【摘要】：目的：原發(fā)性肝癌（Hepatocellular carcinoma, HCC)是世界第三大導致死亡的惡性腫瘤。HCC發(fā)生的其中一個危險因素是乙型肝炎病毒（Hepatitis B virus, HBV）感染。目前全世界約有4億人感染HBV，中國約占1/3。HBV基因組由部分環(huán)狀的雙鏈DNA組成，全長約為3.2kb，基因組包含4個開放閱讀框架，分別為C、P、S和X區(qū)。乙型肝炎病毒X基因（Hepatitis B virus X, HBx）編碼154個氨基酸，在病毒的復制等方面發(fā)揮重要作用。大量的研究報道表明，，HBx蛋白在HCC的發(fā)生中發(fā)揮重要的作用。作為一個多功能的調(diào)節(jié)因子，HBx能夠和細胞核內(nèi)的轉(zhuǎn)錄因子相互作用，從而影響細胞的轉(zhuǎn)錄活性。此外，HBx還可涉及一些信號轉(zhuǎn)導通路，例如：Wnt/β-catenin, Ras/MAPK, SAPK/JNK,NF-κB, FAK等。 Wnt/β-catenin信號通路調(diào)控著細胞的衰老、死亡、增殖、轉(zhuǎn)移，以及在決定胚胎形態(tài)等方面都發(fā)揮著重要的作用。大量的研究表明，Wnt信號通路的異�；罨梢詤⑴c腫瘤的發(fā)生，例如：肺癌、結(jié)腸癌等。以前也有研究表明，Wnt信號通路的異�；罨部蓪е赂伟┑陌l(fā)生。在肝癌的發(fā)生中，除了Wnt/β-catenin信號通路組成部分的基因突變外，一些表觀遺傳修飾也可導致這條信號通路的異常活化。分泌卷曲相關(guān)蛋白（Secreted frizzled-related proteins, SFRPs)是一種Wnt信號的拮抗因子。SFRPs家族共有5個分泌性糖蛋白家族成員，包括SFRP1~5，由約300個氨基酸殘基組成。有研究報道表明，由于SFRPs啟動子區(qū)甲基化導致其表達的下調(diào)，在HBV相關(guān)的HCC中發(fā)揮重要作用。相反，回復SFRP1的表達，可以有效抑制腫瘤細胞的增殖和遷移能力。這些數(shù)據(jù)表明，SFRPs基因的表觀沉默表達，在由Wnt/β-catenin信號通路異常活化導致的HCC中發(fā)揮重要作用。 也有研究報道表明，HBx也能夠激活Wnt/β-catenin這條信號通路，但是其在表觀沉默SFRPs從而導致肝癌的發(fā)生等方面，目前還未見相關(guān)的報道。本研究擬在細胞水平和腫瘤組織標本中觀察HBx對SFRPs的表觀調(diào)控，從而進一步研究HBx導致腫瘤發(fā)生的分子機制。 方法：收集臨床肝癌組織標本，提取癌組織DNA、RNA和總蛋白，通過RT-PCR、western blot檢測腫瘤組織與癌旁組織中SFRP1和SFRP5的表達情況，進一步采用甲基化特異性PCR（Methylation specific PCR,MSP）及亞硫酸鹽測序PCR（Bisulfite sequencing PCR, BSP），檢測肝癌組織中SFRP1、SFRP5啟動子區(qū)的甲基化表達情況。同時觀察感染HBx的腫瘤細胞株SFRP1、SFRP5啟動子活性變化，進一步證實HBx能否調(diào)控SFRP1、SFRP5的表達。通過體外功能實驗研究回復SFRP1、SFRP5或干擾甲基化修飾酶Dnmt1后肝癌細胞的增殖和遷移能力變化，探討HBx蛋白調(diào)控SFRP1、SFRP5的分子機制。 結(jié)果：我們在重慶醫(yī)科大學附屬第二醫(yī)院收集到35例原發(fā)性肝癌組織標本，分別提取腫瘤和癌旁組織的總RNA、DNA、總蛋白，觀察到與癌旁組織相比，腫瘤組織Wnt信號抑制分子SFRP1、SFRP5的表達明顯下調(diào)，甲基化特異性PCR以及亞硫酸鹽測序PCR實驗均表明腫瘤組織與癌旁組織相比，SFRP1和SFRP5啟動子區(qū)甲基化修飾顯著增高。體外構(gòu)建成功含SFRP1和SFRP5區(qū)的啟動子截短突變報告質(zhì)粒，采用雙熒光素酶報告基因法檢測，結(jié)果表明SFRP1、SFRP5啟動子區(qū)在轉(zhuǎn)錄起始位點400bp左右啟動子活性最強。感染HBx后，SFRP1、SFRP5的啟動子活性明顯低于對照組。在體外功能實驗中，平板克隆形成實驗、MTS實驗、Brdu摻入實驗、結(jié)晶紫實驗均可證實，穩(wěn)定表達HBx的肝癌細胞系，HBx蛋白可以明顯促進肝癌細胞增殖，并且干擾Dnmt1或者回復SFRP1、SFRP5能夠抑制肝癌細胞的增殖作用。遷移實驗結(jié)果表明，HBx蛋白能夠增強肝癌細胞系Huh7細胞的遷移的能力，干擾Dnmt1或者回復SFRP1和SFRP5的表達能夠抑制肝癌細胞的遷移能力。 結(jié)論：乙型肝炎病毒X蛋白通過降低SFRP1、SFRP5的啟動子活性，明顯下調(diào)Wnt信號抑制分子SFRP1、SFRP5的表達。HBx促進腫瘤細胞的增殖和遷移能力與活化DNA甲基轉(zhuǎn)移酶下調(diào)SFRPs的表達進而活化Wnt信號相關(guān)。
[Abstract]:Objective: Hepatocellular carcinoma (HCC) is the third largest cause of death in the world, one of the risk factors for the occurrence of.HCC in malignant tumors is hepatitis B virus (Hepatitis B virus, HBV) infection. At present, about 400 million people all over the world are infected with HBV, and China's 1/ 3.HBV genome is composed of some circular double stranded DNA. For 3.2kb, the genome contains 4 open reading frames, which are C, P, S and X. The hepatitis B virus X gene (Hepatitis B virus X, HBx) encodes 154 amino acids, which plays an important role in the replication of the virus. HBx can interact with the transcription factors in the nucleus and affect the transcriptional activity of the cells. In addition, HBx may also involve some signal transduction pathways, such as Wnt/ beta -catenin, Ras/MAPK, SAPK/JNK, NF- kappa B, FAK and so on.
Wnt/ beta -catenin signaling pathway regulates cell aging, death, proliferation, metastasis, and plays an important role in determining embryo morphology. A large number of studies have shown that abnormal activation of Wnt signaling pathway may be involved in the occurrence of tumor, such as lung cancer and colon cancer. Previous studies have shown that the abnormal activity of the Wnt signaling pathway In addition to the genetic mutation of the Wnt/ beta -catenin signaling pathway, some epigenetic modifications can also lead to abnormal activation of the signal pathway in the occurrence of liver cancer. The secretory curl related protein (Secreted frizzled-related proteins, SFRPs) is a.SFRPs family of the antagonistic factor of Wnt signal. A total of 5 secretory glycoprotein family members, including SFRP1~5, composed of about 300 amino acid residues, have been reported to show that SFRPs promoter region methylation leads to down regulation of its expression and plays an important role in HBV related HCC. On the contrary, response to SFRP1 expression can effectively inhibit the proliferation and migration of tumor cells. The data suggest that the apparent silencing of SFRPs gene plays an important role in HCC induced by abnormal activation of Wnt/ beta -catenin signaling pathway.
There are also reports that HBx can also activate the Wnt/ beta -catenin signaling pathway, but it has not yet been reported in terms of the apparent silence of SFRPs leading to the occurrence of liver cancer. This study is intended to observe the apparent regulation of HBx on SFRPs at the cell level and tumor tissue, thus further studying the genesis of the tumor by HBx. Molecular mechanism.
Methods: the DNA, RNA and total protein of the carcinoma tissue were collected and the expression of SFRP1 and SFRP5 in the tumor tissues and adjacent tissues were detected by RT-PCR and Western blot, and the methylation specific PCR (Methylation specific PCR, MSP) and sulfite sequencing were used to detect the liver cancer. The expression of methylation in the promoter region of SFRP1 and SFRP5 in the tissue. Meanwhile, the changes in the activity of SFRP1 and SFRP5 promoter in the tumor cell line infected with HBx were observed, and the expression of HBx could be further proved to regulate the expression of SFRP1, SFRP5. The proliferation and migration ability of the hepatoma cells after SFRP1, SFRP5, or interfering methylated enzyme Dnmt1 were studied. To explore the molecular mechanism of HBx protein regulating SFRP1 and SFRP5.
Results: 35 specimens of primary liver cancer were collected from the Second Hospital Affiliated to Chongqing Medical University, and the total RNA, DNA and total protein were extracted from the tumor and para cancer tissues. Compared with the para cancer tissue, the Wnt signal inhibitor SFRP1, the expression of SFRP5, the methylation specific PCR and the sulfite sequencing PCR were observed. The experiment showed that the methylation modification of SFRP1 and SFRP5 promoter regions was significantly higher than that of the para cancerous tissue. In vitro, a successful promoter of SFRP1 and SFRP5 region was successfully constructed, and the double luciferase reporter gene was used to detect the promoter. The results showed that the promoter region of SFRP1 and SFRP5 promoter region was activated around the transcription start site 400bp. After infection of HBx, the promoter activity of SFRP1 and SFRP5 was significantly lower than that of the control group. In the function experiment in vitro, the plate cloning experiment, the MTS experiment, the Brdu incorporation experiment, the crystal violet experiment could prove that the HBx cell line could be steadily expressed, and the HBx protein could obviously promote the proliferation of hepatoma cells, and interfered Dnmt1 or reverting SFRP1, SFRP5. It can inhibit the proliferation of hepatoma cells. The migration test results show that HBx protein can enhance the migration ability of Huh7 cells in the hepatoma cell line, and the interference of Dnmt1 or the expression of SFRP1 and SFRP5 can inhibit the migration of hepatoma cells.
Conclusion: hepatitis B virus X protein can reduce the promoter activity of SFRP1, SFRP5, and obviously down regulate the Wnt signal inhibitor SFRP1. SFRP5 expression.HBx promotes the proliferation and migration of tumor cells, which is related to activating the expression of DNA methyltransferase and reducing the expression of SFRPs and activating Wnt signal.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R512.62

【共引文獻】

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