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Purpose: Our research aimed to evaluate whether proto-oncogene serine/threonine-protein kinase Pim-1 (Pim1) inactivation could attenuate asthma by promoting runt-related transcription factor 3 (Runx3) expression and explore the underlying molecular mechanism., Method: Phorbol 12-myristate 13-acetate (PMA, 50 nM) was used to induce inflammation in BEAS-2B human airway epithelial cells. ELISA and immunofluorescence double staining confirmed inflammation modelling and differential expression of Pim1 and Runx3. Pim1 inhibitor (SGI-1776) and Runx3 siRNA (siRunx3) were used in this study. Apoptosis, inflammation, MUC5AC protein expression, Pim1 kinase and Runx3 protein expression, and PI3K/AKT/nuclear factor-κB (NF-κB) pathway-associated protein expression were also assessed by flow cytometry, immunofluorescence and western blot. The effects of Pim1 inactivation on airway inflammation, pathological injury and mucus secretion in wild-type and Runx3 knockout mice were observed by in vivo experiments., Results: The results of the in vitro experiments showed that PMA stimulation causes BEAS-2B cell apoptosis and promotes the MUC5AC expression. In addition, PMA stimulation activated the PI3K/AKT/NF-κB pathway. SGI-1776 treatment partially reversed these effects, whereas siRunx3 attenuated the effects of SGI-1776 on PMA-stimulated BEAS-2B cells. In vivo experiments showed that in Runx3-KO asthmatic mice, inhibition of Pim1 kinase had less effect on airway inflammation, pathological injury and mucus secretion. Meanwhile, Pim1 kinase expression was higher in Runx3-KO asthmatic mice than in wild-type asthmatic mice. Furthermore, inhibition of Pim1 kinase inhibited activation of the PI3K/AKT/NF-κB pathway, whereas these effects were attenuated in Runx3-KO mice., Conclusion: Our results suggest that Pim1 inactivation can ameliorate airway inflammation and mucus hypersecretion through upregulation of Runx3 and the effect could be mediated through modulation of the PI3K/AKT/NF-κB pathway., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: RUNX3 is hypermethylated in multiple cancers. TIMP2 also functions as a regulator of tumors. However, there are only very few reports on the association of methylation of RUNX3 and TIMP2 with lung cancer (LC) in peripheral blood., Methods: 426 LC patients and 428 age- and sex-matched healthy controls were recruited. DNA methylation in blood was semi-quantitively assessed by mass spectrometry. For the association analysis, binary logistic regression analysis adjusted covariant was applied, and ORs were presented as per +10% methylation., Results: Hypermethylation of CpG&#95;1, CpG&#95;5 and CpG&#95;8 in RUNX3 was significantly associated with LC (ORs = 1.45, 1.35 and 1.35, respectively, adjusted p  < 0.05), and even stage I LC. The association between the three RUNX3 CpG sites and LC was enhanced by increased age (> 55 years, ORs ranged from 1.43 to 1.75, adjusted p  < 0.05), male gender (ORs ranged from 1.47 to 1.59, adjusted p  < 0.05) and tumor stage (stage II&III&IV, ORs ranged from 1.86 to 3.03, adjusted p  < 0.05)., Conclusions: This study suggests a significant association between blood-based RUNX3 hypermethylation and LC, especially in elder people, in males and in LC patients with advanced stage.

Radiation enteritis remains a major challenge for radiotherapy against abdominal and pelvic malignancies. Nevertheless, there is no approved effective therapy to alleviate irradiation (IR)-induced gastrointestinal (GI) toxicity. In the current study, Cannabidiol (CBD) was found to mitigate intestinal injury by GPX4-mediated ferroptosis resistance upon IR exposure. RNA-sequencing was employed to investigate the underlying mechanism involved in the radio-protective effect of CBD, wherein runt-related transcription factor 3 (RUNX3) and its target genes were changed significantly. Further experiment showed that the transactivation of GPX4 triggered by the direct binding of RUNX3 to its promoter region, or by stimulating the transcriptional activity of NF-κB via RUNX3-mediated LILRB3 upregulation was critical for the anti-ferroptotic effect of CBD upon IR injury. Specially, CBD was demonstrated to be a molecular glue skeleton facilitating the heterodimerization of RUNX3 with its transcriptional chaperone core-biding factor β (CBFβ) thereby promoting their nuclear localization and the subsequent transactivation of GPX4 and LILRB3. In short, our study provides an alternative strategy to counteract IR-induced enteritis during the radiotherapy on abdominal/pelvic neoplasms., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Runt-related transcription factor (RUNX) family members play critical roles in the development of multiple organs. Mammalian RUNX family members, consisting of RUNX1, RUNX2, and RUNX3, have distinct tissue-specific expression and function. In this study, we examined the spatiotemporal expression patterns of RUNX family members in developing kidneys and analyzed the role of RUNX1 during kidney development. In the developing mouse kidney, RUNX1 protein was strongly expressed in the ureteric bud (UB) tip and weakly expressed in the distal segment of the renal vesicle (RV), comma-shaped body (CSB), and S-shaped body (SSB). In contrast, RUNX2 protein was restricted to the stroma, and RUNX3 protein was only expressed in immune cells. We also analyzed the expression of RUNX family members in the cynomolgus monkey kidney. We found that expression patterns of RUNX2 and RUNX3 were conserved between rodents and primates, whereas RUNX1 was only expressed in the UB tip, not in the RV, CSB, or SSB of cynomolgus monkeys, suggesting a species differences. We further evaluated the roles of RUNX1 using two different conditional knockout mice: Runx1 f/f :HoxB7-Cre and Runx1 f/f :R26-CreER T2 and found no abnormalities in the kidney. Our findings showed that RUNX1, which is mainly expressed in the UB tip, is not essential for kidney development., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Motoko Yanagita reports financial support was provided by Japan Agency for Medical Research and Development. Motoko Yanagita reports a relationship with Mitsubishi Tanabe Pharma Corporation, Boehringer Ingelheim that includes: funding grants. None has patent pending to none. none If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: Understanding the immune mechanisms associated with liver transplantation (LT), particularly the involvement of tissue-resident memory T cells (TRMs), represents a significant challenge. Methods: This study employs a multi-omics approach to analyse liver transplant samples from both human (n = 17) and mouse (n = 16), utilizing single-cell RNA sequencing, bulk RNA sequencing, and immunological techniques. Results: Our findings reveal a comprehensive T cell-centric landscape in LT across human and mouse species, involving 235,116 cells. Notably, we found a substantial increase in CD8 + TRMs within rejected grafts compared to stable ones. The elevated presence of CD8 + TRMs is characterised by a distinct expression profile, featuring upregulation of tissue-residency markers (CD69, CXCR6, CD49A and CD103 +/- ,), immune checkpoints (PD1, CTLA4, and TIGIT), cytotoxic markers (GZMB and IFNG) and proliferative markers (PCNA and TOP2A) during rejection. Furthermore, there is a high expression of transcription factors such as EOMES and RUNX3. Functional assays and analyses of cellular communication underscore the active role of CD8 + TRMs in interacting with other tissue-resident cells, particularly Kupffer cells, especially during rejection episodes. Conclusions: These insights into the distinctive activation and interaction patterns of CD8 + TRMs suggest their potential utility as biomarkers for graft rejection, paving the way for novel therapeutic strategies aimed at enhancing graft tolerance and improving overall transplant outcomes., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

With the increasing age of the population worldwide, the incidence rate of Parkinson's disease (PD) is increasing annually. Currently, the treatment strategy for PD only improves clinical symptoms. No effective treatment strategy can slow down the progression of the disease. In the present study, whole transcriptome sequencing was used to obtain the mRNA and miRNA expression profiles in a PD mouse model, which revealed the pathogenesis of PD. The transcription factor RUNX3 upregulated the miR-186-3p expression in the PD model. Furthermore, the high miR-186-3p expression in PD can be targeted to inhibit the DAT expression, resulting in a decrease in the dopamine content of dopaminergic neurons. Moreover, miR-186-3p can be targeted to inhibit the IGF1R expression and prevent the activation of the IGF1R-P-PI3K-P-AKT pathway, thus increasing the apoptosis of dopaminergic neurons by regulating the cytochrome c-Bax-cleaved caspase-3 pathway. Our research showed that the RUNX3-miR-186-3p-DAT-IGF1R axis plays a key role in the pathogenesis of PD, and miR-186-3p is a potential target for the treatment of PD., (© 2024. The Author(s).)

Aim: The impaired function of tubular mitochondria is critical in diabetic kidney disease (DKD) progression. RUNX3 is down-regulated in DKD models. We intend to explore the effects of RUNX3 on mitochondrial dysfunction and renal tubule injury in DKD and related mechanisms., Methods: The development of diabetes models involved injecting mice with streptozotocin while treating HK-2 cells with high glucose (HG). By using immunohistochemical techniques, the renal localizations of RUNX3 were identified. Levels of adenosine triphosphate (ATP), mitochondrial membrane potential, and biochemical index were detected by appropriate kits. Reactive oxygen species (ROS) generation was assessed with dihydroethidium and MitoSOX Red staining. Apoptosis was assessed by flow cytometry and TUNEL. RUNX3 ubiquitination was measured., Results: RUNX3 was mainly present in renal tubules. Overexpressing RUNX3 increased Mfn2, Mfn1, ATP levels, and mitochondrial membrane potential, reduced Drp1 and ROS levels and cell apoptosis, as well as Cyt-C release into the cytoplasm. RUNX3 overexpression displayed a reduction in urinary albumin to creatinine ratio, Hemoglobin A1c, serum creatinine, and blood urea nitrogen. Overexpressing TLR4 attenuated the inhibitory effect of RUNX3 overexpression on mitochondrial dysfunction and cell apoptosis. HG promoted RUNX3 ubiquitination and SMURF2 expression. RUNX3 knockdown cancelled the inhibitory effect of SMURF2 on mitochondrial dysfunction and cell apoptosis., Conclusion: SMURF2 interference inhibits RUNX3 ubiquitination and TLR4/NF-κB signalling pathway, thereby alleviating renal tubule injury., (© 2024 Asian Pacific Society of Nephrology.)

NK cells are cytotoxic innate immune cells involved in antitumor immunity, and they provide a treatment option for patients with acute myeloid leukemia (AML). In this issue of the JCI, Cubitt et al. investigated the role of CD8α, a coreceptor present on approximately 40% of human NK cells. IL-15 stimulation of CD8α- NK cells induced CD8α expression via the RUNX3 transcription factor, driving formation of a unique induced CD8α (iCD8α+) population. iCD8α+ NK cells displayed higher proliferation, metabolic activity, and antitumor cytotoxic function compared with preexisting CD8α+ and CD8α- subsets. Therefore, CD8α expression can be used to define a potential dynamic spectrum of NK cell expansion and function. Because these cells exhibit enhanced tumor control, they may be used to improve in NK cell therapies for patients with AML.

Inflammatory bowel disease (IBD) is a chronic and progressive inflammatory intestinal disease that affects people around the world. The primary cause of IBD is an imbalance in the host immune response to intestinal flora. Several human genes, including IL10, STAT3, IRGM, ATG16L1, NOD2 and RUNX3, are associated with inappropriate immune responses in IBD. It has been reported that homozygous Runx3-knockout (ko) mice spontaneously develop colitis. However, the high mortality rate in these mice within the first two weeks makes it challenging to study the role of Runx3 in colitis. To address this issue, a spontaneous colitis (SC) mouse model carrying a C-terminal truncated form of Runx3 with Tyr319stop point mutation has been generated. After weaning, SC mice developed spontaneous diarrhea and exhibited prominent enlargement of the colon, accompanied by severe inflammatory cell infiltration. Results of immunofluorescence staining showed massive CD4 + T cell infiltration in the inflammatory colon of SC mice. Colonic IL-17A mRNA expression and serum IL-17A level were increased in SC mice. CD4 + T cells from SC mice produced stronger IL-17A than those from wildtype mice in Th17-skewing conditions in vitro. In addition, the percentages of Foxp3 + Treg cells as well as the RORγt + Foxp3 + Treg subset, known for its role in suppressing Th17 response in the gut, were notably lower in colon lamina propria of SC mice than those in WT mice. Furthermore, transfer of total CD4 + T cells from SC mice, but not from wildtype mice, into Rag1-ko host mice resulted in severe autoimmune colitis. In conclusion, the C-terminal truncated Runx3 caused autoimmune colitis associated with Th17/Treg imbalance. The SC mouse model is a feasible approach to investigate the effect of immune response on spontaneous colitis., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Cancer-associated fibroblasts (CAFs) are the primary stromal cells found in tumor microenvironment, and display high plasticity and heterogeneity. By using single-cell RNA-seq technology, researchers have identified various subpopulations of CAFs, particularly highlighting a recently identified subpopulation termed antigen-presenting CAFs (apCAFs), which are largely unknown., Methods: We collected datasets from public databases for 9 different solid tumor types to analyze the role of apCAFs in the tumor microenvironment., Results: Our data revealed that apCAFs, likely originating mainly from normal fibroblast, are commonly found in different solid tumor types and generally are associated with anti-tumor effects. apCAFs may be associated with the activation of CD4+ effector T cells and potentially promote the survival of CD4+ effector T cells through the expression of C1Q molecules. Moreover, apCAFs exhibited highly enrichment of transcription factors RUNX3 and IKZF1, along with increased glycolytic metabolism., Conclusions: Taken together, these findings offer novel insights into a deeper understanding of apCAFs and the potential therapeutic implications for apCAFs targeted immunotherapy in cancer., Competing Interests: The reviewer LH declared a shared parent affiliation, with no collaboration, with the authors JC, RC, JH to the handling editor at the time of the review., (Copyright © 2024 Chen, Chen and Huang.)

The choice of developing thymocytes to become CD8+ cytotoxic or CD4+ helper T cells has been intensely studied, but many of the underlying mechanisms remain to be elucidated. Recent multiomics approaches have provided much higher resolution analysis of gene expression in developing thymocytes than was previously achievable, thereby offering a fresh perspective on this question. Focusing on our recent studies using CITE-seq (cellular indexing of transcriptomes and epitopes) analyses of mouse thymocytes, we present a detailed timeline of RNA and protein expression changes during CD8 versus CD4 T cell differentiation. We also revisit our current understanding of the links between T cell receptor signaling and expression of the lineage-defining transcription factors ThPOK and RUNX3. Finally, we propose a sequential selection model to explain the tight linkage between MHC-I versus MHC-II recognition and T cell lineage choice. This model incorporates key aspects of previously proposed kinetic signaling, instructive, and stochastic/selection models.

Class switch recombination (CSR) diversifies the effector functions of antibodies and involves complex regulation of transcription and DNA damage repair. Here, we show that the deubiquitinase USP7 promotes CSR to immunoglobulin A (IgA) and suppresses unscheduled IgG switching in mature B cells independent of its role in DNA damage repair, but through modulating switch region germline transcription. USP7 depletion impairs Sα transcription, leading to abnormal activation of Sγ germline transcription and increased interaction with the CSR center via loop extrusion for unscheduled IgG switching. Rescue of Sα transcription by transforming growth factor β (TGF-β) in USP7-deleted cells suppresses Sγ germline transcription and prevents loop extrusion toward IgG CSR. Mechanistically, USP7 protects transcription factor RUNX3 from ubiquitination-mediated degradation to promote Sα germline transcription. Our study provides evidence for active transcription serving as an anchor to impede loop extrusion and reveals a functional interplay between USP7 and TGF-β signaling in promoting RUNX3 expression for efficient IgA CSR., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Breast cancer severely affects women health. 70% of breast cancer are estrogen receptor positive. Breast cancer stem cells are a group of tumor with plasticity, causing tumor relapse and metastasis. RUNX3 is a tumor suppressor frequently inactivated in estrogen receptor positive breast cancer. However, the mechanism of how RUNX3 is involved in the regualation of cancer stem cell traits in estrogen receptor positive breast cancer remains elusive. In this study, we utilized cut-tag assay to investigate the binding profile RUNX3 in BT474 and T47D cell, and confirmed EXOSC4 as the bona-fide target of RUNX3; RUNX3 could bind to the promoter are of EXOSC4 to suppress its expression. Furthermore, EXOSC4 could increase the colony formation, cell invasion and mammosphere formation ability of breast cancer cells and upregulate the the expression of SOX2 and ALDH1. Consistent with these findings, EXOSC4 was associated with poorer survival for Luminal B/Her2 breast cancer patiens. At last, we confirmed that EXOSC4 mediated the tumor suppressive role of RUNX3 in breast cancer cells. In conclusion, we demonstrate that RUNX3 directly binds to the promoter region of EXOSC4, leading to the suppression of EXOSC4 expression and exerting a tumor-suppressive effect in estrogen receptor postivive breast cancer cells., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Long noncoding RNA MIR17HG was involved with the progression of non-small-cell lung cancer (NSCLC), but specific mechanisms of MIR17HG-mediated immune escape of NSCLC cells were still unknown. The present study investigated the function of MIR17HG on regulatory T cell (Treg)-mediated immune escape and the underlying mechanisms in NSCLC. Expression of MIR17HG and miR-17-5p in NSCLC tissue samples were detected using quantitative real-time PCR (qRT-PCR). A549 and H1299 cells were transfected with sh-MIR17HG, miR-17-5p inhibitor, or sh-MIR17HG + miR-17-5p inhibitor, followed by cocultured with Tregs. Cell proliferation was measured using 5-ethynyl-20-deoxyuridine (Edu) staining assay and cell counting kit-8 (CCK-8) assay. Flow cytometry was used for determining positive numbers of FOXP3 + CD4 + /CD25 + /CD8 + Tregs. Through subcutaneous injection with transfected A549 cells, a xenograft nude mouse model was established. Weights and volumes of xenograft tumors were evaluated. Additionally, the expressions of immune-related factors including transforming growth factor beta (TGF-β), vascular endothelial growth factor A (VEGF-A), interleukin-10 (IL-10), IL-4, and interferon-gamma (IFN-γ) in cultured cells, were evaluated by enzyme-linked immunosorbent assay and western blot analysis. Then, miR-17-5p was decreased and MIR17HG was enhanced in both NSCLC tissues and cell lines. MIR17HG knockdown significantly suppressed cell proliferation, tumorigenicity, and immune capacity of Tregs in A549 and H1299 cells, whereas sh-MIR17HG significantly reduced expression levels of VEGF-A, TGF-β, IL-4, and IL-10 but promoted the IFN-γ level in vitro and in vivo. Moreover, downregulation of miR-17-5p significantly reversed the effects of sh-MIR17HG. Additionally, we identified that runt- related transcription factor 3 (RUNX3) was a target of miR-17-5p, and sh-MIR17HG and miR-17-5p mimics downregulated RUNX3 expression. In conclusion, downregulation of MIR17HG suppresses tumorigenicity and Treg-mediated immune escape in NSCLC through downregulating the miR-17-5p/RUNX3 axis, indicating that this axis contains potential biomarkers for NSCLC., (© 2024 Wiley Periodicals LLC.)

We have recently reported that transcription factor Runx3 is required for pulmonary generation of CD8 + cytotoxic T lymphocytes (CTLs) that play a crucial role in the clearance of influenza A virus (IAV). To understand the underlying mechanisms, we determined the effects of Runx3 knockout (KO) on CD8 + T cell local expansion and phenotypes using an inducible general Runx3 KO mouse model. We found that in contrast to the lungs, Runx3 general KO promoted enlargement of lung-draining mediastinal lymph node (mLN) and enhanced CD8 + and CD4 + T cell expansion during H1N1 IAV infection. We further found that Runx3 deficiency greatly inhibited core 2 O-glycosylation of selectin ligand CD43 on activated CD8 + T cells but minimally affected the cell surface expression of CD43, activation markers (CD44 and CD69) and cell adhesion molecules (CD11a and CD54). Runx3 KO had a minor effect on lung effector CD8 + T cell death by IAV infection. Our findings indicate that Runx3 differently regulates CD8 + T cell expansion in mLNs and lungs by H1N1 IAV infection. Runx3 is required for CD43 core 2 O-glycosylation on activated CD8 + T cells, and the involved Runx3 signal pathway may mediate CD8 + T cell phenotype for pulmonary generation of CTLs.

Objective: Asthma is a chronic heterogeneous airway disease, and imbalanced T-helper type 1 (Th1) and Th2 cell-mediated inflammation contribute to its pathogenesis. Although it has been suggested that androgen and estrogen were involved in development of asthma, the underlying mechanisms remained largely unclear. Studies have demonstrated that Runx3 could promote naive CD4 + T cells to differentiate into Th1 cells. Hence, our study aimed to explore the potential regulatory mechanism of androgen and estrogen on asthma via modulating Runx3. Methods: First, clinical assessments and pulmonary function tests were conducted on 35 asthma patients and 24 healthy controls. The concentrations of androgen, estrogen, and androgen estrogen ratios were assessed in peripheral blood samples of asthma patients and healthy controls. Then, a murine asthma model was established to explore the effects of estrogen and androgen (alone or in combination) on asthma. Third, an in vitro assay was used to explore the mechanism of combination of androgen and estrogen in asthma. Results: We observed decreased androgen and increased estrogen levels in asthma patients compared with healthy controls. In mice with experimental asthma, there were increased serum concentrations of estrogen and decreased serum concentrations of androgen, intervention with combination of androgen and estrogen alleviated airway inflammations, increased Runx3 expressions and elevated Th1 differentiation. In CD4+ T cells co-cultured with bronchial epithelial cells (BECs), treatment with androgen plus estrogen combination promoted Th1 differentiation, which was mitigated by Runx3 knockdown in BECs and enhanced by Runx3 overexpression. Conclusion: These findings suggest that androgen estrogen combination modulate the Th1/Th2 balance via regulating the expression of Runx3 in BECs, thereby providing experimental evidence supporting androgen and estrogen combination as a novel therapy for asthma., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Objectives: Previous reports have indicated that the methylation profile in peripheral blood mononuclear cells (PBMCs) in different genes and loci is altered in colorectal cancer (CRC). Regarding the high mortality rate and silent nature of CRC, screening and early detection can meaningfully reduce disease-related deaths. Therefore, for the first time, we aimed to evaluate the early non-invasive diagnosis of CRC via quantitative promoter methylation analysis of RUNX3 and RASSF1A genes in PBMCs., Materials and Methods: In the present study, we analyzed the methylation status of two important tumor suppressor genes including RUNX3 and RASSF1A in 70 CRC patients and 70 non-malignant subjects using methylation-quantification of endonuclease-resistant DNA (MethyQESD), and a bisulfite conversion-independent method., Results: RUNX3 was significantly hypermethylated in PBMCs of CRC patients compared to healthy controls (P < 0.001). By determining the efficient cutoff value, the sensitivity, and specificity of RUNX3 promoter methylation for CRC diagnosis reached 84.28% and 77.14%, respectively. The receiver operating characteristic (ROC) curve analyses demonstrated that RUNX3 promoter methylation has high accuracy (areas under the curve [AUC] = 0.840, P < 0.001) for discriminating CRC subjects from healthy individuals. Moreover, RUNX3 methylation levels in PBMCs progressively increased with the stage of the disease (P < 0.001). Although the amount of RASSF1A promoter methylation was not significantly different between CRC patients and controls as well as in different stages of the disease (P > 0.05)., Conclusion: Our findings confirmed that PBMCs are reliable sources of methylation analysis for CRC screening, and RUNX3 promoter methylation can be used as a promising biomarker for early diagnosis of CRC., (Copyright © 2023 Copyright: © 2023 Journal of Cancer Research and Therapeutics.)

Macrophage-derived lipid-laden foam cells from the subendothelium play a crucial role in the initiation and progression of atherosclerosis. However, the molecule mechanism that regulates the formation of foam cells is not completely understood. Here, we found that SLAMF7 was upregulated in mice bone marrow-derived macrophages and RAW264.7 cells stimulated with oxidized low-density lipoprotein (ox-LDL). SLAMF7 promoted ox-LDL-mediated macrophage lipid accumulation and M1-type polarization. SLAMF7 deficiency reduced serum lipid levels and improved the lesions area of carotid plaque and aortic arch in high-fat diet-fed ApoE -/- mice. In response to ox-LDL, SLAMF7 downregulated NR4A1 and upregulated RUNX3 through transcriptome sequencing analysis. Overexpression NR4A1 reversed SLAMF7-induced lipid uptake and M1 polarization via inhibiting RUNX3 expression. Furthermore, RUNX3 enhanced foam cell formation and M1-type polarization. Taken together, the study suggested that SLAMF7 play contributing roles in the pro-atherogenic effects by regulating NR4A1-RUNX3., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Oncogenic K-RAS mutations occur in approximately 25% of human lung cancers and are most frequently found in codon 12 (G12C, G12V, and G12D). Mutated K-RAS inhibitors have shown beneficial results in many patients; however, the inhibitors specifically target K-RAS G12C and acquired resistance is a common occurrence. Therefore, new treatments targeting all kinds of oncogenic K-RAS mutations with a durable response are needed. RUNX3 acts as a pioneer factor of the restriction (R)-point, which is critical for the life and death of cells. RUNX3 is inactivated in most K-RAS -activated mouse and human lung cancers. Deletion of mouse lung Runx3 induces adenomas (ADs) and facilitates the development of K-Ras -activated adenocarcinomas (ADCs). In this study, conditional restoration of Runx3 in an established K-Ras -activated mouse lung cancer model regressed both ADs and ADCs and suppressed cancer recurrence, markedly increasing mouse survival. Runx3 restoration suppressed K-Ras -activated lung cancer mainly through Arf-p53 pathway-mediated apoptosis and partly through p53-independent inhibition of proliferation. This study provides in vivo evidence supporting RUNX3 as a therapeutic tool for the treatment of K - RAS -activated lung cancers with a durable response.

Purpose: The aim of this study was to assess the clinical significance of RUNX3 gene hypermethylation in the pathogenetic mechanisms of breast cancer in women, taking into account its cohypermethylation with the BRCA1 gene., Methods: This study included 74 women with newly diagnosed breast cancer (samples from female primary breast carcinomas and paired peripheral blood samples) and 62 women without oncological pathology-control group (peripheral blood samples). Epigenetic testing for hypermethylation status studying was performed in all samples on freshly collected material with the addition of a preservative before the storage and DNA isolation., Results: Hypermethylation of the RUNX3 gene promoter region was detected in 71.6% samples of breast cancer tissue and in 35.13% samples of blood. The RUNX3 gene promoter region hypermethylation was significantly higher among breast cancer patients compared to the control group. The frequency of cohypermethylation in RUNX3 and BRCA1 genes was significantly increased in breast cancer tissues compared to the blood of patients., Conclusion: A significantly increased frequency of the hypermethylation of the RUNX3 gene promoter region and its cohypermethylation with the BRCA1 gene promoter region was found in tumor tissue and blood samples from patients with breast cancer, in contrast to the control group. The identified differences indicate the importance of further investigations of suppressor genes cohypermethylation in patients with breast cancer. Further large-scale studies are needed to find out whether the detected hypermethylation and cohypermethylation of the RUNX3 gene promoter region will have an impact on the treatment strategy in patients., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Osteosarcoma (OS) is characterized by TP53 mutations in humans. In mice, loss of p53 triggers OS development, and osteoprogenitor-specific p53-deleted mice are widely used to study the process of osteosarcomagenesis. However, the molecular mechanisms underlying the initiation or progression of OS following or parallel to p53 inactivation remain largely unknown. Here, we examined the role of transcription factors involved in adipogenesis (adipo-TFs) in p53-deficient OS and identified a novel tumor suppressive molecular mechanism mediated by C/ebpα. C/ebpα specifically interacts with Runx3, a p53 deficiency-dependent oncogene, and, in the same manner as p53, decreases the activity of the oncogenic axis of OS, Runx3-Myc, by inhibiting Runx3 DNA binding. The identification of a novel molecular role for C/ebpα in p53-deficient osteosarcomagenesis underscores the importance of the Runx-Myc oncogenic axis as a therapeutic target for OS., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Vagal sensory neurons monitor mechanical and chemical stimuli in the gastrointestinal tract. Major efforts are underway to assign physiological functions to the many distinct subtypes of vagal sensory neurons. Here, we use genetically guided anatomical tracing, optogenetics, and electrophysiology to identify and characterize vagal sensory neuron subtypes expressing Prox2 and Runx3 in mice. We show that three of these neuronal subtypes innervate the esophagus and stomach in regionalized patterns, where they form intraganglionic laminar endings. Electrophysiological analysis revealed that they are low-threshold mechanoreceptors but possess different adaptation properties. Lastly, genetic ablation of Prox2 and Runx3 neurons demonstrated their essential roles for esophageal peristalsis in freely behaving mice. Our work defines the identity and function of the vagal neurons that provide mechanosensory feedback from the esophagus to the brain and could lead to better understanding and treatment of esophageal motility disorders., Competing Interests: Declaration of interests S.D.L. is a consultant for Kallyope., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

MYC is one of the most commonly dysregulated proto-oncogenes in cancer. MYC promotes cancer initiation and maintenance by regulating multiple biological processes, such as proliferation and stem cell function. Here, we show that developmental regulator RUNX3 targets MYC protein for rapid degradation through the glycogen synthase kinase-3 beta-F-box/WD repeat-containing protein 7 (GSK3β-FBXW7) proteolytic pathway. The evolutionarily conserved Runt domain of RUNX3 interacts directly with the basic helix-loop-helix leucine zipper of MYC, resulting in the disruption of MYC/MAX and MYC/MIZ-1 interactions, enhanced GSK3β-mediated phosphorylation of MYC protein at threonine-58 and its subsequent degradation via the ubiquitin-proteasomal pathway. We therefore uncover a previously unknown mode of MYC destabilization by RUNX3 and provide an explanation as to why RUNX3 inhibits early-stage cancer development in gastrointestinal and lung mouse cancer models., (© 2023. The Author(s).)

The RUNX transcription factors are frequently dysregulated in human cancers, suggesting their potential as attractive targets for drug treatment. However, all three transcription factors have been described as both tumor suppressors and oncogenes, indicating the need to determine their molecular mechanisms of action. Although RUNX3 has long been considered a tumor suppressor in human cancers, several recent studies have shown that RUNX3 is upregulated during the development or progression of various malignant tumors, suggesting it may act as a "conditional" oncogene. Resolving this paradox and understanding how a single gene can exhibit both oncogenic and tumor-suppressive properties is essential for successful drug targeting of RUNX. This review describes the evidence for the activities of RUNX3 in human cancer and proposes an explanation for the duality of RUNX3 involving the status of p53. In this model, p53 deficiency causes RUNX3 to become oncogenic, leading to aberrant upregulation of MYC.

The RUNX family of transcription factors, including RUNX1, RUNX2, and RUNX3, are key regulators of development and can function as either tumor suppressors or oncogenes in cancer. Emerging evidence suggests that the dysregulation of RUNX genes can promote genomic instability in both leukemia and solid cancers by impairing DNA repair mechanisms. RUNX proteins control the cellular response to DNA damage by regulating the p53, Fanconi anemia, and oxidative stress repair pathways through transcriptional or non-transcriptional mechanisms. This review highlights the importance of RUNX-dependent DNA repair regulation in human cancers.

RUNX3 is a transcription factor with regulatory roles in cell proliferation and development. While largely characterized as a tumor suppressor, RUNX3 can also be oncogenic in certain cancers. Many factors account for the tumor suppressor function of RUNX3, which is reflected by its ability to suppress cancer cell proliferation after expression-restoration, and its inactivation in cancer cells. Ubiquitination and proteasomal degradation represent a major mechanism for the inactivation of RUNX3 and the suppression of cancer cell proliferation. On the one hand, RUNX3 has been shown to facilitate the ubiquitination and proteasomal degradation of oncogenic proteins. On the other hand, RUNX3 can be inactivated through the ubiquitin-proteasome system. This review encapsulates two facets of RUNX3 in cancer: how RUNX3 suppresses cell proliferation by facilitating the ubiquitination and proteasomal degradation of oncogenic proteins, and how RUNX3 is degraded itself through interacting RNA-, protein-, and pathogen-mediated ubiquitination and proteasomal degradation.

A cell cycle is a series of events that takes place in a cell as it grows and divides. At the G 1 phase of cell cycle, cells monitor their cumulative exposure to specific signals and make the critical decision to pass through the restriction (R)-point. The R-point decision-making machinery is fundamental to normal differentiation, apoptosis, and G 1 -S transition. Deregulation of this machinery is markedly associated with tumorigenesis. Therefore, identification of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in tumor biology. RUNX3 is one of the genes frequently inactivated in tumors by epigenetic alterations. In particular, RUNX3 is downregulated in most K-RAS -activated human and mouse lung adenocarcinomas (ADCs). Targeted inactivation of Runx3 in the mouse lung induces adenomas (ADs), and markedly shortens the latency of ADC formation induced by oncogenic K-Ras . RUNX3 participates in the transient formation of R-point-associated activator (RPA-RX3-AC) complexes, which measure the duration of RAS signals and thereby protect cells against oncogenic RAS. This review focuses on the molecular mechanism by which the R-point participates in oncogenic surveillance.

CAR-T therapy has shown successful in the treatment of certain types of hematological malignancy, while the efficacy of CAR-T cell in treating solid tumors has been limited due to the exhaustion of CAR-T caused by the tumor microenvironment in solid tumors. Therefore, improving the exhaustion of CAR-T cell is one of the inspiring strategies for CAR-T treatment of solid tumors. As an important regulator in T cell immunity, the transcription factor RUNX3 not only negatively regulates the terminal differentiation T-bet gene, reducing the ultimate differentiation of T cells, but also increases the residency of T cells in non-lymphoid tissues and tumors. By overexpressing RUNX3 in CAR-T cells, we found that increasing the expression of RUNX3 maintained the low differentiation of CAR-T cells, further improving the exhaustion of CAR-T cells during antigen stimulation. In vitro, we found that RUNX3 could reduce the release of cytokines while maintaining CAR-T cells function. In re-challenge experiments, CAR-T cells overexpressing RUNX3 (Runx3-OE CAR-T) were safer than conventional CAR-T cells, while RUNX3 could also maintain the anti-tumor efficacy of CAR-T cells in vivo. Collectively, we found that Runx3-OE CAR-T cells can improve CAR-T phenotype and reduce cytokines release while maintaining CAR-T cells function, which may improve the safety of CAR-T therapy in clinical trials., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Disturbances in immune regulation, intestinal dysbiosis and inflammation characterize ankylosing spondylitis (AS), which is associated with RUNX3 loss-of-function variants. ZAP70 W163C mutant (SKG) mice have reduced ZAP70 signaling, spondyloarthritis and ileitis. In small intestine, Foxp3 + regulatory T cells (Treg) and CD4 + CD8αα + TCRαβ + intraepithelial lymphocytes (CD4-IEL) control inflammation. TGF-β and retinoic acid (RA)-producing dendritic cells and MHC-class II + intestinal epithelial cells (IEC) are required for Treg and CD4-IEL differentiation from CD4 + conventional or Treg precursors, with upregulation of Runx3 and suppression of ThPOK. We show in SKG mouse ileum, that ZAP70 W163C or ZAP70 inhibition prevented CD4-IEL but not Treg differentiation, dysregulating Runx3 and ThPOK. TGF-β/RA-mediated CD4-IEL development, T-cell IFN-γ production, MHC class-II + IEC, tissue-resident memory T-cell and Runx3-regulated genes were reduced. In AS intestine, CD4-IEL were decreased, while in AS blood CD4 + CD8 + T cells were reduced and Treg increased. Thus, genetically-encoded TCR signaling dysfunction links intestinal T-cell immunodeficiency in mouse and human spondyloarthropathy., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest exists., (Copyright © 2022. Published by Elsevier Inc.)

Hepatocellular carcinoma (HCC) is one common cancer in the world. Previous studies have shown that miR-17 family members are elevated in most tumors and promote tumor progression. However, there is no comprehensive analysis of the expression and functional mechanism of the microRNA-17 (miR-17) family in HCC. The aim of this study is to comprehensively analyze the function of the miR-17 family in HCC and the molecular mechanism of its role. Bioinfoimatics analysis of the miR-17 family expression profile and its relationship to clinical significance using The Cancer Genome Atlas (TCGA) database, and this result was confirmed using quantitative real-time polymerase chain reaction. miR-17 family members were tested for functional effects through transfection of miRNA precursors and inhibitors, and monitoring cell viability and migration by cell count and wound healing assays. In addition, we using dual-luciferase assay and Western blot demonstrated the targeting relationship between the miRNA-17 family and RUNX3. These members of miR-17 family were highly expressed in HCC tissues, and the overexpression of the miR-17 family promoted the proliferation and migration of SMMC-7721 cells, whereas treatment with anti-miR17 inhibitors caused the opposite effects. Notably, we also found that inhibitors anti-each member of miR-17 can suppress the expression of the entire family member. In addition, they can bind to the 3' untranslated region of RUNX3 to regulate its expression at the translational level. Our results proved that miR-17 family has oncogenic characteristics, overexpression every member of the family contributed to HCC cell proliferation and migration by reducing the translation of RUNX3.

Respiratory viruses pose a continuing and substantive threat to human health globally. Host innate and adaptive immune responses are the critical antiviral defense mechanisms to control viral replication and spread. The present study is designed to determine the role of transcription factor Runx3 in the host immune response to influenza A virus (IAV) infection. As Runx3 is required for embryonic development, we generated an inducible Runx3 global knockout (KO) mouse model and found that Runx3 KO in adult C57BL/6 mice minimally affected thymic function under normal conditions and survival was at least 250 days post Runx3 deletion. We applied the mouse model to IAV infection and found that Runx3 KO resulted in a huge reduction (>85%) in numbers of total and antigen-specific pulmonary CD8 + cytotoxic T cells during IAV infection, while it had a minor effect on pulmonary generation of CD4 + T cells. To our surprise, this general KO of Runx3 did not significantly alter viral clearance and animal survival following IAV infection. Interestingly, we found that Runx3 KO significantly increased the numbers of pulmonary innate immune cells such as macrophages and neutrophils and the production of pro-inflammatory cytokines during IAV infection. We further found that Runx3 was strongly detected in CCR2 + immune cells in IAV-infected mouse lungs and was induced in activated macrophages and dendritic cells (DCs). As pulmonary CD8 + cytotoxic T cells play a central role in the clearance of IAV, our findings suggest that Runx3 KO may enhance host innate immunity to compensate for the loss of pulmonary CD8 + cytotoxic T cells during IAV infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hao, Kundu, Shetty, Tucker, Idell and Tang.)

Background: MicroRNA-20a (miR-20a) is dysregulated in many types of malignancies, including human hepatocellular carcinoma (HCC), but its expression level and functional significance in HCC are still disputed. We aimed to study the role of miR-20a-5p in HCC and its downstream molecular mechanisms., Methods: We used real-time polymerase chain reaction to detect the expression of miR-20a-5p and runt-related transcription factor 3 ( RUNX3 ) in HCC and paraneoplastic tissue, transfected Huh7 and highly metastatic human hepatocellular carcinoma (MHCC97H) cells. A live cell workstation was used to observe the proliferation and migration of transfected cells. The invasiveness of transfected cells was verified by Transwell assay. Cell apoptosis was detected by flow cytometry. The expression levels of proteins after transfection were measured using simple western immunoblot measurements. Gene expression profiles between HCC and normal samples were obtained from The Cancer Genome Atlas. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment results were processed by the database for annotation, visualization and integrated discovery. Potential target genes of miR-20a-5p were predicted to further investigate how miR-20a-5p regulates epithelial-mesenchymal transition (EMT) in HCC., Results: MiR-20a-5p was significantly highly expressed in HCC tissues, and overexpression of miR-20a-5p significantly promoted HCC cell proliferation, migration, and invasion and inhibited apoptosis in vitro. The protein expression of E-cadherin was decreased and that of vimentin was increased after overexpression of miR-20a-5p in HCC cells. We discovered the intersection of genes from miRDB, miR TarBase, and TargetScan, obtained 397 target genes and finally focused on RUNX3. RUNX3 was not only reduced in HCC specimens but also drastically reduced in HCC cells overexpressing miR-20a-5p. RUNX3 expression decreased with elevated miR-20a-5p, which activated downstream EMT signaling and promoted cell proliferation, migration, and invasion., Conclusions: Since RUNX3 is involved in EMT in HCC, as proven by previous research, our findings provide further evidence for a novel regulatory pathway comprising the miR-20a/RUNX3/EMT axis that upregulates EMT signaling and enhances the migration of HCC cells., (Copyright © 2022 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.)

Ulcerative colitis (UC) results from a complex interplay between the environment, gut microbiota, host genetics, and immunity. Runt-related transcription factor 3 (RUNX3) regulates Th1/Th2 balance and, thus, the synthesis of cytokines and inflammation. We aimed to analyze the dependence of RUNX3 promoter 2 ( P2 ) methylation level on: age, sex, body mass index (BMI), C-reactive protein (CRP), serum albumin, disease duration, Pediatric Ulcerative Colitis Activity Index (PUCAI), the Paris classification, and exposure to medications. This multicenter, cross-sectional study recruited hospitalized children with UC. Methylation of RUNX3 P2 was measured with methylation-sensitive restriction enzymes in the whole blood DNA. Sixty-four children were enrolled, with a mean age of 14.5 ± 2.8 years. Half of them were female (51.6%), and the average BMI Z-score was -0.44 ± 1.14. The mean methylation of RUNX3 P2 was 54.1 ± 13.3%. The methylation level of RUNX3 P2 did not correlate with age, sex, nutritional status, CRP, albumin, PUCAI, or the extent of colitis (Paris E1-E4). RUNX3 P2 methylation did not differ between patients recruited within two and a half months of diagnosis and children who had UC for at least a year. Current or past exposure to biologics, immunosuppressants, or steroids was not associated with RUNX3 P2 methylation. Methylation of RUNX3 promoter 2 in whole blood DNA does not seem to be associated with the characteristics of UC in children.

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Dysregulation of messenger RNAs (mRNA) has been recognized to be associated with HCC carcinogenesis and development. Polypeptide GalNAc Transferase 6 (GALNT6), an O-type glycosyltransferase, has been confirmed as tumor promoter in different cancers. However, the function of GALNT6 in HCC remains to be studied., Methods: RT-qPCR and western blot experiments were, respectively, performed for evaluating RNA expressions and protein levels. Supported by bioinformatics analysis, mechanism assays were conducted for validating the potential relation between different genes. Functional assays were implemented to analyze HCC cell migration and invasion after different transfections., Results: GALNT6 was aberrantly upregulated in HCC cells. Knockdown of GALNT6 could repress HCC cell migration and invasion. RUNX3 was verified to bind to GALNT6 promoter and activate GALNT6 transcription. GALNT6 depletion led to inhibited O-glycosylation and aggravated degradation of MUC1. MUC1 overexpression could rescue the impeded HCC cell migration and invasion induced by GALNT6 knockdown., Conclusion: To sum up, GALNT6 transcriptionally activated by RUNX3 mediated the O-glycosylation of MUC1, thus exerting promoting influence on HCC cell migration and invasion., Competing Interests: The authors have declared that no competing financial interest exists., (Copyright © 2022 Fei Gao and Gang Zheng.)

RUNX3 is a transcription factor dysregulated in acute myeloid leukemia (AML). However, its role in normal myeloid development and leukemia is poorly understood. Here we investigate RUNX3 expression in both settings and the impact of its dysregulation on myelopoiesis. We found that RUNX3 mRNA expression was stable during hematopoiesis but decreased with granulocytic differentiation. In AML, RUNX3 mRNA was overexpressed in many disease subtypes, but downregulated in AML with core binding factor abnormalities, such as RUNX1::ETO. Overexpression of RUNX3 in human hematopoietic stem and progenitor cells (HSPC) inhibited myeloid differentiation, particularly of the granulocytic lineage. Proliferation and myeloid colony formation were also inhibited. Conversely, RUNX3 knockdown did not impact the myeloid growth and development of human HSPC. Overexpression of RUNX3 in the context of RUNX1::ETO did not rescue the RUNX1::ETO-mediated block in differentiation. RNA-sequencing showed that RUNX3 overexpression downregulates key developmental genes, such as KIT and RUNX1, while upregulating lymphoid genes, such as KLRB1 and TBX21. Overall, these data show that increased RUNX3 expression observed in AML could contribute to the developmental arrest characteristic of this disease, possibly by driving a competing transcriptional program favoring a lymphoid fate., (© 2022. The Author(s).)

Colorectal cancer (CRC) is one of the most prevalent and lethal malignancies. Exploring the underlying molecular mechanisms is very helpful for the development of new therapy. Here, we investigated the function of circMETTL3/miR-107/PER3 in CRC. Human CRC tissues from diagnosed CRC patients and six CRC cell lines, one normal human colon cell line were used. qRT-PCR and western blotting were performed to determine expression levels of RUNX3, circMETTL3, miR-107, PER3, and proliferation-, and migration-related proteins. CCK-8, colony formation assay, transwell assay, and scratch wound assay were utilized to assess CRC cell proliferation and invasion. ChIP, EMSA, biotin-pull down, RIP assay, and dual luciferase reporter assay were performed to validate interactions of RUNX3/METTL3 promoter, circMETTL3/miR-107, and miR-107/PER3. FISH was used to characterize circMETTL3. MSP was employed to measure methylation level. Nude mouse xenograft model was used to determine the effects on tumor growth and metastasis in vivo. RUNX3, circMETTL3, and PER3 were diminished while miR-107 was elevated in CRC tissues and cells. Low levels of RUNX3 and circMETTL3 correlated with poor prognosis of CRC. Overexpression of RUNX3, circMETTL3, or PER3 suppressed while miR-107 mimics promoted, CRC cell proliferation and invasion, as well as tumor growth and metastasis in vivo. Mechanistically, RUNX3 bound to METTL3 promoter and activated circMETTL3 transcription. circMETTL3 directly bound with miR-107 which targeted PER3. circMETTL3/miR-107 regulated CRC cell proliferation and invasion via PER3. CircMETTL3, transcriptionally activated by RUNX3, restrains CRC development and metastasis via acting as a miR-107 sponge to regulate PER3 signaling., (© 2022. The Author(s).)

Objective: To explore the role of Runt-related transcription factor 3 (RUNX3) in metabolic regulation of trastuzumab-resistant gastric cancer cells and investigate the mechanism of RUNX3 knockdown-mediated reversal of trastuzumab resistance., Methods: We performed a metabolomic analysis of trastuzumab-resistant gastric cancer cells (NCI N87R) and RUNX3 knockdown cells (NCI N87R/RUNX3) using ultra performance liquid chromatography (UPLC) coupled with Q Exactive Focus Orbitrap mass spectrometry (MS). Multivariate combined with univariate analyses and MS/MS ion spectrums were used to screen the differential variables. MetaboAnalyst 5.0 database was employed for pathway enrichment analysis. Differential metabolites-genes regulatory relationships were constructed based on OmicsNet database. The changes in GSH/GSSG and NADPH/NADP ratios in NCI N87R/RUNX3 cells were measured using detection kits., Results: The metabolic profile of NCI N87R cells was significantly altered after RUNX3 knockdown, with 81 differential metabolites identified to contribute significantly to the classification, among which 43 metabolites were increased and 38 were decreased ( P < 0.01). In NCI N87R cells, RUNX3 knockdown resulted in noticeable alterations in 8 pathways involving glutamine metabolism, glycolysis, glycerophospholipid, nicotinate-nicotinamide and glutathione metabolism, causing also significant reduction of intracellular GSH/GSSG and NADPH/NADP ratios ( P < 0.01). The differential metabolites-genes network revealed a regulatory relationship between the metabolic molecules and genes., Conclusion: RUNX3 reverses trastuzumab resistance in gastric cancer cells by regulating energy metabolism and oxidation-reduction homeostasis and may serve as a potential therapeutic target for trastuzumab-resistant gastric cancer.

Dermatomyositis (DM) is a systemic autoimmune disease. It's known that the number of regulatory T (Treg) cells was decreased in DM. Besides, Treg cells were increased after treatment in DM patients. Forkhead box P3 (Foxp3) is specifically expressed in Treg cells and Runt-related transcription factor (RUNX3) could regulate Foxp3 transcription. And our previous experiment showed that Homeobox D gene cluster antisense growth-associated long noncoding RNA (HAGLR) was up-regulated in DM patients. Here, we aimed to explore whether HAGLR regulated the differentiation of Treg cells through RUNX3-mediated transcription of Foxp3, thus affecting the progression of DM. The levels of HAGLR, Foxp3, and RUNX3 were examined by quantitative real-time PCR. The protein levels of Foxp3 and RUNX3 were examined by western blot. The proportions of Treg cells in CD4 + T cells were detected by flow cytometry. Hematoxylin and eosin staining was conducted to observe the histopathological changes of the muscle. RNA pull-down assay was performed to detect the interaction between HAGLR and RUNX3. A dual-luciferase reporter gene assay was conducted to examine the effect of HAGLR on the transcriptional regulation of Foxp3 by RUNX3. HAGLR was up-regulated and Foxp3 was down-regulated in DM patients. Besides, RUNX3 protein levels were decreased in DM patients, while its mRNA levels did not change significantly. The proportion of Treg cells was down-regulated in DM patients. In addition, interference with HAGLR could increase the levels of Foxp3, RUNX3 protein level, and the proportion of Treg cells. Besides, there was an interaction between HAGLR and RUNX3. We also found that knockdown of HAGLR and RUNX3 restored the increased Treg cells induced by HAGLR knockdown alone. In vivo experiments indicated that injection with adv-HAGLR increased Treg cell proportion and attenuated DM development. Interference with HAGLR could increase the protein levels of RUNX3, high levels of RUNX3 further promoted the expression of the Foxp3, thus restoring the number of Treg cells and easing the development of DM., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Targeting glutamine metabolism is previously indicated as a potential and attractive strategy for gastric cancer (GC) therapy. However, the underlying mechanisms responsible for the modification of glutamine metabolism in GC cells have not been fully elucidated. Accordingly, the current study sought to investigate the physiological mechanisms of RUNX3-mediated circDYRK1A in glutamine metabolism of GC., Methods: Firstly, GC tissues and adjacent normal tissues were obtained from 50 GC patients to determine circDYRK1A expression in GC tissues. Next, the binding affinity among RUNX3, circDYRK1A, miR-889-3p, and FBXO4 was detected to clarify the mechanistic basis. Moreover, GC cells were subjected to ectopic expression and knockdown manipulations of circDYRK1A, miR-889-3p, and/or FBXO4 to assay GC cell malignant phenotypes, levels of glutamine, glutamic acid, and α-KG in cell supernatant and glutamine metabolism-related proteins (GLS and GDH). Finally, nude mice were xenografted with GC cells to explore the in vivo effects of circDYRK1A on the tumorigenicity and apoptosis., Results: circDYRK1A was found to be poorly expressed in GC tissues. RUNX3 was validated to bind to the circDYRK1A promoter, and circDYRK1A functioned as a miR-889-3p sponge to up-regulate FBXO4 expression. Moreover, RUNX3-upregulated circDYRK1A reduced levels of glutamine, glutamic acid, and α-KG, and protein levels of GLS and GDH, and further diminished malignant phenotypes in vitro. Furthermore, in vivo experimentation substantiated that circDYRK1A inhibited the tumorigenicity and augmented the apoptosis in GC., Conclusion: In conclusion, these findings highlighted the significance and mechanism of RUNX3-mediated circDYRK1A in suppressing glutamine metabolism in GC via the miR-889-3p/FBXO4 axis., (© 2022. The Author(s).)

Background: Accumulating evidence has indicated that runt-related transcription factor 3 ( RUNX3 ) gene polymorphism (rs7528484) is associated with an alimentary system cancer risk. However, the role of rs7528484 in colorectal cancer is still unclear. The present study aimed to explore the association between rs7528484 and colorectal cancer susceptibility in a Chinese Han population., Material and Methods: We firstly investigated the effect of the polymorphism rs7528484 in distal promoter of RUNX3 polymorphism on colorectal cancer risk in a Chinese Han population comprising 427 colorectal cancer patients and 503 controls. We then carried out a phenotype-genotype association analysis to validate its influence on the adjacent gene RUNX3 ., Results: Logistic regression analysis demonstrated that the T allele of rs7528484 was significantly associated with an increased risk for colorectal cancer occurrence in our case-control study (odds ratio = 1.33; 95% confidence interval = 1.09-1.65; P  = 0.005). In stratified analysis, the susceptibility of colorectal cancer in the T allele carriers increased among the smokers, III and IV tumor stage, and at the rectum. Furthermore, the T allele was significantly correlated with lower expression of RUNX3 in vitro., Conclusion: In summary, the current case-control and genotype-phenotype study provides convincing evidence that functional RUNX3 polymorphism (rs7528484) is related to colorectal cancer risk and is a plausible marker for the prediction of colorectal cancer.

The response of naive CD8 + T cells to their cognate antigen involves rapid and broad changes to gene expression that are coupled with extensive chromatin remodeling, but the mechanisms governing these changes are not fully understood. Here, we investigated how these changes depend on the basic leucine zipper ATF-like transcription factor Batf, which is essential for the early phases of the process. Through genome scale profiling, we characterized the role of Batf in chromatin organization at several levels, including the accessibility of key regulatory regions, the expression of their nearby genes, and the interactions that these regions form with each other and with key transcription factors. We identified a core network of transcription factors that cooperated with Batf, including Irf4, Runx3, and T-bet, as indicated by their colocalization with Batf and their binding in regions whose accessibility, interactions, and expression of nearby genes depend on Batf. We demonstrated the synergistic activity of this network by overexpressing the different combinations of these genes in fibroblasts. Batf and Irf4, but not Batf alone, were sufficient to increase accessibility and transcription of key loci, normally associated with T cell function. Addition of Runx3 and T-bet further contributed to fine-tuning of these changes and was essential for establishing chromatin loops characteristic of T cells. These data provide a resource for studying the epigenomic and transcriptomic landscape of effector differentiation of cytotoxic T cells and for investigating the interdependency between transcription factors and its effects on the epigenome and transcriptome of primary cells.

Abdominal aortic aneurysm (AAA) has been associated with the dysfunction of vascular smooth muscle cells (VSMCs) and extracellular matrix (ECM) remodelling. Runt-related transcription factor 3 (RUNX3) has been reported to be up-regulated in aneurysmal aorta samples compared with normal aorta. However, its function in VSMCs and the mechanism of function remains unknown. Therefore, our study aimed to investigate the role of RUNX3 in ECM remodelling and VSMC function, and further explore the underlying mechanism. Our results verified that RUNX3 was increased in aortic samples of AAA compared with healthy controls. Overexpression vectors of RUNX3 (ov-RUNX3) and siRNA of RUNX3 (si-RUNX3) were transfected into Human aortic smooth muscle cells (HAoSMCs). The results indicated that ov-RUNX3 promoted cell proliferation, migration, and MMP-2/3/9 secretion, and suppressed TIMP-1, collagen I/III, SM22, MYH11 and CNN1 expression in HAoSMCs. The silencing of RUNX3 has the opposite effect. Furthermore, we found that RUNX3 targets TGF-β1 and suppressed its transcription. The silencing of TGF-β1 increased cell proliferation, migration and MMP-2/3/9 expression, and inhibited TIMP-1, Collagen I/III, SM22, MYH11 and CNN1 expression. In addition, TGF-β1 reversed the effect of RUNX3 overexpression on HAoSMCs. Hence, our study indicated that RUNX3 promotes cell proliferation, migration, and ECM remodelling through suppressing TGF-β1., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The underlying mechanisms of thymocyte development and lineage determination remain incompletely understood, and the emerging evidences demonstrated that RNA binding proteins (RBPs) are deeply involved in governing T cell fate in thymus. Serine/arginine-rich splicing factor 1 (SRSF1), as a classical splicing factor, is a pivotal RBP for gene expression in various biological processes. Our recent study demonstrated that SRSF1 plays essential roles in the development of late thymocytes by modulating the T cell regulatory gene networks post-transcriptionally, which are critical in response to type I interferon signaling for supporting thymocyte maturation. Here, we report SRSF1 also contributes to the determination of the CD8 + T cell fate. By specific ablation of SRSF1 in CD4 + CD8 + double positive (DP) thymocytes, we found that SRSF1 deficiency impaired the maturation of late thymocytes and diminished the output of both CD4 + and CD8 + single positive T cells. Interestingly, the ratio of mature CD4 + to CD8 + cells was notably altered and more severe defects were exhibited in CD8 + lineage than those in CD4 + lineage, reflecting the specific function of SRSF1 in CD8 + T cell fate decision. Mechanistically, SRSF1-deficient cells downregulate their expression of Runx3 , which is a crucial transcriptional regulator in sustaining CD8 + single positive (SP) thymocyte development and lineage choice. Moreover, forced expression of Runx3 partially rectified the defects in SRSF1-deficient CD8 + thymocyte maturation. Thus, our data uncovered the previous unknown role of SRSF1 in establishment of CD8 + cell identity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ji, Bao, Yuan, Qi, Wang, You, Yu, Liu, Cui, Wang, Liu, Guo, Feng, Chen, Kang and Yu.)

Research Question: What is the mechanism of hypermethylation of runt-related transcription factor 3 (RUNX3) in the eutopic endometrium of endometriosis as biomarker in the malignant transformation of endometriosis?, Design: Methylation-specific polymerase chain reaction was used to analyse the methylation status of RUNX3 in endometriosis-associated ovarian cancer (EAOC). Primary eutopic endometrial stromal cells (ESC) were isolated from the uteri of patients with ovarian endometriosis. After RUNX3 knockdown by RNA interference technology or ESC treated with oestradiol, the proliferation and invasion ability were evaluated in ESC by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and transwell assays., Results: The frequency of methylation of RUNX3 in neoplastic tissue in the EAOC group was significantly higher than that in the ectopic endometrium of the endometriosis group (P < 0.001), and the frequency of methylation of RUNX3 in the eutopic endometrium of the EAOC group was significantly higher than that in the endometriosis group (P < 0.001). However, there was no significant difference in the eutopic endometrium when compared between the endometriosis group and the control endometrium group (P = 0.233). Silencing RUNX3 promoted the proliferation and invasion of ESC (P < 0.001 and P < 0.001). Following intervention with oestrogen, it was observed that the oestradiol group showed higher levels of RUNX3 methylation (P < 0.001) and DNA methyltransferase 1 (DNMT1) mRNA and protein expression (P < 0.001 and P < 0.001), and lower RUNX3 mRNA and protein expression when compared with the ESC group (P < 0.001 and P < 0.001)., Conclusion: This study demonstrated that hypermethylation of the RUNX3 was related to the malignant transformation of endometriosis and that this process was related to corresponding changes in the eutopic endometrium. Furthermore, the 'oestrogen-DNMT1' signalling pathway may induce the hypermethylation of RUNX3 to promote the malignant transformation of endometriosis., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background & Aims: RUNX transcription factors play pivotal roles in embryonic development and neoplasia. We previously identified the single missense mutation R122C in RUNX3 from human gastric cancer. However, how RUNX3 R122C mutation disrupts stem cell homeostasis and promotes gastric carcinogenesis remained unclear., Methods: To understand the oncogenic nature of this mutation in vivo, we generated the RUNX3 R122C knock-in mice. Stomach tissues were harvested, followed by histologic and immunofluorescence staining, organoid culture, flow cytometry to isolate gastric corpus isthmus and nonisthmus epithelial cells, and RNA extraction for transcriptomic analysis., Results: The corpus tissue of RUNX3 R122C/R122C homozygous mice showed a precancerous phenotype such as spasmolytic polypeptide-expressing metaplasia. We observed mucous neck cell hyperplasia; massive reduction of pit, parietal, and chief cell populations; as well as a dramatic increase in the number of rapidly proliferating isthmus stem/progenitor cells in the corpus of RUNX3 R122C/R122C mice. Transcriptomic analyses of the isolated epithelial cells showed that the cell-cycle-related MYC target gene signature was enriched in the corpus epithelial cells of RUNX3 R122C/R122C mice compared with the wild-type corpus. Mechanistically, RUNX3 R122C mutant protein disrupted the regulation of the restriction point where cells decide to enter either a proliferative or quiescent state, thereby driving stem cell expansion and limiting the ability of cells to terminally differentiate., Conclusions: RUNX3 R122C missense mutation is associated with the continuous cycling of isthmus stem/progenitor cells, maturation arrest, and development of a precancerous state. This work highlights the importance of RUNX3 in the prevention of metaplasia and gastric cancer., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: As a tumor suppressor or oncogenic gene, abnormal expression of RUNX family transcription factor 3 (RUNX3) has been reported in various cancers., Introduction: This study aimed to investigate the role of RUNX3 in melanoma., Methods: The expression level of RUNX3 in melanoma tissues was analyzed by immunohistochemistry and the Oncomine database. Based on microarray datasets GSE3189 and GSE7553, differentially expressed genes (DEGs) in melanoma samples were screened, followed by functional enrichment analysis. Gene Set Enrichment Analysis (GSEA) was performed for RUNX3. DEGs that co-expressed with RUNX3 were analyzed, and the transcription factors (TFs) of RUNX3 and its co-expressed genes were predicted. The protein-protein interactions (PPIs) for RUNX3 were analyzed utilizing the GeneMANIA database. MicroRNAs (miRNAs) that could target RUNX3 expression, were predicted., Results: RUNX3 expression was significantly up-regulated in melanoma tissues. GSEA showed that RUNX3 expression was positively correlated with melanogenesis and melanoma pathways. Eleven DEGs showed significant co-expression with RUNX3 in melanoma, for example, TLE4 was negatively co-expressed with RUNX3. RUNX3 was identified as a TF that regulated the expression of both itself and its co-expressed genes. PPI analysis showed that 20 protein-encoding genes interacted with RUNX3, among which 9 genes were differentially expressed in melanoma, such as CBFB and SMAD3. These genes were significantly enriched in transcriptional regulation by RUNX3, RUNX3 regulates BCL2L11 (BIM) transcription, regulation of I-kappaB kinase/NFkappaB signaling, and signaling by NOTCH. A total of 31 miRNAs could target RUNX3, such as miR-326, miR-330-5p, and miR-373-3p., Conclusion: RUNX3 expression was up-regulated in melanoma and was implicated in the development of melanoma., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Osteosarcoma (OS) in human patients is characterized by genetic alteration of TP53. Osteoprogenitor-specific p53-deleted mice (OS mice) have been widely used to study the process of osteosarcomagenesis. However, the molecular mechanisms responsible for the development of OS upon p53 inactivation remain largely unknown. In this study, we detected prominent RUNX3/Runx3 expression in human and mouse p53-deficient OS. Myc was aberrantly upregulated by Runx3 via mR1, a consensus Runx site in the Myc promoter, in a manner dependent on p53 deficiency. Reduction of the Myc level by disruption of mR1 or Runx3 knockdown decreased the tumorigenicity of p53-deficient OS cells and effectively suppressed OS development in OS mice. Furthermore, Runx inhibitors exerted therapeutic effects on OS mice. Together, these results show that p53 deficiency promotes osteosarcomagenesis in human and mouse by allowing Runx3 to induce oncogenic Myc expression., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Objective To investigate the expression and correlation of Runt-related transcription factor 3(RUNX3)and enhancer of zeste homolog 2(EZH2)in rectal cancer,and to reveal the relationship between the expression of RUNX3 and EZH2 and the sensitivity of XELOX regimen to neoadjuvant chemotherapy in locally advanced rectal cancer patients. Methods The carcinoma and paracancerous tissues of 31 patients with rectal adenocarcinoma and no preoperative antitumor therapy were selected as cancer group and paracancer group,respectively.The relative mRNA levels of RUNX3 and EZH2 in the two groups were measured by real-time quantitative reverse transcription-polymerase chain reaction,and the protein levels were determined by immunohistochemical assay.The expression of RUNX3 and EZH2 was compared between cancer tissue and paracancerous tissue.The pre-treatment wax blocks of 26 patients with locally advanced rectal cancer who received 3 cycles of XELOX regimen as neoadjuvant chemotherapy before surgery were selected as the pre-neoadjuvant therapy group,and the postoperative pathological wax blocks were selected as the post-neoadjuvant treatment group.Tumor regression grade(TRG)was determined to evaluate the efficacy of neoadjuvant therapy.Immunohistochemical assay was used to detect the protein levels of RUNX3 and EZH2 in the two groups,and then the relationship between the expression patterns of the two proteins and the efficacy of neoadjuvant chemotherapy was analyzed. Results Compared with paracancerous tissue,the cancer tissue showed down-regulated mRNA level and reduced positive protein expression rate of RUNX3,while up-regulated mRNA level( P =0.001)and increased positive protein expression rate of EZH2( P =0.022).The mRNA levels of RUNX3 and EZH2 in the cancer group were negatively correlated( r =-0.599, P =0.000).Twelve patients who received neoadjuvant chemotherapy reached TRG0-TRG2,and the overall effective rate of neoadjuvant chemotherapy was 46.15%(12/26).Compared with pre-neoadjuvant therapy group,the post-neoadjuvant therapy group had increased positive expression rate of RUNX3 protein( P =0.163)and decreased positive expression rate of EZH2 protein( P =0.095).In the pre-neoadjuvant therapy group,the effective rate of neoadjuvant chemotherapy was 75.00%(9/12)in patients with positive RUNX3 expression,77.78%(7/9)in patients with negative EZH2 expression,and 100%(7/7)in patients with positive expression of RUNX3 and negative expression of EZH2.Multivariate Logistic regression showed that the expression of RUNX3 protein was the factor influencing the efficacy of neoadjuvant chemotherapy. Conclusions The positive expression rate of RUNX3 in cancer tissue was lower than that in paracancerous tissue,while that of EZH2 showed the opposite trend.Neoadjuvant chemotherapy may affect the expression of RUNX3 and EZH2 in rectal cancer.The patients with high RUNX3 expression and low EZH2 expression in locally advanced rectal cancer were more sensitive to neoadjuvant chemotherapy with XELOX regimen.

Objectives: Runx3, a member of the Runx family of transcription factors, has been studied as a tumour suppressor and key player of organ development. In a previous study, we reported differentiation failure and excessive angiogenesis in the liver of Runx3 knock-out (KO) mice. Here, we examined a function of the Runx3 in liver, especially in iron metabolism., Methods: We performed histological and immunohistological analyses of the Runx3 KO mouse liver. RNA-sequencing analyses were performed on primary hepatocytes isolated from Runx3 conditional KO (cKO) mice. The effect of Runx3 knock-down (KD) was also investigated using siRNA-mediated KD in functional human hepatocytes and human hepatocellular carcinoma cells., Result: We observed an iron-overloaded liver with decreased expression of hepcidin in Runx3 KO mice. Expression of BMP6, a regulator of hepcidin transcription, and activity of the BMP pathway were decreased in the liver tissue of Runx3 KO mice. Transcriptome analysis on primary hepatocytes isolated from Runx3 cKO mice also revealed that iron-induced increase in BMP6 was mediated by Runx3. Similar results were observed in Runx3 knock-down experiments using HepaRG cells and HepG2 cells. Finally, we showed that Runx3 enhanced the activity of the BMP6 promoter by responding to iron stimuli in the hepatocytes., Conclusion: In conclusion, we suggest that Runx3 plays important roles in iron metabolism of the liver through regulation of BMP signalling., (© 2021 The Authors. Cell Proliferation published by John Wiley & Sons Ltd.)

Background: Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is essential for bone formation, and its dysfunction is reported to be associated with osteoporosis (OP). Recent researches have determined that lncRNA PART1 participates in the pathogenesis of multiple diseases. However, its role in modulating osteogenic differentiation of hBMSCs is unclear., Methods: PART1, miR-185-5p, and RUNX3 levels were assessed via RT-qPCR. The protein levels of OCN, OSN, and COL1A1 were measured by western blotting. The osteoblastic phenotype was evaluated via ALP activity and ARS staining. The relationship between miR-185-5p and PART1 or RUNX3 was validated by luciferase reporter, RIP assays., Results: PART1 and RUNX3 expression were enhanced during hBMSC osteogenic differentiation. PART1 deletion decreased OCN, OSN, and COL1A1 levels and weakened ALP activity, but promoted the apoptosis of hBMSCs. Moreover, PART1 served as a ceRNA to influence the RUNX3 level via targeting miR-185-5p. In addition, RUNX3 was verified to activate the transcription of PART1 in hBMSCs. Finally, rescue assays indicated that suppression of miR-185-5p or addition of RUNX3 partially abolished the effects of PART1 knockdown on the levels of OCN, OSX, and COL1A1 levels, ALP activity, and apoptosis., Conclusion: Our study elaborated that PART1/miR-185-5p/RUNX3 feedback contributed to osteogenic differentiation and inhibited the hBMSCs apoptosis, suggesting that PART1 might be a novel target for OP treatment.