细胞周期调控蛋白Nip在DNA损伤修复中的生物学功能和分子机制研究

发布时间：2018-10-24 22:16

【摘要】：正常情况下,环境中多种有害因素,包括电离辐射(ionizing radiation, IR)、紫外线照射(ultraviolet irradiation, UV)和化学致癌剂等都可能导致细胞DNA损伤。随着生物进化,生物界产生了多种DNA修复途径,例如碱基切除修复(base excision repair, BER)、核苷酸切除修复(nucleotide excision repair, NER)、同源重组(homologous recombination, HR)和非同源末端连接(no n-ho mo logo us end joining, NHEJ)等。DNA损伤修复对维护基因组完整性至关重要。DNA修复异常很可能导致基因组的不稳定性和细胞的恶性转化,进而导致肿瘤的发生。对DNA修复的长期深入研究,为临床肿瘤治疗提供了大量依据。DNA修复机制复杂多样,是科学界的研究热点之-中心体蛋白N1p (ninein-like protein)是我室通过酵母双杂交实验获得的与BRCA1相关的蛋白。多项研究结果表明,Nlp能参与微管生发、纺锤体形成、中心体成熟以及胞质分裂等多种生物学过程。虽然Nlp在细胞周期中发挥重要的调控功能,是有丝分裂进程所必需的。但是它在其他方面的功能还不清楚。本课题的前期工作中,我们用UV照射肺癌细胞系H1299后,发现细胞内Nlp的定位发生明显改变。这提示我们Nlp可能参与UV导致的DNA损伤应答。为了验证该假设,我们通过宿主细胞再活化反应(HCR)和CPDs移除速率检测的方法,发现Nlp能够促进核苷酸切除修复(nucleotide excision repair, NER)通路的活性。为了进一步研究Nlp的功能,我们利用两种细胞系、Nlp转基因小鼠与对照鼠MEF和新生小鼠皮肤分别进行了UV照射后的细胞活性、克隆形成、流式凋亡以及原位凋亡检测,发现Nlp具有保护细胞抵抗紫外线辐射引起的细胞凋亡的功能。为了探索其相关机制,我们通过UV局部照射的方法发现UV照射后Nlp定位到DNA损伤位点上。之后,通过核蛋白抽提、western blotting、co-IP、GST pull-down等方法,发现UV照射可导致Nlp向细胞核内转运。Nlp的入核受NPM1调控,且与p53无关。Nlp的C末端(1030-1382)介导了其与NPM1相互作用及其入核。另外,Nlp能够分别与XPA和ERCC1相互作用,并且促进两者结合,从而发挥促进DNA损伤修复的功能。除此之外,鉴于UV辐射与人皮肤癌的发生有明确相关性,我们通过免疫组化的方法,发现人皮肤癌样本中Nlp蛋白表达水平明显低于正常对照组。这揭示了Nlp表达异常很可能参与人类皮肤癌的发生发展过程。综上所述,本研究发现并证明了细胞周期调控蛋白Nlp是核苷酸切除修复(NER)通路中一个新的且重要的成员,从而为了解核苷酸切除修复通路的调控机制提供了新的见解,为肿瘤的治疗提供了新的思路。
[Abstract]:Under normal circumstances, many harmful factors in the environment, including ionizing radiation (ionizing radiation, IR), ultraviolet irradiation of (ultraviolet irradiation, UV) and chemical carcinogens, may lead to cell DNA damage. As organisms evolve, the biological world produces a variety of DNA repair pathways. For example, base excision repair (base excision repair, BER), nucleotide excision repair (nucleotide excision repair, NER), homologous recombination (homologous recombination, HR) and non-homologous terminal junction (no n-ho mo logo us end joining, NHEJ), etc.) DNA damage repair is essential to maintain genomic integrity. DNA repair abnormality Which is likely to lead to genomic instability and malignant cell transformation. This in turn leads to tumorigenesis. The long-term in-depth study of DNA repair provides a large number of evidences for clinical tumor treatment. The mechanism of DNA repair is complex and diverse. Centrosomal protein N1p (ninein-like protein) is a protein related to BRCA1 obtained by yeast two-hybrid experiment in our laboratory. Many studies have shown that Nlp is involved in many biological processes, such as microtubule formation, spindle formation, centrosomal maturation and cytokinesis. Although Nlp plays an important regulatory role in cell cycle, it is necessary for mitosis. But its other functions are unclear. In our earlier work, we found that the localization of Nlp in lung cancer cell line H1299 was significantly changed after irradiation with UV. This suggests that our Nlp may be involved in the DNA damage response induced by UV. In order to verify this hypothesis, we found that Nlp can promote the activity of nucleotide excision repair (nucleotide excision repair, NER) pathway through the detection of (HCR) and CPDs removal rate of the host cell reactivation reaction. In order to further study the function of Nlp, we used two kinds of cell lines, Nlp transgenic mice, control mice, MEF and newborn mice skin, respectively, to observe the cell activity, clone formation, flow rate apoptosis and in situ apoptosis after UV irradiation. It was found that Nlp could protect cells from apoptosis induced by ultraviolet radiation. In order to explore its mechanism, we found that Nlp was located on the site of DNA damage after UV irradiation by means of local UV irradiation. Then, by means of nuclear protein extraction, western blotting,co-IP,GST pull-down and other methods, it was found that UV irradiation could induce the translocation of Nlp into the nucleus. The nucleation of Nlp was regulated by NPM1, and the C terminal of Nlp (1030-1382) mediated its interaction with NPM1 and its nuclear entry. In addition, Nlp can interact with XPA and ERCC1, and promote the combination of them, thus promoting the function of DNA damage repair. In addition, in view of the clear correlation between UV radiation and the occurrence of human skin cancer, we found that the expression of Nlp protein in human skin cancer samples was significantly lower than that in the normal control group by immunohistochemical method. This suggests that abnormal Nlp expression may be involved in the occurrence and development of human skin cancer. To sum up, this study has found and proved that the cell cycle regulatory protein Nlp is a new and important member of nucleotide excision repair (NER) pathway, thus providing a new understanding of the regulation mechanism of nucleotide excision repair pathway. It provides a new idea for the treatment of tumor.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R730.5

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