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Context Obesity is a major health problem associated with severe comorbidities, including type-2 diabetes and cancer, wherein microRNAs might be useful as diagnostic/prognostic tools or therapeutic targets. Objective To explore the differential expression pattern of microRNAs in obesity and their putative role in obesity-related comorbidities such as insulin resistance. Methods An Affymetrix-miRNA array was performed in plasma samples from normoweight (n=4/BMI&25) and obese subjects (n=4/BMI>30). The main changes were validated in two independent cohorts (n=221/n=18). Additionally, in silico approaches were performed and in vitro assays applied in tissue samples and prostate (RWPE-1) and liver (HepG2) cell-lines. Results 26 microRNAs were altered (p&0.01) in plasma of obese subjects compared to controls using the Affymetrix-miRNA array. Validation in ampler cohorts revealed that miR-4454 levels were consistently higher in obesity, associated with insulin-resistance (HOMA-IR/insulin) and modulated by medical (metformin/statins) and surgical (bariatric surgery) strategies. miR-4454 was highly expressed in prostate and liver tissues and its expression was increased in prostate and liver cells by insulin. In vitro, overexpression of miR-4454 in prostate cells resulted in decreased expression levels of INSR, GLUT4, and phosphorylation of AMPK/AKT/ERK, as well as in altered expression of key spliceosome components (ESRP1/ESRP2/RBM45/RNU2) and insulin-receptor splicing variants. Conclusions Obesity was associated to an alteration of the plasmatic miRNA landscape, wherein miR-4454 levels were higher, associated with insulin-resistance and modulated by obesity-controlling interventions. Insulin regulated miR-4454, which, in turn may impair the cellular response to insulin, in a cell type-dependent manner (i.e. prostate gland), by modulating the splicing process.

Introduction Altered splicing landscape is an emerging cancer hallmark; however, the dysregulation and implication of the cellular machinery controlling this process (spliceosome components and splicing factors) in hepatocellular carcinoma (HCC) is poorly known. This study aimed to comprehensively characterize the spliceosomal profile and explore its role in HCC. Methods Expression levels of 70 selected spliceosome components and splicing factors and clinical implications were evaluated in two retrospective and six in silico HCC cohorts. Functional, molecular and mechanistic studies were implemented in three cell lines (HepG2, Hep3B and SNU‐387) and preclinical Hep3B‐induced xenograft tumours. Results Spliceosomal dysregulations were consistently found in retrospective and in silico cohorts. EIF4A3, RBM3, ESRP2 and SRPK1 were the most dysregulated spliceosome elements in HCC. EIF4A3 expression was associated with decreased survival and greater recurrence. Plasma EIF4A3 levels were significantly elevated in HCC patients. In vitro EIF4A3‐silencing (or pharmacological inhibition) resulted in reduced aggressiveness, and hindered xenograft‐tumours growth in vivo, whereas EIF4A3 overexpression increased tumour aggressiveness. EIF4A3‐silencing altered the expression and splicing of key HCC‐related genes, specially FGFR4. EIF4A3‐silencing blocked the cellular response to the natural ligand of FGFR4, FGF19. Functional consequences of EIF4A3‐silencing were mediated by FGFR4 splicing as the restoration of non‐spliced FGFR4 full‐length version blunted these effects, and FGFR4 inhibition did not exert further effects in EIF4A3‐silenced cells. Conclusions Splicing machinery is strongly dysregulated in HCC, providing a source of new diagnostic, prognostic and therapeutic options in HCC. EIF4A3 is consistently elevated in HCC patients and associated with tumour aggressiveness and mortality, through the modulation of FGFR4 splicing.

Background Obesity is a metabolic-chronic disease with important associated morbidities and mortality. Bariatric-surgery is the most effective treatment for maintaining long-term weight-loss in severe obesity and consequently for decreasing obesity-related complications, including chronic inflammation. Aim To explore changes in components of the inflammasome-machinery after bariatric-surgery and their relations with clinical/biochemical-parameters at baseline and six-months after bariatric-surgery. Patients and methods 22 patients with morbid-obesity that underwent bariatric-surgery (sleeve-gastrectomy and roux-en-Y gastric bypass) were included. Epidemiological/clinical/anthropometric/biochemical evaluation was performed at baseline and six-months after bariatric-surgery. Inflammasome-components and inflammatory-associated factors [NOD-like-receptors (NLRs); inflammasome-activation-components; cytokines and inflammation/apoptosis-related components; and cell-cycle and DNA-damage regulators) were evaluated in peripheral-blood mononuclear-cells (PBMCs) at baseline and six-months after bariatric-surgery. Clinical-molecular correlations/associations were analyzed. Functional parameters (lipid-accumulation/viability/apoptosis) were analyzed in response to specific inflammasome-components silencing in liver HEPG2-cells-). Results A profound dysregulation of inflammasome-components after bariatric-surgery was found, especially in NOD-like-receptors, cell-cycle and DNA-damage regulators. Several components were associated to baseline metabolic comorbidities including type-2-diabetes (CCL2/CXCR1/SIRT1), hypertension (AIM2/ASC/P2RX7) and dyslipidemia (CXCL3/NLRP7), and displayed changes in their molecular profile six-months after bariatric-surgery. Gene-expression fingerprint of certain factors (NLRC4/NLRP12/CXCL3/CCL8/TLR4) accurately differentiated pre- and post-operative PBMCs. Most changes were independent of the performed surgical technique. Silencing of NLRC4/NLRP12- resulted in altered lipid-accumulation, apoptosis-rate and cell-viability in HEPG2-cells. Conclusion Bariatric-surgery induces a profound alteration in gene-expression pattern of components of the inflammasome-machinery in PBMCs. Expression and changes of certain inflammasome-components are associated to baseline metabolic comorbidities, including type-2-diabetes, and may be related to the improvement and reversion of some obesity-related comorbidities after bariatric-surgery.

Hepatocellular carcinoma (HCC) pathogenesis is associated with alterations in splicing machinery components (spliceosome and splicing factors) and aberrant expression of oncogenic splice variants. We aimed to analyze the expression and potential role of the spliceosome component PRPF8 (pre-mRNA processing factor 8) in HCC. PRPF8 expression (mRNA/protein) was analyzed in a retrospective cohort of HCC patients (n = 172 HCC and nontumor tissues) and validated in two in silico cohorts (TCGA and CPTAC). PRPF8 expression was silenced in liver cancer cell lines and in xenograft tumors to understand the functional and mechanistic consequences. In silico RNAseq and CLIPseq data were also analyzed. Our results indicate that PRPF8 is overexpressed in HCC and associated with increased tumor aggressiveness (patient survival, etc.), expression of HCC-related splice variants, and modulation of critical genes implicated in cancer-related pathways. PRPF8 silencing ameliorated aggressiveness in vitro and decreased tumor growth in vivo. Analysis of in silico CLIPseq data in HepG2 cells demonstrated that PRPF8 binds preferentially to exons of protein-coding genes, and RNAseq analysis showed that PRPF8 silencing alters splicing events in multiple genes. Integrated and in vitro analyses revealed that PRPF8 silencing modulates fibronectin (FN1) splicing, promoting the exclusion of exon 40.2, which is paramount for binding to integrins. Consistent with this finding, PRPF8 silencing reduced FAK/AKT phosphorylation and blunted stress fiber formation. Indeed, HepG2 and Hep3B cells exhibited a lower invasive capacity in membranes treated with conditioned medium from PRPF8-silenced cells compared to medium from scramble-treated cells. This study demonstrates that PRPF8 is overexpressed and associated with aggressiveness in HCC and plays important roles in hepatocarcinogenesis by altering FN1 splicing, FAK/AKT activation and stress fiber formation.

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The dysregulation of the splicing process has emerged as a novel hallmark of metabolic and tumor pathologies. In breast cancer (BCa), which represents the most diagnosed cancer type among women worldwide, the generation and/or dysregulation of several oncogenic splicing variants have been described. This is the case of the splicing variants of HER2, ER, BRCA1 or, the recently identified by our group, In1-ghrelin and SST5TMD4, which exhibit oncogenic roles, increasing the malignancy, poor prognosis and resistance to treatment of BCa. This altered expression of oncogenic splicing variants has been closely linked with the dysregulation of the elements belonging to the macromolecular machinery that controls the splicing process (spliceosome components and the associated splicing factors). In this review, we compile the current knowledge demonstrating the altered expression of splicing variants and spliceosomal components in breast cancer, showing the existence of a growing body of evidence supporting the close implication of the alteration in the splicing process in mammary tumorigenesis.