Egr-1 Levels Research Articles

Transcriptome meta-analysis and validation to discovery of hub genes and pathways in focal and segmental glomerulosclerosis

BackgroundFocal segmental glomerulosclerosis (FSGS), a histologic pattern of injury in the glomerulus, is one of the leading glomerular causes of end-stage renal disease (ESRD) worldwide. Despite extensive research, the underlying biological alterations causing FSGS remain poorly understood. Studying variations in gene expression profiles offers a promising approach to gaining a comprehensive understanding of FSGS molecular pathogenicity and identifying key elements as potential therapeutic targets. This work is a meta-analysis of gene expression profiles from glomerular samples of FSGS patients. The main aims of this study are to establish a consensus list of differentially expressed genes in FSGS, validate these findings, understand the disease’s pathogenicity, and identify novel therapeutic targets.MethodsAfter a thorough search in the GEO database and subsequent quality control assessments, seven gene expression datasets were selected for the meta-analysis: GSE47183 (GPL14663), GSE47183 (GPL11670), GSE99340, GSE108109, GSE121233, GSE129973, and GSE104948. The random effect size method was applied to identify differentially expressed genes (meta-DEGs), which were then used to construct a regulatory network (STRING, MiRTarBase, and TRRUST) and perform various pathway enrichment analyses. The expression levels of several meta-DEGs, specifically ADAMTS1, PF4, EGR1, and EGF, known as angiogenesis regulators, were analyzed using quantitative reverse transcription polymerase chain reaction (RT-qPCR).ResultsThe identified 2,898 meta-DEGs, including 665 downregulated and 669 upregulated genes, were subjected to various analyses. A co-regulatory network comprising 2,859 DEGs, 2,688 microRNAs (miRNAs), and 374 transcription factors (TFs) was constructed, and the top molecules in the network were identified based on degree centrality. Part of the pathway enrichment analysis revealed significant disruption in the angiogenesis regulatory pathways in the FSGS kidney. The RT-qPCR results confirmed an imbalance in angiogenesis pathways by demonstrating the differential expression levels of ADAMTS1 and EGR1, two key angiogenesis regulators, in the FSGS condition.ConclusionIn addition to presenting a consensus list of differentially expressed genes in FSGS, this meta-analysis identified significant distortions in angiogenesis-related pathways and factors in the FSGS kidney. Targeting these factors may offer a viable strategy to impede the progression of FSGS.

Effects of psilocybin, psychedelic mushroom extract and 5-hydroxytryptophan on brain immediate early gene expression: Interaction with serotonergic receptor modulators.

Background: Immediate early genes (IEGs) are rapidly activated and initiate diverse cellular processes including neuroplasticity. We report the effect of psilocybin (PSIL), PSIL-containing psychedelic mushroom extract (PME) and 5-hydroxytryptophan (5-HTP) on expression of the IEGs, cfos, egr1, and egr2 in mouse somatosensory cortex (SSC). Methods: In our initial experiment, male C57Bl/6j mice were injected with PSIL 4.4mg/kg or 5-HTP 200mg/kg, alone or immediately preceded by serotonergic receptor modulators. IEG mRNA expression 1hour later was determined by real time qPCR. In a replication study a group of mice treated with PME was added. Results: In our initial experiment, PSIL but not 5-HTP significantly increased expression of all three IEGs. No correlation was observed between the head twitch response (HTR) induced by PSIL and its effect on the IEGs. The serotonergic receptor modulators did not significantly alter PSIL-induced IEG expression, with the exception of the 5-HT2C antagonist (RS102221), which significantly enhanced PSIL-induced egr2 expression. 5-HTP did not affect IEG expression. In our replication experiment, PSIL and PME upregulated levels of egr1 and cfos while the upregulation of egr2 was not significant. Conclusions: We have shown that PSIL and PME but not 5-HTP (at a dose sufficient to induce HTR), induced a significant increase in cfos and egr1 expression in mouse SSC. Our findings suggest that egr1 and cfos expression may be associated with psychedelic effects.

ATF3/EGR1 regulates myocardial ischemia/reperfusion injury induced autophagy and inflammation in cardiomyocytes.

Myocardial ischemia/reperfusion injury (MIRI) is an irreversible adverse event during the management of coronary heart disease that lacks effective controls. The underlying mechanism of MIRI still requires further investigation. Recent studies have suggested that overexpression of ATF3 protects against MIRI by regulating inflammatory responses, ferroptosis, and autophagy. The downstream target of ATF3, EGR1, also showed cardioprotective properties against MIRI by promoting autophagy. Therefore, further investigating the effect of ATF3/EGR1 pathway on MIRI-induced inflammation and autophagy is needed. Cardiomyocyte MIRI model was established by challenging H9C2 cells with hypoxia/reoxygenation (H/R). The ATF3 overexpression-H/R cell model by transfecting ATF3 plasmid into the H9C2 cell line. The transcription levels of ATF3 and EGR1 were determined using RT-qPCR, the levels of TNF-α and IL-6 were determined using ELISA kits, the protein expression of LC3 I, LC3 II, and P62 was determined via WB, and microstructure of H9C2 cell was observed by transmission electron microscopy (TEM). Overexpression of ATF3 significantly downregulated Egr1 levels, indicating that EGR1 might be the target of ATF3. By upregulating ATF3 levels, the extracellular levels of the inflammatory cytokines TNF-α and IL-6 significantly decreased, and the protein expression of the autophagy markers LC3 I, LC3 II, and P62 significantly increased. TEM results revealed that the cell line in the H/R-ATF3 group exhibited a higher abundance of autophagosome enclosures of mitochondria. The results indicated that ATF3/EGR1 may alleviate inflammation and improve autophagy in an H/R-induced MIRI model of cardiomyocytes.

Decreased FGF23 inhibits placental angiogenesis via the ERK1/2-EGR-1 signaling pathway in preeclampsia

PurposeThis study aimed to investigate the expression of fibroblast growth factor 23 (FGF23) in pregnant women with preeclampsia and elucidate its role in promoting placental angiogenesis through the ERK1/2-EGR-1 signaling pathway. MethodsSerum FGF23 levels were measured by ELISA in healthy pregnant women and patients with preeclampsia during the first, second, and third trimesters of pregnancy. Wound healing, Transwell, and tube formation assays were performed to investigate the effects of FGF23 on cell migration, invasion and tube formation. The expression of vascular endothelial growth factor A (VEGF-A) and its upstream signaling molecules, p-ERK, and EGR-1, in placental tissues was detected by RT-qPCR and western blotting. Additionally, the effect of FGF23 on VEGF-A, p-ERK, and EGR-1 expression was further explored in vitro. ResultsSerum FGF23 levels increased with gestational age. During the third trimester, the control group exhibited a more pronounced increase in FGF23 levels than the preeclampsia group. Administering exogenous FGF23 promoted trophoblast cell migration, invasion and enhanced tube formation in vascular endothelial cells. The expression levels of VEGF-A, p-ERK, and EGR-1 in the placental tissues were significantly lower in the preeclampsia group than in the control group. In vitro experiments confirmed that FGF23 up-regulated VEGF-A expression through the p-ERK/EGR-1 signaling pathway. ConclusionThe serum level of FGF23 decreased in pregnant women with preeclampsia, inhibiting the ERK1/2-EGR-1 pathway and resulting in decreased expression of VEGF-A, thereby inhibiting placental angiogenesis. This could be a potential mechanism involved in the progression of preeclampsia.

Ladostigil Reduces the Adenoside Triphosphate/Lipopolysaccharide-Induced Secretion of Pro-Inflammatory Cytokines from Microglia and Modulate-Immune Regulators, TNFAIP3, and EGR1.

Treatment of aging rats for 6 months with ladostigil (1 mg/kg/day) prevented a decline in recognition and spatial memory and suppressed the overexpression of gene-encoding pro-inflammatory cytokines, TNFα, IL1β, and IL6 in the brain and microglial cultures. Primary cultures of mouse microglia stimulated by lipopolysaccharides (LPS, 0.75 µg/mL) and benzoyl ATPs (BzATP) were used to determine the concentration of ladostigil that reduces the secretion of these cytokine proteins. Ladostigil (1 × 10-11 M), a concentration compatible with the blood of aging rats in, prevented memory decline and reduced secretion of IL1β and IL6 by ≈50%. RNA sequencing analysis showed that BzATP/LPS upregulated 25 genes, including early-growth response protein 1, (Egr1) which increased in the brain of subjects with neurodegenerative diseases. Ladostigil significantly decreased Egr1 gene expression and levels of the protein in the nucleus and increased TNF alpha-induced protein 3 (TNFaIP3), which suppresses cytokine release, in the microglial cytoplasm. Restoration of the aberrant signaling of these proteins in ATP/LPS-activated microglia in vivo might explain the prevention by ladostigil of the morphological and inflammatory changes in the brain of aging rats.

Investigation of Early Growth Response Protein (EGR) Levels in Patients with Fibromyalgia Syndrome: A Cross-Sectional Study.

Fibromyalgia is a soft tissue rheumatism characterized by chronic and widespread musculoskeletal pain at specific points in the body. In this study, we aimed to investigate the relationship between Early Growth Response (EGR1, EGR2, and EGR3) protein levels in patients with Fibromyalgia Syndrome (FMS) and healthy controls. In our studies, 76 FMS patient group and 78 healthy control group who were newly diagnosed with primary FMS according to the 2010 American College of Rheumatology criteria for fibromyalgia in Sivas Cumhuriyet University Hospital, Physical Therapy, and Rehabilitation were used. Venous blood samples were taken from both groups for the measurement of EGR1, EGR2, and EGR3 protein plasma levels, and protein levels were determined using ELISA methods. Statistical parametric test assumptions were compared using the Independent Student's t-test. In addition, specificity, sensitivity, and AUC values were calculated with the ROC curve. The relationship between plasma EGR1 protein levels of FMS patients and control groups was statistically significant (p=0.001). EGR1 protein levels were found to be lower in the patient group diagnosed with FMS compared to the control group. It has been suggested that EGR1 protein levels can be important in the diagnosis of FMS disease.

Inducible Keratinocyte Specific FGFR2 Deficiency Inhibits UVB-Induced Signaling, Proliferation, Inflammation, and Skin Carcinogenesis

A potential role for fibroblast growth factor receptor 2 (FGFR2) in cutaneous squamous cell carcinoma (cSCC) has been reported. To demonstrate the specific role of FGFR2 in UVB-induced skin carcinogenesis and development of cSCC, we generated a keratinocyte specific, tamoxifen inducible mouse model of FGFR2 deficiency. In this mouse model, topical application of 4-hydroxy tamoxifen led to the induction of Cre recombinase to delete FGFR2 in epidermal keratinocytes of both male and female transgenic mice. Analysis of epidermal protein lysates isolated from FGFR2 deficient mice exposed to UVB showed significant reductions of phospho-FGFR (pFGFR; Y653/654) and phospho-fibroblast growth factor receptor substrate 2α as well as downstream effectors of mTORC1 signaling. Phosphorylation of signal transducer and activators of transcription 1/3 was significantly reduced as well as levels of IRF-1, DUSP6, early growth response 1, and PD-L1 compared to the control groups. Keratinocyte-specific ablation of FGFR2 also significantly inhibited epidermal hyperproliferation, hyperplasia, and inflammation after exposure to UVB. Finally, keratinocyte-specific deletion of FGFR2 significantly inhibited UVB-induced cSCC formation. Collectively, the current data demonstrate an important role of FGFR2 in UVB-induced oncogenic signaling as well as development of cSCC. In addition, the current preclinical findings suggest that inhibition of FGFR2 signaling may provide a previously unreported strategy to prevent and/or treat UVB-induced cSCC.

Time-course transcriptome analysis of lungs from mice infected with inhaled aerosolized Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia (SMA) has emerged as an important pathogen capable of causing an opportunistic and nosocomial infection. We performed RNA sequencing (RNA-seq) of lung tissues from mice with pulmonary SMA infection over time via aerosolized intratracheal inhalation to investigate transcription profile changes in SMA-infected lungs. A mouse model of acute lethal SMA pneumonia was established in this study using aerosolized intratracheal inhalation, laying the groundwork for future SMA research. RNA-seq was then used to create a transcriptional profile of the lungs of the model mice at 0, 4, 12, 24, 48, and 72 hours post-infection (hpi). Mfuzz time clustering, weighted gene coexpression network analysis (WGCNA), and Immune Cell Abundance Identifier for mouse (ImmuCellAI-mouse) were used to analyze RNA-seq data. A gradual change in the lung transcriptional profile was observed, which was consistent with the expected disease progression. At 4 hpi, the expression of genes related to the acute phase inflammatory response increased, as predicted abundance of innate immune cells. At this stage, an increased demand for energy was also observed, including an increase in the expression of genes involved in circulation, muscle function and mitochondrial respiratory chain function. The expression of genes associated with endoplasmic reticulum stress (ERS) and autophagy increased at 24 hpi. Unlike the number of natural killer (NK) cells following most bacterial lung infections, the abundance of NK cells decreased following infection with SMA. The expression levels of Cxcl10, Cd14, Gbp5, Cxcr2, Tnip1, Zc3h12a, Egr1, Sell and Gbp2 were high and previously unreported in SMA pneumonia, and they may be important targets for future studies. To our knowledge, this is the first study to investigate the pulmonary transcriptional response to SMA infection. The findings shed light on the molecular mechanisms underlying the pathogenesis of SMA pneumonia, which may aid in the development of therapies to reduce the occurrence of SMA pulmonary infection.

Co-expression network analysis for the identification of potential prostate cancer genes and in vitro confirmation of their expression in cell model in the presence of Staphylococcal tsst-1 gene.

Prostate cancer has arisen as an important life-threatening malignancy in males worldwide. Therefore, it is important to study underlying molecular pathways to be able to proposed appropriate a novel pathway of apoptosis in prostate cancer. This study aimed to explore the molecular effects of Staphylococcal tsst-1 gene on PC3 cell line apoptosis by in silico and in vitro studies. In this work, the differential expression of genes in prostate cancer was predicted by analyzing the public dataset GSE132063. Then, the pcDNA3.1 (+) vector was used to transfer tsst-1 gene to the PC3 cells and its effects was investigated using flow cytometry and qPCR. Co-expression network analysis indicated that lncRNAs had strong relationship with apoptosis genes in prostate cancer. Results of protein-protein docking indicated that BCL2L11, GRAMD3 and EGR1 interacted with tsst-1. Finally, the flow cytometry results showed that transfection by pcDNA3.1 (+)- tsst-1 could increase cellular death rates (48.15%) compared with the pcDNA3.1 (+) groups (6.35%); and the expression levels of GRAMD3, EGR1, BCL2L11 and PLAC4 were dysregulated in tsst-1 -transfected PC3 compared with empty-transfected PC3 (p < .05). In conclusion, our data will provide a novel landscape to understanding the mechanism of Staphylococcal tsst-1 gene on the PC3 cells apoptosis pathways.

Increased CTGF expression in alveolar epithelial cells by cyclic mechanical stretch: Its mechanism and the therapeutic effect of pirfenidone

The mechanisms of fibrosis onset and development remain to be elucidated. However, it has been reported that mechanical stretch promotes fibrosis in various organs and cells, and may be involved in the pathogenesis of pulmonary fibrosis. We demonstrated that ventilator-induced lung hyperextension stimulation in mice increased the expression of connective tissue growth factor (CTGF), a profibrotic cytokine, in lung tissue. Increased CTGF expression induced by cyclic mechanical stretch (CMS) was also observed in vitro using A549 human alveolar epithelial cells. Pathway analysis revealed that the induction of CTGF expression by CMS involved MEK phosphorylation. Furthermore, early growth response 1 (Egr-1) was identified as a transcription factor associated with CTGF expression. Finally, the antifibrotic drug pirfenidone significantly reduced CTGF expression, MEK phosphorylation, and Egr-1 levels induced by CMS. Thus, our results demonstrated that profibrotic cytokine CTGF induced by CMS may be a therapeutic target of pirfenidone.

Targeting Hub Genes Involved in Muscle Injury Induced by Jumping Load Based on Transcriptomics.

The purpose of this study was to find hub genes that may play key roles in skeletal muscle injury induced by jumping load. Twelve female Sprague Dawley rats were divided into the normal control (NC) group and the jumping-induced muscle injury (JI) group. After 6 weeks of jumping, transmission electron microscopy, hematoxylin-eosin staining, transcriptomics sequencing and genes analysis, interaction network prediction of multiple proteins, real-time PCR detection, and Western blotting were performed on gastrocnemius muscles from NC and JI groups. As compared with NC rats, excessive jumping can result in notable structural damage and inflammatory infiltration in JI rats. A total of 112 differentially expressed genes were confirmed in NC rats versus JI rats, with 59 genes upregulated and 53 genes downregulated. Using the online String database, four hub genes in the transcriptional regulatory network were targeted, including FOS, EGR1, ATF3, and NR4A3. All expression levels of FOS, EGR1, ATF3, and NR4A3 mRNAs were decreased in JI rats compared with NC rats (p < 0.05 or p < 0.01). All expression levels of c-Fos, EGR1, ATF3, and NOR1 proteins were upregulated in JI rats (p < 0.01, p < 0.05, p > 0.05, and p < 0.01, respectively). Collectively, these findings indicate that FOS, EGR1, ATF3, and NR4A3 genes may be functionally important in jumping-induced muscle injury.

An unusual signal transducer GIV/Girdin engages in the roles of adipocyte-derived hormone leptin in liver fibrosis

Obese patients usually have hyperleptinemia and are prone to develop liver fibrosis. Leptin is intimately linked to liver fibrogenesis, a multi-receptor-driven disease. Gα-Interacting Vesicle-associated protein (GIV) functions as a multimodular signal transducer and a guanine nucleotide exchange factor for Gi controling key signalings downstream of diverse receptors. This study aimed to examine the roles of GIV in leptin-caused liver fibrosis and employed the culture-activated hepatic stellate cells (HSCs) and leptin-deficient mice, respectively. Results indicated that leptin upregulated GIV expression in HSCs. GIV was involved in leptin-induced HSC activation and liver fibrosis. GIV mediated leptin regulation of TIMP1, MMP9, PDGFβ receptor and TGFβ receptor and was required for leptin stimulating the pathways of Erk1/2, Akt1, and Smad3. GIV was also a mediator for leptin-regulation of Cyclin D1 and Caspase-3 activity but GIV reduced Caspase-3 level independently of leptin in vivo. Erk1/2 signaling, Egr1 and c-Jun were associated with the effect of leptin on GIV expression in HSCs. Leptin-induced Erk1/2 signaling increased Egr1 and p-c-Jun levels and promoted their binding to GIV promoter at the sites between −190 bp and −180 bp and between −382 bp and − 376 bp, respectively. Egr1 knockdown lessened leptin-upregulation of GIV in vitro and in vivo. In human cirrhotic livers, the increase in GIV protein level parallelled with the elevated p-Erk1/2 and Egr1 levels in HSCs. In summary, the unusual signal transducer GIV was identified as an important mediator in leptin-induced liver fibrosis. GIV may have significant implications in liver fibrosis progression of obese patients with hyperleptinaemia.

EGR1 knockdown confers protection against ferroptosis and ameliorates intervertebral disc cartilage degeneration by inactivating the MAP3K14/NF-κB axis

This study explored whether EGR1-MAP3K14-NF-κB axis regulated ferroptosis and IVD cartilage generation. EGR1 and MAP3K14 expression levels were determined in CEP tissues of IVDD patients and intermittent cyclic mechanical tension (ICMT)-treated CEP cells. After EGR1 and MAP3K14 were altered in ICMT-treated CEP cells, the expression levels of degeneration- and ferroptosis-related proteins were measured. Binding relationship between EGR1 and MAP3K14 was evaluated. Additionally, the impacts of EFR1 knockdown on ferroptosis and cartilage degeneration in vivo were analyzed. EGR1 and MAP3K14 were overexpressed in clinical samples and cell models of IVDD. In IVDD cell models, EGR1 knockdown reduced ferroptosis and cartilage degeneration, which was reversed by MAP3K14 overexpression or Erastin treatment. NF-κB pathway inhibition nullified these effects of sh-EGR1 + oe-MAP3K14 treatment. EGR1 knockdown inhibited ferroptosis and relieved CEP degeneration via MAP3K14-NF-κB axis inactivation in vivo. Collectively, our findings highlighted that EGR1 promoted ferroptosis and IVD cartilage degeneration through MAP3K14-NF-κB axis.

Early growth response-1 is a new substrate of the GSK3β-FBXW7 axis

EGR1, a short-lived transcription factor, regulates several biological processes, including cell proliferation and tumor progression. Whether and how EGR1 is regulated by Cullin-RING ligases (CRLs) remains elusive. Here, we report that MLN4924, a small molecule inhibitor of neddylation, causes EGR1 accumulation by inactivating SCFFBXW7 (CRL1), which is a new E3 ligase for EGR1. Specifically, FBXW7 binds to EGR1 via its consensus binding motif/degron, whereas cancer-derived FBXW7 mutants showed a much reduced EGR1 binding. SiRNA-mediated FBXW7 knockdown caused EGR1 accumulation, whereas FBXW7 overexpression reduced EGR1 levels. Likewise, FBXW7 knockdown significantly extended EGR1 protein half-life, while FBXW7 overexpression promotes polyubiquitylation of wild-type EGR1, but not EGR1-S2A mutant with the binding site abrogated. GSK3β kinase is required for the FBXW7-EGR1 binding, and for enhanced EGR1 degradation by wild type FBXW7, but not by FBXW7 mutants. Likewise, GSK3β knockdown or treatment with GSK3β inhibitor significantly increased the EGR1 levels and extended EGR1 protein half-life, while reducing EGR1 polyubiquitylation. Hypoxia exposure reduces the EGR1 levels via enhancing the FBXW7-EGR1 binding, and FBXW7-induced EGR1 polyubiquitylation. Biologically, EGR1 knockdown suppressed cancer cell growth, whereas growth stimulation by FBXW7 knockdown is partially rescued by EGR1 knockdown. Thus, EGR1 is a new substrate of the GSK3β-FBXW7 axis, and the FBXW7-EGR1 axis coordinately regulates growth of cancer cells.

Mots-c Attenuates Myocardial Mitochondrial Damage Through Inhibition Of Ccn1/erk1/2/egr1 Pathway

Mitochondrial dysfunction is considered as a major contributor to cardiac systolic and diastolic dysfunction in diabetes. As a newly identified mitochondria derived peptide, MOTS-c plays a vital role in the regulation of metabolic homeostasis and was shown to be closely related to type 2 diabetes (T2D). We hypothesized that MOTS-c could prevent myocardial mitochondrial damage and improve cardiac structure and function in T2D. PURPOSE: To explored the effects and molecular mechanisms of MOTS-c on mitochondrial damage and cardiac dysfunction in diabetes. METHODS: Thirty male Sprague Dawley rats were randomly divided into three groups: control (C, n = 10), diabetes (D, n = 10) and diabetes plus MOTS-c treatment (DM, n = 10). Diabetic rats induced by high-fat high-sugar diet combined with a low-dose streptozotocin (30 mg/kg) injection. Rats in DM group were injected with MOTS-c at a dose of 0.5 mg/kg/day and 7 days/week for 8 weeks. Echocardiography was used to measured cardiac systolic and diastolic function, and transmission electron microscopy (TEM) was used to observed myocardial ultrastructure. RNA sequencing was used to determin differentially expressed genes (DEGs). Gene Ontology (GO) were selected for functional enrichment analysis to screen out the related pathways and genes, and qRT-PCR was used to measure the mRNA expression levels of CCN1, ERK1/2 and EGR1. RESULTS: MOTS-c treatment significantly prevented mitochondrial swelling and vacuoles and repaired the disorder of myocardial fiber arrangement. There was no significant differences in ejection fraction (EF), E-wave peak, A-wave peak and E/A ratio between DM and C groups (all p > 0.05). 8-week MOTS-c treatment altered 47 causative genes in myocardium of diabetic rats. The results of GO enrichment analysis showed that CCN1 and EGR1 were highly expressed and appeared in the apoptosis related terms. CCN1 was included in the ERK1/2 cascade with the highest significance. The mRNA expression of CCN1, ERK2 and EGR1 were significantly decreased after MOTS-c treatment (p < 0.01). CONCLUSIONS: MOTS-c improves myocardial mitochondrial damage and attenuates the cardiac pathological remodeling and dysfunction. CCN1/ERK1/2/EGR1 pathway may play an important role in MOTS-c preventing myocardial damage in diabetes.

Integrated bioinformatical analysis, machine learning and in vitro experiment-identified m6A subtype, and predictive drug target signatures for diagnosing renal fibrosis.

Renal biopsy is the gold standard for defining renal fibrosis which causes calcium deposits in the kidneys. Persistent calcium deposition leads to kidney inflammation, cell necrosis, and is related to serious kidney diseases. However, it is invasive and involves the risk of complications such as bleeding, especially in patients with end-stage renal diseases. Therefore, it is necessary to identify specific diagnostic biomarkers for renal fibrosis. This study aimed to develop a predictive drug target signature to diagnose renal fibrosis based on m6A subtypes. We then performed an unsupervised consensus clustering analysis to identify three different m6A subtypes of renal fibrosis based on the expressions of 21 m6A regulators. We evaluated the immune infiltration characteristics and expression of canonical immune checkpoints and immune-related genes with distinct m6A modification patterns. Subsequently, we performed the WGCNA analysis using the expression data of 1,611 drug targets to identify 474 genes associated with the m6A modification. 92 overlapping drug targets between WGCNA and DEGs (renal fibrosis vs. normal samples) were defined as key drug targets. A five target gene predictive model was developed through the combination of LASSO regression and stepwise logistic regression (LASSO-SLR) to diagnose renal fibrosis. We further performed drug sensitivity analysis and extracellular matrix analysis on model genes. The ROC curve showed that the risk score (AUC = 0.863) performed well in diagnosing renal fibrosis in the training dataset. In addition, the external validation dataset further confirmed the outstanding predictive performance of the risk score (AUC = 0.755). These results indicate that the risk model has an excellent predictive performance for diagnosing the disease. Furthermore, our results show that this 5-target gene model is significantly associated with many drugs and extracellular matrix activities. Finally, the expression levels of both predictive signature genes EGR1 and PLA2G4A were validated in renal fibrosis and adjacent normal tissues by using qRT-PCR and Western blot method.

M6A Regulator-Mediated RNA Methylation Modification Patterns Regulate the Immune Microenvironment in Osteoarthritis.

Epigenetic regulation, particularly RNA n6 methyl adenosine (m6A) modification, plays an important role in the immune response. However, the regulatory role of m6A in the immune microenvironment in osteoarthritis (OA) remains unclear. Accordingly, we systematically studied RNA modification patterns mediated by 23 m6A regulators in 38 samples and discussed the characteristics of the immune microenvironment modified by m6A. Next, we constructed a novel OA m6A nomogram, an m6A-transcription factor-miRNA network, and a drug network. Healthy and OA samples showed distinct m6A regulatory factor expression patterns. YTHDF3 expression was upregulated in OA samples and positively correlated with type II helper cells and TGFb family member receptors. Furthermore, three different RNA modification patterns were mediated by 23 m6A regulatory factors; in Mode 3, the expression levels of YTHDF3, type II T helper cells, and TGFb family member receptors were upregulated. Pathways related to endoplasmic reticulum oxidative stress and mitochondrial autophagy showed a strong correlation with the regulatory factors associated with Mode 3 and 23 m6A regulatory factors. Through RT-qPCR we validated that SREBF2 and EGR1 as transcription factors of YTHDF3 and IGF2BP3 are closely associated with the development of OA, hsa-miR-340 as a miRNA for YTHDF3 and IGF2BP3 was involved in the development of OA, we also detected the protein expression levels of IGF2BP3, YTHDF3, EGR1 and SREBF2 by western blotting, and the results were consistent with PCR. Overall, the constructed nomogram can facilitate the prediction of OA risk.

MiR-let-7c-3p targeting on Egr-1 contributes to the committed differentiation of leukemia cells into monocyte/macrophages.

In preliminary experiments, it was found that the expression of early growth response-1 (Egr-1) was upregulated during the committed differentiation of leukemia cells into monocytes/macrophages. The cross-analysis of gene chip detection and database prediction indicated that Egr-1 was associated with upstream microRNA (miR)-let-7c-3p, thus the present study focused on the role of the miR-let-7c-3p/Egr-1 signaling axis in the committed differentiation of leukemia cells into monocytes/macrophages. Phorbol 12-myristate 13-acetate (PMA) was used to induce the directed differentiation of human K562 leukemia cells into monocytes/macrophages and the differentiation of K562 leukemia cells was determined by cell morphology observation and expression of differentiation antigens CD11b and CD14 by flow cytometry. The expression levels of Egr-1 and miR-let-7c-3p were detected by reverse transcription-quantitative PCR and the protein expression of Egr-1 was detected by western blotting. The effect of Egr-1 on the differentiation of K562 cells was detected by short interfering (si)RNA interference assay. A dual-luciferase reporter assay was used to detect target binding of miR-let-7c-3p on the 3′UTR of Egr-1. Cell transfection of miR-let-7c-3p mimics and inhibitors was used to modulate the expression of miR-let-7c-3p, as indicated by RT-qPCR assays. Western blotting was also used to examine the effect of miR-let-7c-3p on Egr-1 expression. The PMA-induced differentiation of K562 cells was transfected with miR-let-7c-3p and the expression of differentiation antigen was detected by flow cytometry. A differentiation model of K562 leukemia cells into monocytes/macrophages was induced by PMA, which was indicated by morphological observations and upregulation of CD11b and CD14 antigens. The gene or protein expression of Egr-1 was significantly higher compared with that of the control group, while the expression of miR-let-7c-3p was significantly lower compared with that of the control group. siRNA interference experiments showed that the expression of cell differentiation antigen CD14 in the 100 µg/ml PMA + si-Egr-1 group was significantly lower compared with that in the 100 µg/ml PMA + si-ctrl group. The dual luciferase reporter gene results showed that the luciferase activity of the co-transfected mimic and Egr-1 WT groups was significantly lower than that of the NC control group, while the luciferase activity of the co-transfected mimic and Egr-1 MUT groups was comparable to that of the NC control group. Therefore, the dual-luciferase reporter gene assay confirmed that miR-let-7c-3p can target Egr-1. Western blotting showed that the expression of Egr-1 following transfection with miR-let-7c-3p inhibitor was significantly higher compared with that of the negative control and the expression of Egr-1 after transfection with miR-let-7c-3p mimic was significantly lower than that of the negative control. Following exposure to PMA, the expressions of CD11b and CD14 in the miR-let-7c-3p inhibitor group were significantly higher than those in the miR-let-7c-3p NC group, as indicated by CD11b and CD14 respectively. In conclusion, miR-let-7c-3p could bind to the 3′UTR of Egr-1 and negatively regulated Egr-1 expression. The miR-let-7c-3p/Egr-1 signaling axis was closely associated with the committed differentiation of K562 cells from leukemia cells to monocytes/macrophages.

Elevation of EGR1/zif268, a Neural Activity Marker, in the Auditory Cortex of Patients with Schizophrenia and its Animal Model.

The family of epidermal growth factor (EGF) including neuregulin-1 are implicated in the neuropathology of schizophrenia. We established a rat model of schizophrenia by exposing perinatal rats to EGF and reported that the auditory pathophysiological traits of this modelsuch as prepulse inhibition, auditory steady-state response, and mismatch negativity are relevant to those of schizophrenia. We assessed the activation status of the auditory cortex in this model, as well as that in patients with schizophrenia, by monitoring the three neural activity-induced proteins: EGR1 (zif268), c-fos, and Arc. Among the activity markers, protein levels of EGR1 were significantly higher at the adult stage in EGF model rats than those in control rats. The group difference was observed despite an EGF model rat and a control rat being housed together, ruling out the contribution of rat vocalization effects. These changes in EGR1 levels were seen to be specific to the auditory cortex of this model. The increase in EGR1 levels were detectable at the juvenile stage and continued until old ages but displayed a peak immediately after puberty, whereas c-fos and Arc levels were nearly indistinguishable between groups at all ages with an exception of Arc decrease at the juvenile stage. A similar increase in EGR1 levels was observed in the postmortem superior temporal cortex of patients with schizophrenia. The commonality of the EGR1 increase indicates that the EGR1 elevation in the auditory cortex might be one of the molecular signatures of this animal model and schizophrenia associating with hallucination.

Regulatory mechanism of downregulation of SOD1 expression on cardiomyocyte function.

Many diseases are clinically related to oxidative stress. Obstructive sleep apnea (OSA) is a common disease with oxidative stressin clinical practice, which is mostly associated with cardio-cerebrovascular diseases. It has been shown that the level of oxidative stress increases and the level of antioxidant copper zinc superoxide dismutase (SOD1) decreases in intermittent hypoxia (IH). SOD1 is one of the key antioxidant enzymes in organisms, and it can also be used as a signal transmission controller. Its abnormal expression further affects organ functions, but the specific mechanism is not yet fully clear. We downregulated the SOD1 gene in H9C2 cell line, using high-throughput RNA sequencing (RNA-seq) to find differentially expressed genes (DEGs) related to cardiomyocyte function by using GO and KEGG databases to annotate, enrich and analyze the metabolic pathways of DEGs. Through the analysis of these functional gene changes, we can understand the regulation of SOD1 downregulation on cardiomyocyte function. The results found 213 DEGs, of which 135 genes were upregulated and 78 genes were downregulated. The upregulated DEGs were mainly enriched in biological processes such as transcriptional regulation and metabolism. The expression levels of EGR1 and NR1D1 exceeded 1 in the samples. EGR1 was reported to be involved in oxidative stress and cardiac hypertrophy, and NR1D1played an important regulatory role in regulating inflammatory responses and reducing ROS production. The biological processes involved in downregulated DEGs mainly involve metabolism and redox processes. Among them, SCD1 and CCL2 genes were highly expressed among the genes involved in the redox process involved in SOD1. SCD1 is an important player in the regulation of cardiometabolic processes; downregulation of CCL2 reduces atherosclerosis. We found that the TNF signaling pathway, NOD-like receptor signaling pathway, and chemokine signaling pathway, which were enriched in KEGG analysis, were all associated with inflammation, and the CXCL1 and CCL7 genes are all related to inflammation. The gene and signaling pathways involved in oxidative stress and inflammatory response process regulated by SOD1 were demonstrated. SOD1 may affect the function of the heart by affecting myocardial contraction, inflammation, lipid metabolism, and other pathways. It is inferred that they may also play a role in the process of OSA-related myocardial injury, which is worthy of attention and further study.