【摘要】 目的:检测微小RNA-380-5p(mi R-380-5p)在人宫颈癌组织和细胞系中的表达,探讨其抑制宫颈癌C33A细胞增殖和迁移的作用机制。方法:收集2016年12月至2017年7月同济医学院附属武汉中心医院妇产科手术切除的16例宫颈癌患者癌组织和对应的癌旁组织标本,以及宫颈癌细胞系HCC94、C33A、Hela、SiHa和人子宫颈上皮永生化细胞H8,用qPCR法检测miR-380-5p在癌及癌旁组织、4种宫颈癌细胞及H8细胞中的表达。用脂质体转染技术将miR-380-5p mimi(c实验组)和miR-NC(阴性对照组)瞬时转染C33A细胞,用qPCR法检测转染后细胞中miR-380-5p表达,CCK-8法和Transwell小室法分别检测转染细胞的增殖和迁移能力。用生物信息学软件TargetScan预测miR-380-5p的下游基因,用双荧光素酶报告基因实验验证miR-380-5p对下游基因Ras同源基因家族成员A(Ras homolog gene family member A,RHOA)的结合作用,qPCR法和Western blotting检测miR-380-5p下游基因RHOA的表达。结果:宫颈癌组织中miR-380-5p表达水平明显低于癌旁组织(P<0.01),宫颈癌细胞系中miR-380-5p表达水平均明显低于H8细胞(P<0.05),以C33A细胞中的表达水平最低(P<0.01)。与阴性对照组比较,转染miR-380-5p mimic能显著抑制C33A细胞的增殖(P<0.05)和迁移能力(P<0.01),且下调RHOA、ROCK1、ROCK2、CDK2和N-cadherin蛋白的表达(均P<0.01)。生物信息学软件预测RHOA可能为miR-380-5p的调控基因,双荧光素酶报告基因实验结果显示miR-380-5p可与RHOA 3’-非编码区(UTR)特异性结合,miR-380-5p可明显下调RHOA基因的表达(P<0.01)。结论:miR-380-5p在宫颈癌组织及细胞系中低表达,过表达miR-380-5p可能通过下调RHOA及其下游蛋白的表达抑制宫颈癌C33A细胞的增殖和迁移。更多还原

【Abstract】 Objective: To investigate the expression of microRNA-380-5 p(miR-380-5 p) in cervical cancer tissues and cell lines, and to explore the mechanism of miR-380-5 p inhibiting the proliferation and migration of cervical cancer cells. Methods: 16 pairs of cervical cancerous tissues and corresponding para-cancerous tissues were collected from the Department of Obstetrics and Gynecology, the Affiliated Wuhan Central Hospital of Tongji Medical College from December 2016 to July 2017; in addition, cervical cancer cell lines(HCC94, C33 A, Hela, SiHa) and human cervical epithelial immortalized H8 cells were also collected for this study. The expression of miR-380-5 p in above mentioned tissues and cell lines was detected by Real-time quantitative polymerase chain reaction(qPCR). miR-380-5 p mimic(experimental group) and miR-NC(negative control group) were transiently transfected into C33 A cells by lipofection,and q PCR was used to detect the expression of miR-380-5 p in the transfected cells. Cell proliferation and migration were evaluated by cell counting kit(CCK-8) and Transwell assay. Bioinformatics software TargetScan predicted the downstream genes of miR-380-5 p,and dual luciferase reporter assay was used to verify the binding of miR-380-5 p to the downstream gene RHOA(Ras homolog gene family member A). qPCR and Western blotting were used to detect the expression of miR-380-5 p downstream gene-RHOA. Results:The expression level of miR-380-5 p in cervical cancer tissues and cell lines was significantly lower than that in para-cancerous tissues and normal cervical epithelial H8 cells(P<0.01); and the expression in C33 A cells was the lowest(P<0.01). Compared with the negative control group, the miR-380-5 p mimic transfection singnificantly inhibited the proliferation(P<0.05) and migration ability of C33 Acells(P<0.01), and down-regulated protein expressions of RHOA, ROCK1, ROCK2, CDK2 and N-cadherin(all P<0.01). Bioinformatics software predicted that RHOA may be a downstream gene of miR-380-5 p, and dual luciferase reporter assay proved the specific binding of miR-380-5 p to the 3’UTR of RHOA(P<0.01). miR-380-5 p could significantly down-regulate RHOA gene expression(P<0.01). Conclusion: miR-380-5 p is low-expressed in cervical cancer cell lines. Over-expression of miR-380-5 p may inhibit the proliferation and migration of cervical cancer C33 A cells by down-regulating the expression of RHOA gene and its downstream proteins.更多还原