Liu Yanhong, Jiang Yuanyuan, Xu Lidong, Qu Chongxing, Zhang Lei, Xiao Xingguo, Chen Wenxia, Li Kunkun, Liang Qianping, Wu Huili
Department of Gastroenterology, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, People's Republic of China.
Cancer Manag Res. 2020 Jul 17;12:5945-5956. doi: 10.2147/CMAR.S245941. eCollection 2020.
Gastric cancer (GC) is a severe threat to human life, with high incidence and mortality. Circular RNAs (circRNAs) play crucial roles in the progression of GC. This study attempted to investigate the potential role of circ-NRIP1 and associated action mechanisms in GC cells.
The expression of circ-NRIP1 and miR-186-5p was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability, apoptosis, and migration were assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, flow cytometry assay, and transwell assay, respectively. Cellular glycolysis, including cellular glucose uptake, lactate, and ATP/ADP ratios, was also detected by commercial assay kits. The protein levels of hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2) were quantified by Western blot. The relationship between miR-186-5p and circ-NRIP1 or myosin heavy chain 9 (MYH9) was predicted by the online bioinformatics tool, starBase, and verified by dual-luciferase reporter assay. Xenograft tumor model was used to evaluate biological function in vivo.
The expression of circ-NRIP1 was up-regulated in tissues of GC patients and cells, as well as negatively associated with that of miR-186-5p in tissues. circ-NRIP1 knockdown inhibited cell proliferation, migration, and glycolysis, but induced apoptosis in HGC-27 and AGS cells. circ-NRIP1 competitively targeted miR-186-5p, and MYH9 was a target of miR-186-5p. miR-186-5p knockdown inverted the bio-function effects and glycolytic activation from circ-NRIP1 silencing in HGC-27 and AGS cells. Meanwhile, MYH9 overexpression could rescue the effects of miR-186-5p. Besides, miR-186-5p knockdown inverted the expression pattern of si-circ-NRIP1 transfection in GC cells. Additionally, in vivo experiments confirmed that sh-circ-NRIP1 inhibited tumor growth.
circ-NRIP1 accelerated the glycolysis and GC progression by modulating MYH9 via miR-186-5p, suggesting that circ-NRIP1 was a promising biomarker for the treatment of GC.
胃癌（GC）对人类生命构成严重威胁，发病率和死亡率都很高。环状RNA（circRNAs）在胃癌进展中发挥着关键作用。本研究试图探讨circ-NRIP1在胃癌细胞中的潜在作用及相关作用机制。
采用定量实时聚合酶链反应（qRT-PCR）检测circ-NRIP1和miR-186-5p的表达。分别通过3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-H-四氮唑溴盐（MTT）法、流式细胞术和Transwell实验评估细胞活力、凋亡和迁移情况。还通过商业检测试剂盒检测细胞糖酵解情况，包括细胞葡萄糖摄取、乳酸和ATP/ADP比值。采用蛋白质免疫印迹法对己糖激酶2（HK2）和丙酮酸激酶M2（PKM2）的蛋白水平进行定量分析。利用在线生物信息学工具starBase预测miR-186-5p与circ-NRIP1或肌球蛋白重链9（MYH9）之间的关系，并通过双荧光素酶报告基因实验进行验证。采用异种移植瘤模型在体内评估生物学功能。
circ-NRIP1在胃癌患者组织和细胞中的表达上调，且在组织中与miR-186-5p的表达呈负相关。敲低circ-NRIP1可抑制HGC-27和AGS细胞的增殖、迁移和糖酵解，但诱导细胞凋亡。circ-NRIP1竞争性靶向miR-186-5p，且MYH9是miR-186-5p的靶标。敲低miR-186-5p可逆转HGC-27和AGS细胞中circ-NRIP1沉默所产生的生物学功能效应和糖酵解激活。同时，过表达MYH9可挽救miR-186-5p的作用效果。此外，敲低miR-186-5p可逆转GC细胞中si-circ-NRIP1转染后的表达模式。另外，体内实验证实sh-circ-NRIP1可抑制肿瘤生长。
circ-NRIP1通过miR-186-5p调节MYH9，从而加速糖酵解和胃癌进展，提示circ-NRIP1有望成为胃癌治疗的生物标志物。
