Sorafenib is a major nonsurgical option for patients with advanced hepatocellular carcinoma (HCC); however, its clinical efficacy is largely undermined by the acquisition of resistance. The aim of this study was to identify the key lncRNA involved in the regulation of the sorafenib response in HCC. A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) single-guide RNA (sgRNA) synergistic activation mediator (SAM)-pooled lncRNA library was applied to screen for the key lncRNA regulated by sorafenib treatment. The role of the identified lncRNA in mediating the sorafenib response in HCC was examined in vitro and in vivo. The underlying mechanism was delineated by proteomic analysis. The clinical significance of the expression of the identified lncRNA was evaluated by multiplex immunostaining on a human HCC microtissue array. CRISPR/Cas9 lncRNA library screening revealed that Linc01056 was among the most downregulated lncRNAs in sorafenib-resistant HCC cells. Knockdown of Linc01056 reduced the sensitivity of HCC cells to sorafenib, suppressing apoptosis in vitro and promoting tumour growth in mice in vivo. Proteomic analysis revealed that Linc01056 knockdown in sorafenib-treated HCC cells induced genes related to fatty acid oxidation (FAO) while repressing glycolysis-associated genes, leading to a metabolic switch favouring higher intracellular energy production. FAO inhibition in HCC cells with Linc01056 knockdown significantly restored sensitivity to sorafenib. Mechanistically, we determined that PPARα is the critical molecule governing the metabolic switch upon Linc01056 knockdown in HCC cells and indeed, PPARα inhibition restored the sorafenib response in HCC cells in vitro and HCC tumours in vivo. Clinically, Linc01056 expression predicted optimal overall and progression-free survival outcomes in HCC patients and predicted a better sorafenib response. Linc01056 expression indicated a low FAO level in HCC. Our study identified Linc01056 as a critical epigenetic regulator and potential therapeutic target in the regulation of the sorafenib response in HCC.

中文翻译：

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lncRNA LINC01056 缺失导致 HCC 中索拉非尼耐药

索拉非尼是晚期肝细胞癌（HCC）患者的主要非手术选择；然而，其临床疗效在很大程度上因耐药性的获得而受到削弱。本研究的目的是确定参与 HCC 索拉非尼反应调节的关键 lncRNA。应用成簇规则间隔短回文重复序列 (CRISPR)/CRISPR 相关蛋白 9 (Cas9) 单向导 RNA (sgRNA) 协同激活介体 (SAM) 混合 lncRNA 文库筛选索拉非尼治疗调控的关键 lncRNA。在体外和体内检查了所鉴定的 lncRNA 在介导 HCC 索拉非尼反应中的作用。通过蛋白质组学分析描述了潜在的机制。通过对人 HCC 微组织阵列进行多重免疫染色来评估所鉴定的 lncRNA 表达的临床意义。 CRISPR/Cas9 lncRNA 文库筛选显示，Linc01056 是索拉非尼耐药 HCC 细胞中下调最明显的 lncRNA 之一。 Linc01056 的敲低降低了 HCC 细胞对索拉非尼的敏感性，在体外抑制细胞凋亡，并在体内促进小鼠肿瘤生长。蛋白质组学分析显示，索拉非尼处理的 HCC 细胞中的 Linc01056 敲低诱导了与脂肪酸氧化 (FAO) 相关的基因，同时抑制了糖酵解相关基因，从而导致有利于更高细胞内能量产生的代谢转换。敲除 Linc01056 后，FAO 对 HCC 细胞的抑制显着恢复了对索拉非尼的敏感性。从机制上讲，我们确定 PPARα 是控制 HCC 细胞中 Linc01056 敲低后代谢转换的关键分子，事实上，PPARα 抑制恢复了体外 HCC 细胞和体内 HCC 肿瘤中索拉非尼的反应。临床上，Linc01056 表达可预测 HCC 患者的最佳总体生存和无进展生存结果，并预测更好的索拉非尼反应。 Linc01056 表达表明 HCC 中的 FAO 水平较低。我们的研究确定 Linc01056 是 HCC 索拉非尼反应调节中的关键表观遗传调节剂和潜在治疗靶点。