p53-p21通路抑制组蛋白甲基转移酶NSD2表达的机制及NSD2在DNA损伤中的功能研究

发布时间：2018-08-31 18:27

【摘要】：NSD2是一种在黑色素瘤等多种肿瘤中高表达的组蛋白甲基转移酶,其在Wolf-Hirschhorn syndrome(WHS)和多发性骨髓瘤(multiple myeloma,MM)中表达失控的原因已经得到了较好的阐明,而NSD2在其他多种肿瘤中的表达失调的原因和机制还未有报道。本论文以p53野生型的人黑色素瘤细胞系92-1作为细胞模型,首先通过X-ray、UV、依托泊苷和羟基脲四种DNA损伤处理,证明DNA损伤后NSD2表达下调是一种普遍现象。接着采用依托泊苷和羟基脲两种药物处理,结合RNA干扰技术(RNA interference,RNAi)技术,通过荧光定量PCR和蛋白质免疫印迹,证明p53直接抑制NSD2的转录,而不是通过其下游基因p21来执行此项功能。实验结果暗示细胞衰老时p21有可能介导NSD2蛋白的降解,通过应用蛋白酶体抑制剂MG-132和敲低APC/C激活因子cdh1,阐明了一条细胞衰老中介导NSD2降解的信号通路:p53-p21-APC/C~(cdh1)-蛋白酶体。通过比较NSD2在羟基脲处理和X-ray辐照处理后DNA损伤应答(DNA damage response,DDR)的差异,发现敲低NSD2使羟基脲处理引起的p-CHK2和γ-H2AX水平升高得到显著抑制,而对X-ray辐照处理引起的p-CHK2和γ-H2AX水平升高则没有明显影响,这说明NSD2在不同的DNA损伤响应通路发挥的功能不同。以上三点结论之前文献均未见报道,也是本论文的创新之处。
[Abstract]:NSD2 is a highly expressed histone methyltransferase in melanoma and other tumors. The reasons for its uncontrolled expression in Wolf-Hirschhorn syndrome (WHS) and multiple myeloma (multiple myeloma,MM) have been well elucidated. However, the cause and mechanism of NSD2 expression imbalance in other tumors have not been reported. In this paper, the p53 wild-type human melanoma cell line 92-1 was used as the cell model. Firstly, four kinds of DNA damage treatments, X-raybuv, etoposide and hydroxyurea, proved that the down-regulation of NSD2 expression was a common phenomenon after DNA injury. Then, etoposide and hydroxyurea were treated with RNA interference technique (RNA interference,RNAi), fluorescence quantitative PCR and Western blot were used to prove that p53 could directly inhibit the transcription of NSD2. This function is not performed through the downstream gene p21. The results suggested that p21 might mediate the degradation of NSD2 protein during cell senescence. By using proteasome inhibitor MG-132 and knockdown APC/C activator cdh1, a signal pathway of NSD2 degradation mediated by cell senescence was elucidated. The signal pathway: p53-p21-APC-C ~ (cdh1) -proteasome was elucidated by p53-p21-APC-C ~ (cdh1) -proteasome. By comparing the difference of DNA damage response (DNA damage response,DDR between hydroxyurea treatment and X-ray irradiation, it was found that knockdown of NSD2 significantly inhibited the increase of p-CHK2 and 纬 -H2AX levels induced by hydroxyurea treatment. However, there was no significant effect on p-CHK2 and 纬 -H2AX levels induced by X-ray irradiation, which indicated that NSD2 had different functions in different DNA damage response pathways. The above three conclusions have not been reported before, which is the innovation of this paper.
【学位授予单位】：中国科学院研究生院(近代物理研究所)
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R730.2

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