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PubMed2026

Engineering extracellular vesicles for targeted siRNA delivery: Advances, therapeutic applications, and clinical translation.

Small interfering RNA (siRNA) therapeutics have emerged as a transformative approach for sequence-specific gene silencing, offering the potential to treat a broad spectrum of diseases by selectively suppressing disease-associated genes. However, the clinical translation of siRNA remains limited by rapid enzymatic degradation, poor cellular uptake, inadequate endosomal escape, and off-target effects, necessitating the development of efficient delivery systems. Extracellular vesicles (EVs) have gained considerable attention as natural nanocarriers owing to their excellent biocompatibility, low immunogenicity, intrinsic targeting capability, and ability to protect therapeutic cargo while traversing complex biological barriers. This review comprehensively discusses the biological characteristics of EVs, the molecular basis of RNA interference, and the major challenges associated with siRNA delivery [Fig. 1]. Recent advances in EV engineering, including cargo-loading strategies such as electroporation, sonication, extrusion, parent-cell engineering, and microfluidic approaches, together with surface functionalization using peptides, antibodies, aptamers, and hybrid nanoplatforms, are critically evaluated for improving targeting specificity and intracellular delivery. Furthermore, the therapeutic applications of engineered EV-mediated siRNA delivery in cancer, neurological disorders, liver diseases, cardiovascular diseases, inflammatory disorders, and infectious diseases are systematically summarized, highlighting their potential to enhance gene silencing while minimizing systemic toxicity. Current challenges related to large-scale manufacturing, cargo-loading efficiency, standardization, quality control, regulatory approval, and clinical translation are also discussed, together with emerging technologies involving synthetic biology, genome engineering, artificial intelligence, and multifunctional hybrid vesicles. Overall, engineered extracellular vesicles represent a highly versatile and biologically inspired platform for targeted siRNA delivery, providing a promising foundation for the development of next-generation precision RNA therapeutics and accelerating the clinical translation of gene-silencing strategies.

Extracellular vesicle engineering↗
PubMed2025

Global siRNA screen identifies human host factors critical for SARS-CoV-2 replication and late stages of infection.

Defining the subset of cellular factors governing SARS-CoV-2 replication can provide critical insights into viral pathogenesis and identify targets for host-directed antiviral therapies. While a number of genetic screens have previously reported SARS-CoV-2 host dependency factors, most of these approaches relied on utilizing pooled genome-scale CRISPR libraries, which are biased toward the discovery of host proteins impacting early stages of viral replication. To identify host factors involved throughout the SARS-CoV-2 infectious cycle, we conducted an arrayed genome-scale siRNA screen. Resulting data were integrated with published functional screens and proteomics data to reveal (i) common pathways that were identified in all OMICs datasets-including regulation of Wnt signaling and gap junctions, (ii) pathways uniquely identified in this screen-including NADH oxidation, or (iii) pathways supported by this screen and proteomics data but not published functional screens-including arachionate production and MAPK signaling. The identified proviral host factors were mapped into the SARS-CoV-2 infectious cycle, including 32 proteins that were determined to impact viral replication and 27 impacting late stages of infection, respectively. Additionally, a subset of proteins was tested across other coronaviruses revealing a subset of proviral factors that were conserved across pandemic SARS-CoV-2, epidemic SARS-CoV-1 and MERS-CoV, and the seasonal coronavirus OC43-CoV. Further studies illuminated a role for the heparan sulfate proteoglycan perlecan in SARS-CoV-2 viral entry and found that inhibition of the non-canonical NF-kB pathway through targeting of BIRC2 restricts SARS-CoV-2 replication both in vitro and in vivo. These studies provide critical insight into the landscape of virus-host interactions driving SARS-CoV-2 replication as well as valuable targets for host-directed antivirals.

Humans↗
PubMed2026

A small viral protein suppresses immune amplification by two distinct mechanisms.

Diverse viral suppressors of RNA interference (RNAi) and RNA silencing (VSRs) interact directly with core protein and/or RNA components of the host RNAi pathway. However, the specific counter-defense function of any VSR biochemical activity is fully validated only when it is shown as essential for viral infection in the wild-type but not mutant hosts defective in antiviral RNAi. Here, we investigated the role of VSR activities for direct binding to small-interfering RNA duplexes (siRNA), long double-stranded RNA (dsRNA), or RNA-dependent RNA polymerase 1 (RDR1) during plant infection by wild-type and mutant cucumber mosaic virus (CMV), a positive-strand RNA virus expressing the 110-residue 2b protein as its VSR. We demonstrate that a C-terminally truncated 2b mutant (2b1-93) active in direct binding to siRNA and dsRNA, but not RDR1, was able to suppress the amplification of virus-derived siRNAs (vsiRNA) and antiviral RNAi mediated by RDR6, but not RDR1. By contrast, an N-terminally truncated 2b mutant (2b18-110) inactive in direct binding to siRNA or dsRNA was able to suppress vsiRNA amplification and antiviral RNAi mediated by RDR1, but not RDR6, and was less effective to promote systemic CMV infection and disease development than 2b1-93. Together, our results show that whereas RDR1 suppression requires direct binding of VSR-2b to RDR1, but not siRNA or dsRNA, RDR6 suppression depends on direct binding to siRNA and dsRNA, but not RDR1. Therefore, CMV, through its VSR-2b, suppresses two parallel vsiRNA amplification pathways by distinct molecular mechanisms, and this unique property may account for the unusually wide host range of CMV.IMPORTANCEHost amplification of antiviral immunity is essential for robust control of viral infections. However, little is known about the mechanisms that viruses have evolved to suppress immune amplification in plants. Here, we characterized whole plant infection by cucumber mosaic virus (CMV) with its viral suppressor of RNA interference (RNAi) mutated to become inactive in direct binding to small-interfering RNA duplexes (siRNA), long double-stranded RNA (dsRNA), or RNA-dependent RNA polymerase 1 (RDR1). We demonstrate maximal suppression of both RDR1- and RDR6-mediated antiviral RNAi amplification by the CMV 2b protein, a viral suppressor of RNAi (VSR). Notably, whereas RDR1 suppression requires direct binding of 2b to RDR1 but not siRNA or dsRNA, RDR6 suppression depends on direct binding to siRNA and dsRNA, but not RDR1. Our findings reveal a novel counter-defense strategy evolved by a wide host range positive-strand RNA virus to suppress two pathways of immune amplification by distinct mechanisms.

Cucumovirus↗
PubMed2025

Argonaute 2 targets viral transcripts but not genomes of RNA viruses during antiviral RNA interference in Drosophila.

RNA interference (RNAi) mediated by the small interfering RNA (siRNA) pathway is a major antiviral mechanism in insects. This pathway is triggered when double-stranded RNA (dsRNA) produced during virus replication is recognized by Dicer-2, leading to the formation of virus-derived siRNA duplexes. These siRNAs are loaded onto the programmable nuclease Argonaute-2 (AGO2), with one strand serving as a guide to target and cleave fully complementary sequences of viral RNAs. While siRNAs are generated from viral dsRNA, the specific viral RNA species targeted for silencing during RNA virus replication remains unclear. In this study, we characterized the primary viral RNA targets of the Drosophila siRNA pathway during infections caused by negative and positive RNA viruses, namely Vesicular stomatitis virus (VSV) and Sindbis virus (SINV). Our findings reveal that polyadenylated transcripts of VSV and SINV are the major targets of silencing by the siRNA pathway during infection, likely when they are poised for translation. Consistent with earlier findings, we show that AGO2 is associated with ribosomes in control and virus infected cells. Therefore, we propose that the inhibition of the replication of RNA viruses in Drosophila results from the silencing of incoming viral transcripts, facilitated by the association of AGO2 with ribosomes.

Animals↗
PubMed2026

Dnmt3b and Dnmt3l knockdown reduces blastocyst development in early mouse embryos.

A one-cell embryo called a zygote develops into a blastocyst through several successive cell divisions and lineage specification, this process is called early embryo development. Both embryonic genome activation (EGA) and the first lineage specification during early embryonic development depend on tightly coordinated epigenomic organization. Regulation of the epigenome is primarily governed by DNA methylation mediated through DNA methyltransferase (Dnmt) enzymes. Dnmt1 is responsible for the maintenance of methylation during cellular division, while Dnmt3a/Dnmt3b enzymes play a role in the establishment of de novo methylation particularly during gametogenesis and early embryo development. Despite its lack of catalytic activity, Dnmt3l functions as a cofactor enhancing Dnmt3a/3b activity. Dnmt3b deficiency results in global hypomethylation and ultimately embryonic lethality. In this study, we aim to elucidate the effect of Dnmt3b and Dnmt3l silencing on early embryo development. For this purpose, our experimental groups were established using an in vitro mouse embryo development model: control, Dnmt3b small interfering RNA (siRNA), Dnmt3l siRNA, and a nontargeting siRNA group. Following gene silencing at the one-cell stage, embryonic developmental competence, the expression pattern of nonsilenced Dnmt enzymes, global DNA methylation levels, and transcriptome profiles were analyzed at the blastocyst stage. Dnmt3b/3l silencing resulted in decreased global DNA methylation and Dnmt1/3a expression, and reduced blastocyst rate. Differentially expressed genes included those involved in X-chromosome inactivation (Xist), transcriptional regulation (Rn7sk), translation (Eef1a1, Eef2), trophoblast development (Hsd3b1), compaction (Gja1), and oxidative phosphorylation (CYTB, COX1, mt-Rnr1). Our findings indicate that siRNA-mediated knockdown of Dnmt3b and Dnmt3l is associated with reduced blastocyst development, impaired embryo quality, and alterations in DNA methylation-related processes during early embryonic development.

Animals↗
PubMed2026

Systematic identification of germ granule proteins reveals specialized roles in RNAi and small RNA inheritance.

Biomolecular condensates, such as germ granules, organize RNAi pathways critical for fertility and genome regulation. However, the protein composition and functional contributions of these condensates remain poorly defined. Here, we applied TurboID proximity labeling to the Caenorhabditis elegans germ granule protein SIMR-1, integrating mass spectrometry with genetic screening, CRISPR-based tagging, and small RNA sequencing. This systematic approach identified several previously uncharacterized germ granule proteins that contribute to fertility, germline immortality, exogenous RNAi, and transgenerational inheritance. Small RNA sequencing of 21 mutants revealed broad and class-specific defects in siRNA and miRNA biogenesis, with distinct factors associated with defects in WAGO-class 22G-RNAs, CSR-class 22G-RNAs, or histone-directed small RNAs. Among these, we identified PINT-1, a highly disordered protein that directly interacts with and is recruited to germ granules by the PIWI Argonaute PRG-1. PINT-1 is required for piRNA-dependent and -independent secondary siRNA biogenesis and germline development. Comparative genomics revealed that PINT-1 has coevolved with PRG-1 across clade V nematodes, with a conserved structured N terminus and a rapidly diverging repeat-rich intrinsically disordered region. Together, our findings expand the germ granule proteome and reveal how distinct condensate components contribute to specialized functions within the small RNA pathways, while highlighting an evolutionarily coadapted PIWI interactor critical for siRNA biogenesis.

Animals↗
PubMed2026

Transcriptomic and metabolomic analyses revealed the action mechanism of nesfatin-1 gene on glucolipid metabolism during early development stage of largemouth bass.

Nesfatin-1 has biological roles including the suppression of food intake and the regulation of glucose and lipid metabolism. However, the information available regarding nesfatin-1 in the glycolipid metabolism in the early development stage of fish is still limited. In order to investigate the role of the nesfatin-1 gene in the early development stage of the largemouth bass (Micropterus salmoides), the nesfatin-1 gene was knocked down using siRNA interference technology. Then, we evaluated its mRNA expression levels, transcriptomes and metabolomes. The mRNA expression levels of nesfatin-1 gene were appreciably decreased at 48 h, 72 h and 96 h after injection of nesfatin-1 siRNA in the early development stage. The omics results revealed that knockdown of the nesfatin-1 gene induced 1833 differentially expressed genes (DEGs) and 2370 differentially expressed metabolites (DEMs). Bioinformatic analysis enriched the most affected molecular pathways (sphingolipid metabolism, fatty acid elongation, amino sugar and nucleotide sugar metabolism and biosynthesis of unsaturated fatty acids) and metabolic pathways (biosynthesis of unsaturated fatty acids, sphingolipid metabolism and amino sugar and nucleotide sugar metabolism) in early development stage of largemouth bass. In amino sugar and nucleotide sugar metabolism, increased expression levels of genes such as chic, chs1, and gck genes, alongside decreased expression levels of the chia.1 gene, resulted in significantly elevated concentrations of N-Acetyl-D-glucosamine, β-d-fructose 6-phosphate, β-d-Fructose, D-mannose 6-phosphate, d-glucose, d-glucose 1-phosphate, UDP-glucose, and UDP-glucuronate, whilst the concentration of UDP-N-acetyl-α-D-glucosamine was markedly reduced. Therefore, the nesfatin-1 gene may influence the early development stage of largemouth bass by affecting signaling pathways associated with glycolipid metabolism. Our findings further expand the understanding of molecular mechanisms of the nesfatin-1 gene, and provide further theoretical support for the initial breeding and feed adaptation of largemouth bass.

Animals↗
PubMed2026

Emerging Nucleic Acid-Based Therapies for Hypercholesterolemia with Focus on a New Modality, Liver-Directed miR-30c Analog C2.

Despite major advances in lipid-lowering therapies, a significant unmet need remains, particularly for patients with homozygous familial hypercholesterolemia (HoFH), severe heterozygous familial hypercholesterolemia (HeFH), and those who fail to achieve guideline-recommended LDL-C targets. Nucleic acid-based therapeutics have emerged as a transformative approach for treating hypercholesterolemia. Antisense oligonucleotides and small interfering RNAs (siRNAs) have demonstrated durable hepatic gene silencing and have led to approved therapies, while gene replacement and in vivo genome-editing strategies offer the potential for long-lasting, and possibly one-time, interventions. In parallel, microRNAs (miRNAs) have attracted increasing interest because of their ability to coordinately regulate multiple genes involved in lipoprotein metabolism, cholesterol transport, and lipid homeostasis. Human genetic studies further support the importance of miRNA-mediated regulation, exemplified by a rare ~2.5 kb deletion in the distal LDLR 3'UTR ("del2.5") that disrupts miRNA-binding sites and is associated with lifelong low LDL-C levels. This review summarizes recent advances, mechanisms of action, clinical progress, and remaining challenges across antisense oligonucleotides, siRNAs, gene therapy, genome editing, and emerging miRNA-based therapeutics for hypercholesterolemia. As an example of the latter approach, the liver-directed miR-30c analog C2 has demonstrated preclinical activity by coordinately reducing hepatic lipoprotein secretion and lipogenesis while enhancing cholesterol elimination, resulting in reduced LDL-C and atherosclerosis. However, it must be noted that these findings remain preclinical, and further optimization of delivery, pharmacokinetics, safety, and long-term efficacy will be required before clinical evaluation. Continued advances in RNA chemistry, targeted delivery, and genome engineering are expected to further expand the therapeutic landscape for dyslipidemia and cardiovascular disease.

Humans↗
PubMed2025

MED12-STAT1-TAP2 axis regulates CD8 + T cell cytotoxicity and mediates immunotherapy outcome in non-small cell lung cancer.

Although immunotherapy for late-stage non-small cell lung carcinoma (NSCLC) has been clinically utilized, its prognosis remains highly heterogeneous, prompting us to investigate novel predictive immunotherapy biomarkers for NSCLC. We analyzed the correlations between MED12 nonsynonymous mutations and survival, clinical, genomic, transcriptomic information, and immune infiltration information through data mining across multiple datasets. We also investigated the mechanism of MED12 using luciferase assay, Western blot, ChIP-PCR, and siRNA. MED12 is significantly associated with survival in completely independent immunotherapy datasets, including MSKCC (N = 350), Naiyer2015 (N = 34), our own (N = 295) and the pan-cancer dataset, but not in the TCGA dataset, where patients received non-immunotherapy regimens. Mutations in MED12 showed no significant correlation with known metrics (TMB, IPS/CTLA4/PD1 status, PD-1/PD-L1 expression, and TCR/BCR status) or DNA Damage Repair (DDR) pathway mutations, yet they carried independent prognostic information according to the Cox multivariate regression. On the other hand, MED12 mutation is significantly associated with multiple immune-related pathways and immune infiltration of CD8 + T cells and activated NK cells. Lactate dehydrogenase assay revealed that knockdown of TAP2 restored the upregulation of CD8 + T cell cytotoxicity triggered by MED12 knockdown. ChIP-PCR, luciferase assay and siRNA knock down assay indicate that MED12 binds to the promoter region of STAT1 to suppress its transcription, while the transcription factor STAT1 promotes the transcription of TAP2, thus inhibiting the antigen processing and presentation. Collectively, MED12 mutation is an independent and valuable biomarker for predicting the response to immune checkpoint inhibitor (ICI)therapy in NSCLC by modulating CD8 + T cell cytotoxicity via the STAT1/TAP2 axis.

Humans↗
PubMed2025

Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour.

The basolateral amygdala (BLA) serves in the evaluation of reward. However, the causal molecular substrates in the BLA necessary for reward seeking behaviour are largely unknown. Reward conditioning induces long-lasting changes in epienzymes in limbic areas, including the amygdala. The current study probed the role of histone arginine methylation as a novel epigenetic mechanism in neuropeptide Y (NPY) gene regulation in the BLA during reward and reinforcement. For reward conditioning, adult Wistar rats were trained to self-administer sucrose pellets in a nose-poke operant chamber. Reward conditioning increased protein arginine methyltransferase 4 (PRMT4) and NPY in the BLA. Moreover, after operant conditioning, histone arginine methylation (H3R17me2a) and PRMT4 occupancy at the NPY promoter were heightened. PRMT4 was predominantly colocalised in the nucleus of the NPY-expressing cells in the BLA. Intra-BLA administration of specific siRNA or inhibitor of PRMT4 after conditioning waned the nose-poke activity, which was further reinstated during the subsequent 5 days. These effects of PRMT4 repression were correlated with the NPY expression and H3R17me2a levels at the NPY promoter. Furthermore, NPY peptide administration after PRMT4 siRNA or inhibitor infusion in BLA restored the nose-poke activity. PRMT4 is known to interact with CREB-binding protein (CBP). Therefore, co-occupancy of PRMT4 and CBP resulted in heightened histone acetylation (H3K14ac) in the conditioned rats. The current study suggests a pivotal role of PRMT4-mediated histone arginine methylation in NPY gene expression in the amygdala necessary for the reward-seeking behaviour.

Animals↗
PubMed2026

Chromatin-binding protein HMGN1 promotes HCC tumorigenesis via histone methylation-induced RALB transcriptional suppression.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, with metastasis being the primary cause of its high mortality. The chromatin-binding protein, high mobility group nucleosome binding domain 1 (HMGN1), has been implicated in tumour progression, but its specific role and mechanism in HCC metastasis remain unclear. This study investigates the function of HMGN1 and its potential as a therapeutic target. Analysis of patient samples confirms an upregulation of HMGN1 in HCC tissues, correlating with advanced disease and poor prognosis. Functional assays demonstrate that HMGN1 promotes HCC metastasis in vitro and in vivo. Mechanistically, integrated RNA sequencing and chromatin immunoprecipitation sequencing analyses reveal that HMGN1 binds to the promoter of RAS-like proto-oncogene B (RALB) gene, recruiting the repressive histone mark H3K9me2 to epigenetically silence its transcription and drive metastasis. Therapeutically, a nanoparticle delivery system for siRNA against HMGN1 effectively silences its expression and inhibits metastasis in orthotopic liver xenograft tumour models. Our findings establish HMGN1 as a key epigenetic driver of HCC metastasis and highlight siRNA-nanoparticle targeting of HMGN1 as a promising precision therapeutic strategy.

Humans↗
PubMed2020

Glucocorticoids mobilize macrophages by transcriptionally up-regulating the exopeptidase DPP4.

Glucocorticoids are potent endogenous anti-inflammatory molecules, and their cognate receptor, glucocorticoid receptor (GR), is expressed in nearly all immune cells. Macrophages are heterogeneous immune cells having a central role in both tissue homeostasis and inflammation and also play a role in the pathogenesis of some inflammatory diseases. Paradoxically, glucocorticoids have only a limited efficacy in controlling the resolution of these macrophage-related diseases. Here, we report that the transcriptomes of monocyte-like THP-1 cells and macrophage-like THP-1 cells (THP1-MΦ) have largely conserved gene expression patterns. In contrast, the differentiation to THP1-MΦ significantly altered the sensitivity of gene transcription to glucocorticoids. Among glucocorticoid-regulated genes, we identified the exopeptidase dipeptidyl peptidase-4 (DPP4) as a critical glucocorticoid-responsive gene in THP1-MΦ. We found that GR directly induces DPP4 gene expression by binding to two glucocorticoid-responsive elements (GREs) within the DPP4 promoter. Additionally, we show that glucocorticoid-induced DPP4 expression is blocked by the GR antagonist RU-486 and by GR siRNA transfection and that DPP4 enzyme activity is reduced by DPP4 inhibitors. Of note, glucocorticoids highly stimulated macrophage mobility; unexpectedly, DPP4 mediated the glucocorticoid-induced macrophage migration, and siRNA-mediated knockdowns of GR and DPP4 blocked dexamethasone-induced THP1-MΦ migration. Moreover, glucocorticoid-induced DPP4 activation was also observed in proinflammatory M1-polarized murine macrophages, as well as peritoneal macrophages, and was associated with increased macrophage migration. Our results indicate that glucocorticoids directly up-regulate DPP4 expression and thereby induce migration in macrophages, potentially explaining why glucocorticoid therapy is less effective in controlling macrophage-dominated inflammatory disorders.

Animals↗
PubMed2026

CD109 exhibits a dynamic expression pattern in coronary endothelium and endocardial-derived valve mesenchyme during heart development with preserved morphogenesis following endothelial-specific deletion.

BACKGROUND: CD109 encodes a GPI-linked glycoprotein that acts as a signaling modulator in the TGF-β pathway. CD109 has emerged in several genome-wide association studies as linked to coronary artery disease, myocardial infarction, and angina pectoris. Heterozygous loss-of-function mutations in CD109 have also been reported in patients with congenital heart defects, suggesting potential developmental relevance, though CD109 has never been investigated in the context of cardiovascular development. We previously identified Cd109 upregulation in murine atrioventricular valves undergoing myxomatous degeneration following a reduction of epicardial-derived cells. Here, we characterize Cd109 expression in the murine cardiovascular system and assess its function during development using in vitro and in vivo approaches. RESULTS: We found that Cd109 is strongly expressed in the endothelium of the coronary vasculature and in endocardial-derived subpopulations in the atrioventricular valves. This expression persists through key stages in cardiovascular development. Western blotting and immunostaining confirm endothelial expression in heart and lung tissues. siRNA-mediated knockdown of CD109 in primary human endothelial cells led to dysregulation of vascular development pathways and decreased tube formation capacity. We generated endothelial-specific Cd109 knockout mice, eliminating Cd109 expression from heart and lung tissues without overt consequences for atrioventricular valve or coronary vascular morphogenesis during heart development. CONCLUSION: CD109 exhibits a highly dynamic spatiotemporal expression pattern during cardiovascular development, with enriched expression in coronary endothelial cells and endocardial-derived subpopulations in the valves. Despite this striking developmental expression pattern, previously reported human genetic associations with cardiovascular diseases, and endothelial-associated phenotypes following siRNA-mediated CD109 knockdown in a primary human endothelial cell line, endothelial/endocardial-lineage deletion of Cd109 did not produce overt abnormalities in atrioventricular valve or coronary vascular morphogenesis during embryonic development. Collectively, these findings identify CD109 as a useful marker of coronary endothelial and endocardial-derived valve cell populations and suggest that CD109 may function in a context-dependent or modulatory manner rather than as an essential regulator of cardiovascular morphogenesis under normal developmental conditions.

CD109↗
PubMed2026

Targeted Epigenetic Silencing of Jumonji Domain-Containing Protein 3 Alleviates Nuclear Factor-Kappa B-Mediated Inflammation in Familial Mediterranean Fever.

BACKGROUND: Familial Mediterranean fever (FMF) is an inherited autoinflammatory condition caused by variants in the MEFV gene encoding pyrin, the essential component of the NLRP3/NF-κB complex of inflammasomes. Deregulation of nuclear factor-kappa B (NF-κB), a key proinflammatory mediator, leads to chronic inflammation in autoinflammatory/autoimmune diseases. Epigenetic modulation offers a new approach to regulate inflammasome activity, with Jumonji domain-containing protein 3 (JMJD3) being a promising target for managing inflammatory illnesses. GSK-J4 is a selective inhibitor of JMJD3, restricting pro-inflammatory cytokines and inflammation. AIM: Our research aimed to elucidate the role of JMJD3 and the NF-κB-JMJD3 signaling pathways in regulating inflammation in an in vitro model, and to investigate GSK-J4's effect in inhibiting inflammasome activation in primed peripheral blood mononuclear cells (PBMCs) isolated from FMF cases. METHODS: PBMCs were cultured and primed with LPS, and then treated with GSK-J4. JMJD3 knockdown was achieved using siRNA interference. Cellular inflammatory dynamics were assessed by Western blotting (WB) and ELISA. The qRT-PCR was used for gene expression quantification. Untreated cells served as a negative control. RESULTS: Our results showed significantly downregulated gene expression of NF-κB, NLRP3, and inflammatory cytokines in GSK-J4-treated cells compared to untreated cells, as confirmed by ELISA. WB reported a reduction of NF-κB in induced cells following GSK-J4 treatment. Knocking down JMJD3 also showed decreased levels of JMJD3, NF-κB, and inflammatory cytokines, indicating its proinflammatory role. CONCLUSION: The study showed that selective inhibition or silencing of JMJD3 significantly suppressed the inflammasome in FMF cases, suggesting its role as a therapeutic target for alleviating inflammation in various autoinflammatory diseases.

Humans↗
PubMed2026

Identifying transcription factors controlling the basal expression of human MRP4 highlights a substantial role for Sp1.

The multidrug resistance protein 4 (MRP4/ABCC4) is a versatile efflux pump, known to transport several drugs but also signaling molecules such as cyclic nucleotides and lipid mediators. Based on this substrate spectrum and its broad tissue distribution, MRP4 plays a significant physiological and pathophysiological role in both the cardiovascular and oncological fields. However, the determinants of its gene expression are still incompletely defined. This study aimed to identify key regulatory elements and transcription factors that are essential for basal MRP4 expression. Using luciferase reporter assays with a series of 5'-deletion constructs, we identified a region upstream of the transcription start site as crucial for basal expression across diverse cell types. This region is evolutionary highly conserved and contains putative binding sites for Sp1 and Ets transcription factors. Site-directed mutagenesis of both binding elements resulted in a significant decrease in the promoter activity in HeLa and megakaryoblastic M07e cells. The binding of Sp1 to this region was further confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Finally, siRNA knockdown of Sp1 led to a significant decrease in MRP4 protein levels and function. In summary, we show that Sp1 binds to the MRP4 promoter and plays an essential role in the basal expression of MRP4, with Ets factors also potentially cooperating in this regulation.

Humans↗
PubMed2026

RNAi in the Rhizarian Phytopathogen Plasmodiophora brassicae: The Causal Agent of Clubroot Disease in Cruciferous Crops.

Although RNA interference (RNAi) is widespread and functionally important across eukaryotes, RNAi pathways are diverse or even lost in some lineages. Rhizaria represents a major and distinct eukaryotic supergroup that includes Plasmodiophora brassicae (Pb), the causal agent of cruciferous clubroot disease, yet RNAi in this lineage remains poorly understood. Here, we characterized an unusual RNAi pathway in Pb. Small RNA sequencing across five representative Pb life stages revealed abundant siRNAs and miRNAs characterized by a predominant 21-nt length, phased genomic distribution, 2-nt 3' overhangs, and a strong 5'-cytidine bias. Three Pb miRNAs were further validated by northern blotting and stem-loop RT-qPCR. Genome analysis identified two canonical AGO homologs, PbAGO1 and PbAGO2, but no Dicer homologs, except for an RNase III-containing Drosha-like protein, PbDRL. Functional analyses showed that PbAGO1 and PbAGO2 mediate gene silencing, whereas PbDRL is required for sRNA biogenesis. Further, the cell wall component chitin was identified from Pb zoosporangia during the early infection and RNAi interfering with its biosynthesis in transgenic plants of Arabidopsis and Brassica napus blocked Pb early infection and conferred broad-spectrum resistance. Our study uncovers an unusual RNAi pathway in Rhizaria and provides a promising strategy to control cruciferous clubroot disease.

Plasmodiophora brassicae↗
PubMed2025

USP7 Inhibitors Destabilize EBNA1 and Suppress Epstein-Barr Virus Tumorigenesis.

Epstein-Barr virus (EBV) is a ubiquitous human ɣ-herpesvirus implicated in various malignancies, including Burkitt's lymphoma and gastric carcinomas. In most EBV-associated cancers, the viral genome is maintained as an extrachromosomal episome by the EBV nuclear antigen-1 (EBNA1). EBNA1 is considered to be a highly stable protein that interacts with the ubiquitin-specific protease 7 (USP7). Here, we show that pharmacological inhibitors and small interfering RNA (siRNA) targeting USP7 reduce EBNA1 protein levels in a proteosome-dependent manner. Proteomic analysis revealed that USP7 inhibitor GNE6776 altered the EBNA1 protein interactome, including disrupting USP7 association with EBNA1. GNE6776 also inhibited EBNA1 binding to EBV oriP DNA and reduced viral episome copy number. Transcriptomic studies revealed that USP7 inhibition affected chromosome segregation and mitotic cell division pathways in EBV+ cells. Finally, we show that GNE6776 selectively inhibited EBV+ gastric and lymphoid cell proliferation in cell culture and slowed EBV+ tumor growth in mouse xenograft models. These findings suggest that USP7 inhibitors perturb EBNA1 stability and function and may be exploited to treat EBV latent infection and tumorigenesis.

Ubiquitin-Specific Peptidase 7↗
PubMed2026

UBE2D4 Upregulation Promotes Cuproptosis Sensitivity in Colorectal Cancer.

BACKGROUND: Cuproptosis, a copper-dependent form of regulated cell death, represents a potential therapeutic vulnerability in colorectal cancer (CRC). However, the regulatory mechanisms governing cuproptosis in CRC remain largely unknown. METHODS: UBE2D4 expression was analyzed in the TCGA-COAD cohort and validated in CRC cell lines (HCT116, HT29) and normal colon epithelial cells (FHC) by qRT-PCR and western blot. Paired CRC and adjacent normal tissues (n = 5) were also examined by western blot. UBE2D4-knockdown HT29 cells were generated by transient siRNA transfection to assess cell viability (CCK-8), migration (wound healing assay), and expression of cuproptosis-related genes (DLAT, HSP70, LIAS) under copper overload conditions (elesclomol+CuSO4). RESULTS: UBE2D4 was significantly upregulated in CRC tissues and cell lines compared to normal controls. In paired clinical samples, western blot confirmed that UBE2D4 protein expression was elevated in tumor tissues, accompanied by increased DLAT, HSP70 and LIAS. Copper overload induced typical cuproptotic mitochondrial morphology and triggered a marked upregulation of UBE2D4, DLAT, and HSP70, alongside downregulation of LIAS. UBE2D4 silencing had no effect on baseline cell viability or migration but significantly rescued cells from copper-induced cytotoxicity. Genetically, UBE2D4 knockdown specifically attenuated the copper-induced elevation of DLAT, while restoring HSP70 and LIAS to near-baseline levels. CONCLUSION: These findings identify UBE2D4 as a genetically upregulated and functionally significant gene in colorectal cancer. Its upregulation correlates with altered expression of cuproptosis-related genes, particularly DLAT, suggesting that UBE2D4 expression status may represent a genetic determinant of cuproptosis sensitivity in CRC. This study provides a genetic basis for stratifying CRC patients who might benefit from copper-based therapeutic strategies.

Humans↗