基因表觀遺傳機制調控麩胱甘肽轉移酶在肺癌細胞及早期非小細胞肺癌患者中的表現之研究

背景:麩胱甘肽轉移酶 (Glutathione S-transferase, GST) 是一種解毒酵素。GST-M2的低表現在肺癌細胞可以被偵測到；然而，GST-M2在肺癌細胞中調控機制並不清楚。在本研究中，我們探討在肺癌細胞中的GST-M2的表觀調控機制(epigenetic mechanism)。方法:在肺癌細胞以DNA甲基轉移酵素 (DNA methyltransferase；DNMT)抑制劑5’-aza-2’-deoxycytidine (5’-Aza-dC) 處理後，我們評估GST-M2的啟動子甲基化；Reporter activity assay、Chromatin immunoprecipitation (ChIP)、electrophoretic mobility-shift assay (EMSA) 以及shRNA 試驗被使用來測試是否Sp1甲基化影響結合在GST-M2 啟動子上，並且調控GST-M2的轉譯。即時定量PCR (real-timePCR)也被運用來決定73名非小細胞肺癌(non-small cell lung cancer [NSCLC])組織中，GST-M2與DNMT-3b mRNA程度。在肺癌細胞中，藉由5’-Aza-dC的處理，GST-M2的基因表現被回復；在肺癌細胞及非小細胞肺癌組織中，GST-M2也表現出高度的啟動子甲基化程度。結果:在肺癌細胞中，CpG甲基化減弱了Sp1結合至GST-M2啟動子。在肺部正常細胞中，Sp1的剔除減少了GST-M2的表現；並且在肺癌細胞中，DNMT-3b的靜默則增加了GST-M2的表現。此外，在具有低GST-M2表現的肺部腫瘤中相較於具有高GST-M2的肺部腫瘤中，DNMT-3b表現是顯著較高的，特別是女性與第一期病患分組。結論:GST-M2的表觀靜默能夠被辨識出Sp1調控的GST-M2轉譯表現，並且代表著導致在肺癌細胞和第一期NSCLC病患中所觀察到的GST-M2減少表現之機制。

關鍵字

麩胱甘肽轉移酶肺癌；專一性蛋白1 ； DNA甲基化； DNA 甲基轉移酶

並列摘要

BACKGROUND:Glutathione S-transferases M2 (GST-M2) is a detoxifying enzyme. Low expression of GST-M2 in lung cancer cells was detected. However, little is known about the regulation of GST-M2 in lung cancer cells. In this study, we investigated the epigenetic regulatory mechanisms of GST-M2 in lung cancer cells. METHODS:We evaluated the promoter methylation of GST-M2 following the DNA methyltransferase (DNMT) inhibitor, 5’-aza-2’-deoxycytidine (5’-aza-dC) treatment in lung cancer cells. Reporter activity assay, Chromatin immunoprecipitation (ChIP), electrophoretic mobility-shift assay (EMSA) and small interfering RNA (siRNA) assays were used to detect whether methylation of Sp1 affects the binding to the GST-M2 promoter and regulates GST-M2 transcription. Real-time PCR was applied to determine GST-M2 and DNMT-3b mRNA levels in 73 non-small cell lung cancer (NSCLC) tissues. RESULTS:GST-M2 expression was restored by the treatment with 5’-aza-dC in lung cancer cells. GST-M2 also exhibited high frequency of promoter hypermethylation in lung cancer cells and NSCLC tumor tissues. CpG hypermethylation abated Sp1 binding to the GST-M2 promoter in lung cancer. Knockdown of Sp1 in lung normal cells reduced GST-M2 expression, and silencing of DNMT-3b increased GST-M2 expression in lung cancer cells. In addition, DNMT-3b expression was significantly higher in lung tumors with low GST-M2 expression than that in lung tumors with high GST-M2 expression, especially in female and stage I groups. CONCLUSIONS: Epigenetic silencing of GST-M2 could be distinguished Sp1 mediated GST-M2 transcriptional expressions and represent the mechanism leading to the decreased expression of GST-M2 observed in lung cancer cells and stage I/II NSCLC patients.

並列關鍵字

Glutathione S-transferases ； lung cancer ； specificity protein 1 ； DNA methylation ； DNA methyltransferase

參考文獻

Al Rashid ST, Dellaire G, Cuddihy A, Jalali F, Vaid M, Coackley C, Folkard M, Xu Y, Chen BP, Chen DJ, Lilge L, Prise KM, Bazett Jones DP, Bristow RG (2005) Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo. Cancer Res 65(23): 10810-10821

Albino AP, Huang X, Jorgensen E, Yang J, Gietl D, Traganos F, Darzynkiewicz Z (2004) Induction of H2AX phosphorylation in pulmonary cells by tobacco smoke: a new assay for carcinogens. Cell Cycle 3(8): 1062-1068

Alexandrov K, Rojas M, Satarug S (2010) The critical DNA damage by benzo(a)pyrene in lung tissues of smokers and approaches to preventing its formation. Toxicol Lett 198(1): 63-68

Asakura T, Sasagawa A, Takeuchi H, Shibata S, Marushima H, Mamori S, Ohkawa K (2007) Conformational change in the active center region of GST P1-1, due to binding of a synthetic conjugate of DXR with GSH, enhanced JNK-mediated apoptosis. Apoptosis 12(7): 1269-1280

Attwood JT, Yung RL, Richardson BC (2002) DNA methylation and the regulation of gene transcription. Cell Mol Life Sci 59(2): 241-257

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基因表觀遺傳機制調控麩胱甘肽轉移酶在肺癌細胞及早期非小細胞肺癌患者中的表現之研究

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