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学科主题生物化学与分子生物学
顺铂诱导的细胞衰老过程中p21与p16表达上调的机制
刘玲
2016-05-12
学位类型硕士
导师倪菊华
专业医学生物化学与分子生物学
授予单位北京大学
授予地点北京大学基础医学院
学位硕士
关键词顺铂 细胞早衰 p21 p16 DNA甲基化
其他题名Mechanisms Underlying the Upregulation of p21 and p16 in Cisplatin-induced Cellular Senescence
分类号R73-36
页数46
摘要

低剂量顺铂可通过上调p21与p16表达而诱导肿瘤细胞衰老,但其作用机制不明。本研究以HeLa细胞系为主要研究体系,对低剂量顺铂诱导细胞衰老过程中p21与p16的上调机制做了探讨。

用低剂量顺铂处理HeLa细胞,通过细胞衰老相关的β-半乳糖苷酶染色实验发现细胞被诱导衰老。在顺铂诱导HeLa细胞衰老的过程中,借助蛋白质免疫印迹技术,可见DNA甲基转移酶DNMT1蛋白水平降低,p21与p16蛋白水平升高。DNA甲基化分析实验揭示,低剂量顺铂处理可降低p21与p16的启动子甲基化水平。在HeLa细胞中敲减DNMT1,通过蛋白质免疫印迹与实时定量PCR实验发现,p21与p16的蛋白质与mRNA水平均有显著升高,说明p21与p16是DNMT1的靶分子。进一步研究发现,顺铂对DNMT1蛋白水平的降低作用与其激活p38MAPK有关。用p38MAPK特异性抑制剂SB203580预处理细胞,可部分逆转顺铂对DNMT1蛋白水平以及p21与p16启动子甲基化的降低作用,进而部分逆转顺铂对p21与p16表达的诱导。此外,我们发现抑制p38MAPK活性也可部分逆转顺铂诱导的HeLa细胞早衰。

另一方面,我们在HeLa细胞中敲减RNA甲基转移酶NSun2,通过蛋白质免疫印迹实验发现p21与p16的蛋白水平有明显上升。通过NCBI数据库检索与质谱分析,我们发现NSun2分子存在潜在的p38MAPK磷酸化位点,但体外磷酸化实验未发现p38MAPK可磷酸化NSun2,因此需要更多的研究来探明两者的关系。

总之,我们的结果表明,顺铂可通过p38MAPK信号通路下调由DNMT1介导的p21与p16启动子甲基化水平,进而上调二者的表达。这些结果为解析低剂量顺铂诱导肿瘤细胞衰老的信号转导机制提供了实验依据。

英文摘要

Low-dose cisplatin induces cellular senescence of cancer cells by elevating the expression of p21 and p16. However, the mechanisms underlying cisplatin-induced upregulation of p21 and p16 are not fully elucidated. In the present study, HeLa cells were employed to explore the regulatory machanisms controlling the upregulation of p21 and p16 in low-dose cisplatin-induced cellular senescence.

Proved by SA-β-gal staining, we found that low-dose cisplatin induced HeLa cells senescence. Western blot analysis showed that treatment of HeLa cells by low-dose cisplatin decreased the protein levels of DNMT1 and raised the protein levels of p21 and p16. Methylation-specific PCR revealed that low-dose cisplatin reduced the promoter methylation of p21 and p16. The combination of knockdown of DNMT1 in HeLa cells with Western blot analysis and real-time quantitative PCR demonstrated that the protein and mRNA levels of p21 and p16 were dramatically improved, indicating that p21 and p16 were targets of DNMT1. Activation of p38MAPK is responsible for the reduction of DNMT1 in response to low-dose cisplatin, since pre-incubating cells with p38MAPK inhibitor SB203580 could partly reverse the effect of ciaplatin in reducing the protein levels of DNMT1 and the promoter methylation of p21 and p16, thereby reducing the upregulation of mRNA levels of p21 and p16. Furthermore, inhibition of p38MAPK by SB203580 reversed cisplatin-induced premature senescence.

On the other hand, the combination of knockdown of RNA methyltransferase NSun2 in HeLe cells with Western blot analysis showed that silencing NSun2 obviously improved the protein levels of p21 and p16. In addiation, searching in the datebase of National Center for Biotechnology Information (NCBI) and mass spectrum analysis revealed that NSun2 contained potential sites phosphorylated by p38MAPK. However, the in vitro phosphorylation analysis failed to prove that. It may require more researches to study this point.

In sum, we have demonstrated that cisplatin reduces the promoter methylation of p21 and p16 mediated by DNMT1 via activating p38MAPK, which in turn, increases the expression of p21 and p16 in premature senescence. These studies provide evidence for elucidating the mechanisms underlying cisplatin-induced cellular senescence.

语种中文
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文献类型学位论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/149409
专题北京大学基础医学院
北京大学第三临床医学院_儿科
作者单位北京大学基础医学院
第一作者单位北京大学基础医学院
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刘玲. 顺铂诱导的细胞衰老过程中p21与p16表达上调的机制[D]. 北京大学基础医学院. 北京大学,2016.
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