Biology subjects

Licata, G.

Publications and source records attributed to Licata, G..

6 recordsLinked to original sources

bioRxiv2023

Comprehensive IsomiR sequencing profile of human pancreatic islets and EndoC-βH1 beta-cells

Aims/HypothesisMiRNAs play a crucial role in regulating the islet transcriptome, influencing beta cell functions and pathways. Emerging evidence suggests that during biogenesis a single miRNA locus can generate various sequences, known as isomiRNAs (isomiRs). However, a comprehensive profiling analysis of isomiRs in human pancreatic islets and beta cells is still lacking. This study aims to unveil the isomiRs expression profile in Laser Capture Microdissected (LCM) human pancreatic islets (HI) from non-diabetic donors and in the human beta cell line EndoC-{beta}H1, in order to shed light on novel molecular mechanisms governing beta cell function. MethodsRNA was extracted from LCM HI deriving from n=19 non-diabetic donors and from EndoC-{beta}H1 beta cells. Small RNA-seq was performed. Data were processed with the sRNAbench online pipeline for miRNAs/isomiRs quantification. Results were further validated using an external miRNA-seq database (isomiRdb). ResultsIn both HI and EndoC-{beta}H1, isomiRs accounted for a substantial proportion of total miRNA reads (HI: 59.4+/-1.9%; EndoC-{beta}H1: 43.8+/-0.6%). Among isomiRs, the most prevalent types were 3-end modifications, including trimming (HI=71.8+/-2.8%; EndoC-{beta}H1=55.8+/-1.0%) and extension (HI: 12.1+/-1.9%; EndoC-{beta}H1: 17.4+/-0.9%), followed by non-templated addition (HI: 9.8+/-0.9%; EndoC-{beta}H1: 14.0+/-1.2%). The analysis of the composition of the n=10 most expressed miRNAs highlighted a significant contribution of reads assigned to isomiRs. For instance, the most abundant miRNA, miR-375-3p, resulted from 59.7+/-2.4% of canonical and 40.3+/-2.4% of isomiRs in EndoC-{beta}H1 and from 45.3+/-2.0% of canonical and 54.7+/-2.0% of isomiR reads in HI. Interestingly, miR-7-5p, a beta cell-specific miRNA, was predominantly expressed as an isomiR both in EndoC-{beta}H1 (65.3+/-2.7%) and in HI (82.4+/-1.4%). To identify a reliable beta cell isomiR signature, common sequences detected in HI and EndoC-{beta}H1 were filtered based on their contribution to total miRNA expression, ultimately resulting in a set of 46 isomiRs. The expression of the isomiR signature in beta cells was further evaluated using an external database, isomiRdb, which contains small-RNA sequencing data from 99 different human cell types. This analysis revealed a significant enrichment of 11 out of the 46 isomiRs in beta cells compared to other cell types. The signature was functionally characterized through regression analysis with clinical and metabolic parameters related to beta cell function in non-diabetic individuals, demonstrating a significant negative correlation between basal insulin secretion and isomiR-411-5p, but not with its corresponding canonical miRNA. Conclusion/InterpretationThis study provides a comprehensive profile of isomiR expression in pancreatic islets and beta cells, highlighting the potential significance of isomiRs as novel regulators of beta cell function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/566223v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@145f36borg.highwire.dtl.DTLVardef@1701433org.highwire.dtl.DTLVardef@13e5866org.highwire.dtl.DTLVardef@1dbe50_HPS_FORMAT_FIGEXP M_FIG C_FIG Research in ContextO_ST_ABSWhat is already know about this subject?C_ST_ABS- isomiRs are sequence variants of microRNAs (miRNAs) and may have distinct functional role respect to the canonical sequence. - isomiRs show cell/tissue specificity and are involved in multiple disease contexts. What is the key question?- What is the profile of isomiRs in human pancreatic islets (HI) and in beta cells? - Do isomiRs have a functional role in beta cells? What are the new findings?- IsomiRs represent a relevant fraction of total miRNAs in HI and beta cells. - 3end miRNA sequence modifications are the major fraction of isomiRs in beta cells. - A set of n=11 isomiRs, more expressed than their canonical miRNAs, are enriched in beta cells compared to the other human cell types. - Specific isomiRs are associated with beta cell glucose sensitivity and basal insulin secretion. How might this impact on clinical practice in the foreseeable future?- A comprehensive profile of isomiRs in beta cells may improve our understanding of molecular mechanisms driving beta cell function and dysfunction. - A highly detailed and granular view of miRNAs sequence variants and their expression levels may help in the design of novel therapeutic RNA-based strategies.

molecular biology↗

bioRxiv2021

Increased expression of viral sensor MDA5 in pancreatic islets and in hormone-negative endocrine cells in recent onset type 1 diabetic donors

The interaction between genetic and environmental factors determines the development of type 1 diabetes (T1D). Some viruses are capable of infecting and damaging pancreatic {beta}-cells, whose antiviral response could be modulated by specific viral RNA receptors and sensors such as Melanoma Differentiation Associated gene 5 (MDA5), encoded by the IFIH1 gene. MDA5 has been shown to be involved in pro-inflammatory and immunoregulatory outcomes, thus determining the response of pancreatic islets to viral infections. Although the function of MDA5 has been previously well explored, a detailed immunohistochemical characterization of MDA5 in pancreatic tissues of non-diabetic and T1D donors is still missing. In the present study we used multiplex immunofluorescence imaging analysis to characterize MDA5 expression and distribution in pancreatic tissues obtained from 22 organ donors (10 non-diabetic autoantibody-negative, 2 autoantibody-positive, 8 recent-onset and 2 long-standing T1D). In non-diabetic control donors, MDA5 was expressed both in - and in {beta}-cells. The colocalization rate imaging analysis showed that MDA5 was preferentially expressed in -cells. In T1D donors, we observed an increased colocalization rate MDA5-glucagon respect to MDA5-insulin in comparison to non-diabetic controls; such increase was more pronounced in recent onset respect to long standing T1D donors. Of note, an increased colocalization rate MDA5-glucagon was found in insulin-deficient-islets (IDI) respect to insulin containing islets (ICI). Strikingly, in T1D donors we detected the presence of MDA5-positive/hormones-negative endocrine islet-like clusters, putatively deriving from dedifferentiation or neogenesis phoenomena. These clusters were exclusively identified in recent onset donors and not detected in autoantibody-positive non-diabetic or T1D long-standing ones. In conclusion, we showed that MDA5 is preferentially expressed in -cells and its expression is increased in recent onset T1D donors. Finally, we observed that MDA5 may also characterize the phenotype of dedifferentiated or newly forming islet cells, thus opening to novel roles for MDA5 in pancreatic endocrine cells.

immunology↗

bioRxiv2021

Outer Membrane Vesicles from the gut microbiome contribute to tumor immunity by eliciting cross-reactive T cells

The gut microbiome plays a key role in cancer immunity. One proposed mechanism is through the elicitation of T cells, which incidentally recognize neo-epitopes arising from cancer mutations ("molecular mimicry (MM)" hypothesis). To support MM, Escherichia coli Nissle was engineered with the SIINFEKL epitope (OVA) and orally administered to C57BL/6 mice. The treatment elicited OVA-specific CD8+ T cells in the lamina propria and inhibited the growth of OVA-B16F10 tumors. Importantly, the administration of Outer Membrane Vesicles (OMVs) engineered with different T cell epitopes elicited epitope-specific T cells and inhibited tumor growth. Microbiome shotgun sequencing and TCR sequencing provided evidence that cross-reacting T cells were induced at the mucosal level and subsequently reached the tumor site. Overall, our data support the role of MM in tumor immunity, assign a new role to OMVs and pave the way to new probiotics/OMV-based anti-cancer immunotherapies.

microbiology↗

bioRxiv2021

Reduced miR-184-3p expression occurring in Type 2 diabetic pancreatic islets protects β-cells from lipotoxic and proinflammatory apoptosis via a CRTC1-dependent mechanism

Loss of functional {beta}-cell mass in Type 2 diabetes (T2D) involves molecular mechanisms including {beta}-cell apoptosis, dysfunction, and/or dedifferentiation. MicroRNA miR-184-3p has been demonstrated to be involved in multiple {beta}-cell functions including insulin secretion, proliferation and survival. However, downstream targets and upstream regulators of miR-184-3p have not yet been fully elucidated. Here, we showed that levels of miR-184-3p are reduced in human T2D pancreatic islets and that its reduction protected {beta}-cells from lipotoxic- and inflammatory-induced apoptosis. Interestingly, CREB-Transcriptional Coactivator-1 (CRTC1) is a direct target of miR-184-3p and indeed its expression is upregulated in human T2D pancreatic islets. The downregulation of miR-184-3p in {beta}-cells induced the upregulation of CRTC1 both at mRNA and protein level. Of note, miR-184-3p protection effect was dependent on CRTC1, since its silencing in human {beta}-cells abrogates the protective mechanism exerted by miR-184-3p inhibition. Additionally, we found that the {beta}-cell specific transcription factor NKX6.1, whose DNA binding sites were predicted to be present in human and mouse MIR184 gene promoter sequence, was reduced in T2D human pancreatic islets, in line with miR-184-3p downregulation, and was positively correlated with microRNA expression. Using chromatin immunoprecipitation analysis and mRNA silencing experiments, we demonstrated that NKX6.1 directly controls both human and murine miR-184 expression. In conclusion, we found that miR-184-3p expression is controlled by the {beta}-cell specific transcription factor NKX6.1 and that miR-184-3p reduction protects {beta}-cells from apoptosis through the upregulation of its target gene CRTC1.

cell biology↗

bioRxiv2020

SARS-CoV-2 receptor Angiotensin I-Converting Enzyme type 2 is expressed in human pancreatic islet β-cells and is upregulated by inflammatory stress

Increasing evidence demonstrated that the expression of Angiotensin I-Converting Enzyme type 2 (ACE2), is a necessary step for SARS-CoV-2 infection permissiveness. In the light of the recent data highlighting an association between COVID-19 and diabetes, a detailed analysis aimed at evaluating ACE2 expression pattern distribution in human pancreas is still lacking. Here, we took advantage of INNODIA network EUnPOD biobank collection to thoroughly analyse ACE2, both at mRNA and protein level, in multiple human pancreatic tissues and using several methodologies. Using multiple reagents and antibodies, we showed that ACE2 is expressed in human pancreatic islets, where it is preferentially expressed in subsets of insulin producing {beta}-cells. ACE2 is also is highly expressed in pancreas microvasculature pericytes and moderately expressed in rare scattered ductal cells. By using different ACE2 antibodies we showed that a recently described short-ACE2 isoform is also prevalently expressed in human {beta}-cells. Finally, using RT-qPCR, RNA-seq and High-Content imaging screening analysis, we demonstrated that pro-inflammatory cytokines, but not palmitate, increases ACE2 expression in the {beta}-cell line EndoC-{beta}H1 and in primary human pancreatic islets. Taken together, our data indicate a potential link between SARS-CoV-2 and diabetes through putative infection of pancreatic microvasculature and/or ductal cells and/or through direct {beta}-cell virus tropism.

cell biology↗

bioRxiv2019

Glutamine Synthetase-1 induces autophagy-lysosomal degradation of Huntingtin aggregates and ameliorates animal motility in a Drosophila model for HD

Glutamine Synthetase1 (GS1) is an enzyme that catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia as a key element of the glutamate glutamine cycle, a complex physiological process occurring between glia and neurons, necessary to control the homeostasis of glutamate. Using a Drosophila model for Huntingtons disease, we report that expression of GS1 in neurons ameliorates the motility defects of animals expressing the mutant Httex1-Q93 form of the huntingtin gene. At the cellular level, expression of GS1 increases the basal level of autophagy and significantly reduces the size of the toxic Htt-Q93 protein aggregates. In addition, we found that expression of GS1 prevents TOR localization at the lysosomal membrane and reduction in the phosphorylation of its effector S6K. This study reveals a novel function for GS1 in neurons linking its activity to the inhibition of TOR signaling and autophagy. The identification of novel pharmacological regulators of autophagy is of particular interest considering its beneficial role in controlling neuronal health and counteracting the detrimental effects of toxic aggregates of proteinopathies including Huntingtons disease.

cell biology↗