肝细胞癌中转录抑制因子ZHX2靶基因的分析研究

发布时间：2018-08-21 08:51

【摘要】：肝癌是全世界癌症死亡的重要原因。其发病率和死亡率一直位居于恶性肿瘤前列。肝癌往往在中晚期才被确诊,而且病情进展迅速,常常失去了有效的治疗机会。肝癌的治疗手段一般包括手术、放疗、化疗和综合治疗等。现在的治疗方式多采用手术切除,但是术后复发率很高。分子靶向治疗是新兴的治疗手段,靶向药物能够提高肝癌的治疗效果,但是长远生存率仍然改观不大,而且靶向药物受益人群有限。因此,为肝癌的诊断和治疗寻求有效的新靶标是势在必行的。目的:转录抑制因子ZHX2作为ZHX蛋白家族成员之一,在人体内广泛存在,不仅在体内各种组织中有表达,而且对多种疾病中的关键基因表达具有调控作用。本课题组前期研究表明,ZHX2在肝癌组织和肝癌细胞系中低表达,提示可能为肝癌的抑制因子并且发现在肝癌组织和细胞系中发现ZHX2可以和细胞因子NF-YA结合调控下游基因的表达,从而抑制肝癌的发生发展。国内外多项研究报道ZHX2可以影响其他基因对肝癌发生发展的调控,但是具体调控机制仍不明确。因此,针对肝癌中转录抑制因子ZHX2调控的机制进行初步探讨研究。分析肝细胞癌中转录抑制因子ZHX2转录调控特征,获得ZHX2的靶基因;研究ZHX2和其靶基因在肝细胞癌中的表达及其临床病理特征的关系,探讨ZHX2与靶基因间的调控关系。方法:首先采用RT-PCR方法检测肝癌细胞株HepG2、SMM7721、Be17402和正常肝细胞株L02中ZHX2 mRNA表达的情况。接着利用ZHX2抗体进行染色质免疫共沉淀(chromatin immunoprecipitation, ChIP)实验,将富集到的DNA样品进行高通量测序,得到肝细胞肝癌中ZHX2潜在靶基因,然后利用生物信息学进行分析。最后通过免疫组织化学技术检测ZHX2和得到的靶基因PTEN、P53在肝细胞癌组织中的表达,并分析它们与临床病理学特征的关系。结果:ZHX2mRNA在3种肝癌细胞株中均有表达。对ChIP富集得到的DNA进行分析,共得到1981个ZHX2结合位点的peaks,含有3093个结合的基因。ZHX2的peaks主要分布在1、2、3、5、7号染色体,在基因元件上的分布主要在基因间区、内含子区、转录起始位点上游和启动子区。对ZHX2结合在启动子区的232个基因进行基因功能(gene ontology, GO)注释,发现ZHX2参与了与生物调节、代谢进程和分子调节功能等有关的生物功能,和形成血液微粒、近心异染色质等有关的细胞组分,在分子功能上与GTP酶的激活、调节和其他酶的激活有关的过程。然后进行KEGG pathway分析显示其中一些靶基因可能富集于4条通路,分别是3条传染性疾病的通路和1条吞噬通路。相对于肝癌癌旁组织,ZHX2在肝细胞癌组织中低表达(P=0.042),与血清AFP值相关(P=0.041); PTEN在癌组织中低表达(P=0.000),分别与肿瘤的分化程度和血清AFP值有关(P=0.025,P=0.028); P53在癌组织中高表达(P=0.000),与肿瘤的分化程度有关(P=0.045)。ZHX2 与 PTEN 存在相关性(r=0.258,P=0.031)。结论:本研究是首次采用ChIP-Seq技术从全基因组水平解析ZHX2在肝癌中的转录调控的靶基因。发现ZHX2除了与启动子区域结合外,还可以和基因间区的远距离调控区域结合调控靶基因。对启动子区域的靶基因进行基因富集分析显示结合的靶基因与代谢过程、生物调控过程等有关;富集通路分析提示富集靶基因可能影响了传染性疾病和吞噬相关调控。ZHX2与PTEN、P53在肝细胞癌发生发展中起作用。PTEN结合于ZHX2的启动子区域,且ZHX2与PTEN存在正相关性,提示两者相互作用对HCC进行调节,为进一步研究ZHX2在肝癌中的具体调控机制提供依据。
[Abstract]:Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide. Its morbidity and mortality have always been at the forefront of malignant tumors. HCC is often diagnosed in the middle and advanced stages, and the disease progresses rapidly, often losing the opportunity for effective treatment. Molecular targeted therapy is a new therapeutic method. Targeted drugs can improve the therapeutic effect of hepatocellular carcinoma, but the long-term survival rate is still not improved, and the beneficiaries of targeted drugs are limited. Therefore, it is imperative to seek effective new targets for the diagnosis and treatment of hepatocellular carcinoma. As one of the members of ZHX protein family, transcription inhibitor ZHX2 exists extensively in human body. It is not only expressed in various tissues of the body, but also regulates the expression of key genes in various diseases. Previous studies in this group showed that ZHX2 is low expressed in hepatocellular carcinoma tissues and hepatocellular carcinoma cell lines, suggesting that ZHX2 may be an inhibitor of hepatocellular carcinoma. ZHX2 can bind with cytokine NF-YA to regulate the expression of downstream genes in hepatocellular carcinoma tissues and cell lines, thus inhibiting the development of hepatocellular carcinoma. To investigate the mechanism of ZHX2 regulation, we analyzed the transcriptional regulation characteristics of ZHX2 in hepatocellular carcinoma and obtained the target gene of ZHX2. To study the expression of ZHX2 and its target gene in hepatocellular carcinoma and their relationship with clinicopathological characteristics, and to explore the regulatory relationship between ZHX2 and target gene. Methods The expression of ZHX2 mRNA in hepatocellular carcinoma cell lines HepG2, SMM7721, Be17402 and normal liver cell line L02 was detected. Then the ZHX2 antibody was used to carry out chromatin immunoprecipitation (ChIP) assay. The enriched DNA samples were sequenced at high throughput to obtain the potential target gene of ZHX2 in hepatocellular carcinoma, and then the ZHX2 gene was generated. Finally, the expression of ZHX2 and PTEN, P53 in hepatocellular carcinoma was detected by immunohistochemistry and their relationship with clinicopathological features was analyzed.Results: ZHX2 mRNA was expressed in all three hepatocellular carcinoma cell lines. ZHX2 peaks are mainly distributed on chromosomes 1, 2, 3, 5 and 7, and are mainly distributed in the intergenic region, intron region, upstream of transcription initiation site and promoter region. Biological functions related to biological regulation, metabolic processes and molecular regulatory functions, and cell components related to the formation of blood particles, myocardial heterochromatin, etc. are involved in the molecular functions of GTP enzyme activation, regulation and other enzyme activation. Then KEGG pathway analysis shows that some of the target genes may be enriched in 4. The expression of ZHX2 was lower in HCC tissues (P = 0.042) than in adjacent tissues (P = 0.041), and the expression of PTEN was lower in cancer tissues (P = 0.000), which was related to tumor differentiation and serum AFP value (P = 0.025, P = 0.028), respectively. ZHX2 was associated with PTEN (r = 0.258, P = 0.031). CONCLUSION: This study is the first time that ChIP-Seq technique was used to analyze the target gene of ZHX2 transcriptional regulation in hepatocellular carcinoma at the whole genome level. Distance-regulated regions bind to regulatory target genes.Gene enrichment analysis of the target genes in the promoter region showed that the binding target genes were related to metabolic processes and biological regulatory processes.Enrichment pathway analysis suggested that enrichment of target genes might affect the regulation of infectious diseases and phagocytosis.ZHX2 and PTEN,P53 were involved in the occurrence and development of hepatocellular carcinoma. PTEN binds to the promoter region of ZHX2, and there is a positive correlation between ZHX2 and PTEN, suggesting that they interact to regulate HCC, providing a basis for further study of the specific regulatory mechanism of ZHX2 in hepatocellular carcinoma.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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