Background and Aims There is growing evidence that single‐stranded, circular RNA (circRNA) plays a key role in the development of certain cancers, including hepatocellular carcinoma (HCC). It is less clear, however, what role circRNA plays in HCC metastasis. Approach and Results In this study, through circRNA sequencing, we identified a circRNA: circASAP1 (a circRNA derived from exons 2 and 3 of the ASAP1 gene, hsa_circ_0085616), which is associated with pulmonary metastasis after curative resection in patients with HCC. CircASAP1 was overexpressed in HCC cell lines with high metastatic potential and in metastatic HCCs. In vitro, circASAP1 promoted cell proliferation, colony formation, migration, and invasion, and in vivo, it enhanced tumor growth and pulmonary metastasis. Mechanism studies showed that circASAP1 acts as a competing endogenous RNA for microRNA 326 (miR‐326) and microRNA 532‐5p (miR‐532‐5p), both of which are tumor suppressors in HCC. We found that mitogen‐activated protein kinase (MAPK) 1 and colony stimulating factor (CSF)‐1 were direct common targets for microRNA 326 (miR‐326) and microRNA 532‐5p (miR‐532‐5p), which were regulated by circASAP1. CircASAP1 promotes HCC cell proliferation and invasion by regulating miR‐326/miR‐532‐5p‐MAPK1 signaling and, furthermore, mediates tumor‐associated macrophage infiltration by regulating the miR‐326/miR‐532‐5p‐CSF‐1 pathway. Clinical HCC samples exhibited a positive correlation between circASAP1 expression and levels of CSF‐1, MAPK1, and CD68+ tumor‐associated macrophages, all of which were predictive of patient outcomes. Conclusion We identified circASAP1 as a key regulator of HCC metastasis that acts on miR‐326/miR‐532‐5p‐MAPK1/CSF‐1 signaling and serves as a prognostic predictor in patients with HCC.

Although circular RNAs (circRNA) are known to modulate tumor initiation and progression, their role in hepatocellular carcinoma (HCC) metastasis remains poorly understood. Here, three metastasis-associated circRNAs identified in a previous circRNA-sequencing study were screened and validated in two HCC cohorts. CircRPN2 was downregulated in highly metastatic HCC cell lines and HCC tissues with metastasis. Patients with HCC with lower circRPN2 levels displayed shorter overall survival and higher rates of cumulative recurrence. Mechanistic studies in vitro and in vivo revealed that circRPN2 binds to enolase 1 (ENO1) and accelerates its degradation to promote glycolytic reprogramming through the AKT/mTOR pathway, thereby inhibiting HCC metastasis. CircRPN2 also acted as a competing endogenous RNA for miR-183–5p, which increases forkhead box protein O1 (FOXO1) expression to suppress glucose metabolism and tumor progression. In clinical samples, circRPN2 expression negatively correlated with ENO1 and positively correlated with FOXO1, and expression of circRPN2, either alone or in combination with ENO1 and FOXO1, was a novel indicator of HCC prognosis. These data support a model wherein circRPN2 inhibits HCC aerobic glycolysis and metastasis via acceleration of ENO1 degradation and regulation of the miR-183–5p/FOXO1 axis, suggesting that circRPN2 represents a possible therapeutic target in HCC. Significance: The circRNA circRPN2 is a potential prognostic biomarker and therapeutic target in hepatocellular carcinoma that suppresses aerobic glycolysis and metastasis.