稳定过表达XAF1基因对A2780卵巢癌细胞生物学功能的影响

Effect of stable overexpression of XAF1 gene on biological characteristics of ovarian cancer A2780 cells

目的构建稳定过表达XAF1基因A2780卵巢癌细胞株,并观察XAF1基因对卵巢癌细胞增殖、凋亡、细胞周期及对紫杉醇敏感性的影响。方法分别将质粒pcDNA3.1(+)和pcDNA3.1(+)-XAF1转染至卵巢癌细胞A2780,通过质粒抗性标记(遗传霉素G418)筛选得到阴性对照细胞株(A2780/Negative control,A2780/NC)和稳定表达XAF1的细胞株(A2780/XAF1);通过行细胞克隆形成实验和CCK8实验检测细胞增殖及对紫杉醇的敏感性,流式细胞仪检测细胞周期及细胞凋亡。结果成功构建了稳定表达XAF1的卵巢癌细胞A2780/XAF1,细胞形态无明显变化;与A2780/NC相比,A2780/XAF1克隆形成能力能力更低(P=0.0016),细胞贴壁后第1天和第3天增殖活性更低(P=0.009,0.0035),两组细胞的细胞周期分布差异存在统计学意义(P<0.0001),两两比较结果显示A2780/XAF1组G2-M期细胞百分比显著增加(P<0.001)。在无凋亡刺激、无血清培养及紫杉醇诱导下,A2780/XAF1的凋亡率均比A2780/NC更高(P<0.001);在不同紫杉醇浓度作用下,A2780/XAF1的增殖活性均显著低于A2780/NC(P<0.001),且A2780/XAF1的紫杉醇半数抑制浓度显著低于A2780/NC。结论成功构建稳定表达XAF1的卵巢癌细胞A2780/XAF1;XAF1调控卵巢癌细胞A2780的增殖、凋亡及细胞周期,并增加了卵巢癌对紫杉醇的敏感性。

Objective To construct an ovarian cancer cell line stably overexpressing XAF1 gene and observe the effects of XAF1 gene overexpression on proliferation,apoptosis,cell cycle and sensitivity to paclitaxel of the cells.Methods Ovarian cancer A2780 cells were transfected with the plasmids pcDNA3.1(+)or pcDNA3.1(+)-XAF1,and the cells stably Over expressing XAF1(A2780/XAF1 cells)were screened using G418.Cell clone formation assay and CCK8 assay were used to evaluate the changes in proliferation and paclitaxel sensitivity of the transfected cells,and cell cycle and apoptosis of the cells were analyzed using flow cytometry.Results We successfully obtained A2780/XAF1 cells stably overexpressing XAF1,which exhibited no significant changes in cell morphology.Compared with the negative control cells(A2780/NC),A2780/XAF1 cells had lowered clone formation ability(P=0.0016)and attenuated proliferative activity on the first(P=0.009)and third(P=0.0035)days after cell adherence with also a significantly increased percentage of cells in G2-M phase(P<0.001).A2780/XAF1 cells showed significantly higher apoptosis rates than A2780/NC cells in the absence of apoptotic stimulation,in serum-free culture or following paclitaxel induction(P<0.001).The proliferative activity of A2780/XAF1 cells was significantly lower than that of A2780/NC cells after exposure to different paclitaxel concentrations(P<0.001).The half inhibitory concentration of paclitaxel was significantly lower in A2780/XAF1 than in A2780/NC cells.Conclusion Overexpression of XAF1 significantly inhibits the proliferation,induces cell cycle arrest,promotes apoptosis,and increases paclitaxel sensitivity in ovarian cancer cells.