微小RNA-18a-3p靶向调控HEPN1及对骨肉瘤细胞迁移侵袭的影响  

Targeted regulation of HEPN1 by microRNA-18a-3p and its effect on migration and invasion of osteosarcoma cells

作　　者：李涛[1] 李娜[1,2] 李佳[1] 王磊[1] LI Tao;LI Na;LI Jian;WANG Lei(Department of Orthopedics, Qinghai Provincial People's Hospital, Xining 810000, China)

机构地区：[1]青海省人民医院骨三科,西宁810000 [2]青海省人民医院神经内科,810000

出　　处：《临床肿瘤学杂志》2020年第5期412-417,共6页Chinese Clinical Oncology

摘　　要：目的探讨微小RNA-18a-3p(miR-18a-3p)对肝细胞癌下调蛋白1(HEPN1)的靶向调控作用及对骨肉瘤(OS)细胞侵袭迁移的影响。方法从美国国家生物技术信息中心(NCBI)的GEO数据库中下载基因表达谱OS样本数据集GSE65071的series matrix数据,分析OS患者和健康对照的血浆miR-18a-3p水平;采用实时定量PCR检测OS细胞MG63和成骨细胞hFOB 1.19的miR-18a-3p和HEPN1 mRNA水平,Western blotting检测HEPN1蛋白水平;脂质体介导miR-18a-3p抑制物转染MG63细胞(Inhibitor组),同时设NC组(转染阴性对照序列)和对照组(仅予脂质体处理而未行转染),活细胞计数(CCK-8)法、划痕实验和Transwell小室实验检测细胞活力、划痕愈合率和穿膜细胞数以评估增殖和迁移侵袭能力,构建pmirGLO载体并借助双荧光素酶报告检测系统验证miR-18a-3p与HEPN1间的靶向关系。结果GSE65071数据集显示,20例OS患者的血浆miR-18a-3p水平为7.262±0.494,高于15例健康对照的6.005±0.081(P<0.05)。与hFOB 1.19细胞相比,MG63细胞的miR-18a-3p水平升高至3.361±0.257,而HEPN1 mRNA水平降低至0.571±0.068,差异有统计学意义(P<0.05);与对照组和NC组相比,Inhibitor组的miR-18a-3p水平降低,而HEPN1 mRNA和蛋白水平升高(P<0.05)。双荧光素酶报告基因实验结果显示,miR-18a-3p模拟物可抑制转染野生型HEPN13′端非翻译区质粒的细胞相对荧光素酶活性(P<0.05),但对转染突变型质粒的细胞无影响(P>0.05)。与对照组和NC组相比,Inhibitor组转染后24 h的增殖活力有降低趋势(P>0.05),而转染48~96 h的增殖活力降低(P<0.05);Inhibitor组的划痕愈合率和穿膜细胞数分别为(22.339±4.172)%和(153.673±7.570)个,均低于对照组的(63.007±3.841)%和(365.336±10.772)个及NC组的(65.670±3.101)%和(366.335±13.596)个(P<0.05)。对照组和NC组上述指标的差异无统计学意义(P>0.05)。结论miR-18a-3p在OS中高表达且发挥促癌的作用,下调其表达可抑制OS细胞的增殖和迁移侵袭能力,可�Objective To investigate the targeted regulation of hepatocellular carcinoma,down-regulated 1(HEPN1)by microRNA-18a-3p(miR-18a-3p)and its effect on migration and invasion of osteosarcoma(OS)cells.Methods The series matrix data of GSE65071 was downloaded from the GEO database of National Biotechnology Information Center(NCBI),and the plasma levels of miR-18a-3p in OS patients and healthy controls were analyzed.Levels of miR-18a-3p and HEPN1 mRNA in OS cells MG63 and osteoblast hFOB 1.19 were detected by real-time quantitative PCR.Protein level of HEPN1 was detected by Western blotting.Liposome mediated transfection of miR-18a-3p inhibitor into MG63 cells(Inhibitor group)was carried out.Cells transfected with negative control sequence were set as NC group and cells only challenged with liposome were set as Control group.Cell viability,scratch healing rate and number of transmembrane cells were measured by CCK-8,scratch test and Transwell chamber test to evaluate the proliferation,migration and invasion ability.The pmirGLO vector was constructed and the targeting relationship between miR-18a-3p and HEPN1 was verified by Dual-Luciferase Report Assay System.Results GSE65071 data set showed that the plasma miR-18a-3p level of 20 OS patients was 7.262±0.494,higher than 6.005±0.081 of 15 healthy controls(P<0.05).Compared with hFOB 1.19 cells,miR-18a-3p level of MG63 cells increased to 3.361±0.257,while the HEPN1 mRNA level decreased to 0.571±0.068(P<0.05).Compared with Control group and NC group,the miR-18a-3p level of Inhibitor group decreased,while the mRNA and protein level of HEPN1 increased(P<0.05).The results of Dual-Luciferase Report Assay System showed that miR-18a-3p mimics could inhibit the relative luciferase activity of cells transfected with plasmids carrying wild-type HEPN13′untranslated region(P<0.05),but had no effect on cells transfected with mutant plasmids(P>0.05).Compared with Control group and NC group,there was a decreasing trend in term of the proliferation activity at 24 h after transfection

关 键 词：骨肉瘤 微小RNA-18a-3p 迁移 侵袭 肝细胞癌下调蛋白1 

分 类 号：R738.6[医药卫生—肿瘤]