Affiliated Hospital of Guizhou Medical University, Guiyang, China.
The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Hospital, Kunming, China.
BMC Mol Cell Biol. 2023 Mar 31;24(1):14. doi: 10.1186/s12860-022-00450-5.
Recent studies have reported that the circadian transcription factor aryl hydrocarbon receptor nuclear translocator like 2 (ARNTL2) promotes the metastatic progression of lung adenocarcinoma. However, the molecular mechanisms of ARNTL2 in non-small cell lung cancer (NSCLC) cell growth and proliferation remain to be explored.
The expression of ARNTL2 and acyl-CoA thioesterase 7 (ACOT7) in lung cancer patients was analyzed based on TCGA database. Gain-of-function of ARNTL2 and ACOT7 was conducted by transfecting the cells with plasmids or lentivirus. Knockdown assay was carried out by siRNAs. Western blot and qRT-PCR were performed to check the protein and mRNA expression. Dual luciferase and ChIP-qPCR assay was applied to check the interaction of ARNTL2 on ACOT7's promoter sequence. Triglyceride level, MDA production, the activity of casapase 3 to caspase 7, and lipid ROS were measured by indicated assay kit. Cellular function was detected by CCK8, colony formation and flow cytometry analysis of cell death and cell cycle.
We demonstrated that ARNTL2 upregulation of ACOT7 was critical for NSCLC cell growth and proliferation. Firstly, overexpression of ARNTL2 conferred the poor prognosis of LUAD patients and supported the proliferation of NSCLC cells. Based on molecular experiments, we showed that ARNTL2 potentiated the transcription activity of ACOT7 gene via direct binding to ACOT7's promoter sequence. ACOT7 high expression was correlated with the worse prognosis of LUAD patients. Gain-of-function and loss-of-function experiments revealed that AOCT7 contributed to NSCLC cell growth and proliferation. ACOT7 regulated the apoptosis and ferroptosis of NSCLC cells, while exhibited no effect on cell cycle progression. ACOT7 overexpression also potentiated fatty acid synthesis and suppressed lipid peroxidation. Lastly, we showed that ARNTL2 knockdown and overexpression inhibited and promoted the cellular triglyceride production and subsequent cell proliferation, which could be reversed by ACOT7 overexpression and knockdown.
Our study illustrated the oncogenic function of ARNTL2/ACOT7 axis in the development of NSCLC. Targeting ARNTL2 or ACOT7 might be promising therapeutic strategies for NSCLC patients with highly expressed ARNTL2.
最近的研究报告称，昼夜节律转录因子芳香烃受体核转位蛋白样 2（ARNTL2）促进肺腺癌的转移进展。然而，ARNTL2 在非小细胞肺癌（NSCLC）细胞生长和增殖中的分子机制仍有待探索。
根据 TCGA 数据库分析肺癌患者中 ARNTL2 和酰基辅酶 A 硫酯酶 7（ACOT7）的表达。通过转染质粒或慢病毒来实现 ARNTL2 和 ACOT7 的功能获得。通过 siRNA 进行敲低实验。通过 Western blot 和 qRT-PCR 检查蛋白和 mRNA 的表达。应用双荧光素酶和 ChIP-qPCR 测定来检查 ARNTL2 对 ACOT7 启动子序列的相互作用。通过指示的测定试剂盒测量甘油三酯水平、MDA 产生、 caspase 3 到 caspase 7 的活性以及脂质 ROS。通过 CCK8、集落形成和细胞死亡和细胞周期的流式细胞术分析来检测细胞功能。
我们证明了 ARNTL2 对 ACOT7 的上调对于 NSCLC 细胞的生长和增殖至关重要。首先，ARNTL2 的过表达赋予 LUAD 患者不良预后，并支持 NSCLC 细胞的增殖。基于分子实验，我们表明 ARNTL2 通过直接结合 ACOT7 的启动子序列增强了 ACOT7 基因的转录活性。ACOT7 的高表达与 LUAD 患者的预后不良相关。功能获得和功能丧失实验表明，AOCT7 促进 NSCLC 细胞的生长和增殖。ACOT7 调节 NSCLC 细胞的凋亡和铁死亡，但对细胞周期进程没有影响。ACOT7 的过表达还增强了脂肪酸合成并抑制了脂质过氧化。最后，我们表明 ARNTL2 的敲低和过表达抑制和促进了细胞内甘油三酯的产生和随后的细胞增殖，这可以通过 ACOT7 的过表达和敲低来逆转。
我们的研究说明了 ARNTL2/ACOT7 轴在 NSCLC 发展中的致癌功能。针对 ARNTL2 或 ACOT7 可能是 ARNTL2 高表达的 NSCLC 患者有前途的治疗策略。
