HPD在肺癌中异常表达的转录调控机制研究

发布时间：2018-03-09 00:05

本文选题：HPD　切入点：肺癌　出处：《安徽医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：4-羟苯丙酮酸二加氧酶(4-Hydroxyphenylpyruvate Dioxygenase, HPD),是一种参与酪氨酸代谢途径的代谢酶,主要在肝脏和肾脏表达,在其他组织呈现极低水平表达或不表达。前期研究表明HPD的表达异常及突变与可导致酪氨酸血症,此外在肝损伤过程中也发现HPD表达上调,提示HPD可能是肝损伤的重要标志物。我们实验室前期利用组织芯片、qRT-PCR和Western-Blotting等技术意外发现HPD在肺癌组织及细胞中高表达,而在癌旁组织中表达水平较低。但关于HPD在肺癌中异常表达的分子机制及其对肺癌细胞行为影响的研究,目前尚无任何报道。为进一步确证HPD与肺癌的关系,本课题进行了以下两方面的研究：第一部分：检测敲低HPD表达对化疗药物诱导的肺癌细胞系A549凋亡的影响。首先将前期设计的HPD RNA干涉片段克隆到慢病毒表达载体上,然后通过瞬转筛选得到干涉效果较明显的干涉载体,并包装病毒颗粒,感染肺癌细胞系,经嘌呤筛选得到敲低HPD的A549稳定细胞株及对照稳定株,用抗肿瘤药物Gefitinib处理以上各种稳定株,检测其凋亡情况。第二部分：构建了HPD基因启动子荧光素酶报告基因载体并进行了转录活性分析,初步探索了HPD的转录调控特点。首先利用UCSC在线数据库分析人HPD基因组结构,并获得其5'端上游启动子区域-2000 bp-+39bp的DNA)序列。以人基因组DNA为模板,克隆到pGL3-Basic载体上,设计不同引物,进一步构建系列缺失体,最后通过荧光素酶报告基因实验分析其转录活性。第三部分：通过生物信息学数据库预测HPD基因启动子序列上潜在的转录因子结合位点,并结合文献报道初步探索了HNF1α (Hepatocyte nuclear factor1α),HNF4a (Hepatocyte nuclear factor la)和Nrf2 (Nuclear factor, erythroid 2-like 2)等转录因子对HPD基因的转录调控作用。本论文发现敲低HPD表达增强了肺癌细胞A549对化疗药物Gefitinib的敏感性,构建了HPD启动子报告基因载体,明确了其在肺癌中特异表达的关键结构域,并发现HNF1α和Nrf2对HPD基因表达的调控作用。本论文初步揭示了HPD在肺癌中异常高表达的调控机制,为深入研究HPD的基因表达调控奠定了基础。
[Abstract]:4-Hydroxyphenylpyruvate Dioxygenase (HPDD), a metabolic enzyme involved in tyrosine metabolism, is expressed mainly in liver and kidney. Previous studies have shown that abnormal expression and mutation of HPD may lead to tyrosinemia, and that the expression of HPD is up-regulated during liver injury. The results suggest that HPD may be an important marker of liver injury. In our laboratory, we found that HPD was overexpressed in lung cancer tissues and cells by using tissue microarray qRT-PCR and Western-Blotting techniques. However, there is no report on the molecular mechanism of abnormal expression of HPD in lung cancer and its effect on the behavior of lung cancer cells. In order to further confirm the relationship between HPD and lung cancer, In this paper, the following two aspects were studied: the first part: the effect of low HPD expression on apoptosis of lung cancer cell line A549 induced by chemotherapeutic drugs was detected. Firstly, the previously designed HPD RNA interference fragment was cloned into lentivirus expression vector. Then the interference vector with obvious interference effect was obtained by transient screening, and the virus particles were packaged and infected with lung cancer cell line. The stable cell line A549 with low HPD knockout and the control stable cell line were obtained by purine screening. The stable strains were treated with anti-tumor drug Gefitinib and their apoptosis was detected. Part two: the luciferase reporter gene vector of HPD gene promoter was constructed and the transcriptional activity was analyzed. The transcriptional regulatory characteristics of HPD were preliminarily explored. Firstly, the genomic structure of human HPD was analyzed by UCSC online database, and the 5'end upstream promoter region -2000bp-39bp DNA sequence was obtained. Using human genomic DNA as template, it was cloned into pGL3-Basic vector. Different primers were designed to construct a series of deletions. Finally, the transcriptional activity of luciferase reporter gene was analyzed by experiments. Part three: the potential transcription factor binding sites on the promoter sequence of HPD gene were predicted by bioinformatics database. The transcriptional regulation of HPD gene by transcription factors such as HNF1 伪 nuclear nuclear factor1 伪 HNF4a Hepatocyte nuclear factor la. and Nrf2 nuclear factor, erythroid 2-like 2) was preliminarily explored. It was found that the low expression of HPD enhanced the sensitivity of lung cancer cell A549 to the chemotherapeutic drug Gefitinib. The HPD promoter reporter gene vector was constructed, the key domain of its specific expression in lung cancer was identified, and the regulatory effect of HNF1 伪 and Nrf2 on the expression of HPD gene was found. In this paper, the regulatory mechanism of abnormal overexpression of HPD in lung cancer was revealed. It lays a foundation for further study on the regulation of HPD gene expression.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2

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