KLF4 Research Articles

Suppression of KLF5 basal expression in oral carcinoma-derived cells through three intact CREB1-binding sites in the silencer region

ObjectivesKrüppel-like factor (KLF)5, which is overexpressed in carcinomas such as oral cancer, inhibits epidermal differentiation. KLF5 induces dedifferentiation of carcinoma cells, which effectuates carcinoma progression; nevertheless, the regulatory mechanism affecting the transcription of the KLF5 gene remains ambiguous. MethodsTranscriptional activity of the KLF5 silencer, specifically the 425-bp region (425-region), was examined using reporter assays. An additional analysis was conducted to assess the impact of the minimal essential region (MER) of KLF5 on its basal expression. The affinity of cAMP responsive element binding protein 1 (CREB1) for three potential CREB1-binding sites in the 425-region was analyzed using DNA pull-down and quantitative chromatin immunoprecipitation assays. Reporter assays employing a human oral squamous carcinoma cell line, HSC2, transfected with small interfering RNA or complementary DNA for CREB1, were performed to investigate the effect of CREB1 binding sites on MER activity. ResultsThe 425-region exhibited no transcriptional activity and suppressed MER transcriptional activity. This region encodes three putative CREB1-binding sites, and CREB1 demonstrated equal binding affinity for all three sites. The deletion of each of these binding sites reduced CREB1 precipitation and enhanced MER activity. Endogenous CREB1 knockdown and overexpression elevated and reduced MER activity, respectively, at the intact sites. Conversely, site deletion hampered and improved MER activity upon CREB1 knockdown and overexpression, respectively. ConclusionsSuppression of KLF5 basal expression via CREB1 binding to the 425-region requires all three CREB1-binding sites to remain intact in oral carcinoma cells. Consequently, deletion of the CREB1-binding site relieves suppression of KLF5 basal expression.

Reprogramming to alleviate Alzheimer’s disease through ameliorating microglial inflammation and neuronal damage

AbstractBackgroundAlzheimer’s disease (AD) is a neurodegenerative disease, characterized by glial cell dysfunction and progressive neuron loss, leading to varying degrees of neuropathology and cognition impairment. Aging and epigenetic alterations accompany the progression of AD and disrupt cellular functions; therefore, whether reprogramming could restore epigenetic information while maintaining cellular identity remains unclear.MethodThe polycistronic expression cassette contained multiple transcription factors of the Oct4, Sox2, and Klf4 genes (OSK) that were cloned into a coexpression vector (Ad‐OSK). The effects of OSK on cognitive function and neuropathology were explored in 4‐month‐old and 6‐month‐old APP/PS1 transgenic AD model mice via stereotaxic injection into the bilateral hippocampus.ResultAd‐OSK maintained microglial and neuronal cellular identities while attenuating microglia‐induced neuroinflammation and neuronal apoptosis. Ectopic expression of OSK in mouse brain tissue alleviates and restores cognitive function, youthful neuronal pathogenesis, Aβ plaque deposition, microglial activation, and myelin damage in early stage and later stage AD mice, respectively. The DNA demethylation process is required for the beneficial effects of OSK induced reprogramming in brain health.ConclusionThese results suggest that brain tissues preserve youthful epigenetic information, which is primarily stored by DNA demethylation during OSK reprogramming and can be accessed to improve brain health in AD mice. Our findings provide the foundation for the future use of OSK adenovirus or mRNA medicine to treat AD.

Study on the TGF-β Ligand Trap Enhances Erythroid Differentiation in HEL Cell By Inhibiting Smad3 Phosphorylation

Objective: Anemia is the most common clinical manifestation of MF, and its pathogenesis may be closely related to the over activation of TGF-β signaling pathway. This study aims to establish an erythroid differentiation model by inducing HEL cells with hemin and investigate the mechanism of the TGF-β ligand trap luspatercept enhances erythroid differentiation in HEL cell. Methods: 1.ELISA was used to detect the levels of GDF11 in peripheral blood plasma in patients with MF and healthy people. 2.The erythroid differentiation model was established by inducing HEL cells with hemin. The differentiation and proliferation levels of cells were detected by benzidine staining and CCK8. The expression levels of mRNA and protein in the cells were detected by RNA-seq and TMT proteomic analysis, and verified by flow cytometry, RT-PCR and WB. 3.HEL cells were induced with 40 μM hemin as a model of erythroid differentiation. 100ng/mL GDF11 interfered with the over activation of TGF-β signal pathway in differentiated HEL cells. 5μg/mL luspatercept blocked the activation of TGF-β signaling pathway by GDF11. Divided them into three groups, named: hemin group , GDF11 group, and luspatercept group. WB was used to detect the protein levels of pSmad3 and Smad4 in nuclear. Benzidine staining and flow cytometry were used to detect the levels of cell differentiation. RT-PCR and WB were used to detect the expression levels of GATA-1 and KLF1. The changes of RNA expression were detected by RNA-seq and verified by WB. 4.All experimental data were analyzed by SPSS 22.0 statistical software. Results: 1.The GDF11 levels of plasma of MF patients were higher than those of normal people ( P=0.0411). 2.The erythroid differentiation model of HEL cells induced with 40 μM hemin was successfully constructed. The results of RNA-seq and TMT proteome analysis showed that compared with the control group, the expression of hematopoietic related genes such as GATA-1, KLF1 and EPOR in hemin group were up-regulated, and the intracellular proteins related to erythroid differentiation such as HBγ- 2, HB-α and CD235a were up-regulated. Flow cytometry indicated that the expression of CD235a in hemin group was significantly higher than that of the control group (33.83% vs81%, P<0.01). RT-PCR and WB experiment showed that the expression of GATA-1 and KLF1 in hemin group were higher than those in control group. 3.WB showed that compared with hemin group, the hyperphosphorylation of smad3 and Smad4 in nuclear levels were increased in GDF11 group. And luspatercept could inhibit the hyperphosphorylation of Smad3 and reduce Smad4 in nuclear levels of GDF11 group. Luspatercept could inhibit GDF11 mediated activation of TGF-β signaling pathway in HEL cells. 4.Compared with hemin group, the benzidine positivity rate of cells and the expression of CD235a on the cell surface in GDF11 group were significantly decreased (benzidine: 13.0% vs 9%, P<0.01; CD235a: 16.8% vs 33.83%, P<0.01), and the expression of GATA-1 and KLF1 were also decreased. GDF11 could inhibit the erythroid differentiation of HEL cells. 5.Compared with GDF11 group, the benzidine positivity rate of cells and the expression of CD235a on the cell surface in luspatercept group were significantly increased (benzidine: 29.0% vs0%, P<0.01; CD235a: 31.1% vs 16.8%, P<0.01), and the expression of GATA-1 and KLF1 were increased. Luspatercept could reverse the inhibition of GDF11 on the erythroid differentiation of HEL cells. 6.Compared with the hemin group, the expression of GATA-1, KLF1, EPOR and other genes related to hematopoiesis in GDF11 group were down-regulated, and the differentially expressed genes were related to the biological functions of ribosomal metabolism process, chromosome regulation, erythropoiesis, oxygen binding, oxidoreductase activity and so on, mainly involving erythroid differentiation, oxidative phosphorylation, iron metabolism, TGF- β, JAK-STAT and other channels. Luspatercept could restore the expression of GATA-1, KLF1, EPOR and other genes by inhibiting the pathway over-activated by GDF11. Conclusion: Hemin could induce the erythroid differentiation of HEL cells. The TGF-β ligand trap luspatercept could inhibit GDF11-mediated hyperphosphorylation of Smad3 in HEL cells and promote the erythroid differentiation of HEL cells. Luspatercept could be a therapeutic tool for patients with myelofibrosis associated anemia.

Macrophage KLF15 prevents foam cell formation and atherosclerosis via transcriptional suppression of OLR-1

BackgroundMacrophage-derived foam cells are a hallmark of atherosclerosis. Scavenger receptors, including lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (OLR-1), are the principal receptors responsible for the uptake and modification of LDL, facilitating macrophage lipid load and the uptake of oxidized LDL by arterial wall cells. Krüppel-like factor 15 (KLF15) is a transcription factor that regulates the expression of genes by binding to the promoter during transcription. Therefore, this study aimed to investigate the precise role of macrophage KLF15 in atherogenesis. MethodsWe used two murine models of atherosclerosis: mice injected with an adeno-associated virus (AAV) encoding the Asp374-to-Tyr mutant version of human PCSK9, followed by 12 weeks on a high-fat diet (HFD), and ApoE−/−‐ mice on a HFD. We subsequently injected mice with AAV-KLF15 and AAV-LacZ to assess the role of KLF15 in the development of atherosclerosis in vivo. Oil Red O, H&E, and Masson's trichome staining were used to evaluate atherosclerotic lesions. Western blots and RT-qPCR were used to assess protein and mRNA levels, respectively. ResultsWe determined that KLF15 expression was downregulated during atherosclerosis formation, and KLF15 overexpression prevented atherosclerosis progression. KLF15 expression levels did not affect body weight or serum lipid levels in mice. However, KLF15 overexpression in macrophages prevented foam cell formation by reducing OLR-1-meditated lipid uptake. KLF15 directly targeted and transcriptionally downregulated OLR-1 levels. Restoration of OLR-1 reversed the beneficial effects of KLF15 in atherosclerosis. ConclusionMacrophage KLF15 transcriptionally downregulated OLR-1 expression to reduce lipid uptake, thereby preventing foam cell formation and atherosclerosis. Thus, our results suggest that KLF15 is a potential therapeutic target for atherosclerosis.

Glimepiride, a novel soluble epoxide hydrolase inhibitor, protects against heart failure via increasing epoxyeicosatrienoic acids

BackgroundEpoxyeicosatrienoic acids (EETs), which exert multiple endogenous protective effects, are hydrolyzed into less active dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). However, commercial drugs related to EETs or sEH are not yet in clinical use. MethodsFirstly, the plasma concentration of EETs and DHETs of 316 patients with heart failure (HF) were detected and quantitated by liquid chromatography-tandem mass spectrometry. Then, transverse aortic constriction (TAC)-induced HF was introduced in cardiomyocyte-specific Ephx2−/− mice. Moreover, Western blot, real-time PCR, luciferase reporter, ChIP assays were employed to explore the underlying mechanism. Finally, multiple sEH inhibitors were designed, synthesized, and validated in vitro and in vivo. ResultsThe ratios of DHETs/EETs were increased in the plasma from patients with HF. Meanwhile, the expression of sEH was upregulated in the heart of patients and mice with HF, especially in cardiomyocytes. Cardiomyocyte-specific Ephx2−/− mice ameliorated cardiac dysfunction induced by TAC. Consistently, Ephx2 knockdown protected Angiotensin II (AngII)-treated cardiomyocytes via increasing EETs in vitro. Mechanistically, AngII could enhance the expression of transcript factor Krüppel-like factor 15 (KLF15), which in turn upregulated sEH. Importantly, glimepiride was identified as a novel sEH inhibitor, which benefited from the elevated EETs during HF. ConclusionsGlimepiride attenuates HF in mice in part by increasing EETs.Clinical trial identifier:NCT03461107 (https://clinicaltrials.gov).

The GR-KLF15 axis promotes suppression of hepatic lipogenesis during fasting.

Fasting leads to many physiological changes in peripheral tissues, including the liver, where suppression of de novo lipogenesis through inhibition of sterol regulatory element-binding protein 1 (SREBP-1) expression and/or activity is a key adaptation to preserve glucose for maintenance of blood glucose levels. Yoshinori Takeuchi and colleagues provide novel mechanistic insights into the regulation of SREBP-1 expression during fasting and highlight the importance of the hypothalamic-pituitary-adrenal axis and, particularly, glucocorticoid-induced binding of the glucocorticoid receptor to enhancer regions of the KLF15 (Kruppel-like factor 15) gene as a novel mechanism underlying the suppression of SREBP-1 during fasting.

Mechanisms of Breast Cancer Stem Cell Specification and Self-Renewal Mediated by Hypoxia-Inducible Factor 1.

Many advanced human cancers contain regions of intratumoral hypoxia, with O2 gradients extending to anoxia. Hypoxia-inducible factors (HIFs) are activated in hypoxic cancer cells and drive metabolic reprogramming, vascularization, invasion, and metastasis. Hypoxia induces breast cancer stem cell (BCSC) specification by inducing the expression and/or activity of the pluripotency factors KLF4, NANOG, OCT4, and SOX2. Recent studies have identified HIF-1-dependent expression of PLXNB3, NARF, and TERT in hypoxic breast cancer cells. PLXNB3 binds to and activates the MET receptor tyrosine kinase, leading to activation of the SRC non-receptor tyrosine kinase and subsequently focal adhesion kinase, which promotes cancer cell migration and invasion. PLXNB3-MET-SRC signaling also activates STAT3, a transcription factor that mediates increased NANOG gene expression. Hypoxia-induced NARF binds to OCT4 and serves as a coactivator by stabilizing OCT4 binding to the KLF4, NANOG, and SOX2 genes and by stabilizing the interaction of OCT4 with KDM6A, a histone demethylase that erases repressive trimethylation of histone H3 at lysine 27, thereby increasing KLF4, NANOG, and SOX2 gene expression. In addition to increasing pluripotency factor expression by these mechanisms, HIF-1 directly activates expression of the TERT gene encoding telomerase, the enzyme required for maintenance of telomeres, which is required for the unlimited self-renewal of BCSCs. HIF-1 binds to the TERT gene and recruits NANOG, which serves as a coactivator by promoting the subsequent recruitment of USP9X, a deubiquitinase that inhibits HIF-1α degradation, and p300, a histone acetyltransferase that mediates acetylation of H3K27, which is required for transcriptional activation.

Krüppel-like Factor 15 Suppresses Ferroptosis by Activating an NRF2/GPX4 Signal to Protect against Folic Acid-Induced Acute Kidney Injury.

Acute kidney injury (AKI) is a common and serious disease with high morbidity and mortality, and its pathophysiological mechanisms are not fully understood. Increasing evidence suggests an important role of ferroptosis in AKI. Krüppel-like factor 15 (KLF15) is a transcription factor involved in several metabolic diseases, but its role in AKI and ferroptosis remains unclear. In this study, we explored the potential role of KLF15 using a folic acid-induced AKI model. Our study showed that KLF15 expression was reduced in kidney tissues of AKI mice, and KLF15 knockout exacerbated folic acid-induced ferroptosis and kidney injury. In vitro studies revealed that the ferroptosis inducer erastin significantly suppressed KLF15 expression in human tubular epithelial cells. Notably, the overexpression of KLF15 attenuated ferroptosis, as evidenced by a decrease in the lipid peroxidation marker of malondialdehyde and the upregulation of glutathione peroxidase 4 (GPX4), while KLF15 knockdown with shRNA exerted the opposite effect. Mechanistically, KLF15 stabilized the protein of nuclear factor erythroid 2-related factor 2 (NRF2) and subsequently increased the GPX4 level. Collectively, KLF15 plays an important role in the modulation of ferroptosis in AKI and may be a potential therapeutic target for treating AKI.

GR-KLF15 pathway controls hepatic lipogenesis during fasting.

During periods of fasting, the body undergoes a metabolic shift from carbohydrate utilization to the use of fats and ketones as an energy source, as well as the inhibition of de novo lipogenesis and the initiation of gluconeogenesis in the liver. The transcription factor sterol regulatory element-binding protein-1 (SREBP-1), which plays a critical role in the regulation of lipogenesis, is suppressed during fasting, resulting in the suppression of hepatic lipogenesis. We previously demonstrated that the interaction of fasting-induced Kruppel-like factor 15 (KLF15) with liver X receptor serves as the essential mechanism for the nutritional regulation of SREBP-1 expression. However, the underlying mechanisms of KLF15 induction during fasting remain unclear. In this study, we show that the glucocorticoid receptor (GR) regulates the hepatic expression of KLF15 and, subsequently, lipogenesis through the KLF15-SREBP-1 pathway during fasting. KLF15 is necessary for the suppression of SREBP-1 by GR, as demonstrated through experiments using KLF15 knockout mice. Additionally, we show that GR is involved in the fasting response, with heightened binding to the KLF15 enhancer. It has been widely known that the hypothalamic-pituitary-adrenal (HPA) axis regulates the secretion of glucocorticoids and plays a significant role in the metabolic response to undernutrition. These findings demonstrate the importance of the HPA-axis-regulated GR-KLF15 pathway in the regulation of lipid metabolism in the liver during fasting.

INTEGRATIVE ANALYSIS OF DIFFERENTIALLY REGULATED PROTEINS IN MENINGIOMA

Abstract AIMS Meningiomas are common primary intracranial tumours that arise from meninges. loss of chromosome 22q is common genetic event observed in meningiomas. About 50% of meningiomas harbour mutations in NF2 gene that resides on chromosome 22. Moreover, frequent mutations have been observed in AKT1, KLF4, TRAF7, and SMO genes. Meningiomas are divided into different genomic subgroups that express unique proteomic profiles. This study aims to investigate genomic background of meningioma and to determine the impact of mutations on underlying protein expression to identify druggable targets. METHOD Total 96 meningioma samples were sequenced using Illumina TruSight Oncology 500 panel. The raw sequence data was analyzed using TSO500 App. The variant data was annotated using Cancer Genome Interpreter and ANNOVAR. Clinical enrichment analysis was performed to investigate common pathways. Genomic data was correlated with LC-MS based proteomic data to determine variants effect on downstream processes. RESULTS Results showed an average of 1083 total and 7 driver mutations per sample. NF2 gene was found among highly mutated genes in tumour samples. Almost all NF2 mutations were frameshift, splice site and nonsense mutations, previously reported to be associated with high rate of multiple and recurring meningiomas. Additionally, driver mutations were identified in known meningioma genes including AKT1, KLF4, and TRAF7 along with previously unpublished mutations in ERBB2, MET, ARID5B, etc. The results correlate with tumour location, grade, and histopathological subtype. CONCLUSIONS The study identified common as well as unique driver mutations in meningioma samples. After functional validation, novel driver mutations could further enhance personalized treatment for meningioma patients.

The transcriptional regulator KLF15 is necessary for myoblast differentiation and muscle regeneration by activating FKBP5

Successful muscle regeneration following injury is essential for functional homeostasis of skeletal muscles. Krüppel-like factor 15 (KLF15) is a metabolic transcriptional regulator in the muscles. However, little is known regarding its function in muscle regeneration. Here, we examined microarray datasets from the Gene Expression Omnibus database, which indicated downregulated KLF15 in muscles from patients with various muscle diseases. Additionally, we found that Klf15 knockout (Klf15KO) impaired muscle regeneration following injury in mice. Furthermore, KLF15 expression was robustly induced during myoblast differentiation. Myoblasts with KLF15 deficiency showed a marked reduction in their fusion capacity. Unbiased transcriptome analysis of muscles on day 7 postinjury revealed downregulated genes involved in cell differentiation and metabolic processes in Klf15KO muscles. The FK506-binding protein 51 (FKBP5), a positive regulator of myoblast differentiation, was ranked as one of the most strongly downregulated genes in the Klf15KO group. A mechanistic search revealed that KLF15 binds directly to the promoter region of FKBP5 and activates FKBP5 expression. Local delivery of FKBP5 rescued the impaired muscle regeneration in Klf15KO mice. Our findings reveal a positive regulatory role of KLF15 in myoblast differentiation and muscle regeneration by activating FKBP5 expression. KLF15 signaling may be a novel therapeutic target for muscle disorders associated with injuries or diseases.

Role of lipid metabolism gene KLF4 in osteoarthritis.

Osteoarthritis (OA) is a common degenerative disease of joints, which can appear in almost any joint of the body. Therefore, the widespread occurrence of this disease has a huge impact on the lives of patients around the world. As an important part of metabolism, lipid metabolism is closely related to the occurrence and development of osteoarthritis. We screened UGCG and KLF4 based on weighted co-expression network analysis (WGCNA) and SVM-REF analysis. The data from Gene Expression Omnibus (GEO) and single-cell data verified the expression of these two genes. We analyzed KLF4-related genes and established a diagnosis model of OA related to lipid metabolism through the least absolute shrinkage and selection operator (LASSO) analysis. RT-PCR was used to verify the expression of KLF4 in osteoarthritis. Ten important lipid metabolism related genes (LMRGs) in OA were obtained. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that they are involve in the formation of immune microenvironment in osteoarthritis. CIBERSORT analysis revealed that there were significant differences in the immune microenvironment between osteoarthritis patients and normal controls. RT-PCR results showed that the expression of KLF4 in OA samples was lower than that in normal samples. The diagnostic model can be used to diagnose OA patients well. Overall, we demonstrated the potential relationship between the abnormal lipid metabolism and the pathological process of OA. Finally, we identified KLF4 as our significant LMRG and constructed a KLF4-related scoring model to accurately diagnose OA. In conclusion, therapy strategies targeting on regulating lipid metabolism may become a key factor in treating OA. Key Points (a) We identified the significant LMRG KLF4 and constructed a novel KLF4-related scoring model for the accuracy diagnosis of OA. (b) The potential relationship between lipid metabolism and the immune microenvironment in OA was demonstrated in our research. (c) The relationship of lipid metabolism and OA has been further improved in our research and provided novel insight for the diagnosis and therapy for OA patients.

Cooperative contributions of the klf1 and klf17 genes in zebrafish primitive erythropoiesis

Krüppel-like transcription factors (Klfs), which are characterized by the three conserved C-terminal zinc fingers, are involved in various biological processes, such as haematopoiesis and angiogenesis. However, how the Klf family of transcription factors cooperate in organogenesis remains elusive. During zebrafish embryogenesis, both klf1 and klf17 are expressed in the intermediate cell mass (ICM), where primitive erythroid cells are produced. Using CRISPR–Cas9 genome editing technology, we established klf1-klf17 double mutant zebrafish to investigate the functionally interactive roles of the klf1 and klf17 genes. The klf1-klf17 mutant exhibited a diminished number of circulating primitive erythroid cells at 2 days postfertilization (dpf), while klf1 or klf17 single mutants and wild-type embryos produced comparable numbers of primitive erythroid cells. Circulating erythroid cells from the klf1-klf17 mutant possessed larger nuclei at 2 dpf than wild-type cells, suggesting the impairment of primitive erythroid cell maturation. The expression of the erythroid cell maturation markers band3 and mitoferrin, but not the haematopoietic progenitor markers c-myb and scl, was decreased in the klf1-klf17 mutant at 1 dpf. Thus, these results illustrate the cooperative function of klf1 and klf17 in the maturation processes of zebrafish primitive erythroid cells.

Genetic Variants Associated With Hidradenitis Suppurativa

Hidradenitis suppurativa (HS) is a common and severely morbid chronic inflammatory skin disease that is reported to be highly heritable. However, the genetic understanding of HS is insufficient, and limited genome-wide association studies (GWASs) have been performed for HS, which have not identified significant risk loci. To identify genetic variants associated with HS and to shed light on the underlying genes and genetic mechanisms. This genetic association study recruited 753 patients with HS in the HS Program for Research and Care Excellence (HS ProCARE) at the University of North Carolina Department of Dermatology from August 2018 to July 2021. A GWAS was performed for 720 patients (after quality control) with controls from the Add Health study and then meta-analyzed with 2 large biobanks, UK Biobank (247 cases) and FinnGen (673 cases). Variants at 3 loci were tested for replication in the BioVU biobank (290 cases). Data analysis was performed from September 2021 to December 2022. Main outcome measures are loci identified, with association of P < 1 × 10-8 considered significant. A total of 753 patients were recruited, with 720 included in the analysis. Mean (SD) age at symptom onset was 20.3 (10.57) years and at enrollment was 35.3 (13.52) years; 360 (50.0%) patients were Black, and 575 (79.7%) were female. In a meta-analysis of the 4 studies, 2 HS-associated loci were identified and replicated, with lead variants rs10512572 (P = 2.3 × 10-11) and rs17090189 (P = 2.1 × 10-8) near the SOX9 and KLF5 genes, respectively. Variants at these loci are located in enhancer regulatory elements detected in skin tissue. In this genetic association study, common variants associated with HS located near the SOX9 and KLF5 genes were associated with risk of HS. These or other nearby genes may be associated with genetic risk of disease and the development of clinical features, such as cysts, comedones, and inflammatory tunnels, that are unique to HS. New insights into disease pathogenesis related to these genes may help predict disease progression and novel treatment approaches in the future.

Evaluation of the effect of boron derivatives on cardiac differentiation of mouse pluripotent stem cells

BackgroundThe heart is one of the first organs to form during embryonic development and has a very important place. So much that the formation of a functional heart is completed on the 55th day of human development and the 15th day of mouse development. Myocardial, endocardial and epicardial cells, which are derived from the mesoderm layer, are the cells that form the basis of the heart. Cardiac development, like other embryonic developments, is tightly controlled and regulated by various signaling pathways. The WNT signaling pathway is the most studied of these signaling pathways and the one with the clearest relationship with heart development. It is known that boron compounds and the Wnt/β-catenin pathway are highly correlated. Therefore, this study aimed to investigate the role of boron compounds in heart development as well as its effect on pluripotency of mouse embryonic stem cells for the first time in the literature. MethodsToxicity of boron compounds was evaluated by using MTS analysis and obtained results were supported by morphological pictures, Trypan Blue staining and Annexin V staining. Additionally, the possible boron-related change in pluripotency of embryonic stem cells were analyzed with alkaline phosphatase activity and immunocytochemical staining of Oct4 protein as well as gene expression levels of pluripotency related OCT4, SOX2 and KLF4 genes. The alterations in the embryonic body formation capacity of mouse embryonic stem cells due to the application boron derivatives were also evaluated. Three linage differentiation was conducted to clarify the real impact of boron compounds on embryonic development. Lastly, cardiac differentiation of mESCs was investigated by using morphological pictures, cytosolic calcium measurement, gene expression and immunocytochemical analysis of cardiac differentiation related genes and in the presence of boron compounds. ResultsObtained results show that boron treatment maintains the pluripotency of embryonic stem cells at non-toxic concentrations. Additionally, endodermal, and mesodermal fate was found to be triggered after boron treatment. Also, initiation of cardiomyocyte differentiation by boron derivative treatments caused an increased gene expression levels of cardiac differentiation related TNNT2, Nkx2.5 and ISL-1 gene expression levels. ConclusionThis study indicates that boron application, which is responsible for maintaining pluripotency of mESCs, can be used for increased cardiomyocyte differentiation of mESCs.

Effects of branched-chain amino acid supplementation on skeletal muscle proteolytic pathways after exercise-induced muscle injury in rats

To explore the role of branched-chain amino acid(BCAA) supplementation on muscle damage and the regulation of Krüppel-like factor 15(KLF15) and nuclear factor kappa B(NF-κB) mediated proteolytic pathways after an acute eccentric exercise. Male SD rats were divided into placebo group(PLA) and BCAA group(BCAA) randomly, 32 rats per group. Both group were then placed into subgroups: placebo and pre-exercise group(PC), placebo and immediately after exercise group(PE), placebo and 6 h after exercise group(PE6), placebo and 12 h after exercise group(PE12), BCAA and pre-exercise group(BC), BCAA and immediately after exercise group(BE), BCAA and 6 h after exercise group(BE6), BCAA and 12 h after exercise group(BE12), 8 rats per group. Rats in BCAA groups were supplied with BCAA(1 g/(kg·d·BW), 3 days) before the exercise day and placebo groups with equal volume of distilled water. The exercised groups performed a 2 h eccentric exercise on treadmill(16 m/min, -16° slope). Blood and gastrocnemius were collected according to the time points. RT-qPCR was used to measure the mRNA expression of KLF15, NF-κB, FoxO1, Atrogin-1 and MuRF1 in gastrocnemius. (1) No damage was found in myocytes of BC and PC group. The process of morphological damage in BCAA group was relatively faster. (2) The mRNA expression levels of KLF15, FoXO1, Atrogin-1 and MuRF1 in PE were higher than those in PC(P&lt;0.05, P&lt;0.01), NF-κB and Atrogin-1 in PE12 were higher than those in PC(P&lt;0.05). The mRNA expression levels of FoXO1 in BE were higher than those in BC(P&lt;0.05). Compared with PE, the mRNA expression levels of KLF15, Atrogin-1 and MuRF1 in BE were lower(P&lt;0.05, P&lt;0.01), NF-κB and Atrogin-1 in BE12 were lower than those in PE12(P&lt;0.05). The level of serum 3-MH in PE12 group was higher than that in PC group(P&lt;0.05). The proteolysis of skeletal muscle after high-intensity eccentric exercise is mediated by two different pathways: KLF15 and NF-κB, whose activation is time-dependent. BCAA may reduce skeletal muscle proteolysis by lowering the level of gene transcription in the KLF15 and NF-κB related protein degradation pathway, which occurs immediately after exercise.

MiR-145-5p suppresses cell proliferation by targeting IGF1R and NRAS genes in multiple myeloma cells

Abstract Objectives Multiple myeloma (MM) is a common hematological cancer. Hence, it is important to conduct further studies investigating the molecular mechanisms in detail that contributes to myeloma genesis. In addition to genetic changes, epigenetic factors such as miRNAs may influence the expression of myeloma-related genes. Methods Our study aimed to detect genes closely related to MM and miRNAs involved in the cancer process by changing the expression of these genes with bioinformatics tools and in vitro methods. Bioinformatics approaches identified hub miRNAs in our study that may have a role in the expression change of genes connected to myeloma. The functional impacts of the chosen miRNA on RPMI8226 and U266 cell lines and the effect of this miRNA on the expression changes of putative target genes were investigated. Results The viability of miR-145-5p transfected cells was found to decrease compared to control cells and the expression of IGF1R and NRAS genes were found to be significantly suppressed in both cell lines at mRNA level. Decreased levels of the IGF1R and NRAS genes were confirmed in miR-145-5p transfected cells at the protein level as well as compared to control cells. In addition, IGF1R/miR-145-5p interaction was demonstrated via luciferase reporter assay. However, expression levels of EGFR, KLF4, IRS1, CDK4 and CDK6 candidate genes had no statistically significant difference in miR-145-5p transfected cells compared to control cells. Conclusions Mir-145-5p was demonstrated to act as a tumor suppressor miRNA and inhibit the proliferation in MM cell lines via targeting IGF1R and NRAS.

Genome-Wide Analysis of the KLF Gene Family in Chicken: Characterization and Expression Profile.

The kruppel-like factor (KLF) gene family is a group of transcription factors containing highly conserved zinc-finger motifs, which play a crucial role in cell proliferation and differentiation. Chicken has been widely used as a model animal for analyzing gene function, however, little is known about the function of the KLF gene family in chickens. In this study, we performed genome-wide studies of chicken KLF genes and analyzed their biological and expression characteristics. We identified 13 KLF genes from chickens. Our phylogenetic, motif, and conserved domain analyses indicate that the KLF gene family has remained conserved through evolution. Synteny analysis showed the collinear relationship among KLFs, which indicated that they had related biomolecular functions. Interaction network analysis revealed that KLFs worked with 20 genes in biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that KLF2 was involved in Apelin and Forkhead Box O (FOXO) signaling pathways. Moreover, qPCR showed that 13 KLF genes were expressed in the nine selected tissues and displayed various gene expression patterns in chickens. RNA-seq showed that KLF3 and KLF10 genes were differentially expressed in the normal and high-fat diet fed groups, and KLF4, KLF5, KLF6, KLF7, KLF9, KLF12, and KLF13 genes were differentially expressed between undifferentiated and differentiated chicken preadipocytes. Besides, RNA-seq also showed that KLF genes displayed different expression patterns in muscle at 11 and 16 embryonic days old, and in 1-day-old chickens. These results indicated that the KLF genes were involved in the development of muscle and fat in chickens. Our findings provide some valuable reference points for the subsequent study of the function of KLF genes.

Identification of a novel KLF1 gene promoter variation (c.-251C > G) in a Thai blood donor with In(Lu) phenotype.

The authors have disclosed no conflicts of interest.

KLF15 Transcriptionally Activates ATG14 to Promote Autophagy and Attenuate Damage of ox-LDL-Induced HAECs.

Kruppel-like factor 15 (KLF15) is involved in many cardiovascular diseases and is abnormally expressed in atherosclerosis (AS), but the regulatory mechanism of KLF15 in AS has not been reported so far. RT-qPCR was used to detect the expression of KLF15 and ATG14 in AS patients. Subsequently, human aortic endothelial cells (HAECs) were induced by oxidized low densitylipoprotein (ox-LDL), and the expression of KLF15 in model cells was detected. KLF15 was overexpressed in cells by lipofection transfection, and then CCK8, flow cytometry, Western blot, ELISA, and related assay kits were used to detect cell viability, apoptosis, inflammatory response as well as oxidative stress, respectively. The targeted regulatory relationship between KLF15 and autophagy-related 14 (ATG14) was detected by ChIP and luciferase reporter assays. Following ATG14 silencing in KLF15-overexpressing cells, immunofluorescence and Western blot were used to detect the autophagy. Finally, after the addition of 3-Methyladenine (3-MA), an autophagy inhibitor, the aforementioned experiments were conducted again to further explore the mechanism. The expression of KLF15 and ATG14 were decreased in AS patients and ox-LDL-induced HAECs. Overexpression of KLF15 protected ox-LDL-induced HAECs from damage, which might be achieved through transcriptional regulation of ATG14. In addition, KLF15 could promote autophagy through transcriptional activation of ATG14. KLF15 transcriptionally activated ATG14 to promote autophagy and attenuate damage of ox-LDL-induced HAECs.