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.

EGR1 transcriptionally regulates SVEP1 to promote proliferation and migration in human coronary artery smooth muscle cells.

A low-frequency variant of sushi, von Willebrand factor type A, EGF, and pentraxin domain-containing protein 1 (SVEP1) is associated with the risk of coronary artery disease, as determined by a genome-wide association study. SVEP1 induces vascular smooth muscle cell proliferation and an inflammatory phenotype to promote atherosclerosis. In the present study, qRT‒PCR demonstrated that the mRNA expression of SVEP1 was significantly increased in atherosclerotic plaques compared to normal tissues. Bioinformatics revealed that EGR1 was a transcription factor for SVEP1. The results of the luciferase reporter assay, siRNA interference or overexpression assay, mutational analysis and ChIP confirmed that EGR1 positively regulated the transcriptional activity of SVEP1 by directly binding to its promoter. EGR1 promoted human coronary artery smooth muscle cell (HCASMC) proliferation and migration via SVEP1 in response to oxidized low-density lipoprotein (ox-LDL) treatment. Moreover, the expression level of EGR1 was increased in atherosclerotic plaques and showed a strong linear correlation with the expression of SVEP1. Our findings indicated that EGR1 binding to the promoter region drive SVEP1 transcription to promote HCASMC proliferation and migration.

Iguratimod suppresses sclerostin and receptor activator of NF-κB ligand production via the extracellular signal-regulated kinase/early growth response protein 1/tumor necrosis factor alpha pathway in osteocytes and ameliorates disuse osteoporosis in mice.

Disuse osteoporosis is a prevalent complication among patients afflicted with rheumatoid arthritis (RA). Although reports have shown that the antirheumatic drug iguratimod (IGU) ameliorates osteoporosis in RA patients, details regarding its effects on osteocytes remain unclear. The current study examined the effects of IGU on osteocytes using a mouse model of disuse-induced osteoporosis, the pathology of which crucially involves osteocytes. A reduction in distal femur bone mass was achieved after 3 weeks of hindlimb unloading in mice, which was subsequently reversed by intraperitoneal IGU treatment (30 mg/kg; five times per week). Histology revealed that hindlimb-unloaded (HLU) mice had significantly increased osteoclast number and sclerostin-positive osteocyte rates, which were suppressed by IGU treatment. Moreover, HLU mice exhibited a significant decrease in osteocalcin-positive cells, which was attenuated by IGU treatment. In vitro, IGU suppressed the gene expression of receptor activator of NF-κB ligand (RANKL) and sclerostin in MLO-Y4 and Saos-2 cells, which inhibited osteoclast differentiation of mouse bone marrow cells in cocultures. Although IGU did not affect the nuclear translocation or transcriptional activity of NF-κB, RNA sequencing revealed that IGU downregulated the expression of early growth response protein 1 (EGR1) in osteocytes. HLU mice showed significantly increased EGR1- and tumor necrosis factor alpha (TNFα)-positive osteocyte rates, which were decreased by IGU treatment. EGR1 overexpression enhanced the gene expression of TNFα, RANKL, and sclerostin in osteocytes, which was suppressed by IGU. Contrarily, small interfering RNA-mediated suppression of EGR1 downregulated RANKL and sclerostin gene expression. These findings indicate that IGU inhibits the expression of EGR1, which may downregulate TNFα and consequently RANKL and sclerostin in osteocytes. These mechanisms suggest that IGU could potentially be used as a treatment option for disuse osteoporosis by targeting osteocytes.

Blocking EGR1/TGF-ß1 and CD44s/STAT3 Crosstalk Inhibits Peritoneal Metastasis of Gastric Cancer.

Peritoneal metastasis (PM) continues to limit the clinical efficacy of gastric cancer (GC). Early growth response 1 (EGR1) plays an important role in tumor cell proliferation, angiogenesis and invasion. However, the role of EGR1 derived from the tumor microenvironment in reshaping the phenotypes of GC cells and its specific molecular mechanisms in increasing the potential for PM are still unclear. In this study, we reported that EGR1 was significantly up-regulated in mesothelial cells from GC peritoneal metastases, leading to enhanced epithelial-mesenchymal transformation (EMT) and stemness phenotypes of GC cells under co-culture conditions. These phenotypes were achieved through the transcription and secretion of TGF-ß1 by EGR1 in mesothelial cells, which could regulate the expression and internalization of CD44s. After being internalized into the cytoplasm, CD44s interacted with STAT3 to promote STAT3 phosphorylation and activation, and induced EMT and stemness gene transcription, thus positively regulating the metastasis of GC cells. Moreover, TGF-ß1 secretion in the PM microenvironment was significantly increased compared with the matched primary tumor. The blocking effect of SHR-1701 on TGF-ß1 was verified by inhibiting peritoneal metastases in xenografts. Collectively, the interplay of EGR1/TGF-ß1/CD44s/STAT3 signaling between mesothelial cells and GC cells induces EMT and stemness phenotypes, offering potential as a therapeutic target for PM of GC.

EGR1 Regulates SHANK3 Transcription at Different Stages of Brain Development.

The expression levels of SHANK3 are associated with autism spectrum disorder (ASD). The dynamic changes in SHANK3 expression during different stages of brain development may impact the progression of ASD. However, no studies or detailed analyses exploring the upstream mechanisms that regulate SHANK3 expression have been reported. In this study, we employed immunofluorescence to examine the expression of SHANK3 in brain organoids at various stages. Our results revealed elevated levels of SHANK3 expression in brain-like organoids at Day 60. Additionally, we utilized bioinformatics software to predict and analyze the SHANK3 gene's transcription start site. Through the dual luciferase reporter gene technique, we identified core transcription elements within the SHANK3 promoter. Site-directed mutations were used to identify specific transcription sites of SHANK3. To determine the physical binding of potential transcription factors to the SHANK3 promoter, we employed electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Our findings demonstrated that the transcription factor EGR1 regulates SHANK3 expression by binding to the transcription site of the SHANK3 promoter. Although this study did not investigate the pathological phenotypes of human brain organoids or animal model brains with EGR1 deficiency, which could potentially substantiate the findings observed for SHANK3 mutants, our findings provide valuable insights into the relationship between the transcription factor, EGR1, and SHANK3. This study contributes to the molecular understanding of ASD and offers potential foundations for precise targeted therapy.

EGR1 suppresses HCC growth and aerobic glycolysis by transcriptionally downregulating PFKL.

BACKGROUND: Hepatocellular Carcinoma (HCC) is a matter of great global public health importance; however, its current therapeutic effectiveness is deemed inadequate, and the range of therapeutic targets is limited. The aim of this study was to identify early growth response 1 (EGR1) as a transcription factor target in HCC and to explore its role and assess the potential of gene therapy utilizing EGR1 for the management of HCC. METHODS: In this study, both in vitro and in vivo assays were employed to examine the impact of EGR1 on the growth of HCC. The mouse HCC model and human organoid assay were utilized to assess the potential of EGR1 as a gene therapy for HCC. Additionally, the molecular mechanism underlying the regulation of gene expression and the suppression of HCC growth by EGR1 was investigated. RESULTS: The results of our investigation revealed a notable decrease in the expression of EGR1 in HCC. The decrease in EGR1 expression promoted the multiplication of HCC cells and the growth of xenografted tumors. On the other hand, the excessive expression of EGR1 hindered the proliferation of HCC cells and repressed the development of xenografted tumors. Furthermore, the efficacy of EGR1 gene therapy was validated using in vivo mouse HCC models and in vitro human hepatoma organoid models, thereby providing additional substantiation for the anti-cancer role of EGR1 in HCC. The mechanistic analysis demonstrated that EGR1 interacted with the promoter region of phosphofructokinase-1, liver type (PFKL), leading to the repression of PFKL gene expression and consequent inhibition of PFKL-mediated aerobic glycolysis. Moreover, the sensitivity of HCC cells and xenografted tumors to sorafenib was found to be increased by EGR1. CONCLUSION: Our findings suggest that EGR1 possesses therapeutic potential as a tumor suppressor gene in HCC, and that EGR1 gene therapy may offer benefits for HCC patients.

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, IL1ß, 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 IL1ß 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.

Transcriptome Profiling of Cardiac Glycoside Treatment Reveals EGR1 and Downstream Proteins of MAPK/ERK Signaling Pathway in Human Breast Cancer Cells.

Cardiac glycosides (CGs) constitute a group of steroid-like compounds renowned for their effectiveness in treating cardiovascular ailments. In recent times, there has been growing recognition of their potential use as drug leads in cancer treatment. In our prior research, we identified three highly promising CG compounds, namely lanatoside C (LC), peruvoside (PS), and strophanthidin (STR), which exhibited significant antitumor effects in lung, liver, and breast cancer cell lines. In this study, we investigated the therapeutic response of these CGs, with a particular focus on the MCF-7 breast cancer cell line. We conducted transcriptomic profiling and further validated the gene and protein expression changes induced by treatment through qRT-PCR, immunoblotting, and immunocytochemical analysis. Additionally, we demonstrated the interactions between the ligands and target proteins using the molecular docking approach. The transcriptome analysis revealed a cluster of genes with potential therapeutic targets involved in cytotoxicity, immunomodulation, and tumor-suppressor pathways. Subsequently, we focused on cross-validating the ten most significantly expressed genes, EGR1, MAPK1, p53, CCNK, CASP9, BCL2L1, CDK7, CDK2, CDK2AP1, and CDKN1A, through qRT-PCR, and their by confirming the consistent expression pattern with RNA-Seq data. Notably, among the most variable genes, we identified EGR1, the downstream effector of the MAPK signaling pathway, which performs the regulatory function in cell proliferation, tumor invasion, and immune regulation. Furthermore, we substantiated the influence of CG compounds on translational processes, resulting in an alteration in protein expression upon treatment. An additional analysis of ligand-protein interactions provided further evidence of the robust binding affinity between LC, PS, and STR and their respective protein targets. These findings underscore the intense anticancer activity of the investigated CGs, shedding light on potential target genes and elucidating the probable mechanism of action of CGs in breast cancer.

EGR1/LINC00839/SOX5 axis modulates migration, invasion and Gemcitabine resistance of bladder cancer cells.

BACKGROUND: Bladder cancer is one of the most common malignant tumors of the urinary system, and its incidence is increasing worldwide. However, the underlying mechanisms that trigger migration, invasion and chemotherapy resistance are unclear. RESULTS: Bioinformatics analysis of bladder cancer cohort indicated that LINC00839 is deregulated in bladder cancer. LINC00839 was validated and highly expressed in bladder cancer patients and cell lines. In addition, LINC00839 induced the migration, invasion and Gemcitabine resistance of bladder cancer cells. We identified that the transcription factor EGR1 directly repressed LINC00839 and thereby suppressed the migration and invasion of bladder cancer cells. Furthermore, LINC00839 interacted with miR-142, which subsequently regulated the expression of SOX5, a well-studied oncogene and targeted by miR-142. In addition, EGR1 served as a suppressive transcription factor of SOX5. Therefore, EGR1 directly or indirectly regulates SOX5 via LINC00839/miR-142 axis. LINC00839 induced Gemcitabine resistance by promoting autophagy. CONCLUSIONS: EGR1, LINC00839/miR-142 and SOX5 form a coherent feed-forward loop that modulates the migration, invasion and Gemcitabine resistance of bladder cancer.

Isogenic pairs of induced-pluripotent stem-derived endothelial cells identify DYRK1A/PPARG/EGR1 pathway is responsible for Down syndrome-associated pulmonary hypertension.

Down syndrome (DS) is the most prevalent chromosomal disorder associated with a higher incidence of pulmonary arterial hypertension (PAH). The dysfunction of vascular endothelial cells (ECs) is known to cause pulmonary arterial remodeling in PAH, although the physiological characteristics of ECs harboring trisomy 21 (T21) are still unknown. In this study, we analyzed the human vascular ECs by utilizing the isogenic pairs of T21-induced pluripotent stem cells (iPSCs) and corrected disomy 21 (cDi21)-iPSCs. In T21-iPSC-derived ECs, apoptosis and mitochondrial reactive oxygen species (mROS) were significantly increased, and angiogenesis and oxygen consumption rate (OCR) were significantly impaired as compared with cDi21-iPSC-derived ECs. The RNA-sequencing identified that EGR1 on chromosome 5 was significantly upregulated in T21-ECs. Both EGR1 suppression by siRNA and pharmacological inhibitor could recover the apoptosis, mROS, angiogenesis, and OCR in T21-ECs. Alternately, the study also revealed that DYRK1A was responsible to increase EGR1 expression via PPARG suppression, and that chemical inhibition of DYRK1A could restore the apoptosis, mROS, angiogenesis, and OCR in T21-ECs. Finally, we demonstrated that EGR1 was significantly upregulated in the pulmonary arterial ECs from lung specimens of a patient with DS and PAH. In conclusion, DYRK1A/PPARG/EGR1 pathway could play a central role for the pulmonary EC functions and thus be associated with the pathogenesis of PAH in DS.

Evaluating Gene Expression and Methylation Profiles of TCF4, MBP, and EGR1 in Peripheral Blood of Drug-Free Patients with Schizophrenia: Correlations with Psychopathology, Intelligence, and Cognitive Impairment.

Discovery and validation of new, reliable diagnostic and predictive biomarkers for schizophrenia (SCZ) are an ongoing effort. Here, we assessed the mRNA expression and DNA methylation of the TCF4, MBP, and EGR1 genes in the blood of patients with SCZ and evaluated their relationships to psychopathology and cognitive impairments. Quantitative real-time PCR and quantitative methylation-specific PCR methods were used to assess the expression level and promoter DNA methylation status of these genes in 70 drug-free SCZ patients and 72 healthy controls. The correlation of molecular changes with psychopathology and cognitive performance of participants was evaluated. We observed downregulation of TCF4 and upregulation of MBP mRNA levels in SCZ cases, relative to controls in our study. DNA methylation status at the promoter region of TCF4 demonstrated an altered pattern in SCZ as well. Additionally, TCF4 mRNA levels were inversely correlated with PANSS and Stroop total errors and positively correlated with WAIS total score and working memory, consistent with previous studies by our group. In contrast, MBP mRNA level was significantly positively correlated with PANSS and Stroop total errors and inversely correlated with WAIS total score and working memory. These epigenetic and expression signatures can help to assemble a peripheral biomarker-based diagnostic panel for SCZ.

EGR1 functions as a new host restriction factor for SARS-CoV-2 to inhibit virus replication through the E3 ubiquitin ligase MARCH8.

IMPORTANCE: Emerging vaccine-breakthrough severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight an urgent need for novel antiviral therapies. Understanding the pathogenesis of coronaviruses is critical for developing antiviral drugs. Here, we demonstrate that the SARS-CoV-2 N protein suppresses interferon (IFN) responses by reducing early growth response gene-1 (EGR1) expression. The overexpression of EGR1 inhibits SARS-CoV-2 replication by promoting IFN-regulated antiviral protein expression, which interacts with and degrades SARS-CoV-2 N protein via the E3 ubiquitin ligase MARCH8 and the cargo receptor NDP52. The MARCH8 mutants without ubiquitin ligase activity are no longer able to degrade SARS-CoV-2 N proteins, indicating that MARCH8 degrades SARS-CoV-2 N proteins dependent on its ubiquitin ligase activity. This study found a novel immune evasion mechanism of SARS-CoV-2 utilized by the N protein, which is helpful for understanding the pathogenesis of SARS-CoV-2 and guiding the design of new prevention strategies against the emerging coronaviruses.

EGR1 Promotes Ovarian Hyperstimulation Syndrome Through Upregulation of SOX9 Expression.

Angiogenesis is strongly associated with ovarian hyperstimulation syndrome (OHSS) progression. Early growth response protein 1 (EGR1) plays an important role in angiogenesis. This study aimed to investigate the function and mechanism of EGR1 involved in OHSS progression. RNA-sequencing was used to identify differentially expressed genes. In vitro OHSS cell model was induced by treating KGN cells with human chorionic gonadotropin (hCG). In vivo OHSS model was established in mice. The expression levels of EGR1, SOX1, and VEGF were determined by Quantitative Real-Time polymerase chain reaction (qRT-PCR), Western blot, immunofluorescence staining, and immunochemistry assay. The content of VEGF in the culture medium of human granulosa-like tumor cell line (KGN) cells was accessed by the ELISA assay. The regulatory effect of EGR1 on SRY-box transcription factor 9 (SOX9) was addressed by luciferase reporter assay and chromatin immunoprecipitation. The ERG1 and SOX9 levels were significantly upregulated in granulosa cells from OHSS patients and there was a positive association between EGR1 and SOX9 expression. In the ovarian tissues of OHSS mice, the levels of EGR1 and SOX9 were also remarkedly increased. Treatment with hCG elevated the levels of vascular endothelial growth factor (VEGF), EGR1, and SOX9 in KGN cells. Silencing of EGR1 reversed the promoting effect of hCG on VEGF and SOX9 expression in KGN cells. EGR1 transcriptionally regulated SOX9 expression through binding to its promoter. In addition, administration of dopamine decreased hCG-induced VEGF in KGN cells and ameliorated the progression of OHSS in mice, which were companied with decreased EGR1 and SOX9 expression. EGR1 has a promoting effect on OHSS progression and dopamine protects against OHSS through suppression of EGR1/SOX9 cascade. Our findings may provide new targets for the treatment of OHSS.

EGR1-mediated metabolic reprogramming to oxidative phosphorylation contributes to ibrutinib resistance in B-cell lymphoma.

The use of Bruton tyrosine kinase inhibitors, such as ibrutinib, to block B-cell receptor signaling has achieved a remarkable clinical response in several B-cell malignancies, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Acquired drug resistance, however, is significant and affects the long-term survival of these patients. Here, we demonstrate that the transcription factor early growth response gene 1 (EGR1) is involved in ibrutinib resistance. We found that EGR1 expression is elevated in ibrutinib-resistant activated B-cell-like subtype DLBCL and MCL cells and can be further upregulated upon ibrutinib treatment. Genetic and pharmacological analyses revealed that overexpressed EGR1 mediates ibrutinib resistance. Mechanistically, TCF4 and EGR1 self-regulation induce EGR1 overexpression that mediates metabolic reprogramming to oxidative phosphorylation (OXPHOS) through the transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation increases intracellular adenosine triphosphate production, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with metformin or IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting EGR1-mediated metabolic reprogramming to OXPHOS with metformin or IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.

Tenogenic differentiation of human tendon-derived stem cells induced by long non-coding RNA LINCMD1 via miR-342-3p/EGR1 axis.

BACKGROUND: Tendon-derived stem cells (TDSCs) are proposed as a potential cell-seed for the treatment of tendon injury due to their tenogenic differentiation potential. In this work, we defined the action of long non-coding RNA (lncRNA) muscle differentiation 1 (LINCMD1) in tenogenic differentiation of human TDSCs (hTDSCs). METHODS: Quantitative real-time PCR (qRT-PCR) was used to assess the levels of LINCMD1, microRNA (miR)-342-3p, and early growth response-1 (EGR1) mRNA. Cell proliferation was detected by the XTT colorimetric assay. Protein expression was quantified by western blot. hTDSCs were grown in an osteogenic medium to induce osteogenic differentiation, and the extent of osteogenic differentiation was assessed by Alizarin Red Staining (ARS). The activity of alkaline phosphatase (ALP) was measured by the ALP Activity Assay Kit. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to evaluate the direct relationship between miR-342-3p and LINCMD1 or EGR1. RESULTS: Our results showed that enforced expression of LINCMD1 or suppression of miR-342-3p accelerated the proliferation and tenogenic differentiation and reduced osteogenic differentiation of hTDSCs. LINCMD1 regulated miR-342-3p expression by binding to miR-342-3p. EGR1 was identified as a direct and functional target of miR-342-3p, and knockdown of EGR1 reversed the effects of miR-342-3p suppression on cell proliferation and tenogenic and osteogenic differentiation. Furthermore, the miR-342-3p/EGR1 axis mediated the regulation of LINCMD1 on hTDSC proliferation and tenogenic and osteogenic differentiation. CONCLUSION: Our study suggests the induction of LINCMD1 in tenogenic differentiation of hTDSCs through miR-342-3p/EGR1 axis.

Lentinan induces apoptosis of mouse hepatocellular carcinoma cells through the EGR1/PTEN/AKT signaling axis.

Lentinan (LNT) isolated from Lentinus edodes is a vital host defense potentiator previously utilized as an adjuvant in cancer therapy. The present study investigated the effect of LNT on the mouse hepatocellular carcinoma (HCC) cell line Hepa1­6 and its possible mechanism. Mouse HCC apoptosis and its potential associated mechanism were then explored using in vitro and in vivo approaches. For in vitro approaches, the effect of LNT on the proliferation of Hepa1­6 cells was investigated by Cell Counting Kit­8 assay. Annexin V­FITC staining and flow cytometry were applied to explore HCC apoptosis. Western blotting was used to analyze related proteins, such as EGR1, phosphatase and tensin homolog (PTEN), phosphorylated protein kinase B (p­Akt), protein kinase B (Akt), B lymphocyte­2 (Bcl­2), Bcl2 family­associated X protein (Bax), etc. Cellular immunofluorescence staining was employed to assess the localization and expression of EGR1 and PTEN in nuclear and cytoplasmic fractions of Hepa1­6 cells. The association between EGR1 and PTEN was explored by EGR1 overexpression in cell lines. For in vivo methods, a mouse model of diethylnitrosamine (DEN)­induced primary liver cancer was established using C57BL/6 mice to investigate the inhibitory effect of LNT on liver cancer. Histopathology of liver tissue from mice was detected by hematoxylin­eosin staining and immunohistochemical assay. In vitro and in vivo results showed that LNT can inhibit the proliferation and promote the apoptosis of mouse HCC cells. Besides, LNT increased the expression of EGR1 in Hepa1­6 cells, which is translocated to the nucleus to function as a transcriptional factor. EGR1 then activates the expression of the tumor suppressor PTEN, thereby inhibiting the activation of the AKT signaling pathway. These data revealed a novel anti­tumor mechanism by which LNT can induce apoptosis to inhibit mouse HCC progression through the EGR1/PTEN/AKT axis. These results provide a scientific basis for the potential use of LNT in drug development and clinical applications associated with primary liver cancer.

Lysophosphatidic Acid Induces Podocyte Pyroptosis in Diabetic Nephropathy by an Increase of Egr1 Expression via Downregulation of EzH2.

Podocyte damage and renal inflammation are the main features and pathogenesis of diabetic nephropathy (DN). Inhibition of lysophosphatidic acid (LPA) receptor 1 (LPAR1) suppresses glomerular inflammation and improves DN. Herein, we investigated LPA-induced podocyte damage and its underlying mechanisms in DN. We investigated the effects of AM095, a specific LPAR1 inhibitor, on podocytes from streptozotocin (STZ)-induced diabetic mice. E11 cells were treated with LPA in the presence or absence of AM095, and the expression of NLRP3 inflammasome factors and pyroptosis were measured. A chromatin immunoprecipitation assay and Western blotting were performed to elucidate underlying molecular mechanisms. Gene knockdown by transfecting small interfering RNA was used to determine the role of the transcription factor Egr1 (early growth response protein 1) and histone methyltransferase EzH2 (Enhancer of Zeste Homolog 2) in LPA-induced podocyte injury. AM095 administration inhibited podocyte loss, NLRP3 inflammasome factor expression, and cell death in STZ-induced diabetic mice. In E11 cells, LPA increased NLRP3 inflammasome activation and pyroptosis via LPAR1. Egr1 mediated NLRP3 inflammasome activation and pyroptosis in LPA-treated E11 cells. LPA decreased H3K27me3 enrichment at the Egr1 promoter in E11 cells by downregulating EzH2 expression. EzH2 knockdown further increased LPA-induced Egr1 expression. In podocytes from STZ-induced diabetic mice, AM095 suppressed Egr1 expression increase and EzH2/H3K27me3 expression reduction. Collectively, these results demonstrate that LPA induces NLRP3 inflammasome activation by downregulating EzH2/H3K27me3 and upregulating Egr1 expression, resulting in podocyte damage and pyroptosis, which may be a potential mechanism of DN progression.

Neuroprotective microRNA-381 Binds to Repressed Early Growth Response 1 (EGR1) and Alleviates Oxidative Stress Injury in Parkinson's Disease.

As a common and disabling disease of the elderly, the standard therapies of Parkinson's disease (PD) fail to curb the ongoing neurodegeneration, thus calling for newer strategies. This work was conducted to examine the effect of microRNA-381 (miR-381) on oxidative stress injury to dopaminergic neurons in PD in vivo and in vitro. We established an in vivo mouse model of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and an in vitro cell model of PD by treating dopaminergic neuron MN9D cells with 1-methyl-4-phenylpyridinium (MPP+). It was established that miR-381 was poorly expressed in the substantia nigra pars compacta (SNc) of MPTP-lesioned mice. The motor function of MPTP-lesioned mice was evaluated in the presence of ectopic miR-381 expression, and oxidative stress and dopaminergic neuron injury were also characterized. Restoration of miR-381 was demonstrated to diminish oxidative stress and damage in dopaminergic neurons, accompanied by enhanced motor functions. Mechanistically, the putative binding sites of miR-381 were retrieved through the starBase database, and the luciferase activity assay confirmed that it bound to EGR1 and repressed its expression, which then upregulated the expression of PTEN and p53. The neuroprotective effects of miR-381 on the motor function and dopaminergic neuronal damage were counteracted by ectopic EGR1 expression. Together, this study indicates that the binding of miR-381 to EGR1 upregulates PTEN/p53 to alleviate PD, which provides novel insights for a neuroprotective mechanism in PD.

CNN2 silencing inhibits colorectal cancer development through promoting ubiquitination of EGR1.

Colorectal cancer (CRC) is one of the most commonly diagnosed malignant tumors of the digestive tract. H2-calponin (CNN2), an actin cytoskeleton-binding protein, is an isoform of the calponin protein family whose role in CRC is still unknown. Research based on clinical samples showed the up-regulation of CNN2 in CRC and its association with tumor development, metastasis, and poor prognosis of patients. Both in vitro loss-of-function and gain-of-function experiments showed that CNN2 participates in CRC development through influencing malignant cell phenotypes. In vivo, xenografts formed by CNN2 knockdown cells also showed a slower growth rate and smaller final tumors. Furthermore, EGR1 was identified as a downstream of CNN2, forming a complex with CNN2 and YAP1 and playing an essential role in the CNN2-induced regulation of CRC development. Mechanistically, CNN2 knockdown down-regulated EGR1 expression through enhancing its ubiquitination, thus decreasing its protein stability in a YAP1-dependent manner. In summary, CNN2 plays an EGR1-dependent promotion role in the development and progression of CRC, which may be a promising therapeutic target for CRC treatment.

N6-methyladenosine reader YTHDF2 promotes multiple myeloma cell proliferation through EGR1/p21cip1/waf1/CDK2-Cyclin E1 axis-mediated cell cycle transition.

Multiple myeloma (MM) is the second most common hematological malignancy. N6-methyladenosine (m6A) is the most abundant RNA modification. YTH domain-containing family protein 2 (YTHDF2) recognizes m6A-cotaining RNAs and accelerates degradation to regulate cancer progression. However, the role of YTHDF2 in MM remains unclear. We investigated the expression levels and prognostic role of YTHDF2 in MM, and studied the effect of YTHDF2 on MM proliferation and cell cycle. The results showed that YTHDF2 was highly expressed in MM and was an independent prognostic factor for MM survival. Silencing YTHDF2 suppressed cell proliferation and caused the G1/S phase cell cycle arrest. RNA immunoprecipitation (RIP) and m6A-RIP (MeRIP) revealed that YTHDF2 accelerated EGR1 mRNA degradation in an m6A-dependent manner. Moreover, overexpression of YTHDF2 promoted MM growth via the m6A-dependent degradation of EGR1 both in vitro and in vivo. Furthermore, EGR1 suppressed cell proliferation and retarded cell cycle by activating p21cip1/waf1 transcription and inhibiting CDK2-cyclinE1. EGR1 knockdown could reverse the inhibited proliferation and cell cycle arrest upon YTHDF2 knockdown. In conclusion, the high expression of YTHDF2 promoted MM cell proliferation via EGR1/p21cip1/waf1/CDK2-cyclin E1 axis-mediated cell cycle transition, highlighting the potential of YTHDF2 as an effective prognostic biomarker and a promising therapeutic target for MM.