Department of Oncology, Xinqiao Hospital, Army Medical University, Chongqing, China.
Key Laboratory of Immunotherapy, Xinqiao Hospital, Army Military Medical University, Chongqing, China.
Dis Markers. 2022 Aug 22;2022:7527996. doi: 10.1155/2022/7527996. eCollection 2022.
Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) has been reported to be overexpressed in non-small-cell lung cancer (NSCLC) and to correlate with malignant proliferation. However, the mechanism of high MTHFD2 expression in NSCLC has not been clarified.
qPCR, western blot, and immunofluorescence experiments were used to measure the expression of related mRNAs and proteins. Cell apoptosis was measured by flow cytometry and TUNEL assays. The CCK-8 assay was used to determine cell viability. Flow cytometry was used to analyze the cell cycle. ROS, HO, MDA, SOD, and NADPH/NADP were evaluated by relevant assay kits. Transfection of siRNA or vectors was used to downregulate or upregulate gene expression. Dual-luciferase reporter gene assays were used to evaluate the regulated relationship between MTHFD2 and ATF4 or MYC.
MTHFD2 was highly expressed in NSCLC cells. Knockdown of MTHFD2 inhibited proliferation and increased apoptosis. Furthermore, oxidative factors significantly increased, while antioxidant factors significantly decreased in NSCLC cells with MTHFD2 knockdown, indicating that MTHFD2 was involved in NSCLC progression through the redox pathway. Although MTHFD2 was downregulated with ATF4 silencing, the dual-luciferase reporter assay suggested that ATF4 did not directly mediate MTHFD2 transcription. Further studies revealed that MYC had a transcriptional effect on MTHFD2 and was also regulated by ATF4. PCR, and western blotting experiments with ATF4 knockdown and MYC overexpression as well as ATF4 overexpression and MYC knockdown proved that ATF4 stimulated MTHFD2 through MYC mediation.
ATF4 promoted high expression of MTHFD2 in NSCLC dependent on MYC.
亚甲基四氢叶酸脱氢酶 2（MTHFD2）在非小细胞肺癌（NSCLC）中过表达，并与恶性增殖相关。然而，NSCLC 中 MTHFD2 高表达的机制尚不清楚。
采用 qPCR、western blot 和免疫荧光实验检测相关 mRNAs 和蛋白的表达。通过流式细胞术和 TUNEL 检测细胞凋亡。CCK-8 法检测细胞活力。流式细胞术分析细胞周期。用相关试剂盒评估 ROS、HO、MDA、SOD 和 NADPH/NADP。通过 siRNA 或载体转染下调或上调基因表达。双荧光素酶报告基因检测评估 MTHFD2 与 ATF4 或 MYC 之间的调控关系。
MTHFD2 在 NSCLC 细胞中高表达。敲低 MTHFD2 抑制增殖并增加凋亡。此外，NSCLC 细胞中 MTHFD2 敲低后氧化因子显著增加，抗氧化因子显著减少，表明 MTHFD2 通过氧化还原途径参与 NSCLC 进展。虽然 ATF4 沉默下调了 MTHFD2，但双荧光素酶报告基因检测表明 ATF4 并未直接介导 MTHFD2 转录。进一步研究表明，MYC 对 MTHFD2 具有转录效应，并且也受到 ATF4 的调控。用 ATF4 敲低和 MYC 过表达以及 ATF4 过表达和 MYC 敲低进行 PCR 和 western blot 实验证明，ATF4 通过 MYC 介导促进 NSCLC 中 MTHFD2 的高表达。
ATF4 通过 MYC 促进 NSCLC 中 MTHFD2 的高表达。
