CagA诱导的miR-155上调靶向KLF4促进胃粘膜上皮细胞的恶性转化

发布时间：2018-04-20 16:20

本文选题：胃癌 + 幽门螺旋杆菌　；参考：《安徽医科大学》2017年硕士论文

【摘要】：【实验研究背景】在全世界范围内,胃癌发病率高,大约是癌症总发病率的8%,是癌症致死的主要恶性疾病之一,是常见的恶性肿瘤。更为重要的是,约有70%的胃癌相关的死亡病例均位于不发达国家,尤其是东亚地区。在中国,肺癌、乳腺癌、胃癌、肝癌、结肠癌是男性和女性中5种最常见的高发肿瘤。胃癌的发生是宿主基因多态性、我们日常饮食或是吸烟等环境因素、表观遗传学改变等多种危险因素共同作用的结果。目前,幽门螺旋杆菌长期慢性感染胃粘膜上皮被认为是导致胃恶性肿瘤发生的最危险的因素,尤其是胃腺癌。但是幽门螺旋杆菌的致病机制并不清楚,已经报道的幽门螺旋杆菌如何与宿主基因相互作用影响胃癌的发生发展非常少。目前仍然有许多关键性的问题需要进一步探索。最近研究表明,细胞毒素相关抗原A蛋白(CagA)和细胞空泡毒素A(VacA)是幽门螺旋杆菌当中致病性最强的毒力因子,研究最为广泛。进入细胞内的CagA蛋白能够异常激活NF-κB、β-catenin等信号通路,促进细胞增殖、促炎性细胞因子产生及相关miRNA的产生等细胞反应,参与致癌过程。虽然全世界有将近一半的人感染了幽门螺旋杆菌,却只有3%的感染人群最终发展为胃癌。这就说明幽门螺旋杆菌感染能否致病还依赖于与宿主基因的相互作用。表观遗传学包括点突变、剪切、复制、重组、与肿瘤相关基因的甲基化等等的改变,以及肿瘤抑癌基因的失活是从炎症转变为癌症的重要分子机制。胃癌的发生是幽门螺旋杆菌感染与细胞内抑癌基因之间相互作用的结果。miRNA是一类小的,非编码的RNAs,它通过直接与靶mRNA 3′UTR结合,负向调控与肿瘤发生相关的癌基因或者抑癌基因的m RNA水平,促进癌症的发展。广泛的参与到了基因的转录后调控。有报道研究,miR-155是免疫反应和炎症反应的关键调控因素。但是,miR-155在幽门螺旋杆菌感染导致的胃疾病当中发挥了怎样的功能作用并不清楚,已有的报道也有相矛盾的地方。KLF4是一个含有锌指结构的转录因子。我们前期对KLF4进行了研究,实验结果显示,过表达KLF4明显的增加了细胞的凋亡；体内实验说明过表达KLF4抑制了胃癌增殖和侵袭,KLF4在胃癌中具有抑癌基因的作用。胃癌组织中KLF4蛋白表达较正常胃粘膜上皮明显降低,KLF4的表达和功能作用与胃癌的发生发展关系密切。但是KLF4在胃癌中表达异常的分子机制却不清楚。我们提出了以下假设:KLF4在胃癌中表达异常是不是由于幽门螺旋杆菌感染引起的?幽门螺旋杆菌中致病性最强的毒力因子CagA是如何与宿主基因KLF4相互作用,导致KLF4在胃粘膜上皮细胞异常表达?KLF4在胃癌中表达异常是否可能由于miRNA靶向干扰KLF4表达?为了探索抑癌基因KLF4在胃癌中表达降低可能存在的分子机制,本课题在细胞水平上和临床病例相结合的方式进行实验验证。【实验研究方法】1、Western blot检测胃粘膜上皮细胞和胃癌细胞中KLF4蛋白表达。将HA标记的WT-CagA质粒转染进入GES-1细胞,通过转染不同的时间、不同的剂量,检测GES-1细胞中KLF4蛋白和m RNA表达。2、免疫组化方法检测胃癌组织和癌旁组织中KLF4蛋白表达。3、将HA标记的WT-CagA质粒转染进入GES-1细胞,细胞行为学实验检测CagA对GES-1细胞恶性生物学行为的影响。4、搜集33例体检者和胃癌患者血清标本,提取血清中miRNA,用RT-PCR的方法检测miR-155表达的差异性；原位杂交方法检测胃癌组织和癌旁组织中miR-155表达。5、RT-PCR检测胃粘膜上皮细胞和胃癌细胞株中miR-155的表达。将HA标记的WT-CagA质粒转染进入GES-1中,提取细胞中总RNA。用普通PCR的方法,琼脂糖凝胶电泳观察CagA对miR-155表达的影响。6、在GES-1细胞中瞬时转染miR-155-3p minic和miR-155-5p-minic,普通PCR检测其对KLF4 mRNA影响；通过转染不同的时间、不同的剂量,Western blot检测其对KLF4蛋白表达的影响；WT-KLF4 luciferase reporter和MUT-KLF4 luciferase reporter与miR-155 minic/NC共转染,48h后检测荧光素酶活性,从而对miR-155下游靶点的预测进行验证。7、建立稳定表达miR-155-5p-negative controlmiR-155-5p minic的胃粘膜上皮细胞株GES-1和胃癌细胞株AGS。建立稳定表达miR-155-5p-negativemiR-155-5p-inhibitor的胃癌细胞株SGC-7901。通过western blot实验分析稳定表达miR-155和敲低miR-155的细胞株中,KLF4蛋白表达。【实验研究结果】1、与正常的胃粘膜上皮细胞相比,胃癌细胞株当中KLF4蛋白表达明显降低；高表达CagA后很明显的降低了KLF4蛋白和m RNA表达,并且呈现出时间和剂量依赖性的抑制；2、免疫组化结果显示,胃癌组织当中KLF4的表达明显降低,且幽门螺旋杆菌感染的胃癌组织KLF4表达降低更为明显。3、幽门螺旋杆菌毒力因子CagA的高表达促进了胃粘膜上皮细胞的增殖、迁移、平板克隆等恶性生物学行为的改变。4、与正常人的血清相比,胃癌患者血清标本中miR-155的表达增高,尤其在TNM分期Ⅱ期和Ⅲ期更为明显；并且miR-155表达的升高与幽门螺旋杆菌感染密切相关。miR-155在胃癌组织中表达明显高于癌旁组织。5、细胞水平上,在胃上皮细胞GES-1和恶性程度相对较低的胃癌细胞株AGS表达相对较低,在恶性程度较高的胃癌细胞株中miR-155表达水平相对较高；高表达CagA诱导了miR-155表达上调；6、荧光素酶报告基因证实KLF4可能是miR-155的靶基因。7、成功构建了稳定表达miR-155-5p-negative controlmiR-155-5p minic的胃粘膜上皮细胞株GES-1和胃癌细胞株AGS。建立了稳定表达miR-155-5p-negativemiR-155-5p-inhibitor的胃癌细胞株SGC-7901。Western blot结果显示miR-155可能负向调控KLF4表达。【结论】CagA可能通过诱导miR-155表达,靶向干扰KLF4转录及翻译,抑制KLF4表达,从而促进胃粘膜上皮的恶性转化。
[Abstract]:[background] the incidence of gastric cancer is high around the world, about 8% of the total cancer incidence, one of the major malignant diseases fatal to cancer and a common malignant tumor. More importantly, about 70% of the cancer related deaths are in the underdeveloped countries, especially in East Asia. In China, lung cancer, and breast cancer, Gastric cancer, liver cancer, and colon cancer are the 5 most common high incidence tumors in men and women. The occurrence of gastric cancer is the result of host gene polymorphism, environmental factors such as our daily diet or smoking, epigenetic changes and other risk factors. The most dangerous factors for the occurrence of gastric malignant tumors, especially gastric adenocarcinoma. However, the pathogenesis of Helicobacter pylori is not clear. It is reported that the interaction of Helicobacter pylori and host genes affects the development of gastric cancer very little. There are still many key problems that need further exploration. Recent studies have shown that Cytotoxin related antigen A (CagA) and cell vacuolating toxin A (VacA) are the most virulent among Helicobacter pylori, the most widely studied. The CagA proteins entering the cell can activate abnormal activation of NF- kappa B, beta -catenin and other signaling pathways, promote cell proliferation, proinflammatory cytokines production and related miRNA production. Although nearly half of the world is infected with Helicobacter pylori, only 3% of the infected people eventually develop into gastric cancer. This suggests that the infection of Helicobacter pylori is also dependent on the interaction with the host gene. Epigenetics includes point mutations, shear, replication, recombination, and tumor related groups. Changes in methylation, and the inactivation of tumor suppressor genes are important molecular mechanisms from inflammation to cancer. The occurrence of gastric cancer is the result of the interaction between Helicobacter pylori infection and intracellular tumor suppressor genes.MiRNA is a small, non coded RNAs, which is regulated by a direct combination with the target mRNA 3 'UTR. M RNA levels associated with oncogenesis, or tumor suppressor genes, promote cancer development. Extensive involvement in post transcriptional regulation of genes. MiR-155 is a key regulator of the immune response and inflammatory response. However, what is the function of miR-155 in the gastric disease caused by Helicobacter pylori infection The effect is not clear, the existing reports also have a contradictory place.KLF4 is a transcriptional factor containing the zinc finger structure. We studied the KLF4 earlier. The experimental results showed that overexpression of KLF4 significantly increased the apoptosis of the cells; in vivo experiments showed that overexpression of KLF4 inhibited the proliferation and invasion of gastric cancer, and KLF4 had tumor suppressor in gastric cancer. The expression of KLF4 protein in gastric cancer tissue is significantly lower than that of normal gastric mucosa, and the expression and function of KLF4 are closely related to the development of gastric cancer. However, the molecular mechanism of abnormal expression of KLF4 in gastric cancer is not clear. We put forward the following hypothesis: the abnormal expression of KLF4 in gastric cancer is not due to the Helicobacter pylori How is the strongest virulence factor CagA in Helicobacter pylori caused by the interaction of the host gene KLF4 and the abnormal expression of KLF4 in the epithelial cells of the gastric mucosa? Is the abnormal expression of KLF4 in gastric cancer possibly due to the miRNA targeting KLF4 expression? In order to detect the reduction of the expression of the tumor suppressor gene KLF4 in gastric cancer, the possible survival of the tumor suppressor gene KLF4 may exist. In the molecular mechanism, the subject was tested in a combination of cell level and clinical case. [experimental method] 1, Western blot was used to detect the expression of KLF4 protein in gastric epithelial cells and gastric cancer cells. HA labeled WT-CagA plasmid was transfected into GES-1 cells, and different doses were detected by transfection at different time. The expression of KLF4 protein and m RNA in GES-1 cells was.2, and the expression of KLF4 protein in gastric cancer tissues and adjacent tissues was detected by immunohistochemistry. The WT-CagA plasmids labeled with HA were transfected into GES-1 cells. The effect of CagA on the malignant biological behavior of GES-1 cells was detected by cell behavior test. 33 cases of physical examination and gastric cancer patients were collected and extracted. MiRNA in serum was used to detect the difference in expression of miR-155 by RT-PCR. In situ hybridization was used to detect the expression of miR-155 in gastric cancer tissue and adjacent tissues, and the expression of miR-155 in gastric epithelial cells and gastric cancer cell lines was detected by RT-PCR. The WT-CagA plasmid labeled with HA was transfected into GES-1, and the general PCR method was extracted. The effect of CagA on the expression of miR-155 was observed by agarose gel electrophoresis,.6, miR-155-3p minic and miR-155-5p-minic were transiently transfected in GES-1 cells. Ordinary PCR detected its effect on KLF4 mRNA. Luciferase reporter was co transfected with miR-155 minic/NC, and the activity of luciferase was detected after 48h, thus the prediction of the target of the downstream target of miR-155 was verified by.7, and the stable expression of GES-1 and gastric cancer cell strain of the gastric mucosa epithelial cell line, which expressed the miR-155-5p-negative controlmiR-155-5p minic, was established. The gastric cancer cell line SGC-7901. of itor analyzed the expression of KLF4 protein in the cell lines expressing miR-155 and low miR-155 by Western blot test. [experimental results] 1, the expression of KLF4 protein in gastric carcinoma cell lines decreased significantly compared with normal gastric mucosal epithelial cells, and KLF4 protein and M decreased after high expression of CagA. The expression of RNA was expressed in time and dose dependent inhibition. 2, the results of immunohistochemistry showed that the expression of KLF4 in gastric carcinoma tissue decreased obviously, and the expression of KLF4 in gastric cancer tissues of Helicobacter pylori decreased more obviously.3. The high expression of CagA of Helicobacter pylori promoted the proliferation and migration of gastric mucosa epithelial cells. The changes in the malignant biological behavior such as the flat clones.4, compared with the normal human serum, the expression of miR-155 in the serum samples of the gastric cancer patients was increased, especially in the stage II and stage III of the TNM stage, and the expression of miR-155 was closely related to the Helicobacter pylori infection and the expression of.MiR-155 in gastric cancer tissues was significantly higher than that of the para cancerous tissue.5. On the cell level, the expression of AGS in gastric epithelial cell GES-1 and the relatively low malignancy of gastric cancer cell line was relatively low, and the expression level of miR-155 was relatively high in high malignant gastric cancer cells; high expression CagA induced the up-regulated expression of miR-155; 6, the luciferase reporter gene confirmed that KLF4 might be the target gene.7 of miR-155. The gastric mucosal epithelial cell line GES-1 and the gastric cancer cell line that stably expressed miR-155-5p-negative controlmiR-155-5p minic were constructed, and the gastric cancer cell line AGS. was established to stabilize the expression of miR-155-5p-negativemiR-155-5p-inhibitor in the gastric cancer cell line SGC-7901.Western blot results showed that miR-155 may be negatively regulated KLF4 expression. [Conclusion] CagA may be induced by lure. The expression of miR-155 can interfere with KLF4 transcription and translation, inhibit KLF4 expression, and promote malignant transformation of gastric epithelial cells.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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