Small Regulatory Molecules Research Articles

Dennd2c Negatively Controls Multinucleation and Differentiation in Osteoclasts by Regulating Actin Polymerization and Protrusion Formation.

Osteoclasts are bone-resorbing multinucleated giant cells formed by the fusion of monocyte/macrophage lineages. Various small GTPases are involved in the multinucleation and differentiation of osteoclasts. However, the roles of small GTPases regulatory molecules in osteoclast differentiation remain unclear. In the present study, we examined the role of Dennd2c, a putative guanine nucleotide exchange factor for Rab GTPases, in osteoclast differentiation. Knockdown of Dennd2c promoted osteoclast differentiation, resorption, and expression of osteoclast markers. Morphologically, Dennd2c knockdown induced the formation of larger osteoclasts with several protrusions. In contrast, overexpression of Dennd2c inhibited the multinucleation and differentiation of osteoclasts, bone resorption, and the expression of osteoclast markers. Dennd2c-overexpressing macrophages exhibited spindle-shaped mononuclear cells and long thin protrusions. Treatment of Dennd2c-overexpressing cells with the Cdc42 inhibitor ML-141 or the Rac1 inhibitor 6-thio-GTP prevented protrusion formation. Moreover, treatment of Dennd2c-overexpressing cells with the actin polymerization inhibitor latrunculin B restored multinucleated and TRAP-positive osteoclast formation. These results indicate that Dennd2c negatively regulates osteoclast differentiation and multinucleation by modulating protrusion formation in macrophages.

Biochemistry of Redox-Active Sulphur Compounds in Mammalian Cells and Approaches to Detecting Them

The discovery of new classes of regulatory molecules in human and animal metabolism always leads to a large-scale study of their properties in the context of biochemistry, physiology, and pharmacology. About 20 years ago, hydrogen sulfide (H2S) and its derivatives – active sulfur forms (ASFs): persulfides, polysulfides, nitrosothiols, sulfenic acids, etc. – became one of such classes of molecules. The participation of ASFs in a variety of physiological and pathological processes, such as regulation of vascular tone, inflammation, long-term potentialization in the central nervous system, etc., has been shown. Changes in ASF levels or patterns of modification of their targets are associated with a wide range of pathologies: cardiovascular, oncologic, neurodegenerative, and others. For a part of these processes, mechanisms have been studied that involve direct modification of regulatory (NF-κB, Keap1) or effector (GAFD, eNOS, TRPA1) proteins through reactions of cysteine residues and metal-containing centers with APS. The presence of different regulated enzymatic systems producing APS and numerous molecular targets allows us to consider H2S and its derivatives as an important class of small regulatory molecules. H2S is counted among the so-called “gas transmitters”, along with nitric oxide(II) and carbon monoxide. Over the last 20 years, a huge amount of data on the biochemistry of these compounds and approaches to their study has been accumulated.

Hepatocyte aquaporin 8-mediated water transport facilitates bile dilution and prevents gallstone formation in mice

Although water channel aquaporin-8 (AQP8) has been implicated in hepatic bile formation and liver diseases associated with abnormal bile flow in human and animal studies, direct evidence of its involvement in bile secretion is still lacking. This study aimed to determine the role of AQP8 in bile secretion and gallstone formation. We generated various transgenic knock-in and knockout mouse models and assessed liver AQP8 expression by immunostaining and immunoblotting, hepatic bile secretion by cannulation of the common bile duct, cholesterol gallstone formation by feeding a high-fat lithogenic diet, and identified regulatory small molecules by screening the organic fractions of cholagogic Chinese herbs and biochemical characterization. We identified a novel expression pattern of AQP8 protein in the canalicular membrane of approximately 50% of the liver lobules. AQP8-deficient mice exhibited impaired hepatic bile formation, characterized by the secretion of concentrated bile with a lower flow rate and higher levels of bile lipids than that of wild-type littermates. AQP8-/- mice showed accelerated gallstone formation, which was rescued by AAV-mediated hepatic expression of AQP8 or AQP1. Moreover, we identified a small molecule, scutellarin, that upregulates hepatocyte AQP8 expression in vitro and in vivo. In AQP8+/+ mice, scutellarin significantly increased bile flow, decreased bile lipid concentrations, and prevented gallstone formation compared to AQP8-/- mice. Molecular studies revealed that scutellarin promoted the ubiquitination and degradation of HIF-1α, a transcriptional negative regulator of AQP8, by disrupting its interactions with HSP90. AQP8 plays a crucial role in facilitating water transport and bile dilution during hepatic bile formation, thereby mitigating gallstone formation in mice. Small-molecule intervention validated hepatocyte AQP8 as a promising drug target for gallstone therapy. The incidence of gallstone disease is high, and current drug treatments for gallstones are very limited, necessitating the identification of novel drug targets for developing new drugs with universal applicability. To our knowledge, this is the first study to provide direct evidence that hepatic water channel AQP8 plays a key role in bile dilution and gallstone formation. Modulation of hepatic water transport may provide a universal therapeutic strategy for all types of gallstone diseases.

Parkinson's Disease and MicroRNAs: A Duel Between Inhibition and Stimulation of Apoptosis in Neuronal Cells.

Parkinson's disease (PD) is one of the most prevalent diseases of central nervous system that is caused by degeneration of the substantia nigra's dopamine-producing neurons through apoptosis. Apoptosis is regulated by initiators' and executioners' caspases both in intrinsic and extrinsic pathways, further resulting in neuronal damage. In that context, targeting apoptosis appears as a promising therapeutic approach for treating neurodegenerative diseases. Non-coding RNAs-more especially, microRNAs, or miRNAs-are a promising target for the therapy of neurodegenerative diseases because they are essential for a number of cellular processes, including signaling, apoptosis, cell proliferation, and gene regulation. It is estimated that a substantial portion of coding genes (more than 60%) are regulated by miRNAs. These small regulatory molecules can have wide-reaching consequences on cellular processes like apoptosis, both in terms of intrinsic and extrinsic pathways. Furthermore, it was recommended that a disruption in miRNA expression levels could also result in perturbation of typical apoptosis pathways, which may be a factor in certain diseases like PD. The latest research on miRNAs and their impact on neural cell injury in PD models by regulating the apoptosis pathway is summarized in this review article. Furthermore, the importance of lncRNA/circRNA-miRNA-mRNA network for regulating apoptosis pathways in PD models and treatment is explored. These results can be utilized for developing new strategies in PD treatment.

The Impact of miR-155-5p on Myotube Differentiation: Elucidating Molecular Targets in Skeletal Muscle Disorders.

MicroRNAs are small regulatory molecules that control gene expression. An emerging property of muscle miRNAs is the cooperative regulation of transcriptional and epitranscriptional events controlling muscle phenotype. miR-155 has been related to muscular dystrophy and muscle cell atrophy. However, the function of miR-155 and its molecular targets in muscular dystrophies remain poorly understood. Through in silico and in vitro approaches, we identify distinct transcriptional profiles induced by miR-155-5p in muscle cells. The treated myotubes changed the expression of 359 genes (166 upregulated and 193 downregulated). We reanalyzed muscle transcriptomic data from dystrophin-deficient patients and detected overlap with gene expression patterns in miR-155-treated myotubes. Our analysis indicated that miR-155 regulates a set of transcripts, including Aldh1l, Nek2, Bub1b, Ramp3, Slc16a4, Plce1, Dync1i1, and Nr1h3. Enrichment analysis demonstrates 20 targets involved in metabolism, cell cycle regulation, muscle cell maintenance, and the immune system. Moreover, digital cytometry confirmed a significant increase in M2 macrophages, indicating miR-155's effects on immune response in dystrophic muscles. We highlight a critical miR-155 associated with disease-related pathways in skeletal muscle disorders.

Effects of polyamines and indole on the expression of ribosome hibernation factors in Escherichia coli at the translational level.

Polyamines and indole are small regulatory molecules that are involved in the adaptation to stress in bacteria, including the regulation of gene expression. Genes, the translation of which is under the regulatory effects of polyamines, form the polyamine modulon. Previously, we showed that polyamines upregulated the transcription of genes encoding the ribosome hibernation factors RMF, RaiA, SRA, EttA and RsfS in Escherichia coli. At the same time, indole affected the expression at the transcriptional level of only the raiA and rmf genes. Ribosome hibernation factors reversibly inhibit translation under stress conditions, including exposure to antibiotics, to avoid resource waste and to conserve ribosomes for a quick restoration of their functions when favorable conditions occur. In this work, we have studied the influence of indole on the expression of the raiA and rmf genes at the translational level and regulatory effects of the polyamines putrescine, cadaverine and spermidine on the translation of the rmf, raiA, sra, ettA and rsfS genes. We have analyzed the mRNA primary structures of the studied genes and the predicted mRNA secondary structures obtained by using the RNAfold program for the availability of polyamine modulon features. We have found that all of the studied genes contain specific features typical of the polyamine modulon. Furthermore, to investigate the influence of polyamines and indole on the translation of the studied genes, we have constructed the translational reporter lacZ-fusions by using the pRS552/λRS45 system. According to the results obtained, polyamines upregulated the expression of the rmf, raiA and sra genes, the highest expression of which was observed at the stationary phase, but did not affect the translation of the ettA and rsfS genes, the highest expression of which took place during the exponential phase. The stimulatory effects were polyamine-specific and observed at the stationary phase, when bacteria are under multiple stresses. In addition, the data obtained demonstrated that indole significantly inhibited translation of the raiA and rmf genes, despite the stimulatory effect on their transcrip- tion. This can suggest the activity of a posttranscriptional regulatory mechanism of indole on gene expression.

Water deprivation-induced hypoxia and oxidative stress physiology responses in respiratory organs of the Indian stinging fish in near coastal zones.

Water deprivation-induced hypoxia stress (WDIHS) has been extensively investigated in numerous fish species due to their adaptation with accessory respiratory organs to respire air but this has not been studied in Indian stinging fish Heteropneustes fossilis. Data regarding WDIHS-induced metabolism in accessory respiratory organ (ARO) and gills and its relationship with oxidative stress (OS) in respiratory organs of air-breathing fish H. fossilis, are limited. So, this study aimed to investigate the effects of WDIHS (0, 3, 6, 12, and 18 h) on hydrogen peroxide (H2O2) as reactive oxygen species (ROS), OS, redox regulatory enzymes, and electron transport enzymes (ETC) in ARO and gills of H. fossilis. Fish were exposed to air for different hours (up to 18 h) against an appropriate control, and ARO and gills were sampled. The levels of oxygen saturation in the body of the fish were assessed at various intervals during exposure to air. Protein carbonylation (PC) and thiobarbituric acid reactive substances (TBARS) were used as OS markers, H2O2 as ROS marker, and various enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), along with the assessment of complex enzymes (I, II, III, and V) as well as the levels of ascorbic acid (AA) and the reduced glutathione (GSH) were quantified in both the tissues. Discriminant function analyses indicate a clear separation of the variables as a function of the studied parameters. The gills exhibited higher levels of GSH and H2O2 compared to ARO, while ARO showed elevated levels of PC, TBARS, AA, SOD, CAT, and GPx activities compared to the gills. The activities of GR and ETC enzymes exhibited similar levels in both the respiratory organs, namely the gills, and ARO. These organs experienced OS due to increased H2O2, TBARS, and PC levels, as observed during WDIHS. Under WDIHS conditions, the activity/level of CAT, GPx, GR, and GSH decreased in ARO, while SOD activity, along with GR, GSH, and AA levels decreased in gills. However, the activity/level of SOD and AA in ARO and CAT in gills was elevated under WDIHS. Complex II exhibited a positive correlation with WDIHS, while the other ETC enzymes (complex I, III, and V) activities had negative correlations with the WDIHS. The finding suggests that ARO is more susceptible to OS than gills under WDIHS. Despite both organs employ distinct redox regulatory systems to counteract this stress, their effectiveness is hampered by the inadequacy of small redox regulatory molecules and the compromised activity of the ETC, impeding their ability to effectively alleviate the stress induced by the water-deprivation condition.

Human Breast Milk miRNAs: Their Diversity and Potential for Preventive Strategies in Nutritional Therapy.

The endogenous miRNAs of breast milk are the products of more than 1000 nonprotein-coding genes, giving rise to mature small regulatory molecules of 19-25 nucleotides. They are incorporated in macromolecular complexes, loaded on Argonaute proteins, sequestrated in exosomes and lipid complexes, or present in exfoliated cells of epithelial, endothelial, or immune origins. Their expression is dependent on the stage of lactation; however, their detection depends on progress in RNA sequencing and the reappraisal of the definition of small RNAs. Some miRNAs from plants are detected in breast milk, opening the possibility of the stimulation of immune cells from the allergy repertoire. Each miRNA harbors a seeding sequence, which targets mRNAs, gene promoters, or long noncoding RNAs. Their activities depend on their bioavailability. Efficient doses of miRNAs are estimated to be roughly 100 molecules in the cytoplasm of target cells from in vitro and in vivo experiments. Each miRNA is included in networks of stimulation/inhibition/sequestration, driving the expression of cellular phenotypes. Three types of stress applied during lactation to manipulate miRNA supply were explored using rodent offspring: a foster mother, a cafeteria diet, and early weaning. This review presents the main mature miRNAs described from current mothers' cohorts and their bioavailability in experimental models as well as studies assessing the potential of miR-26 or miR-320 miRNA families to alter offspring phenotypes.

Plant miRNA integrated functions in development and reproduction

Plant development and reproduction are complex processes during which an individual fulfills its life cycle, starting from germination and the elaboration of new organs and growth, leading to the formation of reproductive structures and ultimately terminating in the production of the next generation. These mechanisms are the result of a long evolutionary history that has led to sophisticated regulatory mechanisms involving multiple levels of regulators. MicroRNAs (miRNAs) are a class of small regulatory molecules that play a pivotal role in regulatory networks by negatively controlling target genes. Since miRNA very first identification twenty years ago, they have attracted much interest for their role as essential regulators of plant development. In this review, we propose a comprehensive and critical analysis of the importance of miRNAs during plant development and reproduction. We begin by presenting the current understanding of miRNAs’ evolutionary history, biogenesis, mode of action, position in regulatory networks, and their potential as mobile molecules, exploring how these aspects contribute to their functions in plant development and reproduction. Then, we explore the genetic strategies employed to effectively analyze their roles, with an emphasis on recent advancements resulting from genome editing techniques. Next, we focus on miRNA contributions to four crucial processes: growth, organ patterning and identity, life cycle progression and reproduction. Through this analysis, the importance of miRNAs during plant development and reproduction emerges, which we finally discuss in light of the current view miRNAs’ roles during animal development.

Abstract 2332: VSTM2A modulation of immune response in colorectal cancer through abrogating PD-L1 and PD-1 interaction

Abstract Background: Checkpoint immunotherapy using humanized antibody is a new therapeutic strategy against advanced colorectal cancer (CRC). Nonetheless, it shows low tumor penetration due to the large molecule size. Therefore, small immune regulatory molecules are actively sought. Our previous study showed that a small secretory protein VSTM2A suppressed Wnt signalling pathway through inducing LRP6 lysosome dependent degradation. However, its role in immune modulation is largely unknown. Methods: Clinicopathological features were analyzed using CRC patient cohort. Functional biology of VSTM2A was investigated using T cell-antigen presenting cell (APC) co-culture models and knockout mice model. Liquid chromatography with tandem mass spectrometry (LC-MS) was carried out for mechanistic investigation. Results: Gene ontology analysis of colon RNA-seq dataset from TCGA/GTEx revealed significant enrichment of adaptive immune response and lymphocyte activation with high VSTM2A expression. CRC patients with inferior VSTM2A expressions were associated with lower immunoscores in both early and late stages of CRC. In addition, our immunohistochemical examinations of 208 in-house CRC cases further pinpoints to a considerable fraction of VSTM2A-negative cases showing avoidance of spontaneous CD8a+ cells infiltration (Pearson's chi-squared test P < 0.001, φ = 0.24) and worse clinical outcome. LC-MS assay followed by reciprocal immunoprecipitation revealed that VSTM2A interacted with the IgV motif of immune checkpoint protein programmed cell death 1 ligand 1 (PD-L1). We further found that VSTM2A recognized PD-L1 with a dissociation constant (Kd) of 2.6 nM. Recombinant VSTM2A dose dependently suppressed the interaction of PD-L1 and PD-1 with IC50 of 4.51 nM, indicating VSTM2A is a potential intrinsic PD-L1 antagonist. Moreover, our in-vitro mechanistic studies revealed that VSTM2A protein could block the interaction of PD-1/PD-L1, and induced NFAT-RE luciferase activity dose-dependently using a bioluminescent cell-based PD-1/PD-L1 blockade bioassay. VSTM2A could dramatically activate the interleukin-2 production from DO11.10 T cell in the presence non-T cell spherocyte isolated from BALB/c mice and OVA323-339 peptide. Furthermore, our in-vivo study revealed that VSTM2A triggered a distinct increase in CD45+CD3+ T cells infiltration in the syngeneic MC38 mouse model. Vstm2a+/- knockout mice challenged with azoxymethane and dextran sodium sulfate showed significantly high CRC incidence and tumor burden in comparison to wild-type littermates. Tumor from Vstm2a+/- mice showed significantly less CD8+ T cell infiltration. Conclusions: our study demonstrate the tumor extrinsic role of VSTM2A in modulation of microenvironment through abrogating PD-L1/PD-1 interaction. Silencing of VSTM2A in CRC could suppress T cell activation and infiltration in the tumor sites. Citation Format: Yujuan Dong, Yunfei Zhou, Nathalie Wong, Jun Yu, Siu Man Simon Ng. VSTM2A modulation of immune response in colorectal cancer through abrogating PD-L1 and PD-1 interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2332.

Sexual differentiation in dioecious cucurbits - A molecular perspective

Understanding the evolution and genetics of sex differences is significant in both pure and applied research. Sex determination in the family Cucurbitaceae is complex owing to the existence of multiple sexual phenotypes and evolutionary changes between different systems as well as variability in chromosome numbers and structures. Dioecy (or the separate sexed condition) is widespread in angiosperms and the recent availability of molecular markers, next-generation based genome sequencing and small RNA profiling had advanced understanding of the evolution and genetics of sexual dimorphism in several dioecious plant species. Many cucurbits including some economically important species are dioecious. In this review, we summarize knowledge about the molecular mechanisms associated with sexual differentiation in dioecious cucurbits including sexlinked regions and genes identified using molecular markers. We also discuss the roles of small regulatory molecules, phytohormones and transposon variation affecting sex phenotypes in dioecious cucurbits for commercial exploitation.

Diurnal small RNA expression and post-transcriptional regulation in young and old Drosophila melanogaster heads

Background: MicroRNAs are a class of small (~22nt) endogenous RNAs that regulate target transcript expression post-transcriptionally. Previous studies characterized age-related changes in diurnal transcript expression but it is not understood how these changes are regulated, and whether they may be attributed in part to changes in microRNA expression or activity with age. Diurnal small RNA expression changes with age were not previously studied. Methods: To interrogate changes in small RNA expression with age, we collected young (5 day) and old (55 day) Drosophila melanogaster around-the-clock and performed deep sequencing on size-selected RNA from whole heads. Results: We found several microRNAs with changes in rhythmicity after aging, and we investigated microRNAs which are differentially expressed with age. We found that predicted targets of differentially expressed microRNAs have RNA-binding and transcription factor activity. We used a previously published method to identify mRNA transcripts which show evidence of microRNA targeting that is altered after aging, and found several that are involved in muscle development and maintenance. Finally, we identified novel microRNAs using the random-forest-based method miRWoods, which surprisingly also discovered transfer RNA-derived fragments. Conclusions: We showed a decrease in global microRNA expression and a corresponding increase in piRNA expression during aging. We also found an increase in rhythmicity of Drosophila small RNAs during aging, including microRNAs, piRNA clusters, and novel transfer RNA-derived fragments. To our knowledge this is the first study examining diurnal small RNA expression around the clock in young and old Drosophila, and as such it paves the way for future research on changes in small RNA regulatory molecules in the context of aging.

Seasonal Variation of Water Quality Modulated Redox Regulatory System in the Apple Snail Pila globosa and Its Use as a Bioindicator Species in Freshwater Ecosystems across India

Studies on oxidative stress physiology on molluscs to monitor the aquatic environment, influenced by pollutants and stressors are very limited in general and in Indian apple snails, P. globosa in particular. The main focus of the present study was to establish the baseline data on a redox regulatory system in P. globosa sampled across the Indian subcontinent. Snails were sampled from different zones of India in three seasons (rainy, summer and winter) and the redox regulatory system (levels of antioxidant enzyme activities and small redox regulatory molecules) and oxidative stress indicator (lipid peroxidation) were analyzed. The observed elevated lipid peroxidation level in the summer season accompanied with elevated pH, salinity and temperature indicates induction of stress. In the summer season, the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) enzymes were augmented, whereas the level of the free -SH group and the activities of glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were found to be increased. Similarly, correlation analysis between the antioxidant system and aquatic parameters revealed that SOD, CAT and GR were influenced by pH as well as salinity, whereas CAT was strongly correlated with temperature. Collectively, our data indicate the use of P. globosa as a model organism to monitor and access the freshwater environment by determining the redox regulatory status of this animal.

Small molecules playing big roles: Tuning material properties of nucleolar condensates

Living cells are enriched with fluidic materials that are full of activity. Biomolecules such as proteins, lipids, and nucleic acids, as well as small regulatory molecules like ATP, need to be organized and regulated in such a crowded milieu to perform cellular functions. Cells achieve this functional coherence by compartmentalizing their constituents into membrane-bound organelles. Intense research over the past decade has revealed that cells can also accomplish intracellular compartmentalization using membrane-less organelles or biomolecular condensates that are formed via phase separation of proteins and nucleic acids (1).

Insights into functional connectivity in mammalian signal transduction pathways by pairwise comparison of protein interaction partners of critical signaling hubs.

Growth factors and cytokines activate signal transduction pathways and regulate gene expression in eukaryotes. Intracellular domains of activated receptors recruit several protein kinases as well as transcription factors that serve as platforms or hubs for the assembly of multi-protein complexes. The signaling hubs involved in a related biologic function often share common interaction proteins and target genes. This functional connectivity suggests that a pairwise comparison of protein interaction partners of signaling hubs and network analysis of common partners and their expression analysis might lead to the identification of critical nodes in cellular signaling. A pairwise comparison of signaling hubs across several related pathways might reveal novel signaling modules. Analysis of protein interaction connectome by Venn (PIC-Venn) of transcription factors STAT1, STAT3, NFKB1, RELA, FOS, and JUN, and their common interaction network suggested that BRCA1 and TSC22D3 function as critical nodes in immune responses by connecting the signaling hubs into signaling modules. Transcriptional regulation of critical hubs may play a major role in the lung epithelial cells in response to SARS-CoV-2 and in COVID-19 patients. Mutations and differential expression levels of these critical nodes and modules in pathological conditions might deregulate signaling pathways and their target genes involved in inflammation. Biological connectivity emerges from the structural connectivity of interaction networks across several signaling hubs in related pathways. The main objectives of this study are to identify critical hubs, critical nodes, and modules involved in the signal transduction pathways of innate and adaptive immunity. Application of PIC-Venn to several signaling hubs might reveal novel nodes and modules that can be targeted by small regulatory molecules to simultaneously activate or inhibit cell signaling in health and disease.

Neuropeptides in Cancer: Friend and Foe?

Neuropeptides are small regulatory molecules found throughout the body, most notably in the nervous, cardiovascular, and gastrointestinal systems. They serve as neurotransmitters or hormones in the regulation of diverse physiological processes. Cancer cells escape normal growth control mechanisms by altering their expression of growth factors, receptors, or intracellular signals, and neuropeptides have recently been recognized as mitogens in cancer growth and development. Many neuropeptides and their receptors exist in multiple subtypes, coupling with different downstream signaling pathways and playing distinct roles in cancer progression. The consideration of neuropeptide/receptor systems as anticancer targets is already leading to new biological and diagnostic knowledge that has the potential to enhance the understanding and treatment of cancer. In this review, recent discoveries regarding neuropeptides in a wide range of cancers, emphasizing their mechanisms of action, signaling cascades, regulation, and therapeutic potential, are discussed. Current technologies used to manipulate and analyze neuropeptides/receptors are described. Applications of neuropeptide analogs and their receptor inhibitors in translational studies and radio-oncology are rapidly increasing, and the possibility for their integration into therapeutic trials and clinical treatment appears promising.

MiR-26a Reduces Inflammatory Responses via Inhibition of PGE2 Production by Targeting COX-2.

MicroRNAs are small non-coding RNA regulatory molecules that play an important role in the development and function of immune cells. MicroRNA-26a (miR-26a) exhibits anti-inflammatory immune effects on immune cells. However, the exact mechanism by which miR-26a plays an anti-inflammatory role remains unclear. Here, we report that miR-26a reduces inflammatory response via inhibition of prostaglandin E2 (PGE2) production by targeting cyclooxygenase-2 (COX-2). We found that miR-26a was downregulated in vitro and in vivo. The miR-26a mimic significantly decreased COX-2 protein levels, further inhibiting pro-inflammatory cytokine production in LPS-stimulated macrophages. We predicted that miR-26a could potentially target COX-2 in LPS-stimulated macrophages. Computational algorithms showed that the 3′-UTR of COX-2 mRNA contains a binding site for miR-26a. This putative targeting relationship between miR-26a and COX-2 was further confirmed by a dual-reporter gene assay. The anti-inflammatory effects of the miR-26a mimic were diminished by PGE2 supplementation. Importantly, miR-26a mimics protected mice from lethal endotoxic shock and attenuated pro-inflammatory cytokine production. Collectively, these results suggest that miR-26a may function as a novel feedback negative regulator of the hyperinflammatory response and as a drug target for the progression of inflammation.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10753-022-01631-2.

Biogenesis and mechanisms of microRNA‐mediated gene regulation

Endogenous RNAs that control posttranscriptional gene expression are microRNAs (miRNAs). These small regulatory molecules play a crucial role in certain biological processes and their expression is often strictly regulated. They are small 21-24 nucleotide molecules that act as major regulators of gene expression at the posttranscriptional level. One of the mechanisms by which miRNAs control the gene expression is to interact the interaction of the seeds with the 3'-end andand more seldom the 5'-end of mRNA transcribed by the target genes. miRNAs have been identified as important cytoplasmic regulators of gene expression. miRNAs function as posttranscriptional regulators of their messenger RNA (mRNA) targets by mRNA degradation and/or translational repression. It is becoming evident, however, that miRNAs have nuclear functions as well. About the cell type, the physiological state of the body, and various external factors, the following explanation will summarize the complex multilevel regulation of miRNA expression. A better understanding of the molecular mechanisms controlling miRNA expression will provide clarification of the variations in the expression of protein-coding genes.

Probing the Interactions of Splicing Regulatory Small Molecules and Proteins with U1 snRNP Using NMR Spectroscopy.

Alternative RNA splicing is an essential part of gene expression that not only increases the protein diversity of metazoan but also provides an additional layer of gene expression regulation. The U1 small ribonucleoparticle (U1 snRNP) plays an essential role in seeding spliceosome assembly and its binding on weak 5'-splice sites is regulated by transient interactions with splicing factors. Recent progress in allele specific splicing correction has shown the therapeutic potential offered by small molecule splicing modifiers that specifically promotes the recruitment of U1 snRNP to modulate alternative splicing and gene expression. Here, we described a method to reconstitute U1 snRNP in vitro and to study labile interactions with protein or synthetic splicing factors using solution state NMR spectroscopy. This approach allowed us to validate direct interactions between splicing regulators and U1 snRNP and could also be useful for the screening of small molecules acting on splicing regulation.

MiR-F4-C12 Functions on the Regulation of Adipose Accumulation by Targeting PIK3R1 in Castrated Male Pigs.

Simple SummaryMicroRNAs play crucial roles in regulating adipogenesis and fat storage; their role in regulating castrated male pig adipose growth is worth elucidating. Four nine-fold differentially expressed miRNAs were selected to investigate their functions on the regulation of adipose development based on our previous study. In 3T3-L1 cells and backfat tissues of castrated and intact male pigs, miR-F4-C12 was identified as involved in the adipose development using qRT-PCR and oil O staining. PIK3R1 was proposed by the TargetScan, miRDB and starBase as a target of miR-F4-C12 and verified through a dual-luciferase reporter assay and Western blot. These results revealed that miR-F4-C12 may regulate adipose accumulation in castrated male pigs by targeting PIK3R1. Our data provide a valuable foundation to understand the molecular mechanisms involved in adipose tissue metabolism to castration-induced sex hormone deficiency.MicroRNAs (miRNAs) constitute small regulatory molecules for a wide array of biological activities (18~24 nucleotides in length), including adipogenesis and adipose deposition. Their effect is, however, incompletely defined in inducing fat accumulation in castrated male pigs. Based on our study, four nine-times miRNAs were selected to examine their functions in adipose formation activities. In 3T3-L1 cells and backfat tissues of castrated and intact male pigs, miR-F4-C12 was identified as a factor in adipose development utilizing quantitative real-time PCR (qRT-PCR). Further, miR-F4-C12 was identified to promote fat development, suggesting that miR-F4-C12 was involved in adipogenesis. Moreover, phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) was proposed by the TargetScan, miRDB and starBase as a target of miR-F4-C12 and verified through a two-luciferase reporter assay. The over-expression of miR-F4-C12 dramatically decreases the PIK3R1 protein level in 3T3-L1 cells. The mRNA and protein levels of PIK3R1 in castrated pigs are reduced relative to intact pigs, providing further evidence that PIK3R1 is involved in regulating adipose accumulation. These results suggest that miR-F4-C12 involves adipose development and may regulate subcutaneous adipose tissue accumulation by targeting PIK3R1 in castrated male pigs.