MicroRNA-10b对宫颈癌细胞侵袭迁移能力的影响及机制研究

The Effects of MicroRNA-10b on the Invasion and Migration in the Cervical Carcinoma Cells and Its Possible Mechanism

目的:探讨微小RNA-10b(microRNA-10b)对宫颈癌细胞侵袭迁移能力的影响及机制。方法:实时定量聚合酶链反应(real-time PCR)检测microRNA-10b在不同宫颈癌细胞及正常宫颈上皮细胞中的表达情况;脂质体转染microRNA-10b模拟物(microRNA-10b mimics,即mimics组)及microRNA无关序列(microRNA-10b NC,即NC组)至Caski细胞,转染microRNA-10b抑制物(microRNA-10b inhibitor,即inhibitor组)及microRNA inhibitor无关序列(microRNA-10b NC inhibitor,即NC inhibitor组)至He La细胞,转染12 h后,real-time PCR法检测转染效率。采用Transwell试验和划痕试验分别检测microRNA-10b对Caski和He La细胞侵袭迁移能力的影响;real-time PCR及蛋白质印迹(Western blotting)检测各组细胞中胰岛素样生长因子1受体(IGF-1R)mRNA和蛋白表达水平;荧光素酶报告基因试验检测microRNA-10b对IGF-1R基因表达的调控机制。结果:MicroRNA-10b在宫颈癌细胞中表达水平低于正常宫颈上皮细胞(P<0.05)。MicroRNA-10b表达增加能够降低Caski细胞的侵袭迁移能力(P<0.05),抑制IGF-1R的mRNA和蛋白表达(均P<0.05);而microRNA-10b表达下降能够增强He La细胞的侵袭迁移能力(P<0.05),增加IGF-1R的mRNA和蛋白表达(P均<0.05)。在HEK-293T细胞中,microRNA-10b表达增加能够减弱含有IGF-1R基因3′非翻译区(3′-UTR)的报告质粒的荧光素酶活性(P<0.05)。结论:在宫颈癌细胞中,microRNA-10b通过下调IGF-1R基因表达,抑制细胞侵袭迁移能力。

Objective:To investigate the role of microRNA-10b in the cervical carcinoma and explore its possible mechanism. Methods:The expression levels of microRNA-10b in cervical carcinoma cell lines and the normal cervical epithelial cells were detected by the real-time polymerase chain reaction(real-time PCR). The microRNA-10b mimics/NC was transfected into the Caski cells and the microRNA-10b inhibitor/NC inhibitor was transfected into the He La cells by the liposome. The Transwell assay and wound-healing assay were used to determine the effects of microRNA-10b on the invasion and migration ability in the Caski and He La cells. The mRNA and protein levels of insulin-like growth factor 1 receptor(IGF-1R) were analyzed by the real-time PCR and the Western blotting assay, respectively. Luciferase reporter assay was applied to detect the regulatory mechanism of microRNA-10b in the IGF-1R gene. Results:The expression levels of microRNA-10b in the cervical carcinoma cell lines were much lower than those in the normal cervical epithelial cells( P<0.05). The up-regulation of microRNA-10b could decrease the invasion and migration ability of Caski cells(P<0.05), and inhibit the mRNA and protein levels of IGF-1R(P<0.05), while the downregulation of microRNA-10b could increase the invasion and migration ability of He La cells(P<0.05), and increase the mRNA and protein levels of IGF-1R(P<0.05). Besides, the up-regulation of microRNA-10b could decrease the luciferase activity of the reporter vector(containing the 5'untranslated region of IGF-1R gene) in the HEK-293 T cells(P <0.05). Conclusions:In cervical carcinoma cells, microRNA-10b could inhibit the cell invasion and migration capability, which may be the result of the down-regulation of IGF-1R gene.