肺腺癌(LUAD)是对公众健康的严重威胁,并伴随着全球发病率和死亡率的增加。神经元PAS结构域蛋白2(NPAS2)已被证实为LUAD的癌基因;然而,对其分子机制知之甚少。这里,在LUAD细胞系和16HBE细胞中检测到NPAS2的表达水平。进行了功能增益和功能丧失实验。细胞计数试剂盒-8,集落形成,流式细胞术,进行伤口愈合和Transwell测定以评估细胞增殖,凋亡,移民和入侵,分别。通过耗氧率(OCR)评估葡萄糖代谢的重编程,复杂的活动,乳酸生产和葡萄糖消耗。通过蛋白质印迹检查关键蛋白的表达。我们证明了LUAD细胞系中NPAS2和β-抑制素-1(ARRB1)的异常上调。发现ARRB1是NPAS2的关键转录因子,在NPAS2的启动子区域内具有结合位点,从而引起其转录激活。功能实验表明,NPAS2耗竭通过抑制细胞增殖显著抑制A549细胞的恶性行为,迁移,侵袭和上皮间质转化及促进细胞凋亡。同时,NPAS2耗竭增加了OCR和复合物的活性(I,II,III和V),并减少了A549细胞中的乳酸产生和葡萄糖摄取,表明NPAS2消耗抑制有氧糖酵解,伴有糖酵解酶表达降低。然而,ARRB1过表达部分恢复了NPAS2敲低引起的变化。总之,我们的研究表明,ARRB1可以转录激活NPAS2,促进恶性活动和糖酵解,并最终促进LUAD的发展,证明了治疗LUAD的新治疗策略。
Lung adenocarcinoma (LUAD) is a serious threat to public health and is accompanied by increased morbidity and mortality worldwide. Neuronal PAS domain protein2 (NPAS2) has been confirmed as an oncogene in LUAD; however, little is known about its molecular mechanism. Here, the expression level of NPAS2 was detected in LUAD cell lines and 16HBE cells. Gain- and loss-of-function experiments were performed. Cell Counting Kit-8, colony formation, flow cytometry, wound-healing and Transwell assays were conducted to assess cell proliferation, apoptosis, migration and invasion, respectively. Reprogramming of glucose metabolism was evaluated via oxygen consumption rate (OCR), complexes activities, lactic production and glucose consumption. The expression of critical proteins was examined by western blot. We demonstrated aberrant upregulation of NPAS2 and β-arrestin-1 (ARRB1) in LUAD cell lines. ARRB1 was found to be a critical transcription factor of NPAS2 with binding sites within the promoter region of NPAS2, thereby causing its transcriptional activation. Functional experiments revealed that NPAS2 depletion significantly inhibited the malignant behaviours of A549 cells by suppressing cell proliferation, migration, invasion and epithelial-mesenchymal transition and promoting cell apoptosis. Meanwhile, NPAS2 depletion increased OCR and activities of complexes (I, II, III and V), and reduced lactic acid production and glucose uptake in A549 cells, indicating that NPAS2 depletion inhibited aerobic glycolysis, accompanied by reduced expression of glycolytic enzymes. However, the changes caused by NPAS2 knockdown were partly restored by ARRB1 overexpression. In conclusion, our study suggests that ARRB1 could transcriptionally activate NPAS2, facilitating malignant activities and glycolysis, and ultimately promoting the progression of LUAD, proving a novel therapeutic strategy for the treatment of LUAD.