CPSF3 modulates the balance of circular and linear transcripts in hepatocellular carcinoma

Abstract Background: CircRNAs play an important role in tumorigenesis and metastasis, and their expression is comprehensively downregulated in hepatocellular carcinoma (HCC). Previous studies have shown that the polyadenylation signal1 is critical for circRNA expression, while the 3’-end processing complex participates in circRNA cyclization. As a core executor of 3’-end cleavage, we hypothesized that CPSF3 modulates the circularization of circRNA. Methods: The clinical prognostic correlation was analyzed by TCGA database and tissue microarrays. Cytological experiments were performed to determine the role of CPSF3 in the proliferation and migration of HCC cells. The Red/GFP dual fluorescent reporter system was employed to explore the mechanism of RNA back-splicing regulated by CPSF3 protein and PAS sequences. The circRNAs regulated by CPSF3 were screened by RNA-seq and further validated by real-time PCR and dot blotting. Changes in the activity of downstream pathways were explored by various molecular experiments. Finally, the safety and efficacy of the CPSF3 inhibitor JTE-607 were verified by in vitro cytology experiments and in vivo xenograft nude mouse models. Results: CPSF3 was highly expressed in HCC cells and promoted the proliferation and migration of HCC cells. Its high level of expression predicted poor prognosis in HCC patients. Mechanistic study revealed that CPSF3 enhanced RNA cleavage, thereby reducing circRNA levels, increasing linear mRNA levels, and ultimately inhibiting miRNA-mediated gene silencing. Furthermore, the chemical inhibition of CPSF3 by JET-607 suppressed the proliferation of HCC cells both in vitro and in vivo. Conclusions: Our findings indicate that the increased expression of CPSF3 in HCC promotes the shift of pre-mRNA from circRNA to linear mRNA, thereby disrupting miRNA-mediated gene silencing and ultimately leading to uncontrolled cell proliferation. JTE-607, a CPSF3 inhibitor, exerted a therapeutic effect on HCC by blocking CPSF3 activity.