探討SOX21在腦瘤的表觀基因調控

Epigenetic Regulation of SOX21 in Brain Tumor

王敦吾(Tun-Wu Wang)

指導教授 ： 蕭淑惠

國立中正大學/理學院/生命科學系分子生物研究所/碩士(2016年)

摘要

神經膠質母細胞瘤 (glioblastoma)是成人中最致命的primary brain tumor之一。儘管接受了完整的化學與放射線療程，病人平均存活時間只有15個月。SOX21 (SRY (Sex Determining Region Y)-Box 21)是transcription factor，屬於 SOX family的一個成員。在研究中指出，神經細胞發育的階段SOX21 protein 會促進分化的過程，藉由與SOX2 protein 進行 counteracting。同時，SOX21被發現專一的表達在先導神經細胞和神經細胞，而非神經膠質細胞和星狀細胞。有研究報導SOX21在一些人類的神經膠質母細胞瘤的cell lines中無法被檢測到，將SOX21過度表達在這些cell lines可以抑制 in vivo中的細胞 progression，藉由與SOX2形成complexes，根據以上關聯性SOX21可能是治療神經膠質母細胞瘤的新目標之一。
為了要確認SOX21在不同種類細胞的專一性表達，是否跟 brain tumor的 progression有關。我們利用兩種不同種類的brain tumor cell lines，分別是U87 glioblastoma與IMR32 neuroblastoma。因為異常的DNA甲基化是一種被大家所熟悉的機制會造成gene silencing。我們首先利用methylation specific PCR (MSP)來確定SOX21 promoter 在U87 和 IMR32 細胞中的甲基化程度。結果顯示在U87細胞中SOX21 promoter有高度甲基化而IMR32細胞則完全沒有甲基化。接下來利用RT-PCR來驗證mRNA的表現量是否與MSP結果相符。發現IMR32細胞中有被檢測到豐富的SOX21 mRNA表達而U87細胞中則沒有。接下來我們利用bisulfite sequencing去更進一步確認SOX21 promoter的甲基化程度，發現在選擇的clones中U87細胞有42%的CpG site有被甲基化，而IMR32完全沒有被甲基化。我們的Western-blotting 結果也可以驗證，U87細胞中沒有被檢測到SOX21 protein，IMR32則反之。此外我們也使用腦瘤病人的檢體來測試(MSP 與 qMSP)SOX21在transcription start site區域的甲基化程度，結果與細胞株的實驗完全相符，可以支持我們在細胞實驗的結果。從我們的結果可以指出，DNA的高度甲基化是影響SOX21在不同種類的 brain tumor 有差異表達量的機制之一。
目前，我們利用轉染pCMV-AC-SOX21-GFP載體來過量表達SOX21在U87細胞中。我們使用螢光顯微鏡觀察是否有表達GFP的報導基因，與RT-PCR確認SOX21 mRNA的表達。我們觀察到已過量表達SOX21的U87細胞型態有明顯的改變，此外我們從流式細胞儀的實驗結果得知U87細胞的細胞周期也隨著過量表達SOX21而改變。

關鍵字

表觀基因體學 ； 腦瘤 ； DNA甲基化修飾

並列摘要

Glioblastoma is the most fatal primary brain tumor in adults. Despite maximum treatment, patients only have a median survival time of 15 months. SOX21 (SRY (Sex Determining Region Y)-Box 21) is a transcription factor that belongs to the SOX family. During development, SOX21 protein is reported to promote neuronal differentiation by counteracting the activity of SOX2 in neuronal cells. Concordantly, SOX21 is found to be specifically expressed in neural precursor cells and neuronal cells, but not in glial cells and astrocytes. Studies in an array of human glioblastoma cell lines reported that SOX21 is undetectable in these cells, but overexpression of SOX21 could cause reduced cell number. SOX21 is also shown to inhibit glioma progression in vivo by forming complexes with SOX2, implicating that SOX21 may be a therapeutic target for glioblastoma.
To determine the mechanism underlying the cell type-specific expression and the role of SOX21 is brain tumor progression, we use U87 glioblastoma cells and IMR32 neuroblastoma cells to address these questions. Because aberrant DNA hypermethylation is a well-known epigenetic mechanism to cause gene silencing, we first used methylation specific PCR (MSP) to determine the methylation level of SOX21 promoter in U87 and IMR32 cells. Our data reveal that while SOX21 is hypermethylated in U87 cells, it is unmethylated in IMR32 cells. Then RT-PCR was applied to validate the MSP result. Consistently, SOX21 is undetected in U87 cells, but its expression is abundant in IMR32 cells. We next utilized bisulfite sequencing to further confirm the methylation of SOX21 promoter, and we found that 42% of the cloned CpG dinucleotides are methylated in U87 cells, but none of these dinucleotides are methylated in IMR32 cells. Our Western-blotting results also show that SOX21 protein is expressed in IMR32 cells, but not in U87 cells. Furthermore, we confirmed that SOX21 is hypermethylated in the human brain tumor specimens by both MSP and qMSP. Our data thus demonstrate that DNA hypermethylation is one of the mechanisms responsible for the cell-type specific expression of SOX21 in brain tumor.
To elucidate the role of SOX21 in brain tumor, SOX21 is overexpressed in U87 cells by transfecting the pCMV-AC-SOX21-GFP plasmid. We found U87 cells with overexpressed SOX21 exhibit altered morphology and cell cycle, suggesting that SOX21 could affect the proliferation and differentiation of brain tumor.

並列關鍵字

SOX21 ； Epigenetics ； DNA Methylation ； Brain Tumor

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