Overexpression Of Runx2 Research Articles

Overexpression of RUNX1 mitigates dexamethasone-induced impairment of osteogenic differentiation and oxidative stress injury in bone marrow mesenchymal stem cells by promoting alpha-2 macroglobulin transcription

Overexpression of RUNX1 mitigates dexamethasone-induced impairment of osteogenic differentiation and oxidative stress injury in bone marrow mesenchymal stem cells by promoting alpha-2 macroglobulin transcription

MiR-600 mediates EZH2/RUNX3 signal axis to modulate breast cancer cell viability and sorafenib sensitivity.

Breast cancer (BC) ranks as the most prevalent gynecologic tumor globally. Abnormal expression of miRNAs is concerned with the development of cancers such as BC. However, little is known about the role of miR-600 in BC. This work aimed to explore the role of miR-600 in the malignant progression and sorafenib sensitivity of BC cells. Expression and interaction of miR-600/EZH2/RUNX3 were analyzed by bioinformatics. qRT-PCR was utilized to assay RNA expression of miR-600 and mRNA expression of EZH2/RUNX3. The binding relationship between miR-600 and EZH2 was tested by dual luciferase assay and RNA immunoprecipitation (RIP). The effects of miR-600/EZH2/RUNX3 axis on the malignant behavior and sorafenib sensitivity of BC cells were detected by CCK-8 and colony formation assay. Low expression of miR-600 and RUNX3 in BC was found by bioinformatics and molecular assays. High expression of EZH2 in BC was negatively correlated with RUVX3. Dual luciferase assay and RIP demonstrated that MiR-600 could bind to EZH2. Cell assays displayed that miR-600 knockdown could foster the malignant progression of BC cells and reduce the sensitivity of BC cells to sorafenib. EZH2 knockdown or RUNX3 overexpression could offset the effect of miR-600 inhibitor on the malignant behavior and sorafenib sensitivity of BC cells. MiR-600 can hinder the malignant behavior of BC cells and foster sensitivity of BC cells to sorafenib via EZH2/RUNX3 axis, exhibiting themiR-600/EZH2/RUNX3 axis as a feasible therapeutic target for BC patients.

Runx1 is upregulated by STAT3 and promotes proliferation of neonatal rat cardiomyocytes.

Though it is well known that mammalian cardiomyocytes exit cell cycle soon after birth, the mechanisms that regulate proliferation remain to be fully elucidated. Recent studies reported that cardiomyocytes undergo dedifferentiation before proliferation, indicating the importance of dedifferentiation in cardiomyocyte proliferation. Since Runx1 is expressed in dedifferentiated cardiomyocytes, Runx1 is widely used as a dedifferentiation marker of cardiomyocytes; however, little is known about the role of Runx1 in the proliferation of cardiomyocytes. The purpose of this study was to clarify the functional significance of Runx1 in cardiomyocyte proliferation. qRT-PCR analysis and immunoblot analysis demonstrated that Runx1 expression was upregulated in neonatal rat cardiomyocytes when cultured in the presence of FBS. Similarly, STAT3 was activated in the presence of FBS. Interestingly, knockdown of STAT3 significantly decreased Runx1 expression, indicating Runx1 is regulated by STAT3. We next investigated the effect of Runx1 on proliferation. Immunofluorescence microscopic analysis using an anti-Ki-67 antibody revealed that knockdown of Runx1 decreased the ratio of proliferating cardiomyocytes. Conversely, Runx1 overexpression using adenovirus vector induced cardiomyocyte proliferation in the absence of FBS. Finally, RNA-sequencing analysis revealed that Runx1 overexpression induced upregulation of cardiac fetal genes and downregulation of genes associated with fatty acid oxidation. Collectively, Runx1 is regulated by STAT3 and induces cardiomyocyte proliferation by juvenilizing cardiomyocytes.

Runx2 Overexpression Enhances Potency and Counteracts Exhaustion in CD8+ CAR T Cells

Chimeric antigen receptor (CAR) T cells are highly effective in treating B cell malignancies, but 50% of patients eventually relapse, often due to progressive dysfunction of the remaining CAR T cell population. While several others have implicated the bZIP transcription factor (TF) family in CAR T cell exhaustion (Seo et al. Nat Immunol. 2021, Lynn et al. Nature. 2019, Zhang et al. Cancer Cell. 2022), here we reveal a novel role for the TF Runx2 in mediating resistance to exhaustion and enhancing potency of CD8+ CAR T cells (CAR8). We have previously shown that CAR8 derived from naïve or memory T cell subsets maintain functional features of the T cell population from which they were derived (DeGolier et al. ASH. 2021). We predicted that an epigenomic and transcriptomic comparison of CAR8 derived from naïve or memory cells could reveal drivers of T cell differentiation and function. To precisely control T cell antigen experience, and to study T cell persistence in the absence of xenogeneic GvHD, we chose to use a syngeneic murine model, with adoptive transfer of E2A-PBX B cell acute lymphoblastic leukemia treated by anti-murine CD19 CAR T cells (Qin et al. Blood. 2018). We used a well-characterized ovalbumin vaccination model to produce memory T cells with which to generate memory-derived CAR8, and concurrently generated naïve-derived CAR8 from naïve hosts (Ahonen et al. J Exp Med. 2004). Comparative ATACseq of naïve CD8+ T cells to memory CD8+ T cells at 28 days post-vaccination revealed differential chromatin accessibility at target loci for Runx family transcription factors. These differences were maintained upon transduction of these T cell populations with a CAR, and after reinfusion into leukemia-bearing hosts. While the Runx family contains three members, RNAseq analysis uncovered that only Runx2 transcripts were highly enriched in memory over naïve cells prior to CAR-transduction but showed that gene expression levels converged upon CAR transduction and reinfusion. Additionally, broad comparisons of chromatin accessibility and transcriptomic profiles to published datasets showed that naïve-derived CAR8 progressively became more “memory-like”, while memory-derived CAR8 became more “effector-like.” We hypothesized that establishing Runx2 expression in naïve-derived CD8+ CAR T cells (CAR8 ND) could enhance the existing “memory-like” state of these T cells and boost T cell potency and anti-leukemia response. Indeed, overexpression of Runx2 in naïve-derived CAR8 (Runx2-CAR8 ND) strongly enhanced leukemia clearance (p<0.05) in mice treated with a sub-curative 1e5 CAR+ T cell dose. While there was no difference in the PD1+ proportion (p = 0.47), indicating similar activation and/or exposure to target antigen, mice treated with Runx2-CAR8 ND exhibited a lower proportion of PD1+/CD39+ cells (p<0.001) as well as a dramatically reduced proportion of PD1+/TOX+ cells (p<0.001), suggesting that Runx2 overexpression counteracts the differentiation trajectory toward terminal exhaustion. Additionally, Runx2-CAR8 ND showed enhanced T cell proportions in the marrow at 11 days post-CAR infusion (p<0.01), with >2-fold higher levels of Runx2-CAR8 ND compared to control CAR8 ND. Finally, in mice treated with a curative 2e6 CAR+ T cell dose, Runx2-CAR8 ND demonstrated a trend toward increased persistence in the blood at 19 days post-CAR infusion, relative to control CAR8 ND (p = 0.053). In conclusion, a comparison of naïve and memory-derived CD8+ CAR T cells at the epigenetic level revealed Runx2 overexpression in naïve-derived CAR T cells as a novel strategy to enhance the anti-leukemia response. Preliminary findings suggest Runx2-CAR8 ND show enhanced T cell proportions and decreased leukemic burden, as well as favorable exhaustion phenotypes. Future studies will expand our characterization of Runx2-CAR8 ND to better understand the effects of Runx2 on CAR T cell epigenetic profile, function and persistence in the bone marrow microenvironment, as well as test the effects of Runx2 overexpression in human CAR T cells. We anticipate that Runx2 overexpression will prove to be a useful strategy in enhancing the durability of CAR-mediated leukemia remissions.

RUNX1 predicts poor prognosis and correlates with tumor progression in clear cell renal carcinoma

BackgroundRunt-related transcription factor 1 (RUNX1), also called acute myeloid leukaemia 1, is a member of RUNX family of transcription factors. This family is composed of evolutionarily conserved transcription factors that function as critical lineage determinants in various tissues, however its function in cancer development and clinical significance in RCC are still unknown. MethodsWe used paraffin-embedded tumor tissues from 100 patients and fresh-harvested and paired adjacent normal renal tissues from 15 RCC patients who underwent primary surgical resection in Xijing Hospital between 2018 and 2022. The expression level of RUNX1 was evaluated by immunohistochemistry and Western Blot. RUNX1 promoted tumor cells proliferation, migration and invasion were verified by CCK-8, wound-healing and transwell assays. Finally, we constructed a xenografts model of the 786-O cell lines to observe the effect of RUNX1 on tumorigenesis in vivo. ResultsTCGA database showed higher RUNX1 expression levels in KIRC (kidney renal clear cell carcinoma). In overall survival analysis, RCC patients with higher RUNX1 expression level would have a shorter survival period than those with lower expression. Similarly, immunohistochemical results of our cohort also showed that RUNX1 was over-expression in cancer tissues than in corresponding non-cancer tissues. We also proved this result at protein level by western-blot. Meanwhile, prognostic and OS analyses of our cohort showed that the RUNX1 expression level was an individual prognostic factor in RCC patients. CCK-8, wound-healing and transwell assays proved that the overexpression of RUNX1 in Caki-1 cells promoted the proliferation, migration and invasion of the cells. Knocking down RUNX1 in 786-O cells inhibited the proliferation, migration and invasion of cells. The experimental results of xenografts model in nude mice showed that the knockdown of RUNX1 in 786-O cells slowed down the growth of tumor. ConclusionRUNX1 is a poor prognostic factor of clear cell renal carcinoma, which may provide a novel therapeutic target for ccRCC.

RUNX1 promotes proliferation and migration in non-small cell lung cancer cell lines via the mTOR pathway.

RUNX1, a member of the RUNX family of metazoan transcription factors, participates in the regulation of differentiation, proliferation, and other processes involved in growth and development. It also functions in the occurrence and development of tumors. However, the role and mechanism of action of RUNX1 in non-small cell lung cancer (NSCLC) are not yet clear. We used a bioinformatics approach as well as in vitro and in vivo assays to evaluate the role of RUNX1 in NSCLC as the molecular mechanisms underlying its effects. Using the TCGA, GEO, GEPIA (Gene Expression Profiling Interactive Analysis), and Kaplan-Meier databases, we screened the differentially expressed genes (DEGs) and found that RUNX1 was highly expressed in lung cancer and was associated with a poor prognosis. Immunohistochemical staining based on tissue chips from 110 samples showed that the expression of RUNX1 in lung cancer tissues was higher than that in adjacent normal tissues and was positively correlated with lymph node metastasis and TNM staging. In vitro experiments, we found that RUNX1 overexpression promoted cell proliferation and migration functions and affected downstream functional proteins by regulating the activity of the mTOR pathway, as confirmed by an analysis using the mTOR pathway inhibitor rapamycin. In addition, RUNX1 affected PD-L1 expression via the mTOR pathway. These results indicate that RUNX1 is a potential therapeutic target for NSCLC.

Long Noncoding RNA TALAM1 Is a Transcriptional Target of the RUNX2 Transcription Factor in Lung Adenocarcinoma.

Lung cancer is the leading cause of cancer death worldwide. It has been reported that genetic and epigenetic factors play a crucial role in the onset and evolution of lung cancer. Previous reports have shown that essential transcription factors in embryonic development contribute to this pathology. Runt-related transcription factor (RUNX) proteins belong to a family of master regulators of embryonic developmental programs. Specifically, RUNX2 is the master transcription factor (TF) of osteoblastic differentiation, and it can be involved in pathological conditions such as prostate, thyroid, and lung cancer by regulating apoptosis and mesenchymal-epithelial transition processes. In this paper, we identified TALAM1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) as a genetic target of the RUNX2 TF in lung cancer and then performed functional validation of the main findings. We performed ChIP-seq analysis of tumor samples from a patient diagnosed with lung adenocarcinoma to evaluate the target genes of the RUNX2 TF. In addition, we performed shRNA-mediated knockdown of RUNX2 in this lung adenocarcinoma cell line to confirm the regulatory role of RUNX2 in TALAM1 expression. We observed RUNX2 overexpression in cell lines and primary cultured lung cancer cells. Interestingly, we found that lncRNA TALAM1 was a target of RUNX2 and that RUNX2 exerted a negative regulatory effect on TALAM1 transcription.

Abstract P3031: The Cross Talk Between Increased Abdominal Adiposity And Calcification Of Aortic Valve

Calcified aortic valve disease (CAVD) is the most prevalent valvular heart disease in western populations affecting nearly 2.5% of individuals over 65. Currently, there is no viable pharmacological treatment to stop the disease's progression or activate mineral regression. The only effective therapy to treat CAVD is surgical valve replacement or trans-catheterization. Obesity is the major common nutritional disorder worldwide and is one of the important risk factors for CAVD. It has been shown that DLK1-/- mice have increased abdominal adiposity. Indeed Dlk1 plays an essential role in regulating adipocytes differentiation and proliferation. Recently, it has been shown that there is an association between fat tissue accumulation and an increased risk of aortic stenosis. We hypothesize that dysregulation of DLK1 stimulates adiposity, increases circulating lipids, enhances valve lipids retention, and stimulates calcification. Herein, our objective is to examine the expression of DLK1 in calcified aortic valves and its impact on lipid retention. Method: We examined the effect of Dlk1 loss of function on lipid retention in aortic valve tissue in ApoE-/- Dlk1-/- mice fed with a high-fat diet and in human explanted aortic valves using immuno-staining. Results: The complete deletion of DLK-1 in ApoE-/- mice were associated with an increased oxidized-LDL deposit in aortic valve tissue. In addition, circulating cholesterol (420 ± 8 mg/dl vs 250± 12 mg/dl, P=0.002) and triglyceride concentration (200 ± 3 mg/dl vs 50± 10 mg/dl, P<0.001) were higher in ApoE-/- DLK1-/- compared to apoE-/- mice. An increased valvular calcification accompanied these changes, as shown by the over-expression of RUNX2 and the increased aortic jet velocity in ApoE-/- DLK1-/- mice. Interestingly the overexpression of DLK1 in isolated human valve cells decreased calcification and lipid retention in vitro. Conclusion: This study showed the significant role of Dlk1-derived abdominal adiposity in valve-lipid retention and calcification.

Runx2 activates hepatic stellate cells to promote liver fibrosis via transcriptionally regulating Itgav expression

AbstractBackgrounds and aimsAs a central event during liver fibrosis, hepatic stellate cells (HSC) have been thought to be a potential therapeutic target for liver fibrosis. Previous studies have shown that runt‐related transcription factor 2 (Runx2) is associated with the development of non‐alcoholic fatty liver disease, while its specific role in HSC activation and hepatic fibrosis remains elusive.Approach and resultsIn this study, we found that Runx2 expression was significantly upregulated in human liver fibrosis with different aetiologies. Runx2 expression was also gradually elevated in mouse liver during fibrosis, and Runx2 was mainly expressed in the activated HSC. Knockdown of Runx2 in HSC markedly alleviated CCl4‐induced, 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine‐induced or methionine‐choline deficient (MCD)‐induced liver fibrosis, while hepatic overexpression of Runx2 via HBAAV‐Runx2 or VA‐Lip‐Runx2 injection exacerbated CCl4‐induced liver fibrosis. In vitro analysis demonstrated that Runx2 promoted HSC activation and proliferation, whereas Runx2 knockdown in HSC suppressed these effects. RNA‐seq and Runx2 ChIP‐seq analysis demonstrated that Runx2 could promote integrin alpha‐V (Itgav) expression by binding to its promoter. Blockade of Itgav attenuated Runx2‐induced HSC activation and liver fibrosis. Additionally, we found that cytokines (TGF‐β1, PDGF, EGF) promote the expression and nuclear translocation of Runx2 through protein kinase A (PKA) in HSC.ConclusionsRunx2 is critical for HSC activation via transcriptionally regulating Itgav expression during liver fibrosis, and may be a promising therapeutic target for liver fibrosis.

Holothurin A Inhibits RUNX1-Enhanced EMT in Metastasis Prostate Cancer via the Akt/JNK and P38 MAPK Signaling Pathway.

Due to the challenge of prostate cancer (PCa) management, there has been a surge in efforts to identify more safe and effective compounds that can modulate the epithelial-mesenchymal transition (EMT) for driving metastasis. Holothurin A (HA), a triterpenoid saponin isolated from Holothuria scabra, has now been characterized for its diverse biological activities. However, the mechanisms of HA in EMT-driven metastasis of human PCa cell lines has not yet been investigated. Moreover, runt-related transcription factor 1 (RUNX1) acts as an oncogene in prostate cancer, but little is known about its role in the EMT. Thus, the purpose of this study was to determine how RUNX1 influences EMT-mediated metastasis, as well as the potential effect of HA on EMT-mediated metastasis in endogenous and exogenous RUNX1 expressions of PCa cell lines. The results demonstrated that RUNX1 overexpression could promote the EMT phenotype with increased EMT markers, consequently driving metastatic migration and invasion in PC3 cell line through the activation of Akt/MAPK signaling pathways. Intriguingly, HA treatment could antagonize the EMT program in endogenous and exogenous RUNX1-expressing PCa cell lines. A decreasing metastasis of both HA-treated cell lines was evidenced through a downregulation of MMP2 and MMP9 via the Akt/P38/JNK-MAPK signaling pathway. Overall, our approach first demonstrated that RUNX1 enhanced EMT-driven prostate cancer metastasis and that HA was capable of inhibiting the EMT and metastatic processes and should probably be considered as a candidate for metastasis PCa treatment.

Distinct domains in the transcription factor Runx3 mediate differential antiviral CD8 T cell phenotypes

Abstract Runx3 is one of three Runt-family transcription factors (TF) that control gene expression by modifying chromatin structure. Upon initial T cell receptor stimulation, Runx3 drives chromatin accessibility found in mature circulating and tissue-resident memory CD8 T cells. Runx3-deficient cells preferentially develop a KLRG1 hiCD127 loterminal effector (TE) phenotype, whereas Runx3 overexpression promotes formation of KLRG1 loCD127 hiclassical memory precursor (MP) cells and KLRG1 hiCD127 hidouble positive (DP) effector cells during the effector phase. Runt-family TFs harbor activation, inhibition, and VWRPY domains that interact with distinct chromatin modifying proteins, but their individual contributions to Runx3-driven phenotypes in effector CD8 T cells are unresolved. Here we examined Runx3-deficient CD8 T cells complemented with wildtype or Runx3-domain deleted mutants during viral infection. The activation and VWRPY domains were each required to repress the TE cell phenotype. However, the activation, but not VWRPY domain, was necessary to repress high T-bet expression. Moreover, the larger gene expression pattern resulting from complementation with the VWRPY mutant was generally similar to that of wildtype Runx3. In addition, we found that wildtype Runx3 repressed the TF Tox and upregulated PD-1 and Tim-3, which required its inhibition domain. Runx3 lacking its inhibition domain also generated more MP cells and more highly induced CD127 compared to wildtype Runx3. These results indicate that distinct domains in Runx3 mediate differential control of gene expression in effector CD8 T cells, and might suggest avenues for targeting Runx3 domains to elicit specific memory T cell phenotypes. Supported by grants from NIAID (R01 AI095634, P01 AI145815)

RUNX1 promotes liver fibrosis progression through regulating TGF-β signalling.

Liver fibrosis is caused by chronic liver injury. There are limited treatments for it, and the pathogenesis is unclear. Therefore, there is an urgent need to explore the pathogenesis of liver fibrosis, and to try to identify new potential therapeutic targets. For this study we used the carbon tetrachloride abdominal injection induced liver fibrosis animal model in mice. Primary hepatic stellate cell isolation was performed by a density-gradient separation method, and this was followed by immunofluorescence stain analyses. Signal pathway analysis was performed by dual-luciferase reporter assay and western blotting. Our results showed that RUNX1 was upregulated in cirrhotic liver tissues compared with normal liver tissues. Besides, overexpression of RUNX1 caused more severe liver fibrosis lesions than control group under CCl4 -induced conditions. Moreover, α-SMA expression in the RUNX1 overexpression group was significantly higher than in the control group. Interestingly, we found that RUNX1 could promote the activation of TGF-β/Smads in a dual-luciferase reporter assay. Thus we demonstrated that RUNX1 could be considered as a new regulator of hepatic fibrosis by activating TGF-β/Smads signalling. Based on this, we concluded that RUNX1 may be developed as a new therapeutic target in the treatment of liver fibrosis in the future. In addition, this study also provides a new insight about the aetiology of liver fibrosis.

Runx2 overexpression promotes bone repair of osteonecrosis of the femoral head (ONFH)

BackgroundRunt-related transcription factor-2 (Runx2) has been considered an inducer to improve bone repair ability of mesenchymal stem cells (MSCs).Methods and resultsTwenty-four rabbits were used to establish Osteonecrosis of the femoral head (ONFH) and randomly devided into four groups: Adenovirus Runx2 (Ad-Runx2) group, Runx2-siRNA group, MSCs group and Model group. At 1 week after model establishment, the Ad-Runx2 group was treated with 5 × 107 MSCs transfected through Ad-Runx2, the Runx2-siRNA group was treated with 5 × 107 MSCs transfected through Runx2-siRNA, the MSCs group was injected with 5 × 107 untreated MSCs, and the Model group was treated with saline. The injection was administered at 1 week and 3 weeks after model establishment. The expression of bone morphogenetic protein 2 (BMP-2), Runx2 and Osterix from the femoral head was detected at 3 and 6 weeks after MSCs being injected, and Masson Trichrome Staining, Gross Morphology, X-ray and CT images observation were used to evaluate the repair effect of ONFH. The data revealed that the expression of BMP-2, Runx2 and Osterix in the Runx2-siRNA group was reduced at 3 weeks compared with the MSCs group, and then the expression further reduced at 6 weeks, but was still higher than the Model group besides Osterix; The expression of these three genes in the Ad-Runx2 group was higher than in the MSCs group. Masson Trichrome Staining, Gross Morphology and X-ray and CT images observation revealed that necrotic femoral head of the MSCs group was more regular and smooth than the Runx2-siRNA group, which has a collapsed and irregular femoral head. In the Ad-Runx2 group, necrotic femoral head was basically completely repaired and covered by rich cartilage and bone tissue.ConclusionsOverexpression of Runx2 can improve osteoblastic phenotype maintenance of MSCs and promote necrotic bone repair of ONFH.

The transcription factor RUNX1 affects the maturation of porcine oocytes via the BMP15/TGF-β signaling pathway

Bone morphogenetic protein 15 (BMP15) is specifically expressed in oocytes in pigs at all stages from early stages to ovulation and has an important role in oocyte maturation. However, there are few reports on the molecular mechanisms by which BMP15 affects oocyte maturation. In this study, we identified the core promoter region of BMP15 using a dual luciferase activity assay and successfully predicted the DNA binding motif of the transcription factor RUNX1. The effect of BMP15 and RUNX1 on oocyte maturation was examined using the first polar body extrusion rate, a reactive oxygen species (ROS) assay and total glutathione (GSH) content at three time points of 12, 24 and 48 h of in vitro culture of porcine isolated oocytes. Subsequently, the effect of the transcription factor RUNX1 on the TGF-β signaling pathway (BMPR1B and ALK5) was further verified using RT–qPCR and Western blotting. We found that the overexpression of BMP15 significantly increased the first polar body extrusion rate (P < 0.01) and total glutathione content of oocytes cultured in vitro for 24 h and decreased reactive oxygen levels (P < 0.01), whereas interference with BMP15 decreased the first polar body extrusion rate (P < 0.01), increased reactive oxygen levels in oocytes cultured in vitro for 24 h (P < 0.01), and decreased glutathione content (P < 0.01). The dual luciferase activity assay and online software prediction showed that RUNX1 is a potential transcription factor binding to the core promoter region (−1203/−1423 bp) of BMP15. Overexpression of RUNX1 significantly increased the expression of BMP15 and oocyte maturation rate, while inhibition of RUNX1 decreased the expression of BMP15 and the oocyte maturation rate. Moreover, the expression of BMPR1B and ALK5 in the TGF-β signaling pathway increased significantly after overexpression of RUNX1, whereas their expression decreased after inhibition of RUNX1. Overall, our results suggest that the transcription factor RUNX1 positively regulates the expression of BMP15 and influences oocyte maturation through the TGF-β signaling pathway. This study provides a theoretical basis for further complementing the BMP15/TGF-β signaling pathway to regulate mammalian oocyte maturation.

Directed differentiation of human iPSCs to functional ovarian granulosa-like cells via transcription factor overexpression.

An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and several types of somatic cells. Among these, granulosa cells play a key role in follicle formation and support for oogenesis. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human induced pluripotent stem cells (hiPSCs), a method of generating granulosa cells has been elusive. Here, we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of hiPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is sufficient to generate granulosa-like cells. Our granulosa-like cells have transcriptomes similar to human fetal ovarian cells and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with hPGCLCs, our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the premigratory to the gonadal stage as measured by induction of DAZL expression. This model system will provide unique opportunities for studying human ovarian biology and may enable the development of therapies for female reproductive health.

Runt-related transcription factor 3, mediated by DNA-methyltransferase 1, regulated Schwann cell proliferation and myelination during peripheral nerve regeneration via JAK/STAT signaling pathway

Schwann cells (SCs) play a crucial role in peripheral nerve injury and regeneration. Recently, RUNX3 was found to be linked with neurological dysfunction. We examined the RUNX3 expression in sciatic nerve stumps with peripheral nerve injury of rats, cyclic adenosine monophosphate (cAMP)-induced SCs. MTT assay was applied to determine the proliferation of SCs. Cell migration and apoptosis were assessed using wound healing assay and flow cytometry. Subsequently, we detected the methylation level of RUNX3 using Methylation-specific PCR assay and verified its regulation by DNMT1. The RUNX3 expressions were increased in sciatic nerve stumps with peripheral nerve injury and cAMP-induced SCs differentiation, which were related to demethylation of its promoter region regulated by DNMT1. RUNX3 knockdown notably suppressed the proliferation and migration, and induced the cell apoptosis of SCs. Silencing of RUNX3 inhibited the cAMP-induced morphological changes of SCs and the increase of myelin-related proteins induced by cAMP in SCs, while RUNX3 overexpression exerted opposite effects. Besides, the overexpression of RUNX3 promoted the activation of JAK/STAT signaling to regulate SCs proliferation and myelination. Meanwhile, DNMT1 overexpression inhibited the expression of RUNX3, and cell proliferation and myelination. In conclusion, RUNX3 mediated by DNMT1 regulated SC proliferation and myelination via JAK/STAT signaling pathway.

RUNX1 Upregulation Causes Mitochondrial Dysfunction via Regulating the PI3K-Akt Pathway in iPSC from Patients with Down Syndrome.

Down syndrome (DS) is the most common autosomal aneuploidy caused by trisomy of chromosome 21. Previous studies demonstrated that DS affected mitochondrial functions, which may be associated with the abnormal development of the nervous system in patients with DS. Runt-related transcription factor 1 (RUNX1) is an encoding gene located on chromosome 21. It has been reported that RUNX1 may affect cell apoptosis via the mitochondrial pathway. The present study investigated whether RUNX1 plays a critical role in mitochondrial dysfunction in DS and explored the mechanism by which RUNX1 affects mitochondrial functions. Expression of RUNX1 was detected in induced pluripotent stem cells of patients with DS (DS-iPSCs) and normal iPSCs (N-iPSCs), and the mitochondrial functions were investigated in the current study. Subsequently, RUNX1 was overexpressed in N-iPSCs and inhibited in DS-iPSCs. The mitochondrial functions were investigated thoroughly, including reactive oxygen species levels, mitochondrial membrane potential, ATP content and lysosomal activity. Finally, RNA-sequencing was used to explore the global expression pattern. It was observed that the expression levels of RUNX1 in DS-iPSCs were significantly higher than those in normal controls. Impaired mitochondrial functions were observed in DS-iPSCs. Of note, overexpression of RUNX1 in N-iPSCs resulted in mitochondrial dysfunction, while inhibition of RUNX1 expression could improve the mitochondrial function in DS-iPSCs. Global gene expression analysis indicated that overexpression of RUNX1 may promote the induction of apoptosis in DS-iPSCs by activating the PI3K/Akt signaling pathway. The present findings indicate that abnormal expression of RUNX1 may play a critical role in mitochondrial dysfunction in DS-iPSCs.

RUNX1 Ameliorates Rheumatoid Arthritis Progression through Epigenetic Inhibition of LRRC15.

Leucine-rich repeat containing 15 (LRRC15) has been identified as a contributing factor for cartilage damage in osteoarthritis; however, its involvement in rheumatoid arthritis (RA) and the underlying mechanisms have not been well characterized. The purpose of this study was to explore the function of LRRC15 in RA-associated fibroblast-like synoviocytes (RA-FLS) and in mice with collagen-induced arthritis (CIA) and to dissect the epigenetic mechanisms involved. LRRC15 was overexpressed in the synovial tissues of patients with RA, and LRRC15 overexpression was associated with increased proliferative, migratory, invasive, and angiogenic capacities of RA-FLS and accelerated release of pro-inflammatory cytokines. LRRC15 knockdown significantly inhibited synovial proliferation and reduced bone invasion and destruction in CIA mice. Runt-related transcription factor 1 (RUNX1) transcriptionally represses LRRC15 by binding to core-binding factor subunit beta (CBF-β). Overexpression of RUNX1 significantly inhibited the invasive phenotype of RA-FLS and suppressed the expression of proinflammatory cytokines. Conversely, the effects of RUNX1 were significantly reversed after overexpression of LRRC15 or inhibition of RUNX1-CBF-β interactions. Therefore, we demonstrated that RUNX1-mediated transcriptional repression of LRRC15 inhibited the development of RA, which may have therapeutic effects for RA patients.

Upregulated TSP1 and CD47 expression in the lung in nitrofen-induced congenital diaphragmatic hernia.

Persistent pulmonary hypertension remains a major cause of mortality and morbidity in congenital diaphragmatic hernia (CDH). The secreted glycoprotein thrombospondin-1 (TSP1), a ligand for receptor CD47, is widely expressed on both systemic and pulmonary vascular cells. TSP1-CD47 signaling has been reported to be one of the pathogeneses of pulmonary hypertension (PH). After creating a nitrofen-induced CDH rat model, fetuses were sacrificed on D17, D19 and D21 and divided into a control group and a CDH group. Quantitative real-time polymerase chain reaction was performed to determine the pulmonary gene expression of TSP1, CD47 and Runx3 (a regulator of TSP1). An immunofluorescence study was performed to evaluate the expression and localization of TSP1, CD47 and Runx3. The relative mRNA expression of pulmonary TSP1, CD47 and Runx3 on D21 was significantly increased in the CDH group (p=0.005, p=0.001, p=0.046, and p=0.002, respectively). The immunofluorescence study also confirmed the overexpression of TSP1, CD47 and Runx3 in the CDH group. Our results provide evidence that TSP1-CD47 signaling is involved in the pathogenesis of PH in a nitrofen-induced CDH model. Our data suggest that anti-CD47 antibodies can be novel therapeutic targets for the treatment of PH in CDH.

Exosomes derived from miR-338-3p-modified adipose stem cells inhibited inflammation injury of chondrocytes via targeting RUNX2 in osteoarthritis

BackgroundOsteoarthritis (OA) is a chronic degenerative disease that is one of the main causes of disability in middle-aged and elderly people. Adipose stem cell (ASC)-derived exosomes (ASC-Exo) could repair cartilage damage and treat OA. MiRNA-338-3p expression was confirmed to play a role in inhibiting proinflammatory cytokines. Herein, we aimed to explore the mechanism by which exosomes derived from miR-338-3p overexpressing ASCs protects chondrocytes from interleukin (IL)-1β-induced chondrocyte change.MethodsExosomes were extracted from ASCs transfected with miR-338-3p or its antisense inhibitor. The ASC-Exos (miR-338-3p silencing/overexpression) were incubated with IL-1β-induced ATDC5 cells, followed by evaluation of the chondrocyte proliferation, degradation, and inflammation injury.ResultsIn vitro results revealed that ASC-Exos inhibited the expression of prostaglandin E2 (PGE2), IL-6, IL-1β, and TNF-α, as well as promoted the proliferation of ATDC5 cells. Moreover, ASC-Exos inhibited inflammation injury and degradation of ATDC5 cells by transferring miR-338-3p. Luciferase reporter assays showed that RUNX2 was a target gene of miR-338-3p. Additionally, RUNX2 overexpression in ATDC5 cells reversed the protective effect of miR-338-3p on chondrocytes. Taken together, this study demonstrated that exosomes secreted from miR-338-3p-modified ASCs were effective in the repair of IL-1β-induced chondrocyte change by inhibiting RUNX2 expression.ConclusionsOur result provided valuable data for understanding the mechanism of ASC-Exos in OA treatment.Graphical abstract