索拉非尼在肝细胞癌（HCC）中的治疗效果因耐药性的发展而受到损害。本研究旨在鉴定在基因组水平上导致索拉非尼耐药的关键 microRNA (miRNA)。应用 CRISPR/Cas9 筛选，然后进行体外和体内功能获得和丧失分析来确定miR-3689a-3p 介导 HCC 中索拉非尼反应。研究了miR-3689a-3p的上下游分子及其作用机制。CRISPR/Cas9筛选发现miR-3689a-3p是索拉非尼敏感HCC中上调最多的miRNA。 miR-3689a-3p 的敲低显着增加了索拉非尼的耐药性，而其过度表达则使 HCC 对索拉非尼治疗的反应敏感。蛋白质组学分析表明，miR-3689a-3p 的作用与铜依赖性线粒体超氧化物歧化酶 1 型 (SOD1) 活性有关。从机制上讲，miR-3689a-3p 靶向超氧化物歧化酶 (CCS) 的细胞内铜伴侣的 3'UTR 并抑制其表达。结果，miR-3689a-3p 破坏了细胞内铜的运输，并减少了 SOD1 介导的线粒体氧化应激的清除，最终导致索拉非尼治疗导致 HCC 细胞死亡。 CCS 过度表达减弱了 HCC 中索拉非尼的反应。临床上，miR-3689a-3p 在 HCC 中下调，并预测 HCC 患者的良好预后。我们的研究结果为 miR-3689a-3p 作为正向调节剂和改善 HCC 索拉非尼治疗的潜在药物靶点提供了全面的证据。版权所有 © 2023作者们。由爱思唯尔有限公司出版。保留所有权利。

Therapeutic outcome of sorafenib in hepatocellular carcinoma (HCC) is undermined by the development of drug resistance. This study aimed to identify the critical microRNA (miRNA) which is responsible for sorafenib resistance at the genomic level.CRISPR/Cas9 screen followed by gain- and loss-of-function assays both in vitro and in vivo were applied to identify the role of miR-3689a-3p in mediating sorafenib response in HCC. The upstream and downstream molecules of miR-3689a-3p and their mechanism of action were investigated.CRISPR/Cas9 screening identified miR-3689a-3p was the most up-regulated miRNA in sorafenib sensitive HCC. Knockdown of miR-3689a-3p significantly increased sorafenib resistance, while its overexpression sensitized HCC response to sorafenib treatment. Proteomic analysis revealed that the effect of miR-3689a-3p was related to the copper-dependent mitochondrial superoxide dismutase type 1 (SOD1) activity. Mechanistically, miR-3689a-3p targeted the 3'UTR of the intracellular copper chaperone for superoxide dismutase (CCS) and suppressed its expression. As a result, miR-3689a-3p disrupted the intracellular copper trafficking and reduced SOD1-mediated scavenge of mitochondrial oxidative stress that eventually caused HCC cell death in response to sorafenib treatment. CCS overexpression blunted sorafenib response in HCC. Clinically, miR-3689a-3p was down-regulated in HCC and predicted favorable prognosis for HCC patients.Our findings provide comprehensive evidence for miR-3689a-3p as a positive regulator and potential druggable target for improving sorafenib treatment in HCC.Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.