Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China.
Public Health College, Harbin Medical University, Harbin, China.
J Cell Biochem. 2018 Jan;119(1):723-735. doi: 10.1002/jcb.26236. Epub 2017 Jul 31.
The study was to evaluate the effect of ten-eleven translocation 1 (TET1) regulating o6-methylguanine-DNA methyltransferase (MGMT) in chemotherapy resistance of oral squamous cell carcinoma (OSCC) stem cells. OSCC stem cells were divided into the blank, negative control (NC), TET1-siRNA, TET1-siRNA + MGMT-OE, and MGMT-OE groups. Methylation-specific polymerase chain reaction (MSP), qRT-PCR and Western blotting were conducted to detect the methylation status of MGMT, expressions of TET1, MGMT, ABCG2, and Oct-4. Cell proliferation, cisplatin chemosensitivity, and cell cycle and apoptosis, were detected using CCK8 and flow cytometry. A chromatin immunoprecipitation (ChIP) assay was employed for detecting the link between TET1 and MGMT gene promoters. In comparison to the NC group, the TET1-siRNA group exhibited increased levels of MGMT methylation, the number of apoptotic cells and cisplatin chemosensitivity consisting of varying concentrations, however, decreased levels of mRNA and protein expressions of TET1 as well as MGMT, cell viability, the number of cells in the S phase, and protein expressions of ABCG2 and Oct-4 were all have diminished amounts. The TET1-siRNA + MGMT-OE and MGMT-OE groups had higher MGMT mRNA and protein expression, as well as increased protein expressions of ABCG2 and Oct-4, greater cell activity, higher number of cells in the S phase, decreased apoptotic rates in cells and decreased cisplatin chemosensitivity with different concentrations. Our study provided evidence that low-expression of TET1 in OSCC stem cells may stimulate MGMT promoter methylation, while inhibiting MGMT mRNA expression, this ultimately strengthens the sensitivity of OSCC stem cells in regards to chemotherapeutics.
该研究旨在评估十号染色体缺失的磷酸酶及张力蛋白同源物 1（TET1）调节 O6-甲基鸟嘌呤-DNA 甲基转移酶（MGMT）在口腔鳞状细胞癌（OSCC）干细胞化疗耐药中的作用。OSCC 干细胞分为空白组、阴性对照组（NC）、TET1-siRNA 组、TET1-siRNA+MGMT-OE 组和 MGMT-OE 组。采用甲基化特异性聚合酶链反应（MSP）、qRT-PCR 和 Western blot 检测 MGMT 的甲基化状态、TET1、MGMT、ABCG2 和 Oct-4 的表达。用 CCK8 和流式细胞术检测细胞增殖、顺铂化疗敏感性以及细胞周期和凋亡。采用染色质免疫沉淀（ChIP）检测 TET1 与 MGMT 基因启动子之间的联系。与 NC 组相比，TET1-siRNA 组 MGMT 甲基化水平升高，不同浓度顺铂化疗敏感性的细胞凋亡数量增加，但 TET1 和 MGMT 的 mRNA 和蛋白表达水平、细胞活力、S 期细胞数量和 ABCG2 和 Oct-4 的蛋白表达水平均降低。TET1-siRNA+MGMT-OE 和 MGMT-OE 组 MGMT mRNA 和蛋白表达水平升高，ABCG2 和 Oct-4 蛋白表达水平升高，细胞活力增强，S 期细胞数量增加，细胞凋亡率降低，不同浓度顺铂化疗敏感性降低。本研究表明，OSCC 干细胞中 TET1 低表达可能刺激 MGMT 启动子甲基化，抑制 MGMT mRNA 表达，从而增强 OSCC 干细胞对化疗药物的敏感性。
