USP7通过调节组蛋白去甲基化酶PHF8的稳定促进乳腺癌的发生与发展

发布时间：2018-08-26 08:45

【摘要】：PHF8属于组蛋白去甲基化家族成员,在脑部发育和神经发育的过程中起到重要作用;同时PHF8还能参与多种病理过程,包括X染色体连锁智力发育迟缓和肿瘤发生。但是PHF8自身的异常调控以及PHF8在癌症发生发展过程中的分子机制尚不明确。在该研究中我们我们通过体内外的去泛素化实验阐明了USP7通过其去泛素化酶活性去除PHF8的泛素基团,从而稳定PHF8。接下来,我们运用RNA-seq技术筛选出两者共同调控转录的靶基因cyclin A2,通过免疫组化、细胞及动物模型的表型实验,证实了PHF8/USP7通过上调cyclin A2的转录从而影响乳腺癌细胞的增殖。进一步,我们发现在DNA损伤时,PHF8与USP7的相互作用有所加强;USP7介导的PHF8稳定性调节可以通过促进细胞的双链损伤修复使细胞抵抗DNA损伤。目的探索PHF8自身异常调控以及PHF8在癌症发生发展过程中的分子机制。结果：1、PHF8与USP7具有直接相互作用。通过免疫亲和纯化与质谱分析鉴定出了与PHF8有相互作用的蛋白质去泛素化酶USP7,并且运用免疫共沉淀和昆虫细胞纯化蛋白在体内、体外验证了它们存在直接相互作;PHF8的C端不含明显结构域的部分与USP7的N端的MATH结构域为二者相互作用所依赖的结构域。2、USP7通过去泛素化酶活性特异性地稳定PHF8蛋白水平。USP7能够通过相互作用与PHF8结合,再通过其自身去泛素化酶活性,催化去除PHF8上多聚泛素链,抑制PHF8通过泛素-蛋白酶体途径发生降解。3、USP7/PHF8调控轴能够正向激活cyclin A2信号通路。PHF8和USP7能够共同激活多个下游靶基因,这些靶基因在细胞周期、DNA复制等通路中发挥重要作用。其中,cyclin A2是USP7和PHF8共同的下游靶基因,USP7和PHF8能够正向调控CCNA2勺转录;PHF8能够直接结合在CCNA2的启动子区域;USP7则通过PHF8间接调节CCNA2的转录。4、PHF8能够调节USP7的转录,两者形成正反馈循环。USP7同时也是PHF8的下游靶基因,能够受到PHF8的正向调节。两者形成正反馈的回路：一方面,USP7在蛋白水平上稳定PHF8的蛋白水平;另一方面,PHF8又可以调节USP7的mRNA水平,促进USP7的转录。两者相互依存,相互影响,相互促进。5、USP7/PHF8调控轴促进乳腺癌细胞的增殖与肿瘤生长。USP7通过去泛素化PHF8上调cyclin A2的表达;该效应在体外促进乳腺癌细胞的生长与增殖,在体内促进乳腺癌的发生与发展。6、USP7和PHF8在多种肿瘤组织中的表达上调,并与乳腺癌的恶性程度呈正相关。USP7和PHF8的蛋白及mRNA水平在乳腺中高表达,并且与乳腺癌的临床分级呈正相关在这些样本中,USP7和PHF8的表达水平与cyclin A2也呈现正相关。7、在DNA损伤反应应答中,USP7介导的PHF8稳定性调节可以促进乳腺癌细胞对抗DNA损伤。在DNA双链损伤发生时,USP7能够通过加强与PHF8的相互作用和增强去除PHF8多聚泛素链,进而增强同源重组修复(HR)和非同源重组末端链接(NHEJ)的修复效率,提高乳腺癌细胞对DNA损伤的耐受能力。结论：综上所述,本课题发现PHF8与USP7在体内、体外有特异的相互作用。USP7能够通过其自身的去泛素化酶活性特异性地稳定PHF8的蛋白水平,抑制PHF8通过泛素-蛋白酶体途径发生降解。USP7介导的PHF8蛋白的稳定性能激活cyclin A2信号通路,从而促进乳腺癌细胞生长和增殖能力。PHF8也能调控USP7的转录活性,正向介导USP7的转录,与USP7形成正反馈回路。在组织样本分析中,我们发现USP7/PHF8/cyclin A2在多种肿瘤中高表达,与乳腺癌的恶性程度分级呈正相关。最后,在DNA损伤反应应答中,USP7和PHF8的相互作用和功能有所增强,并通过增强HR和NHEJ的修复效率提高乳腺癌细胞对抗DNA损伤能力。
[Abstract]:PHF8 is a member of the histone demethylation family and plays an important role in brain development and neurodevelopment. PHF8 is also involved in a variety of pathological processes, including X-linked mental retardation and tumorigenesis. In this study, we demonstrated that USP7 can stabilize PHF8 by removing the ubiquitin group of PHF8 through its activity of ubiquitination enzymes in vivo and in vitro. Next, we used RNA-seq technology to screen the target gene cyclin A2 which co-regulates transcription. Through immunohistochemistry, cell and animal model phenotypic experiments, we identified the two genes. It is confirmed that PHF8/USP7 can affect the proliferation of breast cancer cells by up-regulating the transcription of cyclin A2. Further, we found that the interaction between PHF8 and USP7 was enhanced during DNA damage. USP7-mediated regulation of PHF8 stability can make cells resist DNA damage by promoting the repair of double-strand damage. Results: 1. PHF8 interacted with USP7 directly. Protein deubiquitinase USP7 interacting with PHF8 was identified by immunoaffinity purification and mass spectrometry analysis, and their existence was verified in vitro by immunoprecipitation and insect cell purification in vivo. The MATH domain of the N-terminal of USP7 and the C-terminal of PHF8 are dependent on the interaction of the two domains. 2. USP7 stabilizes the level of PHF8 protein specifically by the activity of deubiquitinase. USP7 can bind to PHF8 through the interaction and catalyze the removal of PHF8 by its own deubiquitinase activity. Polyubiquitin chains inhibit the degradation of PHF8 via ubiquitin-proteasome pathway. 3. USP7/PHF8 regulatory axis can activate cyclin A2 signaling pathway. PHF8 and USP7 can activate multiple downstream target genes, which play an important role in cell cycle, DNA replication and other pathways. Target genes, USP7 and PHF8 can positively regulate CCNA2 spoon transcription; PHF8 can directly bind to the promoter region of CCNA2; USP7 can indirectly regulate CCNA2 transcription through PHF8; and PHF8 can regulate the transcription of USP7 and form a positive feedback loop. USP7 is also a downstream target gene of PHF8 and can be positively regulated by PHF8. The pathway: on the one hand, USP7 stabilizes the protein level of PHF8 at the protein level; on the other hand, PHF8 regulates the mRNA level of USP7 and promotes the transcription of USP7. They are interdependent and mutually reinforcing. 5. USP7/PHF8 axis promotes the proliferation and tumor growth of breast cancer cells. USP7 upregulates cyclin A2 expression through deubiquitination of PHF8. The expression of USP7 and PHF8 was up-regulated in various tumor tissues and positively correlated with the malignant degree of breast cancer. The protein and mRNA levels of USP7 and PHF8 were highly expressed in breast and positively correlated with the clinical grading of breast cancer. The expression levels of USP7 and PHF8 were also positively correlated with cyclin A2 in these samples. In DNA damage response, USP7-mediated regulation of PHF8 stability could promote breast cancer cells to resist DNA damage. To enhance the repair efficiency of homologous recombinant repair (HR) and non-homologous recombinant end-linking (NHEJ) and enhance the tolerance of breast cancer cells to DNA damage. Conclusion: In summary, this study found that PHF8 and USP7 have specific interactions in vivo and in vitro. USP7 can specifically stabilize PHF8 protein through its own ubiquitin-depleting enzyme activity. The stability of PHF8 protein mediated by USP7 can activate cyclin A2 signaling pathway, thereby promoting the growth and proliferation of breast cancer cells. PHF8 can also regulate the transcriptional activity of USP7, mediate the transcription of USP7, and form a positive feedback loop with USP7. It was found that USP7/PHF8/cyclin A2 was highly expressed in various tumors and was positively correlated with the malignant grade of breast cancer. Finally, in DNA damage response, the interaction and function of USP7 and PHF8 were enhanced, and the ability of breast cancer cells to resist DNA damage was enhanced by enhancing the repair efficiency of HR and NHEJ.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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