本文選題：PRMT5　切入點：NF-Y　出處：《暨南大學》2015年博士論文　論文類型：學位論文

【摘要】：PRMT5(Protein arginine methyltransferase 5),即蛋白精氨酸甲基化轉移酶5,它屬于II型甲基化轉移酶,可以對稱性地甲基化組蛋白或者非組蛋白底物的精氨酸殘基,通過表觀遺傳的方式調控靶基因的表達或通過翻譯后甲基化修飾途徑調節(jié)關鍵的信號分子。最近的研究表明,PRMT5在許多人類腫瘤如肺癌、卵巢癌、結腸直腸癌、乳腺癌、黑色素瘤、白血病或淋巴瘤及惡性膠質瘤中呈高表達,且其高表達水平直接與腫瘤的進程及預后差密切相關。值得注意的是,敲低PRMT5可以抑制這些腫瘤細胞的增殖或誘導細胞凋亡,這些研究表明PRMT5可能是潛在的癌基因。然而,腫瘤中PRMT5的表達如何被調控在很大程度上并不清楚。因此,本研究旨在從轉錄及翻譯后水平探討腫瘤細胞中PRMT5高表達的調控機制。第一,我們從轉錄水平探討腫瘤細胞中PRMT5高表達的調控機制。Hu實驗室的前期觀察表明,PRMT5在前列腺癌組織中呈高表達。因此,為了進一步研究PRMT5在前列腺癌中的轉錄激活機制,我們選擇前列腺癌最常見的三個細胞系LNCa P,PC-3及DU 145細胞進行實驗。首先證明在LNCaP細胞中PRMT5的啟動子是雜合子,不同的等位基因(allele)之間具有不同的SNPs(Single Nucletide Polymorphisms)及13 bp Indel(Insertion/Deletion Polymorphisms)基因多態(tài)性。接著,我們利用雙熒光素酶報告基因(Dual-Luciferase Reporter Gene)鑒定找到PRMT5的核心啟動子位于-240 bp區(qū)域以內,并用EMSA(Electrophoresis Mobility Shift Assay)、ChIP(Chromatin Immunoprecipitation)、定點突變、刪減突變等分子生物學方法從多個角度證明,在多個腫瘤細胞系中轉錄因子NF-Y(Nuclear Factor Y)結合到PRMT5核心啟動子的CCAAT盒上進而調控PRMT5的轉錄激活。另外,我們發(fā)現下調PRMT5的表達所導致的細胞增殖抑制直接與NF-YA的敲低有關。更重要的是,研究結果證明PKC-c-Fos信號通路可以通過下調NF-YA的表達而抑制PRMT5的轉錄。鑒于多個PKC(Protein Kinase C)家族的成員被報道在某些腫瘤中表達水平下調,我們推測PKC-c-Fos信號通路的失活可能與某些腫瘤病人中PRMT5的高表達有關,而深入研究PRMT5如何與PKC/c-Fos信號通路的相互交叉將為進一步闡釋PRMT5促腫瘤發(fā)生的角色提供理論依據。第二,我們從翻譯后修飾水平探討PRMT5蛋白表達的調控機制�；谏厦娴慕Y果及我們未發(fā)表的發(fā)現,我們推測PRMT5自身可以被翻譯后修飾。為此,使用Co-IP(Co-Immunoprecipitation)及WB(Western blot)技術證實PRMT5蛋白可以發(fā)生多聚泛素化翻譯后修飾。通過Co-IP及定點突變,證實PRMT5上的多個賴氨酸位點對于PRMT5的多聚泛素化修飾均很重要。接著,使用MS(Mass Spectrometry)、Co-IP、GST下拉、亞細胞共定位、BiFC、定點突變、刪減突變等分子生物學方法,我們找到并證明CHIP(Carboxy terminus of Heat shock cognate 70-Interacting Protein)是與PRMT5相互作用的E3連接酶,后者可以與分子伴侶(chaperones)系統(tǒng)如熱休克蛋白90及70(Hsp90/Hsp70)發(fā)生相互作用,進而通過泛素化依賴的蛋白酶體降解方式調控PRMT5/MEP50(Methylosome protein 50)的表達。另外,我們的結果顯示Hsp90的抑制劑如GA(Geldanamycin)或17-AAG(17-(Allylamino)-17-demethoxygeldanamycin)介導的PRMT5下調依賴CHIP E3連接酶介導的蛋白酶體降解機制,且17-AAG可以協同過表達CHIP對PRMT5表達的抑制作用及誘導細胞凋亡的效應。這些發(fā)現提示在腫瘤組織中CHIP表達水平的下調可能與PRMT5的高表達水平相關。最后,我們證明靶向PRMT5聯合17-AAG干預可作為一個新的方法抑制腫瘤細胞的生長。綜上所述,我們探討了PRMT5的轉錄及翻譯后修飾的調控機制。研究結果表明,轉錄因子NF-Y對激活PRMT5的轉錄很關鍵,且NF-Y的轉錄活性受到PKC/c-Fos信號通路的負調控。另一方面,E3連接酶CHIP與分子伴侶系統(tǒng)相偶聯,介導PRMT5翻譯后的泛素化修飾及蛋白酶體依賴的降解。這些研究將為促進靶向PRMT5及其上游相關信號分子的抗癌治療提供理論和實驗依據,為新抗癌藥物的研發(fā)奠定基礎。
[Abstract]:PRMT5 (Protein arginine methyltransferase 5), namely protein arginine methyltransferase 5, which belongs to the type II methyltransferase, can arginine residues symmetrically methylated histone and non histone substrates, regulation of the key signaling molecule through epigenetic regulation of target gene expression of or through posttranslational methylation pathways. Recent studies show that PRMT5 in many human cancers such as lung cancer, ovarian cancer, colorectal cancer, breast cancer, melanoma, leukemia or lymphoma and malignant glioma showed high expression, and its high expression level directly with tumor progression and poor prognosis are closely related. It is worth noting that knockdown of PRMT5 inhibited tumor cell proliferation or apoptosis, these studies suggest that PRMT5 may be a potential oncogene. However, how to express the tumor in the PRMT5 is regulated to a great extent Is not clear. Therefore, this study aims to explore the regulatory mechanism of PRMT5 expression in tumor cells from the transcription and translation level. First, we investigate the early laboratory observation at the level of transcription regulatory mechanism of.Hu high expression of PRMT5 in tumor cells showed that the expression of PRMT5 in prostate cancer tissues was. Therefore, in order to further transcription the research of PRMT5 in prostate cancer activation mechanism, we choose the most common prostate cancer cell line LNCa three P, PC-3 and DU 145 cells were used. In the first show that the PRMT5 promoter in LNCaP cells is heterozygous for different alleles (allele) with different SNPs (Single Nucletide Polymorphisms) 13 BP and Indel (Insertion/Deletion Polymorphisms) gene polymorphism. Then, we use dual luciferase reporter gene (Dual-Luciferase Reporter Gene) to find the core promoter of PRMT5 identification The promoter is located within -240 BP region, and EMSA (Electrophoresis Mobility Shift Assay), ChIP (Chromatin Immunoprecipitation), point mutation, deletion mutation and other molecular biology methods proved from many angles, in a number of transcription factors in cancer cell lines NF-Y (Nuclear Factor Y) binding to the PRMT5 promoter, CCAAT box transcriptional activation of PRMT5. In addition, we found that the down-regulation of PRMT5 expression caused by the inhibition of cell proliferation directly with NF-YA knockdown. More importantly, the results demonstrated that the transcription of PKC-c-Fos signaling pathway can inhibit PRMT5 by down regulating the expression of NF-YA and in view of multiple PKC (Protein Kinase C) family members the reported level of down regulated expression in some tumors, we hypothesized that the activity may be related to the high expression of PRMT5 in some tumor patients in the PKC-c-Fos signaling pathway, and in-depth study of how PRMT5 And cross the PKC/c-Fos signaling pathway will further explain the role PRMT5 promoting tumorigenesis and provide a theoretical basis. From second, we discuss the regulation mechanism of the level of post-translational modification of PRMT5 protein expression. Based on the above results, we found unpublished, we speculate that PRMT5 itself can be modified after translation. Therefore, the use of Co-IP (Co-Immunoprecipitation) and WB (Western blot) technology confirmed that PRMT5 protein could be polyubiquitinated post-translational modification. Through Co-IP and PRMT5 point mutations, confirmed multiple lysine sites for PRMT5 poly ubiquitination is very important. Then, using the MS (Mass Spectrometry), Co-IP, GST pull-down, subcellular co localization, BiFC, point mutation, deletion mutation and other molecular biological methods, we find that CHIP (Carboxy terminus of Heat shock cognate 70-Interacting Protein) is PRMT5 The interaction of E3 ligase, which can be used with molecular chaperone (chaperones) systems such as heat shock protein 90 and 70 (Hsp90/Hsp70) interact with proteasome degradation through the ubiquitin dependent regulation of PRMT5/MEP50 (Methylosome protein 50) expression. In addition, our results show that GA inhibitors such as Hsp90 (Geldanamycin) or 17-AAG (17- (Allylamino) -17-demethoxygeldanamycin) mediated down-regulation of PRMT5 dependent proteasomal degradation mechanism of CHIP E3 ligase mediated, and 17-AAG can be co overexpression of CHIP on the expression of PRMT5 and inhibition effect by inducing apoptosis. These findings suggest that the expression of CHIP in tumor tissue is related to the down-regulation of PRMT5 and high the expression level. Finally, we show that targeting PRMT5 and 17-AAG intervention can be used as a new method to suppress the growth of tumor cells. To sum up, we discussed PR The regulation mechanism of modification of transcription and translation of MT5. The results show that the transcription of NF-Y on activation of PRMT5 is the key transcription activity and NF-Y negative regulation by PKC/c-Fos signaling pathway. On the other hand, coupled with E3 ligase CHIP and molecular chaperone systems, mediated ubiquitination and proteasomal degradation of PRMT5 translation after the dependent. These studies will provide theoretical and experimental basis for the promotion of anticancer therapy targeting PRMT5 and its upstream signal molecule, lay the foundation for the development of new anticancer drugs.

【學位授予單位】：暨南大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R730.2

【共引文獻】

相關期刊論文 前1條

1 陳萬濤;;E3泛素連接酶CHIP與上皮性癌關系研究[J];口腔頜面外科雜志;2014年06期

相關博士學位論文 前1條

1 李珊;CHIP在骨發(fā)生及肝癌中的作用[D];清華大學;2014年

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本文編號：1622196