6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶4对卵巢癌细胞干性的影响及其机制

Effect and mechanism of PFKFB4 on ovarian cancer cell stemness

目的:探讨6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶4(PFKFB4)对卵巢癌细胞干性的影响及其机制。方法:利用慢病毒系统构建PFKFB4过表达的卵巢癌SKOV3和A2780稳定细胞系。采用Real-time PCR和Western blot检测干性相关转录因子SOX2、OCT4和NANOG的表达,成球实验检测细胞的成球能力,确定PFKFB4对卵巢癌细胞干性的影响;利用自噬激活剂雷帕霉素处理卵巢癌细胞,检测SOX2、OCT4和NANOG的表达及细胞的成球能力,评估自噬对卵巢癌细胞干性的影响;Western blot检测自噬相关蛋白LC3B-Ⅱ和p62的表达,确定PFKFB4对卵巢癌细胞自噬的影响;生物信息学分析卵巢癌中PFKFB4与BNIP3表达的相关性;Real-time PCR和Western blot检测PFKFB4对BNIP3表达的影响。结果:过表达PFKFB4促进SOX2、OCT4和NANOG的表达和卵巢癌细胞的成球能力;雷帕霉素刺激增加SOX2、OCT4和NANOG的表达和卵巢癌细胞的成球能力;过表达PFKFB4增加卵巢细胞自噬水平,包括增加LC3B-Ⅱ蛋白表达、降低p62蛋白表达;生物信息学分析显示,卵巢癌组织中PFKFB4与BNIP3表达高度正相关;过表达PFKFB4增加BNIP3的mRNA和蛋白水平。结论:PFKFB4可能通过激活BNIP3介导的细胞自噬增强卵巢癌细胞的干性。

Objective To investigate the effects of fructose-2-kinase 6-phosphate/fructose-2,6-diphosphatase 4(PFKFB4)on the stemness of ovarian cancer cells and its mechanism.Methods Ovarian cancer SKOV3 and A2780 stable cell lines with PFKFB4 overexpression were constructed by lentivirus system.Real-time PCR and western blot analysis was performed to detect the expression of stemness-related transcription factors SOX2,OCT4 and NANOG.The sphere-forming ability of the cells was detected by sphere formation assay to determine the effect of PFKFB4 on the stemness of ovarian cancer cells.The expression of SOX2,OCT4 and NANOG and the sphere-forming ability of the cells were detected after treatment with rapamycin,an autophagy activator,to evaluate the effect of autophagy on the stemness of ovarian cancer cells.The expression of autophagy-related proteins LC3B-Ⅱand p62 was detected by western blot analysis to determine the effect of PFKFB4 on autophagy of ovarian cancer cells.The correlation between the expression of PFKFB4 and BNIP3 in ovarian cancer was analyzed by bioinformatics.Real-time PCR and western blot analysis was used to detect the effect of PFKFB4 on the expression of BNIP3.Results Overexpression of PFKFB4 promoted the expression of SOX2,OCT4 and NANOG and the sphere-forming ability of ovarian cancer cells.Rapamycin increased the expression of SOX2,OCT4 and NANOG and the sphere-forming ability of ovarian cancer cells.Overexpression of PFKFB4 increased the autophagy level of ovarian cells,including increasing the expression of LC3B-Ⅱprotein and decreasing the expression of p62 protein.Bioinformatics analysis showed that PFKFB4 and BNIP3 expression were highly positively correlated in ovarian cancer.Overexpression of PFKFB4 increased the mRNA and protein levels of BNIP3.Conclusion PFKFB4 may enhance the stemness of ovarian cancer cells by activating BNIP3-mediated autophagy.