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miR-149和miR-320a在乳腺癌肿瘤多药耐药中的作用及机制研究

发布时间:2018-07-14 10:29  文章来源:笔耕文化传播
【摘要】:乳腺癌是现阶段对女性健康的一大重要危害。化疗是治疗癌症的一个最有效的方法,但是在乳腺癌中形成的肿瘤多药耐药(MDR)对治疗肿瘤形成了巨大的障碍。肿瘤多药耐药由多方面的原因造成,例如:药物外排,进入细胞的药物减少和表观遗传修饰。微小RNA(miRNA)是表观遗传修饰的一种,在肿瘤的发生、发展过程中扮演了非常重要的角色。本课题发现并研究了介导乳腺癌化疗耐药的miRNA及其机制。比较乳腺癌化疗耐药细胞系(MCF-7/ADM)及其亲本对照(MCF-7/WT)的小RNA高通量测序结果,得到一批在耐药细胞中表达显著变化的miRNA。以这些miRNA作为出发点,我们主要得出如下结论:1、对高通量测序方法得到的MCF-7/WT和MCF-7/ADM两株细胞的miRNA表达谱进行分析,结果显示miR-149和miR-320a是MCF-7/ADM细胞中显著下调的mi RNA;进一步通过荧光定量PCR方法确认了miR-149和miR-320a在这两株细胞中的相对表达量。结果显示,miR-149和miR-320a在MCF-7/ADM细胞中的表达与MCF-7/WT细胞相比显著下降。用MTT的方法检测mi R-149和miR-320a对MCF-7/ADM细胞药物敏感性的影响。结果显示,在MCF-7/ADM细胞中转染miR-149和miR-320a的模拟物能增加MCF-7/ADM细胞对药物的敏感性。2、深入分析miR-149和miR-320a表达紊乱机制。用启动子活性检测实验发现,miR-149与GPC1共用一个启动子;mi R-320a受独立的启动子调控。甲基化位点检测与亚硫酸盐测序实验发现miR-149和miR-320a在MCF-7/ADM细胞中较MCF-7/WT细胞高度甲基化,从而降低了miR-149和miR-320a的表达。甲基化抑制剂抑制miR-149和miR-320a的甲基化可以提高miR-149和miR-320a的表达。3、对miR-149和mi R-320a的作用机制进行研究。用TargetScan软件对miR-149和miR-320a的靶点进行预测,并在HEK293T细胞中用双荧光报告基因方法验证了NDST1是miR-149的作用靶点之一;miR-320a被证实同时靶向TRPC5及NFATc3。NDST1信号通路激活,介导MCF-7/ADM细胞中的化疗耐药。以miR-320a mimic抑制该mi RNA的低表达,可以通过降低TRPC5及NFATc3的表达,从而显著降低MCF-7/ADM的耐药性。4、在临床乳腺癌穿刺样本中,miR-149和miR-320a亦被发现在化疗耐药的患者中较低地表达,并显著相关于NDST1、TRPC5及NFATc3的高表达。NDST1的高表达乳腺癌患者,其预后较低表达患者显著较差。低表达miR-320a的雌激素受体阳性乳腺癌患者同时也表现出较差的预后。总之,该论文主要发现了miR-149和miR-320a介导乳腺癌化疗耐药的机制,为化疗耐药逆转药物的开发,及临床化疗耐药标志物的建立,提供了理论及实验基础。
[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.
【学位授予单位】:江南大学
【学位级别】:博士
【学位授予年份】:2017
【分类号】:R96

【参考文献】

相关期刊论文 前1条

1 ;Bioinformatic analysis of microRNA biogenesis and function related proteins in eleven animal genomes[J];遗传学报;2009年10期



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