JMJD2B在幽门螺杆菌介导的胃黏膜上皮细胞恶性转化中的作用研究
发布时间：2018-10-29 12:54
【摘要】：背景胃癌是世界范围内最常见的恶性肿瘤之一,其死亡率居于全球癌症相关死亡率的第三位。中国是胃癌高发国家,中国胃癌发病率居世界第二,仅次于日本。胃癌的发生是多因素共同作用的结果,其中幽门螺杆菌(Helicobacter pylori, H. pylori)感染是胃癌的重要致病因素。虽然已有大量研究证实幽门螺杆菌感染与胃癌发生之间的关系,但其确切的致病机制仍不明确。近期研究发现,幽门螺杆菌感染可以调控宿主细胞的表观遗传学修饰的改变,例如组蛋白修饰状态的改变、DNA甲基化等,这些改变与幽门螺杆菌的致癌作用密切相关。组蛋白去甲基化酶JMJD2B,又称KDM4B,是新近发现的JMJD2蛋白家族中的一员,主要靶向组蛋白H3第9位赖氨酸的三甲基(H3K9me3)使其发生去甲基化,调节染色质结构或基因表达,在炎症和多种恶性肿瘤发生中发挥重要的表观遗传学作用。本课题组前期研究发现,JMJD2B通过调控相关的细胞生物学过程在胃癌的发生发展过程中发挥重要的作用。然而,JMJD2B在胃癌细胞中高表达的机制尚不清楚。鉴于幽门螺杆菌感染是胃黏膜恶性转化的重要始动因素,因此,本研究探讨了幽门螺杆菌对JMJD2B的调控作用及机制。另外,研究发现幽门螺杆菌感染可以上调细胞COX2的表达,COX2在幽门螺杆菌致胃黏膜上皮细胞病变的过程中,不仅可以加重炎症反应,还可促进胃癌的发生发展。虽然已有研究揭示COX2在幽门螺杆菌致癌过程中的作用,但幽门螺杆菌激活COX2表达的分子机制仍未完全阐明。近期研究发现COX2的表达受到表观遗传学修饰的调控,如DNA甲基化、组蛋白乙酰化等,与胃癌的进展及预后密切相关。因此,本实验对JMJD2B介导的表观遗传学机制是否参与调控幽门螺杆菌诱导的COX2的表达上调进行了研究。目的探讨组蛋白去甲基化酶JMJD2B在幽门螺杆菌诱导的炎症向胃癌恶性转化过程中的作用及相关分子机制,进一步阐明幽门螺杆菌的致癌机制。方法1.幽门螺杆菌感染调控JMJD2B表达的机制和功能研究：QRT-PCR、western blot和双荧光素酶报告基因等方法检测幽门螺杆菌感染对JMJD2B表达的调控,同时利用克隆形成实验分析JMJD2B对幽门螺杆菌诱导的细胞增殖的影响。利用β-catenin的小干扰RNA转染至细胞,通过QRT-PCR和western blot检测fMJD2B的表达。另外,构建JMJD2B的启动子质粒和突变质粒,通过双荧光素酶报告基因检测β-catenin对JMJD2B表达的调控。2. JMJD2B在幽门螺杆菌诱导的COX2表达上调中的作用及其机制研究：将JMJD2B的小干扰RNA转染至细胞,利用QRT-PCR. western blot和双荧光素酶报告基因等方法检测JMJD2B对COX2表达的调控。通过免疫共沉淀和染色质免疫共沉淀实验研究JMJD2B调控COX2表达的分子机制。3.幽门螺杆菌调控JMJD2B和COX2表达的动物实验：采用灌胃方式将幽门螺杆菌SS1注入C57BL/6J、鼠胃内构建模型,通过QRT-PCR检测小鼠胃组织中的MJD2B和COX2表达水平。4. JMJD2B和COX2在幽门螺杆菌感染的胃组织标本中的表达：收集临床胃炎及胃癌患者的胃组织标本,通过免疫组织化学方法检测JMJD2B和COX2在正常组织、慢性非萎缩性胃炎、慢性萎缩性胃炎及胃癌组织标本中的表达水平,确定JMJD2B和COX2在不同组织中的表达差异,并分析JMJD2B表达与幽门螺杆菌感染以及与COX2表达之间的相关性。结果1.幽门螺杆菌感染上调JMJD2 B表达,这种调控作用不依赖其毒力因子CagA。2. β-catenin调控JMJD2B启动子活性介导幽门螺杆菌感染诱导的JMJD2B的转录表达。3. JMJD2B是幽门螺杆菌诱导的COX2表达上调中的关键调节分子。JMJD2B与NF-κB相互作用,共同结合于COX2启动子区,使组蛋白H3K9me3发生去甲基化反应,转录激活COX2表达。4.动物实验表明,幽门螺杆菌感染的鼠胃黏膜组织中JMJD2B和COX2的表达水平均升高。5.免疫组化结果表明,JMJD2B和COX2在正常组织、慢性非萎缩性胃炎、慢性萎缩性胃炎及胃癌组织标本中的表达水平均逐渐升高,且JMJD2B的表达与幽门螺杆菌的感染状态及COX2的表达均具有相关性。结论幽门螺杆菌感染胃黏膜上皮细胞诱导P-catenin移位入核,通过转录因子LEF/TCF结合至JMJD2B的启动子区,上调JMJD2B的表达。JMJD2B结合于COX2启动子区,使组蛋白H3K9me3发生去甲基化反应,改变染色质构象,招募转录因子NF-κB结合至COX2的启动子区,激活COX2的转录表达,促进了胃黏膜上皮细胞的恶性转化。本研究从细胞水平、动物水平和组织水平三方面探讨了幽门螺杆菌感染调控JMJD2B表达促进胃黏膜上皮细胞恶性转化的相关分子机制,提示JMJD2B有可能作为一种新的胃癌治疗的靶点,有助于胃癌的早期诊断和干预。
[Abstract]:Background Gastric cancer is one of the most common malignancies worldwide and the mortality rate is the third in global cancer-related mortality. China is a high incidence of gastric cancer and the incidence of gastric cancer in China ranks second in the world after Japan. The occurrence of gastric cancer is the result of multifactorial co-action, in which Helicobacter pylori (H. pylori) infection is an important pathogenic factor of gastric cancer. Although there have been a large number of studies confirming the relationship between Helicobacter pylori infection and the occurrence of gastric cancer, the exact pathogenesis of Helicobacter pylori is still unclear. Recent studies have found that Helicobacter pylori infection can regulate the changes in epigenetic modification of host cells, such as changes in histones modification states, DNA methylation, and the like, which are closely related to the carcinogenic effects of H. pylori. Histone de-methyl parathion-2B, also known as KD4B, is one of the newly discovered HMGBJD2 protein family, which mainly targeted the 3-methyl (H3K9me3) of the 9-bit lysine of histones H3 to demethylation, regulate chromatin structure or gene expression, plays an important epigenetic role in the occurrence of inflammation and various malignant tumors. In the previous study, it was found that the cell biology process of Jurkat 2B played an important role in the development of gastric cancer. However, the mechanism of high expression of cyclin 2B in gastric cancer cells is unknown. Helicobacter pylori infection is an important starting factor in malignant transformation of gastric mucosa. In addition, it is found that Helicobacter pylori infection can upregulate the expression of COX2, and COX2 can not only aggravate inflammatory response, but also promote the development of gastric cancer. Although studies have revealed the role of COX2 in the carcinogenesis of Helicobacter pylori, the molecular mechanism of Helicobacter pylori activated COX2 expression has not been fully elucidated. Recently, it has been found that the expression of COX2 is regulated by epigenetic modification, such as DNA methylation, histones and so on. It is closely related to the progression and prognosis of gastric cancer. Therefore, this study conducted a study on whether the epigenetic mechanism mediated by HMGB2B was involved in the regulation of the expression of COX2 induced by H. pylori. Objective To investigate the role and molecular mechanism of histones to the malignant transformation of Helicobacter pylori induced by Helicobacter pylori and to further clarify the carcinogenic mechanism of H. pylori. Method 1. The mechanism and function of Helicobacter pylori infection regulating the expression of Helicobacter pylori 2B were studied: QRT-PCR, Western blot and double luciferase reporter gene. The expression of fMCS2B was detected by QRT-PCR and Western blot. In addition, the promoter plasmid and the mutant plasmid of Jurkat 2B were constructed, and the regulation of the expression of Jurkat 2B was detected by double luciferase reporter gene. The role of cyclin 2B in the upregulation of COX2 expression induced by Helicobacter pylori and its mechanism were studied: the small interfering RNA of Jurkat 2B was transfected into the cells and QRT-PCR was used. Western blot and luciferase reporter gene assay were used to detect the regulation of COX2 expression. The molecular mechanism of COX2 expression was studied by immune co-precipitation and chromatin immune co-precipitation experiment. The results showed that Helicobacter pylori SS1 was injected into C57BL/ 6J mice by gavage, and the expression levels of MCS2B and COX2 in gastric tissues of mice were detected by QRT-PCR. The expression of Jurkat 2B and COX2 in gastric tissue specimens of Helicobacter pylori infection: the collection of gastric tissue specimens of clinical gastritis and gastric cancer patients, the detection of COX2 in normal tissues and chronic non-atrophic gastritis by immunohistochemistry. The levels of expression in the specimens of chronic atrophic gastritis and gastric cancer were determined. The differences in the expression of cyclin 2B and COX2 in different tissues were determined, and the correlation between the expression of cyclin 2B and the infection of Helicobacter pylori and the expression of COX2 was analyzed. Result 1. Helicobacter pylori infection upregulated the expression of cyclin JD2 B, which did not depend on its virulence factor CagA. 2. Gene-catenin regulates the transcription and expression of cyclin 2B induced by Helicobacter pylori infection by the promoter activity of Jurkat 2B promoter. Jurkat 2B is a key regulatory molecule in the upregulation of COX2 expression induced by Helicobacter pylori. Jurkat 2B interacts with NF-Sepharose B and binds to the COX2 promoter region, so that the histones H3K9me3 are subjected to methylation reaction, and the transcription activates COX2 expression. Animal experiments showed that the expression levels of cyclin 2B and COX2 in gastric mucosa tissues of Helicobacter pylori infection increased. The results showed that the expression level of cyclin 2B and COX2 in normal tissues, chronic non-atrophic gastritis, chronic atrophic gastritis and gastric cancer tissue specimens increased gradually, and the expression of Jurkat 2B was correlated with the infection status of Helicobacter pylori and the expression of COX2. Conclusions Helicobacter pylori infection of gastric mucosa epithelial cells induces P-catenin migration into the nucleus, and the transcription factor LEF/ TCF binds to the promoter region of Jurkat 2B, and the expression of Jurkat 2B is up-regulated. Jurkat 2B binds to the COX2 promoter region to demethylation the histones H3K9me3, changes chromatin conformation, and recruits the transcription factor NF-5B to the promoter region of COX2, activates the transcription expression of COX2, and promotes the malignant transformation of gastric mucosa epithelial cells. In this study, we discussed the molecular mechanism of Helicobacter pylori infection regulated by Helicobacter pylori infection and promoted the malignant transformation of gastric epithelial cells from three aspects: cell level, animal level and tissue level. It is helpful for early diagnosis and intervention of gastric cancer.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2
本文编号：2297758
[Abstract]:Background Gastric cancer is one of the most common malignancies worldwide and the mortality rate is the third in global cancer-related mortality. China is a high incidence of gastric cancer and the incidence of gastric cancer in China ranks second in the world after Japan. The occurrence of gastric cancer is the result of multifactorial co-action, in which Helicobacter pylori (H. pylori) infection is an important pathogenic factor of gastric cancer. Although there have been a large number of studies confirming the relationship between Helicobacter pylori infection and the occurrence of gastric cancer, the exact pathogenesis of Helicobacter pylori is still unclear. Recent studies have found that Helicobacter pylori infection can regulate the changes in epigenetic modification of host cells, such as changes in histones modification states, DNA methylation, and the like, which are closely related to the carcinogenic effects of H. pylori. Histone de-methyl parathion-2B, also known as KD4B, is one of the newly discovered HMGBJD2 protein family, which mainly targeted the 3-methyl (H3K9me3) of the 9-bit lysine of histones H3 to demethylation, regulate chromatin structure or gene expression, plays an important epigenetic role in the occurrence of inflammation and various malignant tumors. In the previous study, it was found that the cell biology process of Jurkat 2B played an important role in the development of gastric cancer. However, the mechanism of high expression of cyclin 2B in gastric cancer cells is unknown. Helicobacter pylori infection is an important starting factor in malignant transformation of gastric mucosa. In addition, it is found that Helicobacter pylori infection can upregulate the expression of COX2, and COX2 can not only aggravate inflammatory response, but also promote the development of gastric cancer. Although studies have revealed the role of COX2 in the carcinogenesis of Helicobacter pylori, the molecular mechanism of Helicobacter pylori activated COX2 expression has not been fully elucidated. Recently, it has been found that the expression of COX2 is regulated by epigenetic modification, such as DNA methylation, histones and so on. It is closely related to the progression and prognosis of gastric cancer. Therefore, this study conducted a study on whether the epigenetic mechanism mediated by HMGB2B was involved in the regulation of the expression of COX2 induced by H. pylori. Objective To investigate the role and molecular mechanism of histones to the malignant transformation of Helicobacter pylori induced by Helicobacter pylori and to further clarify the carcinogenic mechanism of H. pylori. Method 1. The mechanism and function of Helicobacter pylori infection regulating the expression of Helicobacter pylori 2B were studied: QRT-PCR, Western blot and double luciferase reporter gene. The expression of fMCS2B was detected by QRT-PCR and Western blot. In addition, the promoter plasmid and the mutant plasmid of Jurkat 2B were constructed, and the regulation of the expression of Jurkat 2B was detected by double luciferase reporter gene. The role of cyclin 2B in the upregulation of COX2 expression induced by Helicobacter pylori and its mechanism were studied: the small interfering RNA of Jurkat 2B was transfected into the cells and QRT-PCR was used. Western blot and luciferase reporter gene assay were used to detect the regulation of COX2 expression. The molecular mechanism of COX2 expression was studied by immune co-precipitation and chromatin immune co-precipitation experiment. The results showed that Helicobacter pylori SS1 was injected into C57BL/ 6J mice by gavage, and the expression levels of MCS2B and COX2 in gastric tissues of mice were detected by QRT-PCR. The expression of Jurkat 2B and COX2 in gastric tissue specimens of Helicobacter pylori infection: the collection of gastric tissue specimens of clinical gastritis and gastric cancer patients, the detection of COX2 in normal tissues and chronic non-atrophic gastritis by immunohistochemistry. The levels of expression in the specimens of chronic atrophic gastritis and gastric cancer were determined. The differences in the expression of cyclin 2B and COX2 in different tissues were determined, and the correlation between the expression of cyclin 2B and the infection of Helicobacter pylori and the expression of COX2 was analyzed. Result 1. Helicobacter pylori infection upregulated the expression of cyclin JD2 B, which did not depend on its virulence factor CagA. 2. Gene-catenin regulates the transcription and expression of cyclin 2B induced by Helicobacter pylori infection by the promoter activity of Jurkat 2B promoter. Jurkat 2B is a key regulatory molecule in the upregulation of COX2 expression induced by Helicobacter pylori. Jurkat 2B interacts with NF-Sepharose B and binds to the COX2 promoter region, so that the histones H3K9me3 are subjected to methylation reaction, and the transcription activates COX2 expression. Animal experiments showed that the expression levels of cyclin 2B and COX2 in gastric mucosa tissues of Helicobacter pylori infection increased. The results showed that the expression level of cyclin 2B and COX2 in normal tissues, chronic non-atrophic gastritis, chronic atrophic gastritis and gastric cancer tissue specimens increased gradually, and the expression of Jurkat 2B was correlated with the infection status of Helicobacter pylori and the expression of COX2. Conclusions Helicobacter pylori infection of gastric mucosa epithelial cells induces P-catenin migration into the nucleus, and the transcription factor LEF/ TCF binds to the promoter region of Jurkat 2B, and the expression of Jurkat 2B is up-regulated. Jurkat 2B binds to the COX2 promoter region to demethylation the histones H3K9me3, changes chromatin conformation, and recruits the transcription factor NF-5B to the promoter region of COX2, activates the transcription expression of COX2, and promotes the malignant transformation of gastric mucosa epithelial cells. In this study, we discussed the molecular mechanism of Helicobacter pylori infection regulated by Helicobacter pylori infection and promoted the malignant transformation of gastric epithelial cells from three aspects: cell level, animal level and tissue level. It is helpful for early diagnosis and intervention of gastric cancer.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2
【相似文献】
相关硕士学位论文 前1条
1 韩凤娟;JMJD2B在幽门螺杆菌介导的胃黏膜上皮细胞恶性转化中的作用研究[D];山东大学;2016年
，本文编号：2297758
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2297758.html
