摘要
目的探讨5-氮杂-2′-脱氧胞苷(5-Aza-2’-deoxycytidine,5-Aza-CdR)预处理人子宫内膜样癌(Endometrioid carcinoma,EC)JEC细胞后,雌激素受体(Estrogen receptor,ER)拮抗剂他莫昔芬(Tamoxifen,TAM)、氟维司群(ICI-182780,ICI)对JEC细胞生长、形态改变及p57^(kip2)蛋白表达的影响。方法将JEC细胞用5-Aza-CdR处理后,用亚硫酸氢盐测序(Bisulfite sequencing PCR,BSP)法检测细胞中p57^(kip2)启动子区的甲基化率。将JEC细胞分为6组:对照组、雌二醇(β-estradiol,E_(2))组、TAM组、ICI组、E_(2)+TAM组、E_(2)+ICI组,待5-Aza-CdR处理后,用甲基偶氮唑盐比色(MTT)法检测各组细胞培养24、48、72 h的增殖活性;用光镜和电镜观察培养72 h时细胞的形态改变,用蛋白印迹(Western blot)法检测培养72 h时细胞中p57^(kip2)蛋白的表达情况。结果用5-Aza-CdR处理后,JEC细胞中p57^(kip2)启动子区甲基化率从81.1%降为76.2%。与对照组比较,细胞增殖活性在E_(2)组均升高,在ICI组、E_(2)+TAM组、E_(2)+ICI组则均降低,且均在24 h改变最明显(P<0.05)。对照组细胞呈圆形或类圆形,细胞游离缘见少量微绒毛;与对照组相比,E_(2)组细胞密度明显升高,E_(2)+TAM组密度明显降低;在TAM组、ICI组、E_(2)+TAM组、E_(2)+ICI组中,可见更多的分泌泡,在TAM组、E_(2)+ICI组则可见明显的凋亡小体。与对照组比较,在E_(2)组中p57^(kip2)蛋白表达减少(P<0.05),在ICI组、E_(2)+TAM组、E_(2)+ICI组中p57^(kip2)蛋白表达均增高(P<0.05),以E_(2)+TAM组增高最明显。结论5-Aza-CdR可下调JEC细胞中p57^(kip2)基因启动子区的高甲基化水平,从而恢复抑癌因子p57^(kip2)的生物活性,逆转TAM对JEC细胞的弱雌激素样作用,增强ICI对JEC细胞增殖的抑制作用;还可能协同TAM、ICI上调p57^(kip2)蛋白的表达水平,拮抗E_(2)对p57^(kip2)蛋白水平的下调作用,达到削弱CDK的激酶活性,从而发挥负向调控细胞周期、抑制JEC细胞进一步生长的作用。
Objective To investigate the effects of estrogen receptor(ER)antagonist tamoxifen(TAM)and ICI-182780(ICI)on cell proliferation,morphology and the cell-cycle inhibitor p57^(kip2)protein expression in human endometrioid carcinoma JEC cells after demethylation agent 5-Aza-2’-deoxycytidine(5-Aza-CdR)pretreatment.Methods JEC cells were pretreated with 5-Aza-CdR followed by the treatments withβ-estradiol(E_(2)),TAM,ICI,E_(2)+TAM,E_(2)+ICI,and vehicle,respectively.Cell proliferation was determined by MTT assay at 24,48 and 72 h.Cell morphology was examined at 72 h under light and electron microscopy.The expression of p57^(kip2)protein was determined at 72 h by Western blot analysis.Results The methylation rate of p57^(kip2)protein was decreased by 5-Aza-CdR pretreatment from 81.1%to 76.2%.E_(2)increased cell proliferation.Cell proliferation was decreased in ICI,E_(2)+TAM and E_(2)+ICI groups,especially at 24 h.Control cells were round with a bit of microvilli on the edge of cells.The cell density was increased by E_(2)and decreased in E_(2)+TAM groups.More secretory vesicles were observed in TAM,ICI,E_(2)+TAM and E_(2)+ICI groups.Apoptotic bodies were found in TAM and E_(2)+ICI groups.Compared to control group,E_(2)decreased the expression of p57^(kip2)protein,while p57^(kip2)protein was increased in ICI,E_(2)+TAM and E_(2)+ICI groups.Conclusion 5-Aza-CdR decreases the hyper-methylation of the promoter region of p57^(kip2)gene and enhances the anti-proliferation effect of ICI and TAM against E_(2).Thus,5-Aza-CdR could act synergistically with TAM and ICI to decrease the kinase activity of CDK and negatively regulate cell cycle to inhibit estrogen-responsive JEC cells growth.
作者
钟栎
周俊
张春英
周正平
Zhong Yue;Zhou Jun;Zhang Chunying;Zhou Zhengping(Department of Pathology,the Affiliated Hospital of Zunyi Medical University,Zunyi Guizhou 563099,China;Department of Electron Microscopy,Zunyi Medical University,Zunyi Guizhou 563099,China;Department of Pathology,Zunyi Medical University,Zunyi Guizhou 563099)
出处
《遵义医科大学学报》
2021年第1期1-8,共8页
Journal of Zunyi Medical University
基金
贵州省科技合作计划资助项目(NO:黔科合基础[2017]1216)。
