靶向NAC-1基因的siRNA对卵巢癌HO8910细胞生长及其Wnt/β-catenin信号途径的影响

Effects of specific small interfering RNA against nucleus accumbens-1 gene on cell proliferation of ovarian cancer cells and Wnt / β-catenin signaling pathway

目的 :研究NAC-1调节卵巢癌HO8910细胞生长增殖的机制。方法 NAC-1基因特异性小干扰RNA(siRNA)处理HO8910细胞,然后检测细胞增殖率、细胞周期、以及NAC-1蛋白、WNT信号途径β-链蛋白、低密度脂蛋白受体相关蛋白6(LRP6)基因及其下游靶基因cyclin D1和survivin基因表达。结果 NAC-1基因的特异性siRNA不仅显著抑制HO8910细胞中NAC-1 mRNA和蛋白的表达,明显抑制细胞增殖,细胞周期被阻滞于G1期,而且显著抑制其Wnt/β-catenin信号途径活性:转染48 h,β-catenin、cyclin D1、survivin蛋白水平显著下降,cyclin D1、survivin基因mRNA表达水平显著下降,与空白对照组相比较有显著差异(P<0.01)。结论 NAC-1通过调节β-catenin蛋白水平增强WNT信号途径活性、促进卵巢癌HO8910细胞增殖。

Objective To investigate the mechanism of nucleus accumbens-1（Nac1 or NAC-1）protein on the proliferation of ovarian cancer cell line HO8910. Methods Specific NAC-1 siRNAs and one negative siRNA were individually transfected into HO8910 cells for 48 h . Levels of NAC-1, β-catenin, LRP6, cyclinD1, and survivin mRNA and protein were analyzed by real-time fluorescence quantitative PCR （qRT-PCR） and Western blotting, respectively. The cell proliferation was determined by MTT assay and plate clone formation assay. The distribution of cell cycle was assessed by flow cytometry. Results After NAC-1 siRNA treatment for 48 h, not only the NAC-1 gene was silenced in HO8910 cells, the cell growth was inhibited, and cell cycle arrested in G1 phase, but also the activity of Wnt/β-catenin signaling decreased significantly, which levels of β-catenin, cyclinD1, and survivin proteins, cyclinD1 and survivin mRNA all decreased, as compared with the control groups, the difference was statistically significant. However, NAC-1 gene silencing in ovarian carcinoma cells HO8910 did not affect levels of p-LRP6 protein, LRP6 mRNA and β -catenin mRNA, as compared with the control groups, the difference was not statistically significant. Conclusion NAC-1 enhance the activity of WNT signaling pathway through the regulation of β -catenin protein level, promote ovarian cancer HO8910 cell proliferation.