摘要
目的分析长链非编码RNA酪氨酸蛋白激酶跨膜受体1反义RNA 1(lncRNA ROR1-AS1)通过靶向微小RNA-504(miR-504)对肝癌细胞MHCC97H增殖、迁移和侵袭的影响。方法收集2019年6月—2021年5月于内蒙古医科大学附属医院肝胆外科收治肝癌患者32例的癌组织及癌旁组织。肝癌细胞MHCC97H分为si-ROR1-AS1组、si-NC组、miR-504 mimics组、miR-NC组、si-ROR1-AS1+anti-miR-504组及si-ROR1-AS1+anti-miR-NC组,并进行相应质粒转染。qRT-PCR法检测组织样本及细胞中ROR1-AS1、miR-504的表达;克隆形成实验和MTT法检测细胞增殖;Transwell检测细胞迁移、侵袭;Western-blot法检测细胞周期蛋白依赖性激酶抑制剂1A(p21)、基质金属蛋白酶2(MMP-2)、MMP-9及上皮型钙黏附素(E-cadherin)水平;双荧光素酶报告实验检测ROR1-AS1与miR-504的调控关系。结果肝癌患者癌组织中ROR1-AS1水平高于癌旁组织,miR-504水平低于癌旁组织(t=11.544、10.905,P均<0.001)。与si-NC组比较,si-ROR1-AS1组MHCC97H细胞克隆形成数、细胞存活率、迁移细胞数、侵袭细胞数及MMP-2、MMP-9水平降低(t=8.978、9.647、8.444、13.282、10.026、12.006,P均<0.001),miR-504、p21、E-cadherin水平上升(t=10.527、9.722、12.901,P均<0.001)。与miR-NC组比较,miR-504 mimics组MHCC97H细胞克隆形成数、细胞存活率、迁移细胞数、侵袭细胞数及MMP-2、MMP-9水平降低(t=8.831、9.680、8.187、12.480、10.026、10.954,P均<0.001),p21、E-cadherin蛋白水平升高(t=9.418、12.614,P均<0.001)。双荧光素酶报告实验显示,ROR1-AS1可靶向调控miR-504。与si-ROR1-AS1+anti-miR-NC组比较,si-ROR1-AS1+anti-miR-504组细胞克隆形成数、细胞存活率、迁移细胞数、侵袭细胞数及MMP-2、MMP-9水平升高(t=7.064、7.012、6.746、10.222、7.213、7.982,P均<0.001),p21、E-cadherin蛋白水平下降(t=4.841、7.120,P均<0.001)。结论干扰ROR1-AS1表达可抑制肝癌MHCC97H细胞的增殖、迁移及侵袭,其具体机制可能与靶向作用miR-504水平有关。
Objective To analyze the effect of antisense RNA 1(lncRNA ROR1-AS1)targeting microRNA-504(miR-504)on the proliferation,migration and invasion of human hepatoma cell line MHCC97H by long chain non-coding RNA tyrosine protein kinase transmembrane receptor 1.Methods The cancer tissues and adjacent tissues of 32 patients with liver cancer were collected from the Department of Hepatobiliary Surgery of the Affiliated Hospital of Inner Mongolia Medical University from June 2019 to May 2021.MHCC97H cells were divided into si-ROR1-AS1 group,si-NC group,miR-504 mimics group,miR-NC group,si-ROR1-AS1+anti-miR-504 group and si-ROR1-AS1+anti-miR-NC group,and the corresponding plasmids were transfected.The expression of ROR1-AS1 and miR-504 in tissue samples and cells was detected by qRT-PCR.Cell proliferation was detected by clonogenic assay and MTT assay.Transwell detected cell migration and invasion.The levels of cyclin-dependent kinase inhibitor 1A(p21),matrix metalloproteinase-2(MMP-2),MMP-9 and E-cadherin were detected by Western-blot method.The regulatory relationship between ROR1-AS1 and miR-504 was detected by double luciferase report experiment.Results The level of ROR1-AS1 in cancer tissue of patients with liver cancer was higher than that in paracancerous tissue,and the level of miR-504 was lower than that in paracancerous tissue(t=11.544,10.905,P<0.001).Compared with si-NC group,the number of MHCC97H cell clones,cell survival rate,number of migrating cells,number of invasive cells,and levels of MMP-2 and MMP-9 in si-ROR1-AS1 group decreased(t=8.978,9.647,8.444,13.282,10.026,12.006,P<0.001),and the levels of miR-504,p21,E-cadherin increased(t=10.527,9.722,12.901,P<0.001).Compared with the miR-NC group,the number of MHCC97H cell clones,cell survival rate,number of migrating cells,number of invasive cells and the level of MMP-2 and MMP-9 in the miR-504 mimics group decreased(t=8.831,9.680,8.187,12.480,10.026,10.954,P<0.001),and the level of p21 and E-cadherin protein increased(t=9.418,12.614,P<0.001).The double luciferase report experiment showed that ROR1-AS1 could target the regulation of miR-504.Compared with si-ROR1-AS1+anti-miR-NC group,si-ROR1-AS1+anti-miR-504 group increased the number of cell clones,cell survival rate,number of migrating cells,number of invasive cells,and levels of MMP-2 and MMP-9(t=7.064,7.012,6.746,10.222,7.213,7.982,P<0.001),while p21 and E-cadherin protein levels decreased(t=4.841,7.120,P<0.001).Conclusion Interference with the expression of ROR1-AS1 can inhibit the proliferation,migration and invasion of hepatoma MHCC97H cells.The specific mechanism may be related to the targeted miR-504 level.
作者
乌吉斯古楞
张文华
张彤
Wu Jisiguleng;Zhang Wenhua;Zhang Tong(Hepatobiliary Surgery,the Affiliated Hospital of Inner Mongolia Medical University,Inner Mongolia,Hohhot 010050,China)
出处
《疑难病杂志》
CAS
2023年第2期138-143,共6页
Chinese Journal of Difficult and Complicated Cases
基金
内蒙古自治区教育厅自然科学重点项目(NJZZ22654)。
