丝裂霉素C调控miR-200b启动子甲基化诱导成纤维细胞凋亡

发布时间：2018-11-01 11:32

【摘要】：目的:探讨丝裂霉素C(MMC)调控mi R-200b表达诱导人成纤维细胞凋亡的潜在机制。方法:体外培养人硬膜外瘢痕组织中提取的成纤维细胞,用不同浓度的MMC处理后,采用Real-time PCR检测mi R-200b表达变化,TUNEL染色分析成纤维细胞凋亡情况,Western blot检测凋亡相关蛋白cleaved-PARP、Bax、Bcl-2表达的变化,亚硫酸氢盐(BSP)法测定mi R-200b启动子区域甲基化程度的改变。结果:Real-time PCR结果显示MMC处理后,成纤维细胞中mi R-200b表达量显著下降,差异具有统计学意义(P0.05),TUNEL染色结果表明MMC能够显著地诱导人成纤维细胞的凋亡,差异具有统计学意义(P0.05)。Western blot结果显示MMC能够升高cleaved-PARP、Bax表达,降低Bcl-2表达。MMC处理后mi R-200b启动子区域甲基化程度发生了变化,随着药物浓度的增加,甲基化程度不断增加。结论:MMC通过下调mi R-200b诱导人成纤维细胞凋亡,可能是通过促进其启动子区甲基化实现的。
[Abstract]:Aim: to investigate the potential mechanism of mitomycin C (MMC) regulating the expression of mi R-200b in inducing apoptosis of human fibroblasts. Methods: fibroblasts extracted from human epidural scar tissue were cultured in vitro. After treated with different concentrations of MMC, the expression of mi R-200b was detected by Real-time PCR, and the apoptosis of fibroblasts was analyzed by TUNEL staining. The expression of apoptosis-related protein (cleaved-PARP,Bax,Bcl-2) was detected by Western blot, and the methylation degree of mi R-200b promoter was determined by (BSP) method. Results: Real-time PCR results showed that the expression of mi R-200b in fibroblasts decreased significantly after MMC treatment, and the difference was statistically significant (P0.05), TUNEL staining showed that MMC could significantly induce apoptosis of human fibroblasts. The difference was statistically significant (P0.05). Western blot results showed that MMC could increase the expression of cleaved-PARP,Bax and decrease the expression of Bcl-2. The methylation degree of mi R-200b promoter region changed with the increase of drug concentration after MMC treatment. The degree of methylation is increasing. Conclusion: MMC induces apoptosis of human fibroblasts by down-regulating mi R-200b, which may be achieved by promoting methylation of its promoter region.
【作者单位】：扬州大学临床医学院(苏北人民医院)骨科;
【基金】：国家自然科学基金青年项目(编号:81301550) 江苏省六大人才高峰资助项目(编号:2015-WSN-108)
【分类号】：R68

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