沉默DAXX基因表达促进人卵巢癌细胞凋亡和衰老

Silencing DAXX Gene Promotes Apoptosis and Senescence in Cultured Ovarian Cancer Cells

DAXX是Fas死亡结构域相关蛋白(Fas death-associated protein,DAXX),可与Fas死亡受体的死亡域结合.通过激活c-Jun NH2末端激酶通路,DAXX增强Fas介导的凋亡,同时也增强转录生长因子β依赖的凋亡.本研究通过免疫组化检测人正常卵巢组织和卵巢癌组织中DAXX蛋白表达,然后通过Tet-on诱导体系构建DAXX基因沉默的慢病毒,感染卵巢癌细胞后,加入嘌呤霉素筛选稳定表达的OV2008细胞,四环素诱导DAXX基因沉默,通过免疫印迹和定量PCR检测DAXX基因干扰效果,MTT检测细胞增殖,克隆形成实验检测克隆形成能力,免疫印迹和免疫荧光方法检测DNA损伤蛋白的变化,SA-β-gal检测细胞衰老.结果表明,在人正常卵巢组织中DAXX低表达,而在人卵巢癌组织中DAXX高表达,随着四环素浓度的增加DAXX的表达量降低,DAXX基因沉默抑制卵巢癌OV2008细胞的增殖和克隆形成.免疫印迹结果显示,DAXX基因沉默可诱导DNA损伤相关蛋白p-H2AX和p-CHK2高表达.SA-β-gal检测结果表明,DAXX基因沉默可诱导OV2008细胞发生衰老,免疫印迹检测发现,p21和p27在DAXX沉默的卵巢癌细胞中高表达.综上结果,DAXX沉默可以抑制卵巢癌细胞的增殖和克隆形成,同时也促进卵巢癌细胞发生DNA损伤和衰老.该研究为卵巢癌的基因治疗提供新的思路.

DAXX was initially identified as a pro-apoptotic protein that bound to the death domain of the CD95 death receptor. By activating the c-Jun NH2-terminal kinase-pathway, DAXX was showed to enhance both CD95-mediated and transforming growth factor-β-dependent apoptosis. In this study, we constructed a tetracycline-inducible （tet-on） DAXX silencing lentiviral vector, packaged lentiviral particles, then infected 0V2008 cells, cells were selected and cultured in complete cell culture medium along with either puromycin （lμg/mL） for 7 - 10 days. DAXX was efficiently knocked down in these cells with tetracycline. DAXX protein and gene expression were detected by Western blotting and Q-PCR,cells proliferation and clone number of DAXX-depleted 0V2008 cells were detected by MTT assay and colony forming assay. DNA damage protein expression of DAXX-depleted cells was detected by Western blotting and immunofluorescence. Senescent cells were detected by SA-β-galactosidase staining. The results showed that tetracycline induced dose-dependent decrease of DAXX. DAXX depletion significantly inhibited the proliferation and colony forming of OV2008 cells, as assessed by MTT proliferation assay and colony forming assay. Western blotting results showed that p-H2AX and p-CHK2 expressions were significantly increased after DAXX silencing. DAXX silencing induced senescence were determined by SA-β-gal staining. Western blotting results showed that p21 and p27 expressions were significantly increased after DAXX silencing. The results showed that DAXX silencing inhibited ovarian cancer cell proliferation, colony forming, and promoted DNA damage insults and senescence. Our results give new idea in the field of gene therapy for ovarian cancer.