Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. Prognosis is poor, and therapeutic options are limited. MicroRNAs (miRNAs) have emerged as potential therapeutic molecules against cancer. Here, we investigated the therapeutic efficacy of miR-199a-3p, an miRNA highly expressed in normal liver and downregulated in virtually all HCCs. The therapeutic value of miR-199a-3p mimic molecules was assayed in the TG221 mouse, a transgenic model highly predisposed to the development of liver cancer. Administration of miR-199a-3p mimics in the TG221 transgenic mouse showing liver cancer led to a significant reduction of number and size of tumor nodules compared to control animals. In vivo delivery confirmed protein downregulation of the miR-199a-3p direct targets, mechanistic target of rapamycin (MTOR) and p21 activated kinase 4 (PAK4), ultimately leading to the repression of FOXM1. Remarkably, the anti-tumor activity of miR-199a-3p mimics was comparable to that obtained with sorafenib. These results suggested that miR-199a-3p may be considered a promising HCC therapeutic option.

Hepatocellular carcinoma (HCC) is the most common liver cancer and second leading cause of cancer related death worldwide. Most HCCs occur in a damaged cirrhotic background and it may be difficult to discriminate between regenerative nodules and early HCCs. No dependable molecular biomarker exists for the early detection of HCC. MicroRNAs (miRNAs) have attracted attention as potential blood-based biomarkers. To identify circulating miRNAs with diagnostic potential in HCC, we performed preliminary RNAseq studies on plasma samples from a small set of HCC patients, cirrhotic patients and healthy controls. Then, out of the identified miRNAs, we investigated miR-101-3p, miR-106b-3p, miR-1246 and miR-411-5p in plasma of independent HCC patients’ cohorts. The use of droplet digital PCR (ddPCR) confirmed the aberrant levels of these miRNAs. The diagnostic performances of each miRNA and their combinations were measured using Receiver Operating Characteristic (ROC) curve analyses: a classifier consisting of miR-101-3p, miR-1246 and miR-106b-3p produced the best diagnostic precision in plasma of HCC vs. cirrhotic patients (AUC = 0.99). A similar performance was found when the levels of miRNAs of HCC patients were compared to healthy controls (AUC = 1.00). We extended the analyses of the same miRNAs to serum samples. In serum of HCC vs. cirrhotic patients, the combination of miR-101-3p and miR-106b-3p exhibited the best diagnostic accuracy with an AUC = 0.96. Thus, circulating miR-101-3p, miR-106b-3p and miR-1246, either individually or in combination, exhibit a considerable potential value as diagnostic biomarkers of HCC.

Metformin is a hypoglycaemic agent used to treat type 2 diabetes mellitus (DM2) patients, with a broad safety profile. Since previous epidemiological studies had shown that the incidence of hepatocellular carcinoma (HCC) decreased significantly in metformin treated DM2 patients, we hypothesised that intervention with metformin could reduce the risk of neoplastic transformation of hepatocytes. HCC is the most common primary liver malignancy and it generally originates in a background of liver fibrosis and cirrhosis. In the present study, we took advantage of a transgenic mouse (TG221) characterized by microRNA-221 overexpression, with cirrhotic liver background induced by chronic administration of carbon tetrachloride (CCl4). This mouse model develops fibrosis, cirrhosis and liver tumours that become visible in 100% of mice at 5-6 months of age. Our results demonstrated that metformin intervention improves liver function, inhibits hepatic stellate cell (HSC) activation, reduces liver fibrosis, depletes lipid accumulation in hepatocytes, halts progression to decompensated cirrhosis and abrogates development HCC in CCl4 challenged transgenic mouse model. The study establishes the rationale for investigating metformin in cirrhotic patients regardless of concomitant DM2 status. virus-associated HCC [4, 5]. However, non-viral causes 39 namely alcoholic or non-alcoholic fatty liver disease 40 (NAFLD) and non-alcoholic steatohepatitis Q6 (NASH) 41 account for nearly 46% liver cancer deaths [6]. Addition-42 ally, increasing epidemic of obesity and type 2 diabetes 43 mellitus (DM2), both important risk factors of NAFLD and 44 NASH, might emerge as dominant risk factors for HCC 45 incidence in future [7, 8]. Hence, prevention programs can 46 have an important impact in decreasing HCC occurrence. 47 Metformin is a first-line dimethylbiguanide hypogly-48 caemic agent used to treat DM2 patients, with a broad safety 49 profile. Abundant epidemiological data have indicated that 50 incidence of several cancers, including HCC decreased * Massimo Negrini

Most hepatocellular carcinomas (HCCs) arise in the context of chronic liver disease and/or cirrhosis. Thus, chemoprevention in individuals at risk represents an important but yet unproven approach. In this study, we investigated the ability of microRNA (miRNA)-based molecules to prevent liver cancer development in a cirrhotic model. To this end, we developed a mouse model able to recapitulate the natural progression from fibrosis to HCC, and then we tested the prophylactic activity of an miRNA-based approach in the model. The experiments were carried out in the TG221 transgenic mouse, characterized by the overexpression of miR-221 in the liver and predisposed to the development of liver tumors. TG221 as well as wild-type mice were exposed to the hepatotoxin carbon tetrachloride (CCl4) to induce chronic liver damage. All mice developed liver cirrhosis, but only TG221 mice developed nodular lesions in 100% of cases within 6 months of age. The spectrum of lesions ranged from dysplastic foci to carcinomas. To investigate miRNA-based prophylactic approaches, anti-miR-221 oligonucleotides or miR-199a-3p mimics were administered to TG221 CCl4-treated mice. Compared to control animals, a significant reduction in number, size, and, most significantly, malignant phenotype of liver nodules was observed, thus demonstrating an important prophylactic action of miRNA-based molecules. In summary, in this article, we not only report a simple model of liver cancer in a cirrhotic background but also provide evidence for a potential miRNA-based approach to reduce the risk of HCC development.

Background Health surveillance of murine colonies employed for scientific purposes aim at detecting unwanted infection that can affect the well-being of animals and personnel, and potentially undermine scientific results. In this study, we investigated the use of a next-generation sequencing (NGS) metagenomic approach for monitoring the microbiota composition and uncovering the possible presence of pathogens in mice housed in specific pathogen-free (SPF) or conventional (non-SPF) facilities. Results Analysis of metagenomic NGS assay through public and free algorithms and databases allowed to precisely assess the composition of mouse gut microbiome and quantify the contribution of the different microorganisms at the species level. Sequence analysis allowed the uncovering of pathogens or the presence of imbalances in the microbiota composition. In several cases, fecal pellets taken from conventional facilities were found to carry gene sequences from bacterial pathogens (Helicobacter hepaticus, Helicobacter typhlonius, Chlamydia muridarum, Streptococcus pyogenes, Rodentibacter pneumotropicus, Citrobacter rodentium, Staphylococcus aureus), intestinal protozoa (Entamoeba muris, Tritrichomonas muris, Spironucleus muris) nematoda (Aspiculuris tetraptera, Syphacia obvelata), eukaryotic parasites (Myocoptes musculinus) and RNA virus (Norwalk virus). Thus, the use of NGS metagenomics can reduce the number of tests required for the detection of pathogens and avoid the use of sentinel mice. Conclusions In summary, in comparison with standard approaches, which require multiple types of test, NGS assay can detect bacteria, fungi, DNA and RNA viruses, and eukaryotic parasites from fecal pellets in a single test. Considering the need to protect animal well-being and to improve the success and reproducibility of preclinical studies, this work provides the proof-of-concept that the use of NGS metagenomics for health monitoring of laboratory mice is a feasible and dependable approach, that is able to broaden the current concept of health monitoring of laboratory mice from “pathogen surveillance” to a more inclusive “microbiota surveillance”.