本文關(guān)鍵詞： AP-2α miRNA 相互作用蛋白 藥物敏感性 膀胱癌　出處：《湖南師范大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：膀胱癌是泌尿系統(tǒng)最常見(jiàn)的惡性腫瘤。對(duì)于膀胱癌患者的治療,一般采用切除手術(shù),必要時(shí)結(jié)合化療或(和)放療及輔助抗癌藥物。盡管如此,有些患者的治療效果仍然不是很好,其主要原因是我們對(duì)膀胱癌潛在致病機(jī)理了解的比較少。許多研究表明,轉(zhuǎn)錄因子AP-2α是一個(gè)癌癥抑制因子。在膀胱癌、乳腺癌等癌癥病人中轉(zhuǎn)錄因子AP-2α的表達(dá)水平與化療敏感性正相關(guān)。本論文系統(tǒng)地研究AP-2α在膀胱癌中的作用及上下游調(diào)控網(wǎng)絡(luò)。AP-2α主要作為轉(zhuǎn)錄因子發(fā)揮功能。為了研究AP-2α調(diào)控的下游靶基因,我們?cè)谌笔P-2α表達(dá)的膀胱癌細(xì)胞系UM-UC-3中過(guò)表達(dá)AP-2α,然后進(jìn)行轉(zhuǎn)錄組測(cè)序。一共鑒定到差異表達(dá)基因247個(gè),其中27個(gè)上調(diào),220個(gè)下調(diào),然后對(duì)它們進(jìn)行生物信息學(xué)分析來(lái)研究它們?cè)诎螂装┲凶饔�。同時(shí),我們還用RT-PCR驗(yàn)證了4個(gè)潛在的AP-2α靶基因,發(fā)現(xiàn)1個(gè)表達(dá)上調(diào),3個(gè)表達(dá)下調(diào),這與轉(zhuǎn)錄組測(cè)序結(jié)果是一致的。為了研究AP-2α對(duì)miRNA的轉(zhuǎn)錄調(diào)控,我們?cè)诎螂装┘?xì)胞系UM-UC-3中過(guò)表達(dá)AP-2α之后,進(jìn)行了小RNA測(cè)序。一共鑒定到差異表達(dá)miRNA 41個(gè),其中3個(gè)表達(dá)上調(diào),38個(gè)表達(dá)下調(diào)。AP-2α的功能受其相互作用蛋白的影響。為了發(fā)現(xiàn)新的AP-2α相互作用蛋白,我們采用免疫共沉淀結(jié)合質(zhì)譜技術(shù)來(lái)尋找新的AP-2α相互作用蛋白。首先我們對(duì)高表達(dá)AP-2α的正常膀胱上皮細(xì)胞系SV-HUC-1蛋白進(jìn)行交聯(lián),然后用AP-2α抗體和對(duì)照抗體IgG進(jìn)行免疫沉淀,差異條帶通過(guò)質(zhì)譜鑒定。一共鑒定到118個(gè)蛋白。同時(shí),我們還對(duì)其中一個(gè)蛋白質(zhì)Ran進(jìn)行了熒光免疫共定位來(lái)驗(yàn)證其與AP-2α的相互作用,結(jié)果表明Ran確實(shí)與AP-2α存在相互作用。為了研究miRNA對(duì)AP-2α基因的調(diào)控,我們通過(guò)生物信息學(xué)分析調(diào)控AP-2α的miRNA。其中,miR-193a-5p能夠結(jié)合到轉(zhuǎn)錄因子AP-2αmRNA的編碼區(qū)。進(jìn)一步研究發(fā)現(xiàn),在膀胱癌細(xì)胞系UM-UC-3中抑制miR-193a-5p可以提高AP-2α的表達(dá)水平,而在正常膀胱上皮細(xì)胞系SV-HUC-1中過(guò)表達(dá)mi R-193a-5p則抑制AP-2α的表達(dá)。同時(shí),我們的研究還發(fā)現(xiàn)mi R-193a-5p通過(guò)抑制AP-2α的表達(dá)來(lái)降低膀胱癌細(xì)胞對(duì)化療藥物順鉑的敏感性。本文系統(tǒng)分析了膀胱癌細(xì)胞中AP-2α調(diào)控的靶基因和miRNA、AP-2α相互作用蛋白及miRNA對(duì)AP-2α的調(diào)控。這些研究加深了我們對(duì)AP-2α功能的理解,為揭示膀胱癌的致病分子機(jī)理、尋找新的生物標(biāo)記物奠定了基礎(chǔ)。
[Abstract]:Bladder cancer is the most common malignant tumor in the urinary system. For patients with bladder cancer, resection surgery is commonly used, combined with chemotherapy or / and radiotherapy and adjuvant anticancer drugs when necessary. Some patients are still not very effective, the main reason is that we know less about the underlying pathogenesis of bladder cancer. Many studies show that. Transcription factor AP-2 偽 is a cancer suppressor in bladder cancer. The expression of transcription factor AP-2 偽 was positively correlated with chemosensitivity in patients with breast cancer and other cancers. In this paper, we systematically studied the role of AP-2 偽 in bladder cancer and the main regulatory network. AP-2 偽. To study the downstream target genes regulated by AP-2 偽. We overexpressed AP-2 偽 in bladder cancer cell line UM-UC-3 without AP-2 偽 expression, and then sequenced the transcription sequence. A total of 247 differentially expressed genes were identified, 27 of which were up-regulated. There were 220 down-regulated genes, and then bioinformatics analysis was performed to study their role in bladder cancer. At the same time, four potential AP-2 偽 target genes were identified by RT-PCR. One expression was up-regulated and three down-regulated, which was consistent with the results of transcriptome sequencing. In order to study the transcriptional regulation of AP-2 偽 on miRNA. After overexpression of AP-2 偽 in bladder cancer cell line UM-UC-3, we sequenced the small RNA. A total of 41 differentially expressed miRNA were identified, 3 of which were up-regulated. The function of down-regulated .AP-2 偽 expression was affected by its interaction protein. In order to find new AP-2 偽 interaction protein. We used co-immunoprecipitation and mass spectrometry to search for new AP-2 偽 interacting proteins. Firstly, we cross-linked the SV-HUC-1 protein of normal bladder epithelial cell line with high expression of AP-2 偽. Then the AP-2 偽 antibody and the control antibody IgG were used for immunoprecipitation, and the differential bands were identified by mass spectrometry. A total of 118 proteins were identified. One of the proteins, Ran, was also co-located by fluorescence immunoassay to verify its interaction with AP-2 偽. The results showed that Ran actually interacted with AP-2 偽. In order to study the regulation of AP-2 偽 gene by miRNA. We regulate the miRNAs of AP-2 偽 by bioinformatics analysis, in which miR-193a-5p can bind to the coding region of transcription factor AP-2 偽 mRNA. Inhibition of miR-193a-5p in bladder cancer cell line UM-UC-3 can increase the expression of AP-2 偽. The overexpression of mi R-193a-5p in normal bladder epithelial cell line SV-HUC-1 inhibited the expression of AP-2 偽. Our research also found that mi. R-193a-5p reduces the sensitivity of bladder cancer cells to cisplatin by inhibiting the expression of AP-2 偽. The target genes and miRN regulated by AP-2 偽 in bladder cancer cells were systematically analyzed. A. These studies have deepened our understanding of the function of AP-2 偽 and the regulation of AP-2 偽 by miRNA in order to elucidate the pathogenesis of bladder cancer. The search for new biomarkers laid the foundation.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.14

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