EB病毒相关胃癌中RASSF1A基因甲基化状态和表达的研究

发布时间：2018-07-24 20:08

【摘要】：EB病毒(Epstein-Barr virus,EBV)是疱疹病毒属γ疱疹病毒亚科成员,是重要的DNA肿瘤病毒。EBV感染与部分胃癌的关系已经得到确认,但EBV相关胃癌(EBV-associated gastric carcinoma,EBVa GC)的发病机制尚未完全明了。近来研究表明EBV可能通过诱导某些抑癌基因启动子区Cp G岛高甲基化而使该基因功能失活参与胃癌的发生发展。目的:明确EBV阳性和阴性胃癌细胞系以及EBVaGC和EBV阴性胃癌(EBV-negatived gastric carcinoma,EBVn GC)组织中RASSF1A基因启动子区甲基化状态以及对其表达的影响,为进一步明确Ras相关结构域家族基因1A(ras associated domain family gene1A,RASSF1A)基因在EBVaGC发生中的作用及作用机制提供实验基础。方法:采用甲基化特异性PCR(Methylation-Specific PCR,MSP)和基于焦亚硫酸氢盐修饰的基因组测序法(Bisulfite genomic sequencing,BGS)检测5-氮杂-2'-脱氧胞苷(5-Aza-2’deoxycytidine,5-Aza-dc)处理前后8种胃癌细胞系以及胃癌组织中RASSF1A基因启动子区甲基化状态;实时荧光定量PCR(Real time quantitative PCR,real time q PCR)检测EBV阴性及阳性细胞系中RASSF1Am RNA的转录表达;免疫组化技术检测EBVa GC和EBVn GC组织中RASSF1A蛋白表达。结果:(1)MSP检测结果显示4种EBV阳性胃癌细胞系(GT38,GT39,SNU719,AGS-EBV)RASSF1A基因启动子区呈未甲基化状态,电泳结果为U型,4种EBV阴性胃癌细胞系(HGC-27,MKN-45,SGC7901,AGS)RASSF1A基因启动子区呈高甲基化状态,电泳结果为M型。BGS检测RASSF1A基因启动子区的86个Cp G位点甲基化状态,结果显示:4种EBV阳性胃癌细胞系除少量位点发生甲基化外,其余位点均未发生甲基化,甲基化率均小于10%;而4种EBV阴性胃癌细胞系则表现为几乎所有位点的甲基化,甲基化率均达90%以上。(2)MSP检测结果显示:36例EBVa GC组织中有28例为M型(28/36,77.8%),M+U型为8例(8/36,22.2%);41例EBVn GC组织中U型有33例(33/41,80.5%),M+U型为8例(8/41,19.5%)。BGS结果显示:36例EBVa GC平均甲基化率达86%以上;41例EBVn GC平均甲基化率为22.3%。(3)经去甲基化试剂5-Aza-dc处理后,4种EBV阴性细胞系MSP电泳结果均发生变化,由原来的M型转变成M+U型;而4种EBV阳性细胞系MSP电泳结果仍为U型。(4)EBV阳性胃癌细胞系RASSF1A基因的转录表达水平均高于阴性胃癌细胞系。5-Aza-dc作用后,EBV阴性胃癌细胞RASSF1A基因的转录表达水平明显升高,EBV阳性胃癌细胞系则无明显变化。(5)EBVa GC和EBVn GC组织中RASSF1A蛋白表达在EBVa GC和EBVn GC组织中无显著差异(P0.05)。结论:(1)EBV阳性和EBV阴性胃癌细胞系中RASSF1A基因启动子区甲基化状态不同,提示EBV感染可导致RASSF1A基因启动子区甲基化状态发生改变。(2)RASSF1A基因启动子区的高甲基化可抑制该基因的转录表达;去甲基化试剂5-Aza-dc处理可使RASSF1A基因启动子区去甲基化,使得该基因表达水平升高。(3)EBVa GC和EBVn GC中组织中RASSF1A基因启动子区甲基化状态不同,提示EBV感染可能是导致RASSF1A基因启动子区域甲基化的原因;但其与EBV阳性细胞系RASSF1A基因启动子区低甲基化现象相左的结果需要我们进一步深入研究加以证实。
[Abstract]:EB virus (Epstein-Barr virus, EBV) is a member of the subfamily of herpes simplex virus (HSV), an important DNA tumor virus.EBV infection and some gastric cancer, but the pathogenesis of EBV related gastric cancer (EBV-associated gastric carcinoma, EBVa GC) has not been fully understood. The methylation of the Cp G island of the promoter region of the oncogene makes the gene function inactivation to participate in the development of gastric cancer. Objective: to clarify the methylation status of the promoter region of the EBV positive and negative gastric cancer cell lines and the EBVaGC and EBV negative gastric carcinoma (EBV-negatived gastric carcinoma, EBVn GC) and the effect on its expression. The Ras related domain family gene 1A (RAS associated domain family gene1A, RASSF1A) gene provides an experimental basis for the role and mechanism of the gene in the occurrence of EBVaGC. G, BGS) detected the methylation status of the RASSF1A gene promoter region of 8 gastric cancer cell lines and gastric cancer tissues before and after the treatment of 5- heterozygous -2'- deoxycytidine (5-Aza-2 'deoxycytidine, 5-Aza-dc), and the real-time fluorescent quantitative PCR (Real time quantitative) was used to detect the transcription of the negative and positive cell lines. The expression of RASSF1A protein in the tissues of EBVa GC and EBVn GC was detected by histochemical technique. Results: (1) the results of MSP detection showed that the promoter region of the 4 EBV positive gastric cancer cell lines (GT38, GT39, SNU719, AGS-EBV) was not methylation state, and the electrophoresis results were type, 4 kinds of negative gastric cancer cell lines were in the promoter region. The methylation status of the 86 Cp G loci in the promoter region of the RASSF1A gene was detected by M type.BGS. The results showed that the 4 EBV positive gastric cancer cell lines had no methylation and the methylation rate was less than 10% except for a small number of loci methylation, while the 4 EBV negative gastric cancer cell lines were almost all sites. Methylation and methylation rates were above 90%. (2) MSP detection results showed that 28 of the 36 EBVa GC tissues were M (28/36,77.8%), M+U in 8 (8/36,22.2%), 33 in EBVn GC tissue in 41 cases, and 8 for M+U type. After the rate of 22.3%. (3) was treated with the demethylation reagent 5-Aza-dc, the results of the MSP electrophoresis of the 4 EBV negative cell lines were all changed, from the original M type to the M+U type, while the MSP electrophoresis results of the 4 EBV positive cell lines were still U type. (4) the RASSF1A gene expression level of the EBV positive gastric cancer cell lines was higher than that of the negative gastric cancer cell line.5-Aza-dc. After use, the transcriptional expression level of RASSF1A gene in EBV negative gastric cancer cells was significantly increased, and there was no significant change in EBV positive gastric cancer cell lines. (5) there was no significant difference in the expression of RASSF1A protein in EBVa GC and EBVn GC tissues in EBVa GC and EBVn GC tissues. (1) the promoter region of the gene promoter region was methylated in the positive and negative gastric cancer cell lines. It is suggested that EBV infection can lead to a change in the methylation status of the promoter region of the RASSF1A gene. (2) the hypermethylation of the promoter region of the RASSF1A gene inhibits the transcriptional expression of the gene, and the demethylation agent 5-Aza-dc treatment can demethmethylation of the promoter region of the RASSF1A gene, making the gene expression level higher. (3) EBVa GC and EBVn G The methylation status of the promoter region of the RASSF1A gene in the tissue of C is different, suggesting that EBV infection may be the cause of the methylation of the promoter region of the RASSF1A gene, but the results from the dismethylation of the RASSF1A gene promoter region of the EBV positive cell line need to be further confirmed by our further study.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

【参考文献】