miR-155在结肠癌中的功能及作用机制的研究

发布时间：2018-07-08 10:46

本文选题：miR-155 + HMG-box转录因子1(HBP1)　；参考：《吉林大学》2016年博士论文

【摘要】：结肠癌是一种起源于大肠上皮组织的常见恶性肿瘤,发病率逐年增高,预后较差。研究发现mi RNA能够在肿瘤中通过影响癌基因或者肿瘤抑制基因,发挥多种生物学功能。在正常情况下,癌症中mi RNA的下调抑制原癌基因的表达。相反,也有肿瘤中高表达的mi RNA被发现并且抑制肿瘤抑制基因的表达,因此mi RNA基因既可充当肿瘤抑制基因又可充当癌基因角色,基于mi RNA疗法很可能转化为一种有效的结肠癌治疗策略。大量证据表明mi RNA如mi R-155与结肠癌病因学和生物学关系极其密切,而且mi R-155在多种肿瘤预防、诊断、治疗及预后等临床方面表现出良好的应用前景。然而,mi R-155在结肠癌中作用的分子机制并不清楚。结肠癌中信号通路中最常见的改变是Wnt途径的激活。Wnt/β-catenin信号通路在结肠癌早期发生中扮演重要角色。因此,本研究的目的是研究mi R-155在结肠癌中的功能及作用机制,探索mi R-155在结肠癌组织及细胞中的可能靶标。方法:1.应用实时荧光定量PCR和(或)Northern blot检测结肠癌组织及细胞的mi R-155以及Wnt/β-catenin信号通路相关的靶基因Axin2,CD44和LGR5的表达水平。2.利用小鼠结肠癌异种移植瘤模型考察mi R-155对小鼠结肠癌肿瘤生长的影响。3.Western blot方法检测细胞的增殖生物标志物Ki-67,HBP1,β-catenin蛋白表达水平。4.利用TOPFlash报告基因检测mi R-155对结肠癌细胞Wnt/β-catenin信号通路的影响。5.流式细胞仪检测细胞周期,用Mod Fit软件进行细胞周期分析。6.MTT检测细胞活性,计算细胞增殖率。7.利用生物信息学方法在线数据库Target Scan(http://www.targetscan.org)对mi R-155可能的靶基因进行预测。8.利用报告基因载体对mi R-155靶m RNA验证。9.斯皮尔曼分析方法分析mi R-155水平与结肠癌患者生存期相关性。结果:实时荧光定量PCR及Northern blot检测发现与配对的癌旁正常结肠组织相比,结肠癌组织中mi R-155的表达水平显著升高,结肠癌细胞系中mi R-155的表达水平较正常结肠上皮细胞系显著升高。结肠癌细胞转染mi R-155抑制剂后,增殖率显著下降,并且阻断Wnt/β-catenin信号通路。抑制mi R-155表达减慢小鼠结肠癌异种移植瘤的增长。生物信息学方法及报告基因载体确定HMG-box转录因子1(HBP1)为mi R-155新的靶分子,进而介导Wnt/β-catenin信号通路。细胞转染mi R-155抑制剂后抑制HBP1,β-catenin蛋白及Wnt/β-catenin信号通路相关的靶基因Axin2,CD44和LGR5的表达。另外,斯皮尔曼分析结果表明血清中高表达mi R-155结肠癌患者生存时间缩短。结论:mi R-155高表达可促进结肠癌的肿瘤生长,它可能通过HBP1介导Wnt/β-catenin通路激活,参与肿瘤的发生发展过程。因此,mi R-155可能成为一种很有应用前景的结肠癌临床治疗药物。
[Abstract]:Colon cancer is a common malignant tumor originating from large intestine epithelium. The incidence of colon cancer is increasing year by year and the prognosis is poor. It has been found that mi RNA can play a variety of biological functions by influencing oncogene or tumor suppressor gene in tumor. Under normal conditions, the down-regulation of mi RNA inhibits the expression of proto-oncogenes in cancer. On the contrary, there are also high-expressed mi RNA in tumors that are found to inhibit the expression of tumor suppressor genes, so that mi RNA genes can act as both tumor suppressor genes and oncogenes Mi RNA-based therapy is likely to translate into an effective strategy for colon cancer treatment. A great deal of evidence shows that mi RNA, such as miR-155, is closely related to the etiology and biology of colon cancer, and miR-155 has a good prospect in the prevention, diagnosis, treatment and prognosis of many kinds of tumors. However, the molecular mechanism of the action of MMI R-155 in colon cancer is unclear. The most common change in the signaling pathway in colon cancer is the activation of the Wnt pathway. Wnt/ 尾 -catenin signaling pathway plays an important role in the early development of colon cancer. Therefore, the purpose of this study was to study the function and mechanism of miR-155 in colon cancer, and to explore the possible target of mi R-155 in colon cancer tissues and cells. Method 1: 1. Real-time fluorescent quantitative PCR and / or Northern blot were used to detect the expression levels of MIR-155 and Wnt- 尾 -catenin signaling pathway related target genes Axin2CD44 and LGR5 in colon cancer tissues and cells. The effect of miR-155 on tumor growth of mouse colon cancer xenografts was studied. 3. Western blot assay was used to detect the expression level of Ki-67 blot and 尾 -catenin protein. The effect of miR-155 on the Wnt- 尾 -catenin signaling pathway in colon cancer cells was detected by using Topflash reporter gene. Flow cytometry was used to detect cell cycle, cell cycle analysis was performed by Mod fit software. 6. MTT was used to detect cell activity and cell proliferation rate was calculated. Target scan (http://www.targetscan.org), an online database of bioinformatics, was used to predict the possible target gene of mi R-155. The reporter gene vector was used to validate the target mRNA of mi R-155. The correlation between MI-155 level and survival time of colon cancer patients was analyzed by Spelman analysis. Results: Real-time fluorescent quantitative PCR and Northern blot analysis showed that the expression level of mi R-155 in colon cancer tissues was significantly higher than that in matched adjacent normal colon tissues. The expression level of mi R 155 in colon cancer cell line was significantly higher than that in normal colon epithelial cell line. After transfection with mi R 155 inhibitor, the proliferation rate of colon cancer cells decreased significantly, and Wnt / 尾 -catenin signaling pathway was blocked. Inhibition of mi R 155 expression slowed down the growth of xenografts in mouse colon cancer. Bioinformatics method and reporter gene vector confirmed that HMG-box transcription factor 1 (HBP1) was a new target molecule of mi R-155, which mediated the Wnt- 尾 -catenin signaling pathway. The expression of HBP1, 尾 -catenin protein and the target genes axin2pCD44 and LGR5 related to Wnt- 尾 -catenin signaling pathway were inhibited after transfection of mi R-155 inhibitor. In addition, Spelman analysis showed that the survival time of colon cancer patients with high expression of mi R 155 was shortened. Conclusion the high expression of 1: mi R-155 can promote the growth of colon cancer. It may play an important role in tumorigenesis and progression through the activation of Wnt- 尾 -catenin pathway mediated by HBP1. Therefore, MMI R-155 may be a promising clinical therapy for colon cancer.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.35

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