Wnt5a通过PI3K/Akt/GSK3β信号通路调控人卵巢癌SKOV3/VCR细胞对长春新碱的敏感性

Wnt5a modulates vincristine resistance through PI3K/Akt/GSK3β signaling pathway in human ovarian carcinoma SKOV3/VCR cells

本研究旨在探究Wnt5a对人卵巢癌SKOV3细胞长春新碱(vincristine,VCR)耐药性的影响,并探讨其分子机制。采用浓度梯度递增法构建耐药株SKOV3/VCR细胞,将人WNT5A基因的干扰质粒转染SKOV3/VCR细胞并筛选出稳定干扰Wnt5a的细胞株,用RT-PCR检测Wnt5a的mRNA表达水平,用CCK-8法检测细胞活力,用流式细胞术检测细胞凋亡,用Western blot法检测Wnt5a、MDR1、Survivin、β-catenin、Akt、p-Akt(S473)、GSK3β和p-GSK3β(Ser9)的蛋白表达水平。结果显示,SKOV3/VCR细胞中Wnt5a、MDR1、β-catenin和Survivin蛋白的表达水平、Akt和GSK3β蛋白的磷酸化水平以及Wnt5a mRNA表达水平均显著高于其亲代SKOV3细胞;WNT5A基因沉默使VCR抑制SKOV3/VCR细胞活力的IC50从38.412降至9.283 mg/L,提高SKOV3/VCR细胞凋亡率并协同增强VCR诱导的细胞凋亡(P<0.05),下调MDR1、β-catenin和Survivin蛋白的表达水平(P<0.05),抑制Akt和GSK3β蛋白磷酸化(P<0.05);此外,PI3K抑制剂LY294002也可下调SKOV3/VCR细胞中MDR1、β-catenin和Survivin蛋白表达水平,并降低Akt和GSK3β蛋白磷酸化水平(P<0.05)。以上结果提示,沉默WNT5A基因可在体外逆转人卵巢癌耐药株SKOV3/VCR细胞耐药性,作用机制可能与其抑制PI3K/Akt/GSK3β/β-catenin通路,继而下调MDR1和Survivin蛋白表达有关。

The aim of this study was to investigate the effect of Wnt5 a on the vincristine(VCR) resistance in human ovarian carcinoma SKOV3 cells and its possible mechanism. The drug-resistant SKOV3/VCR cells were established by stepwise exposure to VCR, and then the SKOV3/VCR cells were stably transfected with specific shRNA interference plasmid vector targeting for Wnt5 a. The mRNA expression level of Wnt5 a was measured by RT-PCR. CCK-8 assay was used to detect the cell viability of SKOV3/VCR cells. The apoptosis was analyzed by flow cytometry. The protein expression levels of Wnt5 a, MDR1, Survivin,β-catenin, Akt, p-Akt(S473),GSK3β and p-GSK3β(Ser9) were detected by Western blot. The result showed that SKOV3/VCR cells had significantly higher protein expression levels of Wnt5 a, MDR1, Survivin and β-catenin, phosphorylation levels of Akt and GSK3β, and mRNA expression level of Wnt5 a, compared with SKOV3 cells(P < 0.05). WNT5 A gene silencing significantly increased the sensitivity of SKOV3/VCR cells to VCR, the IC50 of VCR being decreased from 38.412 to 9.283 mg/L(P < 0.05), synergistically enhanced VCR-induced apoptosis of SKOV3/VCR cells(P < 0.05), down-regulated the protein expression levels of MDR1,β-catenin and Survivin(P < 0.05), and inhibited phosphorylation of Akt and GSK3β(P < 0.05). Meanwhile, LY294002(PI3 K inhibitor) decreased the protein expression levels of MDR1,β-catenin and Survivin, as well as the phosphorylation levels of Akt and GSK3β in SKOV3/VCR cells(P < 0.05). These results suggest that WNT5 A gene silencing reverses VCR resistance in SKOV3/VCR cells possibly through blocking the PI3 K/Akt/GSK3β/β-catenin signaling pathway, and thus down-regulating the protein expression levels of MDR1 and Survivin.