Li ZhenWei, Wang HaiJiu, Wang Cheng, Wang ZhiXin, Xie Zhi, Liu JinMing
Department of Hepatopancreatobiliary Surgery, Qinghai University Affiliated Hospital, Xining City, Qinghai Province, China; Qinghai Provincial Key Laboratory of Hydatid Disease Research, Xining City, Qinghai Province, China.
Department of Hepatopancreatobiliary Surgery, Qinghai University Affiliated Hospital, Xining City, Qinghai Province, China.
Clinics (Sao Paulo). 2025 Jun 3;80:100697. doi: 10.1016/j.clinsp.2025.100697. eCollection 2025.
To investigate the mechanism by which circHIPK3 enhances FASN stability by inhibiting the ubiquitinated degradation of ALYREF on Gallbladder Cancer (GBC).
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.
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.
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.
探讨环状HIPK3通过抑制ALYREF在胆囊癌（GBC）中的泛素化降解来增强脂肪酸合酶（FASN）稳定性的机制。
采用RT-qPCR检测GBC组织中环状HIPK3的表达，并分析其与GBC患者临床病理特征的关系。进行CCK-8和集落形成实验研究GBC细胞的增殖，流式细胞术检测细胞凋亡，管腔形成实验检测血管生成，Transwell实验研究细胞迁移和侵袭。进行裸鼠移植瘤实验。采用RT-qPCR和蛋白质免疫印迹法检测环状HIPK3、FASN和SKP2。进行泛素化实验检测ALYREF的泛素化情况，采用RNA下拉实验和RNA免疫沉淀实验研究环状HIPK3、ALYREF和FASN之间的结合关系。
环状HIPK3在GBC组织中上调，其表达升高与TNM分期和淋巴结转移相关。环状HIPK3敲低通过抑制体外增殖、血管生成、迁移和侵袭能力，同时促进细胞凋亡，从而抑制GBC进展，体内肿瘤生长也随之减少。环状HIPK3通过抑制泛素化介导的降解来稳定ALYREF。ALYREF敲低模拟了环状HIPK3缺失的肿瘤抑制作用，而ALYREF过表达则挽救了环状HIPK3敲低诱导的恶性表型抑制。环状HIPK3通过ALYREF稳定FASN mRNA。过表达FASN可抵消环状HIPK3下调对细胞血管生成和凋亡的作用。
环状HIPK3通过抑制ALYREF泛素化降解来增强FASN稳定性，促进血管生成，并抑制GBC细胞凋亡。本研究将环状HIPK3定位为GBC中有前景的预后生物标志物和治疗靶点，ALYREF和FASN成为该致癌途径中的关键机制节点。
