MicroRNA-335在胃癌中的表达及其表观遗传调控机制的研究

发布时间：2018-06-29 12:51

本文选题：甲基化 + MicroRNA-335　；参考：《南昌大学》2016年硕士论文

【摘要】：目的:探讨胃癌转移中表达下调MicroRNA-335是否存在表观遗传调控的机制,并通过干预实验进行验证。同时在胃癌中预测并验证MicroRAN-335参与胃癌侵袭和转移的靶向基因。方法:生物信息学平台验证理论的可能性,实时定量PCR检测胃癌细胞株中和胃组织中MicroRNA-335表达水平;重亚硫酸盐测序PCR(BSP)和甲基化特异性PCR(MSP)检测二者MicroRNA-335启动子区的甲基化状态;采用去甲基化方法(5-Azadeoxycytidine)处理胃癌细胞株,分别用实时定量PCR和BSP法检测处理后的MicroRNA-335表达的变化及其甲基化的变化;利用多个生物靶向在线软件(microRNA;Targetscan;Pic Tar;Rnahybrid)分析miR-335可能的靶基因及靶向3’-UTR的位点,实时定量PCR和Western Blot对预测出的靶基因做mRNA和蛋白水平的验证,利用脂质体转染法将hsa-miR-335模拟物及miR-335抑制剂及其对应的阴性对照、空白对照组分别转入人胃癌SGC-7901和BGC-823细胞系,进一步验证这种靶向关系。结果:(1)MicroRNA-335基因序列的启动子区具有稳定的CpG岛:生物信息学网站(UCSC、NCBI、Mirbase)综合对比分析MicroRNA-335基因上游5000bp范围的序列,用确定的基因序列进行启动子和CpG岛搜索,Cpgislands发现2个甲基岛,Methprimer和Cpgplot分别有4个和3个甲基岛,三者重叠率为88.9%,对比分析甲基岛和启动子区域存在交汇。(2)人胃癌细胞系中MicroRNA-335表达降低。通过5-Azadeoxycytidine去甲基化预处理,人胃癌细胞系中miR-335表达升高。胃癌组织中miR-335表达降低,尤以有腹膜转移的组织表达最低。(3)人胃癌细胞系中miR-335的启动子区CpG岛高甲基化,通过5-Azadeoxycytidine去甲基化预处理,miR-335启动子区甲基化水平降低。胃癌中miR-335的启动子区CpG岛高甲基化与miR-335表达存在相关性。通过甲基化特异性PCR(MSP)检测,胃癌组织中miR-335有更高的甲基化,且高甲基化信号与胃癌TNM分期、淋巴结转移、腹膜转移有明显的相关性。胃癌组织中,高甲基化的组织呈现较低的miR-335表达水平。(4)生物信息学预测GTPase activating protein 1(RASA1)可能是miR-335作用于胃癌侵袭转移的靶基因。实时定量PCR及Western Blot初步检测胃癌中尤其是转移组织中RASA1表达升高,外生高表达miR-335后RASA1表达显著减低。结论:(1)胃癌转移中表达下调MicroRNA-335受其启动子CpG岛高甲基化的调控。(2)胃癌组织中miR-335启动子区高甲基化信号与胃癌侵袭转移相关临床病理因素有显著相关性。(3)miR-335可能通过调节RASA1基因表达发挥抑制胃癌侵袭转移作用。
[Abstract]:Aim: to investigate the mechanism of epigenetic regulation of down-regulation of microRNA-335 in gastric cancer metastasis and verify it by intervention experiment. At the same time, microRAN-335 gene involved in invasion and metastasis of gastric cancer was predicted and verified in gastric cancer. Methods: bioinformatics platform was used to test the possibility of microRNA-335 expression in gastric cancer cell lines and gastric tissues by real-time quantitative PCR, and the methylation status of microRNA-335 promoter region was detected by heavy sulfite sequencing PCR (BSP) and methylation specific PCR (MSP). Gastric cancer cell lines were treated with demethylation method (5-Azadeoxycytidine). The changes of microRNA-335 expression and methylation were detected by real-time quantitative PCR and BSP-PCR, respectively. The possible target genes of miR-335 and the sites targeting 3H-UTR were analyzed by microRNA-TargetscanPic tag Rnahybrid. The mRNA and protein levels of the predicted target genes were verified by real-time quantitative PCR and Western Blot. The hsa-miR-335 mimics, miR-335 inhibitors and their corresponding negative controls were transfected into human gastric cancer SGC-7901 and BGC-823 cell lines by liposome transfection. Results: (1) the promoter region of the microRNA-335 gene sequence had stable CpG island: a comprehensive comparative analysis of the 5000bp region upstream of the microRNA-335 gene. The promoter and CpG islands were used to search for two methyl islands, Methprimer and Cpgplot, which were found to have 4 and 3 methyl islands, respectively. The overlap rate among the three groups was 88.9, which was compared and analyzed. (2) the expression of microRNA-335 was decreased in human gastric cancer cell line. The expression of miR-335 in human gastric cancer cell line was increased by 5-Azadeoxycytidine demethylation preconditioning. The expression of miR-335 was decreased in gastric cancer tissues, especially in the tissues with peritoneal metastasis. (3) the promoter region of miR-335 was hypermethylated, and the methylation level of the promoter region of miR-335 was decreased by 5-Azadeoxycytidine demethylation preconditioning. The expression of miR-335 was correlated with CpG island hypermethylation in the promoter region of miR-335 in gastric cancer. The results of methylation specific PCR (MSP) showed that miR-335 had higher methylation in gastric cancer tissues, and the hypermethylation signal was significantly correlated with TNM stage, lymph node metastasis and peritoneal metastasis. In gastric cancer tissues, the hypermethylated tissues showed a low expression level of miR-335. (4) Bioinformatics predicted that GTPase activating protein 1 (RASA1) might be the target gene of miR-335 acting on invasion and metastasis of gastric cancer. The expression of RASA1 in gastric carcinoma, especially in metastatic tissues, was detected by real-time quantitative PCR and Western blot, but the expression of RASA1 was significantly decreased after miR-335 was overexpressed. Conclusion: (1) the down-regulation of microRNA-335 in gastric cancer metastasis is regulated by the hypermethylation of its promoter CpG island. (2) there is a significant correlation between the hypermethylation signal of miR-335 promoter and the clinicopathological factors associated with invasion and metastasis of gastric cancer. Regulation of RASA1 gene expression inhibits invasion and metastasis of gastric cancer.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

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