Insulin-like Growth Factor 2 mRNA-binding Protein 1 Research Articles

METTL14 knockdown attenuates neuron injury and improves function recovery after spinal cord injury via regulating FGF21 in a m6A-IGF2BP1 dependent mechanism.

Fibroblast growth factor 21 (FGF21) and Methyltransferase-like 14 (METTL14) have been identified to be involved in spinal cord injury (SCI). However, whether FGF21 functioned in SCI via METTL14-induced N6-methyladenosine (m6A) modification remains unclear. PC12 cells were exposed to lipopolysaccharide (LPS) in vitro. qRT-PCR and western blotting analyses were applied to detect the mRNA and protein levels of METTL14, FGF21 and Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1). The CCK-8 assay, EdU assay, flow cytometry and ELISA analysis were used to conduct in vitro functional analyses. Cell ferroptosis was assessed by measuring the levels of Fe2+, reactive oxygen species, glutathione and related regulators. The N6-methyladenosine (m6A) modification profile was analyzed by methylated RNA immunoprecipitation (MeRIP) assay. The interaction between IGF2BP1 and FGF21 was validated using RIP assay. SCI animal models were constructed for in vivo analysis. Levels of FGF21 were decreased in LPS-induced PC12 cells. Functionally, FGF21 overexpression reversed LPS-induced proliferation inhibition, apoptosis, ferroptosis and inflammation in PC12 cells. Mechanistically, METTL14 induced FGF21 m6A modification in SCI cell models, and m6A-binding protein IGF2BP1 was involved in regulating FGF21 expression by METTL14. METTL14 silencing abolished LPS-induced neuronal apoptosis, inflammation and ferroptosis via regulating FGF21. Moreover, METTL14 silencing improved neuronal injury in SCI rat models by modulating FGF21 expression. METTL14 knockdown attenuates neuron injury and improves function recovery after SCI via up-regulating FGF21 in an m6A-IGF2BP1 dependent mechanism, suggesting a useful target for SCI recovery.

METTL14 Promotes the Osteogenic Differentiation of Human Bone Marrow Stromal Cells via m6A-Dependent Stabilization of USP7 mRNA.

Osteoporosis (OP) is a common clinical bone disease that can cause a high incidence of non-stress fractures and is one of the main degenerative diseases that endangers the health and life of middle-aged and older women. The mechanism underlying the abnormal differentiation and function of human bone marrow stem cells (hBMSCs) remains to be elucidated. Cell proliferation and differentiation were determined using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, alkaline phosphatase (ALP) staining, and Alizarin Red Staining. The interaction between insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and ubiquitin-specific protease 7 (USP7) was predicted and validated using bioinformatics approaches, luciferase assays, RNA immunoprecipitation (RIP), and immunoprecipitation (IP). Actinomycin D treatment was used to test the stability of mRNA in the various groups. Methyltransferase-like 14 (METTL14) expression was increased in osteogenic differentiation medium-induced hBSMCs and was associated with enhanced osteogenic differentiation. METTL14 regulated the expression USP7 by modulating its N6-methyladenosine (m6A) level. IGF2BP2 exerted an m6A-dependent effect on USP7 mRNA stability and USP7 increased sirtuin 1 (SIRT1) expression in hBMSCs by enhancing SIRT1 deubiquitination. METTL14 stimulated the osteogenic differentiation of hBMSCs through the m6A-IGF2BP2-USP7 pathway and promoted hBMSCs osteogenic development via SIRT1-Bmi1 signaling. METTL14 stimulated the osteogenic differentiation of hBMSCs by stabilizing USP7 mRNA in an m6A-dependent manner. USP7 was also stabilized by IGF2BP2 and it regulated downstream SIRT1-Bmi1 signaling.

Citrullinated IGF2BP1 promotes rheumatoid synovial aggression via increasing the mRNA stability of SEMA3D

Protein citrullination modification plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA), and anti-citrullinated protein antibodies (ACPAs) are extensively employed for clinical diagnosis of RA. However, there remains limited understanding regarding specific citrullinated proteins and their implications in the progression of RA. In this study, we screen and verify insulin-like growth factor-2 mRNA binding protein 1 (IGF2BP1) as a novel citrullinated protein with significantly elevated citrullinated level in RA. Autoantibodies against citrullinated IGF2BP1 are further detected in serum and synovial fluid samples from RA patients, which are positively correlated with erythrocyte sedimentation rate (ESR) and disease activity score 28 (DAS28). Transcriptomic sequencing and functional verification show that citrullination at the R167 site of IGF2BP1 promotes the proliferation, migration, and invasion of RA fibroblast-like synoviocytes (RA-FLSs) by improving the mRNA stability of Semaphorin 3D (SEMA3D). Experiments in collagen-induced arthritis (CIA) mice, the classical animal model of RA, show that IGF2BP1 R176K point mutation (Igf2bp1R167K/R167K) mice exert reduced inflammatory response, clinical scores, and joint destruction. At a molecular level, citrullination of IGF2BP1 promotes the stability of SEMA3D mRNA by promoting the interaction between IGF2BP1 and its cofactor ELAV-like protein 1 (ELAVL1), thereby promoting the invasiveness of RA-FLSs. In this study, a new citrullinated protein of IGF2BP1 is discovered, and the molecular mechanism of its citrullinated modification promoting the progression of RA disease is elucidated, which provides theoretical basis for the diagnosis and treatment of RA.

N6-methyladenosine RNA methylation regulates microplastics-induced cell senescence in the rainbow trout liver.

Microplastics are prevalent in aquatic ecosystems, impacting various forms of aquatic life, including fish. In this study, Rainbow trout (Oncorhynchus mykiss) were exposed to two concentrations of microplastics (0 and 500μg/L) over a 14-day period, during which a comprehensive analysis was conducted to assess the liver accumulation of microplastics and their effects on oxidative stress, the liver response, and transcriptomics. Our findings indicated that microplastics significantly accumulated in the liver and activated the antioxidant system in fish by enhancing the activity of antioxidant enzymes. Histological lesions were also observed in the liver of the fish. Furthermore, microplastics induced alterations in the expression of hepatic N6-methyladenosine readers, specifically downregulating IGF2BP1 (encoding insulin like growth factor 2 mRNA binding protein 1) and upregulating YTHDF2 (encoding YTH N6-methyladenosine RNA binding protein F2), which in turn decreased mRNA stability and reduced the expression of C-myc and other regulatory factors involved in the cell cycle and proliferation. This sequence of events resulted in slowed cell proliferation, the induction of cell cycle arrest, and the promotion of cellular senescence. This study offers valuable insights into the toxicological mechanisms of microplastics and enhances our understanding of the threats that plastic pollution poses to freshwater organisms.

CircENTPD7 affects the immune escape of non‑small cell lung cancer cells by modulating the IGF2BP2/PD‑L1 axis.

Programmed death ligand 1 (PD-L1), an important immune checkpoint molecule, is abnormally activated in non-small cell lung cancer (NSCLC), which can interact with programmed death 1 to aid cancer cells in evading immune surveillance. Furthermore, tumor driver genes may be involved in the occurrence and development of NSCLC and have a potential role in PD-L1-mediated immune escape mechanisms. Therefore, the present study aimed to assess the behavioral and regulatory mechanisms by which circular RNA ENTPD7 (circENTPD7; hsa_circ_0019421) induces an immune response in the progression of NSCLC cells. In brief, circENTPD7, insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and PD-L1 expression were assessed using reverse transcription-quantitative PCR, and IGF2BP2 and PD-L1 protein expression levels were evaluated using western blotting. Subsequently, the proliferation, migration, invasion and immune escape of NSCLC cells were assessed using a Cell Counting Kit-8 assay, Transwell assay, T cell co-culture assay, FITC ester assay, flow cytometry and ELISA. The experimental results revealed that circENTPD7, IGF2BP2 and PD-L1 levels were significantly elevated in NSCLC cells. CircENTPD7 knockdown suppressed the malignant phenotype of NSCLC cells, whereas overexpression of IGF2BP2 counteracted these effects, indicating that circENTPD7 contributed to the proliferation and metastasis of NSCLC cells by upregulating IGF2BP2. Furthermore, overexpression of IGF2BP2 accelerated the proliferation, metastasis and immune escape processes of NSCLC cells by upregulating PD-L1. Collectively, the results indicate that circENTPD7 contributes to the malignant progression of NSCLC cells by modulating the IGF2BP2/PD-L1 axis-mediated immune escape.

METTL3, m6A modification, and EGR1: interplay affecting myocardial I/R injury outcomes

The occurrence of severe myocardial ischemia/reperfusion (I/R) injury is associated with the clinical application of reestablishment technique for heart disease, and understanding its underlying mechanisms is currently an urgent issue. Prior investigations have demonstrated the potential enhancement of MIRI through EGR1 suppression, although the precise underlying regulatory pathways require further elucidation. The core focus of this investigation is to examine the molecular pathways through EGR1 regulates mitophagy-mediated myocardial cell pyroptosis and its impact on MIRI. Cardiomyocyte hypoxia/reoxygenation (H/R) injury models and mouse models of myocardial I/R injury were used to investigate the involvement of EGR1 in regulating mitophagy-mediated myocardial cell pyroptosis in myocardial I/R injury. The research outcomes demonstrated that under H/R conditions, EGR1 expression was upregulated and inhibited the JAK2/STAT3 pathway, leading to enhanced mitophagy and disrupted mitochondrial fusion/fission dynamics, ultimately resulting in myocardial cell pyroptosis. Further research revealed that the upregulation of EGR1 expression was mediated by methyltransferase like 3 (METTL3)-mediated m6A modification of EGR1 mRNA and depended on the binding of insulin like growth factor 2 mrna binding protein 2 (IGF2BP2) to the N6-methyladenosine (m6A) modification site to enhance mRNA stability. In vivo animal experiments confirmed that METTL3 upregulated EGR1 expression through IGF2BP2 and suppressed activation of the janus kinase 2 (JAK2) /signal transducer and activator of transcription 3 (STAT3) pathway, thereby inhibiting mitophagy, disrupting mitochondrial dynamics, promoting myocardial cell pyroptosis, and exacerbating I/R injury.

Exosome-Shuttled METTL14 From AML-Derived Mesenchymal Stem Cells Promotes the Proliferation and Radioresistance in AML Cells by Stabilizing ROCK1 Expression via an m6A-IGF2BP3-Dependent Mechanism.

Acute myelogenous leukemia (AML)-derived mesenchymal stem cells (MSCs) (AML-MSCs) have been identified to play a significant role in AML progression. The functions of MSCs mainly depend on their paracrine action. Here, we investigated whether AML-MSCs functioned in AML cells by transferring METTL14 (Methyltransferase 14) into AML cells via exosomes. Functional analyses were conducted using MTT assay, 5-ethynyl-2-deoxyuridine assay and flow cytometry. qRT-PCR and western blot analyses detected levels of mRNAs and proteins. Exosomes (exo) were isolated from AML-MSCs by ultracentrifugation. The m6A modification profile was determined by methylated RNA immunoprecipitation (MeRIP) assay. The interaction between Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) and Rho Kinase 1 (ROCK1) was validated using RIP assay. AML-MSCs incubation promoted the proliferation and radioresistance in AML cells. Moreover, AML-MSCs incubation led to increases in m6A levels and METTL14 levels in AML cells. METTL14 was transferred into AML cells by packaging into exosomes of AML-MSCs. The knockdown of METTL14 in AML-MSCs exosomes could reduce the proliferation and radioresistance in AML cells. Mechanistically, METTL14 induced ROCK1 m6A modification and stabilized its expression by an m6A-IGF2BP3-dependent mechanism. Rescue assay showed that ROCK1 overexpression reversed the inhibitory effects of METTL14 silencing in AML-MSCs exosomes on AML cell proliferation and radioresistance. Exosome-shuttled METTL14 from AML-MSCs promoted proliferation and conferred radioresistance in AML cells by stabilizing ROCK1 expression via an m6A-IGF2BP3-dependent mechanism.

The miR-6240 target gene Igf2bp3 promotes myoblast fusion by enhancing myomaker mRNA stability

BackgroundMyoblast fusion plays a crucial role in myogenesis. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) functions as an RNA N6-methyladenosine reader and exerts important roles in various biological processes. While our prior study suggested Igf2bp3 contributes to myogenesis, its molecular regulatory mechanism is largely unclear.MethodsReal-time quantitative polymerase chain reaction (RT-qPCR) and western blot were used for gene expression analysis. siRNA and CRISPRi technologies were conducted to knockdown the expression of Igf2bp3. CRISPR/Cas9 technology was performed to knockout Igf2bp3. The Igf2bp3 overexpression vector was designed using the pcDNA3.1(+) vector. Immunofluorescence detection was employed for subcellular localization and cell differentiation analysis. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) assays were conducted for cell proliferation and fusion detection. The dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were utilized for regulatory mechanism analysis of Igf2bp3.ResultsThe overexpression of Igf2bp3 enhances myoblast fusion while knockdown of Igf2bp3 blocks the formation of myotubes. miR-6240 promotes myoblast proliferation while preventing myoblast differentiation and fusion by targeting the 3′ untranslated rgion (UTR) of Igf2bp3. Notably, the impacts of miR-6240 mimics on myoblast proliferation, differentiation, and fusion can be effectively counteracted by the overexpression of Igf2bp3. Moreover, our findings elucidate a direct interaction between Igf2bp3 and the myoblast fusion factor myomaker (Mymk). Igf2bp3 binds to Mymk to enhance its mRNA stability. This interaction results in increased expression of Mymk and heightened myoblast fusion.ConclusionsOur study unveils Igf2bp3 as a novel post-transcriptional regulator of myoblast fusion through the miR-6240/Mymk axis, significantly contributing to our understanding of skeletal muscle development.Graphical

FOXM1-activated IGF2BP3 promotes cell malignant phenotypes and M2 macrophage polarization in hepatocellular carcinoma by inhibiting ferroptosis via stabilizing RRM2 mRNA in an m6A-dependent manner.

Ferroptosis has a crucial role in human carcinogenesis. N6-methyladenosine (m6A) reader insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) suppresses ferroptosis of hepatocellular carcinoma (HCC) cells. Here, we examined the effects and molecular determinants of IGF2BP3-mediated ferroptosis on malignant behaviors of HCC cells. Ferroptosis was evaluated by measuring the levels of malondialdehyde (MDA), glutathione (GSH), reactive oxygen species (ROS), and lipid ROS. HCC cell malignant phenotypes were evaluated by colony formation assay, wound healing assay, and transwell invasion assay. The CD206+ M2-like macrophages were assessed by flow cytometry. m6A RNA immunoprecipitation (MeRIP) was applied to assess the m6A modification of ribonucleotide reductase regulatory subunit M2 (RRM2). RNA immunoprecipitation (RIP) assay was performed to evaluate the interaction of IGF2BP3 and RRM2. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were conducted to confirm the interaction between forkhead box M1 (FOXM1) and IGF2BP3. Human HCC tumors showed increased expression of IGF2BP3 compared with adjacent normal tissues. Disruption of IGF2BP3 promoted cell ferroptosis. Moreover, disruption of IGF2BP3 hindered HCC cell growth, invasiveness, and motility and impeded THP1-derived macrophage M2 polarization and migration by inducing ferroptosis. Additionally, IGF2BP3 disruption repressed xenograft growth in vivo. Mechanistically, IGF2BP3 enhanced RRM2 mRNA stability and elevated its protein expression by reading its m6A modification. Overexpression of RRM2 reversed sh-IGF2BP3-mediated ferroptosis and weakened sh-IGF2BP3-mediated suppression of HCC cell malignant phenotypes and macrophage M2 polarization. Furthermore, IGF2BP3 was a downstream target of FOXM1, and knockdown of FOXM1 induced ferroptosis and inhibited cell malignant phenotypes by downregulating IGF2BP3. FOXM1-induced IGF2BP3 upregulation promotes HCC cell malignant behaviors and macrophages M2 polarization by repressing ferroptosis via m6A-dependent regulation of RRM2 mRNA. Targeting FOXM1/IGF2BP3/RRM2 to enhance ferroptosis might be exploited as a potent therapeutic strategy for HCC.

Circ_0000284 Is Involved in Arsenite-Induced Hepatic Insulin Resistance Through Blocking the Plasma Membrane Translocation of GLUT4 in Hepatocytes via IGF2BP2/PPAR-γ.

Arsenic exposure can induce liver insulin resistance (IR) and diabetes (DM), but the underlying mechanisms are not yet clear. Circular RNAs (circRNAs) are involved in the regulation of the onset of diabetes, especially in the progression of IR. This study aimed to investigate the role of circRNAs in arsenic-induced hepatic IR and its underlying mechanism. Male C57BL/6J mice were given drinking water containing sodium arsenite (0, 0.5, 5, or 50 ppm) for 12 months. The results show that sodium arsenite increased circ_0000284 expression, decreased insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) and peroxisome proliferator-activated receptor-γ (PPAR-γ), and inhibited cell membrane protein levels of insulin-responsive glucose transporter protein 4 (GLUT4) in the mouse livers, indicating that arsenic exposure causes liver damage and disruptions to glucose metabolism. Furthermore, sodium arsenite reduced glucose consumption and glycogen levels, increased the expression of circ_0000284, reduced the protein levels of IGF2BP2 and PPAR-γ, and inhibited GLUT4 protein levels in the cell membranes of insulin-treated HepG2 cells. However, a circ_0000284 inhibitor reversed arsenic exposure-induced reductions in IGF2BP2, PPAR-γ, and GLUT4 levels in the plasma membrane. These results indicate that circ_0000284 is involved in arsenite-induced hepatic insulin resistance through blocking the plasma membrane translocation of GLUT4 in hepatocytes via IGF2BP2/PPAR-γ. This study provides a scientific basis for finding early biomarkers for the control of arsenic exposure and type 2 diabetes mellitus (T2DM), and discovering new prevention and control measures.

RNF5 exacerbates steatotic HCC by enhancing fatty acid oxidation via the improvement of CPT1A stability.

Non-alcoholic fatty liver disease (NAFLD) is expected to become the leading risk factor for liver cancer, surpassing viral hepatitis. Unlike viral hepatitis-related hepatocellular carcinoma (HCC), the role of excessive nutrient supply in steatotic HCC is not well understood, hindering effective prevention and treatment strategies. Therefore, it is crucial to identify key molecules in the pathogenesis of steatotic HCC, investigate changes in metabolic reprogramming due to excessive fatty acid (FA) supply, understand its molecular mechanisms, and find potential therapeutic targets. Trans-species transcriptome analysis identified Ring Finger Protein 5 (RNF5) as a critical regulator of steatotic HCC. RNF5 upregulation is associated with poor prognosis in steatotic HCC compared to canonical HCC. In vitro and in vivo studies showed that RNF5 exacerbates HCC in the presence of additional FA supply. Lipidomics and transcriptome analyses revealed that RNF5 significantly increases carnitine palmitoyltransferase 1A (CPT1A) mRNA levels and is positively correlated with fatty acid oxidation (FAO). Protein interaction analysis demonstrated that RNF5 promotes K63-type ubiquitination of insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1), enhancing CPT1A mRNA stabilization through m6A modification. Additionally, peroxisome proliferator-activated receptor gamma (PPARγ) was found to activate RNF5 expression specifically in HCC cells. Mechanistically, excessive exogenous FAs reorganize FA metabolism in HCC cells, worsening steatotic HCC via the PPARγ-RNF5-IGF2BP1-CPT1A axis. This study highlights a distinct FA metabolism pattern in steatotic HCC, providing valuable insights for potential therapeutic targets.

N6-methyladenosine modification of SPOP relieves ferroptosis and diabetic cardiomyopathy by enhancing ubiquitination of VDAC3

Understanding the pathogenesis of diabetic cardiomyopathy (DCM), a common microvascular complication affecting the heart, is crucial for identifying new therapeutic targets and intervention strategies for DCM. Our study revealed a significant downregulation in Speckle-type POZ protein (SPOP) expression in DCM, while the overexpression of SPOP improved DCM-induced myocardial dysfunction, injury, fibrosis, hypertrophy, and ferroptosis. Mechanistically, SPOP facilitated the degradation of voltage-dependent anion channel 3 (VDAC3) by enhancing its ubiquitination. M6A demethylase AlkB homolog 5 (ALKBH5) reduced the mRNA stability of SPOP by decreasing m6A modification in its 3′UTR. The m6A reader insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) enhanced the stability of SPOP mRNA through recognition of m6A-modified SPOP 3′UTR. Furthermore, ALKBH5 promoted ferroptosis by inhibiting SPOP-induced VDAC3 degradation, while IGF2BP2 inhibited ferroptosis via activation of SPOP-induced VDAC3 degradation in high glucose-treated neonatal mouse ventricular cardiomyocytes (NMVCs). Overall, our study has unveiled a novel role of SPOP in the pathogenesis of ferroptosis and DCM, thereby significantly advancing our understanding of the involvement of ferroptosis during the progression of DCM. Moreover, this discovery offers promising potential therapeutic interventions targeting DCM.

IGF2BP3/CTCF Axis-Dependent NT5DC2 Promotes M2 Macrophage Polarization to Enhance the Malignant Progression of Lung Squamous Cell Carcinomas.

Lung squamous cell carcinoma (LUSC) is a type of lung cancer that develops in the squamous cells. It is known to be promoted by the activation of various signaling pathways and the dysregulation of key regulatory molecules. One such molecule, 5'-nucleotidase domain containing 2 (NT5DC2), has been identified as a critical regulator in various cancers including lung cancer. However, there are no data regarding its role in LUSC. The mRNA expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), CCCTC-binding factor (CTCF), and NT5DC2 was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR), whereas their protein expression was assessed using a western blotting assay. Cell proliferation was determined using a cell counting kit-8 (CCK-8) assay. Cell apoptosis, CD11b expression, and CD206 expression were analyzed using flow cytometry. Tube formation was assessed through a tube formation assay. Glucose consumption, lactate production, and ATP levels were measured using colorimetric methods. The effect of NT5DC2 on the malignant progression of LUSC cells was analyzed using a xenograft mouse model assay. The levels of transforming growth factor-beta 1 (TGF-β1) and interleukin-10 (IL-10) were detected using enzyme-linked immunosorbent assays. The associations among IGF2BP3, CTCF and NT5DC2 were identified using dual-luciferase reporter assay, RNA immunoprecipitation assay and m6A RNA immunoprecipitation assay. The expression of NT5DC2 was found to be upregulated in LUSC tissues and cells when compared with normal lung tissues and normal human bronchial epithelial cells. Silencing of NT5DC2 inhibited LUSC cell proliferation, tube formation, glycolysis, M2 macrophage polarization, and tumor formation while inducing cell apoptosis. In addition, CTCF was found to transcriptionally activate NT5DC2 in LUSC cells. IGF2BP3 stabilized the mRNA expression of CTCF through m6A methylation. Further, overexpression of CTCF or NT5DC2 attenuated the effects of IGF2BP3 silencing in both NCI-520 and SK-MES-1 cells. The IGF2BP3/CTCF axis-dependent NT5DC2 promotes M2 macrophage polarization, thereby enhancing the malignant progression of LUSC. This study was the first to reveal the role of NT5DC2 in LUSC and the underlying mechanism. The result suggests that targeting the IGF2BP3/CTCF/NT5DC2 axis may have clinical significance in the treatment of LUSC.

Significant association between insulin-like growth factor 2 mRNA-binding protein 2, interleukin-6 polymorphisms, and type 2 diabetes mellitus

Background: The study aimed to detect the association between insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and interleukin-6 (IL-6) polymorphisms among type 2 diabetes mellitus (T2DM). Materials and Methods: This study involved 500 individuals; 250 obese DM cases and 250 healthy controls. The polymerase chain reaction restriction fragment length polymorphism was used to identify the genotype of the IGF2BP2 gene for the small nucleoproteins rs4402960 (G>T) and small nucleoproteins rs800795 (G>C). Results: The results indicated that the mutant C-allele of the single nucleotide polymorphism (SNP) rs1800795 variant was highly significantly associated with the study group (P = 0.002). The occurrence of genotypes (C/G and C/C) versus normal G/G genotype of the IL-6 variant was highly significant (P = 0.004) in the study group (49.2% vs. 50.8%) compared to the control group (62% vs. 38%). Similar findings were observed in SNPs for the rs440960 variant, in which the T-allele indicated a highly significant relationship (P = 0.0001) with the study group; the frequency of G/G genotype versus both (T/T and G/T) genotypes was highly significant (P = 0.0001) in the case group (34% vs. 66%) than in the healthy control group (52% vs. 48%). Conclusion: The current study indicated that IGF2BP2 rs4402960 and IL-6 rs1800795 polymorphism were highly significantly associated with the increased risk of obese T2DM among the Saudi Arabian population and presented a genetic model to screen the high-risk individuals with further validations.

CDK14 is regulated by IGF2BP2 and involved in osteogenic differentiation via Wnt/β-catenin signaling pathway in vitro

AimsCyclin-dependent kinase (CDK) family proteins involve in various cellular processes via regulating the cell cycle; however, their expression during osteogenic differentiation and postmenopausal osteoporosis remains poorly understood. Main methodsUsing bioinformatics, we screened for CDK14 bound to Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and explored its expression in vitro with time-gradient model and in a mouse model of postmenopausal osteoporosis, building on prior research. Subsequently, we investigated its effect on osteoblast proliferation, cell cycle dynamics, and osteogenic differentiation by administering CDK14 siRNA and the covalent inhibitor FMF-04-159-2. Furthermore, we examined the interaction between IGF2BP2 and CDK14. Finally, we validated the regulatory role of CDK14 on the Wnt/β-catenin pathway. Key findingsOur findings demonstrate a time-dependent CDK14 expression patterns during osteogenic differentiation of MC3T3-E1 cell line, with an initial increase followed by gradual decline over time. Notably, CDK14 expression exhibited significant reduction in bone tissue of postmenopausal osteoporosis mouse model. CDK14 inhibition altered osteoblast cell cycle dynamics, significantly reduced cellular proliferation capacity, and impaired osteogenic differentiation ability. IGF2BP2 interacted with CDK14 mRNA, and stabilizing mRNA's structure and inhibiting its degradation. Additionally, CDK14 facilitated Low-density lipoprotein receptor-related protein 6 (LRP6) and Glycogen synthase kinase 3β (GSK3β) phosphorylation, thus regulating β-catenin levels. SignificanceThese findings provide further insight into the molecular mechanisms governing osteoblast proliferation, differentiation and osteoporosis.

Chicoric acid exerts therapeutic effects in DSS-induced ulcerative colitis by targeting the USP9X/IGF2BP2 axis.

Chicoric acid, a hydroxycinnamic acid, exhibits anti-inflammation activities. However, the specific mechanisms underlying the effects of chicoric acid on dextran sulfate sodium (DSS)-induced colitis remain unclear. Here, we aimed to elucidate the molecular mechanisms underlying the protective effects of chicoric acid in DSS-induced colitis. Mice with DSS-induced colitis (UC mice) were treated for a week with chicoric acid. Symptoms of colitis, colonic pathology, inflammation-related indicators, and intestinal mucosal barrier function were evaluated. RNA sequencing was performed on colon tissues to obtain differentially expressed genes. The deubiquitinating enzyme USP9X was selected, and the inhibitory and targeting effects of chicoric acid on USP9X were subsequently determined. In vivo and in vitro, DSS-induced colitis was treated with USP9X inhibitors WP1130 and EOAI3402143. Ubiquitination label-free quantitative proteomic analysis was performed to identify protein peptides that may undergo de-ubiquitination by USP9X. Co-immunoprecipitation (Co-IP), immunohistochemistry and western blotting were used to validate in vivo and in vitro results. Chicoric acid significantly alleviated clinical activity and histological changes, inhibited pro-inflammatory cytokine production and improved integrity of the intestinal barrier in UC mice. Moreover, chicoric acid suppressed USP9X expression in colonic tissues from UC mice. Furthermore, USP9X contributed to promoting the onset of UC and that insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) was deubiquitinated by USP9X. Chicoric acid ameliorated DSS-induced colitis by targeting the USP9X/IGF2BP2 axis, indicating that targeting the USP9X/IGF2BP2 axis presents a promising and innovative therapeutic approach for the treatment of UC.

RNA-binding protein IGF2BP1 is required for spermatogenesis in an age-dependent manner.

Post-transcriptional regulation mediated by RNA binding proteins is crucial for male germline development. Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), an RNA binding protein, is specifically expressed in human and mouse male gonads and is involved in manifold biological processes and tumorigenesis. However, the function of IGF2BP1 in mammalian spermatogenesis remains poorly understood. Herein, we generated an Igf2bp1 conditional knockout mouse model using Nanos3-Cre. Germ cell deficiency of Igf2bp1 in mice caused spermatogenic defects in an age-dependent manner, resulting in decreased numbers of undifferentiated spermatogonia and increased germ cell apoptosis. Immunoprecipitation-mass spectrometry analysis revealed that ELAV-like RNA binding protein 1, a well-recognized mRNA stabilizer, interacted with IGF2BP1. Single cell RNA-sequencing showed distinct mRNA profiles in spermatogonia from conditional knockout versus wide type mice. Further research showed that IGF2BP1 plays a vital role in the modulation of spermatogenesis by regulating Lin28a mRNA, which is essential for clonal expansion of undifferentiated spermatogonia. Thus, our results highlight the crucial effects of IGF2BP1 on spermatogonia for the long-term maintenance of spermatogenesis.

METTL3-mediated m6A modification enhances lncRNA H19 stability to promote endothelial cell inflammation and pyroptosis to aggravate atherosclerosis.

This study explored the impact of N6-methyladenosine (m6A) modification on the regulation of long noncoding RNA (lncRNA) and atherosclerosis progression. An atherosclerosis cell model was established by treating human aortic endothelial cells (HAECs) with oxidized low-density lipoprotein. Additionally, an atherosclerotic animal model was developed using ApoE-/- C57BL/6 male mice fed a high-fat diet. Both models were employed to assess the expression changes of proteins associated with m6A modification. First, the effect of m6A modification writer protein methyltransferase-like 3 (METTL3) knockdown on changes in the level of pyroptosis in HAECs was investigated, and bioinformatic analysis confirmed that lncRNA H19 (H19) was the potential target of m6A modification. RNA-binding protein immunoprecipitation assays were subsequently performed to explore the interaction between H19 and the m6A writer protein METTL3, as well as the reader protein recombinant insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). Finally, the effect of H19 expression on pyroptosis levels in HAECs was evaluated. In the aortas of atherosclerosis mice, overall m6A levels were significantly elevated compared with controls (p < .05), with METTL3 and METTL14 mRNA and protein levels notably increased (p < .05). Similarly, ox-LDL-treated HAECs showed a significant rise in m6A levels, along with increased METTL3 and METTL14 expression (p < .05). METTL3 knockdown in HAECs led to decreased pyroptosis, as evidenced by reduced lactate dehydrogenase release and lower levels of IL-1β, IL-18, and IL-6 (p < .05). Overexpression of H19 reversed these effects, indicating METTL3's role in promoting atherosclerosis by stabilizing H19 through m6A modification. H19 was the primary target lncRNA molecule of METTL3-mediated m6A modification in the pathogenesis of atherosclerosis. METTL3-mediated m6A modification regulated H19 expression, thereby aggravating atherosclerosis by activating pyroptosis.

IGF2BP1 phosphorylation in the disordered linkers regulates ribonucleoprotein condensate formation and RNA metabolism.

The insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) is a conserved RNA-binding protein that regulates RNA stability, localization and translation. IGF2BP1 is part of various ribonucleoprotein (RNP) condensates. However, the mechanism that regulates its assembly into condensates remains unknown. By using proteomics, we demonstrate that phosphorylation of IGF2BP1 at S181 in a disordered linker is regulated in a stress-dependent manner. Phosphomimetic mutations in two disordered linkers, S181E and Y396E, modulate RNP condensate formation by IGF2BP1 without impacting its binding affinity for RNA. Intriguingly, the S181E mutant, which lies in linker 1, impairs IGF2BP1 condensate formation in vitro and in cells, whereas a Y396E mutant in the second linker increases condensate size and dynamics. Structural approaches show that the first linker binds RNAs nonspecifically through its RGG/RG motif, an interaction weakened in the S181E mutant. Notably, linker 2 interacts with IGF2BP1's folded domains and these interactions are partially impaired in the Y396E mutant. Importantly, the phosphomimetic mutants impact IGF2BP1's interaction with RNAs and remodel the transcriptome in cells. Our data reveal how phosphorylation modulates low-affinity interaction networks in disordered linkers to regulate RNP condensate formation and RNA metabolism.

The m6A reader IGF2BP1 contributes to the activation of hepatic stellate cells through facilitating TUBB4B mRNA stabilization.

The m6A reader insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) is involved in multiple pathophysiological processes through enhanced expression of the proteins encoded by their target mRNAs. However, the functional role of IGF2BP1-mediated m6A in liver fibrosis remains elusive. Here, we report that IGF2BP1 is highly expressed in activated hepatic stellate cells (HSCs), the major driver of fibrogenesis, and TUBB4B is identified as a potential target of IGF2BP1 by re-analysis of the RNA-seq, RIP-seq, and m6A-seq data. The relevant findings were subsequently demonstrated by a series of molecular and cellular evidences. The knockdown of IGF2BP1 or TUBB4B and pharmacological inhibition of TUBB4B by mebendazole treatments significantly suppress the proliferation, migration, and activation of HSCs. Mechanistically, IGF2BP1 upregulates TUBB4B expression through stabilizing TUBB4B in an m6A-dependent manner, and TUBB4B induces liver fibrosis by activating the FAK signaling pathway. Collectively, our results indicate that targeting IGF2BP1/TUBB4B/FAK axis in HSCs could be a promising therapeutic approach for liver fibrosis.