乙型肝炎病毒X蛋白抑制Wnt信号通路拮抗因子SFRPs的表达及分子机制的研究

发布时间：2018-07-21 19:45

【摘要】：目的：原发性肝癌（Hepatocellular carcinoma, HCC)是世界第三大导致死亡的恶性肿瘤。HCC发生的其中一个危险因素是乙型肝炎病毒（Hepatitis B virus, HBV）感染。目前全世界约有4亿人感染HBV，中国约占1/3。HBV基因组由部分环状的双链DNA组成，全长约为3.2kb，基因组包含4个开放阅读框架，分别为C、P、S和X区。乙型肝炎病毒X基因（Hepatitis B virus X, HBx）编码154个氨基酸，在病毒的复制等方面发挥重要作用。大量的研究报道表明，，HBx蛋白在HCC的发生中发挥重要的作用。作为一个多功能的调节因子，HBx能够和细胞核内的转录因子相互作用，从而影响细胞的转录活性。此外，HBx还可涉及一些信号转导通路，例如：Wnt/β-catenin, Ras/MAPK, SAPK/JNK,NF-κB, FAK等。 Wnt/β-catenin信号通路调控着细胞的衰老、死亡、增殖、转移，以及在决定胚胎形态等方面都发挥着重要的作用。大量的研究表明，Wnt信号通路的异常活化可以参与肿瘤的发生，例如：肺癌、结肠癌等。以前也有研究表明，Wnt信号通路的异常活化也可导致肝癌的发生。在肝癌的发生中，除了Wnt/β-catenin信号通路组成部分的基因突变外，一些表观遗传修饰也可导致这条信号通路的异常活化。分泌卷曲相关蛋白（Secreted frizzled-related proteins, SFRPs)是一种Wnt信号的拮抗因子。SFRPs家族共有5个分泌性糖蛋白家族成员，包括SFRP1~5，由约300个氨基酸残基组成。有研究报道表明，由于SFRPs启动子区甲基化导致其表达的下调，在HBV相关的HCC中发挥重要作用。相反，回复SFRP1的表达，可以有效抑制肿瘤细胞的增殖和迁移能力。这些数据表明，SFRPs基因的表观沉默表达，在由Wnt/β-catenin信号通路异常活化导致的HCC中发挥重要作用。也有研究报道表明，HBx也能够激活Wnt/β-catenin这条信号通路，但是其在表观沉默SFRPs从而导致肝癌的发生等方面，目前还未见相关的报道。本研究拟在细胞水平和肿瘤组织标本中观察HBx对SFRPs的表观调控，从而进一步研究HBx导致肿瘤发生的分子机制。方法：收集临床肝癌组织标本，提取癌组织DNA、RNA和总蛋白，通过RT-PCR、western blot检测肿瘤组织与癌旁组织中SFRP1和SFRP5的表达情况，进一步采用甲基化特异性PCR（Methylation specific PCR,MSP）及亚硫酸盐测序PCR（Bisulfite sequencing PCR, BSP），检测肝癌组织中SFRP1、SFRP5启动子区的甲基化表达情况。同时观察感染HBx的肿瘤细胞株SFRP1、SFRP5启动子活性变化，进一步证实HBx能否调控SFRP1、SFRP5的表达。通过体外功能实验研究回复SFRP1、SFRP5或干扰甲基化修饰酶Dnmt1后肝癌细胞的增殖和迁移能力变化，探讨HBx蛋白调控SFRP1、SFRP5的分子机制。结果：我们在重庆医科大学附属第二医院收集到35例原发性肝癌组织标本，分别提取肿瘤和癌旁组织的总RNA、DNA、总蛋白，观察到与癌旁组织相比，肿瘤组织Wnt信号抑制分子SFRP1、SFRP5的表达明显下调，甲基化特异性PCR以及亚硫酸盐测序PCR实验均表明肿瘤组织与癌旁组织相比，SFRP1和SFRP5启动子区甲基化修饰显著增高。体外构建成功含SFRP1和SFRP5区的启动子截短突变报告质粒，采用双荧光素酶报告基因法检测，结果表明SFRP1、SFRP5启动子区在转录起始位点400bp左右启动子活性最强。感染HBx后，SFRP1、SFRP5的启动子活性明显低于对照组。在体外功能实验中，平板克隆形成实验、MTS实验、Brdu掺入实验、结晶紫实验均可证实，稳定表达HBx的肝癌细胞系，HBx蛋白可以明显促进肝癌细胞增殖，并且干扰Dnmt1或者回复SFRP1、SFRP5能够抑制肝癌细胞的增殖作用。迁移实验结果表明，HBx蛋白能够增强肝癌细胞系Huh7细胞的迁移的能力，干扰Dnmt1或者回复SFRP1和SFRP5的表达能够抑制肝癌细胞的迁移能力。结论：乙型肝炎病毒X蛋白通过降低SFRP1、SFRP5的启动子活性，明显下调Wnt信号抑制分子SFRP1、SFRP5的表达。HBx促进肿瘤细胞的增殖和迁移能力与活化DNA甲基转移酶下调SFRPs的表达进而活化Wnt信号相关。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.62

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