Department of Organ Transplant,Qi Lu hosptial of Shan Dong university, Jinan, 250012, China.
J Cell Biochem. 2012 Nov;113(11):3419-26. doi: 10.1002/jcb.24218.
Previous studies showed that transient transfection of HCVc improved hTERT expression in hepatoma cell lines and it was noteworthy that phosphorylated signal transducer and activator of transcription 3 (pSTAT3) and DNA methyltransferases (DNMTs) were up regulated simultaneously. This study was designed to investigate the role of epigenetic regulation in the process of hTERT up regulation after HCVc transfection. Q-PCR and Western blot were used to analyze the expression of pSTAT3, DNMT1, and hTERT after the transfection of HCVc in hepatoma cell line Huh7. Proliferation and hTERT activity of Huh7 after HCVc transfection were examined by CCK8 and ELISA, respectively. Then, we blocked the JAK/STAT3 pathway or inhibited DNMT1 expression to investigate the regulation of pSTAT3, DNMT1, and hTERT. Methylation status of the promoter of hTERT gene was monitored by MS-PCR. Cell proliferation, hTERT expression level and activity of hTERT were promoted after HCVc transfection. The expression of pSTAT3 and DNMT1 were up-regulated simultaneously. DNMT1 and hTERT were down-regulated after blocking JAK/STAT3 pathway and the expression of hTERT weakened with DNMT1 inhibition. MS-PCR showed HCVc transfection increased the methylation level of hTERT promoter, and this effect was weakened after blocking the JAK/STAT3 pathway or with the treatment with DNMT1 inhibitor. HCVc transfection improved hTERT expression via epigenetic regulation. JAK/STAT3 pathway could be one of the essential factors in regulating DNMT1 expression during this process.
先前的研究表明，HCVc 的瞬时转染可提高肝癌细胞系中的 hTERT 表达，值得注意的是，磷酸化信号转导和转录激活因子 3（pSTAT3）和 DNA 甲基转移酶（DNMTs）同时上调。本研究旨在探讨 HCVc 转染后 hTERT 上调过程中的表观遗传调控作用。采用 Q-PCR 和 Western blot 分析 HCVc 转染肝癌细胞系 Huh7 后 pSTAT3、DNMT1 和 hTERT 的表达。通过 CCK8 和 ELISA 分别检测 Huh7 转染 HCVc 后的增殖和 hTERT 活性。然后，我们阻断 JAK/STAT3 通路或抑制 DNMT1 表达，以研究 pSTAT3、DNMT1 和 hTERT 的调节作用。通过 MS-PCR 监测 hTERT 基因启动子的甲基化状态。HCVc 转染后细胞增殖、hTERT 表达水平和 hTERT 活性均增强。pSTAT3 和 DNMT1 的表达同时上调。阻断 JAK/STAT3 通路和抑制 DNMT1 表达后，DNMT1 和 hTERT 下调，hTERT 表达减弱。MS-PCR 显示，HCVc 转染增加了 hTERT 启动子的甲基化水平，阻断 JAK/STAT3 通路或用 DNMT1 抑制剂处理后，这种作用减弱。HCVc 转染通过表观遗传调控改善 hTERT 表达。JAK/STAT3 通路可能是调节该过程中 DNMT1 表达的重要因素之一。
