lncRNA PVT1通过miR-1207-5p靶向调控Wnt6/β-catenin2信号通路对高级别浆液性卵巢癌的影响

Effects of lncRNA PVT1 on high-level serous ovarian cancer by targeting the Wnt6/β-catenin2 signaling pathway through miR-1207-5p

目的探讨长链非编码RNA(lncRNA)PTV1对高级别浆液性卵巢癌增殖和迁移能力的影响。方法收集61例高级别浆液性卵巢癌患者的癌组织及相应癌旁正常组织,qRT-PCR检测miR-1207-5p、lncRNA PVT1在高级别浆液性卵巢癌组织、癌旁正常组织及不同细胞系中的表达情况。构建lncRNA PVT1沉默细胞系,分为Ovcar3-siPVT1组、Ovcar3-siNC组、NC组。采用MTT和平板克隆实验、Transwell和划痕实验检测细胞增殖、侵袭和迁移;双荧光素酶报告基因检测验证miR-1207-5p与lncRNA PVT1、Wnt6的靶向关系,StarBase和TargetScan网站预测相应miRNA可靶向结合的基因;Western blotting检测Wnt6/β-catenin2信号通路相关蛋白的表达。构建siPVT1+过表达miR-1207-5p、siPVT1+过表达Wnt6细胞系,以siPVT1+siNC为对照,验证lncRNA PVT1的调控作用。结果qRT-PCR结果显示,高级别浆液性卵巢癌组织中lncRNA PVT1的表达水平显著高于正常癌旁组织(P<0.05)。与NC组和Ovcar3-siNC组相比,Ovcar3-siPVT1组中lncRNA PVT1的表达显著下调,细胞克隆数、侵袭数减少,细胞迁移率降低,Wnt6、β-catenin2蛋白表达水平明显下降,差异均具有统计学意义(P<0.05)。双荧光素酶报告基因检测结果证明miR-1207-5p与lncRNA PVT1、Wnt6具有靶向关系。经StartBase和TargetScan网站预测分析,miR-1207-5p分别与lncRNA PVT1、Wnt6存在靶向结合位点。与siPVT1+NC组相比,siPVT1+过表达miR-1207-5p组和siPVT1+过表达Wnt6组细胞克隆数和迁移数明显增加(P<0.05)。结论lncRNA PVT1在高级别浆液性卵巢癌中高表达。高表达lncRNA PVT1可能通过上调miR-1207-5p表达增强Wnt6/β-catenin2信号通路的活性,从而促进高级别浆液性卵巢癌细胞的增殖、侵袭和迁移。

ObjectiveTo investigate the effects of long non-coding RNA PTV1(lncRNA PTV1) on the proliferation and migration of high-grade serous ovarian cancer.MethodsThe expression of miR-1207-5 p and lncRNA PVT1 was detected by qRT-PCR in 61 cases of high-grade serous ovarian cancer tissues and the corresponding normal tissues. The lncRNA PVT1-silenced cell lines were constructed and divided into Ovcar3-siPVT1, Ovcar3-siNC and NC groups. The MTT and plate cloning experiment, Transwell and scratch test were used to detect the proliferation, invasion and migration of cancer cells. The dual luciferase reporter gene detection was used to verify the targeting relationship of miR-1207-5 p with lncRNA PVT1 and Wnt6. StarBase and TargetScan website was retrieved to predict the target binding sites of the corresponding miRNA. Western blotting was used to detect the expression of proteins of Wnt6/β-catenin2 signaling pathway. The cell lines of siPVT1 + overexpressed miR-1207-5 p and siPVT1 + overexpressed Wnt6 were constructed to verify the regulatory effect of lncRNA PVT1 by using siPVT1 + siNC cells as control.ResultsThe results of qRT-PCR showed that the expression level of lncRNA PVT1 was significantly higher in advanced serous ovarian cancer tissue than in the normal adjacent tissue(P<0.05). Compared with the NC group and Ovcar3-siNC group, the Ovcar3-siPVT1 group showed down-regulated expression of lncRNA PVT1, reduced number of cell cloning and invading, and lowed cell migration rate, as well as significantly decreased expression of Wnt6 and β-catenin2 proteins(P<0.05). Results of dual luciferase reporter gene detection showed that miR-1207-5 p had a targeting relationship with lncRNA PVT1 and Wnt6. The StartBase and TargetScan prediction analysis also showed that miR-1207-5 p had target binding sites to lncRNA PVT1 and Wnt6 respectively. Compared with siPVT1 + NC group, the number of cell cloning and migration was increased significantly in siPVT1 + overexpressed miR-1207-5 p group and siPVT1+overexpressed Wnt6 group(P<0.05).ConclusionlncRNA PVT1 had high expression in high-grade serous ovarian cancer. The highly expressed lncRNA PVT1 may enhance the Wnt6/β-catenin2 signaling pathway by down-regulating the expression of miR-1207-5 p, and hereby promote the proliferation and metastasis of high-grade serous ovarian cancer cells.