【摘要】：肝癌是全世界癌癥死亡的重要原因。其發(fā)病率和死亡率一直位居于惡性腫瘤前列。肝癌往往在中晚期才被確診,而且病情進(jìn)展迅速,常常失去了有效的治療機(jī)會(huì)。肝癌的治療手段一般包括手術(shù)、放療、化療和綜合治療等�，F(xiàn)在的治療方式多采用手術(shù)切除,但是術(shù)后復(fù)發(fā)率很高。分子靶向治療是新興的治療手段,靶向藥物能夠提高肝癌的治療效果,但是長(zhǎng)遠(yuǎn)生存率仍然改觀不大,而且靶向藥物受益人群有限。因此,為肝癌的診斷和治療尋求有效的新靶標(biāo)是勢(shì)在必行的。目的:轉(zhuǎn)錄抑制因子ZHX2作為ZHX蛋白家族成員之一,在人體內(nèi)廣泛存在,不僅在體內(nèi)各種組織中有表達(dá),而且對(duì)多種疾病中的關(guān)鍵基因表達(dá)具有調(diào)控作用。本課題組前期研究表明,ZHX2在肝癌組織和肝癌細(xì)胞系中低表達(dá),提示可能為肝癌的抑制因子并且發(fā)現(xiàn)在肝癌組織和細(xì)胞系中發(fā)現(xiàn)ZHX2可以和細(xì)胞因子NF-YA結(jié)合調(diào)控下游基因的表達(dá),從而抑制肝癌的發(fā)生發(fā)展。國(guó)內(nèi)外多項(xiàng)研究報(bào)道ZHX2可以影響其他基因?qū)Ω伟┌l(fā)生發(fā)展的調(diào)控,但是具體調(diào)控機(jī)制仍不明確。因此,針對(duì)肝癌中轉(zhuǎn)錄抑制因子ZHX2調(diào)控的機(jī)制進(jìn)行初步探討研究。分析肝細(xì)胞癌中轉(zhuǎn)錄抑制因子ZHX2轉(zhuǎn)錄調(diào)控特征,獲得ZHX2的靶基因;研究ZHX2和其靶基因在肝細(xì)胞癌中的表達(dá)及其臨床病理特征的關(guān)系,探討ZHX2與靶基因間的調(diào)控關(guān)系。方法:首先采用RT-PCR方法檢測(cè)肝癌細(xì)胞株HepG2、SMM7721、Be17402和正常肝細(xì)胞株L02中ZHX2 mRNA表達(dá)的情況。接著利用ZHX2抗體進(jìn)行染色質(zhì)免疫共沉淀(chromatin immunoprecipitation, ChIP)實(shí)驗(yàn),將富集到的DNA樣品進(jìn)行高通量測(cè)序,得到肝細(xì)胞肝癌中ZHX2潛在靶基因,然后利用生物信息學(xué)進(jìn)行分析。最后通過(guò)免疫組織化學(xué)技術(shù)檢測(cè)ZHX2和得到的靶基因PTEN、P53在肝細(xì)胞癌組織中的表達(dá),并分析它們與臨床病理學(xué)特征的關(guān)系。結(jié)果:ZHX2mRNA在3種肝癌細(xì)胞株中均有表達(dá)。對(duì)ChIP富集得到的DNA進(jìn)行分析,共得到1981個(gè)ZHX2結(jié)合位點(diǎn)的peaks,含有3093個(gè)結(jié)合的基因。ZHX2的peaks主要分布在1、2、3、5、7號(hào)染色體,在基因元件上的分布主要在基因間區(qū)、內(nèi)含子區(qū)、轉(zhuǎn)錄起始位點(diǎn)上游和啟動(dòng)子區(qū)。對(duì)ZHX2結(jié)合在啟動(dòng)子區(qū)的232個(gè)基因進(jìn)行基因功能(gene ontology, GO)注釋,發(fā)現(xiàn)ZHX2參與了與生物調(diào)節(jié)、代謝進(jìn)程和分子調(diào)節(jié)功能等有關(guān)的生物功能,和形成血液微粒、近心異染色質(zhì)等有關(guān)的細(xì)胞組分,在分子功能上與GTP酶的激活、調(diào)節(jié)和其他酶的激活有關(guān)的過(guò)程。然后進(jìn)行KEGG pathway分析顯示其中一些靶基因可能富集于4條通路,分別是3條傳染性疾病的通路和1條吞噬通路。相對(duì)于肝癌癌旁組織,ZHX2在肝細(xì)胞癌組織中低表達(dá)(P=0.042),與血清AFP值相關(guān)(P=0.041); PTEN在癌組織中低表達(dá)(P=0.000),分別與腫瘤的分化程度和血清AFP值有關(guān)(P=0.025,P=0.028); P53在癌組織中高表達(dá)(P=0.000),與腫瘤的分化程度有關(guān)(P=0.045)。ZHX2 與 PTEN 存在相關(guān)性(r=0.258,P=0.031)。結(jié)論:本研究是首次采用ChIP-Seq技術(shù)從全基因組水平解析ZHX2在肝癌中的轉(zhuǎn)錄調(diào)控的靶基因。發(fā)現(xiàn)ZHX2除了與啟動(dòng)子區(qū)域結(jié)合外,還可以和基因間區(qū)的遠(yuǎn)距離調(diào)控區(qū)域結(jié)合調(diào)控靶基因。對(duì)啟動(dòng)子區(qū)域的靶基因進(jìn)行基因富集分析顯示結(jié)合的靶基因與代謝過(guò)程、生物調(diào)控過(guò)程等有關(guān);富集通路分析提示富集靶基因可能影響了傳染性疾病和吞噬相關(guān)調(diào)控。ZHX2與PTEN、P53在肝細(xì)胞癌發(fā)生發(fā)展中起作用。PTEN結(jié)合于ZHX2的啟動(dòng)子區(qū)域,且ZHX2與PTEN存在正相關(guān)性,提示兩者相互作用對(duì)HCC進(jìn)行調(diào)節(jié),為進(jìn)一步研究ZHX2在肝癌中的具體調(diào)控機(jī)制提供依據(jù)。
[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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R735.7

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本文編號(hào)：2195194