探討癌細胞中FLT4與MGMT基因之甲基化狀態並促進 temozolomide對惡性腦瘤治療的效用

Investigation of Methylation Status of FLT4 and MGMT in Cancers and Enhancement of Temozolomide-Induced Cytotoxicity for Malignant Glioma

陳亦禎(Yi-Chen Chen)

指導教授 ： 戴建國

國立中正大學/理學院/生命科學系生物醫學研究所/碩士(2014年)

摘要

口腔癌在台灣男性最常見的癌症中排名第四，其中絕大多數都屬於口腔鱗狀上皮細胞癌。在癌症的發展過程中，致癌的關鍵因素包含了表觀遺傳的調控如DNA的甲基化修飾可造成抑癌基因不表現。在本研究中，我們利用了一組包含807個基因並且可以分析1505個CpG位點的甲基化微陣列晶片去研究口腔癌檢體。從中發現了FLT4這個基因在大部分口腔癌檢體中都有高度甲基化的現象。此外也使用了焦磷酸定序來驗證 FLT4在口腔癌細胞株以及病患檢體中的甲基化程度，並且發現在口腔癌細胞株及病患檢體中FLT4的甲基化程度很高。另外，我們將口腔癌細胞處理了5-aza-dC後，可使FLT4的甲基化程度降低，且基因表現有回復的情況。接著我們使用即時定量聚合酶連鎖反應來鑑定，也證實FLT4的表現量在口腔癌細胞株及病患檢體中很低，而在正常口腔組織中表現量較高。綜合以上結果，我們發現FLT4基因在口腔癌中高度甲基化並藉此調控基因的表現，或許可以作為一個臨床生物指標來預測和診斷口腔癌。
除了研究口腔癌的甲基化圖譜，我們對於多形惡性神經膠質瘤(glioblastoma multiforme, GBM)中抗藥性基因的甲基化修飾也很有興趣。GBM是腦癌中最常見且最惡性的一種，並且難以用手術完全切除。治療腦瘤時經常合併化療藥物烷化劑「帝盟多」(temozolomide, TMZ)，但是若O6-甲基鳥嘌呤-DNA甲基轉移脢(MGMT)這個基因在GBM中高度表現，會使腦癌對TMZ有抗藥性。為了在多種GBM細胞株中偵測 MGMT的甲基化狀態和基因表現量，我們使用焦磷酸定序、即時定量聚合酶連鎖反應以及西方墨點法等技術來做分析。此外也用siRNA來抑制GBM細胞株中的MGMT基因表現，希望可以減少腦癌細胞對TMZ的抗藥性，並偵測細胞的存活率。之後我們更進一步從GBM細胞株中培養出膠質母細胞瘤起始細胞 (glioma-initiating cells, GICs)，並偵測它的MGMT甲基化狀態以及基因表現量與原始GBM細胞株是否有所差異，最後觀察GICs對於TMZ的抗藥性。綜合以上結果，我們發現MGMT確實會影響GBM對於TMZ的抗藥性，或許可以應用在臨床上，作為GBM病患是否使用TMZ做治療的依據。

關鍵字

口腔癌 ； 腦瘤

並列摘要

Oral cancer is the fourth most common cancer type among men in Taiwan, and about 94% of oral cancers are oral squamous cell carcinoma (OSCC). In cancer pathogenesis, epigenetic modification such as DNA methylation driven gene silencing of tumor suppressor genes is recognized as a key event. Therefore, we employed the Illumina GoldenGate Methylation array, which included 1,505 CpG sites covering 807 genes to analyze oral samples from OSCC patients. Among them, Fms-related tyrosine kinase 4 (FLT4) was highly methylated in most of the OSCC samples. In addition, we employed bisulfite pyrosequencing analysis to validate the methylation level of FLT4 in OSCC lines and patient samples. We found hyper-methylation levels were observed in OSCC lines. We also treated OSCC lines with 5-aza-dC, and found that the methylation level of FLT4 was reduced. Furthermore, we examined FLT4 methylation level in patient samples and found that FLT4 was significantly hyper-methylated in OSCC samples than in normal samples. Next, we used real-time PCR and found the expression level of FLT4 in OSCC lines and OSCC samples were low, but significantly higher expression of FLT4 was found in normal oral tissues. Taken DNA methylation and gene expression profiles together, we found FLT4 was hyper-methylated in oral cancer and thus it may hold diagnostic and predictive value as a biomarker.
In addition to understanding the methylation profile of OSCC, we are also interested in exploring the methylation status of drug-resistant genes in glioblastoma multiforme (GBM). GBM is the most common form of primary brain tumor in adults and is difficult to completely resect by surgery. The chemotherapy drug temozolomide (TMZ) is often used to treat GBM, however, upregulated O6-methylguanine-DNA methyltransferase (MGMT) in some GBM can neutralize the cytotoxic effect of TMZ. We were employed pyrosequencing assay, quantitative RT-PCR and western blotting to check the methylation status and expression level of MGMT in various GBM cell lines. In addition, we knock down MGMT expression with siRNA in GBM cell lines to confirm if TMZ resistance in the cells can be reduced. Moreover, we established the GBM-initiating cells (GICs) from parental GBM cell lines, and check the difference of MGMT methylation status between parental GBM cells and GICs. Then the GICs were determined the effect of TMZ on cell viability. In summary, we found MGMT can influence TMZ efficacy in GBM, and the MGMT expression level might be a therapeutic prediction of response to TMZ.

並列關鍵字

oral cancer ； glioma ； MGMT ； FLT4 ； Methylation ； VEGFR3 ； TMZ

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