ABSTRACT

This study was to explore the function of circular progastricsin (circ-PGC) in NSCLC. The histological morphology of tumor tissues was observed by hematoxylin and eosin (HE) staining. The expression of circ-PGC, miR-532-3p and forkhead box R2 (FOXR2) mRNA was measured by real-time quantitative polymerase chain reaction (RT-qPCR). The protein level of FOXR2 was checked by western blot. In functional analyses, cell viability, colony formation, cell apoptosis, cell migration and cell invasion were investigated using cell counting kit-8 (CCK-8) assay, colony formation assay, flow cytometry assay, wound healing assay and transwell assay. Besides, glycolysis metabolism was assessed by the levels of glucose consumption, lactate production and adenosine triphosphate (ATP) production. The predicted relationship between miR-532-3p and circ-PGC and FOXR2 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The results showed that circ-PGC and FOXR2 were upregulated in NSCLC tissues and cells. Circ-PGC knockdown or FOXR2 knockdown inhibited NSCLC cell viability, colony formation, cell migration, invasion and glycolysis metabolism, and FOXR2 overexpression rescued these inhibitory effects caused by circ-PGC knockdown. MiR-532-3p harbored the same binding site with circ-PGC and FOXR2, and circ-PGC positively regulated FOXR2 expression by targeting miR-532-3p. The expression of β-catenin and c-Myc was decreased in cells after circ-PGC knockdown but recovered with miR-532-3p inhibition or FOXR2 overexpression. Circ-PGC downregulation also inhibited tumor growth in vivo. In conclusion, circ-PGC positively regulated FOXR2 expression by competitively binding to miR-532-3p, thereby promoting the development of NSCLC, and the Wnt/β-catenin signaling pathway might be activated by the circ-PGC/miR-532-3p/FOXR2 network.

摘要

本研究旨在探讨环状前胃素（circ-PGC）在非小细胞肺癌中的作用。通过苏木精和伊红（HE）染色观察肿瘤组织的组织学形态。通过实时定量聚合酶链反应 (RT-qPCR) 测量 circ-PGC、miR-532-3p 和叉头盒 R2 (FOXR2) mRNA 的表达。通过蛋白质印迹检查FOXR2的蛋白质水平。在功能分析中，使用细胞计数试剂盒 8 (CCK-8) 测定、集落形成测定、流式细胞术测定、伤口愈合测定和 transwell 测定研究细胞活力、集落形成、细胞凋亡、细胞迁移和细胞侵袭。此外，通过葡萄糖消耗、乳酸产生和三磷酸腺苷 (ATP) 产生水平评估糖酵解代谢。通过双荧光素酶报告基因分析和 RNA 免疫沉淀 (RIP) 分析验证了 miR-532-3p 与 circ-PGC 和 FOXR2 之间的预测关系。结果显示circ-PGC和FOXR2在NSCLC组织和细胞中上调。Circ-PGC 敲低或 FOXR2 敲低抑制了 NSCLC 细胞活力、集落形成、细胞迁移、侵袭和糖酵解代谢，而 FOXR2 过表达挽救了由 circ-PGC 敲低引起的这些抑制作用。MiR-532-3p 与 circ-PGC 和 FOXR2 具有相同的结合位点，并且 circ-PGC 通过靶向 miR-532-3p 正向调节 FOXR2 的表达。circ-PGC 敲低后细胞中 β-catenin 和 c-Myc 的表达降低，但随着 miR-532-3p 抑制或 FOXR2 过表达而恢复。Circ-PGC 下调也抑制肿瘤生长 结果显示circ-PGC和FOXR2在NSCLC组织和细胞中上调。Circ-PGC 敲低或 FOXR2 敲低抑制了 NSCLC 细胞活力、集落形成、细胞迁移、侵袭和糖酵解代谢，而 FOXR2 过表达挽救了由 circ-PGC 敲低引起的这些抑制作用。MiR-532-3p 与 circ-PGC 和 FOXR2 具有相同的结合位点，并且 circ-PGC 通过靶向 miR-532-3p 正向调节 FOXR2 的表达。circ-PGC 敲低后细胞中 β-catenin 和 c-Myc 的表达降低，但随着 miR-532-3p 抑制或 FOXR2 过表达而恢复。Circ-PGC 下调也抑制肿瘤生长 结果显示circ-PGC和FOXR2在NSCLC组织和细胞中上调。Circ-PGC 敲低或 FOXR2 敲低抑制了 NSCLC 细胞活力、集落形成、细胞迁移、侵袭和糖酵解代谢，而 FOXR2 过表达挽救了由 circ-PGC 敲低引起的这些抑制作用。MiR-532-3p 与 circ-PGC 和 FOXR2 具有相同的结合位点，并且 circ-PGC 通过靶向 miR-532-3p 正向调节 FOXR2 的表达。circ-PGC 敲低后细胞中 β-catenin 和 c-Myc 的表达降低，但随着 miR-532-3p 抑制或 FOXR2 过表达而恢复。Circ-PGC 下调也抑制肿瘤生长 侵袭和糖酵解代谢，以及 FOXR2 过表达挽救了这些由 circ-PGC 敲低引起的抑制作用。MiR-532-3p 与 circ-PGC 和 FOXR2 具有相同的结合位点，并且 circ-PGC 通过靶向 miR-532-3p 正向调节 FOXR2 的表达。circ-PGC 敲低后细胞中 β-catenin 和 c-Myc 的表达降低，但随着 miR-532-3p 抑制或 FOXR2 过表达而恢复。Circ-PGC 下调也抑制肿瘤生长 侵袭和糖酵解代谢，以及 FOXR2 过表达挽救了这些由 circ-PGC 敲低引起的抑制作用。MiR-532-3p 与 circ-PGC 和 FOXR2 具有相同的结合位点，并且 circ-PGC 通过靶向 miR-532-3p 正向调节 FOXR2 的表达。circ-PGC 敲低后细胞中 β-catenin 和 c-Myc 的表达降低，但随着 miR-532-3p 抑制或 FOXR2 过表达而恢复。Circ-PGC 下调也抑制肿瘤生长 circ-PGC 敲低后细胞中 β-catenin 和 c-Myc 的表达降低，但随着 miR-532-3p 抑制或 FOXR2 过表达而恢复。Circ-PGC 下调也抑制肿瘤生长 circ-PGC 敲低后细胞中 β-catenin 和 c-Myc 的表达降低，但随着 miR-532-3p 抑制或 FOXR2 过表达而恢复。Circ-PGC 下调也抑制肿瘤生长在体内。综上所述，circ-PGC通过竞争性结合miR-532-3p正向调节FOXR2表达，从而促进NSCLC的发展，Wnt/β-catenin信号通路可能被circ-PGC/miR-532-3p激活。 /FOXR2 网络。