miRNA-15a通过靶向Wnt3a调控Wnt/β-catenin信号通路抑制卵巢癌的发展和转移

miRNA-15a suppresses ovarian cancer cell proliferation and metastasis by targeting Wnt3a and regulating the Wnt/β-catenin signaling pathway

目的:研究在卵巢癌发展转移进程中miRNA-15a的作用,并探讨其作用机制。方法:收集锦州医科大学附属第一医院2012年5月至2015年6月32例卵巢癌患者的手术切除标本,采用RT-PCR检测miRNA-15a表达。将培养的卵巢癌SKOV-3细胞分为转染miRNA-15a模拟物的实验组和转染无义miRNA的对照组,采用CCK8、Transwell及流式细胞术检测细胞增殖、侵袭和凋亡的变化,采用Western blot法检测相关蛋白表达。通过生物信息学方法预测miRNA-15a靶基因,采用双荧光素酶报告基因实验结合Western blot法验证miRNA-15a对靶基因的调控。检测Wnt3a与miRNA-15a模拟物共转染SKOV-3细胞后对细胞增殖和侵袭的影响。结果:与癌旁组织中的miRNA-15a相对表达量0.692±0.228相比,显著低于卵巢癌组织中的0.911±0.209,差异具有统计学意义(P<0.05)。与对照组细胞增殖能力相比,miRNA-15a过表达显著抑制实验组细胞增殖能力,差异具有统计学意义(P<0.05)。与对照组189.667±14.742相比,miRNA-15a过表达显著抑制实验组的细胞侵袭能力96.667±13.614,差异具有统计学意义(P<0.05)。miRNA-15a过表达实验组的细胞凋亡率27.400%±0.854%显著高于对照组细胞凋亡率6.933%±0.379%,差异具有统计学意义(P<0.05)。miRNA-15a模拟物下调Wnt3a和β-catenin的表达,抑制细胞周期蛋白(Cyclin D1)和血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达,促进E-钙离子依赖的细胞黏附素(E-calcium-dependent adhesion,E-cad)的表达。双荧光素酶报告基因实验和Western blot结果表明miRNA-15a靶向抑制Wnt3a表达。上调Wnt3a能显著降低miRNA-15a对卵巢癌细胞增殖、侵袭和对Wnt/β-catenin信号通路的抑制作用。结论:miRNA-15a通过靶向Wnt3a调控Wnt/β-catenin信号通路的激活以抑制卵巢癌的发展和转移。

Objective：To investigate the role of miRNA-15a in the progression and metastasis of ovarian cancer and to explore the specific related molecular mechanism. Methods：Thirty-two ovarian cancer specimens were collected from the First Affiliated Hospital of Jinzhou Medical University from May 2012 to June 2015. Mi RNA-15a expression in ovarian cancer tissues and corresponding adjacent tissues was detected by RT-PCR. SKOV-3 ovarian cancer cells were transfected with miRNA-15a mimics in the experimental group or control miRNA in the control group, and the effects of miRNA-15a on cell proliferation, invasion, and apoptosis were detected by CCK8,transwell, and flow cytometry assays, respectively. Expression levels of key proteins involved in related pathways were detected by Western blot. Candidate target genes of miRNA-15a were identified by bioinformatics screening and verified by dual luciferase reporter gene and Western blot assays. Wnt3a was co-transfected with miRNA-15a mimic into SKOV-3 cells to detect its effects on cell proliferation, invasion, and signaling. Results：Compared with that in adjacent tissues（0.911±0.209）, miRNA-15a expression was significantly down-regulated in ovarian cancer tissues（0.692±0.228）（P0.05）. Compared with that in the control group, cell proliferation in the experimental group was significantly lower（P0.05）. Moreover, compared with that in the control group（189.667 ± 14.742）, invasive cells in the experimental group were fewer（96.667±13.614）（P0.05）. Compared with that in the control group（6.933%±0.379%）, the apoptotic rate in the experimental group was（27.400%±0.854%）（P0.05）. Furthermore, miRNA-15a mimics down-regulated Wnt3a, β-catenin, cyclin D1, and vascular endothelial growth factor expression and up-regulated E-cad expression. Dual luciferase reporter gene assay and Western blot results indicated that miRNA-15a directly targets Wnt3a. Importantly, we found that up-regulation of Wnt3a significantly decreased the inhibitory effect of miRNA-15a on the proliferation, invasion, and Wnt/β-catenin signaling pathway in SKOV-3 cells. Conclusions：Mi RNA-15a could inhibit ovarian cancer progression and metastasis by directly targeting Wnt3a and regulating the Wnt/β-catenin pathway.