摘要
目的探讨微小RNA-375(miR-375)对erb-b2受体酪氨酸激酶2(ERBB2)表达的靶向作用及卵巢癌细胞侵袭迁移的影响。方法实时定量PCR检测本院2017年1月至2018年1月手术切除的35例卵巢癌组织的miR-375水平。选取卵巢癌细胞SKOV3作为体外实验对象,将细胞分为空白对照组(仅采用脂质体处理)、阴性对照组(转染NC序列)和过表达组(转染miR-375模拟物mimics)。CCK-8法、划痕实验和Transwell小室实验分别评估miR-375差异表达对SKOV3细胞增殖、迁移和侵袭的影响。TargetScan在线预测筛选出miR-375的潜在靶点ERBB2并通过荧光素酶报告基因分析证实两者的靶向关系。结果35例卵巢癌组织的miR-375水平为0.724±0.054,低于正常卵巢组织的1.012±0.044,而ERBB2水平为1.392±0.068,高于正常卵巢组织的1.034±0.061(P<0.05),且卵巢癌组织中miR-375和ERBB2水平呈负相关(r=-0.766,P<0.05)。与其余两组相比,过表达组SKOV3细胞转染后的miR-375水平升高,而增殖活力降低(P<0.05);过表达组的划痕愈合率和穿膜细胞数分别为(40.424±4.738)%和(114.375±13.618)个,均低于空白对照组的(69.214±3.792)%和(251.624±15.372)个及阴性对照组的(72.670±3.536)%和(268.307±10.414)个(P<0.05);荧光素酶报告基因实验结果显示,与ERBB2野生型载体和NC共转染细胞相比,ERBB2野生型载体和miR-375 mimics共转染细胞的荧光素酶活性降低(P<0.05);而ERBB2突变型载体和miR-375共转染细胞的荧光素酶活性较ERBB2突变型载体和NC共转染细胞无变化。与空白对照组和阴性对照组相比,过表达组的MMP-9和ERBB2水平降低(P<0.05)。空白对照组和阴性对照组上述指标的差异无统计学意义(P>0.05)。结论miR-375能够通过负调控其靶点ERBB2,对卵巢癌的发生发展产生抑制作用,有望为卵巢癌的治疗提供理论依据。
Objective To investigate the effects of microRNA-375(miR-375)on the targeted regulation of erb-b2 receptor tyrosine kinase 2(ERBB2)and migration and invasion of ovarian cancer cells.Methods Real-time quantitative PCR was used to detect the miR-375 level in 35 cases of ovarian cancer tissues resected in our hospital from January 2017 to January 2018.Ovarian cancer cell line SKOV3 was selected as the experimental object in vitro.Cells were divided into blank control group(treated with liposome only),negative control group(transfected with NC sequence)and overexpression group(transfected with miR-375 mimics).CCK-8 assay,scratch test and Transwell chamber test were used to evaluate the effects of miR-375 differential expression on proliferation,migration and invasion of SKOV3 cells.The potential target of miR-375 was screened by Targetscan online prediction,and the relationship between miR-375 and ERBB2 was confirmed by luciferase reporter gene analysis.Results The miR-375 level in 35 cases of ovarian cancer tissues was 0.724±0.054,lower than 1.012±0.044 of normal ovarian tissues,while the ERBB2 level was 1.392±0.068,higher than 1.034±0.061 of normal ovarian tissues(P<0.05).There was a negative correlation between miR-375 and ERBB2(r=-0.766,P<0.05).Compared with other two groups,the miR-375 level of SKOV3 cells in overexpression group increased,while the proliferation activity decreased(P<0.05).The wound healing rate and the number of penetrating cells in the overexpression group were(40.424±4.738)%and 114.375±13.618,lower than(69.214±3.792)%and 251.624±15.372 in the blank control group and(72.670±3.536)%and 268.307±10.414 in the negative control group(P<0.05).Results of luciferase reporter gene showed that compared with ERBB2 wild-type vector and NC co-transfection cells,the luciferase activity of ERBB2 wild-type vector and miR-375 mimics co-transfection cells was lower(P<0.05),while the luciferase activity of ERBB2 mutant vector and miR-375 co-transfection cells was not changed compared with ERBB2 mutant vector and NC co-transfection cells.Compared with blank control group and negative control group,levels of MMP-9 and ERBB2 in overexpression group were lower(P<0.05).No significant difference in the above indexes was observed between blank control group and negative control group(P>0.05).Conclusion MiR-375 can inhibit the occurrence and development of ovarian cancer by negatively regulating its target ERBB2,which is expected to provide a theoretical basis for the treatment of ovarian cancer.
作者
祁青玲
王烈宏
曾慧
QI Qingling;WANG Liehong;ZENG Hui(Department of Obstetrics and Gynecology,Qinghai Red Cross Hospital,Xining 810000,China)
出处
《临床肿瘤学杂志》
CAS
北大核心
2020年第9期809-815,共7页
Chinese Clinical Oncology
