miR-369-3p modulates inducible nitric oxide synthase and is involved in regulation of chronic inflammatory response

Dendritic cells are the most important antigen-presenting cells that link the innate and acquired immune system. In our previous study, we identified that the upregulation of miR-369-3p suppresses the LPS-induced inflammatory response, reducing C/EBP-β, TNFα and IL-6 production. With the aim of gaining further insight into the biological function of miR-369-3p during acute inflammatory response, in the present study we identified novel gene targets of miR-369-3p and demonstrated the suppressive ability of these genes on the inflammatory dendritic cells. Bioinformatic analyses revealed that iNOS is a potential target of miR-369-3p. We demonstrated that the ectopic induction of miR-369-3p markedly reduced iNOS mRNA and protein as well as NO production. Moreover, we found that the upregulation of miR-369-3p decreased the release of TNFα, IL-6, IL-12, IL-1α, IL-1β in response to LPS, and increased the production of anti-inflammatory cytokines such as IL-10 and IL-1RA. In addition, LPS-induced nuclear translocation of NF-kB was inhibited by miR-369-3p. Levels of miR-369-3p were decreased in human inflamed regions of human intestine obtained from IBD patients. Our results provide novel additional information on miR-369-3p as a potential core of the signaling regulating the inflammatory response. These findings suggest that miR-369-3p should be considered as a potential target for the future development of new molecular therapeutic approaches

miR-195-5p Regulates Tight Junctions Expression via Claudin-2 Downregulation in Ulcerative Colitis

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation associated with an increased intestinal permeability. Several studies have shown that microRNAs (miRNAs) are involved in the IBD pathogenesis. Here, we aimed to functionally characterize the role of miRNAs in the regulation of intestinal permeability and barrier function. We identified 18 dysregulated miRNAs in intestinal epithelial cells (IECs) from the ulcerative colitis (UC) mice model and control mice. Among them, down-regulated miR-195-5p targeted claudin-2 (CLDN2) and was involved in impaired barrier function. CLDN2 expression levels were increased in UC mice models and negatively correlated with miR-195-5p expression. We demonstrated that gain-of-function of miR-195-5p in colonic epithelial cell lines decreased the CLDN2 levels. This modulation, in turn, downregulated claudin-1 (CLDN1) expression at protein level but not that of occludin. Our data support a previously unreported role of miR-195-5p in intestinal tight junctions’ regulation and suggest a potential pharmacological target for new therapeutic approaches in IBD

Role of microRNAs in the Regulation of Dendritic Cell Generation and Function

Dendritic cells (DCs) are antigen-presenting cells with a key role in immune responses. They act as a link between the innate and adaptive systems and they can induce and maintain immunologic tolerance. DCs are subdivided into conventional and plasmacytoid DCs. These cell subsets originate from the same bone marrow precursors and their differentiation process is determined by several extrinsic and intrinsic factors, such as cytokines, transcription factors, and miRNAs. miRNAs are small non-coding RNAs that play a crucial role in modulating physiological and pathological processes mediated by DCs. miRNA deregulation affects many inflammatory conditions and diseases. The aim of this review was to underline the importance of miRNAs in inflammatory processes mediated by DCs in physiological and pathological conditions and to highlight their potential application for future therapies

The Increase of miR-195-5p Reduces Intestinal Permeability in Ulcerative Colitis, Modulating Tight Junctions’ Expression

Defects in the intestinal epithelial barrier functions characterize inflammatory conditions such as Inflammatory Bowel Disease (IBD). Overexpression of pro-inflammatory cytokines such as TNF-α, IL-1B, IL-6 and INF-γ trigger epithelial damage. These cytokines are due to upregulation of claudin-2 (CLDN2) that form a pore channel, resulting in redistribution of TJs and an alteration of barrier permeability. Recently, we demonstrated that miR-195-5p is able to regulate CLDN2 and indirectly also CLDN1 in intestinal epithelial cells. Now, we aimed to investigate the modulation of miR-195-5p on the expression of CLDN2 and other TJs under inflammatory conditions induced by TNF-α. We demonstrated that miR-195-5p also modulated the expression of CLDN2 levels after stimulation with TNF-α. In addition, we discovered the role of miR-195-5p in the integrity of the intestinal barrier and in promoting the restoration of the intestinal epithelial. Moreover, we established that replacement of miR-195-5p attenuated the colonic inflammatory response in DSS-induced, colitis and it reduced colonic permeability. In conclusion, our data revealed the role of miR-195-5p in intestinal inflammation in ulcerative colitis, suggesting a potential pharmacological target for new therapeutic approaches

miR-195-5p Regulates Tight Junctions Expression via Claudin-2 Downregulation in Ulcerative Colitis

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation associated with an increased intestinal permeability. Several studies have shown that microRNAs (miRNAs) are involved in the IBD pathogenesis. Here, we aimed to functionally characterize the role of miRNAs in the regulation of intestinal permeability and barrier function. We identified 18 dysregulated miRNAs in intestinal epithelial cells (IECs) from the ulcerative colitis (UC) mice model and control mice. Among them, down-regulated miR-195-5p targeted claudin-2 (CLDN2) and was involved in impaired barrier function. CLDN2 expression levels were increased in UC mice models and negatively correlated with miR-195-5p expression. We demonstrated that gain-of-function of miR-195-5p in colonic epithelial cell lines decreased the CLDN2 levels. This modulation, in turn, downregulated claudin-1 (CLDN1) expression at protein level but not that of occludin. Our data support a previously unreported role of miR-195-5p in intestinal tight junctions’ regulation and suggest a potential pharmacological target for new therapeutic approaches in IBD

Identification of a Novel miR-195-5p/PNN Axis in Colorectal Cancer

Pinin (PNN) is a desmosome-associated protein that reinforces the organization of keratin intermediate filaments and stabilizes the anchoring of the cytoskeleton network to the lateral surface of the plasma membrane. The aberrant expression of PNN affects the strength of cell adhesion as well as modifies the intracellular signal transduction pathways leading to the onset of CRC. In our previous studies, we characterized the role of miR-195-5p in the regulation of desmosome junctions and in CRC progression. Here, with the aim of investigating additional mechanisms related to the desmosome complex, we identified PNN as a miR-195-5p putative target. Using a public data repository, we found that PNN was a negative prognostic factor and was overexpressed in colon cancer tissues from stage 1 of the disease. Then, we assessed PNN expression in CRC tissue specimens, confirming the overexpression of PNN in tumor sections. The increase in intracellular levels of miR-195-5p revealed a significant decrease in PNN at the mRNA and protein levels. As a consequence of PNN regulation by miR-195-5p, the expression of KRT8 and KRT19, closely connected to PNN, was affected. Finally, we investigated the in vivo effect of miR-195-5p on PNN expression in the colon of AOM/DSS-treated mice. In conclusion, we have revealed a new mechanism driven by miR-195-5p in the regulation of desmosome components, suggesting a potential pharmacological target for CRC therapy

A Novel Mechanism of Immunoproteasome Regulation via miR-369-3p in Intestinal Inflammatory Response

The immunoproteasome is a multi-catalytic protein complex expressed in hematopoietic cells. Increased expression of immuno-subunits followed by increased proteasome activities is associated with the pathogenesis of IBD. Therefore, the identification of molecules that could inhibit the activities of this complex has been widely studied. microRNAs are small molecules of non-coding RNA that regulate the expression of target genes. Our purpose was to demonstrate that miR-369-3p is able to reduce the expression of the PSMB9 subunit and consequently modulate the catalytic activities of immunoproteasome. After bioinformatics prediction of the gene target of miR-369-3p, we validated its modulation on PSMB9 expression in the RAW264.7 cell line in vitro. We also found that miR-369-3p indirectly reduced the expression of other immunoproteasome subunits and that this regulation reduced the catalytic functions of the immunoproteasome. Increased levels of PSMB9 were observed in colon samples of acute IBD patients compared to the remission IBD group and control group. Our data suggest that miR-369-3p may be a future alternative therapeutic approach to several compounds currently used for the treatment of inflammatory disorders including IBD

Identification of <em>SLC15A4/PHT1</em> Gene Products Upregulation Marking the Intestinal Epithelial Monolayer of Ulcerative Colitis Patients

SLC15A4/PHT1 is an endolysosome-resident carrier of oligopeptides and histidine recently come into view as a key path marker of immune/autoimmune/inflammatory pathways in immune cells. Yet, its emerging role in inflammatory processes directly targeting the gastrointestinal epithelial layer, as in the multifactorial pathophysiology of inflammatory bowel disease (IBD), is poorly investigated. Here, the first identification of SLC15A4/PHT1 gene products in human colonic epithelium of ulcerative colitis (UC) patients is reported, showing protein primarily localized in intracellular vesicle-like compartments. Qualitative and quantitative immunohistochemical analyses of colon biopsies revealed overexpression of SLC15A4/PHT1 protein product in the epithelial layer of UC patients. Results were successfully mirrored in vitro, in spontaneously differentiated enterocyte-like monolayers of Caco-2 cells specifically exposed to DSS (dextran sodium sulphate) to mimic IBD inflammatory onsets. SLC15A4/PHT1 expression and cellular localization were characterized confirming its (dys)regulation traits in inflamed vs. healthy epithelia, strongly hinting the hypothesis of SLC15A4/PHT1 increased function associated with epithelial inflammation in IBD patients

Artificial Intelligence and Complex Network Approaches Reveal Potential Gene Biomarkers for Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and the number of cases is constantly increasing. Early and accurate HCC diagnosis is crucial to improving the effectiveness of treatment. The aim of the study is to develop a supervised learning framework based on hierarchical community detection and artificial intelligence in order to classify patients and controls using publicly available microarray data. With our methodology, we identified 20 gene communities that discriminated between healthy and cancerous samples, with an accuracy exceeding 90%. We validated the performance of these communities on an independent dataset, and with two of them, we reached an accuracy exceeding 80%. Then, we focused on two communities, selected because they were enriched with relevant biological functions, and on these we applied an explainable artificial intelligence (XAI) approach to analyze the contribution of each gene to the classification task. In conclusion, the proposed framework provides an effective methodological and quantitative tool helping to find gene communities, which may uncover pivotal mechanisms responsible for HCC and thus discover new biomarkers