TRANSLATIONAL STUDIES ON MICRORNA IN HEPATOCELLULAR CARCINOMA: FROM PREDICTIVE AND PROGNOSTIC BIOMARKERS TO MOLECULAR EFFECTORS

Introduction Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, represents the seventh most frequent cancer and the fourth leading cause of cancer-related death worldwide. Due to unspecific sign and symptoms along with the inefficacy of current non-invasive diagnostic tools for detecting early stages HCC, majority of patients are diagnosed at advanced stages that is no longer eligible for curative treatments. Moreover, the long-term outcome of treatments remain unsatisfactory. To overcome these problems, a better diagnostic and predictive biomarker might be one of the most feasible strategies. The characteristic of MiRNAs, that are proved to have essential roles in various cancer pathways and found to be stable in biological fluids, holds a potential value as non-invasive clinical biomarkers in HCC. The present study include three tasks whose aims are: • Task 1 - Identify circulating miRNAs as predictor of early HCC occurrence in high-risk population setting, specific in DAA treated chronic HCV patients. • Task 2 - Identify circulating miRNAs as a prognostic non-invasive biomarker after HCC treatments. • Task 3 - Identify putative targets of each potential miRNAs and their cellular involvement in HCC pathway by in silico target prediction methods and in vitro approaches using miRNA transfection methods in HCC cell model. Result and discussion Task 1: We performed a circulating miRNA profiling analysis of cirrhotic patients treated with DAA before and after therapy initiation. Our group of patients consisted of 15 patients developing HCC after DAA treatment (HCC+) compared to 15 patients nor developing HCC (HCC-) within 12 month after SVR. Through microarray and qRT-PCR analysis, we confirmed differently expressed miR-3197 expression between HCC+ vs HCC− patients at T0 (before the initiation of DAA) (p< 0.05) with diagnostic performance of AUC values of 0.75 with a sensitivity of 80% and specificity of 73% in distinguishing both groups at T0, and AUC values of 0.75 with a sensitivity of 86% and specificity of 73% in distinguishing both groups At T1. Taken together, miR-3197 represent a promising tool for the identification of patients at risk such as HCV patients undergo DAA treatment. Task 2: We performed a longitudinal study analyzing the expression of serum miRNAs in the cohort of 105 HCC patients before treatments (T0), one (T1) and six months (T6) after treatments. Patients were then separated based on therapy response (TR), disease-free survival (DFS), and overall survival (OS). High expression of miR-4454 (p=0.02) and miR-4530 (p=0.04), and low expression of miR-4443 (P=0.05) at T0 were significantly associated with complete response to curative treatments and able to distinguish complete responder (CR) from partial and non-responder (PR) with an AUC of 0.84, sensitivity and specificity of 72% and 75%. High expression of miR-4454 (p=0.03) and miR-4530 (p=0.015) were also associated with DFS > 6 months in patients receiving curative therapies. For non-curative treatments (TACE), at T0 we observed the potential of miR-4492 distinguishing CR and PR (p=0.01) with AUC=0.84 and sensitivity and specificity of 84.6% and 71%. For OS, we observed significantly different expression of miR-4507 (p=0.00037) and ), and miR-3185 (p=0.014) to distinguish patients with shorter and longer OS. Higher Expression of miR-4507 and miR-3185 was significantly associated with longer OS with HR of 1.98 (p=0.016) and 2.02 (p=0.0086), respectively. Task 3: We confirmed the presence of some of our miRNAs candidate from task 1 and task 2 in tumoral tissue from HCC patients. We discovered several predicted target genes from in silico prediction approach. This result will be further validated in HCC cellular model using a transfection system with miRNA mimics and inhibitor.