Overexpression Of Early Growth Response 1 Research Articles

EGR1 Promotes Erastin-induced Ferroptosis Through Activating Nrf2-HMOX1 Signaling Pathway in Breast Cancer Cells.

Purpose: Early growth response 1 (EGR1) is a crucial transcription factor composed of zinc finger structures, inhibitory and activating regulatory regions. We identified the biological effect and molecular mechanisms of EGR1 in breast cancer (BC). Methods: We used qRT-PCR, western blot and immunohistochemistry to examine the expression of EGR1 in BC samples. CCK-8 and colony assay were performed to reveal the effect of EGR1 on the proliferation of BC cells. LDH release assay, MCB assay, MDA assay, C-AM assay and TMRE assay were performed to measure the levels of LDH release, GSH, MDA, LIP and mitochondrial membrane potential. The regulation of EGR1 on the expression of Nrf2 and HMOX1 was investigated through Western blot. Xenograft models were conducted to determine the impact of EGR1 overexpression on BC in vivo. Results: The expression of EGR1 was downregulated in BC tissues compared with the normal tissues, and lower expression of EGR1 associated with poorer clinical outcome in BC patients. Through in vitro experiments, we found that EGR1 downregulation facilitated the proliferation of BC cells, and overexpression of EGR1 inhibited the proliferation of BC cells. In addition, EGR1 knockdown alleviated erastin-induced ferroptosis and overexpression of EGR1 facilitated erastin-induced ferroptosis in BC cells. Moreover, overexpression of EGR1 facilitated the anti-tumor effect caused by erastin in vivo. Mechanistically, the phosphorylation levels of Nrf2 and the expression of HMOX1 were reduced due to the downregulation of EGR1, and increased due to the upregulation of EGR1. Additionally, the finding that EGR1 facilitated erastin-induced ferroptosis was alleviated by the inhibition of Nrf2-HMOX1. Conclusion: The expression of EGR1 is downregulated in BC, which is correlated with poor prognosis of BC patients. EGR1 suppresses the proliferation of BC cells and facilitates erastin-induced ferroptosis by activating Nrf2-HMOX1 signaling pathway in BC cells.

Lithocholic acid decreases mRNA stability of nuclear receptor PPARα by upregulating miR-21 expression in hepatoma HepG2 cells

To investigate the regulatory effects of lithocholic acid (LCA) on nuclear receptor peroxisome proliferatoractivated receptor-alpha (PPARα) mRNA stability at the post-transcriptional level. PPARα 3'UTR luciferase reporter gene vectors were constructed and transfected into HepG2 cells to observe the changes in cellular luciferase activity in response to LCA treatments. Bioinformatic prediction and miRNA PCR array technique were used to identify the differentially expressed miRNAs induced by LCA and their potential binding sites on the 3'UTR. The binding sites (Mut1, Mut2 and Mut1+Mut2) were mutated to compare the changes in cellular luciferase activity following LCA treatment. Western blotting and RTqPCR were used to detect the activated signaling pathway and the expression levels of its downstream transcription factors in LCA-treated cells. The changes in PPARα protein expression level were detected in the cells following overexpression of the transcription factors. Treatment with 100 μmol/L LCA significantly reduced luciferase activity of PPARα 3'UTR1 and 3'UTR2 in HepG2 cells by more than 50% (P<0.01) and induced significant upregulation of miR-21 and miR-22, especially the former (by 2.35 folds, P<0.05). Two predicted miR-21-binding sites in the 3'UTR1 were mutated to construct Mut1, Mut2 and Mut1+Mut2 reporter gene vectors. LCA treatment down-regulated 3'UTR1 luciferase activity by 51%, while Mut1, Mut2, and Mut1+Mut2 were down-regulated by 37%, 39%, and 13%, respectively. LCA caused ERK1/2 phosphorylation and activation of the ERK1/2 signaling pathway, and treatment with 100 μmol/L LCA upregulated the expression of transcription factor early growth response 1 (EGR1) by 5.83 folds (P<0.01). Transient overexpression of EGR1 significantly decreased cellular PPARα protein levels (P<0.05). LCA reduces PPARα mRNA stability and thus decreases PPARα mRNA and protein expressions in hepatocytes by activating the ERK1/2 signaling pathway and upregulating EGR1 and miR-21, which targets 3'UTR regulatory region of PPARα mRNA.

The CD2v protein of African swine fever virus inhibits macrophage migration and inflammatory cytokines expression by downregulating EGR1 expression through dampening ERK1/2 activity

African swine fever virus (ASFV) is a highly contagious and deadly virus that leads to high mortality rates in domestic swine populations. Although the envelope protein CD2v of ASFV has been implicated in immunomodulation, the molecular mechanisms underlying CD2v-mediated immunoregulation remain unclear. In this study, we generated a stable CD2v-expressing porcine macrophage (PAM-CD2v) line and investigated the CD2v-dependent transcriptomic landscape using RNA-seq. GO terms enrichment analysis and gene set enrichment analysis revealed that CD2v predominantly affected the organization and assembly process of the extracellular matrix. Wound healing and Transwell assays showed that CD2v inhibited swine macrophage migration. Further investigation revealed a significant decrease in the expression of transcription factor early growth response 1 (EGR1) through inhibiting the activity of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Notably, EGR1 knockout in swine macrophages restricted cell migration, whereas EGR1 overexpression in PAM-CD2v restored the ability of macrophage migration, suggesting that CD2v inhibits swine macrophage motility by downregulating EGR1 expression. Furthermore, we performed chromatin immunoprecipitation and sequencing for EGR1 and the histone mark H3K27 acetylation (H3K27ac), and we found that EGR1 co-localized with the activated histone modification H3K27ac neighboring the transcriptional start sites. Further analysis indicated that EGR1 and H3K27ac co-occupy the promoter regions of cell locomotion-related genes. Finally, by treating various derivatives of swine macrophages with lipopolysaccharides, we showed that depletion of EGR1 decreased the expression of inflammatory cytokines including TNFα, IL1α, IL1β, IL6, and IL8, which play essential roles in inflammation and host immune response. Collectively, our results provide new insights into the immunomodulatory mechanism of ASFV CD2v.

Iguratimod attenuated fibrosis in systemic sclerosis via targeting early growth response 1 expression

BackgroundThe early growth response 1 (EGR1) is a central transcription factor involved in systemic sclerosis (SSc) pathogenesis. Iguratimod is a synthesized anti-rheumatic disease-modifying drug, which shows drastic inhibition to EGR1 expression in B cells. This study is aiming to investigate the anti-fibrotic effect of iguratimod in SSc.MethodsEGR1 was detected by immunofluorescence staining real-time PCR or western blot. Iguratimod was applied in EGR1 overexpressed or knockdown human dermal fibroblast, bleomycin pre-treated mice, tight skin 1 mice, and SSc skin xenografts. RNA sequencing was performed in cultured fibroblast and xenografts to identify the iguratimod regulated genes.ResultsEGR1 overexpressed predominantly in non-immune cells of SSc patients. Iguratimod reduced EGR1 expression in fibroblasts and neutralized changes of EGR1 response genes regulated by TGFβ. The extracellular matrix (ECM) production and activation of fibroblasts were attenuated by iguratimod while EGR1 overexpression reversed this effect of iguratimod. Iguratimod ameliorated the skin fibrosis induced by bleomycin and hypodermal fibrosis in TSK-1 mice. Decreasing in the collagen content as well as the density of EGR1 or TGFβ positive fibroblasts of skin xenografts from naïve SSc patients was observed after local treatment of iguratimod.ConclusionTargeting EGR1 expression is a probable underlying mechanism for the anti-fibrotic effect of iguratimod.

Gene therapy targeting miR‑212‑3p exerts therapeutic effects on MAFLD similar to those of exercise.

Exercise is the main treatment for patients with metabolic‑associated fatty liver disease (MAFLD); however, it may be difficult for some patients to adhere to or tolerate an exercise regime. Thus, finding a treatment alternative to exercise is of particular importance. The authors have previously demonstrated that the high expression of microRNA (miRNA/miR)‑212 promotes lipogenesis invitro. The present study aimed to explore the therapeutic potential, as well as the mechanisms of action of miR‑212 in MAFLD. The expression of miR‑212‑3p, but not that of miR‑212‑5p, was found to be significantly elevated in MAFLD and to be decreased by exercise. Compared with exercise treatment, the inhibition of miR‑212‑3p expression in a mouse model fed a high‑fat diet exerted beneficial effects on MAFLD similar to those of exercise. Conversely, the overexpression of miR‑212‑3p abolished the ameliorative effects of exercise on MAFLD. Fibroblast growth factor 21 (FGF21) and chromodomain helicase DNA binding protein 1 (CHD1) were identified as target genes of miR‑212‑3p in lipid metabolism using bioinformatics analysis. Mechanistically, the inhibition of miR‑212‑3p mimicked the effects of exercise on lipid metabolism by regulating FGF21, but not CHD1. The exercise‑related transcription factor, early growth response 1 (EGR1), was identified upstream of miR‑212‑3p through promoter motif analysis. EGR1 overexpression inhibited miR‑212‑3p expression. The overexpression of miR‑212‑3p abolished the effects of exercise on lipid metabolism by exogenously attenuating the transcriptional repression of EGR1. Moreover, the overexpression of miR‑212‑3p abolished the regulatory effects of EGR1 on FGF21. On the whole, the present study demonstrates that miR‑212‑3p plays a key role in the effects of exercise on MAFLD. The findings presented herein suggest a potential therapeutic effect of targeting miR‑212‑3p in MAFLD.

Inhibition of the MAPK/c-Jun-EGR1 Pathway Decreases Photoreceptor Cell Death in the rd1 Mouse Model for Inherited Retinal Degeneration

Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies that typically results in photoreceptor cell death and vision loss. Here, we explored the effect of early growth response-1 (EGR1) expression on photoreceptor cell death in Pde6brd1 (rd1) mice and its mechanism of action. To this end, single-cell RNA-seq (scRNA-seq) was used to identify differentially expressed genes in rd1 and congenic wild-type (WT) mice. Chromatin immunoprecipitation (ChIP), the dual-luciferase reporter gene assay, and western blotting were used to verify the relationship between EGR1 and poly (ADP-ribose) polymerase-1 (PARP1). Immunofluorescence staining was used to assess PARP1 expression after silencing or overexpression of EGR1. Photoreceptor cell death was assessed using the TUNEL assay following silencing/overexpression of EGR1 or administration of MAPK/c-Jun pathway inhibitors tanzisertib and PD98059. Our results showed differential expression of ERG1 in rd1 and WT mice via scRNA-seq analysis. The ChIP assay demonstrated EGR1 binding to the PARP1 promoter region. The dual-luciferase reporter gene assay and western blotting results revealed that EGR1 upregulated PARP1 expression. Additionally, the TUNEL assay showed that silencing EGR1 effectively reduced photoreceptor cell death. Similarly, the addition of tanzisertib and PD98059 reduced the expression of c-Jun and EGR1 and decreased photoreceptor cell death. Our study revealed that inhibition of the MAPK/c-Jun pathway reduced the expression of EGR1 and PARP1 and prevented photoreceptor cell death. These results highlight the importance of EGR1 for photoreceptor cell death and identify a new avenue for therapeutic interventions in RP.

FOCAD/miR-491-5p, downregulated by EGR1, function as tumor suppressor by inhibiting the proliferation and migration of gastric cancer cells

Gastric cancer is a common malignant tumor in China; however, its carcinogenesis remains unknown. Focadhesin (FOCAD) is a tumor suppressor gene in gliomas, its expression, role, and mechanism in gastric cancer have not been defined. The aim of the present study was to explore the expression pattern of FOCAD in human normal tissues and cancer tissues and elucidate the role and regulatory mechanism of Early Growth Response 1 (EGR1) in FOCAD and its intron, miR-491-5p, in gastric cancer. Immuno histochemical staining revealed that FOCAD is widely and highly expressed in normal gastric mucosa, but is absent in gastric cancer tissue. Based on an association analysis FOCAD expression was found to be negatively associated with lymph node metastasis (P = 0.004); higher FOCAD levels were associated with longer survival in patients with gastric cancer (P = 0.001). MTT, colony, Transwell chamber, and flow cytometry assays revealed that siFOCAD promoted cell proliferation, growth, and migration, and inhibited apoptosis. Furthermore, bioinformatic analysis, Fluorescence reporter gene and chromatin immunoprecipitation analyses confirmed that EGR1 binds to the promoter and negatively regulates FOCAD and miR-491-5p at the transcriptional level. The overexpression of EGR1 was also found to promote cell proliferation, growth, and migration, and inhibit apoptosis. Overall, FOCAD is specifically overexpressed in the gastric mucosa and is significantly downregulated in gastric cancer. To our knowledge, this is the first study to demonstrate that FOCAD is a tumor suppressor, higher FOCAD levels might be a better prognostic marker of gastric cancer, and FOCAD/miR-491-5p may be negatively regulated by EGR1.

Abstract 2777: Characterizing the molecular underpinnings of sorafenib sensitivity in desmoid tumors

Abstract Background: Desmoid tumors (DT) are rare fibroblastic sarcomas that have high recurrence rates. Though we know mutations in the beta-catenin (CTNNB1) gene are commonly observed with dysregulation of the Wnt signaling pathway in patients with DT, the molecular mechanisms driving DT growth remain poorly understood. There is currently no standard systemic treatment for DT. The multi-kinase inhibitor, sorafenib, has been identified as a potential front-line agent for DT after a recent clinical trial demonstrated an objective response in one third of patients. The mechanism for how sorafenib inhibits DT progression is still unclear, so it is unknown which characteristics predict response to the drug. Our work has identified the early growth response-1 (EGR1) gene as a downstream target of sorafenib. Methods: Nine DT cell lines have previously been brought into culture with stable passage. These cell lines were treated with sorafenib at 10 μM. RNA was collected from parental and treated cells and subsequent RNA sequencing was performed. Differential gene expression was performed using R. Appropriate comparisons for multiplicity were performed. Western blots were performed to confirm changes in expression of key genes at the protein level. EGR1 overexpression vector was introduced to the 2 DT cell lines and further treated with sorafenib. Additional banked patient desmoid tumor samples were sequenced to assess EGR1 levels and correlation with the CTNNB1. Results: We first evaluated the effect of sorafenib on CTNNB1 in the DT models. Western blot demonstrated no changes in protein levels of CTNNB1 in the 4 cell lines tested at 72 hours post-treatment. This was further confirmed on RNA-sequencing. To identify additional gene targets of sorafenib, additional differential gene expression analysis was performed resulting in 13 altered genes at an adjusted p &amp;lt; 0.01. The most significantly altered gene, both by p-value and by fold change, was the EGR1 mRNA transcript. EGR1 transcript levels were significantly attenuated by sorafenib treatment (logFC = 2.720, adjusted p-value &amp;lt;&amp;lt; 0.0001). Western blot analysis confirmed this decrease. EGR1-high DT cell line models exhibited faster growth rates and also higher sensitivity to sorafenib. Fifteen additional patient desmoid tumor samples confirmed the steady state of CTNNB1 and high variability of the EGR1 transcript. There was no correlation between the EGR1 and CTNNB1 transcript. Conclusion: This study found that sorafenib does not directly modulate CTNNB1, but does decrease EGR1 expression at both the mRNA and protein level. This suggests that EGR1 expression may be associated with aggressive DT and may also predict responsiveness to treatment with sorafenib. Confirmatory EGR1 modulation experiments are underway and will be provided at time of presentation. Citation Format: Max Devine, Joy Tang, Colin Stets, John Hays, James L. Chen. Characterizing the molecular underpinnings of sorafenib sensitivity in desmoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2777.

EGR1 plays an important role in BMP9-mediated osteoblast differentiation by promoting SMAD1/5 phosphorylation.

Bone morphogenetic proteins (BMPs) are essential regulators of skeletal homeostasis, and BMP9 is the most potently osteogenic among them. Here, we found that BMP9 and BMP2 rapidly induced early growth response 1 (EGR1) protein expression in osteoblasts through MEK/ERK pathway-dependent transcriptional activation. Knock-down of EGR1 using siRNA significantly inhibited BMP9-induced matrix mineralization and osteogenic marker gene expression in osteoblasts. Knock-down of EGR1 significantly reduced SMAD1/5 phosphorylation and inhibited the expression of their transcriptional targets in osteoblasts stimulated by BMP9. In contrast, forced EGR1 overexpression in osteoblasts enhanced BMP9-mediated osteoblast differentiation and SMAD1/5 phosphorylation. An intracellular association between EGR1 and SMAD1/5 was identified using immunoprecipitation assays. These results indicated that EGR1 plays an important role in BMP9-stimulated osteoblast differentiation by enhancing SMAD1/5 phosphorylation.

CircCOL5A1 inhibits proliferation, migration, invasion, and extracellular matrix production of keloid fibroblasts by regulating the miR-877-5p/EGR1 axis

BackgroundCircular RNA (circRNA) has been proved to mediate the biological functions of fibroblasts to participate in the regulation of keloid formation. However, the role of circCOL5A1 in keloid formation remains to be further confirmed. MethodsPrimary keloid fibroblasts were isolated form keloid tissues. The expression of circCOL5A1, microRNA (miR)− 877–5p, and early growth response 1 (EGR1) were determined by quantitative real-time PCR. Transfection experiments were carried out to explore the effects of circCOL5A1, miR-877–5p, and EGR1 on cell functions. Cell proliferation, migration, invasion and apoptosis were detected using cell counting kit 8 assay, colony formation assay, transwell assay and flow cytometry. The protein levels of apoptosis markers, extracellular matrix (ECM) markers and EGR1 were measured by western blot analysis. The mechanism of circCOL5A1 was confirmed by RNA pull-down assay, dual-luciferase reporter assay and RIP assay. ResultsOur data showed that circCOL5A1 was upregulated in keloid tissues and fibroblasts. Silencing of circCOL5A1 had an inhibition effect on proliferation, migration, invasion and ECM production, while had a promotion effect on apoptosis in keloid fibroblasts. MiR-877–5p could be sponged by circCOL5A1, and its overexpression could repress the biological functions of keloid fibroblasts. The rescue experiments showed that miR-877–5p inhibitor could reverse the suppressive effect of circCOL5A1 knockdown on the biological functions of keloid fibroblasts. In addition, EGR1 was a target of miR-877–5p, and its expression was positively regulated by circCOL5A1. The inhibition effect of miR-877–5p on the biological functions of keloid fibroblasts could be abolished by EGR1 overexpression. ConclusionIn summary, circCOL5A1 facilitates keloid fibroblast proliferation, migration, invasion and ECM production through the miR-877–5p/EGR1 axis, thereby potentially promoting keloid formation.

Methylation of the tyrosine hydroxylase gene is dysregulated by cocaine dependence in the human striatum

SummaryCocaine dependence is a chronic, relapsing disorder caused by lasting changes in the brain. Animal studies have identified cocaine-related alterations in striatal DNA methylation; however, it is unclear how methylation is related to cocaine dependence in humans. We generated methylomic profiles of the nucleus accumbens using human postmortem brains from a cohort of individuals with cocaine dependence and healthy controls (n = 25 per group). We found hypermethylation in a cluster of CpGs within the gene body of tyrosine hydroxylase (TH), containing a putative binding site for the early growth response 1 (EGR1) transcription factor, which is hypermethylated in the caudate nucleus of cocaine-dependent individuals. We replicated this finding and found it to be specific to striatal neuronal nuclei. Furthermore, this locus demonstrates enhancer activity which is attenuated by methylation and enhanced by EGR1 overexpression. These results suggest that cocaine dependence alters the epigenetic regulation of dopaminergic signaling genes.

Early Growth Response (EGR)-1 Is a Key Regulator of Human Primary Bone Marrow Stroma Cells with a Dual Role in Proliferation and Hematopoietic Stroma Support Function

Hematopoietic stem cell (HSC) niches are specialized local tissue microenvironments that maintain HSCs and regulate HSC functions. Bone marrow mesenchymal stroma cells (BMSCs) are essential constituents of the HSC niche and they give rise to skeletal lineage cells as well as the hematopoietic stroma. Despite recent progress in the identification of the primary bone fide BMSC in human bone marrow, knowledge regarding the regulation of BMSC proliferation, differentiation and hematopoietic support function is incomplete.We have previously shown that the expression of Early Growth Response 1 (EGR1) in primary BMSCs is substantially higher compared to the non-colony-forming cells and cultured stromal cells, respectively, indicating a possible role of EGR1 in BMSC regulation (Li et al., Stem Cell Reports, 3(6):965-74, 2014). EGR1 encodes a zinc finger transcription factor that exemplifies a group of immediate early response genes, which have diverse functions in cell growth, development and stress response. EGR1 regulates murine HSC proliferation and localization (Min et al., Cell Stem Cell, 2(4):380-91, 2008), but its role in stroma cell regulation has thus far not been investigated. This study therefore aimed to investigate the functional role of EGR1 in human primary BMSCs.Quantitative gene expression analysis demonstrated that EGR1 expression was 126.9 ± 9.1-fold and 2.8 ± 0.2-fold higher in highly CFU-F (colony-forming unit, fibroblast) enriched human primary linneg/CD45neg/CD271pos/CD140aneg bone marrow cells compared to the non-colony forming CD45neg/CD271neg cell population and CD45neg/CD271pos/CD140apos cells with minimum CFU-F activity. Furthermore, we confirmed that EGR1 expression in cultured cells was down-regulated in comparison with primary BMSCs.Multi-lineage differentiation capacity and surface marker expression profiles of BMSCs were not affected by shRNA-induced EGR1 down-regulation. However, colony-forming capacity was enhanced (1.9 ± 0.1-fold) and population doubling times were reduced by EGR1 knockdown compared to scramble controls. Furthermore, the HSC stroma supporting function as measured by production of transplantable HSC in co-culture experiments (4-day serum-free culture supplemented with SCF 25 ng/ml, TPO 25 ng/ml, and FLT3L 25 ng/ml, 1×104 CD34pos seeded cord blood cells) was reduced when EGR1 knockdown cells were used as stroma layer.On the other hand, whereas CFU-F formation was virtually absent in EGR1 overexpressing cells and population doubling times increased by 2.4 ± 0.3-fold, the hematopoietic stroma support function was considerably improved by EGR1 overexpression as indicated by increased percentages and absolute numbers of transplantable CD34posCD90pos cells produced in co-cultures with EGR1 overexpressing BMSCs as stromal cells (12,496 ± 887 for EGR1 overexpression vs 6,867 ± 1242 for GFP control). Accordingly, CD34pos cells expanded on EGR1 overexpressing stroma exhibited improved in-vivo long-term reconstitution levels in NSG mice compared with cells grown on control stroma.To investigate how EGR1 regulates proliferation and hematopoiesis support in BMSCs, transwell experiments were performed showing that both soluble factors and cell-to-cell contact were important for EGR1-mediated hematopoiesis support. Furthermore, proteomic analysis revealed that a group of proteins related to oxidative-reduction processes was downregulated whereas reactive oxygen species (ROS) levels were elevated in EGR1 knockdown cells. In addition, gene expression profiling analysis identified a number of hematopoietic support genes that were upregulated in EGR1 overexpressing cells compared with control cells.In summary, our data indicate that EGR1 is an important BMSC regulator with a dual role on proliferation and hematopoietic stroma support function. EGR1 expression negatively regulated BMSC proliferation while positively regulating hematopoietic support function, which points to its possible role in coordinating the specific functions of BMSC in their different biological contexts. DisclosuresNo relevant conflicts of interest to declare.

EGR1/GADD45α Activation by ROS of Non-Thermal Plasma Mediates Cell Death in Thyroid Carcinoma.

Simple SummaryRecent studies have identified new anti-cancer mechanisms of nonthermal plasma (NTP) in several cancers. However, the molecular mechanisms underlying its therapeutic effect on thyroid cancer have not been elucidated. The objective of this study was to understand the anticancer effects of NTP-activated medium (NTPAM) on thyroid cancer cells and elucidate the signaling mechanisms responsible for NTPAM-induced thyroid cancer cell death.(1) Background: Nonthermal plasma (NTP) induces cell death in various types of cancer cells, providing a promising alternative treatment strategy. Although recent studies have identified new mechanisms of NTP in several cancers, the molecular mechanisms underlying its therapeutic effect on thyroid cancer (THCA) have not been elucidated. (2) Methods: To investigate the mechanism of NTP-induced cell death, THCA cell lines were treated with NTP-activated medium -(NTPAM), and gene expression profiles were evaluated using RNA sequencing. (3) Results: NTPAM upregulated the gene expression of early growth response 1 (EGR1). NTPAM-induced THCA cell death was enhanced by EGR1 overexpression, whereas EGR1 small interfering RNA had the opposite effect. NTPAM-derived reactive oxygen species (ROS) affected EGR1 expression and apoptotic cell death in THCA. NTPAM also induced the gene expression of growth arrest and regulation of DNA damage-inducible 45α (GADD45A) gene, and EGR1 regulated GADD45A through direct binding to its promoter. In xenograft in vivo tumor models, NTPAM inhibited tumor progression of THCA by increasing EGR1 levels. (4) Conclusions: Our findings suggest that NTPAM induces apoptotic cell death in THCA through a novel mechanism by which NTPAM-induced ROS activates EGR1/GADD45α signaling. Furthermore, our data provide evidence that the regulation of the EGR1/GADD45α axis can be a novel strategy for the treatment of THCA.

DNA Methylation of a Novel Regulatory Element Within the Tyrosine Hydroxylase Gene (TH) is Dysregulated by Chronic Cocaine Dependence in the Human Striatum

Cocaine dependence is a chronic, relapsing disorder caused by lasting changes within the brain’s reward circuitry. Methylome-wide studies have identified multiple biological pathways that contain altered DNA methylation dynamics in the striatum of rodents; however, there is a lack of information about the involvement of DNA methylation in cocaine dependence in humans. Here, we generated genome-wide methylomic profiles for the nucleus accumbens, part of the ventral striatum, using human postmortem brains from a cohort of cocaine dependent and healthy controls (n=25 per group). In particular, we have identified hypermethylation of a cluster of CpGs within the gene body of tyrosine hydroxylase gene (TH), which contains a putative binding site for the early growth response 1 (EGR1) transcription factor. We replicated this finding in striatal tissue from an independent cohort (n=18 per group) and found it to be specific to striatal neuronal nuclei. Furthermore, this locus demonstrates enhancer activity in vitro, which is attenuated by methylation and enhanced by EGR1 overexpression. This is the first time that epigenetic dysregulation has been associated with cocaine dependence with respect to dopaminergic signaling and represents an important advancement in our understanding of drug neurobiology in humans.

Caveolin-1 regulates the expression of miR-183 and inhibits the invasion and migration of invasive pituitary adenomas by affecting early growth response 1 (EGR1)/Krueppel-like factor 5 (KLF5) interaction

This study aims to investigate the mechanism through which Caveolin-1 (CAV-1) regulates the expression of micro ribonucleic acid (miR)-183 in invasive pituitary adenoma (IPA) tissues and GH3 cells, and explore the effects of CAV-1 and miR-183 on the invasion and migration ability of GH3 cells. Western blotting was used to detect the expression level of CAV-1, early growth response 1 (EGR1) and Krueppel-like factor 5 (KLF5). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of miR-183. The mechanisms of interaction between CAV-1, EGR1, and KLF5 were studied by immunoprecipitation experiments. Transwell and cell scratch tests were used to determine the invasion and migration ability of GH3 cells. The dual-luciferase reporter gene experiment was used to detect the effects of EGR1 and KLF5 on miR-183 luciferase activity and verify the targeting relationship between miR-183 and ezrin. The expression of CAV-1 was up-regulated. However, following the knockdown of CAV-1, the invasion and migration ability of GH3 cells was significantly inhibited (P<0.05). The expression of miR-183 was down-regulated, but the expression level of miR-183 was markedly increased following the knockdown of CAV-1 (P<0.05). The knockdown of CAV-1 inhibited the nuclear ectopic of the EGR1 protein in GH3 cells. At the same time, the interaction between EGR1 and KLF5 in GH3 cells was significantly inhibited (P<0.05). The luciferase activity of miR-183 increased significantly after overexpression of KLF5 while overexpression of EGR1 and KLF5 had no significant effect on intracellular luciferase activity. Overexpression of miR-183 markedly inhibited the luciferase activity of wild-type EZR and the expression of the EZR protein in GH3 cells. Furthermore, the overexpression of miR-183 or the inhibition of EZR can reduce the invasion and migration ability of GH3 cells. The simultaneous overexpression or inhibition of miR-183 and EZR expression has no obvious effect on the invasion and migration ability of GH3 cells. CAV-1 up-regulates the expression of miR-183 by inhibiting the nuclear ectopic of EGR1 and the interaction between EGR1 and KLF5 in GH3 cells. Also, miR-183 negatively regulates the expression of EZR and inhibits the invasion and migration of GH3 cells.

Lnc003875/miR-363/EGR1 regulatory network in the carcinoma -associated fibroblasts controls the angiogenesis of human placental site trophoblastic tumor (PSTT)

The rare gestational trophoblastic neoplasia placental site trophoblastic tumor (PSTT) frequently demonstrates a high degree of vascularization, which may facilitate the tumor metastasis. However, the underlying mechanisms remain largely unknown. In the present study, we found that early growth response 1 (EGR1) was highly expressed in the carcinoma-associated fibroblasts (CAFs) of PSTT tissues. Further data showed that miR-363 down-regulated EGR1 expression whereas long non-coding RNA NONHSAT003875 (lnc003875) up-regulated EGR1 expression in PSTT derived CAFs. lnc003875 exerted no effect on miR-363 expression, but it recovered the decrease of EGR1 caused by miR-363 mimic. The conditioned media from PSTT CAFs treated with miR-363 mimic abrogated the tube formation capacity of human umbilical vein endothelial cells (HUVECs), which can be partially restored by lnc003875 over-expression. Moreover, over-expression of EGR1 promoted the secretion of Angiopoietin-1 (Ang-1) in PSTT derived CAFs and improved the tube formation of HUVECs, which could be effectively abrogated by Ang-1 siRNAs. In vivo vasculogenesis assay demonstrated that lnc003875/EGR1 in PSTT derived CAFs promoted the vasculogenesis of HUVECs in C57BL/6 mice. Collectively, these findings indicated that lnc003875/miR-363/EGR1/Ang-1 in CAFs may be crucial for the angiogenesis of PSTT.

EGR1 IS NEGATIVELY ASSOCIATED WITH HNSCC CELL INVASION VIA INHIBITION OF MMP9 AND MDM2

Objectives The effect of early growth response-1 (EGR1) on cancer invasion remains controversial depending on the cancer type. EGR1 is known to slow the progression of cancer by inhibiting the expression of MMP2. However, the effect of EGR1 on MMP9, which is important for HNSCC invasion, is disputed. Our aim is to clarify the tumor suppressor role of EGR1 in downregulating MMP9. We also consider MDM2, an enhancer of MMP9 expression. Findings EGR1 mRNA and protein expression were compared in normal and HNSCC tissues using The Cancer Genome Atlas (TCGA) dataset analysis as well as immunohistochemistry (IHC). In vitro cell invasion was performed by two-dimension (2-D) and three-dimension (3-D) spheroids Matrigel invasion assay. TCGA data showed significantly higher EGR1 mRNA levels in nonmetastatic HNSCC tissues than in metastatic tissues. IHC analysis showed significantly higher levels of nuclear EGR1 expression in primary tumor tissues than in paired metastatic lymph node tissues. Transient EGR1 overexpression inhibited the Matrigel invasion of HNSCC cells, as well as decreasing mRNA of MMP9 and MDM2. Consistent with these observations, TCGA data analysis found significantly fewer metastatic patients among a subgroup of a large population presenting higher EGR1 expressions with lower MMP9 and/or MDM2. Conclusions Our data suggests that EGR1 might be a potential candidate to attenuate HNSCC metastasis.

Cellular microRNA bta-miR-2361 inhibits bovine herpesvirus 1 replication by directly targeting EGR1 gene

Bovine herpesvirus 1 (BHV-1) is an economically important pathogen of cattle and has led to significant consequences on the cattle industry worldwide. MicroRNAs (miRNAs) are a class of regulators that play critical roles in virus and host interaction. However, the roles of host miRNAs in BHV-1 infection remain largely unclear. In this study, a set of differentially expressed miRNAs by small RNA deep sequencing were analyzed in the Madin-Darby Bovine Kidney Cells (MDBK) infected with BHV-1 after 12 h, 24 h and 48 h post-infection compared to mock infection, and it was confirmed that bta-miR-2361 was significantly down-regulated. Moreover, bta-miR-2361 mimics transfection could inhibit BHV-1 replication. Combined with up-regulated genes from BHV-1-infected MDBK cells by deep RNA-sequencing and predicted by bioinformatics tools, early growth response 1 (EGR1) was putative target of bta-miR-2361. Furthermore, EGR1 was up-regulated during BHV-1 infection, and overexpression of EGR1 promoted BHV-1 replication whereas knockdown of EGR1 had the opposite effects. Subsequently, the target association between bta-miR-2361 and 3′UTR of EGR1 was further validated using a dual-luciferase reporter assay. In addition, overexpression of bta-miR-2361 resulted in decreased EGR1 mRNA and protein levels. Further mechanistic study showed that EGR1 stimulated BHV-1 UL46 promoter activity, but overexpression of bta-miR-2361 suppressed the production of UL46 gene. Collectively, this is the first study to reveal that bta-miR-2361 as a novel host factor regulates BHV-1 replication via directly targeting the EGR1 gene, which is a transcription factor that regulates viral UL46 gene of BHV-1. These results provide further insight into the study of BHV-1 pathogenesis.

TCF7L2 and EGR1 synergistic activation of transcription of LCN2 via an ERK1/2-dependent pathway in esophageal squamous cell carcinoma cells

High level expression of lipocalin 2 (LCN2) usually indicates poor prognosis in esophageal squamous cell carcinoma (ESCC) and many other cancers. Our previous study showed LCN2 promotes migration and invasion of ESCC cells through a novel positive feedback loop. However, the key transcription activation protein (KTAP) in the loop had not yet been identified. In this study, we first predicted the most probable KTAPs by bioinformatic analysis. We then assessed the transcription regulatory regions in the human LCN2 gene by fusing deletions of its 5′-flanking region to a dual-luciferase reporter. We found that the region −720/−200 containing transcription factor 7-like 2 (TCF7L2) (−273/−209) and early growth response 1 (EGR1) (−710/−616) binding sites is crucial for LCN2 promoter activity. Chromatin immunoprecipitation (ChIP) experiments demonstrated that TCF7L2 and EGR1 bound directly to their binding sites within the LCN2 promoter as KTAPs. Mechanistically, overexpression of TCF7L2 and EGR1 increased endogenous LCN2 expression via the ERK signaling pathway. Treatment with recombinant human LCN2 protein enhanced activation of the ERK pathway to facilitate endogenous LCN2 expression, as well as increase the expression level of TCF7L2 and EGR1. Treatment with the MEK inhibitor U0126 inhibited the activation by TCF7L2 or EGR1 overexpression. Moreover, overexpression of TCF7L2 or EGR1 accelerated the migration and invasion of ESCC cells. A synergistic effect was observed between TCF7L2 and EGR1 in amplifying the induction of LCN2 and enhancing migration and invasion. Taken together, our study indicates that TCF7L2 and EGR1 are the KTAPs of LCN2, within a positive “LCN2 → MEK/ERK → LCN2” path, to promote the migration and invasion of ESCC cells. Based on their clinicopathological significance, LCN2 and its two expression regulators TCF7L2 and ERG1 might be therapeutic targets for ESCC.

The effect of early growth response 1 on levels of Amyloid‐β 40 peptide in U87MG cells

A recent study has shown that early growth response 1 (EGR1) plays a critical role in the β-amyloid cascade and tau hypotheses. In addition, evidence has suggested that EGR1 can regulate levels of amyloid-beta peptides, key molecules in the pathogenesis of Alzheimer's disease (AD). However, whether EGR1 is a deleterious or protective factor in the AD is still controversial. In thispresent study, we constructed an overexpression plasmid, CMV-EGFP-EGR1-Kanamycin, and transfected it into U87MG cells to investigate the effects of EGR1 expression on amyloid-β (1-40) peptide (Aβ40) levels. U87MG cells transfected by CMV-EGFP-EGR1-Kanamycin and CMV-EGFP-Kanamycin were assigned, respectively, to experimental and control groups. Fluorescence microscopy was used to observe transfection efficiencies of the plasmids after 6 hours. EGR1 messenger RNA levels were measured by quantitative reverse transcription polymerase chain reaction. Aβ40 secretion was analyzed byenzyme-linked immunosorbent assay. Expression of the amyloid precursor protein, beta-secretase enzyme, and presenilin 1 proteins were analyzed by Western blot analysis. The results showed that EGR1 overexpression increased Aβ40 secretion in vitro, possibly through increasing BACE1 expression. Based on these results, EGR1 might be a promising therapeutic target for the AD.