Search Results (132)

Objective: This study aimed to elucidate the regulatory mechanisms of the long intergenic non-protein coding RNA 02381 (LINC02381)/microRNA-let-7g-5p (let-7g-5p)/thrombospondin 1 (THBS1) signaling axis in osteosarcoma (OS). Methods: The expression levels of LINC02381, let-7g-5p, and THBS1 were quantified in OS and adjacent normal tissues via reverse transcription quantitative polymerase chain reaction. Their correlations with clinicopathological features were analyzed. Expression patterns were further validated in OS cell lines (143B, U-2OS, Saos-2, MNNG-HOS, MG-63) and normal osteoblast cell line hFOB1.19. The molecular interaction between LINC02381 and let-7g-5p and the targeting relationship of let-7g-5p with THBS1 were verified via dual-luciferase reporter and RNA pull-down assays. Functional effects were assessed using cell counting kit-8, colony formation, Transwell migration, and xenograft tumor models. Results: Compared to adjacent normal tissues, LINC02381 and THBS1 were upregulated in OS tissues (fold change > 3.0, p < 0.001), while let-7g-5p was downregulated (fold change ≈ 0.038, p < 0.001). Similar expression trends were observed in U-2OS cells. Knockdown of LINC02381 or overexpression of let-7g-5p reduced cell proliferation, colony formation, migration, THBS1 expression, and tumor volume (p < 0.001). These inhibitory effects were partially reversed by let-7g-5p inhibitors, restoring cell viability and migration by approximately 70%. Mechanistically, LINC02381 functioned as a competing endogenous RNA (ceRNA), directly binding to let-7g-5p and mitigating its suppression of THBS1. Conclusions:LINC02381 promotes OA progression by acting as a ceRNA for let-7g-5p, thereby upregulating THBS1 expression. This signaling axis represents a potential therapeutic target for OS. Full article

Long non-coding RNAs (lncRNAs) are increasingly recognized as key regulators of gene expression and cellular signaling in cancer. Their functions are primarily mediated through interactions with specific protein partners that modulate chromatin structure, epigenetic remodeling, transcription, and signal transduction. In this review, we explore reports and strategies for the proteomic characterization of lncRNA-associated proteins, particularly emphasizing high-throughput liquid chromatography–mass spectrometry (LC-MS)-based techniques. Affinity-based methods such as RNA pull-down, ChIRP MS, RAP-MS, BioID-MS, and SILAC-MS enable sensitive and specific mapping of lncRNA and protein complexes. These approaches reveal cancer-specific proteomic signatures, post-translational modifications, and mechanistic insights into tumor biology. The use of label-free quantification, bituminization, and crosslinking strategies further enhances the resolution of dynamic RNA–protein networks. Validation tools following bioinformatic analyses, such as Western blotting, immunohistochemistry, immunofluorescence, and ELISA, are used to prioritize and confirm findings. Candidate biomarkers from hepatocellular carcinoma to colorectal and prostate cancers, profiling lncRNA-associated proteins, hold promise for identifying clinically actionable biomarkers and therapeutic targets. This review highlights the translational relevance of lncRNA protein studies and advocates for their broader adoption in oncological research. In LC-MS workflows, proteins bound to lncRNAs are enzymatically digested into peptides, separated via nano-LC, and analyzed using high-resolution tandem MS. Label-free or isotope-labeled methods quantify differential enrichment, followed by bioinformatics-driven pathway annotation. Full article

Background: Duodenal adenocarcinoma (DA) is often insidious due to the low rate of early diagnosis and because the mechanisms that underlie its malignant progression are poorly understood. The tumor microenvironment (TME) plays a crucial regulatory role in promoting tumor malignancy. Hence, this study aimed to identify novel biomarkers for early diagnosis and potential therapeutic targets for DA. Methods: Surgical resection samples and normal tissues from DA patients were collected for RNA sequencing (RNA-seq). The characteristics of TME in DA patients were analyzed, and the differentially expressed long non-coding RNAs (lncRNA) were screened. Functional experiments were performed to verify the relationship between Linc01559, G-rich sequence binding factor 1 (GRSF1), and tumor malignant phenotype. Results: The present study revealed that DA exhibits a significantly upregulated expression of acidic environment markers and a high degree of macrophage infiltration. Further investigation revealed that macrophages upregulate the expression of the long noncoding RNA, Linc01559, in DA through the STAT3/c-MYC signaling pathway, thereby promoting malignant phenotypes such as invasion, metastasis, tumor stemness, and apoptosis. The interaction between GRSF1 and Linc01559 was subsequently confirmed using RNA pulldown-mass spectrometry. It was further revealed that Linc01559 promotes the malignant phenotype of duodenal cancer cells through its interaction with GRSF1. Conclusions: These findings demonstrate that the acidic microenvironment influences the phenotype of DA by regulating the Linc01559–GRSF1 axis. Therefore, these findings provide potential targets for the early detection and treatment of DA. Full article

The cap-binding protein complex (CBC), comprising Cbp20 and Cbp80, is crucial for gene expression, yet its role in the notorious crop pathogen Botrytis cinerea remains unclear. Immunoprecipitation coupled with LC-MS/MS demonstrated that BcCbp20 interacts with BcCbp80. Yeast two-hybrid, GST pull-down, and Split-luciferase complementation assays confirmed that the conserved RNA recognition motif (RRM, 54–127 aa) of BcCbp20 and the N-terminal MIF4G domain (1–370 aa, 1–577 aa) of BcCbp80 constitute the core interaction regions. Genetic transformation experiments revealed that BcCBP80 exerts a more dominant role than BcCBP20 in regulating hyphal morphology, growth rate, conidiophore development, and conidial yield. Furthermore, BcCBP20 and BcCBP80 differentially regulate sclerotium formation to maintain sclerotial quantity. Based on pathogenicity assays, BcCBP80 associated with infection cushion development, with this phenotypic alteration possibly being among the factors correlated with altered pathogenicity. However, the increased sensitivity of ΔBccbp20 to various stress factors may be the primary reason for the diminished pathogenicity. Taken together, these results indicate that BcCBP20 and BcCBP80 play important roles in multiple aspects of B. cinerea growth, development, stress response, and pathogenicity. Full article

High-alkalinity water bodies can disrupt normal ammonia metabolism in fish, leading to ammonia poisoning. In China, there exists a highly tolerant group of Amur ide (Leuciscus waleckii) that can survive in extreme alkaline lakes with alkalinity up to 53.57 mM (pH 9.6), making it an excellent model for elucidating the high-alkalinity tolerance mechanism in fish. We have discovered that this species has evolved a special ammonia excretion mechanism to maintain ammonia efflux in high-alkalinity environments. Compared to the freshwater forms of Amur ide, the ammonia excretion protein RHBG plays a prominent role in the ammonia excretion process of the alkali forms of Amur ide; however, the regulatory mechanism of RHBG expression in fish remains unclear. Through DNA pull-down, RNA-Seq, qPCR, Western blotting, immunofluorescence, and dual-luciferase reporter assays, this study demonstrates that the transcription factor HIF1A can inversely regulate the expression of Rhbg by binding to its promoter region, thereby participating in the high-alkalinity adaptation process of fish. The findings of this study provide a theoretical basis for elucidating the ammonia excretion mechanism and revealing the alkalinity tolerance mechanism in fish. Full article

Host metabolic reprogramming is a critical strategy employed by many viruses to support their replication, and the key metabolic enzyme plays important roles in virus infection. This study investigates the role of pyruvate kinase M2 (PKM2), a glycolytic enzyme with non-canonical functions, in the replication of classical swine fever virus (CSFV). Using PK-15 cells and piglet models, we demonstrate that CSFV infection upregulates PKM2 expression both in vitro and in vivo, creating a proviral environment. knockdown of PKM2 by siRNA reduced CSFV proliferation, while PKM2 overexpression significantly increased virus propagation, which was evaluated by viral protein synthesis, genome replication, and progeny virion production. A direct interaction between PKM2 and CSFV NS5B protein was identified by co-immunoprecipitation and GST-pulldown assays, and PKM2 affected NS5B polymerase activity in a dual-luciferase reporter assay, with PKM2 depletion reducing RdRp function by 50%. Temporal analysis of the first viral replication cycle confirmed PKM2-dependent enhancement of CSFV RNA synthesis. These findings establish PKM2 as a proviral host factor that directly binds NS5B to potentiate RdRp activity, thereby bridging metabolic adaptation and viral genome replication. This study provides new evidence of a glycolytic enzyme physically interacting and enhancing viral polymerase function, offering new information about CSFV–host interaction. Full article

Background/Objectives: PARP inhibitors (PARPi) are pivotal to treating homologous recombination repair-deficient (HRD) cancers, particularly BRCA1/2-mutated ovarian and breast cancers. However, most ovarian and breast cancers harbor wild-type (WT) BRCA1/2, limiting PARPi eligibility. This study aims to identify an approved drug that could induce a BRCAness phenotype, thereby sensitizing WT BRCA cancers to PARPi. Methods: Ovarian and breast cancer cell lines with WT BRCA1/2 were treated with ivosidenib. HR repair efficiency was assessed via RAD51 foci formation and reporter assays. Synthetic lethality with PARPi was evaluated using viability and colony formation assays. Mechanistic studies included RNA-binding protein pulldown, co-immunoprecipitation, and functional analyses of DNA repair pathways. YTHDC2′s role in HR was investigated through siRNA knockdown and rescue experiments. Results: Ivosidenib significantly reduced HR repair efficiency and sensitized cells to PARPi, inducing synthetic lethality. Mechanistically, ivosidenib directly bound YTHDC2, an m6A reader critical for HR. This interaction disrupted YTHDC2′s ability to promote DNA double-strand break repair via HR, evidenced by impaired recruitment of repair proteins (e.g., BRCA1, RAD51) and accumulation of DNA damage (γH2AX foci). YTHDC2 knockdown phenocopied ivosidenib effects, while overexpression rescued HR defects. Conclusions: Ivosidenib induces BRCAness in WT BRCA ovarian and breast cancers by targeting YTHDC2, thereby suppressing HR repair and enhancing PARPi sensitivity. This uncovers a novel, metabolism-independent mechanism of ivosidenib, repositioning it as a therapeutic agent for HRD tumors. These findings propose a strategy to expand PARPi eligibility to WT BRCA cancers, addressing a critical unmet need in oncology. Full article

In the food industry, food spoilage caused by spores is a pressing scientific challenge that needs to be addressed urgently, and spore germination is a key approach to solving this problem. Studies have shown that peptidoglycan-induced spore germination represents a novel mechanism of action, which can bind to the PASTA domain of the serine/threonine kinase PrkC. However, the signaling mechanism of peptidoglycan-induced spore germination remains unclear. This study focuses on Bacillus subtilis, using pull-down experiments to screen for proteins interacting with PrkC. There are 80 interaction proteins of PrkC that were identified in the spore. GO analysis reveals that PrkC-interacting proteins in the spore are mainly involved in metabolic processes, cell part and catalysis. KEGG results indicate that PrkC-interacting proteins in the spore are mainly involved in RNA degradation, quorum sensing, oxidative phosphorylation, etc. Additionally, proteins are categorized into six groups by function based on events that may be associated with post-germination triggered by peptidoglycan-induced activation of the PrkC signaling pathway, including “stimulate translation initiation” and “ATP synthesis and energy metabolism”. The experimental results provide a theoretical basis for further elucidating the signaling mechanism of PrkC, revealing the signaling pathway of peptidoglycan-induced spore germination, and identifying targeted inducers and repressors of spore germination. Full article

Background: Despite advancements in diagnostic efficiency, colorectal cancer (CRC) remains a leading cause of cancer-related mortality, with increasing incidence rates. Circular RNA (circRNA) is a closed-loop, generally stable noncoding RNA that functions as a sponge for microRNAs in CRC. The purpose of this study was to investigate the function and underlying mechanism of circ_RUSC2, a new circRNA, in CRC. The expression levels of circ_RUSC2, miR-661, and TUSC2 were assessed using qRT-PCR, Western blot, and immunohistochemistry. Functional assays, including CCK-8, Transwell, and scratch wound healing, were performed to evaluate cell proliferation, migration, and invasion. RNA pull-down and actinomycin D assays were used to study RNA interactions and stability. In both CRC cells and tissues, miR-661 was markedly elevated, while circ_RUSC2 expression was considerably reduced. Poor differentiation, distant metastases, lymph node metastases, and an advanced stage were all strongly correlated with either miR-661 overexpression or circ_RUSC2 downregulation. circ_RUSC2 was more stable compared to its linear RUSC2 mRNA. CRC cell invasion, migration, and proliferation were suppressed by circ_RUSC2 ectopic expression; this inhibitory effect was restored by a miR-661 mimic. Circ_RUSC2 served as miR-661’s sponge. TUSC2 counteracted the effects of miR-661, which stimulated CRC cell proliferation, migration, and invasion. At the post-transcriptional level, miR-661 controlled the expression of TUSC2 in CRC cells. In comparison to the negative control, circ_RUSC2 expression was markedly reduced, and its half-life was shortened by methyltransferase-like 3 (METTL3) knockdown. Circ_RUSC2 is a stable cytoplasmic circRNA. Circ_RUSC2 inhibits CRC cell malignant phenotypes via the miR-661/TUSC2 axis. The onset and progression of CRC are linked to the downregulation of Circ_RUSC2. circ_RUSC2 might become more stable through N6-methyladenosine (m6A) methylation regulated by METTL3. According to our research, circ_RUSC2 might be a new biomarker and treatment target for CRC. Full article

Non-coding genes, such as microRNA and lncRNA, which have been widely studied, play an important role in the regulatory network of skeletal muscle development. However, the functions and mechanisms of most non-coding RNAs in skeletal muscle regulatory networks are unclear. This study investigated the function and mechanism of miR-34b in muscle growth and development. MiR-34b overexpression and interference tests were performed in C2C12 myoblasts and animal models. It was demonstrated that miR-34b significantly promoted mouse muscle growth and development in vivo, while miR-34b inhibited myoblast proliferation and promoted myoblast differentiation in vitro. Bioinformatics prediction using TargetScan for miRNA target identification and Bibiserv2 for potential miRNA–gene interaction analysis revealed a miR-34b binding site in the SYlSL sequence. The molecular mechanism of miR-34b regulating muscle growth and development was studied by co-transfection experiment, luciferase reporter gene detection, RNA immunoprecipitation, and RNA pull-down. MiR-34b can directly bind to SYISL and AGO2 proteins and regulate the expression of SYISL target genes p21 and MyoG by targeting SYISL, thereby regulating muscle growth and development. This study highlights that, as a novel regulator of myogenesis, miR-34b regulates muscle growth and development by targeting SYISL. Full article

Long non-coding RNAs (lncRNAs) are emerging as critical regulators in honeybee physiology, influencing development, behavior, and stress responses. This study investigates the role of lncRNA LOC113219358 in the immune response and neurophysiological regulation of Apis mellifera brains. Using RNA interference (RNAi) and RNA sequencing (RNA-seq), we demonstrate that silencing lncLOC113219358 significantly alters the expression of 162 mRNA transcripts, including genes associated with detoxification, energy metabolism, and neuronal signaling. Functional enrichment analysis revealed involvement in neuropeptide signaling, ATP synthesis, and oxidative phosphorylation pathways. Acetylcholinesterase (AChE), Glutathione-S-transferase (GST) and cytochrome P450 (CYP450) activities were significantly downregulated with 48 h of RNAi treatment. Additionally, RNA pull-down assays identified 113 proteins interacting with lncLOC113219358, including ATP synthase subunits, heat shock proteins, and major royal jelly proteins, suggesting its role in cellular stress responses and neural activity modulation. These findings provide mechanistic insights into how lncLOC113219358 mediates honeybee responses to environmental stressors, contributing to our understanding of lncRNA-regulated neural and immune functions in pollinators. Full article

Background: Mounting evidence exhibits circRNAs as critical regulators in the progression of many tumors. The regulatory function and potential mechanism by which circ_0008126 in gastric cancer (GC) is unknown. Methods: To validate and analyze the expression levels and clinical values of circ_0008126 in GC patients, the biological phenotypes of circ_0008126 in GC were investigated in vitro and in vivo. The roles and effects of circ_0008126 on miR-502-5p, EIF4A3, and APC in GC cells were explored using rescue experiment, RNA stability assay, RNA pull-down, dual-luciferase reporter, RNA immunoprecipitation (RIP), RNA FISH, immunofluorescence (IF), and TOP/Flash and FOP/Flash assays. Results: Circ_0008126 expression levels were prominently down-regulated in GC tissues and cells. Importantly, low expression of circ_0008126 was relevant to the more lymphatic metastasis, advanced TNM stage, and poor survival period in patients with GC. Functionally, circ_0008126 inhibited GC cell proliferative activity, metastatic ability, and epithelial-mesenchymal transition (EMT) in vitro and vivo. Mechanistically, we verified that EIF4A3 can mediate the formation of circ_0008126, and circ_0008126 could competitively bind miR-502-5p and alleviate its role and effect on APC, thus inactivating the β-catenin pathway in GC. Additionally, circ_0008126 was determined to increase the stability of APC mRNA by interacting with cytoplasmic EIF4A3 protein and then enhancing the APC expression. Conclusions: These data demonstrate that EIF4A3-mediated circ_0008126 could regulate the APC expression and inactivate the β-catenin pathway partly by binding to miR-502-5p and EIF4A3, thus inhibiting the tumorigenesis and development of GC. Full article

Angiopoietin-1 (Ang-1) and its receptor Tie-2 promote vascular integrity and angiogenesis. MicroRNAs (miRNAs) are involved in the regulation of many cellular functions, including endothelial cell (EC) survival, proliferation, and differentiation. Several reports indicate that these effects of miRNAs on EC functions are mediated through the modulation of angiogenesis factor signaling including that of vascular endothelial growth factor (VEGF). To date, very little is known about the roles played by miRNAs in the signaling and angiogenesis promoted by the Ang-1–Tie-2 receptor axis. Our high-throughput screening of miRNAs regulated by Ang-1 exposure in human umbilical vein endothelial cells (HUVECs) has identified miR-1233-3p as a mature miRNA whose cellular levels are significantly downregulated in response to Ang-1 exposure. The expression of miR-1233-3p in these cells is also downregulated by other angiogenesis factors including VEGF, fibroblast growth factor 2 (FGF-2), transforming growth factor β (TGFβ), and angiopoietin-2 (Ang-2). The overexpression of miR-1233-3p in HUVECs using specific mimics significantly attenuated cell survival, migration, and capillary-like tube formation, and promoted apoptosis. Moreover, miR-1233-3p overexpression resulted in reversal of the anti-apoptotic, pro-migration, and pro-differentiation effects of Ang-1. Biotinylated miRNA pull-down assays showed that p53 and DNA damage-regulated 1 (PDRG1) is a direct target of miR-1233-3p in HUVECs. The exposure of HUVECs to Ang-1, angiopoietin-2 (Ang-2), fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), or transforming growth factor β (TGFβ) triggers the regulation of PDRG1 expression. This study highlights that miR-1233-3p exerts inhibitory effects on Ang-1-induced survival, migration, and the differentiation of cultured ECs. Full article

Cardiac development is a complex developmental process. The early cardiac straight tube is composed of an external myocardial layer and an internal endocardial lining. Soon after rightward looping, the embryonic heart becomes externally covered by a new epithelial lining, the embryonic epicardium. A subset of these embryonic epicardial cells migrate and colonize the embryonic myocardium, contributing to the formation of distinct cell types. In recent years, our understanding of the molecular mechanisms that govern proepicardium and embryonic epicardium formation has greatly increased. We have recently witnessed the discovery of a novel layer of complexity governing gene regulation with the discovery of non-coding RNAs. Our laboratory recently identified three distinct lncRNAs, adjacent to the Wt1, Bmp4 and Fgf8 chicken gene loci, with enhanced expression in the proepicardium that are distinctly regulated by Bmp, Fgf and thymosin β4, providing support for their plausible implication in epicardial formation. The expression of lncRNAs was analyzed in different chicken and mouse tissues as well as their subcellular distribution in chicken proepicardial, epicardial, ventricle explants and in different murine cardiac cell types. lncRNA transcriptional regulation was analyzed by using siRNAs and expression vectors of different transcription factors in chicken and mouse models, whereas antisense oligonucleotides were used to inhibit Gm14014 expression. Furthermore, RT-qPCR, immunocytochemistry, RNA pulldown, Western blot, viability and cell migration assays were conducted to investigate the biological functions of Wt1_76127 and Gm14014. We demonstrated that Wt1_76127 in chicken and its putative conserved homologue Gm14014 in mice are widely distributed in different embryonic and adult tissues and distinctly regulated by cardiac-enriched transcription factors, particularly Mef2c and Nkx2.5. Furthermore, silencing assays demonstrated that mouse Gm14014, but not chicken Wt1_76127, is essential for epicardial, but not endocardial or myocardial, cell migration. Such processes are governed by partnering with Myl9, promoting cytoskeletal remodeling. Our data show that Gm14014 plays a pivotal role in epicardial cell migration essential for heart regeneration under these experimental conditions. Full article

Charcot-Marie-Tooth disease type 2N (CMT2N) is an inherited nerve disorder caused by mutations in the alanyl-tRNA synthetase (AlaRS) gene, resulting in muscle weakness and sensory issues. Currently, there is no cure for CMT2N. Here, we found that all five AlaRS mutations in the aminoacylation domain can interact with neuropilin-1 (Nrp1), which is consistent with our previous findings. Interestingly, three of these mutations did not affect alanine activation activity. We then performed a high-throughput screen of 2000 small molecules targeting the prevalent R329H mutant. Using thermal stability assays (TSA), biolayer interferometry (BLI), ATP consumption, and proteolysis assays, we identified Tanshinone I as a compound that binds to and modifies the conformation of the R329H mutant and other CMT-related AlaRS mutants interacting with Nrp1. Molecular docking and dynamic simulation studies further clarified Tanshinone I’s binding mode, indicating its potential against various AlaRS mutants. Furthermore, co-immunoprecipitation (Co-IP) and pull-down assays showed that Tanshinone I significantly reduces the binding of AlaRS mutants to Nrp1. Collectively, these findings suggest that Tanshinone I, by altering the conformation of mutant proteins, disrupts the pathological interaction between AlaRS CMT mutants and Nrp1, potentially restoring normal Nrp1 function. This makes Tanshinone I a promising therapeutic candidate for CMT2N. Full article