摘要
目的探讨长链非编码RNA氧化应激反应丝氨酸丰富1反义RNA 1(lncRNA OSER1-AS1)对肾癌ACHN细胞的增殖、迁移、侵袭的影响及其对微小RNA(microRNA,miR)-612的调控作用。方法2017年1月至2020年3月,采用实时定量反转录聚合酶链反应(RT-qPCR)法检测肾癌组织、癌旁组织中OSER1-AS1、miR-612的表达量;体外培养肾癌细胞ACHN,分别将OSER1-AS1小分子干扰RNA(si-OSER1-AS1)及其阴性对照(si-NC)、miR-612寡核苷酸模拟物(miR-612 mimics)及阴性对照mimic NC序列(miR-NC)、si-OSER1-AS1与miR-612特异性寡核苷酸抑制剂的阴性对照(anti-miR-NC)、si-OSER1-AS1与miR-612特异性寡核苷酸抑制剂(anti-miR-612)转染至ACHN细胞;采用RT-qPCR法检测细胞中OSER1-AS1、miR-612的表达量;采用噻唑蓝(MTT)、Transwell小室实验分别检测细胞增殖、迁移及侵袭能力;双荧光素酶报告实验检测OSER1-AS1、miR-612的靶向关系;蛋白质印迹法(Western blot)检测细胞周期蛋白1(Cyclin D1)、基质金属蛋白酶(MMP)-2、MMP-9、p21蛋白表达量。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。结果肾癌组织OSER1-AS1的表达水平(1.00±0.08比3.37±0.28)高于癌旁组织(t=52.113,P<0.05),miR-612的表达水平(1.00±0.06比0.47±0.04)低于癌旁组织(t=47.062,P<0.05);si-OSER1-AS1组细胞活力(0.66±0.05比0.31±0.03)与Cyclin D1(0.63±0.05比0.25±0.02)、MMP-2(0.82±0.07比0.35±0.03)、MMP-9(0.76±0.06比0.28±0.03)蛋白水平低于si-NC组(t=18.007、21.169、18.514、21.466,P<0.05),si-OSER1-AS1组迁移细胞数[(115.56±9.52)个比(55.99±5.59)个]、侵袭细胞数[(93.41±6.09)个比(46.05±4.35)个]低于si-NC组(t=16.188、18.984,P<0.05),si-OSER1-AS1组p21蛋白水平(0.15±0.02比0.57±0.05)高于si-NC组(t=23.398,P<0.05);miR-612组细胞活力(0.68±0.05比0.39±0.03)与Cyclin D1(0.66±0.05比0.28±0.03)、MMP-2(0.85±0.06比0.46±0.03)、MMP-9(0.78±0.05比0.34±0.02)蛋白水平低于miR-NC组(t=14.920、19.551、17.441、24.512,P<0.05),miR-612组迁移细胞数[(118.76±9.87)个比(64.39±4.65)个]、侵袭细胞数[(99.65±9.12)个比(53.57±3.67)个]低于miR-NC组(t=14.950、14.062,P<0.05),miR-612组p21蛋白水平(0.14±0.02比0.51±0.04)高于miR-NC组(t=24.820,P<0.05);双荧光素酶报告实验证实OSER1-AS1可靶向结合miR-612;抑制miR-612表达可明显逆转干扰OSER1-AS1对细胞增殖、迁移及侵袭的作用。结论干扰OSER1-AS1可通过上调miR-612的表达从而抑制肾癌ACHN细胞增殖、迁移及侵袭。
Objective To explore the effect of long non-coding RNA(lncRNA)oxidative stress responsive serine rich 1 antisense RNA 1(OSER1-AS1)on proliferation,migration and invasion of renal cancer ACHN cells and its regulation on microRNA(miRNA,miR)-612.Methods August 2017 to March 2020.The expression levels of OSER1-AS1 and miR-612 in renal cancer tissue and paracancerous tissue were detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)method.ACHN cells were cultured in vitro,the OSER1-AS1 small interfering RNA(si-OSER1-AS1)and its negative control(si-NC),miR-612 oligonucleotide mimics(miR-612 mimics)and the negative control mimic NC sequence(miR-NC),negative control of si-OSER1-AS1 and miR-612 specific oligonucleotide inhibitor(anti-miR-NC),si-OSER1-AS1 and miR-612 specific oligonucleotide inhibitor(anti-miR-612)was transfected into ACHN cells..RT-qPCR method was used to detect the expression levels of OSER1-AS1 and miR-612 in cells.Methyl thiazolyl tetrazolium(MTT)and Transwell chamber experiments were used to detect cell proliferation,migration and invasion ability.The dual luciferase report experiment was used to detect the targeting relationship of OSER1-AS1 and miR-612.Western blotting was used to detect the expression of Cyclin D1,matrix metalloproteinase(MMP)-2,MMP-9 and p21 proteins.The independent sample t test was used for comparison between two groups,and single-factor analysis of variance was used for comparison between multiple groups.Results The expression level of OSER1-AS1 in renal cancer tissue(1.00±0.08 vs.3.37±0.28)was higher than that in adjacent tissues(t=52.113,P<0.05),and that of miR-612(1.00±0.06 vs.0.47±0.04)lower than in the adjacent tissues(t=47.062,P<0.05).The cell viability in si-OSER1-AS1 group(0.66±0.05 vs.0.31±0.03)and Cyclin D1(0.63±0.05 vs.0.25±0.02),MMP-2(0.82±0.07 vs.0.35±0.03),MMP-9(0.76±0.06 vs.0.28±0.03)protein level were lower than in si-NC group(t=18.007,21.169,18.514,21.466,P<0.05).In si-OSER1-AS1 group the number of migrating cells(115.56±9.52 vs.55.99±5.59),and of invasive cells(93.41±6.09 vs.46.05±4.35)was lower than that in the si-NC group(t=16.188,18.984,P<0.05).In si-OSER1-AS1 group the p21 protein level(0.15±0.02 vs.0.57±0.05)was higher than that in si-NC group(t=23.398,P<0.05).Cell viability in miR-612 group(0.68±0.05 vs.0.39±0.03)and the levels of Cyclin D1(0.66±0.05 vs.0.28±0.03),MMP-2(0.85±0.06 vs.0.46±0.03)],MMP-9(0.78±0.05 vs.0.34±0.02)protein were lower than those in miR-NC group(t=14.920,19.551,17.441,24.512,P<0.05).In miR-612 group the number of migrating cells(118.76±9.87 vs.64.39±4.65),and of of invasive cells(99.65±9.12 vs.53.57±3.67)was less than in miR-NC group(t=14.950,14.062,P<0.05).The level of p21 in miR-612 group(0.14±0.02 vs.0.51±0.04)was higher than that in the miR-NC group(t=24.820,P<0.05).The dual luciferase report experiment confirmed that OSER1-AS1 could target miR-612.Inhibition of miR-612 expression could significantly reverse the effects of OSER1-AS1 on cell proliferation,migration and invasion.Conclusion Interfering with OSER1-AS1 could inhibit the proliferation,migration and invasion of renal cancer ACHN cells by up-regulating the expression of miR-612.
作者
赫志强
王雷
杨锦建
Hao Zhiqiang;Wang Lei;Yang Jinjian(Department of Urology Surgery,Shangqiu People′s Hospital,Shangqiu 476100,China;Department of Urology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)
出处
《中华实验外科杂志》
CAS
北大核心
2021年第3期488-492,共5页
Chinese Journal of Experimental Surgery
