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CEBPA‐IGH, a fusion gene of the immunoglobulin heavy‐chain locus (IGH) and the CCAAT enhancer‐binding protein α (C/EBPα) gene, is recurrently found in B‐ALL cases and causes aberrant expression of C/EBPα, a master regulator of granulocyte differentiation, in B cells. Forced expression of C/EBPα in B cells was reported to cause loss of B‐cell identity due to the inhibition of Pax5, a master regulator of B‐cell differentiation; however, it is not known whether the same mechanism is applicable for B‐ALL development by CEBPA‐IGH. It is known that a full‐length isoform of C/EBPα, p42, promotes myeloid differentiation, whereas its N‐terminal truncated isoform, p30, inhibits myeloid differentiation through the inhibition of p42; however, the differential role between p42 and p30 in ALL development has not been clarified. In the present study, we examined the effect of the expression of p42 and p30 in B cells by performing RNA‐seq of mRNA from LCL stably transfected with p42 or p30. Unexpectedly, suppression of PAX5 target genes was barely observed. Instead, both isoforms suppressed the target genes of MEF2 family members (MEF2s), other regulators of B‐cell differentiation. Similarly, MEF2s target genes rather than PAX5 target genes were suppressed in CEBP‐IGH‐positive ALL (n = 8) compared with other B‐ALL (n = 315). Furthermore, binding of both isoforms to MEF2s target genes and the reduction of surrounding histone acetylation were observed in ChIP‐qPCR. Our data suggest that the inhibition of MEF2s by C/EBPα plays a role in the development of CEBPA‐IGH‐positive ALL and that both isoforms work co‐operatively to achieve it. In the present study, we found that C/EBPα aberrantly expressed in B cells inhibited the function of MEF2 family members by performing RNA‐seq of mRNA of C/EBPα‐transfectants and analyzing RNA‐seq data of 323 clinical samples of ALL. Two isoforms of C/EBPα, p42 and p30, co‐operatively worked for MEF2 inhibition, although it was reported that they worked competitively in AML development.

Metformin has been found to have inhibitory effects on a variety of tumors. However, its effects on non-small cell lung cancer (NSCLC) remain unclear. We demonstrated that metformin could inhibit the proliferation of A549 and H1299 cells. RNA transcriptome sequencing revealed that PDL1 was significantly downregulated in both cell types following treatment with metformin (P < 0.001). Jaspar analysis and chromatin immunoprecipitation showed that CEBPB could directly bind the promoter region of PDL1. Western blotting showed that protein expression of the isoforms CEBPB-LAP*, CEBPB-LAP, and CEBPB-LIP was significantly upregulated and the LIP/LAP ratio was increased. Gene chip analysis showed that PDL1 was significantly upregulated in A549-CEBPB-LAP cells and significantly downregulated in A549-CEBPB-LIP cells (P < 0.05) compared with CEBPB-NC cells. Dual-luciferase reporter gene assay showed that CEBPB-LAP overexpression could promote transcription of PDL1 and CEBPB-LIP overexpression could inhibit the process. Functional assays showed that the changes in CEBPB isoforms affected the function of NSCLC cells. Western blotting showed that metformin could regulate the function of NSCLC cells via AMPK–CEBPB–PDL1 signaling. Animal experiments showed that tumor growth was significantly inhibited by metformin, and atezolizumab and metformin had a synergistic effect on tumor growth. A total of 1247 patients were retrospectively analyzed, including 166 and 1081 patients in metformin and control groups, respectively. The positive rate of PDL1 was lower than that of the control group (HR = 0.338, 95% CI = 0.235–0.487; P < 0.001). In conclusion, metformin inhibited the proliferation of NSCLC cells and played an anti-tumor role in an AMPK–CEBPB–PDL1 signaling-dependent manner.

Obesity is linked to the development of major metabolic disorders such as type 2 diabetes, cardiovascular disease, and cancer. Recent research has focused on the molecular link between obesity and oxidative stress. Obesity impairs antioxidant function, resulting in dramatically increased reactive oxygen levels and apoptosis. In this study, we investigated the effect of IW13 peptide on inhibiting lipid accumulation and regulating the antioxidant mechanism to normalize the lipid metabolism in HFD induced zebrafish larvae. Our results showed that co-treatment with IW13 peptide showed a protective effect in HFD zebra fish larvae by increasing the survival and heart rate. However, IW13 peptide co-treatment reduced triglycerides and cholesterol levels while also restoring the SOD and CAT antioxidant enzymes. In addition, IW13 co-treatment inhibited the formation of lipid peroxidation and superoxide anion by regulating the glutathione level. Also, the results showed that IW13 specifically downregulated the expression of the lipogenic-specific genes (C/EBP-α, SREBP1, and FAS). The findings exhibited that the IW13 peptide with effective antioxidant and anti-obesity activity could act as a futuristic drug to treat obesity and oxidative stress-related diseases.

Background The imbalance of osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is closely related to steroid-induced avascular necrosis of the femoral head (SANFH). We aimed to investigate the epigenetic mechanism of intramedullary fat accumulation and continuous osteonecrosis after glucocorticoid (GC) withdrawal in SANFH. Methods An SANFH model was established in SD rats, which received an intermittent high GC dose for the first 4 weeks followed by an additional 4 weeks without GC. We explored the synergistic effects and mechanisms of C/EBPα and PPARγ on the differentiation of BMSCs by lentivirus-mediated gene knockdown and overexpression assays. A chromatin immunoprecipitation assay was performed to identify epigenetic modification sites on PPARγ in vivo and in vitro. Results In the SANFH model, intramedullary fat was significantly increased, and the transcription factors C/EBPα and PPARγ were upregulated simultaneously in the femoral head. In vitro, C/EBPα promoted adipogenic differentiation of BMSCs by targeting the PPARγ signalling pathway, while overexpression of C/EBPα significantly impaired osteogenic differentiation. Further studies demonstrated that histone H3K27 acetylation of PPARγ played an important role in the epigenetic mechanism underlying SANFH. C/EBPα upregulates the histone H3K27 acetylation level in the PPARγ promoter region by inhibiting HDAC1. Additionally, inhibiting the histone acetylation level of PPARγ effectively prevented adipogenic differentiation, thus slowing the progression of SANFH. Conclusions Our results demonstrate the molecular mechanism by which C/EBPα regulates PPARγ expression by acetylating histones and revealed the epigenetic phenomenon in SANFH for the first time. Graphical abstract

Macranthoside B (MB) is a triterpenoid saponin extracted from Lonicera macranthoides , a traditional Chinese medicine. In the current study, we investigated the anticancer potential of MB in various cancer cells and elucidated its underlying mechanisms. MB exposure inhibited cell proliferation, induced mitochondrial membrane potential (MMP) loss, increased sub-G1 accumulation, and resulted in cleavage of caspase-3 and PARP, which are reflective of apoptosis. In HeLa cells, MB induced down-regulation of SOD2 and GPx1, phosphorylation of Akt and PDK1, and thus promoted ROS-mediated apoptosis. This was further supported by the protection of sub-G1 accumulation, MMP loss, cleavage of caspase-3 and PARP in the presence of N-acetylcys­teine (NAC). Additionally, MB induced cell death via down-regulation of ubiquitin-like with PHD and ring-finger domains 1 (UHRF1) and Bcl-xL. Taken together, this study provides a new insight into the apoptosis-inducing potential of MB, and its molecular mechanisms are associated with an increase in oxidative stress and inhibition of the PDK1/Akt pathway.

Background Understanding how to modulate the microenvironment of tumors that are resistant to immune checkpoint inhibitors represents a major challenge in oncology.Here we investigate the ability of USP7 inhibitors to reprogram the tumor microenvironment (TME) by inhibiting secretion of vascular endothelial growth factor (VEGF) from fibroblasts. Methods To understand the role played by USP7 in the TME, we systematically evaluated the effects of potent, selective USP7 inhibitors on co‐cultures comprising components of the TME, using human primary cells. We also evaluated the effects of USP7 inhibition on tumor growth inhibition in syngeneic models when dosed in combination with immune checkpoint inhibitors (ICIs). Results Abrogation of VEGF secretion from fibroblasts in response to USP7 inhibition resulted in inhibition of tumor neoangiogenesis and increased tumor recruitment of CD8‐positive T‐lymphocytes, leading to significantly improved sensitivity to immune checkpoint inhibitors. In syngeneic models, treatment with USP7 inhibitors led to striking tumor responses resulting in significantly improved survival. Conclusions USP7‐mediated reprograming of the TME is not linked to its previously characterized role in modulating MDM2 but does require p53 and UHRF1 in addition to the well‐characterized VEGF transcription factor, HIF‐1α. This represents a function of USP7 that is unique to fibroblasts, and which is not observed in cancer cells or other components of the TME. Given the potential for USP7 inhibitors to transform “immune desert” tumors into “immune responsive” tumors, this paves the way for a novel therapeutic strategy combining USP7 inhibitors with immune checkpoint inhibitors (ICIs). The oral USP7 inhibitor, ADC‐159, reduces sVEGF from CAFs and impacts tumor vasculature. USP7 inhibition affects HIF‐1α transcriptional modulation, tumor hypoxia and remodeling of the tumor microenvironment creating a permissive immune micro‐climate for infiltrating lymphocytes turning immunologically ‘cold’ tumors, ‘hot’. In preclinical models, combination treatment of ADC‐159 with immunotherapy agents delivers improved anti‐tumor efficacy and survival.

Ischemic stroke (IS) is a common nervous system disease, which is a major cause of disability and death in the world. In present study, we demonstrated a regulatory mechanism of CCAAT/enhancer binding protein-alpha antisense 1 (CEBPA-AS1) in oxygen glucose deprivation/reoxygenation (OGD/R)-induced SH-SY5Y cells, with a focus on neuronal apoptosis. CEBPA-AS1, miR-455, and GPER1 expressions were evaluated by using qRT-PCR and Western blotting. The binding relationship among CEBPA-AS1, miR-455, and GPER1 was determined by a dual luciferase reporter assay. Neuronal viability and apoptosis were examined using MTT and flow cytometry assays, followed by determination of apoptosis-related factors (caspase 3, caspase 8, caspase 9, Bax, and Bcl-2). CEBPA-AS1 and GPER1 levels were upregulated, and miR-455 level was downregulated in the cell model of OGD/R induced. CEBPA-AS1 knockdown increased SH-SY5Y viability and reduced OGD/R-induced apoptosis. CEBPA-AS1 could act as a sponge of miR-455, and CEBPA-AS1 knockdown was found to elevate miR-455 expression. miR-455 overexpression also promoted SH-SY5Y cell viability and rescued them from OGD/R-induced apoptosis by binding to GPER1. GPER1 overexpression or miR-455 inhibition reversed the anti-apoptotic effect of CEBPA-AS1 knockdown. These findings suggest a regulatory network of CEBPA-AS1/miR-455/GPER1 that mediates neuronal cell apoptosis in the OGD model, providing a better understanding of pathogenic mechanisms after IS.

In vitro, the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) mimics the positive effects of insulin on hepatic genes involved in glucose utilization, such as glucokinase (GK) and enzymes of the lipogenic pathway, suggesting that it is a key factor in the control of hepatic glucose metabolism. Decreased glucose utilization and increased glucose production by the liver play an important role in the development of the hyperglycemia in diabetic states. We thus reasoned that if SREBP-1c is indeed a mediator of hepatic insulin action, a hepatic targeted overexpression of SREBP-1c should greatly improve glucose homeostasis in diabetic mice. This was achieved by injecting streptozotocin-induced diabetic mice with a recombinant adenovirus containing the cDNA of the mature, transcriptionally active form of SREBP-1c. We show here that overexpressing SREBP-1c specifically in the liver of diabetic mice induces GK and lipogenic enzyme gene expression and represses the expression of phosphoenolpyruvate carboxykinase, a key enzyme of the gluconeogenic pathway. This in turn increases glycogen and triglyceride hepatic content and leads to a marked decrease in hyperglycemia in diabetic mice. We conclude that SREBP-1c has a major role in vivo in the long-term control of glucose homeostasis by insulin.

Background: CCAAT/enhancer-binding protein- α (CEBPA) is crucial for normal granulopoiesis and is frequently disrupted in acute myeloid leukaemia (AML). Increasing evidence suggests that CEBPA exerts its effects, in parts, by regulating specific microRNAs (miRNAs), as previously shown for miR-223 . The aim of this study was to investigate the genome-wide pattern of miRNAs regulated by CEBPA in myeloid cells. Methods: In Kasumi-1 cells, conditionally expressing CEBPA, we assessed the expression of 470 human miRNAs by microarray analysis. We further investigated the microarray results by qRT-PCR, luciferase reporter assays, and chromatin immunoprecipitation assays. Results: In all, 18 miRNAs were more than two-fold suppressed or induced after CEBPA restoration. Among these 18 miRNAs, we focused on CEBPA-mediated regulation of the tumour-suppressive miR-29b . We observed that miR-29b is suppressed in AML patients with impaired CEBPA function or loss of chromosome 7q. We found that CEBPA selectively regulates miR-29b expression on its miR-29a/b1 locus on chromosome 7q32.3, whereas miR-29b2/c on chromosome 1q32.2 is not affected. Conclusion: This study reports the activation of the tumour-suppressive miR-29b by the haematopoietic key transcription factor CEBPA. Our data provide a rationale for miR-29b suppression in AML patients with loss of chromosome 7q or CEBPA deficiency.

Sterol Regulatory Element Binding Protein-1c Expression and Action in Rat Muscles: Insulin-Like Effects on the Control of Glycolytic and Lipogenic Enzymes and UCP3 Gene Expression Isabelle Guillet-Deniau 1 , Virginie Mieulet 1 , Soazig Le Lay 2 , Younes Achouri 2 , Denis Carré 1 , Jean Girard 1 , Fabienne Foufelle 2 and Pascal Ferré 2 1 UPR 1524 CNRS, Institut Cochin de Génétique Moléculaire, Paris, France 2 Unité INSERM 465, Centre de Recherches Biomédicales des Cordeliers, Université Paris VI, Paris, France Abstract Sterol regulatory element binding protein-1c (SREBP-1c) is a transcription factor that mediates insulin effects on hepatic gene expression. It is itself transcriptionally stimulated by insulin in hepatocytes. Here we show that SREBP-1c mRNA is expressed in adult rat skeletal muscles and that this expression is decreased by diabetes. The regulation of SREBP-1c expression was then assessed in cultures of adult muscle satellite cells. These cells form spontaneously contracting multinucleated myotubes within 7 days of culture. SREBP-1c mRNA is expressed in contracting myotubes. A 4-h treatment with 100 nmol/l insulin increases SREBP-1c expression and nuclear abundance by two- to threefold in myotubes. In cultured myotubes, insulin increases the expression of glycolytic and lipogenic enzyme genes and inhibits the 9-cis retinoic acid-induced UCP3 expression. These effects of insulin are mimicked by adenovirus-mediated expression of a transcriptionally active form of SREBP-1c. We conclude that in skeletal muscles, SREBP-1c expression is sensitive to insulin and can transduce the positive and negative actions of the hormone on specific genes and thus has a pivotal role in long-term muscle insulin sensitivity. Footnotes Address correspondence and reprint requests to Isabelle Guillet-Deniau, Institut Cochin de Génétique Moléculaire, 24 rue du Faubourg St-Jacques, 75014, Paris, France. E-mail: guillet-deniau{at}cochin.inserm.fr . Received for publication 14 August 2001 and accepted in revised form 27 February 2002. EDL, extensor digitorum longus; FAS, fatty acid synthase; HK, hexokinase; PPAR, peroxisome proliferator-activated receptor; SREBP, sterol regulatory element binding protein; STZ, streptozotocin. DIABETES