Department of Radiation Oncology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China.
J Biol Regul Homeost Agents. 2020 Nov-Dec;34(6):2037-2047. doi: 10.23812/20-543-A.
The aim of this study was to screen the differentially expressed circular ribonucleic acid (circRNA) in non-small cell lung cancer (NSCLC) and explore its functional mechanism. Differentially expressed circRNAs in tumor tissues of NSCLC patients were detected via gene microarray and reverse transcriptionquantitative polymerase chain reaction (RT-qPCR). The associaton between their expressions and the clinical phenotypes was explored combined with clinical data. The effect of overexpression of hsa_circ_0004050 on the proliferation of A549 cells was detected via cell counting kit-8 (CCK-8) assay and CFSE assay. The effect of overexpression of hsa_circ_0004050 (human circular ribonucleic acid_0004050) on the apoptosis of A549 cells was detected using the Annexin V-FITC/PI kit. Then the direct-acting miRNAs of hsa_circ_0004050 were screened using bioinformatics software and luciferase reporter assay, and the direct targets of miR- 1233-3p were explored using bioinformatics software and luciferase reporter assay combined with RTqPCR and Western blotting. The effects of overexpression of miR-1233-3p or knockdown of dual specificity phosphatase 9 (DUSP9) on the cell proliferation and apoptosis affected by overexpression of hsa_circ_0004050 were detected. Western blotting was performed to detect the effects of hsa_circ_0004050 on the extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK) signaling pathway. The expression of hsa_ circ_0004050 was significantly lower in tumor tissues than that in para-carcinoma tissues in NSCLC patients. The expression of hsa_circ_0004050 was significantly correlated with TNM stage, tumor size and lymph node metastasis. The results of survival analysis showed that the survival time of patients with a high expression of hsa_circ_0004050 was obviously prolonged. According to the results of phenotype assay, hsa_circ_0004050 could promote apoptosis and inhibit proliferation of A549 cells. In terms of its mechanism, hsa_circ_0004050 could markedly increase the protein expression of DUSP9 via targeting miR-1233-3p in A549 cells, thereby inhibiting the ERK/JNK signaling pathway. Hsa_circ_0004050 may serve as a potential therapeutic target for NSCLC or a biomarker for the diagnosis of NSCLC in the future.
本研究旨在筛选非小细胞肺癌（NSCLC）中差异表达的环状 RNA（circRNA），并探讨其功能机制。通过基因芯片和逆转录定量聚合酶链反应（RT-qPCR）检测 NSCLC 患者肿瘤组织中差异表达的 circRNA。结合临床资料，探讨其表达与临床表型的相关性。通过细胞计数试剂盒-8（CCK-8）检测和 CFSE 检测，检测 hsa_circ_0004050 过表达对 A549 细胞增殖的影响。使用 Annexin V-FITC/PI 试剂盒检测 hsa_circ_0004050 过表达对 A549 细胞凋亡的影响。然后，使用生物信息学软件筛选 hsa_circ_0004050 的直接作用 miRNA，并通过荧光素酶报告基因检测进行验证，结合 RT-qPCR 和 Western blot 检测 miR-1233-3p 的直接靶标。检测过表达 miR-1233-3p 或敲低双特异性磷酸酶 9（DUSP9）对 hsa_circ_0004050 过表达影响的细胞增殖和凋亡的影响。Western blot 检测 hsa_circ_0004050 对细胞外信号调节激酶（ERK）/c-Jun N 末端激酶（JNK）信号通路的影响。NSCLC 患者肿瘤组织中 hsa_ circ_0004050 的表达明显低于癌旁组织。hsa_circ_0004050 的表达与 TNM 分期、肿瘤大小和淋巴结转移显著相关。生存分析结果表明，hsa_circ_0004050 高表达患者的生存时间明显延长。表型检测结果表明，hsa_circ_0004050 可促进 A549 细胞凋亡，抑制增殖。在机制方面，hsa_circ_0004050 可通过靶向 miR-1233-3p 显著增加 A549 细胞中 DUSP9 的蛋白表达，从而抑制 ERK/JNK 信号通路。hsa_circ_0004050 可能成为 NSCLC 的潜在治疗靶点或 NSCLC 的诊断标志物。
