宫颈癌RASSFIA基因启动子与第1外显子区甲基化状态的研究

发布时间：2018-04-21 10:15

本文选题：人乳头瘤病毒 + DNA甲基化　；参考：《青岛大学》2014年硕士论文

【摘要】：人类乳头瘤病毒(human papillomavirus, HPV)的发现使得宫颈癌成为迄今病因最明确的一种癌症,HPV感染干扰经典抑癌基因p53及RB的作用也已确认。HPV也可能通过诱导某些抑癌基因启动子区CpG岛高甲基化而引起该基因表达沉默参与宫颈癌的发生发展,但是HPV如何诱导宿主基因甲基化以及作用于那些基因有待于深入研究。目的探讨4种宫颈癌细胞系及宫颈癌组织中RASSFIA基因启动子及第1外显子区甲基化状态,明确甲基化转移酶抑制剂5-氮杂-2'-脱氧胞苷(5-Aza-2’deoxycytidine,5-Aza-dc)对宫颈癌细胞系RASSFIA基因启动子及第1外显子区甲基化状态及转录表达的影响。方法采用甲基化特异性PCR (Methylation-Specific PCR, MSP)和基于亚硫酸氢盐修饰的基因组测序法(Bisulfite genomic sequencing, BGS)检测5-Aza-dc处理前后4种宫颈癌细胞系、宫颈癌组织以及正常宫颈组织中RASSFIA基因启动子及第1外显子区甲基化状态。同时应用实施荧光定量PCR(Real-time PCR)检测RASSFIA基因的转录表达情况。结果①MSP检测RASSFIA基因启动子及第1外显子区甲基化状态结果显示2种HPV阳性宫颈癌细胞系HeLa, Caski呈U型,2种HPV阴性宫颈癌细胞系HT-3,C-33A呈M型。经5-Aza-dc处理后,HT-3, C-33A细胞系M+U型,而2种HPV阳性细胞系无明显变化。 ②宫颈癌组织和正常宫颈组织中RASSFIA基因启动子及第1外显子区的甲基化率分别为6%(3/48)和0%(0/15),统计学分析表明,宫颈癌组织RASSFIA基因启动子的甲基化率与正常宫颈组织比较无统计学意义(FISH精确概率,P=0.2691)；HPV阳性宫颈癌组织和细胞系以及HPV阴性宫颈癌组织和细胞系中RASSFIA基因启动子与第1外显子的甲基化率分别为2.17%(1/46)和66.7%(4/6),统计学表明,两者之间甲基化率差异有统计学意义(FISH精确概率,P=0.00027)。在HPV感染的宫颈鳞状细胞癌中未发现RASSFIA基因启动子及第1外显子区的甲基化。仅有1例HPV感染的宫颈腺癌中存在RASSFIA基因启动子及第1外显子区的甲基化。 ③BGS结果显示RASSFIA基因启动子及第1外显子区的64个CpG位点基化状态,2种HPV阳性细胞系HeLa及Caski除个别位点发生甲基化外,其余位点均未发生甲基化；而2种HPV阴性宫颈癌细胞系HT-3及C-33A则表现为几乎所有位点的甲基化。两种不同浓度5-Aza-dc作用于HT-3和C-33A细胞系后,其甲基化程度降低。1例HPV阳性、1例HPV阴性宫颈腺癌组织及1例HPV阴性的宫颈鳞癌呈高甲基化状态。 ⑤方差分析显示,两种不同浓度5-Aza-dc作用HPV阴性宫颈癌细胞系HT-3及C-33A在后,RASSFIA基因的转录表达水平差异具有统计学意义(FHT-3=85.598, P=0.00; FC-33A=12.476, P=0.00)。而在HPV阳性宫颈癌细胞系Caski中,两种不同浓度5-Aza-dc作用前后,RASSFIA基因的转录表达水平无统计学意义(Fcaski=1.080,P=0.398)。 ⑥宫颈癌组织与正常宫颈组织中RASSFIA基因的转录表达水平无显著性差异(t=1.908,P=0.069); HPV阳性和HPV阴性宫颈癌组织中RASSFIA基因的转录表达水平也无统计学意义(t=-0.085,P=0.756)。结论①HPV阳性和HPV阴性宫颈癌细胞系中RASSFIA基因启动子及第1外显子区甲基化状态不同。 ②RASSFIA基因启动子及第1外显子区的高甲基化可抑制该基因表达；5-Aza-dc处理可使RASSFIA基因启动子及第1外显子区去甲基化,重新激活基因的表达。 ③仅少数宫颈癌组织中RASSFIA基因启动子及第1外显子区的高甲基化,在HPV感染的宫颈鳞状细胞癌中未发现RASSFIA基因启动子及第1外显子区的高甲基化。 ④RASSFIA基因可能通过遗传学机制或其他表观遗传学机制参与宫颈癌的发生发展,其发挥作用途径及其机制未完全明确,HPV感染与宫颈癌中RASSFIA基因低甲基化之间的关系仍需进一步深入研究。
[Abstract]:The discovery of human papillomavirus (human papillomavirus, HPV) has made cervical cancer the most definitive cancer to date. The effect of HPV infection on the classical tumor suppressor gene p53 and RB has also confirmed that.HPV may also cause the gene expression to be silent in cervical cancer by inducing the Gao Jiaji transformation of certain tumor suppressor genes in the promoter region of CpG island. Occurrence and development, but how HPV can induce the methylation of the host gene and act on those genes need to be further studied.
Objective to investigate the methylation status of RASSFIA gene promoter and 1 exon in 4 cervical cancer cell lines and cervical cancer tissues, and to clarify methylation of the methyltransferase inhibitor 5- -2'- deoxycytidine (5-Aza-2 'deoxycytidine, 5-Aza-dc) for the methylation and transcription of the RASSFIA gene promoter and exon 1 of the cervical cancer cell line. Influence.
Methods the methylation specific PCR (Methylation-Specific PCR, MSP) and hydrogen sulfite modified genome sequencing (Bisulfite genomic sequencing, BGS) were used to detect the methylation status of the 4 cervical cancer cell lines, the cervical cancer tissues and the normal cervical tissues, and the RASSFIA gene promoter and the 1 exon region in the normal cervix tissues. At the same time, PCR Real-time PCR was used to detect the transcription and expression of RASSFIA gene.
Results (1) MSP detection of RASSFIA gene promoter and 1 exon methylation showed that 2 HPV positive cervical cancer cell lines were HeLa, Caski was U, 2 HPV negative cervical cancer cell lines HT-3 and C-33A M type. After 5-Aza-dc treatment, HT-3, and 2 kinds of positive cell lines had no obvious changes.
(2) the methylation rates of RASSFIA gene promoter and 1 exon region in cervical cancer tissues and normal cervix tissues were 6% (3/48) and 0% (0/15) respectively. Statistical analysis showed that the methylation rate of RASSFIA promoter in cervical cancer tissues was not statistically significant (FISH accurate probability, P=0.2691), and HPV positive cervical cancer group. The methylation rates of RASSFIA gene promoter and first exon in HPV negative cervical cancer tissues and cell lines were 2.17% (1/46) and 66.7% (4/6), respectively. Statistics showed that the difference of methylation rates between the two groups was statistically significant (FISH accurate probability, P=0.00027). No RASSFIA based in HPV infected cervical squamous cell carcinoma was found. Methylation of promoters and exon 1 was detected in only 1 cases of HPV infected cervical adenocarcinoma, with methylation of RASSFIA gene promoter and exon 1.
(3) BGS results showed that the RASSFIA gene promoter and the 64 CpG loci in the 1 exon region were based. The 2 HPV positive cell lines, HeLa and Caski, were methylated except for the methylation of the other loci, while the 2 HPV negative cervical cancer cell lines HT-3 and C-33A showed methylation at almost all sites. Two different concentrations were different. After the effect of degree 5-Aza-dc on HT-3 and C-33A cell lines, the degree of methylation decreased in.1 case HPV positive. 1 cases of HPV negative cervical adenocarcinoma tissue and 1 cases of HPV negative cervical squamous cell carcinoma showed hypermethylation status.
(5) analysis of variance analysis showed that the differential expression level of RASSFIA gene was statistically significant (FHT-3=85.598, P=0.00; FC-33A=12.476, P=0.00) after two different concentrations of 5-Aza-dc HPV negative cervical cancer cell lines HT-3 and C-33A (FHT-3=85.598, P=0.00; FC-33A=12.476, P=0.00). The transcriptional expression level was not statistically significant (Fcaski=1.080, P=0.398).
(6) there was no significant difference in the transcriptional expression level of RASSFIA gene in cervical cancer tissues and normal cervical tissues (t=1.908, P=0.069), and there was no statistical significance (t=-0.085, P=0.756) in HPV positive and HPV negative cervical cancer tissues.
Conclusion (1) the methylation status of RASSFIA gene promoter and exon 1 in HPV positive and HPV negative cervical cancer cell lines are different.
The hypermethylation of the RASSFIA gene promoter and the 1 exon can inhibit the gene expression, and the 5-Aza-dc treatment can demethmethylation of the RASSFIA gene promoter and the 1 exon region and reactivate the gene expression.
(3) the hypermethylation of the RASSFIA gene promoter and the 1 exon region in only a few cervical cancer tissues, and the hypermethylation of the RASSFIA gene promoter and the 1 exon region was not found in the HPV infected cervical squamous cell carcinoma.
(4) RASSFIA gene may participate in the development of cervical cancer by genetic mechanism or other epigenetic mechanism. Its mechanism and mechanism are not completely clear. The relationship between HPV infection and RASSFIA gene low methylation in cervical cancer needs further study.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

【参考文献】