精氨酸甲基转移酶PRMT7的自甲基化修饰及其在乳腺癌进程中的作用和机制研究

发布时间：2018-05-02 21:43

本文选题：PRMT7 + 自甲基化修饰　；参考：《东北师范大学》2017年博士论文

【摘要】：蛋白质的翻译后修饰是表观遗传学研究的核心领域之一,主要包括磷酸化修饰、乙酰化修饰、甲基化修饰、SUMO化修饰和泛素化修饰等。蛋白质的翻译后修饰的改变通常都能够影响蛋白质的活性和功能。精氨酸甲基化是一种普遍存在的翻译后修饰,由蛋白精氨酸甲基转移酶(Protein Arginine Methyltransferases,PRMTs)催化,将甲基基团从甲基供体(S-腺苷甲硫氨酸)上转移到精氨酸残基胍基的氮原子上。蛋白质精氨酸甲基化修饰可以激活或抑制基因的转录;参与细胞信号转导、细胞代谢、蛋白质稳定性和DNA损伤修复等过程。近些年的研究表明,异常的精氨酸甲基化修饰与多种疾病密切相关,特别是癌症。PRMT7是发现较晚的PRMTs家族成员,在基因组印记、DNA损伤应答以及细胞分化等过程中发挥着重要的作用。我们以前的研究工作发现,PRMT7在恶性程度高的乳腺癌组织和高转移的乳腺癌细胞中高表达。上调的PRMT7增加了E-cadherin启动子处抑制基因表达的H4R3me2s修饰水平;降低了激活基因表达的H3K4me3修饰水平,组蛋白H3和组蛋白H4的乙酰化修饰水平,抑制E-cadherin的表达。PRMT7的高表达,打破了组蛋白精氨酸甲基化修饰、组蛋白赖氨酸甲基化修饰和组蛋白乙酰化修饰之间的平衡,导致细胞间粘附分子E-cadherin的表达降低,诱发乳腺癌细胞发生上皮细胞间质细胞转换(Epithelial-Mesenchymal Transition,EMT),促进乳腺癌的转移。在本课题的研究中,我们发现PRMT7的第531位精氨酸(PRMT7 R531)在体外和体内均可以发生自甲基化修饰;PRMT7的自甲基化修饰对于PRMT7所介导的EMT及乳腺癌细胞的迁移和侵袭是必须的;利用CRISPR/Cas9技术敲除高转移的MDA-MB-231细胞中本底表达的PRMT7后,细胞的迁移和侵袭能力明显下降,然而再外源过表达野生型的PRMT7恢复了细胞的迁移和侵袭能力,而再外源过表达自甲基化修饰位点突变的PRMT7则没有;随后,小鼠肺转移实验证实,PRMT7 WT同PRMT7R531K相比,明显地促进了乳腺癌细胞MCF7的远端转移;进一步,免疫组化实验显示高表达的甲基化的PRMT7与临床的乳腺癌样本具有一定的相关性,50%III期乳腺癌样本呈现出高表达的甲基化的PRMT7,47.2%三阴性乳腺癌(TNBC)样本中呈现出甲基化的PRMT7的高表达。进一步研究表明,PRMT7的自甲基化修饰增强了PRMT7与转录因子YY1之间的相互作用能力,从而增加了PRMT7到E-cadherin启动子处的招募,导致H4R3me2s水平升高,H3K4me3水平降低,从而抑制E-cadherin的表达,促进乳腺癌转移。综上,我们发现了PRMT7蛋白的一种新的翻译后修饰-自甲基化修饰,这种自甲基化修饰与乳腺癌转移密切相关。该研究为以PRMT7作为乳腺癌治疗的新靶标提供了新的理论基础和实验依据。
[Abstract]:Posttranslational modification of proteins is one of the core areas of epigenetics, including phosphorylation modification, acetylation modification, methylation modification, sumo modification and ubiquitin modification. Changes in post-translational modification of proteins usually affect the activity and function of proteins. Arginine methylation is a common posttranslational modification catalyzed by protein Arginine Methyltransferasesl PRMTs, which transports the methyl group from the methyl donor S- adenosine methionine to the nitrogen atom of the arginine residue guanidine. Protein arginine methylation can activate or inhibit gene transcription, participate in cell signal transduction, cell metabolism, protein stability and DNA damage repair. Recent studies have shown that abnormal arginine methylation is closely associated with many diseases, especially cancer. PRMT7 is a member of the PRMTs family that was discovered later. It plays an important role in DNA damage response and cell differentiation of genomic imprinting. Our previous studies have found that PRMT7 is highly expressed in breast cancer tissues with high malignancy and in breast cancer cells with high metastasis. The up-regulated PRMT7 increased the H4R3me2s modification level of inhibiting gene expression at the E-cadherin promoter, decreased the H3K4me3 modification level of activating gene expression, the acetylation modification level of histone H3 and histone H4, and inhibited the overexpression of E-cadherin. The balance between histone arginine methylation modification, histone lysine methylation modification and histone acetylation modification was broken, and the expression of intercellular adhesion molecule E-cadherin was decreased. Epithelial-mesenchymal transition (EMT) was induced in breast cancer cells to promote the metastasis of breast cancer. In this study, we found that the self-methylation modification of PRMT7 is necessary for the migration and invasion of EMT and breast cancer cells mediated by PRMT7, both in vitro and in vivo. The ability of migration and invasion of MDA-MB-231 cells was significantly decreased after knockout of background expression of PRMT7 in MDA-MB-231 cells with high metastasis by CRISPR/Cas9 technique. However, the ability of migration and invasion was restored by overexpression of wild type PRMT7. However, PRMT7, which was overexpression of automethylation modification site mutation, was not. Subsequently, the mouse lung metastasis test confirmed that PRMT7WT significantly promoted the distal metastasis of MCF7 in breast cancer cells compared with PRMT7R531K. Immunohistochemical analysis showed that high expression of methylated PRMT7 was associated with clinical breast cancer samples. The high expression of methylated PRMT7 was found in 47.2% tri-negative breast cancer samples with high methylation in phase III breast cancer samples. Further studies showed that the self-methylation of PRMT7 enhanced the interaction between PRMT7 and transcription factor YY1, increased the recruitment of PRMT7 to E-cadherin promoter, increased the level of H4R3me2s and decreased the level of H3K4me3, thus inhibited the expression of E-cadherin. Promote metastasis of breast cancer. In conclusion, we have found a new posttranslational modification of PRMT7 protein, self-methylation modification, which is closely related to breast cancer metastasis. This study provides a new theoretical and experimental basis for using PRMT7 as a new target for breast cancer treatment.
【学位授予单位】：东北师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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