A)value of the control group(2.18±0.34)was significantly higher than that of the experimental group(1.29±0.21,t=4.017,P<0.05).The wound healing rate in the control group[(89.38±7.82)%]was significantly higher than that in the experimental group[(51.38±6.95)%,t=5.115,P<0.05].The number of migrating cells in the control group(109.37±9.37)was significantly greater than that in the experimental group(60.18±5.89,t=4.202,P<0.05).LncRNA IRAIN may interact with enhancer of zeste homolog 2(EZH2)protein.The relative expression levels of p15 and p21 proteins in the control group(1.15±0.18,1.30±0.20)was significantly higher than those in the experimental group(0.52±0.15,0.67±0.21,t=3.901,3.729,P<0.05).Conclusion LncRNA IRAIN has low expression in RCC.High expression of lncRNA IRAIN can significantly inhibit the proliferation,migration and invasion of RCC cells by binding EZH2.