Allergic rhinitis (AR) is an upper respiratory tract inflammation disease caused by IgE-mediated reactions against inhaled allergens. The incidence of AR is significantly increasing throughout the world. Hence, more specific, and sensitive gene biomarkers and understanding the underlying pathways are necessary to further explore the AR pathogenesis.To identify gene biomarkers in nasal mucosa and in blood from AR patients which could be used in AR diagnosis.The gene expression profiles of GSE43523 from nasal epithelial cells and GSE75011 from Th2-enriched CD4+ T cells in blood were downloaded from the Gene Expression Omnibus database. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and protein–protein interaction (PPI) network analysis were conducted to investigate the functional changes of genes. The receiver operating characteristic (ROC) curves were used to assess the diagnostic values of the hub genes. Real-time quantitative PCR (RT-qPCR) was performed to validate the hub genes.POSTN in nasal mucosa and PENK and CDC25A in blood was constructed with a good AR predicting performance. The area under the curve (AUC) of the ROC curve of 3 hub genes’ combination was 0.98 for AR diagnosis.Significant differentially enriched gene signatures in AR patients were identified in nasal epithelial cells (n-DEGs) and in blood (t-DEGs). Signatures associated with axoneme, extracellular matrix, collagen fibril organization, cell motility, calcium ion binding, and so on were more enriched in n-DEGs, whereas signatures associated with TNF signaling pathway, detoxification of inorganic compound, and cellular response to corticotropin-releasing hormone stimulus were enriched in t-DEGs. In addition, we identified 8 hub genes and 14 hub genes from n-DEGs and t-DEGs, respectively. The combination of This study utilized gene expression profiles and RT-qPCR validation on nasal mucosa and blood from AR patients to investigate the potential biomarkers for AR diagnosis.The online version contains supplementary material available at 10.1186/s13223-022-00656-4. Allergic rhinitis (AR) is defined as symptoms of nasal obstruction, rhinorrhea, sneezing, and nasal pruritus caused by IgE-mediated reactions against inhaled allergens and involving mucosal inflammation driven by type 2 helper T (Th2) cells . It affeCurrently, next-generation sequencing and bioinformatic analysis have emerged as promising, useful tools for screening genetic alterations involved in the development and progression of diseases, with marked clinical applications ranging from molecular diagnosis to disease classification, patient stratification to therapeutic prediction, and new drug target discovery to response prediction , 8. PrevThe utility of AR biomarkers for clinical trials depends on both sensitivity of the measure but also the practicality of the testing and sampling. Both the collection of nasal epithelial cells and peripheral blood are rapid and minimally invasive procedures. Real-time quantitative PCR (RT-qPCR), as one of the most financially accessible and widely available detection technologies is commonly used for the identification of gene biomarkers both in clinical and research studies.In this study, we aimed to explore the possible molecular mechanisms by integrating databases on nasal epithelial cells and peripheral Th2-enriched CD4+ cells from AR patients and the same cells from healthy controls. We described the transcriptional features and identified gene biomarkers to shed light on AR development at the molecular level and to pave the way toward understanding potential pathogenesis mechanisms to facilitate its diagnosis.The study was approved by the Ethics Committee of Beijing TongRen Hospital, Capital Medical University in accordance with the guidelines of the World Medical Association’s Declaration of Helsinki., and informed consent was obtained from all participants before the study.Dermatophagoides pteronyssinus, D2 stands for Dermatophagoides farinae, and PHAD stands for phadiatop, which suggests a respiratory allergy to inhalant allergens. Subjects were considered “positive” for AR diagnosis more than a cutoff of 0.35 kU/L of serum sIgE. The exclusion criteria are including: (1) co-morbid asthma, eczema, or any other allergic diseases; (2) hypertension, diabetes, or other chronic diseases; or (3) tumor in the nasal cavity or any other inflammatory nasal disease. Details of the subjects’ characteristics are included in Table A total of 20 subjects, including 10 AR patients and 10 healthy controls were recruited in this study. The diagnosis of AR was made according to the Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines , includig, 10 min, at 4 ℃. The 2 mL whole blood samples were collected with tubes with EDTA anticoagulant, then lysis by 10× red blood cell lysis buffer and then washed twice by phosphate buffered saline. Total RNA was extracted from nasal brush samples and blood samples according to the manufacturer’s protocol of TRIzol reagent (Sigma-Aldrich) for real-time quantitative PCR analysis.All subjects underwent nasal brushing of the 1 nare. The brush samples were immediately put into phosphate buffered saline containing 2% bovine serum albumin on ice, underwent RNA isolation after centrifugate at 300https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE43523), a dataset derived from nasal epithelial cells, and GSE75011 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE75011), a dataset derived from Th2-enriched CD4+ T cells from peripheral blood, were obtained from the GEO database. GSE43523, which includes seven samples from patients with seasonal AR (SAR) and five samples from non‑allergic healthy controls, was derived from the GPL6883 platform (Illumina HumanRef-8 v3.0 expression bead chip), and GSE75011, which includes 25 samples from patients with AR and 15 samples from healthy controls, was derived from the GPL16791 platform (Illumina HiSeq 2500) [GSE43523  was performed to compare AR patients and healthy controls with the limma package of R software. Heatmaps and volcano plots of the differentially expressed genes (DEGs) were constructed using the packages pheatmap and ggplot2 of R software [First, background correction and standardization were performed on the original GEO datasets using the packages edgeR and limma of R software . Next, dsoftware .P < 0.05.We analyzed the functions and signaling pathways of the DEGs using MetaCore™ , a computational platform capable of analyzing gene clusters in the context of functional enrichment and pathway analyses. Pathway maps and Gene Ontology (GO) analyses were performed in MetaCore . GO annohttp://string-db.org/) was used to identify the PPIs of the intersecting DEGs of the two datasets, with a combined score > 0.4 used as the threshold for statistically significant interactions [P-value < 0.05 and a κ coefficient of 0.4 were considered the threshold values. Then, the Molecular Complex Detection (MCODE) plugin was used to identify important proteins in the PPI network [STRING (version 11.0) . The relative gene expression level is for target genes relative to reference gene. The gene expression level was calculated as 2(−△△Ct) method, and the △Ct means the Ct of target gene minus the Ct of reference gene. The GAPDH forward primer was 5ʹ-CGGAGTCAACGGATTTGGTC-3ʹ, and GAPDH reverse primer was 5ʹ-TGGGTGGAATCATATTGGAACAT-3ʹ. The POSTN forward primer was 5ʹ-TGCCCAGCAGTTTTGCCCAT-3ʹ, and POSTN reverse primer was 5ʹ-CGTTGCTCTCCAAACCTCTA-3ʹ. The PENK forward primer was 5ʹ-TAATGGAATGTGAAGGTAA-3ʹ, and PENK reverse primer was 5ʹ-GGTTTGCTATTTTCTCTG-3ʹ. The CDC25A forward primer was 5ʹ-TGGAGGTGAAGAACAACA-3ʹ, and CDC25A reverse primer was 5ʹ-GGTCAAGAGAATCAGAATGG-3ʹ.Total RNA was isolated from lysates of nasal brushing cells and white blood cells using TRIzol reagent (Sigma-Aldrich) according to the manufacturer’s protocol. The concentration and purity of the RNA was assessed with a Nanodrop-2000 (Thermo Fisher Scientific). RNase-free water was used to adjust the stock concentration of all RNA samples to 500 ng/μL. Complementary DNA was synthesized from 1.5 μg of total RNA using a High-Capacity cDNA Reverse Transcription Kit with genomic DNA remover . In order to avoid the genomic DNA contamination, total RNA was treated with 4× gDNA remover mix t for 5 min at 37 °C, then for additional 5 min at 85 °C, and stop at 4 °C in a 20 μL reaction system. Then, total RNA with genomic DNA removal was treated with 5× RT mix for 5 min at 25 °C, for 15 min at 42 °C, for 5 s at 85 °C, and stop at 4 °C. All RT-qPCR products were amplified using a SYBR Green I Master Kit  in 10 µL reaction volume on an Applied BiosystemsA receiver operating characteristic (ROC) curve was drawn to calculate the area under the curve (AUC) to discriminate AR patients from normal controls in all AR GEO datasets. SPSS 16.0 for Windows  was used for ROC curve analyses, and other statistical analyses were performed using GraphPad Prism 7.0 software .2FC|> 1, adjusted P-value < 0.05). The same method was used to analyze and identify 165 DEGs (78 up-regulated and 87 down-regulated) in Th2-enriched CD4+ T cells from peripheral blood samples (t-DEGs) in GSE75011 using the edge and limma packages in R.We performed a differential analysis of mRNAs and identified 997 DEGs in nasal epithelial cells from AR patients (n-DEGs), of which 559 were down-regulated and 438 were up-regulated  between AR patients and healthy controls in the heatmap . The top ten significantly enriched pathways in AR (according to the pathway map) are shown in Fig. To explore the potential functions of the DEGs, we generated pathway maps and performed GO functional enrichment analysis in MetaCore , biglycan (BGN), secreted protein acidic and cysteine rich (SPARC), TIMP metallopeptidase inhibitor 3 (TIMP3), collagen type I α2 chain (COL1A2), collagen type III α1 chain (COL3A1), matrix metallopeptidase 9 (MMP9), and periostin (POSTN) and 14 hub t-DEGs, namely, E2F transcription factor 8 (E2F8), denticleless E3 ubiquitin protein ligase homolog (DTL), kinesin family member C1 (KIFC1), proenkephalin (PENK), galanin receptor 2 (GALR2), taste 1 receptor member 3 (TAS1R3), prostaglandin D2 receptor 2 (PTGDR2), C-X3-C motif chemokine receptor 1 (CX3CR1), C–C motif chemokine ligand 20 (CCL20), cyclin dependent kinase inhibitor 1A (CDKN1A), PDZ binding kinase (PBK), cell division cycle 25A (CDC25A), polo like kinase 1 (PLK1), and baculoviral IAP repeat containing 5 (BIRC5).To further explore the connections among the DEGs, PPI networks were constructed with the n-DEGs and t-DEGs. The PPI networks of n-DEGs contained 119 nodes and 160 edges, and the isolated genes (without interactions) were removed. A total of 145 interactions and 94 nodes were screened to establish the PPI networks of t-DEGs  was 0.98  [BGN, one of the small leucine-rich repeat proteoglycans involved in tissue remodeling in inflammatory diseases, showed strong immunoreactivity in mild and severe persistent allergic nasal mucosa [Our study was based on nasal epithelial cells and Th2-enriched CD4+ cells from the peripheral blood of AR patients and nonallergic controls from the GEO database. We identified 997 n-DEGs (438 up-regulated and 559 down-regulated) from nasal epithelial cells and 165 t-DEGs (78 up-regulated and 87 down-regulated) from Th2-enriched CD4+ T cells. In addition, we identified 8 and 14 hub genes from PPI networks of the n-DEGs and t-DEGs that might be used as biomarkers in the pathogenesis of AR. Some of the hub genes have been studied in AR. patients , 23. POSed in AR . PTGDR2 otherapy . A study airway) . The pro airway) , and the airway) . BGN, onl mucosa .In some previous studies, bioinformatic analysis of mRNAs and lncRNAs in AR was performed. For instance, two studies focused on DEGs from the nasal epithelial cells of AR patients , 30. AnoIn our study, MetaCore pathway map analysis showed that NETosis was the most significant differentially enriched pathway between AR patients and healthy controls. Neutrophil extracellular traps are the result of a unique form of cell death that is morphologically characterized by the loss of intracellular membranes before the integrity of the plasma membrane is compromised; this is called NETosis . NETosisAlthough our study demonstrated that combination of multiple gene biomarkers had more accuracy to be diagnostic for AR, there are some major gaps exist in the AR biomarker space. In particular, our study is limited with the small sample size from the AR patients and healthy controls. It’s better to include the data contains the progression of AR before and after treatment. Besides our study, additional work is needed to explore the underlying mechanisms that gene biomarkers involved.POSTN expression in nasal brushings, and the decreasing expressions of CDC25A and PENK in blood in AR or their combination could be used to help AR diagnosis (AUC = 0.98). This study provides new insights into the overall mechanism of AR from the nasal cavity to the blood, but further research is needed to develop novel therapeutic strategies to drug discovery along with medical approaches to clinical treatment of AR.In summary, our study aim to identify genes and pathways in the nasal epithelial cells and peripheral blood of AR patients through publicly available datasets. We identified the DEGs and analyzed their functions through GO analysis and PPI networks and identified the hub genes that might play crucial roles in the pathogenesis of AR. In addition, we validated the hub genes both by GEO database and RT-qPCR in nasal mucosa and in blood from AR patients and the healthy controls. Our study demonstrated that the increasing Additional file 1: Figure S1. Flowchart of our study to identify potential gene markers in AR patients. Table S1. The raw mean Ct value of the nasal brush samples and the blood samples from AR patients and healthy controls. Table S2. The RNA concentration of the nasal brush samples and the blood samples from AR patients and healthy controls.


To determine (1) the effectiveness of rehabilitation for chronic ankle instability as measured by the Star Excursion Balance Test (SEBT) and (2) the relative efficacy and the long-term effects of these rehabilitation interventions.Ten electronic databases were searched (2009-2019).Included articles were randomized controlled trials in English investigating recreational athletes aged ≥18 years with chronic ankle instability. At least 1 functional rehabilitation intervention had to be included and the SEBT test (or the modified version) used as an outcome measure.Two researchers  extracted data regarding participant demographics; intervention characteristics; trial size; and results at baseline, postintervention, and at follow-up, where appropriate.A systematic review and narrative synthesis was conducted. Methodological quality of included studies was assessed using the Cochrane Risk of Bias Tool and the van Tulder scale. The review was registered with PROSPERO (ID: 164468). Ten studies (n=368), 2 high-quality, 1 moderate-quality, and 7 low-quality, were included in the review. Interventions included balance training, strength training, vibration training, and mixed training. Results suggest that rehabilitation of chronic ankle instability that includes wobble board exercises (average percentage change: 14.3%) and hip strengthening exercises (average percentage change: 12.8%) are most effective. Few studies compared different types of rehabilitation for chronic ankle instability. However, improvements on the SEBT suggest that a rehabilitation program focusing on wobble board training and hip strengthening performed 3 times weekly for 4-6 weeks is the optimal rehabilitation program to improve dynamic postural control in recreational athletes with chronic ankle instability.Few studies directly compared different rehabilitation interventions, and there was limited long-term follow-up; therefore, the relative efficacy of different rehabilitation programs remains unclear. However, it seems that rehabilitation of chronic ankle instability should include proprioceptive and strengthening exercises of relatively short duration. Both short-term (6-12wk) and long-term (12+wk) follow-up studies were included.Full-text randomized control trials published in English were included. For the purposes of this review recreational athletes were self-reported or defined as completing at least 20 minutes of moderate to high intensity physical activity 3 times per week.Two researchers  extracted data regarding participant demographics, intervention characteristics, trial size, baseline and postintervention results, and follow-up results where relevant.,Two independent reviewers  assessed each study using the Cochrane Risk of Bias ToolThe data synthesis was conducted following the recommended standards of performance outlined by Eden et alThe minimal detectable change (MDC) values outlined by Munro et al.,,,38,,All studies provided demographic details and included 177 male and 191 female participants  see . Four st,,,,,Control groups included normal activity,,,39,39,,,,39,,,,,47,39,The 10 studies included 2 high-quality,,,,,,Four types of rehabilitation were investigated: balance,37Balance programs included multidirectional hopping,Three studies implemented balance programs, 1 high qualityBurcal et alLinens et al,Strength training included resistance band exercises for the ankle,,Two studies conducted a strength program; both were deemed low-quality because of inadequate allocation concealment and lack of blinding of assessors.Smith et alOne study implemented a vibration program involving single-leg heel raises and single-leg squats on a vibration platform.Three studies adopted a mixed training intervention incorporating general strength and balance training,One study was deemed high quality,Melam et alOne study compared balance and strength training,,,,,,,,,,,,,Five studies involved a 4-week intervention,,,One study completed 1 session per week,40,,,Successful interventions included a frequency of 1 session37Burcal et alThis review aimed to determine (1) the effectiveness of rehabilitation for chronic ankle instability as measured by the SEBT and (2) the relative efficacy and the long-term effects of these rehabilitation interventions. The results suggest that rehabilitation of chronic ankle instability that includes wobble board exercises (average percentage change: 14.3%),,The benefits of wobble board rehabilitation for chronic ankle instability have been well-documented.71,Previous research has reported that those with chronic ankle instability rely more on the hip's contribution during postural control tasks.,,,,,,The suggested optimal frequency is 3 sessions per week (average percentage change: 11.49%). This is supported by 2 high-qualityOnly 1 study compared different rehabilitation typesBurcal et al,This is the only review in the last 10 years that has assessed the effectiveness of different rehabilitation types and suggested optimal rehabilitation parameters. Before this review, Webster and GribbleThe importance of postural control is accepted for many clinical populationsDespite these limitations, this review rigorously evaluated risk of bias within and across the included studies. Furthermore, this is the only review that discusses an optimal rehabilitation program for recreational athletes with chronic ankle instability, thus assisting clinicians regarding the conservative management of chronic ankle instability.Future trials should be adequately powered and focus on meeting the minimum standards to reduce potential threats to bias. There is a need for trials to directly compare different rehabilitation types to provide a definite conclusion regarding the relative efficacy. Future trials should include a sufficient follow-up period to determine the long-term effects of an intervention.This review suggests the optimal rehabilitation parameters required in the management of recreational athletes with chronic ankle instability. Three weekly sessions focusing on wobble board exercises and hip strengthening for 4-6 weeks is suggested. However, the evidence is insufficient for these results to be conclusive and are only suggestions to help guide clinicians in the management of chronic ankle instability.Chronic ankle instability is associated with impaired sensorimotor control, which contributes to deficits in postural control activities. A rehabilitation approach focusing on wobble board exercises and hip strengthening performed 3 times weekly for 4-6 weeks is suggested to help improve dynamic postural control in recreational athletes with chronic ankle instability, at least in the short-term. The lack of long-term follow-up studies prevents definitive conclusions, and the results are suggested as a guideline to assist clinicians in the management of recreational athletes with chronic ankle instability. The long-term effects of the interventions remain unclear and further research is required.


Besides regulating inotropy and chronotropy, α1- and β-adrenergic receptors also regulate metabolic functions in the heart that underlie many cardiac benefits. This review will highlight recent studies that describe how adrenergic receptor-mediated metabolic pathways may be able to restore cardiac energetics to non-failing levels that may offer promising therapeutic strategies.The heart has a reduced capacity to generate sufficient energy when failing, resulting in an energy-starved condition with diminished functions. Studies have identified numerous changes in metabolic pathways in the failing heart that result in reduced oxidation of both glucose and fatty acid substrates, defects in mitochondrial functions and oxidative phosphorylation, and inefficient substrate utilization for the ATP that is produced. Recent early-phase clinical studies indicate that inhibitors of fatty acid oxidation and antioxidants that target the mitochondria may improve heart function during failure by increasing compensatory glucose oxidation. Adrenergic receptors (α Each of these families contain three subtypes , , , and all three families bind epinephrine (Epi) and norepinephrine (NE) with similar affinity. However, they can regulate different physiological processes because they couple to different heterotrimeric and monomeric G-proteins and effector proteins that transduce different signaling pathways (Adrenergic receptors (ARs) are members of the G-Protein Coupled Receptor (GPCR) superfamily that bind adrenaline  and noradrenaline  . They repathways . AR sign1-ARs, particularly the α1A-AR, are recent developments in offering cardioprotective and inotropic benefits during HF. Their roles in regulating metabolic pathways that are altered during HF may be an underappreciated mechanism in their ability to treat HF and is the focus of this review.While β-ARs are well known to be affected during heart failure (HF) and β-AR blockers used in HF treatment, the role of α1- and β-AR subtypes. β-ARs are predominately expressed in the myocyte, followed by the β2-AR subtype [s G-protein to stimulate adenylate cyclase which produces a transient rise in the second messenger cAMP  which cleaves the membrane-bound phosphatidylinositol 4,5-bisphosphate to release inositol triphosphate (IP3) and diacylglycerol (DAG) [1-ARs activate a positive inotropy induced from calcium sensitizing the myofibril filaments [Of the αmyocytes ,18,19    and occurs in the cytosol. Phosphofructokinase 1 is the step that commits glucose to undergoing glycolysis [Various carbohydrates such as glucose, glycogen, and lactate are used by the heart to produce ATP. Glucose is taken up by the myocyte through glucose transporters (GLUTs) GLUT1 and GLUT4 , then meycolysis . If suffAn alternative pathway for glucose utilization is the pentose phosphate pathway (PPP) which is the main generator of nicotinamide adenine dinucleotide phosphate (NADPH) in the cytoplasm . It alsoA loss of that protection from ROS damage in the heart can result from deficiency in G6PD and shown to occur during ischemia , and HF 2) which are oxidized in the electron transport chain  to produce ATP and water. A schematic of fatty acid oxidation in the cardiac myocyte is shown in In the normal adult heart, about 95% of its ATP requirements are due to mitochondrial oxidative phosphorylation with 60–80% of that from fatty acid oxidation ,125,126.As the heart fails, its ATP levels drop by 30–40% during the advanced stages of failure compared to the normal heart ,131,132.The metabolic profiling of induced-HF in various animal models does show considerable variability and the need to be cautious in interpretations of the early metabolic changes. During pressure overload-induced HF, there is a decrease in both fatty acid and glucose oxidation ,143. In Another explanation for the metabolic disparity in the various types of HF models may be dependent upon whether there is failure with preserved ejection fraction (HFpEF) or with reduced ejection fraction (HFrEF) ,149,150.Abnormally high rates of glucose metabolism are one of the earliest metabolic changes that occurs during the development of HF ,164. TheStimulating glucose uptake and oxidation in the heart has been shown to improve the rate of recovery from ischemic damage ,185,186.Inhibition of fatty acid oxidation causes a compensatory increase in glucose oxidation and improves cardiac metabolic efficiency, decreases mitochondrial oxidative capacity, and has also been used for a number of therapeutic strategies in HF . CarnitiSince fatty acid oxidation provides much greater ATP production compared with glucose, therapeutic treatments that revert the heart back to using fatty acids as substrates might also be an option for treating HF, but several models of HF have shown downregulated or have dysfunctional enzymes or proteins involved in the fatty acid oxidation process ,198,199.1-AR agonist, phenylephrine [1- and β-ARs are essential for heart development and metabolism by regulating the embryonic shift from anaerobic glycolysis to aerobic metabolism and oxidative phosphorylation in the heart.ARs regulate many aspects of metabolism in the substrate preference, mobilization, and utilization as it relates to normal heart function and changes that can occur during HF ,200,201.lephrine . Targetelephrine ,203. TheIn general, catecholamines have also been shown to increase glucose uptake, glycolysis, and glucose and fatty acid oxidation in the adult heart ,206,207 1-ARs in metabolism have shown increased gluconeogenesis and ketogenesis in both the liver [1-AR agonists may confer part of their cardioprotective benefits and limit the progression of HF, particularly in the diabetic heart, by metabolically increasing glucose and ketone body availability through enhanced glucogenesis and ketogenesis. Early studies on the role of αhe liver ,215,216 he liver ,218. Thehe liver ,220. Bothe liver ,222, poshe liver  SGLT inhhe liver , unlike he liver ,225. Kethe liver  and morehe liver . Ketoneshe liver ,229,230 he liver , all pro1-AR stimulation can increase glucose substrate availability by increasing its uptake into the heart or in myocytes [1-AR stimulation of PKC activation which increase translocation of GLUT 1 or 4 to the membrane resulting in GLUT activation [1-AR mediated 3H-deoxyglucose uptake was blocked by an inhibitor of GLUT 1 or 4 translocation and by the PKC inhibitor rottlerin or siRNA against PKCδ [1A- and α1B-AR KO mice [1A- or α1B-AR subtypes with constitutively active mutations (CAMs) [1- AR subtypes on metabolism. The α1A-AR and not the α1B-AR subtype appears selective for glucose uptake in the heart as only transgenic CAM α1A- but not CAM α1B-ARs increased glucose uptake in the heart and plasma membrane translocation of both GLUT1 and GLUT4. In congruence, knockout (KO) of the α1A-AR but not α1B-AR mice decreased glucose uptake and GLUT translocation [1A-selective agonist, A61603 also increased glucose uptake into primary myocytes [1A-AR stimulation also increased glucose uptake during glucose-starved conditions of ischemia and protected against annexin V+ apoptosis and increased levels of lactate dehydrogenase [1A-AR-mediated regulation of glucose uptake in the heart would provide the needed increased metabolic requirements in HF but also provide cardioprotective benefits against apoptosis and cell death. Glucose uptake is increased in the heart during ischemia via translocation of the glucose transporters GLUT 1 and GLUT 4 . Both GLmyocytes ,235,236 myocytes ,238,239,myocytes ,243,244.tivation ,245  ,248,249 location ,250. Therogenase ,250. The1A-AR agonists would also not only increase glucose uptake and availability but also increase its oxidation in the heart. Using radioactive tracers of 14C-glucose or 14C-palmitate to measure oxidation rates in primary cardiac myocytes, α1-AR stimulation with phenylephrine increased glucose oxidation which was blocked by an inhibitor to AMP-activated protein kinase (AMPK) [1A-AR agonist treatment may promote energy efficiency and increase the needed ATP in the failing heart by increasing both glucose substrate availability and its subsequent oxidation.Without a concomitant increase in glucose oxidation, an increase in glucose substrate is not energy efficient and of limited benefit in HF. Recently, studies have indicated that αe (AMPK) , under b+ homeostasis [1-ARs have been readily shown to activate AMPK in the heart [1-AR mediated ischemic protection is mediated through PKC and AMPK [1-AR signals that mediates glucose oxidation [1-AR metabolic effects and protective signals in the heart may both be regulated through AMPK.AMPK is a key regulator of energy metabolism and plays a cardioprotective role. AMPK activation is protective in the heart against ischemic damage ,252,253,eostasis ,260,261.eostasis ,263  away from those that induce the blood pressure response. The above review suggests that the αpressure ,302,303.2-AR and not α1-AR agonists commonly contain the imidazoline pharmacophore and, in general, have better selectivity for α2-ARs. α2-AR agonists reduce blood pressure by decreasing NE release at the α2A-AR autoreceptor on sympathetic nerve endings [1-AR [1-AR selective agonists [1A-AR subtype compared to the α1B- or α1D-AR subtypes [1A-AR selective and shown to reduce stress urinary incontinence without a strong response on blood pressure by biasing the signaling towards cAMP and not the IP3/Ca+2 response [1A-AR structured imidazolines to separate cardioprotection from the blood pressure effect would depend upon the therapeutic index or the dose of the drug that can separate therapeutic efficacy from toxic side effects. Dabuzalgron , for example, was taken to Phase ll clinical trials showing improvement in stress urinary incontinence with little or no cardiovascular effect [α endings  and alsogs [1-AR . Howeveragonists ,307,308.subtypes ,310. Theresponse ,313,314 response ,39,80,81r effect  but did r effect .1A-AR agonists are positive allosteric modulators (PAMs) that increase a receptor’s function but does not bind to the orthosteric  site that agonists bind, such as NE [1-ARs with selectivity for the α1A-AR subtype has been developed [Another area of drug development for αch as NE . Allostech as NE . There ach as NE ,318. Theeveloped  that can1-ARs, β1-AR but not β2-AR stimulation increases glycogenolysis by the phosphorylation and activation of glycogen phosphorylase [1-AR agonists can increase anaerobic metabolism when needed [1-ARs appear to increase oxidative metabolism at a greater rate than the β2-AR [1-AR in HF is detrimental. While inotropic interventions for HF might improve short-term hemodynamics, β-AR agonists have long-term effects that have failed to improve outcomes and can even worsen as excessive release of catecholamines induces apoptosis, increased heart rate, and arrhythmias [Similar to αhorylase . Under in needed  and may n needed ,322,323.n needed ,325, butn needed , suggesthe β2-AR  and may hythmias ,329. 3-ARs have been mostly studied in adipose cells to increase cAMP levels, activation of lipase, and thermogenesis [3-AR produces a negative inotropy in opposition to positive inotropy regulated by the β1- or β2-AR [3-AR does not desensitize and may even increase during HF [3-AR agonist may also provide metabolic benefit. However, the role of the β3-AR in HF is controversial. One study suggests that cardiac function can be recovered in HF by blocking the β3-AR which improves the energy efficiency in myocardial tissues by suppressing iNOS expression [3-AR KO mouse model is cardioprotective [3-AR stimulation or cardiac-overexpression is cardioprotective in many models of ischemia, pressure overload and hypertrophy through inducing nitric oxide (NO) [3-AR agonism may still be a viable metabolic therapeutic candidate for HF.βogenesis ,331. Mosor β2-AR ,16, is cor β2-AR , and upror β2-AR ,332. As uring HF ,334,335,pression . A β3-ARotective . In contide (NO) ,343,344.3-AR agonist, mirabegron, can improve glucose homeostasis in insulin-resistant obese humans although this occurs through its function on brown adipose tissue [3-AR agonists improved glucose tolerance, utilization [3-AR agonism is anti-atherosclerotic in apoE KO mice with decreased plasma triglycerides, low density lipoproteins, cholesterol, and increased insulin sensitivity and PPARα and expression in the liver [3-AR agonists can regulate whole body lipid and glucose metabolism. While a clinical trial of mirabegron failed to improve left ventricular ejection fraction in HF [The βe tissue . Mirabege tissue ,346. Mirlization  and insulization  or mice lization ,350. β3-he liver ,352. Theon in HF , there won in HF . 1-ARs in HF and worse outcomes with long-term stimulation, the metabolic effects of β-blockers to treat HF are preferred. β-AR blockers are a standard therapy in the treatment of HF by reducing sympathetic overdrive resulting in increased ejection fraction and survival [1B-AR blockage) in addition to its β-AR blockage, have metabolic effects that contribute to better outcomes in HF, compared with pure β-AR blockers [With the subsequent downregulation of βsurvival . Howeversurvival ,356. As survival ,172,357.survival ,360,361.survival ,360,361.survival ,367,368 blockers ,374,375.1-AR agonists coupling to PPARs, PPAR activators increase mitochondrial oxidative capacity in the heart [1-AR stimulation decreases PPAR activation. Similarly, β1-AR autoantibodies induce a positive inotropy in the heart [2-AR agonist, higenamine, against mitochondrial and respiratory dysfunction is mediated though increased PPARα signals [1-AR selective blockers may be better at increasing mitochondrial function during HF than non-selective β-blockers.Studies have also suggested that β-AR blockers may also increase cardiac mitochondrial respiration ,378,379 he heart  and is che heart . Isoprothe heart ,287, sughe heart  but alsohe heart ,387, apohe heart  and inhihe heart ,390. How signals  (Figure Besides generating ATP, mitochondria are the main produces of ROS, generated during oxidative phosphorylation ,393. Mit1-AR antagonist [1-AR blocking ability of carvedilol has been downplayed in its cardiac protective potential as non-selective α1-AR blockers can increase the morbidity of heart failure [2- versus β1-ARs [1B-ARs versus α1A-ARs and binds with higher affinity at α1-ARs than at β1-ARs [1B-AR. The α1B-AR, unlike the α1A-AR [1B-AR by carvedilol would decrease its maladaptive effects in the heart and not disrupt the metabolic and other cardioprotective benefits of the α1A-AR.The best described β-AR blocker and its effects on cardiac metabolism is carvedilol. Carvedilol is a β-AR antagonist and an αtagonist . The α1- failure . Howevers β1-ARs ,406 and t β1-ARs . A possie α1A-AR ,85,407 ae α1A-AR ,235,250,1A-AR agonism. In clinical studies, carvedilol has better cardioprotective metabolic properties than other β-blockers and may be a preferred treatment for HF. Carvedilol is better than metoprolol in its antioxidative properties ,410,411 1-AR stimulation [1-ARs are downregulated in HF, β-AR blockers may reactivate AMPK pathways to shift metabolism to glucose utilization. The β3-AR is also postulated to mediate its exercise-mediated cardioprotection through activation of AMPK signals [Carvedilol’s mechanism to shift metabolism to glucose utilization is also an AMPK-mediated mechanism, similar to αmulation . AMPK camulation ,426 (Figmulation  and the mulation ,429,430. signals . 1- and β-ARs regulate cardiac metabolism in opposition with α1A-AR agonism and β-AR blockage converging on increasing glucose availability and oxidation, suggesting that a dual action drug might provide the greatest therapeutic benefit. The ability of β-AR antagonists and α1A-AR agonists to regulate, optimize, and shift energy pathways as needed by the heart during failure are also predicted to lead to better outcomes.While inotropic interventions for HF might improve contractile function for the short-term, chronic treatments have failed to improve outcomes. During the progression of HF there is a tremendous amount of metabolic inflexibility  caused b


As an H3K27me3 demethylase and counteracts polycomb-mediated transcription repression, KDM6B has been implicated in the development and malignant progression in various types of cancers. However, its potential roles in esophageal squamous cell carcinoma (ESCC) have not been explored.The expression of KDM6B in human ESCC tissues and cell lines was examined using RT-qPCR, immunohistochemical staining and immunoblotting. The effects of KDM6B on the proliferation and metastasis of ESCC were examined using in vitro and in vivo functional tests. RNA-seq and ChIP-seq assay were used to demonstrate the molecular biological mechanism of KDM6B in ESCC.β, leading to the promotion of C/EBPβ transcription. Besides, we show that GSK-J4, a chemical inhibitor of KDM6B, markedly inhibits proliferation and metastasis of ESCC cells.We show that the expression level of KDM6B increased significantly in patients with lymph node metastasis. Furthermore, we confirmed that KDM6B knockdown reduces proliferation and metastasis of ESCC cells, while KDM6B overexpression has the opposite effects. Mechanistically, KDM6B regulates TNFA_SIGNALING_VIA_NFκB signalling pathways, and H3K27me3 binds to the promoter region of C/EBPβ depending on its H3K27 demethylase activity.The present study demonstrated that KDM6B promotes ESCC progression by increasing the transcriptional activity of C/EBPThe online version contains supplementary material available at 10.1186/s12885-021-08282-w. Esophageal cancer (EC) is one of the most common gastrointestinal malignancies in the world. According to reports in 2018, this disease is ranked as the seventh most frequently diagnosed cancer and the sixth cause of cancer death . TherefoEpigenetic modification plays an important role in tumorigenesis and development.Many reports have confirmed that epigenetic modification plays a crucial role in cancer development. As a major epigenetic mechanisms, histone methylation enables the gene promoter to be accessible or inaccessible to transcription factors, thereby regulating target gene expression , 5. Methβ mediating ESCC cell proliferation and metastasis. Our data, therefore, delineate the biologic function of KDM6B and validate KDM6B may be a novel therapeutic target for ESCC in the future.Here we investigate the biological impact of KDM6B in ESCC. We show that KDM6B is overexpressed in ESCC patients with lymph node metastasis and confirm that KDM6B can promote ESCC cell proliferation, clone formation, G1/S transformation, migration and invasion. Importantly, we demonstrate that KDM6B, in a demethylase-independent fashion, upregulates C/EBP2 and humidified atmosphere at 37 °C. Cells at the third generation of the exponentially growing state were used for all experiments.The human esophageal cancer cell lines  were obtained from the Type Culture Collection of Chinese Academy of Sciences  and cultured in RPMI-1640 medium  supplemented with 10% Fetal Bovine Serum (FBS) (Gibco-BRL), 100 μg/ml streptomycin and 100 units/ml penicillin G  in 5% COSamples were collected from 41 patients with ESCC, who underwent complete surgical resection at the Department of Cardiothoracic Surgery of the Affiliated Hospital of Southwest Medical University from January 2017 to March 2018. The clinicopathological features of the ESCC patients are summarized in Table of KDM6B expression were performed as previously described . SamplesCells were lysed in Lysis buffer  and centrifuged at 4 °C for 15 min. Protein concentrations were measured through BCA Protein Quantitation Assay . Total cell lysates were equally loaded on 8–15% SDS-polyacrylamide gel for running and then transferred to polyvinylidene difluoride (PVDF) membranes . After blocking for 1 h at room temperature with 5% non-fat milk in Phosphate buffered saline with Tween 20 (PBST), the membranes were incubated for overnight with the primary antibodies anti-KDM6B , anti-Tri-Methyl-Histone H3(Lys27) (C36B11) Rabbit mAb , anti- GAPDH , anti- Histone H3 Mouse Monoclonal Antibody . After staining with corresponding horseradish peroxidase (HRP)-linked secondary antibodies, signal detection was performed using a chemiluminescence phototype-HRP Kit  and exposed to film in a dark room.Total RNA from esophageal tissues was extracted with TRIzol  and cDNA was synthesized from 1 μg of total RNA using the ReverTra Ace qPCR RT Master Mix  following the manufacturers’ instruction. The mRNA levels of target genes and the internal standard glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were measured by RT-qPCR in triplicate using the QuantiNova SYBR Green PCR kit  and the StepOne™ real-time PCR System .The specific primers for the genes are listed in Table Total RNA of each sample was extracted using TRIzol Reagent (Invitrogen). Total RNA of each sample was quantified and qualified by Agilent 2100 Bioanalyzer , NanoDrop (Thermo Fisher Scientific Inc.), and 1% agarose gel. We used 1 μg of total and polysome-associated RNA fractions for RNA-seq from two independent biological sample replicates. The RNA-seq was performed in an Illumina HiSeq platform using RNA-seq kit according to the manufacturer’s recommendation .The sequences were processed and analyzed by GENEWIZ.http://www.sigmaaldrich.com/), and the hairpin sequences were cloned into the lentiviral vector pLKO-Tet-On with Puromycinas as a selection marker, shRNAs targeting KDM6B were screened.Gene-specific shRNA sequences were obtained from the Sigma website . And co-transfected into HEK 293 T cells at 70–80% confluency in a 10 cm Petri dish according to the manufacturer’s protocol. At 72 h post-transfection, the supernatant containing virus particles was collected and filtered through a 0.45 μm PVDF Millex syringe filter.Each cell line was subjected to a puromycin tolerance test to determine the optimal concentration for establishing stable cell lines. To generate an shRNA-expressing stable cell line, cells were infected with lentiviral particles with 4 μg/ml polybrene. After 48 h, the cells were selected by 1 μg/ml puromycin (GIBCO) for 5 days. And the knockdown efficiency was tested by WB assay and RT-qPCR.riboFECT™CP  according to the manufacturer’s instructions. Cells were seeded in free-antibiotics media in six-well plates and cultured for 24 h before siRNA transfection. Non-targeting control, siRNA groups were set up when the density of cells reached 50%. Approximately 5 μL of 20 μM siRNA was diluted with 120 μL of 1× riboFECT™ CP buffer and added with 12 μL of riboFECT™ CP reagent. The mixture was incubated for 15 min at room temperature. The prepared riboFECT™ CP mixture was added to 1863 μL of cell culture medium. The culture plate was placed in a CO2 incubator at 37 °C for 48 h, and the cells were then collected. The efficacy of siRNA transfection was evaluated by western blot and real-time quantitative RT–PCR. The siRNA sequences used in this study are as follows:siRNAs against C/EBPβ were siRNA-1:5′-GUAUAUUUUGGGAAUCUUUTT-3′,siRNA-2:GUCUAUGUGUACAGAUGAAUG and non-targeting control siRNA, 5′-UAGCGACUAAACACAUCAA-3′.For rescue experiments, plasmids were transfected on day 1 followed by siRNA transfection on day 2 and observation at 48 h after siRNA transfection.To overexpress KDM6B, ESCC cells were transfected with a plasmid containing the full-length cDNA of the human KDM6B gene, which was obtained from Addgene (plasmid ID #24167). The transfections were performed using LipoFiter™ Liposomal Transfection reagent according to the manufacturer’s instructions. The cells were plated in 6 cm dishes and incubated overnight. When the confluence reached 70–80%, the supernatant of the cell was replaced with fresh RPMI-1640 medium supplemented with 10% FBS, and then transfected with 5 μg of either KDM6B-vector using 16 μl LipoFiter™. The media was replaced 6 h after transfection, and the cells were incubated at 37 °C for 48 h. The overexpress efficiencies were confirmed by western blotting and RT-qPCR assay. The indicated siRNAs were synthesized by Guangzhou Riboio Co., Ltd. (China). Cell transfection was performed using 2 incubator at 37 °C. The culture medium needed replacement 3 d/time, and the culture was stopped when the clone was visible to the naked eye. With the culture medium discarded, the cells were washed two times with PBS, and Cell clones were immobilized by methanol for 15 min and stained with crystal violet solution  for 3 min before photographed and counted. Colonies containing more than 50 cells were counted using a microscope.Cells transfected with the indicated vectors were plated at low density (500 cells per 6-cm plate), incubated in a 5% CO2 incubator at 37 °C for 96 h. After the cells adhered to the wall, With the culture medium discarded, fresh culture medium containing 10 μL CCK-8 reagent  was added for incubation for 2 h. Subsequently, an enzymatic marker  was used to detect the OD value at the wavelength of 450 nm. The cell survival rate was calculated, and the cell growth curve was drawn. The experiment was repeated three times.Cell viability was evaluated using the Cell counting kit-8 (CCK-8) assay. The cells were detached and inoculated into 96-well plates at a density of 1000 cells/well (200 μL in each well). Four wells with cells were set for each group, and blank and control wells were also set. The plate was incubated in a 5% COIn the scratch based wound healing assay  cells we5 cells/ml in serum-free RPMI-1640. A total of 200 μl cell suspension was added into the upper chamber, whilst the lower chamber was treated with 500 μl of RPMI-1640 with 10% FBS at 37 °C for 24 h. The medium was discarded, and non-migrating cells on the top surface of the upper chamber were removed gently using cotton swabs. Migrated cells were fixed with pre-chilled methanol for 30 min then stained with 0.5% crystal violet at room temperature for 20 min. Representative images were taken under an inverted microscope  equipped with a camera .A migration assay was performed using Transwell inserts  that contained a polycarbonate transwell membrane filter . Cells were maintained at a concentration of 5 × 10For the invasion assay, the upper chamber was pre-coated with Matrigel (BD Pharmingen). Ice-cold Matrigel was mixed with ice-cold RPMI-1640 medium at a ratio of 1:8 and spread onto the upper chamber (100 μl/chamber), which was subsequently incubated at 37 °C for 2 h. The following steps, including cell plating, incubation, fixing, and de-staining, were conducted as aforementioned for the migration assay.6 cells). The tumor length (a) and the short diameter (b) were measured using a calliper twice a week, and the tumor growth curve was drawn via V = ab2/2. Three weeks after the injections, the mice were euthanized in a CO2 chamber, followed by cervical dislocation and lack of withdraw reflex to assure animal death. The tumours were removed and photographed.A total of 18 male BALB/C nude mice  were raised in laminar shelves without specific pathogen conditions with constant temperature, constant humidity, and regular disinfection. Bedding, drinking water and feed were replaced regularly under aseptic conditions. When the KYSE150 sh-NC and sh-KDM6B-1 cells were at a logarithmic growth phase, they were subcutaneously injected into the armpit of the forelimb of nude mice  according to the manufacturer’s instructions. Briefly, cells cultured in 15 cm Petri dishes were crosslinked with 1% formaldehyde when reaching 70–80% confluence. Crosslinking was terminated by adding 10 × glycine followed by rinsing the cells twice with cold PBS, and 4 × 107 cells were collected into one 15 ml conical tube. The cell pellet was resuspended and incubated in Buffer A on ice for 10 mins for cell lysis. The nuclei were collected by centrifugation at 3000 rpm for 5 mins at 4 °C and resuspended in Buffer B followed by centrifugation and supernatant removal. The nuclei pellets were resuspended in 1.0 ml Buffer B, and transferred to a 1.5 ml microcentrifuge tube. 7.5 μl of Micrococcal Nuclease was added and incubated for 20 mins at 37 °C to digest the DNA to lengths of approximately 150–300 bp. After the termination of digestion by adding 100 μl of 0.5 M EDTA, the nuclei pellets were collected by centrifuging at 13,000 rpm for 1 min at 4 °C. The nuclear pellets were resuspended in 1 ml ChIP buffer and separated into two tubes, and incubated on ice for 10 mins followed by subsequent sonication of lysates at 9 W, 10 s ON/30 s OFF, nine cycles. Lysates were collected by centrifugation at 10,000 rpm for 10 mins at 4 °C. The supernatant (single nucleosome lysate) was transferred to a new tube, and stored at − 80 °C for the following experiments. 50 μl of lysate was used for the analysis of chromatin digestion.β were: 5′- CATCAAGCAAAACGCTATGGG-3′ and 5′- CTCAGAACCTAGAGCCGGAAA-3′. The IP efficiency was calculated using the equation shown below.Pulldown: The single nucleosome lysate was diluted with ChIP buffer with protease inhibitor cocktail . 10 μl of sample was saved as 2% input. To reduce nonspecific binding, the lysate was pre-cleared with 10% BSA and 30 μl ChIP Grade Protein G Magnetic Beads  at 4 °C with rotation for 1 h. The supernatant was collected by placing the tube on a magnetic separation rack. Antibodies of H3K27me3 or IgG control were added and incubated on rotation at 4 °C overnight followed by incubation with 30 μl of magnetic beads for 2 h at 4 °C. Beads were washed with low salt wash buffer (100 μl 10 × ChIP Buffer in 900 ml water) three times and with high salt wash buffer (100 μl 10 × ChIP Buffer and 70 μl 5 M NaCl in 830 μl water) once on rotation. Then, beads were eluted with 150 μl of ChIP elution buffer for 30 mins at 65 °C with gentle vortexing (1200 rpm) in thermomixer. Eluted supernatant was incubated with 6 μl 5 M NaCl and 2 μl Proteinase K at 65 °C for 2 h in thermomixer to digest the protein. DNA was purified using spin columns, and sent for ChIP-seq or ChIP-PCR analysis. For ChIP-PCR, ll ChIP signals were normalized to the input, and relative fold-change was compared with IgG controls. The primers of C/EBPC [T] = CT = Threshold cycle of PCR. With this method, signals obtained from each immunoprecipitation are expressed as a percent of the total input chromatin.http://www.broadinstitute.org/igv/).ChIP samples were quantified using a Qubit 2.0 Fluorometer  and qualified by Agilent Bioanalyzer 2100. Next-generation sequencing library preparations were constructed following the manufacturer’s protocol (NEBNext® Ultra™II DNA Library Prep Kit for Illumina®). The sequences were processed and analyzed by GENEWIZ. The ChIP-seq peaks were visualized using IGV , and data were expressed as mean ± standard error (SE). GraphPad Prism 6.0  was used to analyze the data. ANOVA and Student’s t test were used for significance analysis. KDM6B mRNA expression between esophageal cancer tissues and normal esophageal tissues, but the overall trend was upregulated  into KDM6B highly expressed cell lines KEYS150 and TE10 to knockdown KDM6B expression. Subsequently, stably transfected cell lines were screened by puromycin for 5 days after 2 days of infection. At the same time, We transfected HA-KDM6B overexpression plasmid for 3 days into KDM6B lowly expressed cell lines Eca9706 and TE11. We use Western blot and RT-PCR to determine the knockdown and overexpress efficiency. Respectively, compared with the sh-NC group, the mRNA and protein expressions of KDM6B were significantly inhibited in cells transfected with sh-KDM6B , and Eca9706 and TE11 transiently transfected with HA-KDM6B overexpression plasmid, the migration rate increased which compared with negative control(p < 0.05). The Transwell assay also revealed similar results when stable infection by the Lv-sh-KDM6B and transient transfected by the HA-KDM6B overexpression plasmid. Lv-sh-KDM6B interference reduced the KYSE150 and TE10 migration and invading cell number, and HA-KDM6B overexpression increased the Eca9706 and TE11 migration and invading cell number, which compared with the control group  of KYSE150 cells under KDM6B knockdown using sh-RNA has been performed. RNA-seq results showed that compared with the control group, after knocking down KDM6B, the number of down-regulated genes and up-regulated genes was 1371 and 542, respectively ,which is shown in the Integrative Genomics Viewer (IGV) software and validated by ChIP–qPCR . The results showed that KDM6B expression was significantly down-regulated Fig. E, and H3p < 0.05). These results suggested that GSK-J4 can weaken ESCC cells’ migration and invasion.As previously stated, knockdown of KDM6B inhibits ESCC cell migration and invasion. We next explore whether the use of GSK-J4 to inhibit KDM6B has the same effect on ESCC. Figure In addition to DNA methylation, the importance of histone methylation in the transcriptional regulation is increasingly appreciated , 32. H3KZEB1, ZEB2, SNAI and other metastasis-related genes [Li et al. found that KDM6B is overexpressed in ESCC and is associated with poor prognosis . We haveed genes . Our resed genes , 40. It ed genes . In receed genes . Besidesed genes . EZH2 ised genes . EZH2 anβ are key regulatory gene for the TNFA_SIGNALING_VIA_NFκB pathway. Yu et al. pointed out that genes such as JUNB, C/EBPβ, and CCNG2 are the target genes of KDM6B. Importantly, C/EBPβ is one of the major transcription factors that recruit KDM6B to the target gene promoter. Almost eliminated the occupancy rate of KDM6B on these target gene promoters [β by demethylating H3K27me3 and promotes the progress of ESCC. The above research enlightens us that some proteins play an important role in the process of KDM6B promoting tumor progression, so how these proteins interact with KDM6B is the focus of our further research.we found that TNFA_SIGNALING_VIA_NFκB, INTERFERON_ GAMMA_RESPONSE, P53, and IL6_JAK_STAT3_SIGNALING pathway were significantly suppressed in KDM6B knockdown KYSE150 as compared with Ctrl cells, and those signaling pathways were known to be closely associated with oncogenesis. KDM6B, JUNB, and C/EBPromoters . Our resAt present, individualized tumor treatment driven by biomarkers has attracted great attention. More and more studies have shown that KDM6B can be used as a biomarker and therapeutic target for tumor diagnosis and prognosis. Our research also found that the KDM6B inhibitor GSK-J4 can inhibit the proliferation and migration of ESCC cells. However, whether KDM6B plays a carcinogenic or anticancer role in certain tumors may depend on the specific tissue and cell type. Therefore, the use of KDM6B as a new treatment requires a broader and deeper understanding of its biological function and molecular mechanism.β depending on its H3K27 demethylase activity. KDM6B demethylase inhibitor, GSK-J4, acts to restore K27 methylation in ESCC cells, while demonstrating potent anti-tumor activity. Thus, we identify KDM6B as an oncogenic factor of ESCC and suggest KDM6B might be a potential molecular target for therapeutic approaches for ESCC.In conclusion, our results suggest that KDM6B was highly overexpressed in metastatic ESCC tissues, and KDM6B promotes ESCC progression by increasing the transcriptional activity of C/EBPAdditional file 1.Additional file 2.


Patients with metastatic prostate cancer (PC) are treated with androgen deprivation therapy (ADT) that initially reduces metastasis growth, but after some time lethal castration-resistant PC (CRPC) develops. A better understanding of the tumor biology in bone metastases is needed to guide further treatment developments. Subgroups of PC bone metastases based on transcriptome profiling have been previously identified by our research team, and specifically, heterogeneities related to androgen receptor (AR) activity have been described. Epigenetic alterations during PC progression remain elusive and this study aims to explore promoter gene methylation signatures in relation to gene expression and tumor AR activity.n = 12), and bone metastasis samples of separate patients with hormone-naive (n = 14), short-term castrated (n = 4) or CRPC (n = 52) disease were analyzed using the Infinium Methylation EPIC arrays, along with gene expression analysis by Illumina Bead Chip arrays (n = 90). AR activity was defined from expression levels of genes associated with canonical AR activity.Genome-wide promoter-associated CpG methylation signatures of a total of 94 tumor samples, including paired non-malignant and malignant primary tumor areas originating from radical prostatectomy samples (AR promoter accompanied with induction of AR mRNA levels. We identified a Methylation Classifier for Androgen receptor activity (MCA) signature, which separated metastases into two clusters (MCA positive/negative) related to tumor characteristics and patient prognosis. The MCA positive metastases showed low methylation levels of genes associated with canonical AR signaling and patients had a more favorable prognosis after ADT. In contrast, MCA negative patients had low AR activity associated with hypermethylation of AR-associated genes, and a worse prognosis after ADT.Integrated epigenome and transcriptome analysis identified pronounced hypermethylation in malignant compared to non-malignant areas of localized prostate tumors. Metastases showed an overall hypomethylation in relation to primary PC, including CpGs in the A promoter methylation signature classifies PC bone metastases into two groups and predicts tumor AR activity and patient prognosis after ADT. The explanation for the methylation diversities observed during PC progression and their biological and clinical relevance need further exploration.The online version contains supplementary material available at 10.1186/s13148-021-01119-0. Most patients with lethal PC develop bone metastases, which are treated with androgen deprivation therapy (ADT). Initially, ADT reduces metastasis growth, but eventually castration-resistant PC (CRPC) develops. Several treatment strategies for metastatic CRPC exist, with the majority aiming at inhibiting androgen receptor (AR) signaling ), capturing the largest variation in the data as a linear combination of the selected transcript levels. Similarly, tumor cell proliferation in metastasis samples was represented by the t[1] scores obtained from PCA of genes predefined to be associated with cell cycle proliferation [RAD54L, CDC20, CENPF, CDKN3, PBK, TOP2A, LCMT2, ASF1B, KIF20A, CDCA8, NUSAP1, PRC1, PLK1, CDCA3, CEP55, CDC2, KIF11, BUB1B, TK1, ASPM, PTTG1, ORC6L, FOXM1, RAD51, CENPM, CDAN1, KIAA0101, MCM10.The AR activity of a metastasis sample was defined by its relative expression levels of genes predefined to be associated with canonical AR activity; escribed . The AR feration ; RAD54L,n = 46), MetB (n = 12), and MetC (n = 8) by PCA and unsupervised clustering based on their transcription profiles, as previously described [The metastasis samples were classified into molecular metastasis subtypes MetA , (ii) inverse correlation between the methylation levels  of CpG sites (mCpGs) and the corresponding transcript levels, and iii) significantly different transcript levels (P < 0.05). In the analysis of promoter methylation levels in relation to AR activity, analysis was based on genes showing (i) positive or negative correlations between mCpGs  and AR activity score , (ii) a considerable difference between samples (stdev > 0.15), and (iii) inverse correlation (> 0.4) between mCpGs  and the corresponding transcript levels. Sets of genes associated with a functional pathway were determined as significantly enriched based on P values representing the probability for a process to arise by chance, considering the numbers of enriched gene products in the data versus the number of genes in the process. P values were adjusted by considering the rank of the process, given the total number of processes in the MetaCore ontology. Enriched pathways were expressed as pathway maps or as process networks, which were created by MetaCore on the basis of both pathway maps and gene ontology processes.Functional enrichment analysis was performed by the MetaCore software . In comparisons between different groups (T and N or M and T), analysis was based on genes showing (i) DM-CpGs  were controlled according to the Benjamini–Hochberg procedure. To evaluate the consistency of the clusters a silhouette analysis was performed using the cluster package  in R.β-values) of CpG sites (mCpGs) were analyzed in pairs of non-malignant (N) and malignant primary tumor (T) areas originating from radical prostatectomy samples of 12 PC patients. In parallel, the mCpG profiles were analyzed in 70 bone metastasis (M) samples of separate patients with hormone-naive , short-term castrated , or castrate-resistant disease   epithelial fraction in N, T, and M samples were 50 (30–50), 80 (70–80), and 70 (50–80)%, respectively P < 0.00, Fig. 1Bn = 442) of the DM genes   corresponding to CpG hypermethylation of the T samples Fig. C. Those n = 23) suggested altered apoptosis response in metastases compared to primary tumors  corresponded to hypomethylation of the M samples  or hypomethylation in combination with gene overexpression  in metastases , and the tetraspanin TSPAN18 showed clear overexpression in metastases, and may deserve further studies, while fold changes for the innate immunity regulator MEFV and the sodium/calcium exchanger SLC8B1 were less convincing. Of special notice was the hypomethylation of the AR gene in the M samples. Although gene amplification and overexpression of the AR in CRPC have been thoroughly described in the literature, less is known about its epigenetic regulation, and the AR was therefore chosen for further exploration.The fact that most of the DM-CpGs seen between M and T were also DM between M and N, suggests that the overlapping CpGs were likely organ-related rather than associated with bone metastasis Fig. C. FurtheAR mRNA overexpression observed in the M samples .To identify mCpGs related to the AR activity within PC metastases, and thus their potential AR dependence, we extracted data for all promoter CpG sites showing positive or negative correlation  to the sample AR activity score. This Methylation Classifier for Androgen receptor activity (MCA) signature consisted of 2970 unique CpGs and separated the M samples into two main clusters named MCA negative and MCA positive   Table . No signge Table .Table 3UThis study integrates genome-wide promoter methylation data with gene expression data and describes consistent changes occurring during PC disease progression from non-malignant prostate epithelium to primary PC and further to bone metastatic disease. In metastatic samples, the study also specifically explores if promoter methylation levels are related to the sample AR activity, defined from transcript levels of genes recently described to differentiate clinically relevant molecular subtypes of PC bone metastases based on AR activity , 10, 12.GSTP1, but also of other genes well known to be hypermethylated in PC, including AOX1, APC, BARHL2, CCDC8, CDKN2A, CYP27A1, EFS, GRASP, HOXA3, HOXC11, HOXD3, KIT, NXK2-1, NXK2-5, PHOX2A, POU3F3, PTGS2, RARB, RHCG, SIX6, TBX15, TMEM106A, WNT2, and ZNF154 [APC, HOXD3, and PTGS2 have previously been suggested to provide prognostic information [ACTA, CNN, DES, TAGLN, and TPN, normally expressed in smooth muscle cells [ANGPT1, STAT3, STAT5, TEK) and cytoskeletal  processes, as well as in regulation of proliferation  and cell adhesion . While reduced cell adhesion is an important hallmark for carcinomas, many of the other functional effects suggested from promoter hypermethylation in PC may be biased not only by the more abundant stroma in the T samples, but also by its different cellular content. While the stroma in the normal prostate is mainly composed of smooth muscle cells, prostate tumors show a progressive loss of smooth muscle cells in the favor of cancer-associated fibroblasts [By pairwise comparisons of 12 tumor areas and adjacent non-malignant tissue isolated from radical prostatectomies, a pronounced hypermethylation was observed in primary PC. This is in line with a wealth of data, summarized in , suggestormation . The lowle cells , but alsroblasts . HypometAR promoter region. A detailed exploration showed a trend of demethylation at several CpGs in the AR promoter, including two alternative transcription start sites that correlated well with induced levels of both AR transcript 1 and 2 in the metastatic samples. High AR mRNA levels in CRPC have been previously associated with AR amplification [AR expression is less well described and studies have also shown inconsistent results [AR promoter hypomethylation in CRPC [AR demethylation may be an earlier cause to AR induction in PC metastases than AR amplification, as AR amplification is generally not observed prior to ADT. Importantly, neither the large variance in AR promoter methylation levels observed among the metastatic samples nor their AR amplification status showed any clear relationship to the tumor sample AR activity.Most of the identified DM-CpGs observed between primary PC and non-malignant prostate tissue were present also in the metastatic tissue. In addition, a unique set of DM-CpGs were observed when going from primary PC to metastases. The large majority of these were hypomethylated and included sites in the fication –36, whil results , 37–40.  in CRPC , but alsSLC45A3, STEAP2, ELK4 and TMPRSS2) may contribute to the AR-driven tumor phenotype seen in the majority of CRPC patients with metastatic disease [n = 63, data not shown).In an attempt to study universal promoter methylation in relation to AR activity within individual metastasis samples, each sample was given an AR activity score related to its expression levels of genes involved in or regulated by canonical AR signaling, and mCpGs positively or negatively correlated to this score were taken further for functional exploration. A Methylation Classifier for Androgen receptor activity (MCA) signature was identified and used to classify the bone metastasis samples. Taken together, our results suggested that low promoter methylation levels of certain AR-regulated genes . The next step will be to verify the suggested therapy-predictive value of the MCA signature. This could be retrospectively done by analyzing methylation signatures in circulating tumor DNA collected from patients prior to treatment for metastatic PC, similarly to what is described in a recent paper by Wu et al. [In conclusion, this study describes general patterns of gene promoter methylation during PC disease progression, and specifically identifies a novel promoter MCA signature that is associated with canonical AR activity in PC bone metastases and to patient prognosis after ADT. Based on this methylation signature, patients with PC bone metastases could be stratified into one of two patient groups; MCA positive patients being likely to respond to ADT and possibly also other AR targeting therapies or MCA negative patients being less responsive to AR targeting therapies, but possibly suitable for treatment with AR inhibitors in combination with epigenetic modulators .Additional file 2: Fig. S2. Schematic flowchart of the pre-processing steps of the HumanMethylation EPIC arrays.Additional file 3: Fig. S3. Silhouette analysis showing the cluster consistencies of the MCA positive and negative clusters in Figure 4A.Additional file 4:Table S1. Differetially methylated CpGs in promoter regions of genes in primary prostate tumor tissue (T) compared to adjacent non-malignant prostate tissue (N), their correlations to corresponding gene expression levels, and the fold change in gene expression levels between sample groups.Additional file 5:  Table S2. Top 10 enriched process networks in localized prostate tumor tissue (T) compared to adjacent non-malignant prostate tissue (N) or in prostate cancer bone metastases (M) compared to localized tumors.Additional file 6:Table S3. Differetially methylated CpGs in promoter regions of genes in bone metastases (M) compared to localized prostate tumors (T), their correlations to corresponding gene expression levels, and the fold change in gene expression levels between sample groups.Additional file 7:Table S4. CpG sites of the AR gene shown in Figure 3A.


The main findings were that stress induces changes in gene expression in the heart and that β2-AR plays a role in this process. The vast majority of genes disregulated by stress were exclusive for only one of the comparisons, indicating that, in the same stressful situation, the profile of gene expression in the heart is substantially different when the β2-AR is active or when it is blocked. Stress induced alterations in the expression of such a large number of genes seems to be part of stress-induced adaptive mechanism.The stress response is adaptive and aims to guarantee survival. However, the persistence of a stressor can culminate in pathology. Catecholamines released as part of the stress response over activate beta adrenoceptors (β-AR) in the heart. Whether and how stress affects the expression of components of the intracellular environment in the heart is still, however, unknown. This paper used microarray to analyze the gene expression in the left ventricle wall of rats submitted to foot shock stress, treated or not treated with the selective β Catecholamines and glucocorticoids, the hallmarks of the stress response, play fundamental roles as physiological regulators in an attempt to maintain homeostasis and adapt to a new condition. The persistence of the stressful situation may even culminate in increased susceptibility to certain diseases. High levels of catecholamines overstimulate adrenoceptors in the cell membrane of almost every organ, including the β-adrenoceptors (β-AR) in the cardiomyocytes1-AR over β2-AR in a proportion of 80:204. Both, β1- and β2-AR couple to G stimulatory (Gs) protein, which stimulates adenylyl cyclase (AC) to convert ATP in 3′–5′-cyclic adenosine monophosphate (cAMP), which then activates protein kinase A (PKA)1. Targets of PKA include L-type calcium channels, T troponin and phospholamban4. The phosphorylation of these proteins contributes an increase in the beating rate, development of tension, and the velocity of relaxation of the cardiomyocytes, thus increasing cardiac output. β2-AR may also couple to G inhibitory (Gi) protein, with effects opposite to those of Gs stimulation. β2-AR-Gi also activates the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, which controls the life and death of cardiomyocytes5.In the human and rodent heart, the main β-AR subtype expressed is β13. Such alterations can also trigger the onset of diseases, such as atherosclerosis, coronary diseases14, hypertension, and heart failure12. Our research group has invested substantial efforts in the investigation of the effects of stress on cardiac reactivity to β-AR stimulation in animal models of stress, mainly the foot shock stress model13. We have reported that isolated atria of rats submitted to foot shock stress show altered sensitivity to the chronotropic and inotropic effects of catecholamines, which is associated with a remodeling of the proportion of β-AR subtypes in the heart. Isolated atria were subsensitive to β1-AR agonists and supersensitive to both β2-AR selective agonists and isoprenaline, a non-selective β-AR agonist. These effects of stress were cancelled in the presence of selective β2-AR antagonists or non-selective β-AR antagonists, suggesting an increase in the β2-AR subtype in the cardiac tissue16. A Western blot approach confirmed the higher expression of β2-AR accompanied by a low expression of β1-AR in the atrium and ventricle of rats submitted to foot shock stress when compared to control non-stressed rats13. A reduced cardiac β1/β2-AR ratio is also involved in other circumstances, such as aging and heart failure. It is considered adaptive since it protects the heart from the cardiotoxic effects of persistent β1-AR overstimulation18.Several studies have already shown the effects of stress in triggering alterations in the cardiovascular system1-AR stimulation, the possible cardioprotective role of prolonged β2-AR activation is still controversial. After the receptor couples with Gi, the Giα subunit inhibits AC, attenuating the signal mediated by β1-AR, while the Giβγ activates several intracellular signals, including PI3K-Akt20. Despite the beneficial effect on the viability of cardiac cells, β2-AR stimulation compromises contractility. Recently, we have demonstrated that the activity of PI3K-Akt is reduced in the heart of foot shock stressed rats even 5 days after the last stress session21. The persistence of this effect indicates the relevance of the intracellular processes triggered by stress in the heart. Moreover, the increase in β2-AR participation in the cardiac stress response and heart failure suggests an important role for β2-AR signaling in cardiac events. However, the complex biological processes contributing to the cardiac response to stress are not yet fully understood.In contrast to the recognized cardiotoxic effect of persistent β2-AR and in the absence of β2-AR signaling due to the pharmacological blockade of the receptor. Our results provide insight into the role played by β2-AR in the differential regulation of gene expression during stress.To contribute to our understanding of the influence of stress on cardiac physiology, we have proposed an evaluation of the gene expression profile in the cardiac tissue of rats submitted to foot shock stress. We have also compared the gene expression profile in the presence of stress-induced upregulation of β2-AR blockade with ICI118,551 did not interfere with this endocrine stress response .In order to identify the genes of which expression was altered in the heart of rats submitted to stress, samples were arranged for microarray so that untreated rats with and without stress were compared. Since β2-AR blockade resulted in the significant disregulation of 114 genes under stress, with 90 being upregulated and 14 downregulated. The functional analysis of the genes with differential expression was performed using IPA software. Interestingly, many non-annotated genes were disregulated in both comparisons, but mainly those in the comparison of the untreated groups , the greatest upregulation was found for the genes coding heat shock proteins (HSPA1A/HSPA1B and HSP90AA1), insulin receptor substrate 2 (IRS2) and ATF3 (activating transcription factor 3), whereas SEZ6L (seizure related 6 homolog like), CCL2 (C–C motif chemokine ligand 2) and APLNR (apelin receptor) underwent downregulation (Table 2-AR: cAMP responsive element modulator (CREM), ERBB receptor feedback inhibitor 1 (ERRFI1), and heat shock protein 90 alpha family class A member (HSP90AA1). The vast majority of disregulated genes were specific for one of the conditions, with 166 annotated genes being disregulated in cardiac tissue when β2-AR was active (ICI-untreated), and 76 when β2-AR was blocked (ICI-treated).There was minimal overlap in the genes disregulated by stress in the two comparisons, as can be seen in the Venn diagram Fig. . Three gAn analysis of the disregulated genes suggests that certain categories of diseases and functions may be triggered or altered in the cardiac tissue of rats submitted to stress  signaling in the context of overstimulation. Moreover, many other genes involved in that same signaling pathway had their expression disregulated under stress, whether or not treated with ICI118,551. RGS1 and RGS16 were upregulated in the untreated groups’ comparison. The RGS family acts as a negative regulator of G protein signaling. By controlling heterotrimeric G proteins they may regulate myocardial hypertrophy and contractility26. RGS1 has been related to the control of inflammation and the immune response28. The stress-induced alteration in the expression of these genes depends on an active β2-AR, since it is unaltered in the group treated with ICI118,551. On the other hand, CREB1 and GPER1 belong to the group of molecules predicted to be upstream regulators activated by stress, independent of β2-AR activation. CREB1, a transcription factor responsive to cAMP, leads to effects similar to CREM24, while GPER1 belongs to the G-protein coupled receptor family that binds estrogen. This receptor activates both the adenylate cyclase-cAMP/PKA signaling pathway 30 and the PI3K-Akt-mTOR signaling pathway31. These two signaling pathways are related to cardioprotection and cell survival20.β-AR activation by catecholamines is the main signal for cAMP generation in cardiac myocytes. The cAMP-dependent transcription factor, CREM, expressed in the myocardium, is involved in the regulation of the expression of various components of the cAMP signaling pathway33. Apelin not only increases inotropy, but also decreases left ventricular pre- and afterload due to its pronounced vasodilation effect34. The inotropic action of apelin is the result of an increase in the availability of intracellular calcium35. It also induces cAMP synthesis, as well as activating the PI3K-Akt signaling pathway33. The concentration of apelin is reduced in the failing heart36 and the contractile function is impaired in cardiomyocytes with knockout for APLNR37. Apelin expression was downregulated under stress when no ICI118,551 treatment was given, although the expression of its receptor (APLNR) was reduced when the β2-AR was blocked.The APLNR gene, also called the APJ receptor gene, encodes the G-protein coupled receptor for apelin, a protein expressed in the cardiovascular system which promotes a positive inotropic effect on the heart as well as angiogenesis and blood vessel relaxation1-AR and apelin seems to be counterbalanced by the upregulation of β2-AR and the action of CREM. Hence, although the proportion of β-AR subtypes is altered, there is no difference in cAMP formation by left atrial membranes of control and foot shock stressed rats stimulated by non-selective agonists13. However, when the membranes are stimulated in the presence of the β2-AR antagonist , the amount of cAMP synthetized by the atrial membranes of stressed rats is lower than that of unstressed ones13. This is probably because the increase in the expression of CREM is not sufficient to sustain the synthesis of cAMP, since β1-AR is downregulated, as has been reported elsewhere13.Therefore, in the heart of untreated stressed rats, the downregulation of β2-AR blockade, the canonical pathway of cAMP signaling is negatively regulated  and catecholamines are known as the stress hormones. The glucocorticoid receptor (GR) is located in the cytoplasm of target cells, bound to a chaperone complex and immunophilins that provide structural stability and function41 by regulating protein folding, proteostasis and intracellular signal transduction. HSP90 and HSP70 are inducible isoforms that present incremented expression after myocardium injury, oxidative stress, and hypoxia. HSP90aa1, the mRNA for HSP90, was upregulated under stress, both with and without β2-AR participation, while HSPa1a, the mRNA for HSP70, was upregulated by stress only under β2-AR blockade. Upregulation of HSP90 and HSP70 has been linked to cardioprotection against injury43. The in vitro overexpression of HSP90aa1 in cardiomyocytes attenuates apoptosis by increasing Bcl-2 expression43. HSP90 is also associated with the survival pathway of PI3K-Akt. The association of HSP90 with Akt leads to phosphorylation and the activation of endothelial nitric oxide synthase42. Under stress, in the presence of β2-AR upregulation, the reduction of fibrosis and apoptosis that was indicated by disease and function categorization, suggests that the modulation of the expression of genes is part of the adaptive mechanisms to stress.The heat shock proteins (HSP) 90 and 70, as well as the proto-oncogene tyrosine-protein kinase (Src), released from the GR complex, also influence cell signaling45. The results presented here have demonstrated that in rats submitted to stress as compared to non-stressed rats, the circulating levels of corticosterone, the predominant GC in rodents, were higher and the ERRFI1 gene expression was upregulated, whether β2-AR is upregulated or blocked. Therefore, a reduction of Akt phosphorylation is expected. Accordingly, a reduced Akt phosphorylation has been reported in the ventricles of rats submitted to the same stress protocol used here21.The activation of GR signaling is also related to the induction of the expression of the gene ERRFI1. This gene encodes a feedback inhibitor of the epidermal growth factor receptor (EGFR) what reduces cardiac hypertrophy2-AR. The changes induced by stress in the β-AR-Gs-AC, GC-GR, and PI3K-Akt signaling pathways are summarized in Fig. 2-AR blockade: ERRFI1, CREM, HSP90aa1, and HSPA1A/A1B are upregulated whereas APLNR is downregulated.The present data have thus suggested that two signaling pathways are the most affected by stress: the β-AR-Gs-AC-cAMP and the PI3K-Akt signaling pathways. Moreover, the modulation of several of their components depends on the presence of the β2-AR, since it appears in the comparisons of both non-ICI treated and ICI treated rats.The changes post stress in cardiac gene regulation also include the progression of the cell cycle, which can be impaired by downregulation of CDK1, and the presence of epigenetic factors, such as the upregulation of the class I histone deacetylase, HDAC8, and, in ICI-treated rats, stress induced upregulation of HSP40, and IRS2. The upregulation of IRS2 suggests that insulin may be an upstream regulator of the stress response, independent of β2-AR. Immunological activation was identified in microarray data in expressed genes, canonical pathways and upstream regulators. The correlation of immunological response with cardiac injury and repair is widely recognized in the failing heart48 and in human coronary heart disease51.An unexpected finding was the recruitment of the immune system to the cardiac tissue of stressed rats, with the profile clearly different for the two groups, as a function of β53. IL-6 was also upregulated in this group. This is a pro-inflammatory cytokine produced by myeloid cells and cardiomyocytes in an autocrine mechanism54, which has been related to cardiac disease and the impairment of cardiac performance51.Among the differently expressed genes in the comparison of non-ICI treated group, IL22RA2 , IL2RA (interleukin 2 receptor antagonist) and IFRD1  were upregulated and are described as regulators of the inflammatory response2-AR. Both are related to acute inflammation in cardiac disease, such as heart failure and myocardial infarction54. IL-1β, TNF-α, and IL-12 are cytokines released by T helper 1 lymphocytes (Th1), which control the cellular immune response. Interestingly, immune cells, including Th1 lymphocytes, express β2-AR and are, therefore, susceptible to catecholamines action55. In immune cells, cAMP-PKA signaling inhibits the transcription of nuclear factor kappa B (NF-κB); through CREB, it activates the transcription of IL-10. This mechanism promotes the differentiation of the Th2 response, which is markedly anti-inflammatory, and also inhibits the development of the Th1 response, which is markedly pro-inflammatory56. The chronic use of β2-AR-selective and nonselective blockers in mice impairs the recruitment of leukocytes to the injured heart and reduces survival57.It has been predicted that the pro-inflammatory cytokines IL-1β and TNF act as upstream regulators of the cardiac stress response in the presence of β2-AR, as previously reported by Moura et al.13, a pro-inflammatory signaling is triggered in the heart of stressed rats. If the upregulation of β2-AR occurs only in the cardiomyocytes or in the resident immune cells as well is, however, unknown at the present time. Indeed, data for the comparison of stressed ICI-treated rats with non-stressed ICI-treated rats did not involve pro-inflammatory genes, which suggests that the treatment with ICI188,551 reduced the development of the Th1 response by blocking β2-AR in the immune cells as well. Several canonical pathways listed in that analysis are related to the immune response, such as the signaling pathway of IL-10, IL-17, CD40, CD27 and IL-6, the acute phase response, and GR signaling pathway. One interesting characteristic of these canonical pathways is the expression of the same gene, NFKBIA, at times in conjunction with MAP2K3 and/or JUN. NFKBIA encodes a potent inhibitor of NF-κB58, which mediates activation of the inflammatory response with important consequences in heart disease. The overexpression of NFKBIA in cardiomyocytes inhibits NF-κB activity, reduces hypertrophy and improves cardiac performance and survival via the Akt signaling pathway59. The presence of NFKBIA in the left ventricle suppresses the expression of NF-κB and attenuates myocardial fibrosis60. Therefore, the expression of NFKBIA mRNA in ICI-treated stressed rats could indicate the presence of a regulatory or anti-inflammatory mechanism in the ventricle of rats submitted to stress when faced with β2-AR blockage.The data reported here thus suggest that in the presence of greater expression of β2-AR clearly plays a role in this process, since the alterations in gene expression that occurred in the presence of this adrenoceptor subtype are completely different from those seen when it is blocked. However, it might be considered that there are many processes between the gene expression and the respective protein synthesis and activity. Indeed, one of the major challenges in all experimental systems is the issue of causality and association. Thus, in order to confirm if the altered gene expression here demonstrated has functional consequences, additional experiments are required. The lack of functional experiments and protein validation is a limitation of this work. Despite that, the great number of significant data presented here suggests that might be an influence of stress in the cardiac cells phenotype, and probably in the heart function.The data has shown that stress induced alterations in the expression of such a large number of genes what seems to be part of adaptive mechanisms. βRattus norvegicus; 24 animals; 250–350 g; 12 weeks-old), obtained from the Center for the Development of Experimental Models (CEDEME), of the Federal University of São Paulo , were housed in standard cages in a temperature-controlled room (22 °C) on a 12:12 h light:dark cycle, with the lights on at 7:00 am. Standard laboratory chow and tap water were available ad libitum. The rats were randomly distributed in two groups as follows: untreated and treated with ICI118,551 (ICI), with two subgroups each: submitted to stress and not submitted to stress. The experimental protocols were approved by the Ethics Committee for Animal Use of the Federal University of São Paulo (CEUA/UNIFESP), protocol number 86613101116, in accordance with the Brazilian National Council for Control of Animal Experimentation , and all study carried out in compliance with the ARRIVE guidelines.Male Wistar rats  were submitted to foot shock sessions; rats in the non-stressed groups  were also placed in the foot shock cage, but did not receive foot shocks. The stress cage was a Plexiglas chamber (26 × 21 × 26 cm) provided with a floor grid made of stainless-steel rods . During the daily 30 min stress sessions, which occurred between 7:30 am and 11:00 am on three consecutive days, foot shocks were delivered by a constant current source controlled by a microprocessor-based instrument. The intensity of the current was 1.0 mA, with duration of 1.0 s, with pulses delivered at random intervals of between 5 and 25 s. The rats were returned to their standard cages after the first and second period in the Plexigas chamber. After the third session, the rats were immediately euthanized by decapitation, in compliance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). The trunk blood was collected in a tube containing EDTA and centrifuged. The plasma was separated and stored at – 80 °C. The hearts were harvested and the left ventricles were isolated and stored at − 80 °C.The foot shock stress protocol was administered as previously described2-AR antagonist63. The rats in the untreated non-stressed control group and those submitted to stress that were not treated with ICI received injections of saline solution, i.p. The foot shock stress sessions began on the third day of the treatment with ICI or saline solution.For 5 days, the rats in the ICI-treated groups, stressed or not, received 500 µg/kg/day, i.p, of ICI118,551 ((±)-1--3-[1-metilletil) amino]-2-butanol; Tocris Bioscience, Bristol, UK), a highly selective βThe plasmatic concentration of corticosterone was determined in all experimental groups (6 rats/group) by enzyme immunoassay (ELISA) using a commercial kit  according to the manufacturer's guidelines.Total RNA was extracted from fragments of up to 100 mg from the left ventricle from all experimental groups (4 rats/group) using TRIzol reagent according to the manufacturer’s instructions . The final volume of the samples was 100 µL of water treated with 0.1% dietilpyrocarbonate ; these were stored at − 80 °C overnight. The mRNA concentration and degree of purity were determined in Nanodrop 2000 c  under 260/280 nm. The RNA was purified using an RNeasy Kit  according to the manufacturer's guidelines. The purified RNA concentration and degree of purity were determined in Nanodrop 2000 c.The gene expression profile was evaluated for the total RNA isolated from the left ventricles of the rats in each of the experimental groups. The two-color microarray-based gene expression analysis was Agilent-074036 SurePrint G3 Rat GE v2 8 × 60 K Microarray G4858A (GEO GPL22145) from Agilent Technologies . There were a total of four samples for each group. Each sample was analyzed once. The analysis was performed at the Center of Excellence—Genomics .Firstly, the total RNA samples were assayed using RNA ScreenTape Analysis in a TapeStation System  to determine RNA quality and concentration (ng/μL). 200 ng of total RNA were used for individual reactions using oligo-dt linked to T7 promoter primer, being the Spike A Mix/Cyanine 3-CTP dye for the non-stressed samples and Spike B Mix/Cyanine 5-CTP dye for the stressed samples. The transcription of cDNA was performed using cDNA Master Mix for 2 h at 40 °C, followed by 15 min at 70 °C. The cDNA served as a template for the next transcription reaction. For the in vitro transcription of cRNA, Cyanine 3-CTP (for the non-stressed samples) and Cyanine 5-CTP (for the stressed samples) were incorporated, using T7 RNA polymerase for 2 h at 40 °C. After this, the samples were stored at − 80 °C. On the following day, the amplified and labeled cRNA was purified using an RNeasy kit, according to the manufacturer's guidelines, and the concentration was determined by RNA ScreenTape Analysis in a TapeStation System. For each microarray, 300 ng portions of each labeled cRNA sample were assembled in pairs of Cyanine 3-CTP (for non-stressed samples) and Cyanine 5-CTP (for stressed samples) incorporation as follows: samples of non-stressed untreated with stressed untreated groups, and samples of non-stressed ICI treated with stressed ICI treated groups. Then they were fragmented for 30 min at 60 °C. The hybridization assembly was prepared according to the Agilent microarray hybridization chamber user guide. The hybridization was performed in the Agilent Microarray Hybridization Quick Assemble Chamber for 17 h at 65 °C at a rotation speed of 10 rpm. The microarray slide was washed with gene expression wash buffers provided by Agilent Technologies, followed by an acetonitrile wash to avoid sediment and dry the slide. Agilent SureScan was used to scan the microarray slide.Data were extracted using Agilent Feature Extraction software. The fluorescence intensity for each spot was corrected by subtracting the background using the standard Feature Extraction algorithm. Then, data was normalized using Lowess algorithm, which is applied for two-color data to compensate dye bias incorporation. The metrics parameters were considered adequate. GeneSpring GX software  was used for differential gene expression analysis. Significant changes were defined using t test against zero on the basis of fold change (≥ 2) and p value (≤ 0.05) cut offs. The log ratio (log two fold change) was taken. The functional significance of the genes with differential expression was determined in Ingenuity Pathway Analysis software  by right-tailed Fisher’s exact test with p value of overlap ≤ 0.05. The z score (≥ 2 or ≤ − 2) was considered.Supplementary Information 1.