MicroRNAs (miRNAs) are small RNAs present in extracellular vesicles (EVs) that, when transferred to a target cell, affect its biological functions. Plant miRNAs regulate the expression of certain mammalian genes. Here, we characterized EVs in fruit and vegetable juice, and their miRNA cargo, and investigated whether such miRNA-containing EVs could be taken up by mammalian enterocytes in vitro. Using filtration and ultra-centrifugation methods, EVs were purified from commercially available and manually squeezed plant juice. EV morphological features and subcellular localization were analyzed using the NanoSight tracking system and electron microscopy. Plant EV miRNA levels were evaluated using quantitative reverse transcription PCR. For the in vitro EV uptake experiments, rat intestinal epithelial cells (IEC6) were used. Plant EVs shared morphological features with mammalian EVs and contained miR156a-5p, miR166a-3p, and miR168a-5p. EVs were present in the cell sap-filled central vacuoles and were taken up by IEC6 cells. Edible plant cells produce EVs that contain various miRNAs and release them into the central vacuole. The exogenous plant EVs are taken up by mammalian enterocytes in vitro. These findings suggest the possibility that exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract.

Non-alcoholic steatohepatitis (NASH) has pathological characteristics similar to those of alcoholic hepatitis, despite the absence of a drinking history. The greatest threat associated with NASH is its progression to cirrhosis and hepatocellular carcinoma. The pathophysiology of NASH is not fully understood to date. In this study, we investigated the pathophysiology of NASH from the perspective of glycolysis and the Warburg effect, with a particular focus on microRNA regulation in liver-specific macrophages, also known as Kupffer cells. We established NASH rat and mouse models and evaluated various parameters including the liver-to-body weight ratio, blood indexes, and histopathology. A quantitative phosphoproteomic analysis of the NASH rat model livers revealed the activation of glycolysis. Western blotting and immunohistochemistry results indicated that the expression of pyruvate kinase muscle 2 (PKM2), a rate-limiting enzyme of glycolysis, was upregulated in the liver tissues of both NASH models. Moreover, increases in PKM2 and p-PKM2 were observed in the early phase of NASH. These observations were partially induced by the downregulation of microRNA122-5p (miR-122-5p) and occurred particularly in the Kupffer cells. Our results suggest that the activation of glycolysis in Kupffer cells during NASH was partially induced by the upregulation of PKM2 via miR-122-5p suppression.

Background and Aims: Metabolic dysfunction‐associated steatotic liver disease (MASLD) affects approximately 25% of the global population and is a major health concern. Liver sinusoidal endothelial cells (LSECs) undergo morphological and functional alterations during MASLD progression, resulting in endotheliopathy characterized by pro‐inflammatory phenotypes. We hypothesize that lipotoxicity synergistically exacerbates LSEC dysfunction in MASLD via IL‐6 trans‐signaling. Methods: Human adipocytes were treated with palmitic acid (PA). Human LSECs were treated with PA and the IL‐6/soluble IL‐6 receptor (sIL‐6R) complex. Western blotting, qRT‐PCR, RNA sequencing, transwell assays, and immunofluorescence were performed to examine inflammatory responses. MASLD model mice were established using a choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) to validate the in vivo results. Alterations in LSEC morphology were examined by western blotting, histology, and electron microscopy. Results: PA promoted IL‐6 secretion from adipocytes. In LSECs, PA increased NFκB phosphorylation, whereas co‐stimulation with PA and IL‐6/sIL‐6R complex synergistically enhanced activator of transcription 3 (STAT3) phosphorylation and upregulated inflammatory cytokines, chemokines, and adhesion molecules, including VCAM1. Furthermore, RNA sequencing showed concurrent NFκB and Janus kinase (JAK)/STAT pathway activation. This synergistic effect promoted neutrophil migration and VCAM1 expression under in vitro conditions. In the MASLD mouse model, hepatic IL‐6 expression was elevated, accompanied by steatosis, increased transaminase levels, and inflammatory cell infiltration, along with increased VCAM1 expression and sinusoidal capillarization characterized by upregulated CD34 and downregulated CD32b and LYVE‐1. Conclusion: Lipotoxicity‐amplified IL‐6 trans‐signaling synergistically exacerbates LSEC endotheliopathy and promotes leukocyte recruitment and liver injury, contributing to MASLD progression and providing novel insights into its pathogenesis.

Polypyrimidine tract‐binding protein 1, an alternative splicing factor, drives cancer‐associated metabolic reprogramming by modulating pyruvate kinase M splicing. Although organ‐abundant microRNAs (miRs) regulate this protein, its role in tumor progression remains unclear. Therefore, we investigated the functional relationship between polypyrimidine tract‐binding protein 1‐associated microRNAs and this protein in pancreatic cancer to identify pancreas‐enriched microRNAs and explore polypyrimidine tract‐binding protein 1 function beyond metabolic regulation. The expression of miR‐216b‐5p, which is typically pancreas‐enriched, was significantly downregulated in clinical pancreatic cancer samples, whereas polypyrimidine tract‐binding protein 1 expression was markedly upregulated. Mechanistically, miR‐216b‐5p directly binds to the 3′ UTR of polypyrimidine tract‐binding protein 1 mRNA, thereby suppressing its expression and inhibiting cancer cell proliferation. Proteomic analysis, integrated with publicly available individual‐nucleotide resolution UV crosslinking and immunoprecipitation data, identified thymidylate synthase as a novel downstream target of polypyrimidine tract‐binding protein 1, likely regulated via splicing‐related RNA‐level mechanisms. Thymidylate synthase expression was also elevated in pancreatic cancer tissues and correlated with poor prognosis. Functional assays revealed that polypyrimidine tract‐binding protein 1 and thymidylate synthase silencing induced cell cycle arrest. Our findings uncover a previously unrecognized miR‐216b‐5p/polypyrimidine tract‐binding protein 1/thymidylate synthase regulatory axis that promotes pancreatic cancer progression and establish polypyrimidine tract‐binding protein 1 as a central molecular regulator of tumor biology beyond its metabolic role.