Chromatin Immunoprecipitation Research Articles

Transcription factor ATF3 aggravates kidney fibrosis by maintaining the state of histone H3 lysine 27 acetylation.

Chronic kidney disease (CKD) is a global health issue, with renal fibrosis being a common pathway in CKD development. Histone modification plays crucial roles in transcriptional regulation, but their pathological functions and mechanisms in CKD are not well understood. We utilized chromatin immunoprecipitation with next-generation DNA sequencing (ChIP-seq) and RNA-seq to evaluate the states and functions of H3 lysine 27 acetylation (H3K27ac) and H3 lysine 4 trimethylation (H3K4me3) in kidney of CKD mice. We identified epigenetic factors regulating H3K27ac through motif analysis. Expression of activating transcription factor 3 (ATF3) in CKD mouse models and patients' kidneys was validated via immunofluorescence staining or Western blot. We further generated the Atf3 deficient (Atf3-/-) mice to explore its effect in kidney function and fibrosis. ChIP-seq of H3K27ac from Atf3-/- CKD mice was employed to validate ATF3's regulatory effects. We explored how ATF3 maintains the state of H3K27ac by integrating the data sources from multiple databases. The states of H3K27ac and H3K4me3 were changed during CKD, and positively correlated with differential gene expression. ATF3 was highly expressed in kidney of both patients and mice with CKD, and co-localized with H3K27ac in genome, epigenetically regulating H3K27ac state. Atf3 deficient in CKD mice significantly ameliorated kidney dysfunction and fibrotic phenotype, and reduced H3K27ac levels at the ATF3 binding sites. Mechanically, ATF3 may recruit the histone acetyltransferases (HATs) network to maintain the H3K27ac state during CKD. ATF3 promotes kidney injury and fibrosis in CKD by maintaining the state of H3k27ac via recruiting HATs network.

Anaerobic metabolism promotes breast cancer survival via Histone-3 Lysine-18 lactylation mediating PPARD axis

Histone lactylation plays a crucial role in cancer progression, but its impact on breast cancer (BC) tumorigenesis is still unclear. We utilized chromatin immunoprecipitation sequencing with H3K18la antibodies, transcriptomics of clinical BC samples, and proteomics and ATAC-seq analyses of in vivo tumors to identify the genes regulated by H3K18la and the transcription factor PPARD. qPCR and Western blot assays were used to detect expressions of molecules. We discovered that H3K18la levels were higher in BC tissues compared to adjacent non-cancerous tissues. H3K18la promoted the expression of PPARD, which in turn influenced the transcription of AKT, but not ILK. ATAC-seq analysis revealed that glycolysis in BC cells enhanced chromatin accessibility. Additionally, we confirmed that HDAC2 and HDAC3 act as “erasers” for H3 lysine lactylation. During the proteomics analysis, AKT-phosphorylation in the aerobic respiration inhibitor group exhibited an apparent disparity and activity. Our study demonstrated that changes in H3K18la in BC and its downstream transcription factor PPARD support cell survival under anaerobic glycolysis conditions. PPARD accelerated cancer proliferation by promoting the transcription and phosphorylation of AKT. This highlights the therapeutic potential of targeting the H3K18la/PPARD/AKT axis in breast cancer, providing new insights into epigenetic regulation and cancer metabolism (Trial registration: The study was approved by the Research Ethics Committee Shandong Provincial Third Hospital (KYLL-2023057; https://www.medicalresearch.org.cn/)).

EZH2 suppresses IR-induced ferroptosis by forming a co-repressor complex with HIF-1α to inhibit ACSL4: Targeting EZH2 enhances radiosensitivity in KDM6A-deficient esophageal squamous carcinoma.

The mutation status of the lysine demethylase 6 A (KDM6A), a gene antagonist to Enhancer of zeste homolog 2 (EZH2), is closely related to the therapeutic efficacy of EZH2 inhibitors in several malignancies. However, the mutational landscape of KDM6A and the therapeutic targetability of EZH2 inhibitors in esophageal squamous carcinoma (ESCC) remain unreported. Here, we found that approximately 9.18% (9/98)of our study ESCC tissues hadKDM6A mutationsofwhich 7cases resulted in a complete loss of expression and consequent loss of demethylase function. We found that KDM6A-deficient ESCC cells exhibited increased sensitivity to EZH2 inhibitor, and the radiosensitizing activity of EZH2 inhibitor was evident in KDM6A-dficient ESCC cells. Further transcriptome analysis revealed that ferroptosis is implicated in the radiosensitizing effect exerted by EZH2 inhibition on KDM6A-deficient ESCC cells. The following Chromatin Immunoprecipitation (ChIP), co-immunoprecipitation, and luciferase reporter assays demonstrated that in KDM6A-deficient ESCC cells, (1) Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) is the target gene for EZH2 to regulate ferroptosis; (2) The IR-induced hypoxia inducible factor 1 subunit alpha (HIF-1α) is a predominant mediator of EZH2 to repress ACSL4; (3) the HRE7-8 regions of the ACSL4 promoter are required for the repressive function of EZH2 on ACSL4; (4) EZH2 regulates ACSL4 by forming a co-repressive complex with HIF-1α. Our study provides preclinical evidence supporting that EZH2 inhibitors may confer therapeutic benefit in KDM6A-deficient ESCC patients.

NRF1 Transcriptionally Regulates USP1-Mediated Deubiquitination of AURKA to Facilitate Osteosarcoma Progression.

The poor prognosis of osteosarcoma (OS) lead to the low survival rate of OS patients. Numerous researches have shown that nuclear factor erythroid 2-like 1 (NRF1) can regulate the development and progression of cancer, but it has not yet been documented in OS. Therefore, the impact of NRF1 on OS progression and specific mechanisms will be investigated in this study. This may provide new ideas for the treatment of OS. The expressions of NRF1, ubiquitin-specific protease 1 (USP1), and aurora kinase A (AURKA) were determined by RT-qPCR and Western blot. CCK-8, colony formation, EdU staining, and Transwell assay were used to detect cell viability, proliferation, and invasion. Apoptosis and cell cycle were detected by flow cytometry. The deubiquitination of AURKA by USP1 was investigated by Co-IP and Western blot. The binding of NRF1 and USP1 promoter was explored by ChIP and dual-luciferase assay. A subcutaneous tumorigenic model in nude mice was used to further explore the effects of NRF1/USP1/AURKA axis on OS in vivo. NRF1, USP1, and AURKA were highly expressed in OS. The proliferation and invasion of OS cells were suppressed and apoptosis enhanced by silencing NRF1 expression. Moreover, knockdown of NRF1 inhibited tumor growth in vivo. USP1 increased AURKA stability by mediating AURKA deubiquitination. NRF1 bound to the promoter of USP1, so as to transcriptionally activate USP1 expression. USP1 overexpression reversed the effects of NRF1 knockdown on USP1 and AURKA expression and on OS cells trends and in vivo tumor growth. NRF1 transcriptionally modulated USP1, which facilitates deubiquitination of AURKA and thereby fostering OS progression.

Replication-IDentifier links epigenetic and metabolic pathways to the replication stress response

Perturbation of DNA replication, for instance by hydroxyurea-dependent dNTP exhaustion, often leads to stalling or collapse of replication forks. This triggers a replication stress response that stabilizes these forks, activates cell cycle checkpoints, and induces expression of DNA damage response genes. While several factors are known to act in this response, the full repertoire of proteins involved remains largely elusive. Here, we develop Replication-IDentifier (Repli-ID), which allows for genome-wide identification of regulators of DNA replication in Saccharomyces cerevisiae. During Repli-ID, the replicative polymerase epsilon (Pol ε) is tracked at a barcoded origin of replication by chromatin immunoprecipitation (ChIP) coupled to next-generation sequencing of the barcode in thousands of hydroxyurea-treated yeast mutants. Using this approach, 423 genes that promote Pol ε binding at replication forks were uncovered, including LGE1 and ROX1. Mechanistically, we show that Lge1 affects replication initiation and/or fork stability by promoting Bre1-dependent H2B mono-ubiquitylation. Rox1 affects replication fork progression by regulating S-phase entry and checkpoint activation, hinging on cellular ceramide levels via transcriptional repression of SUR2. Thus, Repli-ID provides a unique resource for the identification and further characterization of factors and pathways involved in the cellular response to DNA replication perturbation.

Helicobacter pylori SlyD stabilizes TPT1 via hnRNPK and enhances OCT1-mediated CDX2 transcriptional activation to drive gastric intestinal metaplasia

Abstract Background Gastric intestinal metaplasia (GIM) represents an important precancerous lesion in intestinal-type gastric cancer, triggered by persistent Helicobacter pylori (H. pylori) infection. In a previous study, we unveiled SlyD as a novel virulence factor of H. pylori, establishing its role in GIM induction through TPT1. However, the underlying mechanism remains undetermined. Methods Gastric epithelial cells were stimulated with H. pylori 26695, a SlyD inactivated mutant (ΔSlyD), and purified HpSlyD protein, respectively. Real-time qPCR and western blot were subsequently used to assess the expression levels of hnRNPK, TPT1, OCT1, and GIM markers. RNA sequencing was employed to identify differentially expressed genes associated with H. pylori SlyD infection. Protein stability was evaluated using cycloheximide. Molecular interactions were investigated through co-immunoprecipitation, chromatin immunoprecipitation, and dual-luciferase reporter assays. Additionally, molecular docking was utilized to predict TPT1 inhibitors. Immunohistochemistry staining was conducted to validate hnRNPK, TPT1, OCT1, and CDX2 expression in gastric tissue samples from both human and Mongolian gerbils. Results H. pylori SlyD upregulates TPT1 and induces the expression of GIM markers through hnRNPK. The interaction between hnRNPK and TPT1 enhances TPT1 protein stability, with H. pylori SlyD intensifying this association. TPT1 promotes the expression of GIM markers mediated via OCT1, which binds to CDX2 promoter region, thereby modulating its transcriptional activity. Dihydroartemisinin has the potential to target TPT1, inhibiting the H. pylori SlyD-induced expression of GIM markers. Conclusions In vitro and in vivo experiments verified that H. pylori SlyD enhances TPT1 stability through hnRNPK, leading to OCT1-mediated transcriptional activation of CDX2 and the initiation of the GIM process. Our study offers novel perspectives on the pathogenesis of H. pylori-related gastric precancerous conditions.

GADD45α is a direct target of TFEB and contributes to tacrolimus-induced chronic nephrotoxicity.

Tacrolimus-induced chronic nephrotoxicity (TICN) hinders its long-term use, but its mechanism remains unclear. Tacrolimus exerts its pharmacological effect by inhibiting calcineurin and its substrate NFAT. Whether the inhibition of other calcineurin substrates is related to TICN remains to be explored. Transcription factor EB (TFEB), a substrate of calcineurin, plays a crucial role in various homeostasis. Herein, we found that tacrolimus inhibited TFEB nuclear translocation and activity in mouse kidneys and HK-2 cells. Then, TFEB gain- and loss-of-function rescued the effect of tacrolimus in HK-2 cells. Furthermore, TFEB activation both by phosphorylation sites mutation and agonist rescued TICN in mice. To elucidate the mechanism of TFEB, we analyzed ChIP-seq data. Growth arrest and DNA damage-inducible 45α (GADD45α) was identified as a transcriptional target of TFEB via chromatin immunoprecipitation and dual luciferase reporter assays. And then we revealed that GADD45α overexpression rescued DNA damage and kidney injury caused by tacrolimus or TFEB knockdown in vitro, and vise versa. The protective effect of GADD45α against TICN and DNA damage was further demonstrated by overexpressing it in mice. In conclusion, the persistent inhibition of TFEB-GADD45α pathway by tacrolimus contributes to TICN. This study identifies a specific target for intervention of TICN.

Extracellular Vesicles From Dental Pulp Cells Promote Osteogenic Differentiation in Periodontal Ligament Cells.

Periodontal osseous defects are mainly caused by periodontitis, which seriously affects the quality of patient life. Dental pulp cells (DpCs)-derived extracellular vesicles (EVs) can effectively promote tissue regeneration. Homeobox A9 (HOXA9) mRNA is abundant in EVs derived from DSCs, which may be related to promoting alveolar bone regeneration, but the specific mechanism is unclear. We aimed to elucidate the mechanism through which HOXA9 from DPCs-derived EVs can impact the osteogenic differentiation of periodontal ligament cells (PDLCs). DPCs-derived EVs were isolated and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot. Lipopolysaccharide (LPS) was employed to induce the inflammatory environment. Cell viability was assessed by CCK8 assay. Calcium deposition was determined by Alizarin red staining. H3K27ac enrichment in the FLI1 enhancer region and the interaction between C/EBPα, HOXA9, and FLI1 were analyzed by ChIP assay. The interaction between HOXA9 and FLI1 enhancer in 293T cells was analyzed by dual luciferase reporter gene assay. DPCs-derived EVs promoted PDLC osteogenesis under LPS treatment and increased HOXA9 expression in PDLCs. HOXA9 knockdown in DPCs reversed the promoting effect of DPCs-derived EVs on PDLC osteogenic differentiation. HOXA9 from DPCs-derived EVs promoted H3K27ac enrichment in the FLI1 enhancer region by facilitating HOXA9 competitively binding FLI1 enhancer region with C/EBPα. Moreover, HOXA9 from DPCs-derived EVs promoted PDLC osteogenesis by activating the PI3K/AKT pathway through upregulating FLI1. HOXA9 from DPCs-derived EVs promoted PDLC osteogenic differentiation by activating the PI3K/AKT pathway through promoting H3K27ac enrichment in the FLI1 enhancer region and upregulating FLI1. Our study identified a previously unknown mechanism that HOXA9/FLI1 signaling axis participates in the processes of EVs derived from DPCs to treat bone tissue injury. Our research presents a theoretical basis for using EVs derived from DPCs to treat bone tissue injury.

Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3to Inhibit Pulmonary Vascular Remodeling.

The function of super-enhancers (SEs) in pulmonary hypertension (PH), especially in the proliferation of pulmonary artery smooth muscle cells (PASMCs), is currently unknown. We identified SEs-targeted genes in PASMCs with chromatin immunoprecipitation (ChIP)-sequence by H3K27ac antibody and proved that CBP/p300-interacting transactivator with Glu/Asp-rich C-terminal domain, 2 (CITED2) is an SEs-targeted gene through bioinformatics prediction, ChIP-PCR, dual-luciferase reporter gene assays and other experimental methods. We also found that the expression of CITED2 and the transcription factor Forkhead Box J3 (FOXJ3) was reduced in hypoxic mouse PASMCs. In addition, the expression of CITED2 and FOXJ3 also decreased in both the patients with idiopathic pulmonary arterial hypertension (iPAH) and the human PASMCs exposed to hypoxia. The decreased expression of CITED2 was reversed by co-transfection of FOXJ3 and SEs plasmids. Overexpressing of CITED2 attenuated the PASMCs proliferation induced by hypoxia. Lentiviral overexpression of CITED2 also reversed hypoxia-induced pulmonary hypertension mice model. Mechanically, the expression of CITED2 by affecting by FOXJ3, which binding with three SEs located in the about 2000 bp of TSS. In conclusion, we first identified that CITED2 is a kind of SEs-targeted gene, modulated by FOXJ3. The FOXJ3/SEs/CITED2 axis may become a new therapeutic target of PH.

O-GlcNAcylation attenuates ischemia-reperfusion-induced pulmonary epithelial cell ferroptosis via the Nrf2/G6PDH pathway

BackgroundLung ischemia–reperfusion (I/R) injury is a common clinical pathology associated with high mortality. The pathophysiology of lung I/R injury involves ferroptosis and elevated protein O-GlcNAcylation levels, while the effect of O-GlcNAcylation on lung I/R injury remains unclear. This research aimed to explore the effect of O-GlcNAcylation on reducing ferroptosis in pulmonary epithelial cells caused by I/R.ResultsFirst, we identified O-GlcNAc transferase 1 (Ogt1) as a differentially expressed gene in lung epithelial cells of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) patients, using single-cell sequencing, and Gene Ontology analysis (GO analysis) revealed the enrichment of the ferroptosis process. We found a time-dependent dynamic alteration in lung O-GlcNAcylation during I/R injury. Proteomics analysis identified the differentially expressed proteins enriched in ferroptosis and multiple redox-related pathways based on KEGG annotation. Thus, we generated Ogt1-conditional knockout mice and found that Ogt1 deficiency aggravated ferroptosis, as evidenced by lipid reactive oxygen species (lipid ROS), malondialdehyde (MDA), Fe2+, as well as alterations in critical protein expression glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11). Consistently, we found that elevated O-GlcNAcylation inhibited ferroptosis sensitivity in hypoxia/reoxygenation (H/R) injury-induced TC-1 cells via O-GlcNAcylated NF-E2-related factor-2 (Nrf2). Furthermore, both the chromatin immunoprecipitation (ChIP) assay and the dual-luciferase reporter assay indicated that Nrf2 could bind with translation start site (TSS) of glucose-6-phosphate dehydrogenase (G6PDH) and promote its transcriptional activity. As an important rate-limiting enzyme in the pentose phosphate pathway (PPP), elevated G6PDH provided a mass of nicotinamide adenine dinucleotide phosphate (NADPH) to improve the redox state of glutathione (GSH) and eventually led to ferroptosis resistance. Rescue experiments proved that Nrf2 knockdown or Nrf2-T334A (O-GlcNAcylation site) mutation abolished the protective effect of ferroptosis resistance.ConclusionsIn summary, we revealed that O-GlcNAcylation could protect against I/R lung injury by reducing ferroptosis sensitivity via the Nrf2/G6PDH pathway. Our work will provide a new basis for clinical therapeutic strategies for pulmonary ischemia–reperfusion-induced acute lung injury.

ALYREF-Mediated Regulation of TBL1XR1 and KMT2E Synergistically Upregulates APOC1, Contributing to Oxaliplatin Resistance in Esophageal Cancer.

Esophageal cancer (EC) is a rapidly progressing malignancy characterized by a low survival rate and limited treatment success, largely due to late-stage detection, frequent recurrence, and a high propensity for metastasis, despite ongoing advances in therapeutic strategies. While oxaliplatin (L-OHP) is a potent chemotherapeutic agent that induces apoptosis in EC cells, its effectiveness is significantly hindered by the development of resistance. The assessment of gene and protein expression was conducted through a combination of RT-qPCR, Western blot, and IHC staining. Cell viability was assessed using the CCK-8 assay. The interactions among ALYREF, TBL1XR1, KMT2E, and APOC1 were investigated through RIP, ChIP, ChIP-reChIP, RNA pulldown, and dual-luciferase assays. An in vivo mouse model of EC was established. Expression levels of both APOC1 and ALYREF were elevated in L-OHP-resistant EC tissues and cell lines, and their silencing enhanced sensitivity to L-OHP. TBL1XR1 and KMT2E synergistically upregulated APOC1 expression. Moreover, ALYREF recognized the m5C sites on TBL1XR1 and KMT2E mRNAs, stabilizing these transcripts and promoting APOC1 expression. The regulatory role of these interactions was further validated in vivo. This study demonstrated that ALYREF interacted with the m5C sites on TBL1XR1 and KMT2E mRNAs, enhancing their stability and leading to increased transcription of APOC1, which in turn contributed to L-OHP resistance in EC. These findings suggest that targeting APOC1 could be a promising strategy for overcoming L-OHP resistance in EC.

MSC-exosomes pretreated by Danshensu extracts pretreating to target the hsa-miR-27a-5p and STAT3-SHANK2 to enhanced antifibrotic therapy

BackgroundPeritoneal fibrosis (PF) is a serious complication commonly associated with prolonged peritoneal dialysis. Mesenchymal stem cells (MSCs) and their exosomes (Exo) have shown significant therapeutic promise in treating fibrotic conditions. Danshensu (DSS), a bioactive compound from the traditional Chinese herb Danshen reverses fibrosis. This study aims to investigate a novel strategy to enhance the therapeutic efficacy against PF by DSS preconditioning MSCs-derived exosomes (DSS-Exo).MethodsThe in vitro studies included the effects of DSS duration on MSCs, and the characterization of DSS-Exo and Exo, followed by the assessment of RNA and protein expression levels of peritoneal fibrosis markers and inflammatory cytokines levels after treating human peritoneal mesothelial (HMrSV5) cells. In vivo experiments were conducted on a PF mouse model to observe cell morphology, collagen deposition, fibrosis localization, and to evaluate peritoneal functions such as filtration rate, urea nitrogen clearance, peritoneal thickness, and protein leakage. Mechanistic insights were gained through the analysis of the STAT3/HIF-1α/VEGF signaling pathway, tissue dual-fluorescence localization,chromatin immunoprecipitation sequencing (ChIP-seq), and dual-luciferase reporter (DLR) assays. Additionally, the differential expression of miRNAs between DSS-Exo and Exo was explored and validation of key miRNA.ResultsDSS-Exo significantly upregulated E-cadherin, downregulated VEGFA, α-SMA, CTGF and Fibronectin expression in HMrSV5 cells compared to untreated Exo. In vivo studies revealed that DSS-Exo enhanced the ability of Exo to improve peritoneal function,such as the peritoneal filtration rate and urea nitrogen, glucose clearance, while reducing peritoneal thickness and protein leakage, and cell morphology, reduce collagen deposition, and decrease the degree of fibrosis. Mechanistically, these exosomes inhibited the STAT3/HIF-1α/VEGF signaling pathway within peritoneal mesothelial tissues. Furthermore, ChIP-seq and DLR demonstrated that DSS-Exo affected STAT3 directly binds to SHANK2 promoter regions, forming hydrogen bonds between 5 key amino acids such as GLN-344, HIS-332 and 6 key bases such as DG-258, DG-261. miRNA profiling identified DSS-Exo increased hsa-miR-27a-5p_R-1 to regulated STAT3-SHANK2 and modulating the EMT.ConclusionThis study highlighted the innovative use of Danshensu in enhancing MSC-derived exosome therapy for PF. The identification of the hsa-miR-27a-5p_R-1-STAT3-SHANK2 axis may reveal new molecular mechanisms underlying fibrosis, further research is needed to fully elucidate its impact on PF. The integration of Danshensu from traditional Chinese medicine into modern MSC exosome therapy represents a promising frontier in the development of novel treatments for fibrotic diseases.

FoxO1 mediates odontoblast differentiation of hDPSCs via B cell-derived ANGPTL1 in dental caries: A laboratory investigation.

Clinical and invitro evidence indicates that chronic inflammatory responses initiated by dental caries can persist in the dental pulp even after treatment, necessitating the formation of reparative dentin to restore tissue homeostasis and health. Human dental pulp stem cells (hDPSCs) serve as crucial precursors in this reparative process. This study explores the role of B cells and their secreted factor, Angiopoietin Like 1 (ANGPTL1), in promoting hDPSCs differentiation into odontoblasts under carious conditions, with a particular focus on the activation of Forkhead box O1 (FoxO1). Single-cell RNA sequencing (scRNA-Seq) data from the GEO database were analysed to explore cellular interactions and molecular mechanisms in dental pulp. Immunofluorescence staining was used to investigate the expression patterns of B cells or hDPSCs in dental pulp and hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds. The expression levels of ANGPTL1 were quantified using enzyme-linked immunosorbent assay (ELISA). Odontoblast differentiation capacity was assessed by alkaline phosphatase activity, alizarin red S staining, and western blotting analysis. hDPSCs were overexpressed or knocked down FoxO1 with lentiviruses. The regulatory interaction between FoxO1 and the DSPP promoter was evaluated through dual-luciferase reporter assay and chromatin immunoprecipitation assay. Statistical analyses were conducted using Student's t-test or one-way analysis of variance (anova) with a p-value of <.05 considered statistically significant. scRNA-Seq data indicated a significant increase in B cells and ANGPTL1 expression in carious dental pulp. Functional analyses confirmed that ANGPTL1 secreted by B cells activated FoxO1 expression in hDPSCs, enhancing their differentiation into odontoblast-like cells. Blocking ANGPTL1 signalling with a specific antibody reduced FoxO1 expression, indicating a regulatory link between ANGPTL1 and FoxO1. Overexpression of FoxO1 in hDPSCs promoted their differentiation into odontoblasts and facilitated mineralized matrix formation. Mechanistic studies revealed that FoxO1 directly binds to the DSPP promoter, thereby inducing its expression. Our study reveals a novel mechanism in which ANGPTL1 secreted by B cells in a carious environment promotes the odontoblast differentiation of hDPSCs by upregulating FoxO1. This finding highlights a potential therapeutic target for enhancing dental pulp repair and regeneration.

CircHIPK2 recruits SRSF1 to increase TXNIP mRNA stability and promotes autophagy-dependent ferroptosis and apoptosis in myocardial ischemia-reperfusion injury.

Myocardial ischemia/reperfusion injury (MIRI) secondary to acute myocardial infarction (AMI) can lead to cardiomyocyte death and impaired cardiac function. Studies have confirmed that circular RNAs (circRNAs) play an important role in MIRI. In this study, the role and mechanism of circHIPK2 in MIRI were evaluated. Human cardiac myocytes (HCM) were cultured under Hypoxia/Reoxygenation (H/R) condition to establish a MIRI model in vitro. Expression of circHIPK2, SRSF1 and TXNIP was assessed using RT-qPCR. Protein levels of autophagy markers (LC3II/LC3I, Beclin1, p62) and ferroptosis markers (GPX4, FTH1, ACSL4) were detected by Western blot. Cell viability and apoptosis were assessed by CCK-8 and flow cytometry. Levels of oxidative stress markers (MDA, SOD) and inflammatory factors (IL-6, IL-1β, TNF-α) were tested by ELISA assay. Iron concentration was measured with an iron detection kit. Location of circHIPK2 in cells was detected by RNA-nucleosome separation assay. RIP and ChIP assays verified the relationship between circHIPK2, SRSF1 and TXNIP. TXNIP mRNA stability was dertermined by actinomycin D. Infarct area was examined by TTC staining in myocardial ischemia/reperfusion (I/R) mouse model. HE staining evaluated myocardial injury. CircHIPK2 was increased in H/R-induced HCM cells. CircHIPK2 downregulation suppressed oxidative stress, inflammatory factors and autophagy-dependent ferroptosis in HCM cells induced by H/R. Additionally circHIPK2 recruited SRSF1 to target TXNIP and stabilized TXNIP mRNA expression. We further demonstrated that TXNIP upregulation overturned the therapeutic effects of circHIPK2 silencing on H/R model cells. In vivo, downregulation of circHIPK2 improved myocardial dysfunction caused by I/R. Our results demonstrate that circHIPK2 contributes to MIRI through inducing oxidative stress and autophagy-dependent ferroptosis via SRSF1/TXNIP axis, offering new insights into MIRI treatment.

CircMETTL6 Suppresses Ovarian Cancer Cell Growth and Metastasis Through Inhibition of GDF15 Transcription by Disrupting the NONO-POLR2A Complex.

Circular RNAs (circRNAs) are a distinctive class of non-coding RNAs with covalent closed-loop structure, lacking 5' caps and 3' poly(A) tails. These molecules are prevalent in eukaryotes and play keyroles in cancer. Here, the function of a new circRNA, circMETTL6, in ovarian cancer is identified and investigated. The prognostic significance of circMETTL6 is assessed using RNA in situ hybridization. Functional studies involving circMETTL6 overexpression are performed both in vitro and in vivo. Mechanistic investigations are performed using RNA-seq, RNA pull-down, RNA immunoprecipitation, co-immunoprecipitation, chromatin immunoprecipitation, protein degradation assay and dual-luciferase reporter assays. circMETTL6 is significantly downregulated in ovarian cancer, and its lower expression correlates with worse prognosis. Overexpression of circMETTL6 significantly inhibited proliferation, migration, and invasion of ovarian cancer cell in vitro, as well as tumor growth and metastasis in vivo. Mechanistically, circMETTL6 recruited the non-POU domain containing octamer binding protein (NONO) by binding to its Coiled-coil domain and disrupted its binding with RNA polymerase II subunit A (POLR2A), and consequently inhibiting growth differentiation factor 15 (GDF15) transcription, thereby suppressing ovarian cancer progression. These findings establish circMETTL6 as a novel tumor suppressor in ovarian cancer. Targeting the circMETTL6/NONO/GDF15 axis presents a potential therapeutic avenue for ovarian cancer treatment.

Sleep deprivation alters hepatic UGT1A9 and propofol metabolism in mice.

Sleep deprivation (SD) causes circadian misalignment, and circadian clock disruption is associated with metabolic diseases such as obesity, insulin resistance, and diabetes. However, the underlying mechanism for SD-induced circadian clock disruption as well as metabolic enzyme changes is still lacking. Here, we developed SD sensitizes mice with disrupted circadian rhythms to demonstrate the regulation role and mechanism of SD in UDP-glucuronosyltransferases (UGTs) expression and the metabolism of corresponding substrates. We found that UGT Family 1 Member A9 (UGT1A9) expression was significantly decreased in the liver of SD mice, which led to an elevation exposure and prolonged anesthesia effect of propofol, which was attributed to the decreased metabolism. Meanwhile, SD down-regulated basic helix-loop-helix ARNT like 1 (BMAL1) and its target clock genes period circadian clock (Per), cryptochrome circadian regulator (Cry), and nuclear receptor subfamily 1 group D member 1 (Rev-erb) expression in mice. Furthermore, the positive regulation of UGTIA9 mRNA and protein levels by Bmal1 was confirmed in hepatocyte-specific Bmal1-knockout mice (Bmal1-hkO) and Bmal1-overexpressed AML-12 cells. At last, through a combination of promoter analysis, luciferase reporter assay, and chromatin immunoprecipitation (ChIP) assay, it was conducted that Bmal1 regulates Ugtla9 expression by directly binding the -864bp E-box in Ugtla9 promotor or indirectly acting on the Rev-erbα- differentiated embryo chondrocyte 2 (Dec2) axis. In conclusion, our findings suggested that SD can lead to altered drug disposition and effects in vivo, and Bmal1 plays a crucial role in the crosstalk between SD-induced circadian clock disruption and drug metabolism. It initiates a new direction for the understanding of drug efficacy and toxicity changes in SD conditions and provides a scientific basis for improving the rationality of drug use.

Identification of a functional vitamin D response element in the promoter of goose anti-Müllerian hormone gene.

Anti-Müllerian hormone (AMH) plays an important role in avian ovarian follicle development. The high mRNA expression of AMH in avian ovarian prehierarchical follicles helps prevent premature granulosa cell differentiation. Vitamin D3 was reported to downregulate AMH mRNA expression in granulosa cells of prehierarchical follicles in hens; however, the underlying molecular mechanism remains unknown. In this study, AMH mRNA expression was investigated in granulosa cells of prehierarchical follicles in geese under vitamin D3 induction. A potential vitamin D response element (VDRE) present in the goose AMH promoter was identified using luciferase activity, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assays. The results showed that AMH mRNA expressions were downregulated by vitamin D3 in granulosa cells of prehierarchical follicles in geese. A classical VDRE-like sequence was identified in the goose AMH promoter by in silico analyses. Luciferase activity assays revealed that the putative VDRE is a negative regulatory element. Vitamin D receptor (VDR) and retinoic X receptor (RXR) are necessary to decrease the AMH promoter activity induced by vitamin D3. The EMSA and ChIP assays demonstrated that the VDR/RXR complex directly binds to the putative VDRE. These results suggest that vitamin D3 downregulates AMH mRNA expression via a functional VDRE that binds the VDR/RXR heterodimer in goose ovarian prehierarchical follicles. These results provide new insights into the regulatory mechanisms of vitamin D3 in avian ovarian follicular development.

SUMO modified ETV1 promotes M2-polarized tumor-associated macrophage infiltration and cancer progression by facilitating CCL2 transcription in esophageal squamous cell carcinoma cells.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors with a high metastasis rate and a poor prognosis. ETS variant transcription factor 1 (ETV1) plays an important role in multiple malignancies. However, its function in ESCC progression and tumor microenvironment (TME) remains to be explored. In this study, we characterized the role of ETV1 in ESCC process and TME. Gene expression and immune infiltration in ESCC samples from the Cancer Genome Atlas (TCGA) were analyzed. The expression of ETV1 in clinical samples was detected by real-time PCR, western blot and immunohistochemistry staining. Cell growth was detected by CCK-8 and colony formation assays. Macrophage phenotypes were determined using flow cytometry. Immunofluorescence double staining was used to detect the tumor-associated macrophage (TAM) infiltration. The tumor volume was recorded and weighed. Transcriptional activity was measured using dual-luciferase assay, chromatin immunoprecipitation (ChIP) assay and DNA pull-down assay. In this study, through analysis of ESCC samples from TCGA database and the clinic, we noticed that ETV1 was highly expressed in ESCC tumor tissues and was associated with TAM infiltration. Overexpression of ETV1 promoted ESCC cell proliferation in vitro and xenograft tumor growth in nude mice, while ETV1 knockdown elicited the opposite effects. Furthermore, ETV1 upregulation in tumor tissues was found to drive M2 macrophage infiltration both in vitro (transwell assays) and in vivo (xenograft tumor models). C-C motif chemokine ligand 2 (CCL2), a key factor inducing M2 macrophage polarization, was also found to be elevated in ESCC tumor tissues. Mechanism study demonstrated that ETV1 facilitated M2 macrophage infiltration via the transcriptional modulation of CCL2. In addition, the cause of the changes in ETV1 activity and expression was investigated. The E2 small ubiquitin-like modifier (SUMO) binding enzyme UBC9 increased ETV1 activity and expression, indicating the presence of SUMO modification in ETV1. Our data deciphered the mechanism of ETV1-mediated M2 macrophage infiltration in the TME of ESCC, which has important implications for the development of novel prognostic and therapeutic targets to optimize current therapies against ESCC.

Oestrogen suppresses the adipogenesis of fibro/adipogenic progenitors through reactivating the METTL3-ESR1-mediated loop in post-menopausal females.

Post-menopausal women experience more severe muscular fatty infiltration, though the mechanisms remain unclear. The decline in estrogen levels is considered as a critical physiological alteration during post-menopause. Fibro/adipogenic progenitors (FAPs) are identified as major contributors to muscular fatty infiltration. This study aimed to investigate the detailed mechanism underlying the excessive muscular fatty infiltration in postmenopausal females. Supraspinatus muscle samples were collected from female patients with or without menopause, and from mice with or without ovariectomy (OVX), to evaluate muscular fatty infiltration and isolated FAPs. The expressions of (estrogen receptor 1) ESR1, methyltransferase-like 3 (METTL3), and adipogenesis ability in FAPs from post-menopausal women and OVX mice were investigated. RNA sequencing (RNA-Seq) was performed to explore the gene expression profiles and potential mechanisms in FAPs from Pdgfrα-CreERT2; Esr1 knockout (Esr1 KO) mice and Esr1 flox/flox (Esr1 f/f) mice. The interplay of the METTL3-ESR1 mediated loop and its role in regulating adipogenesis in FAPs were investigated using dual luciferase reporter assays, chromatin immunoprecipitation (ChIP), and protein and RNA stability assays. The effects of estrogen supplementation on muscular fatty infiltration and locomotor function in OVX mice were evaluated by immunofluorescent staining and functional analysis. Decreased expression of ESR1/METTL3 and increased adipogenesis ability in FAPs was found in post-menopausal female. METTL3-mediated m6A methylation promoted ESR1 mRNA stability at the post-transcriptional level in FAPs. METTL3-mediated m6A modification promoted ESR1 expression by stabilizing ESR1 mRNA, while ESR1 acted as a transcription factor that enhanced METTL3 transcription in turn. ESR1 also suppressed the transcription of the adipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPARγ), thereby inhibiting adipogenesis in FAPs. Reactivation of the METTL3-ESR1 mediated loop by estrogen alleviated excessive adipogenesis in FAPs from post-menopausal women, and it also reduced muscular fatty infiltration, and improved locomotor function in OVX mice. Excessive muscular fatty infiltration in post-menopausal women arose from the disruption of the METTL3-ESR1 mediated loop of FAPs due to estrogen deficiency. Reactivation of the METTL3-ESR1 mediated loop by estrogen may serve as a novel intervention to inhibit excessive adipogenesis of post-menopausal female FAPs, thereby ameliorating muscular fatty infiltration and improving locomotor function in post-menopausal females. Oestrogen insufficiency disrupted the METTL3ESR1 loop in post-menopausal FAPs, causing excessive muscular fatty infiltration. METTL3-mediated m6A modification stabilized ESR1 mRNA and enhanced ESR1 expression, while increased ESR1 further promoted METTL3 transcription. ESR1 inhibited the transcription of adipogenic factor PPARγ, ameliorating adipogenesis in FAPs. Reactivating the METTL3ESR1 loop via oestrogen in FAPs reduced muscular fatty infiltration and improved locomotor function.

Clofarabine induces tumor cell apoptosis, GSDME-related pyroptosis, and CD8+ T-cell antitumor activity via the non-canonical P53/STING pathway

BackgroundClofarabine (Clo) is a Food and Drug Administration (FDA)-approved drug for the treatment of acute lymphoblastic leukemia; however, its effects on solid tumors remain largely unknown.MethodsIn vitro and in vivo...