Nicola Mosca

Human microRNA hsa-miR-125a-5p interferes with expression of hepatitis B virus surface antigen

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.

Circulating microRNA changes in heart failure patients treated with cardiac resynchronization therapy: responders vs. non-responders

MicroRNAs (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.

MicroRNA-423-5p Promotes Autophagy in Cancer Cells and Is Increased in Serum From Hepatocarcinoma Patients Treated With Sorafenib

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.

Liver microRNA hsa-miR-125a-5p in HBV Chronic Infection: Correlation with HBV Replication and Disease Progression

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 Ishaks 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.

Functional interplay between hepatitis B virus X protein and human miR-125a in HBV infection.

The hepatitis B virus (HBV) is a widespread human pathogen and chronic HBV infection is a major risk factor for hepatocellular carcinoma (HCC). Some cellular microRNAs are emerging as important regulators of virus-host interaction, indirectly or directly modulating HBV replication and pathogenesis. miR-125a binds the viral transcript encoding the surface antigen and interferes with its expression, thus inhibiting viral replication. Intriguingly, liver miR-125a expression has been found increased in patients with high levels of hepatic HBV-DNA. The present study investigates the mechanism by which liver exposure to HBV induces the expression of miR-125a. The analyses were first performed on liver biopsies from HBV patients, showing that the expression of the viral transactivator X protein (HBx) paralleled the increase of miR-125a expression. Then, transfection of HCC cell lines with an HBx-expressing vector showed a substantial increase of miR-125a expression. Overall, the available data depict a self-inhibitory feedback loop in which HBV, through HBx, increases the expression of miR-125a, that in turn interferes with expression of HBV surface antigen, thus repressing viral replication.

A novel splice variant of the human dicer gene is expressed in neuroblastoma cells

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.

Tuning RNA Interference by Enhancing siRNA/PAZ Recognition.

Chemically modified siRNAs were synthesized to enhance the corresponding silencing activities. The introduced modifications endowed siRNAs with high silencing effect, long RNAi persistence, and better serum resistance. Theoretical data allowed us to correlate the observed siRNAs interfering performance with the peculiar interactions with PAZ.

MicroRNA-125a-5p Is a Downstream Effector of Sorafenib in its Antiproliferative Activity Toward Human Hepatocellular Carcinoma Cells†

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.

Molecular mechanisms governing microRNA-125a expression in human hepatocellular carcinoma cells

MicroRNA-125a-5p (miR-125a) is a vertebrate homolog of lin-4, the first discovered microRNA, and plays a fundamental role in embryo development by downregulating Lin-28 protein. MiR-125a is also expressed in differentiated cells where it generally acts as an antiproliferative factor by targeting membrane receptors or intracellular transductors of mitogenic signals. MiR-125a expression is downregulated in several tumors, including hepatocellular carcinoma (HCC) where it targets sirtuin-7, matrix metalloproteinase-11, VEGF-A, Zbtb7a, and c-Raf. In this study, we have isolated the transcription promoter of human miR-125a and characterized its activity in HCC cells. It is a TATA-less Pol II promoter provided with an initiator element and a downstream promoter element, located 3939 bp upstream the genomic sequence of the miRNA. The activity of the promoter is increased by the transcription factor NF-kB, a master regulator of inflammatory response, and miR-125a itself was found to strengthen this activation through inhibition of TNFAIP3, a negative regulator of NF-kB. This finding contributes to explain the increased levels of miR-125a observed in the liver of patients with chronic hepatitis B.

Characterization of a naturally occurring truncated Dicer

Dicer is central to small RNA silencing pathways, thus playing an important role in physiological and pathological states. Recently, a number of mutations in dicer gene have been identified in diverse types of cancer, implicating Dicer in oncogenic cooperation. Here we report on the properties of a rare splice variant of the human dicer gene, occurring in neuroblastoma cells, and not detectable in normal tissues. Due to the skipping of one exon, the alternatively spliced transcript encodes a putative truncated protein, t-Dicer, lacking the dsRNA-binding domain and bearing altered one of the two RNase III catalytic centers. The ability of the exon-depleted t-dicer transcript to be translated in vitro was first investigated by the expression of flagged t-Dicer in human cells. We found that t-dicer transcript could be translated in vitro, albeit not as efficiently as full-length dicer transcript. Then, the possible enzymatic activity of t-Dicer was analyzed by an in vitro dicing assay able to distinguish the enzymatic activity of the individual RNase III domains. We showed that t-Dicer preserved partial dicing activity. Overall, the results indicate that t-dicer transcript could produce a protein still able to bind the substrate and to cleave only one of the two pre-miRNA strands. Given the increasing number of mutations reported for dicer gene in tumours, our experimental approach could be useful to characterize the activity of these mutants, which may dictate changes in selected classes of small RNAs and/or lead to their aberrant maturation.