miR-195-5p as Regulator of γ-Catenin and Desmosome Junctions in Colorectal Cancer.

Desmosomes play a key role in the regulation of cell adhesion and signaling. Dysregulation of the desmosome complex is associated with the loss of epithelial cell polarity and disorganized tissue architecture typical of colorectal cancer (CRC). The aim of this study was to investigate and characterize the effect of miR-195-5p on desmosomal junction regulation in CRC. In detail, we proposed to investigate the deregulation of miR-195-5p and JUP, a gene target that encodes a desmosome component in CRC patients. JUP closely interacts with desmosomal cadherins, and downstream, it regulates several intracellular transduction factors. We restored the miR-195-5p levels by transient transfection in colonic epithelial cells to examine the effects of miR-195-5p on JUP mRNA and protein expression. The JUP regulation by miR-195-5p, in turn, determined a modulation of desmosome cadherins (Desmoglein 2 and Desmocollin 2). Furthermore, we focused on whether the miR-195-5p gain of function was also able to modulate the expression of key components of Wnt signaling, such as NLK, LEF1 and Cyclin D1. In conclusion, we have identified a novel mechanism controlled by miR-195-5p in the regulation of adhesive junctions, suggesting its potential clinical relevance for future miRNA-based therapy in CRC.

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.

The oleic/palmitic acid imbalance in exosomes isolated from NAFLD patients induces necroptosis of liver cells via the elongase-6/RIP-1 pathway.

Excessive toxic lipid accumulation in hepatocytes underlies the development of non-alcoholic fatty liver disease (NAFLD), phenotypically characterized by necrosis and steato-fibrosis, whose molecular mechanism is not yet fully understood. Patients with NAFLD display an imbalanced palmitic (PA) to oleic acid (OA) ratio. Moreover, increasing experimental evidence points out a relevant involvement of the exosomal content in disease progression. Aim of the study was to highlight the PA/OA imbalance within circulating exosomes, the subsequent intracellular alterations, and the impact on NALFD. Liver cells were challenged with exosomes isolated from both healthy subjects and NAFLD patients. The exosomal PA/OA ratio was artificially modified, and biological effects were evaluated. A NAFLD-derived exosomal PA/OA imbalance impacts liver cell cycle and cell viability. OA-modified NAFLD-derived exosomes restored cellular viability and proliferation, whereas the inclusion of PA into healthy subjects-derived exosomes negatively affected cell viability. Moreover, while OA reduced the phosphorylation and activation of the necroptosis marker, Receptor-interacting protein 1 (phospho-RIP-1), PA induced the opposite outcome, alongside increased levels of stress fibers, such as vimentin and fibronectin. Administration of NAFLD-derived exosomes led to increased expression of Elongase 6 (ELOVL6), Stearoyl-CoA desaturase 1 (SCD1), Tumor necrosis factor α (TNF-α), Mixed-lineage-kinase-domain-like-protein (MLKL) and RIP-1 in the hepatocytes, comparable to mRNA levels in the hepatocytes of NAFLD patients reported in the Gene Expression Omnibus (GEO) database. Genetic and pharmacological abrogation of ELOVL6 elicited a reduced expression of downstream molecules TNF-α, phospho-RIP-1, and phospho-MLKL upon administration of NAFLD-derived exosomes. Lastly, mice fed with high-fat diet exhibited higher phospho-RIP-1 than mice fed with control diet. Targeting the Elongase 6-RIP-1 signaling pathway offers a novel therapeutic approach for the treatment of the NALFD-induced exosomal PA/OA imbalance.

Autotaxin inhibitor IOA-289 reduces gastrointestinal cancer progression in preclinical models.

BACKGROUND: Autotaxin (ATX) is a secreted enzyme that converts lysophosphatidylcholine to lysophosphatidic acid (LPA). LPA stimulates cell proliferation and migration and promotes wound repair following tissue damage. ATX levels are directly correlated with stage and grade in several human cancers. Several small molecule ATX inhibitors have been developed in recent years. IOA-289 is a potent ATX inhibitor, developed to treat cancers containing fibrosis. In this study, we tested IOA-289 treatment on different gastrointestinal tract tumor cell lines, in order to evaluate its effects on viability and motility. METHODS: To determine the effects on cell viability and proliferation of treatment with increasing concentrations of IOA-289, we used the crystal violet assay, a clonogenic assay in matrigel, and we evaluated the inhibitor's effect on formation of 3D spheroids in an in vitro model. The effect of IOA-289 on cell cycle phases was analysed with a redox dye reagent. Cell migration capacity was evaluated by wound healing assay and transwell migration assay. To evaluate the pro-apoptotic effect of the inhibitor, cells were stained with Annexin V and immunofluorescence and flow cytometry analysis were performed. An antibody array was also used, to discriminate, in various samples, the differential expression of 43 proteins involved in the apoptosis pathway. RESULTS: We found that IOA-289 is able to inhibit both growth and migration of gastrointestinal tract tumor cell lines, both in 2D (crystal violet assay) and 3D in vitro models (spheroid formation and clonogenic assay in matrigel). This effect is dose-dependent, and the drug is most effective when administered in FBS-free culture medium. The inhibitory effect on cell growth is due to a pro-apoptotic effect of IOA-289. Staining with FITC-conjugated Annexin V showed that IOA-289 induced a dose-dependent increase in fluorescence following incubation for 24 h, and apoptotic cells were also distinguished in flow cytometry using Annexin/PI staining. The antibody array shows that treatment with IOA-289 causes the increased expression of several pro-apoptotic proteins in all tested cell lines. CONCLUSIONS: These results indicate that IOA-289 may be an effective drug for the treatment of tumors of the gastrointestinal tract, particularly those characterized by a high degree of fibrosis.

Dissecting the role of the NADPH oxidase NOX4 in TGF-beta signaling in hepatocellular carcinoma.

The NADPH oxidase NOX4 has been proposed as necessary for the apoptosis induced by the Transforming Growth Factor-beta (TGF-ß) in hepatocytes and hepatocellular carcinoma (HCC) cells. However, whether NOX4 is required for TGF-ß-induced canonical (SMADs) or non-canonical signals is not fully understood yet, neither its potential involvement in other parallel actions induced by TGF-ß. In this work we have used CRISPR Cas9 technology to stable attenuate NOX4 expression in HCC cells. Results have indicated that NOX4 is required for an efficient SMAD2/3 phosphorylation in response to TGF-ß, whereas non-canonical signals, such as the phosphorylation of the Epidermal Growth Receptor or AKT, are higher in NOX4 silenced cells. TGF-ß-mediated inhibition of cell proliferation and viability is attenuated in NOX4 silenced cells, correlating with decreased response in terms of apoptosis, and maintenance of high expression of MYC and CYCLIN D1. These results would indicate that NOX4 is required for all the tumor suppressor actions of TGF-ß in HCC. However, analysis in human HCC tumors has revealed a worse prognosis for patients showing high expression of TGF-ß1-related genes concomitant with high expression of NOX4. Deepening into other tumorigenic actions of TGF-ß that may contribute to tumor progression, we found that NOX4 is also required for TGF-ß-induced migratory effects. The Epithelial-Mesenchymal transition (EMT) program does not appear to be affected by attenuation of NOX4 levels. However, TGF-ß-mediated regulation of cytoskeleton dynamics and focal adhesions require NOX4, which is necessary for TGF-ß-induced increase in the chaperone Hsp27 and correct subcellular localization of Hic-5 within focal adhesions, as well for upregulation of the metalloprotease MMP9. All these results together point to NOX4 as a key element in the whole TGF-ß signaling in HCC cells, revealing an unknown role for NOX4 as tumor promoter in HCC patients presenting activation of the TGF-ß pathway.

Downregulation of γ-Catenin by miR-195-5p Inhibits Colon Cancer Progression, Regulating Desmosome Function.

Desmosomes are essential structures for ensuring tissue functions, and their deregulation is involved in the development of colorectal cancer (CRC). JUP (γ-catenin) is a desmosome adhesion component that also acts as a signaling hub, suggesting its potential involvement in CRC progression. In this context, we recently demonstrated that miR-195-5p regulated JUP and desmosome cadherins expression. In addition, miR-195-5p gain of function indirectly modulated the expression of key effectors of the Wnt pathway involved in JUP-dependent signaling. Here, our purpose was to demonstrate the aberrant expression of miR-195-5p and JUP in CRC patients and to functionally characterize the role of miR-195-5p in the regulation of desmosome function. First, we showed that miR-195-5p was downregulated in CRC tumors compared to adjacent normal tissue. Then, we demonstrated that JUP expression was significantly increased in CRC tissues compared to adjacent normal tissues. The effects of miR-195-5p on CRC progression were assessed using in vitro transient transfection experiments and in vivo miRNA administration. Increased miR-195-5p in colonic epithelial cells strongly inhibits cell proliferation, viability, and invasion via JUP. In vivo gain of function of miR-195-5p reduced the numbers and sizes of tumors and significantly ameliorated the histopathological changes typical of CRC. In conclusion, our findings indicate a potential pharmacological target based on miR-195-5p replacement as a new therapeutic approach in CRC.

Crenigacestat blocking notch pathway reduces liver fibrosis in the surrounding ecosystem of intrahepatic CCA viaTGF-ß inhibition.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is a highly malignant tumor characterized by an intensive desmoplastic reaction due to the exaggerated presence of the extracellular (ECM) matrix components. Liver fibroblasts close to the tumor, activated by transforming growth factor (TGF)-ß1 and expressing high levels of α-smooth muscle actin (α-SMA), become cancer-associated fibroblasts (CAFs). CAFs are deputed to produce and secrete ECM components and crosstalk with cancer cells favoring tumor progression and resistance to therapy. Overexpression of Notch signaling is implicated in CCA development and growth. The study aimed to determine the effectiveness of the Notch inhibitor, Crenigacestat, on the surrounding microenvironment of iCCA. METHODS: We investigated Crenigacestat's effectiveness in a PDX model of iCCA and human primary culture of CAFs isolated from patients with iCCA. RESULTS: In silico analysis of transcriptomic profiling from PDX iCCA tissues treated with Crenigacestat highlighted "liver fibrosis" as one of the most modulated pathways. In the iCCA PDX model, Crenigacestat treatment significantly (p < 0.001) reduced peritumoral liver fibrosis. Similar results were obtained in a hydrodynamic model of iCCA. Bioinformatic prediction of the upstream regulators related to liver fibrosis in the iCCA PDX treated with Crenigacestat revealed the involvement of the TGF-ß1 pathway as a master regulator gene showing a robust connection between TGF-ß1 and Notch pathways. Consistently, drug treatment significantly (p < 0.05) reduced TGF-ß1 mRNA and protein levels in tumoral tissue. In PDX tissues, Crenigacestat remarkably inhibited TGF-ß signaling and extracellular matrix protein gene expression and reduced α-SMA expression. Furthermore, Crenigacestat synergistically increased Gemcitabine effectiveness in the iCCA PDX model. In 31 iCCA patients, TGF-ß1 and α-SMA were upregulated in the tumoral compared with peritumoral tissues. In freshly isolated CAFs from patients with iCCA, Crenigacestat significantly (p < 0.001) inhibited Notch signaling, TGF-ß1 secretion, and Smad-2 activation. Consequently, Crenigacestat also inactivated CAFs reducing (p < 0.001) α-SMA expression. Finally, CAFs treated with Crenigacestat produced less (p < 005) ECM components such as fibronectin, collagen 1A1, and collagen 1A2. CONCLUSIONS: Notch signaling inhibition reduces the peritumoral desmoplastic reaction in iCCA, blocking the TGF-ß1 canonical pathway.

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.

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.

The Tumor Microenvironment Drives Intrahepatic Cholangiocarcinoma Progression.

Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with limited therapeutic options and short overall survival. iCCA is characterized by a strong desmoplastic reaction in the surrounding ecosystem that likely affects tumoral progression. Overexpression of the Notch pathway is implicated in iCCA development and progression. Our aim was to investigate the effectiveness of Crenigacestat, a selective inhibitor of NOTCH1 signaling, against the cross-talk between cancer cells and the surrounding ecosystem in an in vivo HuCCT1-xenograft model. In the present study, a transcriptomic analysis approach, validated by Western blotting and qRT-PCR on iCCA tumor masses treated with Crenigacestat, was used to study the molecular pathways responsive to drug treatment. Our results indicate that Crenigacestat significantly inhibited NOTCH1 and HES1, whereas tumor progression was not affected. In addition, the drug triggered a strong immune response and blocked neovascularization in the tumor ecosystem of the HuCCT1-xenograft model without affecting the occurrence of fibrotic reactions. Therefore, although these data need further investigation, our observations confirm that Crenigacestat selectively targets NOTCH1 and that the desmoplastic response in iCCA likely plays a key role in both drug effectiveness and tumor progression.

Exosomal FZD-7 Expression Is Modulated by Different Lifestyle Interventions in Patients with NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a multifactorial condition characterized from hypertriglyceridemia and hepatic fat accumulation, in the absence of alcohol intake. NAFLD starts as steatosis (NAFL), and the continued injury relative to the toxic fat induces inflammation, steatohepatitis (NASH), and HCC. One of the factors determining liver degeneration during the evolution of NAFLD is a modification of Wnt/Frizzled (FZD) signaling. In particular, an inhibition of Wnt signaling and an overexpression of a specific FZD receptor protein, namely, the FZD7, have been observed in NAFLD. Actually, the prognosis and the follow-up of NAFLD is not easy, and the liver biopsy is the gold standard for an accurate detection of liver fibrosis. In this study, the modulation of the FZD7 expression levels in plasma-derived exosomes of NAFLD-affected patients, before and after specific lifestyle interventions, were experimentally evaluated by Western blotting analysis. The experimental data were analyzed by an accurate statistical study that indicated, in the exosomes derived from plasma of NAFLD patients with moderate or severe steatosis, an average expression level of FZD7 that was significantly higher than healthy subjects at baseline; conversely, the values were normalized after 90 days of specific lifestyle interventions. The overall results suggested that the FZD7 delivered by exosomes represents a good candidate as a new and effective biomarker for diagnosis and prognosis of NAFLD.

CD90 is regulated by notch1 and hallmarks a more aggressive intrahepatic cholangiocarcinoma phenotype.

BACKGROUND: Intrahepatic Cholangiocarcinoma (iCCA) is characterized by a strong stromal reaction playing a role in tumor progression. Thymus cell antigen 1 (THY1), also called Cluster of Differentiation 90 (CD90), is a key regulator of cell-cell and cell-matrix interaction. In iCCA, CD90 has been reported to be associated with a poor prognosis. In an iCCA PDX model, we recently found that CD90 was downregulated in mice treated with the Notch γ-secretase inhibitor Crenigacestat. The study aims to investigate the role of CD90 in relation to the NOTCH pathway. METHODS: THY1/CD90 gene and protein expression was evaluated in human iCCA tissues and xenograft models by qRT-PCR, immunohistochemistry, and immunofluorescence. Notch1 inhibition was achieved by siRNA. THY1/CD90 functions were investigated in xenograft models built with HuCCT1 and KKU-M213 cell lines, engineered to overexpress or knockdown THY1, respectively. RESULTS: CD90 co-localized with EPCAM, showing its epithelial origin. In vitro, NOTCH1 silencing triggered HES1 and THY1 down-regulation. RBPJ, a critical transcriptional regulator of NOTCH signaling, exhibited putative binding sites on the THY1 promoter and bound to the latter, implying CD90 as a downstream NOTCH pathway effector. In vivo, Crenigacestat suppressed iCCA growth and reduced CD90 expression in the PDX model. In the xenograft model, Crenigacestat inhibited tumor growth of HuCCT1 cells transfected to overexpress CD90 and KKU-M213 cells constitutively expressing high levels of CD90, while not affecting the growth of HuCCT1 control cells and KKU-M213 depleted of CD90. In an iCCA cohort, patients with higher expression levels of NOTCH1/HES1/THY1 displayed a significantly shorter survival. CONCLUSIONS: iCCA patients with higher NOTCH1/HES1/THY1 expression have the worst prognosis, but they are more likely to benefit from Notch signaling inhibition. These findings represent the scientific rationale for testing NOTCH1 inhibitors in clinical trials, taking the first step toward precision medicine for iCCA.

A Gene-Based Machine Learning Classifier Associated to the Colorectal Adenoma-Carcinoma Sequence.

Colorectal cancer (CRC) carcinogenesis is generally the result of the sequential mutation and deletion of various genes; this is known as the normal mucosa-adenoma-carcinoma sequence. The aim of this study was to develop a predictor-classifier during the "adenoma-carcinoma" sequence using microarray gene expression profiles of primary CRC, adenoma, and normal colon epithelial tissues. Four gene expression profiles from the Gene Expression Omnibus database, containing 465 samples (105 normal, 155 adenoma, and 205 CRC), were preprocessed to identify differentially expressed genes (DEGs) between adenoma tissue and primary CRC. The feature selection procedure, using the sequential Boruta algorithm and Stepwise Regression, determined 56 highly important genes. K-Means methods showed that, using the selected 56 DEGs, the three groups were clearly separate. The classification was performed with machine learning algorithms such as Linear Model (LM), Random Forest (RF), k-Nearest Neighbors (k-NN), and Artificial Neural Network (ANN). The best classification method in terms of accuracy (88.06 ± 0.70) and AUC (92.04 ± 0.47) was k-NN. To confirm the relevance of the predictive models, we applied the four models on a validation cohort: the k-NN model remained the best model in terms of performance, with 91.11% accuracy. Among the 56 DEGs, we identified 17 genes with an ascending or descending trend through the normal mucosa-adenoma-carcinoma sequence. Moreover, using the survival information of the TCGA database, we selected six DEGs related to patient prognosis (SCARA5, PKIB, CWH43, TEX11, METTL7A, and VEGFA). The six-gene-based classifier described in the current study could be used as a potential biomarker for the early diagnosis of CRC.

Direct and Indirect Effect of TGFß on Treg Transendothelial Recruitment in HCC Tissue Microenvironment.

The balance between anti-tumor and tumor-promoting immune cells, such as CD4+ Th1 and regulatory T cells (Tregs), respectively, is assumed to dictate the progression of hepatocellular carcinoma (HCC). The transforming growth factor beta (TGFß) markedly shapes the HCC microenvironment, regulating the activation state of multiple leukocyte subsets and driving the differentiation of cancer associated fibroblasts (CAFs). The fibrotic (desmoplastic) reaction in HCC tissue strongly depends on CAFs activity. In this study, we attempted to assess the role of TGFß on transendothelial migration of Th1-oriented and Treg-oriented CD4+ T cells via a direct or indirect, CAF-mediated mechanisms, respectively. We found that the blockage of TGFß receptor I-dependent signaling in Tregs resulted in impaired transendothelial migration (TEM) of these cells. Interestingly, the secretome of TGFß-treated CAFs inhibited the TEM of Tregs but not Th1 cells, in comparison to the secretome of untreated CAFs. In addition, we found a significant inverse correlation between alpha-SMA and FoxP3 (marker of Tregs) mRNA expression in a microarray analysis involving 78 HCCs, thus suggesting that TGFß-activated stromal cells may counteract the trafficking of Tregs into the tumor. The apparent dual behavior of TGFß as both pro- and anti-tumorigenic cytokines may add a further level of complexity to the mechanisms that regulate the interactions among cancerous, stromal, and immune cells within HCC, as well as other solid tumors, and contribute to better manipulation of the TGFß signaling as a therapeutic target in HCC patients.

Exosome Released FZD10 Increases Ki-67 Expression via Phospho-ERK1/2 in Colorectal and Gastric Cancer.

Frizzled (FZD) proteins are primary receptors for Wnt signaling that activates the mitogen-activated protein kinase (MAPK) pathways. Dysfunction of Wnt signals with consequently abnormal activation of MAPK3 pathways was found in colorectal cancer (CRC) and gastric cancer (GC). Upregulation of FZD10 protein, localized in the exosomes isolated from plasma of CRC and GC patients, was associated with a poor prognosis. Herein, the expression levels of circulating FZD10 were found to be strongly correlated to their expression levels in the corresponding tissues in CRC and GC patients. Bioinformatic prediction revealed a link between FZD10 and Ki-67 through MAPK3. In both CRC and GC tissues, pERK1/2 levels were significantly increased at more advanced disease stages, and pERK1/2 and Ki-67 were correlated. Silencing of FZD10 in CRC and GC cells resulted in a significant reduction of pERK1/2 and Ki-67 expression, while subsequent treatment with exogenous exosomes partially restored their expression levels. The strong correlation between the expression of Ki-67 in tissues and of FZD10 in exosomes suggests that the exosome-delivered FZD10 may be a promising novel prognostic and diagnostic biomarker for CRC and GC.

Assessment and management of undifferentiated carcinoma with osteoclastic like giant cells of the pancreas: a case report and revision of literature.

BACKGROUND: Undifferentiated carcinoma with osteoclast-like giant cells (UCOGCs) is a rare and aggressive non endocrine pancreatic carcinoma characterized by the presence of osteoclastic giant cells mixed with mononuclear cell. Very few cases have been reported in the literature and the histogenesis is controversial as, at the time of diagnosis, the tumor is often of advanced size and stage and it is difficult to pathologically observe its relationship with the pancreatic duct. CASE PRESENTATION: We present a case of 65-year-old male patient presenting with abdominal pain, nausea, and weight loss, which was treated with surgical resection. Histological examination revealed an undifferentiated pancreatic carcinoma with osteoclast-like giant cells. The patient underwent to a routine pylorus preserving pancreatoduodenectomy. Actually, the patient was in good performance status and disease-free five months. CONCLUSIONS: Based on the present case and limited previous data, further researches preferably with large cohorts are necessary to clarify the pathogenesis of the neoplasm. However, as show in this case, histopathological and immunohistochemically studies are the gold standard for the diagnosis of UCPOGC. Investigation of the genomic alterations in UPOGCs could help to explain the histologic diversity of variant tumor and could provide a genetic basis for prognosis and treatment.

Serum Levels of miR-148b and Let-7b at Diagnosis May Have Important Impact in the Response to Treatment and Long-Term Outcome in IgA Nephropathy.

BACKGROUND/AIMS: Previous studies showed that two microRNAs, let-7b and miR-148, which regulate the O-glycosylation process of IgA1, may predict diagnosis of primary IgA nephropathy (IgAN). The combined analysis of their serum levels in calculated statistical models may act as serum biomarkers for the diagnosis of primary IgAN. In the present study, we aimed to assess their impact not only on clinical and histological findings at onset but also on renal function after a long-term follow-up. PATIENTS AND METHODS: We enrolled 61 Caucasian patients with biopsy-proven IgAN. Serum levels of miR-148b, let-7b, and galactose-deficient IgA1 (Gd-IgA1) at the time of diagnosis were measured using real-time quantitative PCR and enzyme-linked immunosorbent assay using the monoclonal antibody KM55, respectively. Their values along with calculated Models 1 and 2 were correlated with histologic scoring system (Oxford classification system) and with renal function at diagnosis and after 11.9 ± 6.6 years. Fifty-five healthy volunteers were enrolled as controls. RESULTS: No significant correlation was found between miRNA and Gd-IgA1 levels and eGFR and proteinuria at diagnosis. A significant negative association was detected between the presence of crescents and serum levels of let-7b (p = 0.002), miR-148b (p = 0.01), and Models 1 and 2 (p = 0.02 and p = 0.007, respectively). At the end of follow-up, eGFR correlated with let-7b levels (p = 0.01), Model 1 (p = 0.002), and Model 2 (p = 0.004). Patients with fast progression of the renal damage had significantly increased levels of let-7b (p = 0.01), Model 1 (p = 0.003), and Model 2 (p = 0.005) compared to slow progressors, as did those who reached ESKD (p = 0.002, p = 0.001, and p = 0.001, respectively). Results were most prominent in those treated with corticosteroids. Finally, cut off levels in Models 1 and 2 could also predict the renal function outcome after long-term follow-up. CONCLUSIONS: Serum levels of let-7b and miR-148b and their combination, may serve as predictors for long-term renal function outcomes, particularly in patients treated with corticosteroids.

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-ß, 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-1ß 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.