Table_1_circDNMT1 Promotes Malignant Progression of Gastric Cancer Through Targeting miR-576-3p/Hypoxia Inducible Factor-1 Alpha Axis.docx

BackgroundCircular RNAs (circRNAs) regulate multiple malignant behaviors of various types of cancer. The role of circDNMT1, a newly identified circRNA, remains unknown in gastric cancer (GC). This study aimed to elucidate the underlying mechanisms of circDNMT1 in regulating GC progression.MethodsmicroRNA (miRNA) and circRNA expression was detected by quantitative real-time PCR. Western blotting was performed to measure hypoxia inducible factor-1 alpha (HIF-1α) protein expression. Sanger sequencing, gel electrophoresis and fluorescence in situ hybridization were performed to identify the presence of circDNMT1. The clinicopathological features and overall survival of patients were analyzed based on circDNMT1 expression. The proliferation, migration and invasion of GC cells were determined by cell counting kit-8, 5-ethynyl-2’-deoxyuridine, wound healing and transwell assays. Glycolysis of GC cells was detected based on the levels of glucose uptake, the lactate acid, ATP and pyruvic acid production and the extracellular acidification and oxygen consumption rates. The binding sites between miR-576-3p and circDNMT1 or HIF-1α were predicted by online bioinformatic tools and were validated using RNA pull-down and luciferase reporter assays. Xenograft models were established to determine the effects of the circDNMT1/miR-576-3p/HIF-1α axis on GC growth and metastasis in vivo.ResultscircDNMT1 was successfully identified and shown to be overexpressed in GC tissues and cell lines. The expression levels of circDNMT1 were correlated with pathological T stage, pathological TNM stage and shorter survival time of GC patients. circDNMT1 knockdown inhibited the proliferation, migration, invasion and glycolysis of GC cells. circDNMT1 functioned as an oncogenic factor by sponging miR-576-3p. HIF-1α was negatively regulated by miR-576-3p via binding its mRNA 3’ untranslated region. circDNMT1 promoted malignant behaviors and metabolic reprogramming of GC by targeting the miR-576-3p/HIF-1α axis both in vitro and in vivo.ConclusionThese findings demonstrated that circDNMT1 knockdown inhibited GC proliferation, migration, invasion and glycolysis through sponging miR-576-3p/HIF-1α axis. circDNMT1 may be a novel target for GC treatment.

背景：环状RNA（circRNAs）调节多种类型癌症的多种恶性行为。新近鉴定出的环状DNMT1（circDNMT1）在胃癌（GC）中的功能尚不明确。本研究旨在阐明circDNMT1在调节GC进展中的潜在机制。方法：通过定量实时PCR检测microRNA（miRNA）和circRNA的表达。采用Western blotting技术测定低氧诱导因子-1α（HIF-1α）蛋白的表达。通过Sanger测序、凝胶电泳和原位荧光杂交技术鉴定circDNMT1的存在。基于circDNMT1的表达，分析患者的临床病理特征和总生存期。利用细胞计数试剂盒-8、5-乙炔-2'-脱氧尿苷、伤口愈合和Transwell实验确定GC细胞的增殖、迁移和侵袭。基于葡萄糖摄取、乳酸酸、ATP和丙酮酸的产生以及细胞外酸化和氧消耗速率，检测GC细胞的糖酵解。通过在线生物信息学工具预测miR-576-3p与circDNMT1或HIF-1α的结合位点，并利用RNA pull-down和荧光素酶报告实验进行验证。建立异种移植模型，以确定circDNMT1/miR-576-3p/HIF-1α轴对GC生长和转移的体内影响。结果：成功鉴定circDNMT1，并证明其在GC组织和细胞系中过表达。circDNMT1的表达水平与病理T分期、病理TNM分期以及GC患者的生存时间缩短相关。circDNMT1敲低抑制了GC细胞的增殖、迁移、侵袭和糖酵解。circDNMT1通过捕获miR-576-3p发挥致癌因子的作用。miR-576-3p通过结合其mRNA的3'非翻译区负调控HIF-1α。circDNMT1通过靶向miR-576-3p/HIF-1α轴，在体外和体内促进了GC的恶性行为和代谢重编程。结论：这些发现表明，通过捕获miR-576-3p/HIF-1α轴，circDNMT1敲低可抑制GC的增殖、迁移、侵袭和糖酵解。circDNMT1可能成为GC治疗的新靶点。