锌指蛋白403对卵巢上皮IOSE80细胞增殖的影响

Effects of Zinc Finger Protein 403 on the Proliferation of Ovarian Epithelial IOSE80 Cells

目的探讨锌指蛋白403(zinc finger protein 403,ZFP403)在卵巢上皮细胞中的生物学功能及相关机制。方法通过构建ZFP403-shRNA慢病毒,转染卵巢上皮细胞IOSE80,嘌呤霉素抗性筛选得到稳定沉默ZFP403的细胞株;通过qRT-PCR和Western blotting评价ZFP403沉默效果;采用平板集落形成试验评价沉默ZFP403后细胞生长能力的变化;采用软琼脂集落形成试验评价沉默ZFP403后细胞锚着非依赖性生长能力;通过流式细胞术评价沉默ZFP403后对细胞周期的影响;通过Westernblotting试验研究沉默ZFP403后影响细胞周期的相关机制。结果经嘌呤霉素筛选成功构建了ZFP403稳定沉默的细胞株(IOSE80-ZFP403-shRNA1,2)及阴性的对照细胞株(IOSE80-shRNA-NC);平板集落形成试验结果表明沉默ZFP403能明显促进上皮细胞的生长;软琼脂集落形成试验结果表明沉默ZFP403后,上皮细胞具有锚着非依赖性增长能力;流式细胞术结果表明沉默ZFP403能明显促进细胞周期进程;Western blotting结果表明ZFP430通过影响p-cdc2,cdc2,cyclinB1,p21蛋白的表达而影响细胞周期。结论ZFP403通过影响卵巢上皮细胞的异常生长而发挥其生物学功能。ZFP403的表达水平可能影响了卵巢上皮细胞向卵巢癌的转变,具有作为早期卵巢癌临床诊断标志物的可能性。

OBJECTIVE To explore the biological function and related mechanism of zinc finger protein 403(ZFP403)in ovarian epithelial cell.METHODS ZFP403 shRNA lentivirus was constructed and transfected into human ovarian epithelial cell line IOSE80 and screened by puromycin;qRT-PCR and Western blotting were used to detect the silence effect of ZFP403,respectively;the cell growth ability after knockdown of ZFP403 was evaluated by plate colony formation assay;soft agar colony formation assay was used to evaluate the anchorage independent growth ability;the effect of ZFP403 on cell cycle was detected by flow cytometry;Western blotting was used to study the mechanism of ZFP403 on cell cycle related proteins.RESULTS ZFP403 stably silenced cell lines(IOSE80-ZFP403-shRNA1,2)and negative control cell line(IOSE80-shRNA-NC)were successfully constructed after puromycin screening;flow cytometry showed that knockdown of ZFP403 could significantly promote the growth and anchorage independent growth in IOSE80 cells;Western blotting showed that knockdown of ZFP403 could significantly promote cell cycle progression;the expression of p-cdc2,p21 was downregulated and cdc2,cyclinB1 upregulated after ZFP403 knockdown.CONCLUSION ZFP403 promotes the abnormal growth of ovarian epithelial cells.The expression level of ZFP403 may affect the transformation of ovarian epithelial cells to ovarian cancer,which may be used as a clinical diagnostic marker for early ovarian cancer.