CircHIPK3′s dual role in promoting angiogenesis and inhibiting apoptosis through FASN mRNA stabilization in gallbladder cancer

Abstract

Objective  To investigate the mechanism by which circHIPK3 enhances FASN stability by inhibiting the ubiquitinated degradation of ALYREF on Gallbladder Cancer (GBC).

Methods  RT-qPCR was performed to detect circHIPK3 in GBC tissues and to analyze the relationship between circHIPK3 and clinicopathological characteristics of GBC patients. CCK-8 and colony formation assays were performed to study the proliferation of GBC cells, flow cytometry to detect apoptosis, tube formation assay to test angiogenesis, and Transwell assay to study migration and invasion. A nude mice implantation assay was conducted. CircHIPK3, FASN and SKP2 were assessed using RT-qPCR and western blot. Ubiquitination assay was performed to detect ALYREF ubiquitination, and RNA pull-down and RIP to investigate the binding relationship between circHIPK3, ALYREF and FASN.

Results  CircHIPK3 was upregulated in GBC tissues, with elevated expression correlating with TNM stages and lymph node metastasis. CircHIPK3 knockdown suppressed GBC progression by inhibiting proliferation, angiogenesis, migratory and invasive capacity while promoting apoptosis in vitro, paralleled by reduced tumor growth in vivo. CircHIPK3 stabilized ALYREF via inhibiting ubiquitination-mediated degradation. ALYREF knockdown phenocopied the tumor-suppressive effects of circHIPK3 depletion, whereas ALYREF overexpression rescued circHIPK3 knockdown-induced suppression of malignant phenotypes. CircHIPK3 stabilizes FASN mRNA through ALYREF. Overexpressing FASN counteracted the actions of circHIPK3 downregulation on cellular angiogenesis and apoptosis.

Conclusion  CircHIPK3 enhances FASN stability by inhibiting ALYREF ubiquitination degradation, promoting angiogenesis, and inhibiting apoptosis in GBC. This study positions circHIPK3 as a promising prognostic biomarker and therapeutic target in GBC, with ALYREF and FASN emerging as critical mechanistic nodes within this oncogenic pathway.