Lu Huawei, Guo Qingwei, Mao Guozhang, Zhu Junwei, Li Fei
Department of General Thoracic Surgery, Zhoukou Central Hospital, Zhoukou 466000, Henan, People's Republic of China.
Onco Targets Ther. 2020 May 4;13:3717-3728. doi: 10.2147/OTT.S240399. eCollection 2020.
CircLARP4 is reported to act as a tumor suppressor in some cancers. However, the detailed roles and molecular basis of circLARP4 in non-small cell lung cancer (NSCLC) tumorigenesis are still unclear. The aim of the study is to explore the potential roles and molecular basis of circLARP4 in NSCLC tumorigenesis.
qRT-PCR was taken to detect circLARP4 and miR-135b expressions. MTT assay, transwell invasion assay and flow cytometry analysis were applied to evaluate cell proliferation, invasion and apoptosis, respectively. Glycolysis was assessed by measuring hexokinase2 (HK2) expression, glucose consumption and lactate production. Association between circLARP4 and miR-135a was examined by luciferase reporter and RIP assays. The changes of the phosphatase and tension homolog (PTEN)/protein kinase B (AKT)/hypoxia-inducible factor-1α (HIF-1α) pathway were evaluated by Western blot. The nude mouse xenograft models were applied to verify the regulation of circLARP4 in vivo.
CircLARP4 was decreased in NSCLC tissues and cells. CircLARP4 overexpression blocked cell proliferation and invasion, and facilitated apoptosis in NSCLC cells. Meanwhile, circLARP4 overexpression suppressed glycolysis in NSCLC cells, as evidenced by the reduced HK2, glucose consumption and lactate production levels. Further analyses proved a downregulation of miR-135b by circLARP4 in a ceRNA-dependent manner in NSCLC cells. CircLARP4-mediated tumor suppression on NSCLC progression was partially overturned by overexpressing miR-135b. Moreover, we confirmed that circLARP4 had antitumor effect on xenograft tumors and downregulated miR-135b. Furthermore, circLARP4 overexpression inhibited the PTEN/AKT/HIF-1α pathway in NSCLC cells and xenograft tumors by downregulating miR-135b.
Our findings suggested that circLARP4 suppressed NSCLC progression by sponging miR-135b through inactivation of the PTEN/AKT/HIF-1α pathway, which broadens our understanding concerning the roles of circLARP4 in NSCLC tumorigenesis.
据报道，环状LARP4（circLARP4）在某些癌症中发挥肿瘤抑制作用。然而，circLARP4在非小细胞肺癌（NSCLC）肿瘤发生中的具体作用和分子基础仍不清楚。本研究旨在探讨circLARP4在NSCLC肿瘤发生中的潜在作用和分子基础。
采用qRT-PCR检测circLARP4和miR-135b的表达。分别应用MTT法、Transwell侵袭实验和流式细胞术分析评估细胞增殖、侵袭和凋亡。通过检测己糖激酶2（HK2）表达、葡萄糖消耗和乳酸生成来评估糖酵解。通过荧光素酶报告基因实验和RNA免疫沉淀实验检测circLARP4与miR-135a之间的关联。通过蛋白质免疫印迹法评估磷酸酶和张力蛋白同源物（PTEN）/蛋白激酶B（AKT）/缺氧诱导因子-1α（HIF-1α）信号通路的变化。应用裸鼠异种移植模型在体内验证circLARP4的调控作用。
circLARP4在NSCLC组织和细胞中表达降低。circLARP4过表达可阻断NSCLC细胞的增殖和侵袭，并促进其凋亡。同时，circLARP4过表达抑制NSCLC细胞的糖酵解，表现为HK2、葡萄糖消耗和乳酸生成水平降低。进一步分析证明，circLARP4在NSCLC细胞中以ceRNA依赖的方式下调miR-135b。过表达miR-135b部分逆转了circLARP4介导的对NSCLC进展的肿瘤抑制作用。此外，我们证实circLARP4对异种移植肿瘤具有抗肿瘤作用，并下调miR-135b。此外，circLARP4过表达通过下调miR-135b抑制NSCLC细胞和异种移植肿瘤中的PTEN/AKT/HIF-1α信号通路。
我们的研究结果表明，circLARP4通过使PTEN/AKT/HIF-1α信号通路失活，海绵化miR-135b来抑制NSCLC进展，这拓宽了我们对circLARP4在NSCLC肿瘤发生中作用的理解。
