AimsMicroRNAs (miRNAs) play an important role in the pathogenesis of structural alterations of the failing heart through their ability to regulate negatively the expression levels of genes that govern the process of adaptive and maladaptive cardiac remodelling. We studied whether LV reverse remodelling after CRT was associated with changes of circulating miRNAs in patients with heart failure (HF) and dyssynchrony. Methods and resultsA prospective, non-randomized self-control trial was performed in 81 patients with HF eligible for CRT. At baseline, to select the HF miRNA profile, we evaluated the expression of 84 miRNAs (implicated in the pathogenesis of structural alterations of the failing heart) in three groups of patients: healthy subjects (healthy group, n ¼ 15); patients with HF (HF group, n ¼ 81); and patients without HF matched for age, sex, and concomitant disease with HF patients (control group, n ¼ 60). At 12 months, the selected miRNA profile was evaluated in plasma from responder (n ¼ 55) and non-responder HF patients (n ¼ 26) to CRT. In the test cohort, the HF patients were characterized by lower expression of 48 miRNAs (all P , 0.04) as compared with healthy subjects. In the validation cohort, the HF patients were characterized by lower expression of 24 miRNAs (all P , 0.03) as compared with control patients. At 12 months, 55 patients (68%) were considered responders and 26 non-responders to CRT (32%). Responders showed an increase in expression of 19 miRNAs (all P , 0.03) compared with baseline expression, whereas in the non-responders we observed an increase of six miRNAs (all P , 0.05) compared with baseline expression. At follow-up, miRNAs were differentially expressed between responders and non-responders. The responders were characterized by higher expression of five miRNAs (miRNA-26b-5p, miRNA-145-5p, miRNA-92a-3p, miRNA-30e-5p, and miRNA-29a-3p; P , 0.01 for all) as compared with non-responders. ConclusionsIn responders, reverse remodelling is associated with favourable changes in miRNAs that regulate cardiac fibrosis, apoptosis, and hypertrophy.--

MicroRNAs are small non-coding RNAs that modulate gene expression at post-transcriptional level, playing a crucial role in cell differentiation and development. Recently, some reports have shown that a limited number of mammalian microRNAs are also involved in anti-viral defense. In this study, the analysis of the hepatitis B virus (HBV) genome by the computer program MiRanda led to the identification of seven sites that are potential targets for human liver microRNAs. These sites were found to be clustered in a 995-bp segment within the viral polymerase ORF and the overlapping surface antigen ORF, and conserved among the most common HBV subtypes. The HBV genomic targets were then subjected to a validation test based on cultured hepatic cells (HepG2, HuH-7 and PLC/PRF/5) and luciferase reporter genes. In this test, one of the selected microRNAs, hsa-miR-125a-5p, was found to interact with the viral sequence and to suppress the reporter activity markedly. The microRNA was then shown to interfere with the viral translation, down-regulating the expression of the surface antigen. Overall, these results support the emerging concept that some mammalian microRNAs play a role in virus-host interaction. Furthermore, they provide the basis for the development of new strategies for anti-HBV intervention.

Hepatocellular carcinoma (HCC) is the third cause of cancer-related deaths worldwide. Sorafenib is the only approved drug for patients with advanced HCC but has shown limited activity. microRNAs (miRs) have been involved in several neoplasms including HCC suggesting their use or targeting as good tools for HCC treatment. The purpose of this study was to identify novel approaches to sensitize HCC cells to sorafenib through miRs. miR-423-5p was validated as positive regulator of autophagy in HCC cell lines by transient transfection of miR and anti-miR molecules. miR-423-5p expression level was evaluated by real-time polymerase chain reaction (PCR) in sera collected from 39 HCC patients before and after treatment with sorafenib. HCC cells were cotreated with sorafenib and miR-423-5p and the effects on cell cycle, apoptosis, and autophagy were evaluated. Secretory miR-423-5p was upregulated both in vitro and in vivo by sorafenib treatment and its increase was correlated with response to therapy since 75% of patients in which an increase of secretory miR423-5p was found were in partial remission or stable disease after 6 moths from the beginning of therapy. HCC cells transfected with miR-423-5p showed an increase of cell percentage in S-phase of cell cycle paralleled by a similar increase of autophagic cells evaluated at both fluorescence activated cell sorter (FACS) and transmission electron microscopy. Our results suggest the miR423-5p can be used as a useful tool to predict response to sorafenib in HCC patients and is involved in autophagy regulation in HCC cells.

To study in HBsAg chronic carriers the expression of liver hsa-miR-125a-5p and its correlation with liver HBV-DNA values and clinical presentation, 27 consecutive Caucasian, HBsAg/anti-HBe/HBV-DNA-positive patients who were naive to nucleos(t)ide analogues and interferon therapy and had no marker of HCV, HDV or HIV infection and no history of alcohol intake were enrolled. For each patient, liver HBV DNA and liver hsa-miR-125a-5p were quantified by real-time PCR in relation to β-globin DNA or RNU6B, respectively. Liver fibrosis and necroinflammation were graded by applying Ishak's scoring system. Liver hsa-miR-125a-5p was detected in all patients enrolled and a correlation between its concentration and liver HBV DNA was demonstrated (p<0.0001). Higher liver hsa-miR-125a-5p concentrations were observed in patients with HBV-DNA plasma level >103 IU/ml (p<0.02), in those with HAI >6 (p = 0.02) and those with fibrosis score >2 (p<0.02) than in patients with lower scores. Higher HBV-DNA liver concentrations were found in patients with abnormal AST (p = 0.005) and ALT serum levels (p = 0.05), in those with serum HBV DNA higher than 10E3 IU/mL (p = 0.001) and those with fibrosis score >2 (p = 0.02) than in patients with a lower load. By multivariate logistic regression analysis, liver hsa-miR-125a-5p was identified as an independent predictor of disease progression: O.R. = 4.21, C.I. 95%  = 1.08–16.43, p<0.05, for HAI >6; O.R. = 3.12, C.I. 95%  = 1.17–8.27, p<0.05, for fibrosis score >2. In conclusion, in HBsAg/anti-HBe-positive patients, the liver hsa-miR-125a-5p level correlated with liver and plasma HBV-DNA values and was associated to a more severe disease progression.

Sorafenib is an antitumor drug for treatment of advanced hepatocellular carcinoma (HCC). It acts as a multikinase inhibitor suppressing cell proliferation and angiogenesis. Human microRNA-125a-5p (miR-125a) is endowed with similar activities and is frequently downregulated in HCC. Looking for a potential microRNA-based mechanism of action of the drug, we found that sorafenib increases cellular expression of miR-125a in cultured HuH-7 and HepG2 HCC cells. Upregulation of the microRNA inhibited cell proliferation by suppression of sirtuin-7, a NAD(+)-dependent deacetylase, and p21/p27-dependent cell cycle arrest in G1. Later, recruitment of miR-125a in the antiproliferative activity of sorafenib was inquired by modulating its expression in combination with the drug treatment. This analysis showed that intracellular delivery of miR-125a had no additive effect on the antiproliferative activity of sorafenib, whereas a miR-125a inhibitor could counteract it. Finally, evaluation of other oncogenic targets of miR-125a revealed its ability to interfere with the expression of matrix metalloproteinase-11, Zbtb7a proto-oncogene, and c-Raf, possibly contributing to the antiproliferative activity of the drug. J. Cell. Physiol. 232: 1907-1913, 2017. © 2016 Wiley Periodicals, Inc.

Edited by Tamas DalmayKeywords: Dicer miRNA Neuroblastoma Neural differentiation RNase Alternative splicing a b s t r a c t Dicer is a ribonuclease playing a key role in the biogenesis of microRNAs and small interfering RNAs. Here we report the identification of a novel splice variant of human dicer gene, the first one bearing a modified coding sequence. It encodes a truncated protein, t-Dicer that lacks the dsRNA-binding domain and is defective in one of the two RNase III catalytic centers. The splice variant was found in neuroblastoma cells and in cells induced to neuronal differentiation, whereas it was not detectable in other cell lines or in normal tissues. Interestingly, it occurred in primary neuroblastic tumors, mainly in stroma poor neuroblastomas.

BackgroundTherapeutic outcomes using the multikinase inhibitors, sorafenib and regorafenib, remain unsatisfactory for patients with advanced hepatocellular carcinoma (HCC). Thus, new drug modalities are needed. We recently reported the remarkable capacity of miR‐4510 to impede the growth of HCC and hepatoblastoma through Glypican‐3 (GPC3) targeting and Wnt pathway inactivation.MethodsTo identify new targets of miR‐4510, we used a label‐free proteomic approach and reported down‐regulation of RAF proto‐oncogene serine/threonine‐protein kinase (RAF1) by miR‐4510. Because the tumourigenic role of RAF1 in HCC is controversial, we further studied RAF1:miR‐4510 interactions using cellular, molecular as well as functional approaches and a chicken chorioallantoic membrane (CAM) xenograft model.ResultsWe found an increase in RAF1 protein in 59.3% of HCC patients and a specific up‐regulation of its transcript in proliferative tumours. We showed that miR‐4510 inactivates the RAS/RAF/MEK/ERK pathway and reduces the expression of downstream targets (ie c‐Fos proto‐oncogene [FOS]) through RAF1 direct targeting. At a cellular level, miR‐4510 inhibited HCC cell proliferation and migration and induced senescence in part by lowering RAF1 messenger RNA (mRNA) and protein expression. Finally, we confirmed the pro‐tumoural function of RAF1 protein in HCC cells and its ability to sustain HCC tumour progression in vitro and in vivo.ConclusionsIn this work, we confirm that RAF1 acts as an oncogene in HCC and further demonstrate that miR‐4510 acts as a strong tumour suppressor in the liver by targeting many proto‐oncogenes, including GPC3 and RAF1, and subsequently controlling key biological and signalling pathways among which Wnt and RAS/RAF/MEK/ERK signals.

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