NRF2 activation by cysteine as a survival mechanism for triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a very aggressive and heterogeneous group of tumors. In order to develop effective therapeutic strategies, it is therefore essential to identify the subtype-specific molecular mechanisms underlying disease progression and resistance to chemotherapy. TNBC cells are highly dependent on exogenous cystine, provided by overexpression of the cystine/glutamate antiporter SLC7A11/xCT, to fuel glutathione synthesis and promote an oxidative stress response consistent with their high metabolic demands. Here we show that TNBC cells of the mesenchymal stem-like subtype (MSL) utilize forced cystine uptake to induce activation of the transcription factor NRF2 and promote a glutathione-independent mechanism to defend against oxidative stress. Mechanistically, we demonstrate that NRF2 activation is mediated by direct cysteinylation of the inhibitor KEAP1. Furthermore, we show that cystine-mediated NRF2 activation induces the expression of important genes involved in oxidative stress response, but also in epithelial-to-mesenchymal transition and stem-like phenotype. Remarkably, in survival analysis, four upregulated genes (OSGIN1, RGS17, SRXN1, AKR1B10) are negative prognostic markers for TNBC. Finally, expression of exogenous OSGIN1, similarly to expression of exogenous NRF2, can prevent cystine depletion-dependent death of MSL TNBC cells. The results suggest that the cystine/NRF2/OSGIN1 axis is a potential target for effective treatment of MSL TNBCs.

Secreted miR-210-3p, miR-183-5p and miR-96-5p reduce sensitivity to docetaxel in prostate cancer cells.

Docetaxel (DCT) resistance is one of the main factors responsible for treatment failure in metastatic prostate cancer (PCa). Although several mechanisms of DCT resistance have been elucidated, the issue is still far from comprehensive. In this work we show that miR-96-5p, miR-183-5p and miR-210-3p (referred to as sDCTR-miRNAs) are specifically released by DCT resistant (DCTR) PCa clones and decrease the efficacy of DCT in PCa cells when overexpressed. Through bioinformatic analysis, we identified several potential targets of sDCTR-miRNAs' activity including FOXO1, IGFBP3, and PDCD4 known to exert a role in DCT resistance. Additionally, we found that PPP2CB and INSIG1 mediated the ability of sDCTR-miRNAs to reduce the efficacy of DCT. We explored whether secreted sDCTR-miRNAs could affect the phenotype of PCa cells. We found that exposure to exosomes derived from DCTR PCa clones (in which the content of sDCTR-miRNAs was higher than in exosomes from parental cells), as well as exposure to exosome loaded with sDCTR-miRNAs, reduced the cytotoxicity of DCT in PCa cells sensitive to the drug. Finally, we validated circulating miR-183-5p and miR-21-5p as potential predictive biomarkers of DCT resistance in PCa patients. Our study suggests a horizontal transfer mechanism mediated by exosomal miRNAs that contributes to reduce docetaxel sensitivity and highlights the relevance of cell-to-cell communication in drug resistance.

Hooked Up from a Distance: Charting Genome-Wide Long-Range Interaction Maps in Neural Cells Chromatin to Identify Novel Candidate Genes for Neurodevelopmental Disorders.

DNA sequence variants (single nucleotide polymorphisms or variants, SNPs/SNVs; copy number variants, CNVs) associated to neurodevelopmental disorders (NDD) and traits often map on putative transcriptional regulatory elements, including, in particular, enhancers. However, the genes controlled by these enhancers remain poorly defined. Traditionally, the activity of a given enhancer, and the effect of its possible alteration associated to the sequence variants, has been thought to influence the nearest gene promoter. However, the obtainment of genome-wide long-range interaction maps in neural cells chromatin challenged this view, showing that a given enhancer is very frequently not connected to the nearest promoter, but to a more distant one, skipping genes in between. In this Perspective, we review some recent papers, who generated long-range interaction maps (by HiC, RNApolII ChIA-PET, Capture-HiC, or PLACseq), and overlapped the identified long-range interacting DNA segments with DNA sequence variants associated to NDD (such as schizophrenia, bipolar disorder and autism) and traits (intelligence). This strategy allowed to attribute the function of enhancers, hosting the NDD-related sequence variants, to a connected gene promoter lying far away on the linear chromosome map. Some of these enhancer-connected genes had indeed been already identified as contributive to the diseases, by the identification of mutations within the gene's protein-coding regions (exons), validating the approach. Significantly, however, the connected genes also include many genes that were not previously found mutated in their exons, pointing to novel candidate contributors to NDD and traits. Thus, long-range interaction maps, in combination with DNA variants detected in association with NDD, can be used as "pointers" to identify novel candidate disease-relevant genes. Functional manipulation of the long-range interaction network involving enhancers and promoters by CRISPR-Cas9-based approaches is beginning to probe for the functional significance of the identified interactions, and the enhancers and the genes involved, improving our understanding of neural development and its pathology.

Heterozygosity for neuronal ceroid lipofuscinosis predisposes to bipolar disorder.

OBJECTIVE: Bipolar disorder is a heritable chronic mental disorder that causes psychosocial impairment through depressive/manic episodes. Familial transmission of bipolar disorder does not follow simple Mendelian patterns of inheritance. The aim of this study was to describe a large family with 12 members affected by bipolar disorder. Whole-exome sequencing was performed for eight members, three of whom were diagnosed with bipolar disorder, and another reported as "borderline." METHODS: Whole-exome sequencing data allowed us to select variants that the affected members had in common, including and excluding the "borderline" individual with moderate anxiety and obsessive-compulsive traits. RESULTS: The results favored designating certain genes as predispositional to bipolar disorder: a heterozygous missense variant in CLN6 resulted in a "borderline" phenotype that, if combined with a heterozygous missense variant in ZNF92, is responsible for the more severe bipolar disorder phenotype. Both rare missense changes are predicted to disrupt protein function. CONCLUSIONS: Loss of both alleles in CLN6 causes neuronal ceroid lipofuscinosis, a severe progressive childhood neurological disorder. Our results indicate that heterozygous CLN6 carriers, previously reported as healthy, may be susceptible to bipolar disorder later in life if associated with additional variants in ZNF92.

Artificial Intelligence Predictive Models of Response to Cytotoxic Chemotherapy Alone or Combined to Targeted Therapy for Metastatic Colorectal Cancer Patients: A Systematic Review and Meta-Analysis.

Tailored treatments for metastatic colorectal cancer (mCRC) have not yet completely evolved due to the variety in response to drugs. Therefore, artificial intelligence has been recently used to develop prognostic and predictive models of treatment response (either activity/efficacy or toxicity) to aid in clinical decision making. In this systematic review, we have examined the ability of learning methods to predict response to chemotherapy alone or combined with targeted therapy in mCRC patients by targeting specific narrative publications in Medline up to April 2022 to identify appropriate original scientific articles. After the literature search, 26 original articles met inclusion and exclusion criteria and were included in the study. Our results show that all investigations conducted on this field have provided generally promising results in predicting the response to therapy or toxic side-effects. By a meta-analytic approach we found that the overall weighted means of the area under the receiver operating characteristic (ROC) curve (AUC) were 0.90, 95% C.I. 0.80-0.95 and 0.83, 95% C.I. 0.74-0.89 in training and validation sets, respectively, indicating a good classification performance in discriminating response vs. non-response. The calculation of overall HR indicates that learning models have strong ability to predict improved survival. Lastly, the delta-radiomics and the 74 gene signatures were able to discriminate response vs. non-response by correctly identifying up to 99% of mCRC patients who were responders and up to 100% of patients who were non-responders. Specifically, when we evaluated the predictive models with tests reaching 80% sensitivity (SE) and 90% specificity (SP), the delta radiomics showed an SE of 99% and an SP of 94% in the training set and an SE of 85% and SP of 92 in the test set, whereas for the 74 gene signatures the SE was 97.6% and the SP 100% in the training set.

Bridging between Mouse and Human Enhancer-Promoter Long-Range Interactions in Neural Stem Cells, to Understand Enhancer Function in Neurodevelopmental Disease.

Non-coding variation in complex human disease has been well established by genome-wide association studies, and is thought to involve regulatory elements, such as enhancers, whose variation affects the expression of the gene responsible for the disease. The regulatory elements often lie far from the gene they regulate, or within introns of genes differing from the regulated gene, making it difficult to identify the gene whose function is affected by a given enhancer variation. Enhancers are connected to their target gene promoters via long-range physical interactions (loops). In our study, we re-mapped, onto the human genome, more than 10,000 enhancers connected to promoters via long-range interactions, that we had previously identified in mouse brain-derived neural stem cells by RNApolII-ChIA-PET analysis, coupled to ChIP-seq mapping of DNA/chromatin regions carrying epigenetic enhancer marks. These interactions are thought to be functionally relevant. We discovered, in the human genome, thousands of DNA regions syntenic with the interacting mouse DNA regions (enhancers and connected promoters). We further annotated these human regions regarding their overlap with sequence variants (single nucleotide polymorphisms, SNPs; copy number variants, CNVs), that were previously associated with neurodevelopmental disease in humans. We document various cases in which the genetic variant, associated in humans to neurodevelopmental disease, affects an enhancer involved in long-range interactions: SNPs, previously identified by genome-wide association studies to be associated with schizophrenia, bipolar disorder, and intelligence, are located within our human syntenic enhancers, and alter transcription factor recognition sites. Similarly, CNVs associated to autism spectrum disease and other neurodevelopmental disorders overlap with our human syntenic enhancers. Some of these enhancers are connected (in mice) to homologs of genes already associated to the human disease, strengthening the hypothesis that the gene is indeed involved in the disease. Other enhancers are connected to genes not previously associated with the disease, pointing to their possible pathogenetic involvement. Our observations provide a resource for further exploration of neural disease, in parallel with the now widespread genome-wide identification of DNA variants in patients with neural disease.

The FHP01 DDX3X Helicase Inhibitor Exerts Potent Anti-Tumor Activity In Vivo in Breast Cancer Pre-Clinical Models.

Inhibition of DDX3X expression or activity reduces proliferation in cells from various tumor tissues, in particular in breast cancer, and its expression often correlates to tumor aggressiveness. This makes DDX3X a prominent candidate for the design of drugs for novel personalized therapeutic strategies. Starting from an in silico drug discovery approach, a group of molecules has been selected by molecular docking at the RNA binding site of DDX3X. Here, the most promising among them, FHP01, was evaluated in breast cancer preclinical models. Specifically, FHP01 exhibited very effective antiproliferative and killing activity against different breast cancer cell types, among which those from triple-negative breast cancer (TNBC). Interestingly, FHP01 also inhibited WNT signaling, a key tumorigenic pathway already correlated to DDX3X functions in breast cancer model cell lines. Ultimately, FHP01 also caused a significant reduction, in vivo, in the growth of MDA MB 231-derived TNBC xenograft models. Importantly, FHP01 showed good bioavailability and no toxicity on normal peripheral blood mononuclear cells in vitro and on several mouse tissues in vivo. Overall, our data suggest that the use of FHP01 and its related compounds may represent a novel therapeutic approach with high potential against breast cancer, including the triple-negative subtype usually correlated to the most unfavorable outcomes because of the lack of available targeted therapies.

Protocol to analyze circulating small non-coding RNAs by high-throughput RNA sequencing from human plasma samples.

The identification and validation of circulating small non-coding RNA (sncRNA) as biomarkers for disease diagnosis, staging, and response to novel therapies is still a compelling challenge. Pre-analytical variables, such as storage temperature or blood hemolysis, and different analytical approaches affect sncRNA stability, detection, and expression, resulting in discrepancies among studies. Here, we report a systematic standardized protocol to reproducibly analyze circulating sncRNAs, employing high-throughput sncRNA sequencing and qRT-PCR validation, from 200 µL of human plasma samples. For details on the use and execution of this protocol, please refer to Ventriglia et al. (2020), Sebastiani et al. (2017), and Dotta et al. (2018).

High-Throughput Identification of miRNA-Target Interactions in Melanoma Using miR-CATCHv2.0.

MicroRNAs (miRNAs) can regulate the expression of potentially every transcript in the cell, and the definition of miRNA-target interactions is crucial to understand their role in all biological processes. However, the identification of the miRNAs that target a specific mRNA remains a challenge. Here, we describe an innovative method called miR-CATCHv2.0 for the high-throughput identification of the miRNA species bound to an RNA of interest. We also describe how this method can overcome the limitations of the current computational and experimental methods available in this field.

MicroRNA Expression in the Aqueous Humor of Patients with Diabetic Macular Edema.

We identified and compared secreted microRNA (miRNA) expression in aqueous humor (AH) and plasma samples among patients with: type 2 diabetes mellitus (T2D) complicated by non-proliferative diabetic retinopathy (DR) associated with diabetic macular edema (DME) (DME group: 12 patients); T2D patients without DR (D group: 8 patients); and non-diabetic patients (CTR group: 10 patients). Individual patient AH samples from five subjects in each group were profiled on TaqMan Low Density MicroRNA Array Cards. Differentially expressed miRNAs identified from profiling were then validated in single assay for all subjects. The miRNAs validated in AH were then evaluated in single assay in plasma. Gene Ontology (GO) analysis was conducted. From AH profiling, 119 mature miRNAs were detected: 86 in the DME group, 113 in the D group and 107 in the CTR group. miRNA underexpression in the DME group was confirmed in single assay for let-7c-5p, miR-200b-3p, miR-199a-3p and miR-365-3p. Of these four, miR-199a-3p and miR-365-3p were downregulated also in the plasma of the DME group. GO highlighted 54 validated target genes of miR-199a-3p, miR-200b-3p and miR-365-3p potentially implied in DME pathogenesis. Although more studies are needed, miR-200b-3p, let-7c-5p, miR-365-3p and miR-199a-3p represent interesting molecules in the study of DME pathogenesis.

T3 Critically Affects the Mhrt/Brg1 Axis to Regulate the Cardiac MHC Switch: Role of an Epigenetic Cross-Talk.

The LncRNA my-heart (Mhrt) and the chromatin remodeler Brg1 inhibit each other to respectively prevent or favor the maladaptive α-myosin-heavy-chain (Myh6) to ß-myosin-heavy-chain (Myh7) switch, so their balance crucially guides the outcome of cardiac remodeling under stress conditions. Even though triiodothyronine (T3) has long been recognized as a critical regulator of the cardiac Myh isoform composition, its role as a modulator of the Mhrt/Brg1 axis is still unexplored. Here the effect of T3 on the Mhrt/Brg1 regulatory circuit has been analyzed in relation with chromatin remodeling and previously identified T3-dependent miRNAs. The expression levels of Mhrt, Brg1 and Myh6/Myh7 have been assessed in rat models of hyperthyroidism or acute myocardial ischemia/reperfusion (IR) treated with T3 replacement therapy. To gain mechanistic insights, in silico analyses and site-directed mutagenesis have been adopted in combination with gene reporter assays and loss or gain of function strategies in cultured cardiomyocytes. Our results indicate a pivotal role of Mhrt over-expression in the T3-dependent regulation of Myh switch. Mechanistically, T3 activates the Mhrt promoter at two putative thyroid hormone responsive elements (TRE) located in a crucial region that is necessary for both Mhrt activation and Brg1-dependent Mhrt repression. This newly identified T3 mode of action requires DNA chromatinization and is critically involved in mitigating the repressive function of the Brg1 protein on Mhrt promoter. In addition, T3 is also able to prevent the Brg1 over-expression observed in the post-IR setting through a pathway that might entail the T3-mediated up-regulation of miR-208a. Taken together, our data evidence a novel T3-responsive network of cross-talking epigenetic factors that dictates the cardiac Myh composition and could be of great translational relevance.

Variations of circulating miRNA in paediatric patients with Heart Failure supported with Ventricular Assist Device: a pilot study.

Circulating miRNAs (c-miRNAs) are promising biomarkers for HF diagnosis and prognosis. There are no studies on HF pediatric patients undergoing VAD-implantation. Aims of this study were: to examine the c-miRNAs profile in HF children; to evaluate the effects of VAD on c-miRNAs levels; to in vitro validate putative c-miRNA targets. c-miRNA profile was determined in serum of HF children by NGS before and one month after VAD-implant. The c-miRNA differentially expressed were analyzed by real time-PCR, before and at 4 hrs,1,3,7,14,30 days after VAD-implant. A miRNA mimic transfection study in HepG2 cells was performed to validate putative miRNA targets selected through miRWalk database. Thirteen c-miRNAs were modified at 30 days after VAD-implant compared to pre-VAD at NSG, and, among them, six c-miRNAs were confirmed by Real-TimePCR. Putative targets of the validated c-miRNAs are involved in the hemostatic process. The in vitro study confirmed a down-regulatory effect of hsa-miR-409-3p towards coagulation factor 7 (F7) and F2. Of note, all patients had thrombotic events requiring pump change. In conclusion, in HF children, the level of six c-miRNAs involved in the regulation of hemostatic events changed after 30 days of VAD-treatment. In particular, the lowering of c-miR-409-3p regulating both F7 and F2 could reflect a pro-thrombotic state after VAD-implant.

The miR-28-5p Targetome Discovery Identified SREBF2 as One of the Mediators of the miR-28-5p Tumor Suppressor Activity in Prostate Cancer Cells.

miR-28-5p is downregulated in some tumor tissues in which it has been demonstrated to have tumor suppressor (TS) activity. Here, we demonstrate that miR-28-5p acts as a TS in prostate cancer (PCa) cells affecting cell proliferation/survival, as well as migration and invasion. Using the miRNA pull out assay and next generation sequencing, we collected the complete repertoire of miR-28-5p targets, obtaining a data set (miR-28-5p targetome) of 191 mRNAs. Filtering the targetome with TargetScan 7, PITA and RNA22, we found that 61% of the transcripts had miR-28-5p binding sites. To assign a functional value to the captured transcripts, we grouped the miR-28-5p targets into gene families with annotated function and showed that six transcripts belong to the transcription factor category. Among them we selected SREBF2, a gene with an important role in PCa. We validated miR-28-5p/SREBF2 interaction, demonstrating that SREBF2 inhibition affects almost all the tumor processes altered by miR-28-5p re-expression, suggesting that SREBF2 is an important mediator of miR-28-5p TS activity. Our findings support the identification of the targetome of cancer-related miRNAs as a tool to discover genes and pathways fundamental for tumor development, and potential new targets for anti-tumor therapy.

Characterization of pUL5, an HCMV protein interacting with the cellular protein IQGAP1.

Among the Herpesviridae, human cytomegalovirus (HCMV) owns the largest genome and displays a huge coding potential. Here, we characterized the UL5 gene product (pUL5) of the clinical isolate TR strain. The protein was predicted as a 166-amino-acid membrane protein with a theoretical mass of 19 kDa. Recombinant virus expressing pUL5 with a tag allowed the identification of two pUL5 non-glycosylated species of approximately 19 and 9 kDa, expressed with early and late kinetic respectively. Experiments in infection confirmed that the lower molecular weight species was translated from an internal ATG in the UL5 open reading frame. Confocal microscopy analysis showed that pUL5 localized within the assembly compartment, but is not incorporated in the virion, as shown by Western blot on purified viral particles. Finally, pull-down experiments coupled with mass spectrometry analysis identified IQGAP1 as a pUL5 interactor, giving new hints on possible roles of pUL5 during HCMV infection.

miR-182-5p is an evolutionarily conserved Tbx5 effector that impacts cardiac development and electrical activity in zebrafish.

To dissect the TBX5 regulatory circuit, we focused on microRNAs (miRNAs) that collectively contribute to make TBX5 a pivotal cardiac regulator. We profiled miRNAs in hearts isolated from wild-type, CRE, Tbx5lox/+and Tbx5del/+ mice using a Next Generation Sequencing (NGS) approach. TBX5 deficiency in cardiomyocytes increased the expression of the miR-183 cluster family that is controlled by Kruppel-like factor 4, a transcription factor repressed by TBX5. MiR-182-5p, the most highly expressed miRNA of this family, was functionally analyzed in zebrafish. Transient overexpression of miR-182-5p affected heart morphology, calcium handling and the onset of arrhythmias as detected by ECG tracings. Accordingly, several calcium channel proteins identified as putative miR-182-5p targets were downregulated in miR-182-5p overexpressing hearts. In stable zebrafish transgenic lines, we demonstrated that selective miRNA-182-5p upregulation contributes to arrhythmias. Moreover, cardiac-specific down-regulation of miR-182-5p rescued cardiac defects in a zebrafish model of Holt-Oram syndrome. In conclusion, miR-182-5p exerts an evolutionarily conserved role as a TBX5 effector in the onset of cardiac propensity for arrhythmia, and constitutes a relevant target for mediating the relationship between TBX5, arrhythmia and heart development.

RNA sequencing reveals PNN and KCNQ1OT1 as predictive biomarkers of clinical outcome in stage III colorectal cancer patients treated with adjuvant chemotherapy.

Five-year overall survival of stage III colorectal cancer (CRC) patients treated with standard adjuvant chemotherapy (ACHT) is highly variable. Genomic biomarkers and/or transcriptomic profiles identified lack of adequate validation. Aim of our study was to identify and validate molecular biomarkers predictive of ACHT response in stage III CRC patients by a transcriptomic approach. From a series of CRC patients who received ACHT, two stage III extreme cohorts (unfavorable vs. favorable prognosis) were selected. RNA-sequencing was performed from fresh frozen explants. Tumors were characterized for somatic mutations. Validation was performed in stage III CRC patients extracted from two GEO datasets. According to disease-free survival (DFS), 108 differentially expressed genes (104/4 up/downregulated in the unfavorable prognosis group) were identified. Among 104 upregulated genes, 42 belonged to olfactory signaling pathways, 62 were classified as pseudogenes (n = 17), uncharacterized noncoding RNA (n = 10), immune response genes (n = 4), microRNA (n = 1), cancer-related genes (n = 14) and cancer-unrelated genes (n = 16). Three out of four down-regulated genes were cancer-related. Mutational status (i.e., RAS, BRAF, PIK3CA) did not differ among the cohorts. In the validation cohort, multivariate analysis showed high PNN and KCNQ1OT1 expression predictive of shorter DFS in ACHT treated patients (p = 0.018 and p = 0.014, respectively); no difference was observed in untreated patients. This is the first study that identifies by a transcriptomic approach and validates PNN and KCNQ1OT1 as molecular biomarkers predictive of chemotherapy response in stage III CRC patients. After a further validation in an independent cohort, PNN and KCNQ1OT1 evaluation could be proposed to prospectively identify stage III CRC patients benefiting from ACHT.

Systematic evaluation of the microRNAome through miR-CATCHv2.0 identifies positive and negative regulators of BRAF-X1 mRNA.

Here we present miR-CATCHv2.0, an implemented experimental method that allows the identification of the microRNA species directly bound to an RNA of interest. After cross-linking of microRNA::RNA::Ago2 complexes using formaldehyde, the RNA is fragmented using sonication and then subjected to affinity purification using two sets of biotinylated tiling probes (ODD and EVEN). Finally, enriched microRNA species are retrieved by means of small RNA sequencing coupled with an ad hoc analytical workflow. In BRAFV600E mutant A375 melanoma cells, miR-CATCHv2.0 allowed us to identify 20 microRNAs that target X1, the most abundant isoform of BRAF mRNA. These microRNAs fall into different functional classes, according to the effect that they exert (decrease/increase in BRAFV600E mRNA and protein levels) and to the mechanism they use to achieve it (destabilization/stabilization of X1 mRNA or decrease/increase in its translation). microRNA-induced variations in BRAFV600E protein levels are most of the times coupled to consistent variations in pMEK levels, in melanoma cell proliferation in vitro and in sensitivity to the BRAF inhibitor vemurafenib in a xenograft model in zebrafish. However, microRNAs exist that uncouple the degree of activation of the ERK pathway from the levels of BRAFV600E protein. Our study proposes miR-CATCHv2.0 as an effective tool for the identification of direct microRNA-target interactions and, by using such a tool, unveils the complexity of the post-transcriptional regulation to which BRAFV600E and the ERK pathway are subjected in melanoma cells.

Early modifications of circulating microRNAs levels in metastatic colorectal cancer patients treated with regorafenib.

Biomarkers able to improve the cost/benefit ratio are urgently needed for metastatic colorectal cancer patients that are eligible to receive regorafenib. Here, we measured plasma levels of ten circulating microRNAs (c-miRNAs) and we investigated their early changes during treatment, as well as possible correlation with clinical outcome. Ten literature-selected c-miRNAs were quantified by qRT-PCR on plasma samples collected at baseline (d1) and after 15 days of treatment (d15). C-miRNAs showing significant changes were further analyzed to establish correlations with outcome. A decision tree-based approach was employed to define a c-miRNA signature able to predict the outcome. Results achieved in an exploratory cohort were tested in a validation group. In the exploratory cohort (n = 34), the levels of c-miR-21 (p = 0.06), c-miR-141 (p = 0.04), and c-miR-601 (p = 0.01) increased at d15 compared with d1. A c-miRNA signature involving c-miR-21, c-miR-221, and c-miR-760 predicted response to treatment (p < 0.0001) and was significantly associated to PFS (HR = 10.68; 95% CI 3.2-35.65; p < 0.0001). In the validation cohort (n = 36), the increase in c-miR-21 (p = 0.02) and c-miR-601 (p = 0.02) levels at d15 was confirmed, but the associations with outcome were not. Our data indicate that early changes of c-miRNA levels might be influenced by regorafenib treatment. However, further studies are needed to establish the predictive power of such modifications.

Altered Metabolic Profile and Adipocyte Insulin Resistance Mark Severe Liver Fibrosis in Patients with Chronic Liver Disease.

Metabolomics/lipidomics are important tools to identify novel biomarkers associated with liver damage. Patients with chronic liver disease (CLD) and hepatitis C virus (HCV) infection often have alterations in glucose, lipid and protein metabolism. The aim of this study was to evaluate if dysfunctional lipid and amino acid metabolism was associated with fibrosis severity and insulin resistance in CLD/HCV patients. We analyzed the baseline sera of 75 subjects with CLD/HCV infection HCV genotype-1, with proven liver biopsy prior to antiviral treatment. We measured amino acid (AA) and lipid concentration by gas and liquid chromatography-mass spectrometry respectively. Alterations in peripheral glucose metabolism due to insulin resistance (IR) were assesed by HOMA-IR (Glucose x Insulin/22.5), while adipose tissue IR was estimated as (Adipo-IR = Free Fatty Acids x Insulin). Baseline HOMA-IR and Adipo-IR were related to the degree of liver fibrosis. Reduction in ceramides 18:1/22:0, 18:1/24:0, diacylglycerol 42:6 and increased phosphocholine 40:6 were associated with higher fibrosis. Adipo-IR was related to lower levels of lysophosphatidylcholine 14:0 and 18:2 and with higher levels of sphingomyelin 18:2/24:0 and 18:2/24:1. Almost all AA were positively associated with Adipo-IR but not with HOMA-IR. We further confirmed the potential use of metabolomics and lipidomics in CLD/HCV subjects finding novel biomarkers of hepatic fibrosis and show that the adipose tissue IR is associated with more severe liver disease and is an important marker not only of altered lipid but also AA metabolism.

Activation of the interferon type I response rather than autophagy contributes to myogenesis inhibition in congenital DM1 myoblasts.

Congenital myotonic dystrophy type 1 (CDM1) is characterized by severe symptoms that affect patients from birth, with 40% mortality in the neonatal period and impaired skeletal muscle development. In this paper, we examined the relationship between autophagy and abnormal myogenic differentiation of CDM1 myoblasts. We investigated these pathological features at both ultrastructural and molecular levels, utilizing two CDM1 foetal myoblasts, CDM13 and CDM15, with 1800 and 3200 repeats, respectively. The congenital nature of these CDM1 myoblasts was confirmed by the high methylation level at the DMPK locus. Our results indicated that abnormal autophagy was independent of myogenic differentiation, as CDM13 myoblasts differentiated as well as control myoblasts but underwent autophagy like CDM15, displaying impaired differentiation. miRNA expression profiles revealed that CDM15 myoblasts failed to upregulate the complex network of myo-miRNAs under MYOD and MEF2A control, while this network was upregulated in CDM13 myoblasts. Interestingly, the abnormal differentiation of CDM15 myoblasts was associated with cellular stress accompanied by the induction of the interferon type 1 pathway (innate immune response). Indeed, inhibition of the interferon (IFN) type I pathway restores myogenic differentiation of CDM15 myoblasts, suggesting that the inappropriate activation of the innate immune response might contribute to impaired myogenic differentiation and severe muscle symptoms observed in some CDM1 patients. These findings open up the possibility of new therapeutic approaches to treat CDM1.