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

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.

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.

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.

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.

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.

Enhanced Effects of TRAIL-endostatin-based Double-gene-radiotherapy on Suppressing Growth, Promoting Apoptosis and Inducing Cell Cycle Arrest in Vascular Endothelial Cells / 华中科技大学学报（医学）（英德文版）

This study examined the effects of TRAIL-endostatin-based gene-radiotherapy on cellular growth,apoptosis and cell cycle progression in human vascular endothelial cells ECV304 in vitro.The expression of TRAIL and endostatin protein in ECV304 cells was detected by ELISA after the transfection of recombinant plasmid pshuttle-Egrl-shTRAIL-shES and X-ray irradiation.Then MTT assay was used for determining the cellular proliferation,and flow cytometry (FCM) plus Annexin V and propidium iodide (PI) double-staining or PI single-staining were employed for the detection of apoptosis and cell cycle progression.The results showed that expression of TRAIL and endostatin protein exhibited a time- and dose-dependent change in ECV304 cells after pshuttle-Egrl-shTRAIL-shES transfection in conjunction with irradiation.In the TRAIL-endostatin-based single- or double-gene-radiotherapy,the cell viability declined in a time- and dose-dependent manner,the percentage of cells at G2/M phase and apoptotic rate was increased,and the percentage of cells at G0/G1 phase was lowered as compared with those receiving radiotherapy alone.Moreover,TRAIL-endostatin-based double-gene-radiotherapy demonstrated better effects on growth inhibition,promotion of apoptosis and induction of cell cycle arrest in ECV304 cells than single-gene-radiotherapy.

Early Growth Response 1— A Transcription Factor in the crossfire of Signal Transduction Cascades.

Early growth response-1 (Egr-1) is a Cys2-His2-type zinc-finger transcription factor. A broad range of extracellular stimuli is capable of activating Egr-1, thus mediating growth, proliferation, differentiation or apoptosis. Egr-1 is, therefore, participating in the progression of a variety of diseases such as atherosclerosis or cancer. Functional response elements connect Egr-1 to signal transduction cascades targeting Egr-1. Five serum response elements (SRE) have been identified in the promoter region of Egr-1, the binding region of serum response factor (SRF). The Rho/Rho-kinase pathway has been shown to regulate actin reorganization via LIM-kinase mediated cofilin phosphorylation. Recent studies have revealed that the actin binding striated muscle activator of Rho signaling (STARS) promotes translocation of myosin related transcription factors (MRTFs) into the nucleus, leading to SRF activation. The ternary complex factor (TCF) Elk-1 eventually bridges the gap between SRF-mediated gene transcription and the Raf/MEK/ERK pathway. Moreover, the Egr-1 promoter owns two cAMP response elements (CREs), whose relevance for gene expression is still unclear. An Egr-1 binding site (EBS) located on the Egr-1 promoter itself is arguing for a negative feedback mechanism. The acquired knowledge on transcriptional regulation of Egr-1 is not entirely understood. In this review, we highlight upstream and downstream signaling in vitro and in vivo associated with Egr-1.

The influence of EGR-1 on NF-κB in HTLV-1 Tax protein positive cells / 中华微生物学和免疫学杂志

Objective To research the relation of early growth response gene-1(EGR-1) and NF-κB in human T-cell leukemia virus type 1(HTLV-1) Tax protein positive cells. Methods RT-PCR was used to amplify the aimed segments EGR-1 cDNA which was then inserted into an eukaryotic expression plasmid pcDNA3.0 to construct pcDNA3.0-EGR-1. The constructed plasmid was transfected into TaxN and TaxP cells by Tfx-50-mediated transfer method, the expression levels of EGR-1, p65 and Tax mRNA in transfected cells were assay by RT-PCR after 48 h post-transfection, the proteins of EGR-1 and p65 were detected by Western blot after 48 h post-transfection too. The constructed plasmid and pNF-κB-luc reporter gene plasmid was co-transfected into TaxN and TaxP cells by Tfx-50-mediated transfer method, and the activity of luciferase was assay after 48 h post-transfection. Results The results showed that the eukaryotic expression plasmid pcDNA3.0-EGR-1 was successfully constructed. The mRNA and protein expression of EGR-1 could be promoted significantly by Tax. EGR-1 can promote the mRNA and protein expressions of p65 in TaxP cells, the activity of NF-κB was up-regulated by EGR-1 too. Conclusion EGR-1 maybe involve in adult T-cell leukemia(ATL) by increasing the activation of NF-κB.

Combination of two rare mutations causes β -thalassaemia in a Bangladeshi patient

Screening of mutations that cause β-thalassaemia in the Bangladeshi population led to the identification of a patient with a combination of two rare mutations, Hb Monroe and HBB: -92C>G.The β-thalassaemia major male individual was transfusion-dependent and had an atypical β-globin gene cluster haplotype. Of the two mutations, Hb Monroe has been characterized in detail. Clinical effects of the other mutation, HBB: -92C>G,are unknown so far. Bioinformatics analyses were carried out to predict the possible effect of this mutation. These analyses revealed the presence of a putative binding site for Egr1, a transcription factor, within the HBB:-92 region. Our literature survey suggests a close relationship between different phenotypic manifestations of β-thalassaemia and Egr1 expression.

A DNA enzyme targeting Egr-1 inhibits rat vascular smooth muscle cell proliferation by down-regulation of cyclin D1 and TGF-â1

We have demonstrated that a synthetic DNA enzyme targeting early growth response factor-1 (Egr-1) can inhibit neointimal hyperplasia following vascular injury. However, the detailed mechanism of this inhibition is not known. Thus, the objective of the present study was to further investigate potential inhibitory mechanisms. Catalytic DNA (ED5) and scrambled control DNA enzyme (ED5SCR) were synthesized and transfected into primary cultures of rat vascular smooth muscle cells (VSMCs). VSMC proliferation and DNA synthesis were analyzed by the MTT method and BrdU staining, respectively. Egr-1, TGF-â1, p53, p21, Bax, and cyclin D1 expression was detected by RT-PCR and Western blot. Apoptosis and cell cycle assays were performed by FACS. Green fluorescence could be seen localized in the cytoplasm of 70.6 ± 1.52 and 72 ± 2.73 percent VSMCs 24 h after transfection of FITC-labeled ED5 and ED5SCR, respectively. We found that transfection with ED5 significantly inhibited cultured VSMC proliferation in vitro after 24, 48, and 72 h of serum stimulation, and also effectively decreased the uptake of BrdU by VSMC. ED5 specifically reduced serum-induced Egr-1 expression in VSMCs, further down-regulated the expression of cyclin D1 and TGF-â1, and arrested the cells at G0/G1, inhibiting entry into the S phase. FACS analysis indicated that there was no significant difference in the rate of apoptosis between ED5- and ED5SCR-transfected cells. Thus, ED5 can specifically inhibit Egr-1 expression, and probably inhibits VSMC proliferation by down-regulating the expressions of cyclin D1 and TGF-â1. However, ED5 has no effect on VSMC apoptosis.

Construction of human Egr-1 promoter and its response to ionizing radiation in tumor cells / 细胞与分子免疫学杂志

AIM: To construct human Egr-1 promoter luciferase reporter system and study its activity induced by i-onizing radiation. METHODS: Egr-1 promoter was obtained by human genomic PCR and cloned into pGL3-basic vector. After transfection of recombinant plasmid into human tumor cells, the Egr-1 promoter activity induced by ionizing radiation was detected by luciferase reporter assay. RESULTS: The luciferasy reporter system of Egr-1 promoter was successfully constructed. The activity of Egr-1 promoter was substantially increased after different doses of IR and reached to the peak at the time point of 48h after IR. CONCLUSION: The Egr-1 promoter was constructed in this study showed IR inducible activity in tumor cells, laying foundation for the research of radiation, mediated gene therapy.