Inhibition of Proliferation, Migration, and Invasion of Keloid Fibroblasts By miR-183-5p Through Downregulating EGR1 / Inhibición de la Proliferación, Migración e Invasión de Fibroblastos Queloides por miR-183-5p Mediante la Regulación Negativa de EGR1

SUMMARY: Keloid scar is a unique benign fibroproliferative tumor of the human skin. Previously, it was reported that early growth response 1 (EGR1), a transcription factor, promotes keloid fibrosis; however, the mechanism by which EGR1 modulates keloid formation was not elaborated. In this research, the specific function and the microRNA (miRNA) regulatory network of EGR1 in keloids was examined. Keloid fibroblasts (KFs) were transfected with EGR1-small interfering RNA (siEGR1), EGR1-overexpression plasmid (pcDNA3.1-EGR1), and microRNA (miR-183-5p)-mimics to regulate the expression of EGR1 and miR-183-5p. The study employed dual-luciferase reporter assays to explore the targeting regulation of miR-183-5p on EGR1. Additionally, Western blotting, flow cytometry, qRT-PCR, cell count kit-8 (CCK-8), transwell, and wound healing assays, and RNA sequencing were conducted. EGR1 was upregulated in KFs, and EGR1 silencing diminished proliferation, fibrosis, migration, invasion, and apoptosis of cells. In KFs, the expression of miR- 183-5p was reduced, leading to the inhibition of cell proliferation, migration, and invasion. Conversely, it enhanced apoptosis. By targeting EGR1, miR-183-5p partially counteracted the impact of EGR1 on migration, invasion, and fibrosis in KFs. The findings imply that miR-183-5p suppresses keloid formation by targeting EGR1. As a result, EGR1 holds promise as a potential therapeutic target for preventing and treating keloids.

La cicatriz queloide es un tumor fibroproliferativo benigno único de la piel humana. Anteriormente, se informó que la respuesta de crecimiento temprano 1 (EGR1), un factor de transcripción, promueve la fibrosis queloide; sin embargo, no se explicó el mecanismo por el cual EGR1 modula la formación de queloides. En esta investigación, se examinó la función específica y la red reguladora de microARN (miARN) de EGR1 en queloides. Se transfectaron fibroblastos queloides (KF) con ARN de interferencia pequeño de EGR1 (siEGR1), plásmido de sobreexpresión de EGR1 (pcDNA3.1-EGR1) y miméticos de microARN (miR-183-5p) para regular la expresión de EGR1 y miR-183. -5p. El estudio empleó ensayos de indicador de luciferasa dual para explorar la regulación dirigida de miR-183-5p en EGR1. Además, se realizaron pruebas de transferencia Western, citometría de flujo, qRT-PCR, kit de recuento celular-8 (CCK-8), transwell y curación de heridas, y secuenciación de ARN. EGR1 estaba regulado positivamente en KF, y el silenciamiento de EGR1 disminuyó la proliferación, fibrosis, migración, invasión y apoptosis de las células. En KF, la expresión de miR- 183-5p se redujo, lo que llevó a la inhibición de la proliferación, migración e invasión celular. Por el contrario, mejoró la apoptosis. Al apuntar a EGR1, miR-183-5p contrarrestó parcialmente el impacto de EGR1 en la migración, invasión y fibrosis en KF. Los hallazgos implican que miR-183-5p suprime la formación de queloides al apuntar a EGR1. Como resultado, EGR1 es prometedor como objetivo terapéutico potencial para prevenir y tratar los queloides.

Egr-1 Enhances Drug Resistance of Breast Cancer Cells by MDR1 Dependence

Abstract Paclitaxel (PTX) is one of the most effective drugs used in the treatment of breast cancer. Nonetheless, the appearance of MDR1 (multidrug resistance 1) in tumor cells has become a significant hindrance for efficacious chemotherapy. In this study, we show that the expression level of Egr-1 (early growth response gene-1) in cancer tissues (from paclitaxel chemotherapy failure patients) and MCF-7/PTX cells (the breast cancer cell line that was resistant to paclitaxel) was increased. Cell proliferation assay and apoptosis assay revealed that Egr-1 could promote cell growth and inhibit apoptosis in MCF-7/PTX. Mechanistic studies indicated that Egr-1 could bind to the proximal MDR1 promoter and enhance MDR1 transcription. These findings indicate that paclitaxel induced Egr-1 accumulation and upregulated the expression of MDR1, thereby inducing the drug resistance in MCF-7/PTX. Our results suggest a novel pathway by which paclitaxel induces MDR1 expression, possibly illuminating a potential target pathway for the prevention of MDR1-mediated drug resistance.

Roles of c-Fos, EGR-1, PKA, and PKC in cognitive dysfunction in rats after propofol anesthesia

Abstract This study aimed to investigate possible changes in the spatial memory of rats and the expression or activity of EGR-1, c-Fos, PKA, and PKC after propofol anesthesia. Thirty-six Sprague-Dawley rats aged 20 months and 36 Sprague-Dawley rats aged three months were each randomly divided into three groups: the control group, the Morris Water Maze (MWM) group, and the propofol group. In the propofol groups of both young and aged rats, the rats were anesthetized by propofol for two or four hours and then performed the MWM test two days or two weeks after anesthesia to assess cognitive function. EGR-1, c-Fos, PKA, and PKC expressions in the rat hippocampus were determined via immunohistochemistry. For the older rats, the escape latency in the P4h/2d group was significantly prolonged (P < 0.05), and the learning curve was right-shifted in the P4h/2w group (P < 0.05). The expression levels of EGR-1, c-Fos, PKA, and PKC in the MWM groups were significantly higher than those in the control groups (P < 0.05). In the P4h/2d group of aged rats, the expression levels of both PKA and PKC were decreased compared with those of the MWM groups. The decreased expression of both protein kinases may be responsible for the observed impairment after propofol anesthesia

The neuroprotective effect of p-benzoyl salidroside, a derivative of salidroside, on MCAO model rats / 中国药理学通报

Aim To study the neuroprotective effect of p-benzoyl salidroside (pOBz), a derivative of salidroside, on MCAO model rats.Methods ( 1 ) Thirty healthy adult male SD rats were randomly divided into Sham group, MCAO group and MCAO + pOBz group (25, 50, 1(X) mg • kg"1).MCAO model was made by suture-embolus method.The rats were scored for neurological function impairment and weighed every day.pOBz was intraperitoneally injected and administered continuously for two days after preparation of MCAO model.The cerebral infarction volume of rats was detected by MRI.( 2 ) Twenty-four healthy adult male SD rats were randomly divided into Sham group, MCAO group, MCAO + pOBz group (50 mg • kg"1 ) and MCAO + Sal group (50 mg • kg 1 ).The model was made by the suture-embolus method.pOBz was in-traperitoneally injected and administered continuously for one day.Western blot was used to detect the ex pression of NeuN, EGR1 , Bcl-2 and Bax.(3) Eighteen healthy adult male SD rats were randomly divided j j into Sham group, MCAO group and MCAO + pOBz group ( 50 mg • kg 1 ).Administration continued for 2 days.Immunofluorescence staining was used to detect the expression of NeuN.Results Intraperitoneal injection of pOBz for 2 days could reduce the cerebral infarction volume of MCAO rats, improve neurological impairment and increase the expression of NeuN and EGR1 , and the effect was better than that of Sal.pOBz improved Bcl-2/Bax in brain tissues of MCAO rats to the same extent as Sal did.Conclusions pOBz can reduce the volume of cerebral infarction in MCAO rats and has better neuroprotective effect than that of salidroside.

Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo

BACKGROUND: PGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFß1, which plays an important role during luteal regression. METHODS: The current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGFß1 during PGF2α induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2α for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3ßHSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2α induced luteal regression via induction of TGFß1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9. RESULT: The present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2α treatment. Quantification of EGR1 and TGFß1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2α induction. In order to validate the role of PGF2α on stimulating the expression of TGFß1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2α and it was observed that EGR1 KO did not modulate the PGF2α induced expression of TGFß1. In PGF2α treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2α treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2α treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2α-treated wild type luteal cells. CONCLUSION: These results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2α induced TGFß1 signaling for luteolysis.

Effect of Yanghetang on Apoptosis of Triple Negative Breast Cancer Cell Line MDA-MB-231 Based on Egr1/p21 Signaling Pathway / 中国实验方剂学杂志

Objective::To investigate the effect of ancient recipe Yanghetang and its drug-contained serum on apoptosis of triple negative breast cancer cell line MDA-MB-231 based on the signal pathway of early growth response gene 1 (Egr1)/cyclin dependent inhibitor (p21). Method::The drug-containing serum of Yanghetang was prepared from rats, and MDA-MB-231 cells were cultured in vitro. The blank control group was set, and MDA-MB-231 cells in Yanghetang high, middle, and low dose groups (23.4, 11.7, 5.85 g·kg-1) were intervened for 24, 48, 72 h, respectively. After that, the cell counting kit-8(CCK-8) method was used to detect the cell proliferation of each group. The blank control group was set, while MDA-MB-231 cells in Yanghetang high, middle, and low dose groups (23.4, 11.7, 5.85 g·kg-1) were treated for 48 h, and then flow cytometry was used to detect the apoptosis of each group and the distribution of cell cycle. The expression of Egr1 and p21 mRNA in each group was detected by quantificational Real-time polymerase chain reaction (Real-time PCR), while the expression of Egr1 and p21 protein in each group was detected by Western blot. Result::After MDA-MB-231 cells were intervened by Yanghetang for 24, 48, 72 h, MDA-MB-231 cell proliferation was significantly inhibited in Yanghetang high and middle dose groups as compared with the blank control group (P<0.01). After MDA-MB-231 cells were intervened by Yanghetang for 48 h, the apoptosis was significantly increased in Yanghetang high and middle dose groups as compared with the blank control group (P<0.05, P<0.01). In the Yanghetang high, middle dose groups, the proportion of cell cycle G0/G1 phase decreased, and the proportion of G2/M phase increased (P<0.05, P<0.01). The mRNA and protein expressions of Egr1, p21 were increased in Yanghetang high and middle groups (P<0.05, P<0.01). Conclusion::Yanghetang can activate Egr1/p21 signaling pathway in MDA-MB-231 cells, increase the expression of Egr1 and p21, and cause G2/M cell cycle arrest, thereby inducing apoptosis of MDA-MB-231 cells.

Association analysis of EGR1 gene and Alzheimer's disease in Han Chinese people / 上海交通大学学报（医学版）

Objective: To investigate the association between early growth response gene 1 (EGR1) and Alzheimer's disease (AD) in Han Chinese people. Methods: A total of 715 AD patients and 760 health controls were recruited in two independent samples from Eastern China (382 AD patients and 426 normal individuals) and Southwest China (333 AD patients and 334 normal individuals). SNaPshot technique was utilized to analyse the single nucleotide polymorphism (SNP) of rs11743810. A public database was used to explore whether EGR1 gene was differentially expressed in the brain of AD patients and health controls. Then the protein-protein interaction (PPI) assessment was conducted using the STRING database, and the brain eQTL (expression quantitative trait loci) analysis was used to explore the difference in rs11743810 expression between different genotypes in different brain regions. Results: Cross-platform normalized data showed that there was significant difference of EGR1 expression in temporal cortex between AD patients and control subjects (|log2FC|=0.780, P=0.000 before FDR corrected; P=0.001 after FDR corrected). PPI analysis revealed that EGR1 was physically connected with amyloid precursor protein (APP) and clusterin (CLU) protein in the network. However, different genotypes of rs11743810 showed no significant difference in expression in 10 brain regions, and no significant difference in the genotype and allele frequency of rs11743810 between AD patients and controls were found in our two independent samples. Conclusion: The rs11743810 in EGR1 may not be major susceptibility gene site for AD in Han Chinese people.

Neuroprotective effect of salidroside on CoCl2 - Induced hypoxia injury in PC12 cells / 中国药理学通报

Aim: To investigate the neuroprotective effect of salidroside on CoCl2 -induced hypoxia injury in PC 12 cells, and further explore the mechanism of salidroside on inhibiting complement and neuroprotection. Methods: The model of CoCl2-induced hypoxia injury in PC 12 cells was established. The expressions of Egrl, Egr4, C3 and the activity of caspase-3 were assessed with different concentrations of salidroside treatment. The expressions of Egrl, Egr4 and the activity of caspase-3 were tested with C3ar inhibitor and salidroside treatment. The expressions of Bax, Bcl-xl, C3 and the activity of caspase-3 were determined with Egr4 siRNA and salidroside treatment. Results: Salidroside significantly reduced the caspase-3 activity and the protein expression of Bax and C3, and improved the expression of Bcl-xl, Egrl, and Egr4 proteins in CoCl2-induced PC12 cells. Salidroside reduced the caspase-3 activity and the expression of Egrl, Egr4 with C3ar inhibitor treatment. With siRNA interference with the expression of Egr4, salidroside reversed the activity of caspase-3, Bax and Bcl-xl, but the effect on C3 was not obvious. Conclusions: Salidroside has neuroprotective effect on the model of CoCl2 -induced hypoxia injury in PC12 cells, which is related to the inhibition of complement component C3 and the improvement of the expression of Egr4 by salidroside.

Construction of recombinant plasmids Egr1-XPO4 and its synergic inhibition with 5-FU against hepatocarcinoma SK-Hep1 cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To construct recombinant plasmid Egr1-XPO4 and evaluate its synergic inhibition with 5-FU against hepatocarcinoma SK-Hep1 cells. Methods: The XPO4 gene was inserted into vector carrying promoter Egr1 to construct a new recombinant vector, Egr1-XPO4, which was then transfected into human hepatocarcinoma cell line SK-Hep1 and sensitized with chemotherapeutic drug 5-FU. Western blotting was adopted to examine the protein expression of XPO4; CCK assay was used to detect SK-Hep1 cell proliferation after transfection, and Flow Cytometry with Annexin V-FITC/PI staining was used to detect the apoptosis of SK-Hep1 cells. SKHep1 cell xenograft model was constructed on nude mice, and the effect of Egr1-XPO4 in combination with 5-FU on the growth of xenograft was observed. Results: The recombinant plasmid Egr1-XPO4 was successfully constructed.With the sensitization of 5-FU, the expression of XPO4 protein in SK-Hep1 cells was significantly elevated after Egr1-XPO4 transfection, and the evlevation was in a 5FU dose-depend manner.The combined treatment of Egr1-XPO4 and 5-FU produced a significantly stronger inhibition against SKHep1 cell proliferation and greatly promoted apoptosis of SK-Hep1cells compared with 5-FU or pEgr-XPO4 mono-treatment group (all P<0.05). And in vivo antitumor experiment showed that the tumor volume in Egr1-XPO4+5-FU treatment group was significantly smaller than that of Egr1-XPO4 or 5-FU mono-treatment group (P<0.05). Conclusion: The recombinant plasmid Egr1-XPO4 in combination with 5-FU could exertsynergic inhibitionagainst hepatocarcinomaSK-Hep1 cells.

Effect of miR-191 on the proliferation of malignant meningioma cell through EGR1/TP53 signaling pathway / 中华内分泌外科杂志

Objective To study the influence of miR-191 expression on the proliferation of human malignant meningioma cell line IOMM-Lee in vitro and to explore its mechanism.Methods The expression of miR-191 in malignant meningioma tissue,the adjacent normal tissues and human Malignant meningioma cell lines IOMM-Lee and CH157-MN was tested by Realtime PCR.miR-191 inhibitor was transfected in IOMM-Lee cells and MTT assay was employed to detect the cell viability.Bioinformatics prediction software was used in miR-191 target gene predictive analysis and verified by luciferase reporter system.The effect of EGR1 siRNA on the proliferation of IOMM-Lee cells was observed.Prorein interaction database was used to analyze which proteins could interact with EGR1.The effect of inhibition of EGR1 expression on TP53 protein expression was detected.The influence of inhibition of miR-191 expression on EGR1and TP53 expression was observed.Result The expression of miR-191 in malignant meningioma tissue (0.933±0.144) was higher than that in the adjacent normal tissue (0.459±0.104,P＜0.05).The expressiong of miR-191 in humam malignant meningioma cell line IOMM-Lee (1.25±0.07) was higher than that in CH157-MN cell line (0.50±0.14,P＜0.05).The cell proliferation capability was significantly decreased in miR-191 inhibitor group [(0.53±0.02) vs (0.74±0.01),P＜0.05].EGR1 was identified and validated to be a target gene of miR-191.Inhibition of EGR1 gene can promote OMM-Lee cell proliferation (0.83±0.02,0.71 ±0.01,P＜0.05).EGR 1 could positively regulate TP53 protein expression [(13 758.17±57.22) vs (10 239.00±71.30),P＜0.001.miR-191 Inhibition could increase EGR1 [(14 663.00±80.08) vs (11 184.33±153.90),P＜0.001] and TP53 expression [(15 206.17±102.08) vs(11 400.17±97.00),P＜0.001].Conclusion Downregulation of miR-191 can inhibit the proliferation of IOMM-Lee cell,which may be related to the upregulation of EGR1/TP53 signaling pathway.

Effects of intraoperative expansion on expression of stenosis-related genes in saphenous vein graft / 局解手术学杂志

Objective To investigate the effect of high pressure distention on the expression of stenosis-related genes of saphenous vein graft(SVG) during the coronary artery bypass grafting(CABG).Methods The biopsy specimens of saphenous vein collected from 10 patients who have undergone CABG,were divided into expansion group and no-expansion group.Real-time PCR and immunohistochemical staining were performed for examination of mRNA and protein expression of VE-cad,Egr-1,VCAN respectively.Student's t and Chi-square test were used to do statistic analysis.Results The results of RT-PCR showed that the mRNA transcription of Egr-1,VCAN in the expansion group were statistically significantly higher than those in no-expansion group(P<0.05).The mRNA transcription VE-cad in expansion group was statistically significantly lower than that in the no-expansion group(P<0.05).The immunohistochemical staining results showed that the expression of Egr-1 and VCAN in expansion group were significantly stronger than those in no-expansion group,while the expression of VE-cad was significantly lower than no-expansion group.Conclusion The intraoperative expansion of SVG can increase the expression of stenosis-related genes Egr-1 and Versican,and decrease the expression of stenosis-related gene VE-cad,which may be related with the SVG stenosis after CABG.

The molecular mechanism of microRNA-181b in regulatingthe schizophrenia susceptibility gene EGR3 / 西安交通大学学报(医学版)

Objective To verify whether early growth response-3(EGR3) gene is targeted by microRNA-181b using molecular biology methods so as to provide guidance for the subsequent study on microRNA-181b`s role in the molecular mechanisms of schizophrenia.Methods Bioinformatic methods predicted that EGR3 gene is targeted by microRNA-181b.PCR methods amplified the fragment in EGR3 gene 3`UTR including the putative microRNA-181b binding site.Then the sequence was cloned into the pmirGLO luciferase vector.The DNA sequences of the amplified fragments were identified by restriction enzyme digestion and sequencing, and were consistent with the reference sequence from UCSC.This constructed vector was marked as pmirGLO-EGR3 vector.Finally, the pmirGLO vector, the pmirGLO-EGR3 vector, microRNA-181b mimics and negative control (NC) were divided into 5 groups and transfected into HEK393T cells;the luciferase activity was tested by dual luciferase reporter gene assay.Results The results of restriction enzyme digestion and sequencing demonstrated that the PCR fragmentwas successfully cloned into pmirGLO vector.The transfection results showed that the recombinant plasmid was successful transfected into HEK293T under the fluorescence microscope, with transfection efficiency being about 90％.The results of dual luciferase activity assay demonstrated that microRNA-181b significantly decreased the reporter gene`s activity compared with the NC.Conclusion At the cellular level, the schizophrenia susceptibility gene EGR3 was verified to be targeted by micorRNA-181b, which provides a new clue for the subsequent study on microRNA-181b`s role in the molecular mechanisms of schizophrenia.

Early Growth Response-1 Plays a Non-redundant Role in the Differentiation of B Cells into Plasma Cells

Early growth response (Egr)-1 is a Cys2-His2-type zincfinger transcription factor. It has been shown to induce survival and proliferation of immature and mature B cells, respectively, but its role in the differentiation of B cells into plasma cells remains unclear. To examine the effects of Egr-1 deficiency on the activation of B cells, naive B cells from Egr1-/- mice and their wild-type (WT) littermates were activated to proliferate and differentiate, and then assayed by FACS. Proportions of cells undergoing proliferation and apoptosis did not differ between Egr1-/- and WT mice. However, Egr1-/- B cells gave rise to fewer plasma cells than WT B cells. Consistently, Egr1-/- mice produced significantly lower titer of antigen-specific IgG than their WT littermates upon immunization. Our results demonstrate that Egr-1 participates in the differentiation program of B cells into plasma cells, while it is dispensable for the proliferation and survival of mature B cells.

Egr-1 mediating oxidative stress caused by hypoxia/reoxygenation in cardiomyocytes / 中国生化药物杂志

Objective To investigate whether ROS/JNK/Egr-1 signaling pathway was activated in cardiomyocytes after hypoxia/reoxygenation ( H/R).Methods H9c2 cardiomyocytes were grouped randomly as follows: control group, H/R group, control +the ROS donor xanthine oxidase /hypoxanthine (XO/HX) group, H/R +the ROS scavenger edaravones (EDA) group, H/R +the ROS scavenger N-acetyl-L-cysteine (NAC) group, H/R +JNK inhibitor SP60012 group.To establish H9c2 H/R models and the myocardial cells were treated with different concentrations of EDA (2 × 10 -6,2 ×10 -5,2 ×10 -4 M), NAC (5 ×10 -4,2 ×10 -3,8 ×10 -3 M), XO/HX (1mU/mL/1.2 ×10 -4 M , 3mU/mL/3.6 ×10 -4 M, 5mU/mL/6.0 × 10 -4 M) and SP600125 (2 ×10 -5 M).ROS level was measured by flow cytometry, and Egr-1, p-JNK and total JNK protein levels were detected by Western blot.ResuIts ROS levels and Egr-1 protein levels in H/R group were significantly higher than control group (P<0.05).The moderate and high concentrations EDA and NAC of ROS scavenger significantly decreased the high levels of ROS and Egr-1 protein ( P<0.05 ) , but there were no significant differences of low concentration.There was a significant positive correlation between ROS levels and Egr-1 protein (r=0.91,P<0.01).JNK activation levels in each concentrations of XO/HX were significantly higher than control group, and JNK activation increased with the increasing of XO/HX concentrations (P<0.05).JNK activation level in H/R group was higher than control group, after treated by EDA and NAC of ROS scavenger and JNK inhibitor, JNA activation reduced (P<0.05).Egr-1 protein levels in H/R group was higher than that in control group, and JNK inhibitor reduced the expression of Egr-1 protein induced by H/R.ConcIusion H/R activates ROS/Egr-1 signaling pathway in H9c2 cardiomyocytes, and JNK activation plays an important role in this pathway.

Involvement of the Early Growth Response Protein 1 in Vascular Pathophysiology: An Overview.

Hyperactivation of proliferative and growth promoting pathways underlies the progression of vessel remodeling, leading to vascular dysfunction. An upregulation of early growth response protein 1 (Egr-1), a zinc finger transcription factor has been observed in several models of vascular diseases. In the vasculature, Egr-1 expression can be induced by multiple hormonal, metabolic and external stimuli, such as growth factors, cytokines, reactive oxygen species, hyperglycaemia and stretch-induced stress. The structure of the Egr-1 promoter allows both its auto-regulation and its binding with several regulatory transcription cofactors like the serum response factor and the cAMP response element binding protein. Pharmacological and genetic studies have revealed the involvement of several signaling pathways that contribute to the expression of Egr-1. Among them, the mitogen-activated protein kinase pathway has emerged as a predominant signaling cascade that regulates Egr-1 transcription in response to various stimuli. Moreover, targeted deletion of Egr-1 by DNAzymes, antisense oligonucleotides or RNA interference has also helped in defining the importance of Egr-1 in the pathophysiology of vascular diseases. Neointimal formation and expression of genes directly linked with proinflammatory processes have been demonstrated to be enhanced by Egr-1 expression and activity. This review provides an overview on the signaling components implicated in Egr-1 expression and discusses its potential involvement in vascular pathophysiology.

Effects of ionizing radiation on proliferation and invasion of MCF-7 cells transfected with pcDNA3.1-Egr-1-AIF△1-480 plasmid / 吉林大学学报(医学版)

Objective To investigate the influence of truncated apoptosis inducing factor (AIFΔ1-480 ) on the proliferation and invasion of MCF-7 cells,and to clarify the possibility of promoting cancer gene-radiotherapy. Methods The human breast cancer MCF-7 cells were transfected with AIFΔ1-480 recombinant expression vector pcDNA3.1-Egr-1-AIFΔ1-480 (pE-AIFΔ1-480 )mediated by Egr-1;24 h after 2 Gy X-ray irradiation,MTT assay and Transwell invasion assay were performed to measure the changes of cell proliferation and invasion.The MCF-7 cells were diveded into normal control,pcDNA3.1,pE-AIFΔ1-480 ,2 Gy irradiation and pE-AIFΔ1-480+ 2 Gy irradiation groups.Results After transfection and 2 Gy X-ray irradiation,the cells proliferated very fast in normal control, pcDNA3.1 and pE-AIFΔ1-480 groups, and the proliferation regularity was similar. Compared with normal control group,the cell proliferation abilities were significantly decreased in 2 Gy irradiation and pE-AIFΔ1-480 + 2 Gy irradiation groups (P<0.05 ), and it was more obvious in pE-AIFΔ1-480 + 2 Gy irradiation group, and it was significant lower than that in 2 Gy irradiation group (P<0.05).The number of the cells permeating membrane was basically same in normal control,pcDNA3.1 and pE-AIFΔ1-480 groups;compared with normal control group,they were significantly decreased in 2 Gy irradiation and pE-AIFΔ1-480+ 2 Gy irradiation groups(P<0.05 or P<0.01);and it was more significant in pE-AIFΔ1-480+ 2 Gy irradiation group than that in 2 Gy irradiation group (P<0.01). Conclusion AIFΔ1-480 and ionizing radiation could inhibit the proliferation and invasion of human breast cancer MCF-7 cells,both of them have a synergistic effect,and Egr-1 promoter can enhance the suppression effect under radiation conditions.

Inhibitory effect of pshuttle-Egr-1-hSmac plasmid combined with X-ray irradiation on proliferation of breast cancer MDA-MB-435 cells / 吉林大学学报(医学版)

Objective To construct the pshuttle-Egr-1-hSmac plasmid and transfect human breast cancer MDA-MB-435 cells,and to observe its radiotherapy enhancing effect on tumor cells.Methods The empty vector pshuttle and pshuttle-Egr-1-hSmac plasmid were transfected into MDA-MB-435 cells by liposomal.At different time(4,8,12,24 and 48 h)after irradiation with 2.0 Gy X-ray and 24 h after irradiation with 0.5 -5.0 Gy,the total RNA and protein were collected and extracted from these cells to analyze the Smac mRNA and protein expression levels with RT-PCR and Western blotting methods. The cells were divided into control, pshuttle, pshuttle-Egr-1-hSmac,2.0 Gy irradiation group, pshuttle + 2.0 Gy irradiation and pshuttle-Egr-1-hSmac+2.0 Gy irradiation groups.MTT method was used to evaluate cell proliferation,and the cell survival ability was measured with clone formation assay;Annexin Ⅴ/PI double staining and PI single staining were used to examine the apoptosis and cell cycle of MDA-MB-435 cells. Results There was no Smac mRNA expression in MDA-MB-435 cells in control and pshuttle groups,but the Smac mRNA expression levels in MDA-MB-435 cells in pshuttle-Egr-1-hSmac plasmid group were gradually increased with the time prolongation, and reached the maximum at 24 and 48 h;the Smac mRNA expression levels in MDA-MB-435 cells were increased gradually 24 h after irradiation of 0.5 - 5.0 Gy X-ray with the increasing of irradiation doses, and reached the maximum after 2.0 and 5.0 Gy irradiation. The Smac protein expression levels in pshuttle-Egr-1-hSmac plasmid group were increased gradually with the time prolongation,and reached the maximum at 24 h.The Smac protein expression lervels were increased 24 h afer irradiation of 0,0.5,1.0,2.0 and 5.0 Gy X-ray,especially in 5.0 Gy group. The MTT results showed that the A490 values in 2.0 Gy,pshuttle+2.0 Gy and pshuttle-Egr-1-hSmac groups 24, 48,and 72 h after irradiation were lower than those in control group(P<0.01);the A490 values of MDA-MB-435 cells in pshuttle-Egr-1-hSmac group after 1.0-5.0 Gy X-ray irradiation were lower than those in 0 Gy group (P<0.05 or P<0.01);the survival fraction(SF)in pshuttle-Egr-1-hSmac group was lower than those in control group (P<0.01).The percentages of the cells at G0/G1 and S phase in pshuttle-Egr-1-hSmac group were lower than those in 2.0 Gy group(P<0.01),the percentage of the cells at G2/M phase was higher than that in 2.0 Gy group (P<0.01);the apoptotic rate of the cells in pshuttle-Egr-1-hSmac group was higher than that in 2.0 Gy group (P<0.01).Conclusion X-ray irradiation can significantly increase the Smac mRNA and protein expression levels in MDA-MB-435 cells transfected with pshuttle-Egr-1-hSmac plasmid,inhibit the cell survival rate,and induce G2/M arrest and apoptotic increasing;Smac gene combined with radiotherapy could significantly increase the radiosensitivity of breast cancer cells.

Induction of Transcription Factor Early Growth Response Protein 1 during HSV-1 Infection Promotes Viral Replication in Corneal Cells.

Aims: To understand the mechanisms of Early Growth Response Protein 1 (Egr-1) induction upon HSV-1 lytic infection and its roles in regulating viral gene expression and replication. Study Design: Rabbit corneal cell line SIRC and other cell lines were infected by HSV-1 to investigate the Egr-1 induction and its occupancy on the viral genome in different conditions. UV-inactivated HSV-1 and a recombinant virus over-expressing Egr-1 were generated to evaluate the regulatory effects on viral gene expression and replication during the infection. Methodology: Egr-1 induction triggered by viral infection was determined by Western Blot analyses and immune-fluorescent microscopy. Real-time RT-PCR and a novel Cignal™ Reporter Assay were used for quantitative measurement of Egr-1 expression. Chromatin Immuno-precipitation (ChIP) was performed to address the Egr-1 occupancy to the viral regulatory sequences and the influence on viral replication was assessed by plaque assays. Results: Our results indicated that Egr-1 expression requires viral gene expression since the UV-inactivated HSV-1 failed to produce Egr-1 protein. Blockade of viral replication did not block the Egr-1 protein synthesis, supporting the hypothesis that HSV-1 replication was not essential for Egr-1 production. Chromatin immune-precipitation (ChIP) and RT-PCR assays demonstrated that induced Egr-1 was able to interact with key regulatory elements near HSV-1 immediate-early (IE) genes and promote viral gene expression. Recombinant virus overexpressing Egr-1 revealed that Egr-1 enhanced the viral replication and the release of infectious virus. Conclusion: Together these results concluded that HSV-1 triggers the expression of an important host transcription factor Egr-1 via a unique mechanism and benefit the viral gene expression and replication.

Genetic association of the EGR2 gene with bipolar disorder in Korea

The early growth response gene 2 (EGR2) is located at chromosome 10q21, one of the susceptibility loci in bipolar disorder (BD). EGR2 is involved in cognitive function, myelination, and signal transduction related to neuregulin-ErbB receptor, Bcl-2 family proteins, and brain-derived neurotrophic factor. This study investigated the genetic association of the EGR2 gene with BD and schizophrenia (SPR) in Korea. In 946 subjects (350 healthy controls, 352 patients with BD, and 244 with SPR), nine single nucleotide polymorphisms (SNPs) in the EGR2 gene region were genotyped. Five SNPs showed nominally significant allelic associations with BD (rs2295814, rs61865882, rs10995315, rs2297488, and rs2297489), and the positive associations of all except rs2297488 remained significant after multiple testing correction. Linkage disequilibrium structure analysis revealed two haplotype blocks. Among the common identified haplotypes (frequency > 5%), 'T-G-A-C-T (block 1)' and 'A-A-G-C (block 2)' haplotypes were over-represented, while 'C-G-G-T-T (block 1)' haplotype was under-represented in BD. In contrast, no significant associations were found with SPR. Although an extended analysis with a larger sample size or independent replication is required, these findings suggest a genetic association of EGR2 with BD. Combined with a plausible biological function of EGR2, the EGR2 gene is a possible susceptibility gene in BD.

Gene Expression Profiles in Genetically Different Mice Infected with Toxoplasma gondii: ALDH1A2, BEX2, EGR2, CCL3 and PLAU

Toxoplasma gondii can modulate host cell gene expression; however, determining gene expression levels in intermediate hosts after T. gondii infection is not known much. We selected 5 genes (ALDH1A2, BEX2, CCL3, EGR2 and PLAU) and compared the mRNA expression levels in the spleen, liver, lung and small intestine of genetically different mice infected with T. gondii. ALDH1A2 mRNA expressions of both mouse strains were markedly increased at day 1-4 postinfection (PI) and then decreased, and its expressions in the spleen and lung were significantly higher in C57BL/6 mice than those of BALB/c mice. BEX2 and CCR3 mRNA expressions of both mouse strains were significantly increased from day 7 PI and peaked at day 15-30 PI (P<0.05), especially high in the spleen liver or small intestine of C57BL/6 mice. EGR2 and PLAU mRNA expressions of both mouse strains were significantly increased after infection, especially high in the spleen and liver. However, their expression patterns were varied depending on the tissue and mouse strain. Taken together, T. gondii-susceptible C57BL/6 mice expressed higher levels of these 5 genes than did T. gondii-resistant BALB/c mice, particularly in the spleen and liver. And ALDH1A2 and PLAU expressions were increased acutely, whereas BEX2, CCL3 and EGR2 expressions were increased lately. Thus, these demonstrate that host genetic factors exert a strong impact on the expression of these 5 genes and their expression patterns were varied depending on the gene or tissue.