Circular RNA circ_0003028 contributes to tumorigenesis by regulating GOT2 via miR-1298-5p in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a common malignant tumor, with high morbidity and mortality. Circular RNA (circRNA) circ_0003028 was reported to be upregulated in NSCLC. This study is designed to explore the role and mechanism of circ_0003028 on NSCLC progression. In this work, circ_0003028, microRNA-1298-5p (miR-1298-5p), and glutamic oxaloacetic transaminase 2 (GOT2) level were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The localization of circ_0003028 was analyzed by subcellular fractionation assay. Cell proliferation, colony number, cell cycle progression, apoptosis, migration, invasion, and angiogenesis were measured by Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, transwell, and tube formation assays. Protein levels of Beclin1, light chain 3 (LC3)-II/LC3-I, GOT2, proliferating cell nuclear antigen (PCNA) were examined by western blot assay. The binding relationship between miR-1298-5p and circ_0003028 or GOT2 was predicted by circular RNA Interactome or starbase and then verified by dual-luciferase reporter, RNA Immunoprecipitation (RIP), and RNA pull-down assays. The biological role of circ_0003028 on NSCLC tumor growth was examined by the xenograft tumor model in vivo. We reported that circ_0003028 and GOT2 were upregulated, and miR-1298-5p was decreased in NSCLC tissues and cells. Moreover, circ_0003028 knockdown curbed cell proliferative ability, migration, invasion, angiogenesis, and facilitate apoptosis and autophagy in NSCLC cells in vitro. Mechanical analysis discovered that circ_0003028 regulated GOT2 expression by sponging miR-1298-5p. Circ_0003028 silencing hindered the cell growth of NSCLC in vivo. Taken together, circ_0003028 knockdown could suppress NSCLC progression partly by regulating the miR-1298-5p/GOT2 axis, providing an underlying therapeutic target for NSCLC.非小细胞肺癌（Non-small cell lung cancer, NSCLC）是一类高发的恶性肿瘤，具有较高的发病率与死亡率。已有研究表明，环状RNA（circular RNA, circRNA）circ_0003028在非小细胞肺癌组织中呈高表达。本研究旨在探讨circ_0003028在非小细胞肺癌进展中的作用及分子机制。本研究通过实时荧光定量聚合酶链反应（real-time quantitative polymerase chain reaction, RT-qPCR）检测了circ_0003028、微小RNA-1298-5p（microRNA-1298-5p, miR-1298-5p）以及谷草转氨酶2（glutamic oxaloacetic transaminase 2, GOT2）的表达水平；采用亚细胞分级实验分析了circ_0003028的细胞内定位；分别通过细胞计数试剂盒-8（Cell Counting Kit-8, CCK-8）、集落形成实验、流式细胞术、Transwell小室实验及管形成实验检测了细胞增殖能力、集落形成数、细胞周期进程、细胞凋亡、迁移、侵袭及血管生成能力；通过蛋白质印迹实验检测了Beclin1、微管相关蛋白1轻链3（light chain 3, LC3）-Ⅱ/LC3-Ⅰ、GOT2及增殖细胞核抗原（proliferating cell nuclear antigen, PCNA）的蛋白表达水平。通过环状RNA互作组数据库（circular RNA Interactome）或starBase数据库预测了miR-1298-5p与circ_0003028或GOT2的结合位点，并通过双荧光素酶报告基因实验、RNA免疫沉淀（RNA Immunoprecipitation, RIP）及RNA下拉实验验证了二者的靶向结合关系。采用异种移植瘤模型验证了circ_0003028对非小细胞肺癌体内肿瘤生长的生物学作用。本研究发现，circ_0003028与GOT2在非小细胞肺癌组织及细胞系中均呈高表达，而miR-1298-5p的表达水平显著降低。体外实验结果显示，敲低circ_0003028可抑制非小细胞肺癌细胞的增殖、迁移、侵袭及血管生成能力，并促进细胞凋亡与自噬。机制研究表明，circ_0003028可通过海绵吸附miR-1298-5p调控GOT2的表达。体内实验进一步证实，敲低circ_0003028可抑制非小细胞肺癌的体内肿瘤生长。综上，敲低circ_0003028可通过调控miR-1298-5p/GOT2轴抑制非小细胞肺癌的进展，为非小细胞肺癌的临床治疗提供了潜在的新靶点。