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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

吴凤兰, 陈红莲, 胡小伟, 梁利英, 徐宛玲^*

漯河医学高等专科学校医疗系，漯河 462002

摘要

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。 

关键词： Wnt5a; 卵巢癌; 长春新碱; 耐药性; Akt; β-连环蛋白; 多药耐药基因1; 存活素

分类号：R737.31

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

WU Feng-Lan, CHEN Hong-Lian, HU Xiao-Wei, LIANG Li-Ying, XU Wan-Ling^*

Department of Medicine, Luohe Medical College, Luohe 462002, China

Abstract

The aim of this study was to investigate the effect of Wnt5a 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 Wnt5a. The mRNA expression level of Wnt5a 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 Wnt5a, 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 Wnt5a, MDR1, Survivin and β-catenin, phosphorylation levels of Akt and GSK3β, and mRNA expression level of Wnt5a, compared with SKOV3 cells (P < 0.05). WNT5A 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 (PI3K 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 WNT5A gene silencing reverses VCR resistance in SKOV3/VCR cells possibly through blocking the PI3K/Akt/GSK3β/β-catenin signaling pathway, and thus down-regulating the protein expression levels of MDR1 and Survivin.

Key words: Wnt5a; ovarian carcinoma; vincristine; drug resistance; Akt; β-catenin; MDR1; Survivin

收稿日期：2018-06-16　　录用日期：2018-11-12

通讯作者：徐宛玲　　E-mail: 872189991@qq.com

DOI: 10.13294/j.aps.2019.0039

引用本文：

吴凤兰, 陈红莲, 胡小伟, 梁利英, 徐宛玲. 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[J]. 生理学报 2019; 71 (3): 415-423.

WU Feng-Lan, CHEN Hong-Lian, HU Xiao-Wei, LIANG Li-Ying, XU Wan-Ling. Wnt5a modulates vincristine resistance through PI3K/Akt/GSK3β signaling pathway in human ovarian carcinoma SKOV3/VCR cells. Acta Physiol Sin 2019; 71 (3): 415-423 (in Chinese with English abstract).