【摘要】：乳腺癌是現(xiàn)階段對(duì)女性健康的一大重要危害�；熓侵委煱┌Y的一個(gè)最有效的方法,但是在乳腺癌中形成的腫瘤多藥耐藥(MDR)對(duì)治療腫瘤形成了巨大的障礙。腫瘤多藥耐藥由多方面的原因造成,例如:藥物外排,進(jìn)入細(xì)胞的藥物減少和表觀遺傳修飾。微小RNA(miRNA)是表觀遺傳修飾的一種,在腫瘤的發(fā)生、發(fā)展過程中扮演了非常重要的角色。本課題發(fā)現(xiàn)并研究了介導(dǎo)乳腺癌化療耐藥的miRNA及其機(jī)制。比較乳腺癌化療耐藥細(xì)胞系(MCF-7/ADM)及其親本對(duì)照(MCF-7/WT)的小RNA高通量測序結(jié)果,得到一批在耐藥細(xì)胞中表達(dá)顯著變化的miRNA。以這些miRNA作為出發(fā)點(diǎn),我們主要得出如下結(jié)論:1、對(duì)高通量測序方法得到的MCF-7/WT和MCF-7/ADM兩株細(xì)胞的miRNA表達(dá)譜進(jìn)行分析,結(jié)果顯示miR-149和miR-320a是MCF-7/ADM細(xì)胞中顯著下調(diào)的mi RNA;進(jìn)一步通過熒光定量PCR方法確認(rèn)了miR-149和miR-320a在這兩株細(xì)胞中的相對(duì)表達(dá)量。結(jié)果顯示,miR-149和miR-320a在MCF-7/ADM細(xì)胞中的表達(dá)與MCF-7/WT細(xì)胞相比顯著下降。用MTT的方法檢測mi R-149和miR-320a對(duì)MCF-7/ADM細(xì)胞藥物敏感性的影響。結(jié)果顯示,在MCF-7/ADM細(xì)胞中轉(zhuǎn)染miR-149和miR-320a的模擬物能增加MCF-7/ADM細(xì)胞對(duì)藥物的敏感性。2、深入分析miR-149和miR-320a表達(dá)紊亂機(jī)制。用啟動(dòng)子活性檢測實(shí)驗(yàn)發(fā)現(xiàn),miR-149與GPC1共用一個(gè)啟動(dòng)子;mi R-320a受獨(dú)立的啟動(dòng)子調(diào)控。甲基化位點(diǎn)檢測與亞硫酸鹽測序?qū)嶒?yàn)發(fā)現(xiàn)miR-149和miR-320a在MCF-7/ADM細(xì)胞中較MCF-7/WT細(xì)胞高度甲基化,從而降低了miR-149和miR-320a的表達(dá)。甲基化抑制劑抑制miR-149和miR-320a的甲基化可以提高miR-149和miR-320a的表達(dá)。3、對(duì)miR-149和mi R-320a的作用機(jī)制進(jìn)行研究。用TargetScan軟件對(duì)miR-149和miR-320a的靶點(diǎn)進(jìn)行預(yù)測,并在HEK293T細(xì)胞中用雙熒光報(bào)告基因方法驗(yàn)證了NDST1是miR-149的作用靶點(diǎn)之一;miR-320a被證實(shí)同時(shí)靶向TRPC5及NFATc3。NDST1信號(hào)通路激活,介導(dǎo)MCF-7/ADM細(xì)胞中的化療耐藥。以miR-320a mimic抑制該mi RNA的低表達(dá),可以通過降低TRPC5及NFATc3的表達(dá),從而顯著降低MCF-7/ADM的耐藥性。4、在臨床乳腺癌穿刺樣本中,miR-149和miR-320a亦被發(fā)現(xiàn)在化療耐藥的患者中較低地表達(dá),并顯著相關(guān)于NDST1、TRPC5及NFATc3的高表達(dá)。NDST1的高表達(dá)乳腺癌患者,其預(yù)后較低表達(dá)患者顯著較差。低表達(dá)miR-320a的雌激素受體陽性乳腺癌患者同時(shí)也表現(xiàn)出較差的預(yù)后。總之,該論文主要發(fā)現(xiàn)了miR-149和miR-320a介導(dǎo)乳腺癌化療耐藥的機(jī)制,為化療耐藥逆轉(zhuǎn)藥物的開發(fā),及臨床化療耐藥標(biāo)志物的建立,提供了理論及實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Breast cancer is an important hazard to women's health at the present stage. Chemotherapy is one of the most effective treatments for cancer, but multidrug resistance (MDR) in breast cancer is a major obstacle to cancer treatment. Multidrug resistance in tumors is caused by multiple causes, such as drug efflux, drug reduction into cells and epigenetic modification. MicroRNA (miRNA) is an epigenetic modification, which plays a very important role in tumor development. In this study, miRNA mediated chemoresistance in breast cancer and its mechanism were investigated. High throughput sequencing of small RNA in breast cancer chemoresistant cell line (MCF-7 / ADM) and its parent control (MCF-7 / WT) was compared and a batch of miRNAs expressed in resistant cells were obtained. Using these miRNAs as a starting point, we came to the following conclusion: 1, we analyzed the miRNA expression profiles of MCF-7 / WT and MCF-7 / ADM cells obtained by high-throughput sequencing. The results showed that miR-149 and miR-320a were significantly down-regulated mi RNAs in MCF-7 / ADM cells, and the relative expressions of miR-149 and miR-320a in MCF-7 / ADM cells were confirmed by fluorescence quantitative PCR. The results showed that the expression of miR-149 and miR-320a in MCF-7 / ADM cells was significantly lower than that in MCF-7 / WT cells. The effects of miR-149 and miR-320a on the drug sensitivity of MCF-7 / ADM cells were detected by MTT method. The results showed that the mimics transfected with miR-149 and miR-320a in MCF-7 / ADM cells increased the sensitivity of MCF-7 / ADM cells to drugs. The mechanism of miR-149 and miR-320a expression disorder was deeply analyzed. It was found that the promoter miR-149 and GPC1 shared the same promoter, mi R-320a, by independent promoter. The results of methylation site detection and sulfite sequencing showed that miR-149 and miR-320a were highly methylated in MCF-7 / ADM cells compared with MCF-7 / WT cells, thus reducing the expression of miR-149 and miR-320a. The inhibition of the methylation of miR-149 and miR-320a by methylation inhibitor increased the expression of miR-149 and miR-320a. The mechanism of miR-149 and miR-320a was studied. The targets of miR-149 and miR-320a were predicted by TargetScan software, and the double fluorescence reporter gene method was used to verify that NDST1 was one of the action targets of miR-149. It was proved that NDST1 was simultaneously targeted at TRPC5 and NFATc3.NDST1 signaling pathway activation, which mediated chemoresistance in MCF-7 / ADM cells. The low expression of miR-320a mimic could decrease the drug resistance of MCF-7 / ADM by decreasing the expression of TRPC5 and NFATc3, and the expression of miR-149 and miR-320a was also found to be lower in patients with chemotherapeutic resistance. Significant correlation was found in breast cancer patients with high expression of NDST1 / TRPC5 and NFATc3. The prognosis of breast cancer patients with low expression was significantly worse than that of patients with low expression of NDST1 / TRPC5 or NFATc3. Low-expression miR-320a estrogen receptor positive breast cancer patients also showed poor prognosis. In conclusion, the mechanism of miR-149 and miR-320a mediated chemoresistance in breast cancer was found in this paper, which provided a theoretical and experimental basis for the development of chemotherapeutic reversal drugs and the establishment of clinical chemotherapeutic resistance markers.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Bioinformatic analysis of microRNA biogenesis and function related proteins in eleven animal genomes[J];遺傳學(xué)報(bào);2009年10期

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本文編號(hào)：2121357