本文選題：人乳頭瘤病毒 + DNA甲基化��； 參考：《青島大學(xué)》2014年碩士論文

【摘要】：人類乳頭瘤病毒(human papillomavirus, HPV)的發(fā)現(xiàn)使得宮頸癌成為迄今病因最明確的一種癌癥,HPV感染干擾經(jīng)典抑癌基因p53及RB的作用也已確認(rèn)。HPV也可能通過誘導(dǎo)某些抑癌基因啟動(dòng)子區(qū)CpG島高甲基化而引起該基因表達(dá)沉默參與宮頸癌的發(fā)生發(fā)展,但是HPV如何誘導(dǎo)宿主基因甲基化以及作用于那些基因有待于深入研究。 目的探討4種宮頸癌細(xì)胞系及宮頸癌組織中RASSFIA基因啟動(dòng)子及第1外顯子區(qū)甲基化狀態(tài),明確甲基化轉(zhuǎn)移酶抑制劑5-氮雜-2'-脫氧胞苷(5-Aza-2’deoxycytidine,5-Aza-dc)對(duì)宮頸癌細(xì)胞系RASSFIA基因啟動(dòng)子及第1外顯子區(qū)甲基化狀態(tài)及轉(zhuǎn)錄表達(dá)的影響。 方法采用甲基化特異性PCR (Methylation-Specific PCR, MSP)和基于亞硫酸氫鹽修飾的基因組測序法(Bisulfite genomic sequencing, BGS)檢測5-Aza-dc處理前后4種宮頸癌細(xì)胞系、宮頸癌組織以及正常宮頸組織中RASSFIA基因啟動(dòng)子及第1外顯子區(qū)甲基化狀態(tài)。同時(shí)應(yīng)用實(shí)施熒光定量PCR(Real-time PCR)檢測RASSFIA基因的轉(zhuǎn)錄表達(dá)情況。 結(jié)果①M(fèi)SP檢測RASSFIA基因啟動(dòng)子及第1外顯子區(qū)甲基化狀態(tài)結(jié)果顯示2種HPV陽性宮頸癌細(xì)胞系HeLa, Caski呈U型,2種HPV陰性宮頸癌細(xì)胞系HT-3,C-33A呈M型。經(jīng)5-Aza-dc處理后,HT-3, C-33A細(xì)胞系M+U型,而2種HPV陽性細(xì)胞系無明顯變化。 ②宮頸癌組織和正常宮頸組織中RASSFIA基因啟動(dòng)子及第1外顯子區(qū)的甲基化率分別為6%(3/48)和0%(0/15),統(tǒng)計(jì)學(xué)分析表明,宮頸癌組織RASSFIA基因啟動(dòng)子的甲基化率與正常宮頸組織比較無統(tǒng)計(jì)學(xué)意義(FISH精確概率,P=0.2691)；HPV陽性宮頸癌組織和細(xì)胞系以及HPV陰性宮頸癌組織和細(xì)胞系中RASSFIA基因啟動(dòng)子與第1外顯子的甲基化率分別為2.17%(1/46)和66.7%(4/6),統(tǒng)計(jì)學(xué)表明,兩者之間甲基化率差異有統(tǒng)計(jì)學(xué)意義(FISH精確概率,P=0.00027)。在HPV感染的宮頸鱗狀細(xì)胞癌中未發(fā)現(xiàn)RASSFIA基因啟動(dòng)子及第1外顯子區(qū)的甲基化。僅有1例HPV感染的宮頸腺癌中存在RASSFIA基因啟動(dòng)子及第1外顯子區(qū)的甲基化。 ③BGS結(jié)果顯示RASSFIA基因啟動(dòng)子及第1外顯子區(qū)的64個(gè)CpG位點(diǎn)基化狀態(tài),2種HPV陽性細(xì)胞系HeLa及Caski除個(gè)別位點(diǎn)發(fā)生甲基化外,其余位點(diǎn)均未發(fā)生甲基化；而2種HPV陰性宮頸癌細(xì)胞系HT-3及C-33A則表現(xiàn)為幾乎所有位點(diǎn)的甲基化。兩種不同濃度5-Aza-dc作用于HT-3和C-33A細(xì)胞系后,其甲基化程度降低。1例HPV陽性、1例HPV陰性宮頸腺癌組織及1例HPV陰性的宮頸鱗癌呈高甲基化狀態(tài)。 ⑤方差分析顯示,兩種不同濃度5-Aza-dc作用HPV陰性宮頸癌細(xì)胞系HT-3及C-33A在后,RASSFIA基因的轉(zhuǎn)錄表達(dá)水平差異具有統(tǒng)計(jì)學(xué)意義(FHT-3=85.598, P=0.00; FC-33A=12.476, P=0.00)。而在HPV陽性宮頸癌細(xì)胞系Caski中,兩種不同濃度5-Aza-dc作用前后,RASSFIA基因的轉(zhuǎn)錄表達(dá)水平無統(tǒng)計(jì)學(xué)意義(Fcaski=1.080,P=0.398)。 ⑥宮頸癌組織與正常宮頸組織中RASSFIA基因的轉(zhuǎn)錄表達(dá)水平無顯著性差異(t=1.908,P=0.069); HPV陽性和HPV陰性宮頸癌組織中RASSFIA基因的轉(zhuǎn)錄表達(dá)水平也無統(tǒng)計(jì)學(xué)意義(t=-0.085,P=0.756)。 結(jié)論①HPV陽性和HPV陰性宮頸癌細(xì)胞系中RASSFIA基因啟動(dòng)子及第1外顯子區(qū)甲基化狀態(tài)不同。 ②RASSFIA基因啟動(dòng)子及第1外顯子區(qū)的高甲基化可抑制該基因表達(dá)；5-Aza-dc處理可使RASSFIA基因啟動(dòng)子及第1外顯子區(qū)去甲基化,重新激活基因的表達(dá)。 ③僅少數(shù)宮頸癌組織中RASSFIA基因啟動(dòng)子及第1外顯子區(qū)的高甲基化,在HPV感染的宮頸鱗狀細(xì)胞癌中未發(fā)現(xiàn)RASSFIA基因啟動(dòng)子及第1外顯子區(qū)的高甲基化。 ④RASSFIA基因可能通過遺傳學(xué)機(jī)制或其他表觀遺傳學(xué)機(jī)制參與宮頸癌的發(fā)生發(fā)展,其發(fā)揮作用途徑及其機(jī)制未完全明確,HPV感染與宮頸癌中RASSFIA基因低甲基化之間的關(guān)系仍需進(jìn)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.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33

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本文編號(hào)：1781994