Y-box Protein Research Articles

SOX7 inhibits the malignant progression of bladder cancer via the DNMT3B/CYGB axis

Bladder cancer (BCa) stands out as a highly prevalent malignant tumor affecting the urinary system. The Sex determining region Y-box protein family is recognized for its crucial role in BCa progression. However, the effect of Sex determining region Y-box 7 (SOX7) on BCa progression has not been fully elucidated. Herein, RNA-sequencing, western blot (WB), immunohistochemistry (IHC), immunofluorescence (IF) and tissue microarray were utilized to assess SOX7 expression in vitro and in vivo. Additionally, SOX7 expression, prognosis, and SOX7 + cytoglobin (CYGB) score were analyzed using R software. In vitro and vivo experiments were performed with BCa cell lines to validate the effect of SOX7 knockdown and overexpression on the malignant progression of BCa. The results showed that SOX7 exhibits low expression in BCa. It functions in diverse capacities, inhibiting the proliferative, migratory, and invasive capabilities of BCa. In addition, the experimental database demonstrated that SOX7 binds to the promoter of DNA methyltransferase 3 beta (DNMT3B), leading to the transcriptional inhibition of DNMT3B. This subsequently results in a reduced methylation of CYGB promoter, ultimately inhibiting the tumor progression of BCa. SOX7 + CYGB scores were significantly linked to patient prognosis. In conclusion, SOX7 inhibits the malignant progression of BCa via the DNMT3B/CYGB axis. Additionally, the SOX7 + CYGB score is capable of predicting the prognostic outcomes of BCa patients. Therefore, SOX7 and CYGB may play an important role in the progression of bladder cancer, and they can be used as prognostic markers of bladder cancer patients.

METTL3 mediates SOX5 m6A methylation in bronchial epithelial cells to attenuate Th2 cell differentiation in T2 asthma

ObjectiveAsthma, a chronic inflammatory disease in which type 2 T helper cells (Th2) play a causative role in the development of T2 asthma. N6-methyladenosine (m6A) modification, an mRNA modification, and methyltransferase-like 3 (METTL3) is involved in the development of T2 asthma by inhibiting Th2 cell differentiation. Sex determining region Y-box protein 5 (SOX5) is involved in regulating T cell differentiation, but its role in T2 asthma was unclear. The objective of this study was to explore the role of METTL3 and SOX5 in T2 asthma and whether there is an interaction between the two. Materials and methodsAdults diagnosed with T2 asthma (n = 14) underwent clinical information collection and pulmonary function tests. In vivo and in vitro T2 asthma models were established using female C57BL/6 mice and human bronchial epithelial cells (HBE). The expressions of METTL3 and SOX5 were detected by Western blot and qRT-PCR and Western blot. Th2 cell differentiation was determined by flow cytometry and IL-4 level was detected by ELISA. m6A methylation level was determined by m6A quantitative assay. The relationship between METTL3 expression and clinical parameters was determined by Spearman rank correlation analysis. The function of METTL3 and SOX5 genes in asthma was investigated in vitro and in vivo. The RNA immunoprecipitation assay detected the specific interaction between METTL3 and SOX5. ResultsPatients with T2 asthma displayed lower METTL3 levels compared to healthy controls. Within this group, a negative correlation was observed between METTL3 and Th2 cells, while a positive correlation was noted between METTL3 and clinical parameters as well as Th1 cells. In both in vitro and in vivo models representing T2 asthma, METTL3 levels decreased significantly, while SOX5 levels showed the opposite trend. Overexpression of METTL3 gene in HBE cells significantly inhibited Th2 cell differentiation and increased m6A methylation activity. From a mechanism perspective, low METTL3 negatively regulates SOX5 expression through m6A modification dependence, while high SOX5 expression is positively associated with T2 asthma severity. Cell transfection experiments confirmed that METTL3 regulates Th2 cell differentiation and IL-4 release through SOX5. ConclusionsOverall, our results indicate that METTL3 alleviates Th2 cell differentiation in T2 asthma by modulating the m6A methylation activity of SOX5 in bronchial epithelial cells. This mechanism could potentially serve as a target for the prevention and management of T2 asthma.

Glomerular-tubular crosstalk via cold shock Y-box binding protein-1 in the kidney

Glomerular-tubular crosstalk within the kidney has been proposed, but the paracrine signals enabling this remain largely unknown. The cold-shock protein Y-box binding protein 1 (YBX1) is known to regulate inflammation and kidney diseases but its role in podocytes remains undetermined. Therefore, we analyzed mice with podocyte specific Ybx1 deletion (Ybx1ΔPod). Albuminuria was increased in unchallenged Ybx1ΔPod mice, which surprisingly was associated with reduced glomerular, but enhanced tubular damage. Tubular toll-like receptor 4 (TLR4) expression, node-like receptor protein 3 (NLRP3) inflammasome activation and kidney inflammatory cell infiltrates were all increased in Ybx1ΔPod mice. Invitro, extracellular YBX1 inhibited NLRP3 inflammasome activation in tubular cells. Co-immunoprecipitation, immunohistochemical analyses, microscale cell-free thermophoresis assays, and blunting of the YBX1-mediated TLR4-inhibition by a unique YBX1-derived decapeptide suggests a direct interaction of YBX1 and TLR4. Since YBX1 can be secreted upon post-translational acetylation, we hypothesized that YBX1 secreted from podocytes can inhibit TLR4 signaling in tubular cells. Indeed, mice expressing a non-secreted YBX1 variant specifically in podocytes (Ybx1PodK2A mice) phenocopied Ybx1ΔPod mice, demonstrating a tubular-protective effect of YBX1 secreted from podocytes. Lipopolysaccharide-induced tubular injury was aggravated in Ybx1ΔPod and Ybx1PodK2A mice, indicating a pathophysiological relevance of this glomerular-tubular crosstalk. Thus, our data show that YBX1 is physiologically secreted from podocytes, thereby negatively modulating sterile inflammation in the tubular compartment, apparently by binding to and inhibiting tubular TLR4 signaling. Hence, we have uncovered an YBX1-dependent molecular mechanism of glomerular-tubular crosstalk.

Mitochondrial YBX1 promotes cancer cell metastasis by inhibiting pyruvate uptake

Abstract Pyruvate is an essential fuel for maintaining the tricarboxylic acid (TCA) cycle in the mitochondria. However, the precise mole­cular mechanism of pyruvate uptake by mitochondrial pyruvate carrier (MPC) is largely unknown. Here, we report that the DNA/RNA-binding protein Y-box binding protein 1 (YBX1) is localized to the mitochondrial inter-membrane space by its C-terminal domain (CTD) in cancer cells. In mitochondria, YBX1 inhibits pyruvate uptake by associating with MPC1/2, thereby suppressing pyruvate-dependent TCA cycle flux. This association, in turn, promotes MPC-mediated glutaminolysis and histone lactylation. Our findings reveal that the YBX1-MPC axis exhibits a positive correlation with metastatic potential, while does not affect cell proliferation in both cultured cells and tumor xenografts. Therefore, the restricted pyruvate uptake into mitochondria potentially represents a hallmark of metastatic capacity, suggesting that the YBX1-MPC axis is a therapeutic target for combating cancer metastasis.

Genetic and histological relationship between pheromone-secreting tissues of the musk gland and skin of juvenile Chinese forest musk deer (Moschus berezovskii Flerov, 1929).

BACKGROUND: The musk glands of adult male Chinese forest musk deer (Moschus berezovskii Flerov, 1929) (FMD), which are considered as special skin glands, secrete a mixture of sebum, lipids, and proteins into the musk pod. Together, these components form musk, which plays an important role in attracting females during the breeding season. However, the relationship between the musk glands and skin of Chinese FMD remains undiscovered. Here, the musk gland and skin of Chinese FMD were examined using histological analysis and RNA sequencing (RNA-seq), and the expression of key regulatory genes was evaluated to determine whether the musk gland is derived from the skin. METHODS: A comparative analysis of musk gland anatomy between juvenile and adult Chinese FMD was conducted. Then, based on the anatomical structure of the musk gland, skin tissues from the abdomen and back as well as musk gland tissues were obtained from three juvenile FMD. These tissues were used for RNA-seq, hematoxylin-eosin (HE) staining, immunohistochemistry (IHC), western blot (WB), and quantitative real-time polymerase chain reaction (qRT-PCR) experiments. RESULTS: Anatomical analysis showed that only adult male FMD had a complete glandular organ and musk pod, while juvenile FMD did not have any well-developed musk pods. Transcriptomic data revealed that 88.24% of genes were co-expressed in the skin and musk gland tissues. Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis found that the genes co-expressed in the abdomen skin, back skin, and musk gland were enriched in biological development, endocrine system, lipid metabolism, and other pathways. Gene Ontology (GO) enrichment analysis indicated that the genes expressed in these tissues were enriched in biological processes such as multicellular development and cell division. Moreover, the Metascape predictive analysis tool demonstrated that genes expressed in musk glands were skin tissue-specific. qRT-PCR and WB revealed that sex-determining region Y-box protein 9 (Sox9),Caveolin-1 (Cav-1), andandrogen receptor (AR) were expressed in all three tissues, although the expression levels differed among the tissues. According to the IHC results, Sox9 and AR were expressed in the nuclei of sebaceous gland, hair follicle, and musk gland cells, whereas Cav-1 was expressed in the cell membrane. CONCLUSIONS: The musk gland of Chinese FMD may be a derivative of skin tissue, and Sox9, Cav-1, and AR may play significant roles in musk gland development.

17-DMAG ameliorates neuroinflammation and BBB disruption via SOX5 mediated PI3K/Akt pathway after intracerebral hemorrhage in rats

Intracerebral hemorrhage (ICH) can result in secondary brain injury due to inflammation and breakdown of the blood–brain barrier (BBB), which are closely associated with patient prognosis. The potential of the heat shock protein 90 (Hsp90) inhibitor 17-DMAG in promoting neuroprotection has been observed in certain vascular diseases. However, the precise role of 17-DMAG treatment in ICH is not yet fully understood. In this study, we found that treatment with 17-DMAG (5 mg/kg) effectively reduced hematoma expansion and resulted in improved neurological outcomes. Meanwhile, the injection of 17-DMAG had a positive effect on reducing BBB disruption in rats with ICH. This effect was achieved by increasing the levels of BBB tight junction proteins (TJPs) such as zo-1, claudin-5, and occludin. As a result, the leakage of EB extravasation, brain edema and IgG in the peri-hematoma tissue were reduced. Furthermore, the injection of 17-DMAG decreased the infiltration of neutrophils into the brain tissues surrounding the hematoma in ICH rats and also reduced the production of proinflammatory cytokines IL-6 and TNF-α. Next, we used integrative mass spectrometry (MS) and molecular docking analysis to confirm that sex determining region Y-box protein 5 (SOX5) is a potential direct target of 17-DMAG in ICH. SOX5 encodes a positive regulator of the PI3K/Akt axis, and treatment with 17-DMAG resulted in a noticeable increase in SOX5 accumulation. To further investigate the role of SOX5, we employed virus-regulated SOX5 silencing and found that suppressing SOX5 blocked the ability of 17-DMAG to suppress neutrophil trafficking. Additionally, silencing SOX5 blocked the protective effects of 17-DMAG on the BBB by inhibiting PI3K, p-Akt, and BBB TJPs levels, which led to an increase in EB and IgG leakage in the peri-hematoma tissue after ICH. Similarly, when SOX5 was knocked down, the protective effects of 17-DMAG were lost. Overall, the results of our study indicate that the injection of 17-DMAG has the potential to mitigate neuroinflammation and prevent the disruption of the BBB caused by ICH, resulting in improved neurological outcomes in rats. These positive effects are attributed to the regulation of SOX5 and activation of the PI3K/Akt pathway. These findings highlight the possibility of targeting SOX5 and the PI3K/Akt pathway as a novel therapeutic approach for ICH.

Noninvasive Assessment of Renal Fibrosis of Chronic Kidney Disease in Rats by [68Ga]Ga-FAPI-04 Small Animal PET/CT and Biomarkers

Renal fibrosis is the most common pathological feature and common pathway of progression in chronic kidney disease (CKD). We evaluated [68Ga]Ga-FAPI-04 small animal positron emission tomography/computed tomography (PET/CT) and biomarkers as noninvasive assessments of renal fibrosis (RF) in CKD rats to generate new ideas for clinical diagnosis. A rat model of renal fibrosis was administered adenine by gavage (n = 28), and the control group was given 0.9% NaCl by gavage (n = 20). At different time points (weeks 1, 2, 4, and 6), five rats were randomly selected from the two groups for [68Ga]Ga-FAPI-04 small animal PET/CT imaging. At the same time, the expression of Fibroblast activation protein (FAP) in renal tissue and the expression levels of type III procollagen N-terminal peptide (PIIINP), transforming growth factor (TGF-β1), Klotho, and sex-determining region Y-box protein 9 (SOX9) in blood and urine were determined. FAP was highly expressed in the renal tissue of rats in the CKD group and expression increased with the progression of renal fibrosis. [68Ga]Ga-FAPI-04 small animal PET/CT examination showed that the uptake of radioactive tracers in the CKD group was higher than that in the control group, and SUVmax (r = 0.9405) and target-to-background ratio (TBR) (r = 0.9392) were positively correlated with renal fibrosis. The serum levels of PIIINP, TGF-β1, and SOX9 in CKD rats were significantly higher than those in the control group and were positively correlated with RF (r = 0.8234, r = 0.7733, and r = 0.7135, respectively) and SUVmax (r = 0.8412, r = 0.7763, and r = 0.6814, respectively). Compared with the control group, the level of serum Klotho decreased and was negatively correlated with RF (r = -0.6925) and SUVmax (r = -0.6322). Compared with the control group, the levels of PIIINP and TGF-β1 in urine were positively correlated with RF (r = 0.8127 and r = 0.8077, respectively) and SUVmax (r = 0.8400 and r = 0.8177, respectively). Urine Klotho decreased compared with the control group and was negatively correlated with RF (r = -0.5919) and SUVmax (r = -0.5995). The change in urine SOX9 was not statistically significant. In conclusion, compared with renal biopsy, [68Ga]Ga-FAPI-04 small animal PET/CT shows renal fibrosis quickly and noninvasively. PIIINP, TGF-β1, and Klotho in serum and urine may be used as biomarkers of RF, and serum SOX9 is expected to become a new diagnostic biomarker of RF.

DNA binding protein YB-1 is a part of the neutrophil extracellular trap mediation of kidney damage and cross-organ effects

Open-heart surgery is associated with high morbidity, with acute kidney injury (AKI) being one of the most commonly observed postoperative complications. Following open-heart surgery, in an observational study we found significantly higher numbers of blood neutrophils in a group of 13 patients with AKI compared to 25 patients without AKI (AKI: 12.9±5.4×109 cells/L; non-AKI: 10.1±2. 9×109 cells/L). Elevated serum levels of neutrophil extracellular trap (NETs) components, such as dsDNA, histone 3, and DNA binding protein Y-box protein (YB)-1, were found within the first 24 hours in patients who later developed AKI. We could demonstrate that NET formation and hypoxia triggered the release of YB-1, which was subsequently shown to act as a mediator of kidney tubular damage. Experimentally, in two models of AKI mimicking kidney hypoperfusion during cardiac surgery (bilateral ischemia/reperfusion (I/R) and systemic lipopolysaccharide (LPS) administration), a neutralizing YB-1 antibody was administered to mice. In both models, prophylactic YB-1 antibody administration significantly reduced the tubular damage (damage score range 1-4, the LPS model: non-specific IgG control, 0.92±0.23; anti-YB-1 0.65±0.18; and in the I/R model: non-specific IgG control 2.42±0.23; anti-YB-1 1.86±0.44). Even in a therapeutic, delayed treatment model, antagonism of YB-1 ameliorated AKI (damage score, non-specific IgG control 3.03±0.31; anti-YB-1 2.58±0.18). Thus, blocking extracellular YB-1 reduced the effects induced by hypoxia and NET formation in the kidney and significantly limited AKI, suggesting that YB-1 is part of the NET formation process and an integral mediator of cross-organ effects.

Phosphatase protector alpha4 (α4) is involved in adipocyte maintenance and mitochondrial homeostasis through regulation of insulin signaling

Insulin signaling is mediated via a network of protein phosphorylation. Dysregulation of this network is central to obesity, type 2 diabetes and metabolic syndrome. Here we investigate the role of phosphatase binding protein Alpha4 (α4) that is essential for the serine/threonine protein phosphatase 2A (PP2A) in insulin action/resistance in adipocytes. Unexpectedly, adipocyte-specific inactivation of α4 impairs insulin-induced Akt-mediated serine/threonine phosphorylation despite a decrease in the protein phosphatase 2A (PP2A) levels. Interestingly, loss of α4 also reduces insulin-induced insulin receptor tyrosine phosphorylation. This occurs through decreased association of α4 with Y-box protein 1, resulting in the enhancement of the tyrosine phosphatase protein tyrosine phosphatase 1B (PTP1B) expression. Moreover, adipocyte-specific knockout of α4 in male mice results in impaired adipogenesis and altered mitochondrial oxidation leading to increased inflammation, systemic insulin resistance, hepatosteatosis, islet hyperplasia, and impaired thermogenesis. Thus, the α4 /Y-box protein 1(YBX1)-mediated pathway of insulin receptor signaling is involved in maintaining insulin sensitivity, normal adipose tissue homeostasis and systemic metabolism.

SOX5 promotes cell growth and migration through modulating the DNMT1/p21 pathway in bladder cancer.

Bladder cancer (BC) is one of the most prevalent and life-threatening cancers among the male population worldwide. Sex determining region Y-box protein 5 (SOX5) plays important roles in a variety of human cancers. However, little research has been conducted on the function and underlying mechanism of SOX5 in BC. In the present study, we first reveal the increased expression of SOX5 in BC tissues and in vitro cells lines. Second, we discover that inhibition of SOX5 inhibits cell growth and migration but promotes cell apoptosis. Meanwhile, ectopic SOX5 expression stimulates cell growth and migration in BC cells. Then, we show that suppressing SOX5 inhibits the expression of DNA methyltransferase 1 (DNMT1), and that overexpressing DNMT1 alleviates the cell progress of BC cells inhibited by SOX5. Furthermore, we demonstrate that DNMT1 inhibits p21 expression by affecting DNA methylation of the p21 promoter. Collectively, we demonstrate that SOX5 exerts its functions in BC cells by modulating the SOX5/DNMT1/p21 pathway. Finally, we demonstrate that SOX5 knockdown inhibits xenograft tumor growth in vivo. In conclusion, our study elucidates the oncogenic role of SOX5 and its underlying molecular mechanism in BC, and reveals a novel pathway which has the potential to serve as a diagnostic biomarker and therapeutic target for BC.

Long noncoding RNA Gm31629 promotes bone regeneration by maintaining bone marrow mesenchymal stem cells activity.

BackgroundLong noncoding RNA Gm31629 can regulate hypothalamic neural stem cells (htNSCs) senescence and the aging process. However, the effect of Gm31629 on the senescence of bone marrow mesenchymal stem cells (BMSCs) and bone regeneration is unclear. In the present study, we investigated the effects of Gm31629 on the senescence of BMSCs and bone regeneration.MethodsGm31629 knockout (Gm31629-KO) and wild-type (WT) mice were used to establish a bone regeneration model. The Brdu labelling, CCK8 assay, wound healing assay, β-gal staining and osteogenic differentiation assay were used to assess the effects of Gm31629 on the functions of BMSCs. Micro-computed tomography (CT), histochemical and immunohistochemical staining were used to evaluate the ability of bone regeneration. The mimic of Gm31629, theaflavin 3-gallate, was used to investigate its role on the senescence of BMSCs and bone regeneration.ResultsThe expression of Gm31629 reduced in BMSCs of middle-aged mice was compared with that of young mice. The deletion of Gm31629 was sufficient to drive the senescence of BMSCs, resulting in impaired bone regeneration in mice. Mechanistically, Gm31629 could interact with Y-box protein 1(YB-1) and delay its degradation, decreasing the transcription of p16INK4A of BMSCs. We also found that theaflavin 3-gallate could alleviate the senescence of BMSCs and promote bone regeneration in middle-aged mice.ConclusionThese results indicated that Gm31629 played an important role on BMSCs senescence and bone regeneration and provided a therapeutic target to promote bone regeneration.

ERK/RSK-mediated phosphorylation of Y-box binding protein-1 aggravates diabetic cardiomyopathy by suppressing its interaction with deubiquitinase OTUB1

Diabetic cardiomyopathy (DCM) is a major complication of diabetes, but its underlying mechanisms still remain unclear. The multifunctional protein Y-box binding protein-1 (YB-1) plays an important role in cardiac pathogenesis by regulating cardiac apoptosis, cardiac fibrosis, and pathological remodeling, whereas its role in chronic DCM requires further investigation. Here, we report that the phosphorylation of YB-1 at serine102 (S102) was markedly elevated in streptozotocin-induced diabetic mouse hearts and in high glucose-treated cardiomyocytes, whereas total YB-1 protein levels were significantly reduced. Coimmunoprecipitation experiments showed that YB-1 interacts with the deubiquitinase otubain-1, but hyperglycemia-induced phosphorylation of YB-1 at S102 diminished this homeostatic interaction, resulting in ubiquitination and degradation of YB-1. Mechanistically, the high glucose-induced phosphorylation of YB-1 at S102 is dependent on the upstream extracellular signal-regulated kinase (ERK)/Ras/mitogen-activated protein kinase (p90 ribosomal S6 kinase [RSK]) signaling pathway. Accordingly, pharmacological inhibition of the ERK pathway using the upstream kinase inhibitor U0126 ameliorated features of DCM compared with vehicle-treated diabetic mice. We demonstrate that ERK inhibition with U0126 also suppressed the phosphorylation of the downstream RSK and YB-1 (S102), which stabilized the interaction between YB-1 and otubain-1 and thereby preserved YB-1 protein expression in diabetic hearts. Taken together, we propose that targeting the ERK/RSK/YB-1 pathway could be a potential therapeutic approach for treating DCM.

Long noncoding RNA Gm31629 protects against mucosal damage in experimental colitis via YB-1/E2F pathway

Mucosal healing is a key treatment goal for inflammatory bowel disease, and adequate epithelial regeneration is required for an intact gut epithelium. However, the underlying mechanism for mucosal healing is unclear. Long noncoding RNAs (lncRNAs) have been reported to be involved in the development of inflammatory bowel disease. Here, we report that a lncRNA named Gm31629 decreased in intestinal epithelial cells in response to inflammatory stimulation. Gm31629 deficiency led to exacerbated intestinal inflammation and delayed epithelial regeneration in dextran sulfate sodium–induced (DSS-induced) colitis model. Mechanistically, Gm31629 promoted E2F pathways and cell proliferation by stabilizing Y-box protein 1 (YB-1), thus facilitating epithelial regeneration. Genetic overexpression of Gm31629 protected against DSS-induced colitis in vivo. Theaflavin 3-gallate, a natural compound mimicking Gm31629, alleviated DSS-induced epithelial inflammation and mucosal damage. These results demonstrate an essential role of lncRNA Gm31629 in linking intestinal inflammation and epithelial cell proliferation, providing a potential therapeutic approach to inflammatory bowel disease.

RNA-binding protein YBX1 promotes brown adipogenesis and thermogenesis via PINK1/PRKN-mediated mitophagy.

Promoting the thermogenic function of brown adipose tissue (BAT) is a promising strategy to combat obesity and metabolic disorders. While much is known about the transcriptional regulation of BAT activation, however, the underlying mechanism of post-transcriptional control by RNA binding proteins remains largely unknown. Here, we found that RNA binding protein Y-box binding protein 1 (YBX1) expression was abundant in BAT and induced by cold exposure and a β-adrenergic agonist in mice. Loss-of-function experiments showed that YBX1 deficiency inhibited mouse primary brown adipocyte differentiation and thermogenic function. Further study showed that YBX1 positively regulates thermogenesis through enhancing mitophagy. Mechanistically, RNA immunoprecipitation identified that YBX1 directly targeted the transcripts of PTEN-induced kinase 1 (Pink1) and parkin RBR E3 ubiquitin-protein ligase (Prkn), two key regulators of mitophagy. RNA decay assay proved that loss of YBX1 decreased mRNA stability of Pink1 and Prkn, leading to reduced protein expression, thereby alleviating mitophagy and inhibiting thermogenic program. Importantly, in vivo experiments demonstrated that YBX1 overexpression in BAT promoted thermogenesis and mitophagy in mice. Collectively, our results reveal novel insight into the molecular mechanism of YBX1 in post-transcriptional regulation of PINK1/PRKN-mediated mitophagy and highlight the critical role of YBX1 in brown adipogenesis and thermogenesis.

Circular CDC like kinase 1 suppresses cell apoptosis through miR-18b-5p/Y-box protein 2 axis in oral squamous cell carcinoma

ABSTRACT This study aimed to explore the role of circular-CDC like kinase 1 (circ-CLK1) in the pathogenesis of oral squamous cell carcinoma (OSCC). Circ-CLK1 expression levels were detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The effects of circ-CLK1 knockdown on the viability and apoptosis of OSCC cells were determined using the cell counting kit-8 (CCK-8) assay, EdU staining, flow cytometry, and Western blotting. StarBase and TargetScan were used to predict targeting relationships, which were then confirmed by the dual luciferase reporter assay and RNA pull-down assay. We found that the expression of circ-CLK1 was significantly higher in OSCC patients and cell lines. Inhibition of circ-CLK1 reduced the viability and proliferation of OSCC cells while enhancing their apoptosis. However, inhibiting miR-18b-5p or overexpression of Y-box protein 2 (YBX2) can reverse the effect of circ-CLK1 knockdown on OSCC cells. Therefore, circ-CLK1 inhibited the apoptosis of OSCC cells through the miR-18b-5p/YBX2 axis, and these findings suggest that circ-CLK1 could be a potential therapeutic target for OSCC patients.

29-OR: Regulatory Loop of IR Signaling for Adipocyte Mitochondrial Homeostasis

Insulin and IGF1 signaling initiate Tyr phosphorylation of IR/IGF1R and adaptor IRSs, leading to Ser/Thr phosphorylation of PI3K/AKT and MAPK/ERK pathways essential for the maintenance of brown and white adipose tissues (BAT and WAT). Lack of insulin-signaling in adipose tissues caused metabolic disease in mice with hyperglycemia, hyperlipidemia, and fatty liver, but the mice recovered adipose tissues by regenerating adipocytes. We addressed the mechanism of how adipose tissues regenerate using adipocyte-specific and inducible IR/IGF1R knockout mice (Ai-DKO). We identified a reduction of protein phosphatase protector, α4, which can regulate Ser/Thr phosphorylation of down-stream insulin signaling. shRNA-mediated α4 knockdown (KD) in BAT affected not only insulin-stimulated down-stream Ser/Thr phosphorylation of AKT (S473) but also affected up-stream Tyr phosphorylation of IRβ (Y1162/1163) and IRS1 (Y612). Proteomics analysis of α4-binding molecule identified Y-box protein 1 (YBX1), which functions as a transcription factor for Tyr phosphatase PTP1B. We observed increased PTP1B expression in α4-KO preadipocytes. As the impact of α4 in vivo, inducible adipocyte-specific α4 KO (Ai-α4 KO) mice with tamoxifen-inducible Cre-ERT2 transgene showed cold intolerance, diabetes, ectopic lipid accumulation in the liver, and pancreatic islet hyperplasia, with an increase of C18: 0-ceramide in WAT and BAT. RNA-seq showed a marked reduction of genes associated with mitochondrial fatty acid oxidation and the increases in inflammatory cytokine pathways in Ai-α4 KO WAT and BAT, implicating the cause of fat tissue loss was the increased adipocyte apoptosis. The α4 to PTP1B loop is critical for fat tissue maintenance by regulating IR tyrosine phosphorylation. Disclosure M. Sakaguchi: None. S. Okagawa: None. Y. Okubo: None. K. Fukuda: None. M. Igata: None. J. Kawashima: None. T. Kondo: None. E. Araki: Other Relationship; Self; Daiichi Sankyo Company, Limited, Eli Lilly Japan K. K., Mitsubishi Tanabe Pharma Corporation, MSD K. K., Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Pharma Ltd., Sanofi K. K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Speaker’s Bureau; Self; Astellas Pharma Inc., AstraZeneca K. K., Kowa Company, Ltd. Funding Japan Society for the Promotion of Science; Merck Sharp & Dohme Corp; Takeda Foundation

MO430MASS-SPECTROMETRIC IDENTIFICATION OF POST-TRANSLATIONAL GUANIDINYLATED PROTEINS IN THE CONTEXT OF SYSTEMIC LUPUS ERYTHEMATOSUS

Abstract Background and Aims With continuous identification of post-translational modified isoforms of proteins, it is becoming increasingly clear that post-translational modifications limit or modify the biological functions of native proteins are majorly involved in development of various chronic disease. This is mostly due to technically advanced molecular identification and quantification methods, mainly based on mass spectrometry. Mass spectrometry has become one of the most powerful tools for the identification of lipids. Method In this study, we used sophisticated high-resolution mass-spectrometric methods to analyze the soluble ligand of receptor Notch-3, namely the Y-box protein (YB)-1, in serum from systemic lupus erythematosus (SLE) patients. In addition, kidneys of lupus-prone (MRL.lpr) mice were analyzed by mass-spectrometric imaging techniques to identify the underlying pathomechanisms. Serum YB-1 was isolated by chromatographic methods, afterwards digested by trypsin and analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The kidneys were fixed in paraffin, then kidney sections were deparaffinized, tryptic digested and analyzed by mass-spectrometric imaging techniques. Mass-spectrometry of extracellular YB-1 in SLE patient serum revealed post-translational guanidinylation of two lysine’s within the highly conserved cold shock domain (CSD) of the YB-1 protein (YB-1-2G). Patients with increased disease activity and those with active renal involvement (lupus nephritis, LN) had a higher degree of dual-guanidinylation within the CSD. Of note, at least one of these modifications was present in all analyzed LN patients, whereas single-guanidinylated YB-1 was present in only one and double modification in none of the control individuals. Mass-spectrometric imaging analyses specifically localized YB-1-2G and increases Notch-3 expression in kidney sections from MRL.lpr mice. Results The data from this study clearly demonstrate the high potential of high-resolution mass spectrometric methods as well as mass spectrometric imaging techniques to identify pathomechanisms of diseases like SLE/LN.

CircABCC3 knockdown inhibits glioblastoma cell malignancy by regulating miR-770-5p/SOX2 axis through PI3K/AKT signaling pathway

BackgroundCircular RNAs (circRNAs) are found to regulate glioblastoma evolution. However, the role of circ-ATP binding cassette subfamily C member 3 (circABCC3) in glioblastoma process is still unknown. In this study, the effects of circABCC3 on glioblastoma tumorigenesis and underlying mechanism were revealed. MethodsThe expression levels of circABCC3, microRNA-770-5p (miR-770-5p) and sex determining region Y-box protein 2 (SOX2) mRNA were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway-related proteins and SOX2 protein was detected by western blot analysis. Cell proliferation and invasion were severally investigated by cell colony formation and transwell invasion assays. Cell migration was demonstrated by transwell migration and wound-healing assays. Cell apoptosis was revealed by flow cytometry analysis. Tube formation was investigated by tube formation assay. The associated relationship between miR-770-5p and circABCC3 or SOX2 was predicted by starbase or targetscan online database, and identified by dual-luciferase reporter assay, RNA immunoprecipitation assay or RNA pull-down assay. The impacts of circABCC3 knockdown on glioblastoma growth in vivo were revealed by in vivo assay. ResultsCircABCC3 and SOX2 expression were dramatically upregulated, while miR-770-5p expression was apparently downregulated in glioblastoma tissues and cells compared with control groups. CircABCC3 expression was higher in stage III glioblastoma tissues than in stage I + II glioblastoma tissues with close correlation with tumor-node-metastasis (TNM) stage. CircABCC3 absence inhibited cell proliferation, migration, invasion, tube formation and the activation of PI3K/AKT pathway, whereas induced cell apoptosis in glioblastoma. Additionally, circABCC3 acted as a sponge for miR-770-5p, and miR-770-5p targeted SOX2. MiR-770-5p inhibitors impaired the impacts of circABCC3 silencing on glioblastoma progression, angiogenesis and PI3K/AKT pathway. Furthermore, circABCC3 knockdown repressed tumor growth in vivo. ConclusionCircABCC3 regulated glioblastoma development via miR-770-5p/SOX2 axis through PI3K/AKT pathway. This finding lays a theoretical foundation for studying circRNA-directed therapy for glioblastoma.

CircSEC24A promotes IL-1β-induced apoptosis and inflammation in chondrocytes by regulating miR-142-5p/SOX5 axis.

Osteoarthritis (OA) is a common joint disease. Currently, many studies have revealed that circular RNAs (circRNAs) are strongly related to the occurrence and development of diseases. Hence, we aimed to further elucidate the role and molecular mechanism of circRNA SEC24 homolog A, COPII coat complex component (circSEC24A) in OA. Chondrocytes were treated with interleukin-1β (IL-1β) to establish OA cell model in vitro. The expression levels of circSEC24A, microRNA-142-5p (miR-142-5p), and sex-determining region Y-box protein 5 (SOX5) were determined by quantitative real-time polymerase chain reaction. MTT and colony formation assays were used to determine cell proliferation. Cell apoptosis was detected by flow cytometry analysis. The protein levels of inflammatory factors and SOX5 were determined by western blot assay. The relationship between miR-142-5p and circSEC24A or SOX5 was confirmed using dual-luciferase reporter assay and RNA immunoprecipitation assay. CircSEC24A and SOX5 expression were enhanced, while miR-142-5p level was reduced in OA cartilage tissues and chondrocytes. Overexpression of circSEC24A promoted IL-1β-induced injury through decreasing cell proliferation and increasing apoptosis and inflammation in chondrocytes. MiR-142-5p was a direct target of circSEC24A, and its upregulation ameliorated IL-1β-induced injury and abated the effect of oe-circSEC24A in IL-1β-induced chondrocytes. Additionally, SOX5 was a downstream target of miR-142-5p, and its overexpression had a similar role with oe-circSEC24A and reversed the impact of miR-142-5p in IL-1β-induced chondrocytes. CircSEC24A acted as a molecular sponge of miR-142-5p to regulate SOX5 expression in chondrocytes. CircSEC24A aggravated IL-1β-induced injury via modulating miR-142-5p/SOX5 axis, providing possible targets for the clinical diagnosis and treatment of OA.

YBX3 Mediates the Metastasis of Nasopharyngeal Carcinoma via PI3K/AKT Signaling.

The metastasis of nasopharyngeal carcinoma (NPC) is a complex process associated with oncogenic dysfunction, the deciphering of which remains a challenge and requires more in-depth studies. Y-box protein 3 (YBX3) is a DNA/RNA binding protein associated with gene transcription, DNA repair, and the progression of various diseases. However, whether and how YBX3 affects the metastasis of NPC remains unknown. Thus, in this study, we aimed to investigate the role of YBX3 in the metastasis of NPC and determine its underlying mechanism. Interestingly, it was found that the expression of YBX3, which was associated with NPC metastasis, was upregulated in the clinical NPC tissues and cell lines. Moreover, we found that knockdown of YBX3 expression by lentivirus shRNA significantly suppressed NPC cells migration in vitro and metastasis in vivo. Mechanistically, RNA sequencing results suggested that the genes regulated by YBX3 were significantly enriched in cell adhesion molecules, cAMP signaling pathway, calcium signaling pathway, focal adhesion, PI3K/AKT signaling pathway, Ras signaling pathway, Rap1 signaling pathway, NF-κB signaling pathway, and Chemokine signaling pathway. Of these, PI3K/AKT signaling pathway contained the most genes. Accordingly, YBX3 knockdown decreased the activation of PI3K/AKT signaling pathway, thereby inhibit epithelial-to-mesenchymal transition (EMT) and MMP1. These results have demonstrated that YBX3 are involved in the metastasis of NPC through regulating PI3K/AKT signaling pathway, and serve as a potential therapeutic target for patients with NPC.