子宮頸內贅瘤中腫瘤抑制基因和發炎相關基因COX-2的甲基化情形

Promoter Methylation Status of Tumor Suppressor Genes and an Inflammatory-related COX-2 Gene in Cervical Intraepithelial Neoplasia

李建佑(Chien-Yu Li)

指導教授 ： 李瑞年

高雄醫學大學/生命科學院/生物醫學暨環境生物學研究所/碩士(2010年)

https://doi.org/10.6832/KMU.2010.00148

摘要

子宮頸癌 (cervical cancer)是全球女性罹患癌症的第二名，在罹患子宮頸癌症女性中高達99%都檢測到Human Papillomavirus (HPV) DNA的存在，而這些檢測的HPV DNA中，有70%是由HPV16和HPV18所造成的。由HPV感染的細胞有機會發展成子宮頸內贅瘤(cervical intraepithelial neoplasia, CIN)。在病理檢測上，CIN嚴重程度可以依照病人檢體中不正常的細胞比例分成3個不同階段時期來區分成CIN I-CIN III，但是柏氏子宮頸抹片檢查(Papanicolaou smear test)有15-30%的偽陰性。DNA甲基化的改變是屬於癌症較早期發生的事件，而且甲基化的改變常出現在各種癌症發生的早期損傷。我們要鑑定出有希望的甲基化標記(marker)候選者以用於早期檢測和預後的檢查。在許多癌症中發現位於抑癌基因(tumor suppressor gene)的啟動子(promoter)區域出現過度的甲基化(hypermethylation)並促成癌症的生成。另外，發炎反應在腫瘤生長的微環境(tumor microenvironment)中也會促成癌症發展，它會協助malignant cells增生以及存活，促成血管新生現象(angiogenesis)和癌轉移(metastasis)。cox-2基因的過度甲基化現象可能是個有潛力的預後標誌可用於偵測早期子宮頸癌症。因此，本研究主要利用methylation-specific polymerase chain reaction (MSP)和bisulfite sequencing偵測不同時期的子宮頸內贅瘤病人的子宮頸抹片檢體中的甲基化狀態，包含了RARβ、p16和CDH1以及發炎反應相關的COX-2這四個基因。最後，在本研究中發現，正常人及CIN病人的子宮頸細胞檢體中，COX-2 基因都是非甲基化的狀態(unmethylated form)。除此之外，RARβ基因在正常的病人細胞檢體有2.2%的甲基化，在CIN III階段的病人細胞檢體有3.7%的甲基化。CDH1基因在CIN I階段的病人細胞檢體有6.9%甲基化、CIN II有5%甲基化、CIN III有11.1%，
在鱗狀細胞癌的病人細胞檢體有 9%甲基化。只有p16基因的甲基化偵測結果發現，在正常人細胞檢體可偵測到19.2%的甲基化，而隨著子宮頸內贅瘤的嚴重程度，可發現從CIN I到子宮頸癌的檢體中甲基化的比例從39%升高到50%。在p16基因的bisulfite sequencing結果中可發現到10個甲基化MSP產物中的10個CpG位置都呈現甲基化修飾。特別地，在沒有受到HPV感染的檢體中，p16基因的甲基化比例隨著子宮頸內贅瘤越嚴重也隨之增加。然而，p16基因的甲基化比例和HPV感染並沒有關連性。因此，偵測p16基因的啟動子區域的甲基化狀態或許是個有潛力的早期子宮頸癌症的診斷工具，並希望能夠與柏氏子宮頸抹片檢查及HPV病毒檢查一起做為子宮頸檢查工具，以提高檢測的準確性。

關鍵字

子宮頸癌 ； 抑癌基因 ； 甲基化

並列摘要

Cervical cancer is the second most common cancer in women in worldwide. 99% of cervical cancer patients are HPV (Human Papillomavirus) DNA positive, and 70% is caused by HPV16 and HPV18. HPV infected cells maybe developed to premalignant lesions which also called cervical intraepithelial neoplasia (CIN). CIN can be categorized into grades I, II and III depending upon the proportion of the abnormal cells. The Papanicolaou (PAP) test have false negative about 15~30%. DNA methylation changes are an early event in carcinogenesis and are often present in the precursor lesions of various cancers. We wanted to identify promising methylation marker candidates for cervical cancer early detection and prognosis. Hypermethylation in a promoter region of tumor suppressor gene is observed in many cancers and promotes tumorigenesis. On the other hand, inflammation in the tumor microenvironment has many tumor-promoting effects. In previous reports, cox-2 is overexpressed in various cancers and plays an important role in cell adhesion, apoptosis and angiogenesis. Hypermethylation of the cox-2 gene may be a potential prognostic marker in early stage of cervical cancer. We examined the methylation status of tumor suppressor genes, including RARβ, p16 and CDH1, and inflammatory-related COX-2 gene in different stages of CIN by methylation-specific polymerase chain reaction (MSP) and bisulfite sequencing. Finally, the cox-2 genes are observed in unmethylated form in this study from normal to carcinoma specimens. Besides, the methylation frequency of RARβ was observed in 2.2% of normal and 3.7% of CIN III. The methylation frequency of cdh1 genes was observed in 6.9% of CIN I, 5% of CIN II, 11.1% of CIN III, and 9% of squamous cell carcinoma. Only p16 gene has 19.2% methylated frequency in normal specimens and 39-50% methylated frequency from CIN I to carcinoma. The result of bisulfite sequencing indicated that the 10 CpG sites are all methylated in five individuals. Notably, the methylation frequency of p16 is progressively increased during the development of malignant stages in CIN, in the absent of HPV. However, we observed the frequency of p16 gene methylation is independent of the HPV-infection in CIN patients. In conclusion, aberrant promoter methylation analysis on cervical cell specimens of p16 may be a potential diagnostic tool for cervical cancer screening and can be used with cytology and human papillomavirus testing to improve accuracy.

並列關鍵字

cervical cancer:HPV ； tumor suppressor gene ； methylation

參考文獻

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