长链非编码RNA NEAT1调控微小RNA-506/组蛋白-赖氨酸N-甲基转移酶对肾癌786-O细胞生物学行为的影响

Effect of long non-coding RNA nuclear paraspeckle assembly transcript 1 regulating miR-506/EZH2 on biological behavior of renal cell carcinoma 786-O cells

目的:探讨长链非编码RNA NEAT1(lncRNA NEAT1)在肾癌中的表达,以及在肾癌786-O细胞中对增殖、凋亡、迁移和侵袭的作用,以及对微小RNA-506(microRNA-506,miR-506)/组蛋白-赖氨酸N-甲基转移酶(EZH2)信号通路的影响。方法:选取2018年1月至2019年12月武汉大学人民医院收集的38例肾癌组织及癌旁组织样本作为研究对象,采用实时定量反转录聚合酶链反应(RT-qPCR)法检测肾癌组织及相应癌旁正常组织临床样本中lncRNA NEAT1的表达水平;使用在线数据库及采用荧光素酶实验验证NEAT1和miR-506以及EZH2和miR-506的靶向关系;采用噻唑蓝(MTT)法,双染法流式细胞术和细胞迁移侵袭(Transwell)实验观察细胞增殖、凋亡、迁移和侵袭水平的改变;采用RT-qPCR和蛋白质印迹法(Western blot)法分别测miR-506和EZH2蛋白的表达。组间比较采用t检验。结果:lncRNA NEAT1在肾癌组织中表达水平(4.45±0.31)高于癌旁组织(2.39±0.25),差异有统计学意义(t=8.959,P<0.05)。荧光素酶报告实验结果表明NEAT1序列上有miR-506的结合位点,且EZH2是miR-506的下游靶基因。si-NEAT1组24、48和72 h增殖率[(24.67±3.23)%、(40.82±5.03)%、(56.87±6.12)%]明显低于si-NC组[(62.63±4.35)%、(74.18±6.55)%、(87.45±7.21)%],差异有统计学意义(t=-12.135、-6.997、-5.601,P<0.01)。si-NEAT1组凋亡水平[(23.20±1.92)%]显著高于si-NC组[(9.65±1.34)%],差异有统计学意义(t=10.024,P<0.05)。si-NEAT1组迁移和侵袭数量[(52.35±4.76)、(21.63±2.05)个]显著低于si-NC组[(91.23±7.59)、(42.68±3.98)个],差异有统计学意义(t=-7.517,P<0.05;t=-8.144,P<0.05)。RT-qPCR结果显示,si-NC及si-NEAT1两组的miR-506 mRNA表达量分别为1.00±0.00、3.39±0.26,差异有统计学意义(t=15.922,P<0.05)。si-NEAT1组EZH2蛋白表达水平(0.15±0.02)明显低于si-NC组EZH2蛋白表达水平(0.32±0.03),差异有统计学意义(t=-8.167,P<0.05)。结论:LncRNA NEAT1在肾癌中高表达,其可能通过调控miR-506/EZH2信号途径改变肾癌786-O细胞增殖、凋亡、迁移和侵袭的能力。

Objective To investigate the expression of long non-coding RNA(lncRNA)nuclear paraspeckle assembly transcript 1(NEAT1)in renal cell carcinoma tissues,and to observe the roles of lncRNA NEAT1 in the proliferation,apoptosis,migration and invasion in renal cancer A549 cells and the influence on miR-506/EZH2 signaling pathway.Methods From January 2018 to December 2019,a total of 38 samples of renal cancer tissue and the adjacent normal tissue were collected from the Renmin Hospital of Wuhan University.Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to detect the expression of lncRNA NEAT1 in renal cancer tissues and adjacent normal tissues.Online database and luciferase assay were used to verify the targeting relationship between NEAT1 and miR-506,EZH2 and miR-506.Methyl thiazolyl tetrazolium(MTT),Annexin V-fluoresceine isothiocyanate(FITC)/propidium iodide(PI)and Transwell methods were used to detect the changes in the levels of proliferation,apoptosis,migration and invasion.The expression levels of miR-506 and EZH2 mRNA and protein were detected by RT-qPCR and Western blotting,respectively.T test was used for comparison between groups.Results Compared with the expression level of lncRNA NEAT1 in adjacent normal tissues(2.39±0.25),the expression level of lncRNA NEAT1 in renal cancer tissues was significantly increased(4.45±0.31,t=8.959,P<0.05).Luciferase reporter assay showed that there was a miR-506 binding site in NEAT1 sequence,and EZH2 was the downstream target gene of miR-506.Compared with the si-NC group[(62.63±4.35)%,(74.18±6.55)%,(87.45±7.21)%],the proliferation rate in si-NEAT1 group at 24 h,48 h and 72 h[(24.67±3.23)%,(40.82±5.03)%,(56.87±6.12)%]was significantly decreased(t=-12.135,-6.997,-5.601,P<0.05).Compared with si-NC group[(9.65±1.34)%],the apoptosis rate in si-NEAT1 group[(23.20±1.92)%]was significantly increased(t=10.024,P<0.05).Compared with si-NC group(91.23±7.59)and(42.68±3.98),the number of migrating cells and invasive cells in si-NEAT1 group was(52.35±4.76)and(21.63±2.05)with the difference being statistically significant(t=-7.517,P<0.05;t=-8.144,P<0.05).The qRT-PCR showed that the expression of miR-506 mRNA in si-NC and si-NEAT1 groups was(1.00±0.00)and(3.39±0.26)respectively(t=15.922,P<0.05).Compared with si-NC group(0.32±0.03),the expression level of EZH2 in si-NEAT1 group was significantly decreased(0.15±0.02)(t=-8.167,P<0.05).Conclusion LncRNA NEAT1 has a higher expression level in renal cell carcinoma tissues,and it may play an important role in changing the level of cell proliferation,apoptosis,migration and invasion of 786-O cells by regulating the signal pathway of miR-506/EZH2.