<i>circFAM134B</i> is a key factor regulating reticulophagy-mediated ferroptosis in hepatocellular carcinoma

Ferroptosis is an important mode of regulated cell death (RCD). Its inhibition is closely related to therapeutic resistance and poor prognosis in hepatocellular carcinoma (HCC). Previous reports have demonstrated ferroptosis as a biological process highly dependent on selective autophagy, such as ferritinophagy, lipophagy, and clockophagy. Our study also revealed a role for ER-phagy-mediated ferroptosis in HCC cells treated with multi-targeted tyrosine kinase inhibitors (TKIs). In the current study, we found that the homologous circular RNA (circRNA) of the family with sequence similarity 134, member B (FAM134B), hsa_circ_0128505 (was abbreviated as circFAM134B in the present study), was identified to specifically target ER-phagy to promote lenvatinib (LV)-induced ferroptosis using reactive oxygen species (ROS), Fe2+, malondialdehyde (MDA), and western blot (WB) assays in HCC cells. RNA pull-down and mass spectrometry analyses suggested that circFAM134B and FAM134B mRNA were enriched with several common interacting proteins. Among them, poly (A) binding protein cytoplasmic 4 (PABPC4) was identified as the most enriched binding partner. It was proven to be a novel antagonist against the nonsense-mediated mRNA decay (NMD) mechanism. We then applied RNA immunoprecipitation (RIP), RNA pull-down, luciferase reporter, and NMD reporter gene assays to further explore the exact role and underlying mechanism of circFAM134B-PABPC4-FAM134B axis in HCC cells. circFAM134B was confirmed as a sponge that competitively interacted with PABPC4, thereby influencing FAM134B mRNA nonsense decay. Our results provide novel evidences and strategies for the comprehensive treatment of HCC.铁死亡（Ferroptosis）是一类重要的程序性细胞死亡（regulated cell death, RCD）模式。其抑制作用与肝细胞癌（hepatocellular carcinoma, HCC）的治疗耐药及不良预后密切相关。既往研究已证实，铁死亡是高度依赖选择性自噬的生物学过程，例如铁蛋白自噬、脂滴自噬及时钟蛋白自噬。本研究还发现，内质网自噬（ER-phagy）介导的铁死亡在多靶点酪氨酸激酶抑制剂（multi-targeted tyrosine kinase inhibitors, TKIs）处理的肝癌细胞中发挥作用。本研究通过在肝癌细胞中开展活性氧（reactive oxygen species, ROS）、亚铁离子（Fe²+）、丙二醛（malondialdehyde, MDA）及蛋白质印迹（western blot, WB）实验，发现序列相似性家族134成员B（family with sequence similarity 134, member B, FAM134B）的同源环状RNA（circular RNA, circRNA）hsa_circ_0128505（本研究中将其简称为circFAM134B）可特异性靶向内质网自噬，促进仑伐替尼（lenvatinib, LV）诱导的铁死亡。RNA下拉实验与质谱分析结果显示，circFAM134B与FAM134B mRNA可结合多种共有互作蛋白，其中胞质多聚腺苷酸结合蛋白4（poly (A) binding protein cytoplasmic 4, PABPC4）是富集程度最高的结合伴侣。经证实，PABPC4是一种新型的无义介导的mRNA降解（nonsense-mediated mRNA decay, NMD）拮抗剂。随后我们通过RNA免疫沉淀（RNA immunoprecipitation, RIP）、RNA下拉、荧光素酶报告基因及无义介导降解报告基因实验，进一步探究了circFAM134B-PABPC4-FAM134B轴在肝癌细胞中的具体作用及潜在机制。研究证实，circFAM134B可作为海绵分子竞争性结合PABPC4，进而调控FAM134B mRNA的无义降解过程。本研究结果为肝细胞癌的综合治疗提供了全新的研究证据与治疗策略。