Identification of novel microRNA-based therapeutic targets for the treatment of hepatocellular carcinoma: study of the mechanisms involved in metabolic reprogramming, immunity, and sorafenib response

Hepatocellular carcinoma (HCC) represents the third leading cause of cancer-related mortality worldwide, with approximately 40% of patients diagnosed at advanced stages, when systemic therapies are the only available option. Despite the introduction of immunotherapy, a significant proportion of patients remains ineligible; in this setting, sorafenib is still the standard first-line treatment. However, the development of acquired resistance severely limits its efficacy. Although multiple mechanisms underlying sorafenib resistance have been described, including microRNA (miRNA) deregulation, metabolic reprogramming, and tumour microenvironment (TME) alterations, the molecular links among these processes have not been fully elucidated yet. The aims of this work of thesis are to investigate the role of miR-494, miR-22, and miR-30e-3p in HCC progression and response to sorafenib, focusing on their involvement in metabolic rewiring and modulation of the tumour immune microenvironment (TIME), in order to identify novel miRNA-based therapeutic strategies. Bioinformatic analyses and functional assays identified glucose-6-phosphatase catalytic subunit (G6pc) as a direct target of miR-494 and GLUT1 as a target of miR-22, linking both miRNAs to a glycolytic metabolic shift, mitochondrial dysfunction, and oxidative stress in HCC cells. MiR-494 inhibition synergized with sorafenib and metabolic inhibitors, enhancing their antitumour effects. MiR-22 downregulation promoted HIF1α activation, increasing cell growth under hypoxia, and reduced sorafenib sensitivity. Furthermore, miR-30e-3p silencing enhanced CXCL3 expression, tumour growth, and sorafenib resistance in vitro and in vivo, while inducing alterations in immune cell infiltration within the TIME. Overall, these findings identify the miR-494/G6pc, miR-22/GLUT1, and miR-30e-3p/CXCL3 axes as key regulators of HCC metabolism, tumour aggressiveness, drug resistance, and immune modulation, supporting their potential as therapeutic targets in combination with sorafenib for patients ineligible for immunotherapy.