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.

Effect of Survivin Down-Regulation by Egr1-Survivin shRNA Combined with Radiotherapy on Radiosensitivity of Esophageal Squamous Carcinoma / Efecto de la Regulación Negativa de Survivina por shRNA de Egr1-Survivina Combinado con Radioterapia sobre la Radiosensibilidad del Carcinoma Escamoso de Esófago

SUMMARY: This study is to investigate the effect of survivin down-regulation by Egr1-survivin shRNA combined with radiotherapy on the apoptosis and radiosensitivity of esophageal squamous cell carcinoma ECA109 and KYSE150 cells. ECA109 and KYSE150 cells were transfected with Egr1-survivin shRNA, and then treated with radiotherapy. After 24 h, the mRNA and protein levels of Egr1-survivin were detected by qPCR and Western-Blot. Cell cycle and apoptosis were detected by flow cytometry. Western blot also detected levels of cleavaged Caspase 3 and Caspase 9. YM155 was used as a positive control to inhibit survivin expression. The levels of survivin mRNA and protein in ECA109 and KYSE150 cells treated with Egr1-survivin shRNA combined with radiotherapy were significantly lower than those of the blank control group, the empty vector control group, and, the YM155 + radiotherapy group (P<0.05). Meanwhile, after survivin down-regulation, the ratio of G2 to S phase of ECA109 and KYSE150 cells increased significantly, leading to significant G2 and S phase arrest. Additionally, apoptosis of ECA109 and KYSE150 cells increased significantly (P <0.01). Further, protein levels of cleavaged Caspase 3 and Caspase 9 significantly increased in Egr1-survivin shRNA combined with radiotherapy group. Egr1-survivin shRNA combined with radiotherapy can down-regulate survivin expression, which further increases the apoptosis, and enhances the radiosensitivity of ECA109 and KYSE150 cells.

Este estudio tuvo como objetivo investigar el efecto de la regulación negativa de survivina por el shRNA de Egr1-survivina combinado con radioterapia sobre la apoptosis y la radiosensibilidad del carcinoma de células escamosas de esófago Células ECA109 y KYSE150. Las células ECA109 y KYSE150 se transfectaron con shRNA de survivina Egr1 y luego se trataron con radioterapia. Después de 24 h, los niveles de ARNm y proteína de Egr1-survivina se detectaron mediante qPCR y Western-Blot. El ciclo celular y la apoptosis se detectaron mediante citometría de flujo. La transferencia Western también detectó niveles de Caspasa 3 y Caspasa 9 escindidas. Se usó YM155 como control positivo para inhibir la expresión de survivina. Los niveles de ARNm y proteína de survivina en células ECA109 y KYSE150 tratadas con shRNA de survivina Egr1 combinado con radioterapia fueron significativamente más bajos que los del grupo control en blanco, el grupo control de vector vacío y el grupo de radioterapia YM155 + (P <0,05). Mientras tanto, después de la regulación negativa de survivina, la proporción entre las fases G2 y S de las células ECA109 y KYSE150 aumentó significativamente, lo que llevó a una detención significativa de las fases G2 y S. Además, la apoptosis de las células ECA109 y KYSE150 aumentó significativamente (P <0,01). Además, los niveles de proteína de Caspasa 3 y Caspasa 9 escindidas aumentaron significativamente en el shRNA de Egr1- survivina combinado con el grupo de radioterapia. El shRNA de survivina de Egr1 combinado con radioterapia puede regular negativamente la expresión de survivina, lo que aumenta aún más la apoptosis y mejora la radiosensibilidad de las células ECA109 y KYSE150.

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.

Expression of EGR1 gene and location of EGR1 protein in differentiation of bovine skeletal muscle-derived satellite cells / 中国应用生理学杂志

OBJECTIVE@#To investigate the expression of EGR1 gene and the localization of EGR1 protein in bovine skeletal muscle-derived satellite cells (MDSCs), as well as to investigate the mechanism that EGR1 protein enters the nucleus.@*METHODS@#Bovine MDSCs were cultured in differentiation medium for 1 day, 3 days and 5 days, respectively, and each group was triplicate. The expression of EGR1 gene and the localization of EGR1 protein were studied at different differentiation period in MDSCs by qRT-PC and Western blot. Moreover, the changes on the expression of endogenous EGR1 gene and EGR1 proteins were explored by CRISPRi, site-directed mutagenesis and laser confocal method.@*RESULTS@#The results from the qRT-PCR and Western blot showed that the expressions of EGR1 gene on transcription level and translation level were significantly higher in differentiated cells than those in undifferentiated cells. The highest expression was found on the third day after the differentiation, and then began to decline. Immunofluorescence assays showed that EGR1 proteins were preferentially expressed in differentiated MDSCs, and increased along with the increase of number of myotubes. Confocal observation revealed that some EGR1 proteins were transferred into the nucleus in the differentiation of cells, however, the EGR1 proteins would not be detected in the differentiated MDSCs nuclei if a site directed mutagenesis (serine) on EGR1 protein occurred.@*CONCLUSION@#During the differentiation of bovine skeletal muscle satellite cells, the transcriptional level of EGR1 gene is increased, and some EGR1 proteins are transferred into the nucleus. The serine phosphorylation at position 533 of the C terminal of EGR1 protein is necessary for the nucleus transfer.

Specific immediate early gene expression induced by high doses of salicylate in the cochlear nucleus and inferior colliculus of the rat / Expressão específica de genes precoces imediatos induzida por doses elevadas de salicilato no núcleo coclear e colículo inferior de rato

Abstract Introduction: Salicylate at high doses induces tinnitus in humans and experimental animals. However, the mechanisms and loci of action of salicylate in inducing tinnitus are still not well known. The expression of Immediate Early Genes (IEG) is traditionally associated with long-term neuronal modifications but it is still not clear how and where IEGs are activated in animal models of tinnitus. Objectives: Here we investigated the expression of c-fos and Egr-1, two IEGs, in the Dorsal Cochlear Nucleus (DCN), the Inferior Colliculus (IC), and the Posterior Ventral Cochlear Nucleus (pVCN) of rats. Methods: Rats were treated with doses known to induce tinnitus in rats (300 mg/kg i.p. daily, for 3 days), and c-fos and Egr-1 protein expressions were analyzed using western blot and immunocytochemistry. Results: After administration of salicylate, c-fos protein expression increased significantly in the DCN, pVCN and IC when assayed by western blot. Immunohistochemistry staining showed a more intense labeling of c-fos in the DCN, pVCN and IC and a significant increase in c-fos positive nuclei in the pVCN and IC. We did not detect increased Egr-1 expression in any of these areas. Conclusion: Our data show that a high dose of salicylate activates neurons in the DCN, pVCN and IC. The expression of these genes by high doses of salicylate strongly suggests that plastic changes in these areas are involved in the genesis of tinnitus.

Resumo Introdução: Salicilato em doses elevadas induz zumbido nos seres humanos e em animais experimentais. No entanto, os mecanismos e loci de ação do salicilato na indução de zumbido ainda não são bem conhecidos. A expressão dos genes precoces imediatos (GPIs) está tradicionalmente associada a alterações neuronais em longo prazo, mas ainda não está claro como e onde os GPIs são ativados em modelos animais de zumbido. Objetivos: No presente estudo investigamos a expressão de c-fos e Egr-1, dois GPIs, no núcleo coclear dorsal (NCD), colículo inferior (CI) e núcleo coclear ventral posterior (NCVp) de ratos. Métodos: Os ratos foram tratados com doses que, conhecidamente, induzem zumbido em ratos (300 mg/kg IP/dia, por três dias) e as expressões das proteínas c-fos e Egr-1 foram analisadas por meio de Western blot e imunoistoquímica. Resultados: Após a administração de salicilato, a expressão da proteína c-fos aumentou significativamente no NCD, NCVp e CI, quando analisados por Western blot. A coloração imunoistoquímica mostrou uma marcação mais intensa de c-fos no NCD, NCVp e CI e um aumento significativo de núcleos positivos de c-fos no NCVp e CI. Não detectamos aumento da expressão de Egr-1 em qualquer dessas áreas. Conclusão: Nossos dados mostram que uma dose alta de salicilato ativa neurônios no NCD, NCVp e CI. A expressão desses genes por doses altas de salicilato sugere que as alterações plásticas nessas áreas estão envolvidas na gênese do zumbido.

Expressions of inflammatory and fibrogenic factors in perinephric and subcutaneous adipose tissues of patients with adrenocorticotropic hormone-independent Cushing's syndrome / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expressions of inflammation- and fibrosis-related genes in perinephric and subcutaneous adipose tissues in patients with adrenocorticotropic hormone (ACTH)-independent Cushing's syndrome.</p><p><b>METHODS</b>The perinephric and subcutaneous adipose tissues adipose tissues were obtained from 8 patients with ACTH-independent Cushing's syndrome undergoing laparoscopic retroperitoneal adrenalectomy. Real-time PCR was used to detect the mRNA expression levels of interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), matrix metallopeptidase 2 (MMP-2), TIMP metallopeptidase inhibitor 1 (TIMP-1), early growth response 1 (EGR1), CCAAT/enhancer binding protein β(CEBPβ), uncoupling protein 1(UCP-1), PPARγ coactivator 1 alpha (PGC1α) and cell death-inducing DFFA-like effector a (CIDEA).</p><p><b>RESULTS</b>The mRNA level of CIDEA was significantly higher in the perinephric adipose tissue (peri-N) than in the subcutaneous adipose tissue (subQ) (P<0.05). The expressions of CEBPβ, UCP-1, and PGC1α mRNA in the peri-N were similar with those in the subQ. The expressions of IL-6, TIMP1 and EGR1 mRNA in the subQ were significantly higher than those in the peri-N (P<0.05). No significant difference in TNF-α and MMP-2 mRNA levels was found between peri-N and subQ.</p><p><b>CONCLUSION</b>The expression levels of the inflammation- and fibrosis-related genes are higher in the subQ than in the peri-N of patients with ACTH-independent Cushing's syndrome, suggesting that chronic exposure to endogenous hypercortisolism may cause adipose tissue dysfunction.</p>

Effect of DPP4 inhibitor sitagliptin on expressions of early growth response-1 and fibronectin in the kidney of ApoE gene knockout mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of the DPP4 inhibitor sitagliptin on the expressions of early growth response-1 (Egr-1) and fibronectin in the kidney of ApoE gene knockout mice.</p><p><b>METHODS</b>Eight-week-old male ApoE gene knockout mice were randomly divided into sitagliptin + apoE(-/-) group and apoE(-/-) group (n=6), with 6 C57BL mice as the normal control group. After feeding with high-fat diet and drug treatment for 16 weeks, the mice underwent intraperitoneal glucose tolerance test (IPGTT) and were measured for 24-h urinary albumin using ELISA. All the mice were then sacrificed to examine the changes of blood lipid profile and for detection of Egr-1 and fibronectin mRNA and proteins in the renal tissue using real-time PCR and Western blotting.</p><p><b>RESULTS</b>The mice in both apoE(-/-) group and sitagliptin+apoE(-/-) group all showed prominently increased blood lipids as compared with the control group (P<0.05) without significant differences between the two apoE(-/-) groups. The level of HDL was significantly higher in sitagliptin +apoE(-/-) group than in apoE(-/-) group (P<0.001) and control group (P<0.001). IPGTT showed no significant differences in the levels of blood glucose among the 3 groups. The excretion of urinary albumin was increased in apoE(-/-) group compared with the control group (P<0.01), but was significantly lower in sitagliptin+ apoE(-/-) group than in apoE(-/-) group (P<0.01). Real-time PCR and Western blotting showed significantly decreased mRNA and protein expressions of renal cortical Egr-1 and fibronectin in sitagliptin+apoE(-/-) group compared with apoE(-/-) group.</p><p><b>CONCLUSION</b>Sitagliptin can reduce the renal expression of fibronectin by regulating the expression of Egr-1 to achieve renal protection.</p>

Treadmill Exercise Improves Memory Function Depending on Circadian Rhythm Changes in Mice / 대한배뇨장애요실금학회지

PURPOSE: Exercise enhances memory function by increasing neurogenesis in the hippocampus, and circadian rhythms modulate synaptic plasticity in the hippocampus. The circadian rhythm-dependent effects of treadmill exercise on memory function in relation with neurogenesis were investigated using mice. METHODS: The step-down avoidance test was used to evaluate short-term memory, the 8-arm maze test was used to test spatial learning ability, and 5-bromo-2’-deoxyuridine immunofluorescence was used to assess neurogenesis. Western blotting was also performed to assess levels of synaptic plasticity-associated proteins, such as brain-derived neurotrophic factor, tyrosine kinase receptor B, phosphorylated cAMP response element-binding protein, early growth response protein 1, postsynaptic density protein 95, and growth-associated protein 43. The mice in the treadmill exercise at zeitgeber 1 group started exercising 1 hour after sunrise, the mice in the treadmill exercise at zeitgeber 6 group started exercising 6 hours after sunrise, and the mice in the treadmill exercise at zeitgeber 13 group started exercising 1 hour after sunset. The mice in the exercise groups were forced to run on a motorized treadmill for 30 minutes once a day for 7 weeks. RESULTS: Treadmill exercise improved short-term memory and spatial learning ability, and increased hippocampal neurogenesis and the expression of synaptic plasticity-associated proteins. These effects of treadmill exercise were stronger in mice that exercised during the day or in the evening than in mice that exercised at dawn. CONCLUSIONS: Treadmill exercise improved memory function by increasing neurogenesis and the expression of synaptic plasticity-associated proteins. These results suggest that the memory-enhancing effect of treadmill exercise may depend on circadian rhythm changes.

Treadmill Exercise Improves Memory Function Depending on Circadian Rhythm Changes in Mice / 대한배뇨장애요실금학회지

PURPOSE: Exercise enhances memory function by increasing neurogenesis in the hippocampus, and circadian rhythms modulate synaptic plasticity in the hippocampus. The circadian rhythm-dependent effects of treadmill exercise on memory function in relation with neurogenesis were investigated using mice. METHODS: The step-down avoidance test was used to evaluate short-term memory, the 8-arm maze test was used to test spatial learning ability, and 5-bromo-2’-deoxyuridine immunofluorescence was used to assess neurogenesis. Western blotting was also performed to assess levels of synaptic plasticity-associated proteins, such as brain-derived neurotrophic factor, tyrosine kinase receptor B, phosphorylated cAMP response element-binding protein, early growth response protein 1, postsynaptic density protein 95, and growth-associated protein 43. The mice in the treadmill exercise at zeitgeber 1 group started exercising 1 hour after sunrise, the mice in the treadmill exercise at zeitgeber 6 group started exercising 6 hours after sunrise, and the mice in the treadmill exercise at zeitgeber 13 group started exercising 1 hour after sunset. The mice in the exercise groups were forced to run on a motorized treadmill for 30 minutes once a day for 7 weeks. RESULTS: Treadmill exercise improved short-term memory and spatial learning ability, and increased hippocampal neurogenesis and the expression of synaptic plasticity-associated proteins. These effects of treadmill exercise were stronger in mice that exercised during the day or in the evening than in mice that exercised at dawn. CONCLUSIONS: Treadmill exercise improved memory function by increasing neurogenesis and the expression of synaptic plasticity-associated proteins. These results suggest that the memory-enhancing effect of treadmill exercise may depend on circadian rhythm changes.

Prevalência de dor crônica e sua associação com a situação sociodemográfica e atividade física no lazer em idosos de Florianópolis, Santa Catarina: estudo de base populacional / Prevalence of chronic pain and its association with the sociodemographic situation and physical activity in leisure of elderly in Florianópolis, Santa Catarina: population-based study

OBJETIVO: Estimar a prevalência de dor crônica e sua associação com a situação socioeconômica, demográfica e atividade física no lazer em idosos. MÉTODOS: Este estudo é parte do inquérito epidemiológico e transversal de base populacional e domiciliar EpiFloripa Idoso 2009-2010 realizado com 1.705 idosos (≥ 60 anos), residentes em Florianópolis, Santa Catarina. A partir da resposta afirmativa de dor crônica, foram investigadas as associações com as variáveis obtidas por meio de entrevista estruturada. Realizou-se a estatística descritiva, incluindo cálculos de proporções e intervalos de confiança 95% (IC95%). Na análise bruta e ajustada, empregou-se regressão de Poisson, estimando-se as razões de prevalência, com intervalos de confiança de 95% e valores p ≤ 0,05. RESULTADOS: Dentre os idosos investigados, 29,3% (IC95% 26,5 - 32,2) relataram dor crônica. Na análise ajustada, observou-se que as variáveis sexo feminino, menor escolaridade e pior situação econômica ficaram associadas significativamente com maior prevalência de dor crônica; ser fisicamente ativo no lazer ficou associado significativamente com menor prevalência do desfecho. CONCLUSÕES: Percebe-se que a dor crônica é um agravo que acomete considerável parcela de idosos, havendo desigualdades sociais na sua frequência e sendo beneficamente afetada pela atividade física no lazer. É necessário que políticas públicas de saúde subsidiem programas multidisciplinares de controle da dor incluindo a prática regular de atividade física, voltada especificamente à promoção da saúde do idoso, evitando assim que a dor crônica comprometa a qualidade de vida desta população. .

OBJECTIVE: To estimate the prevalence of chronic pain and its association with socioeconomic and demographic status, and leisure physical activity in the elderly population. METHODS: This study is part of an epidemiological cross-sectional population-based household survey called EpiFloripa Elderly 2009-2010, which was conducted with 1,705 elderly individuals (≥ 60 years) residents of Florianópolis, Santa Catarina. From the positive response to chronic pain, the associations with the variables were investigated through a structured interview. Descriptive statistics were conducted, including ratio calculation and 95% confidence intervals. In crude and adjusted analysis, Poisson regression was utilized, estimating prevalence ratios, with 95% confidence intervals and ≤ 0.05 p-values. RESULTS: Among the subjects, 29.3% (IC95% 26.5 - 32.2) reported chronic pain. Adjusted analysis showed that being female, having less years of schooling, and being in worse economic situation were significantly associated with a higher prevalence of chronic pain. Being physically active during leisure time was significantly associated with lower prevalence of the outcome. CONCLUSIONS: Therefore, it is clear that chronic pain affects a considerable amount of elderly individuals. Social inequalities are a harmful influence in these individuals' quality of life, inasmuch as those inequalities increase the frequency with which chronic pain afflicts them. At the same time, physical activity during leisure time decreases chronic pain frequency. It is fundamental that public health policies subsidize multidisciplinary pain management programs, which should include health targeted physical activity for the elderly, thus preventing the decrease in quality of life that chronic pain brings to this population. .

Effects of the Combination of Emodin and 3'-Azido-3'-Deoxythymidine on Proliferation and Apoptosis in leukemia KG-1a cells Transfected with Egr-1 siRNA / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was aimed to investigate the effects of emodin combined with 3'-azido-3'-deoxythymidine (AZT) on proliferation and apoptosis of leukemia cell line KG-1a cells and its mechanism.</p><p><b>METHODS</b>KG-1a cells were transfected with Egr-1 siRNA by electroporation and divided into blank control (KG-1a), nonspecific control (KG-1a/NC) and Egr-1 siRNA (KG-1a/siRNA) groups. Transfection efficiency was tested through fluorescence microscopy and flow cytometry and the transfection effect was detected by using qPCR. The cell proliferation rate was detected with MTT method. After the cells were treated with 10 µmol/L of emodin, 3200 or 1600 µmol/L of AZT and their combinations, the proliferation inhibition rates and the apoptosis rates of cells in 3 groups were detected with MTT method and FCM, respectively.</p><p><b>RESULTS</b>The transfection efficiency of Egr-1 siRNA was found to reach more than 59.21%; as compared with blank control(KG-1a) and nonspectic control(KG-1a/NC), the cell proliferation in Egr-1 siRNA group significantly reduced (P<0.01). The combination of emodin and AZT had considerable synergistic inhibitory effects on proliferation of normal KG-1a cells and nonspecific control(KG-1a NC) cells, but the synergistic effects disappeared after Egr-1 gene silencing.</p><p><b>CONCLUSION</b>The effects of the combination of emodin and AZT on proliferation and apoptosis may be related with Egr-1.</p>

Effect of Guishen Pill on expression levels of Oct-4, MVH, and Egr-1 in mice with diminished ovarian reserve / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of Guishen Pill (GSP) on expression levels of Oct-4, MVH, and Egr-1 in mice with diminished ovarian reserve (DOR).</p><p><b>METHODS</b>Totally 40 female C57BL/6J mice were randomly divided into 4 groups, the normal control group, the model group, the GSP group, and the dehydroepiandrosterone (DHEA) group, 10 in each group. Pregnant mare serum gonadotropin (PMSG), human chorionic gonadotropin (HCG), and prostaglandin F2α (PGF2α) were sequentially administrated to produce superovulation. The DOR model was established by exposing to ozone inhalation. Mice in the GSP group were intragastrically administered with GSP at 0.3 mL. Those in the DHEA group were intragastrically administered with DHEA at 0.3 mL. Equal volume of normal saline was intragastrically administered to mice in the normal control group and the model group. All mice wer treated for 21 days. Serum levels of estrogen (E2), progestogen (P), and anti-Müllerian hormone (AMH) were measured by ELISA. Changes of Oct-4, anti-AMH, and early growth response gene-1 (Egr-1) mRNA in ovaries were dtected by Real-time PCR.</p><p><b>RESULTS</b>Compared with the model group, serum levels of E2, P, and AMH, as well as contents of estrogen receptor (ER), progestogen receptor (PR), MVH, and Oct-4 mRNA significantly increased in the GSP group and the DHEA group (P < 0.05).</p><p><b>CONCLUSION</b>GSP could improve expression levels of Oct-4, MVH, and Egr-1 mRNA in DOR mice and their ovarian function.</p>

Increased Egr-1 binding to promoter induced by histone hyperacetylation promotes gdnf gene transcription / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the mechanism of high transcription of the glial cell-line derived neurotrophic factor (gdnf) gene induced by hyperacetylation of histone H3 lysine 9 (H3K9) at its promoter region II in rat C6 glioma cells.</p><p><b>METHODS</b>The acetylation level of H3K9 at Egr-1 binding site in gdnf gene promoter region II and the binding capacity of Egr-1 to its binding site in gdnf promoter were examined by ChIP-PCR in C6 astroglioma cells and normal rat astrocytes, and its changes were investigated in C6 astroglioma cells after treatment with histone acetyltransferase inhibitor curcumin or deacetylase inhibitor trichostatin A.</p><p><b>RESULTS</b>Compared normal astrocytes, C6 astroglioma cells showed significantly increased acetylation level of H3K9 at Egr-1 binding site in gdnf gene promoter region II and Egr-1 binding capacity (P<0.01). Curcumin treatment significantly reduced H3K9 acetylation level at Egr-1 binding site and decreased both the binding of Egr-1 to promoter region II and gdnf mRNA levels in C6 astroglioma cells (P<0.05). Conversely, increased H3K9 acetylation at the Egr-1 binding site induced by trichostatin A significantly increased the binding of Egr-1 to promoter region II and gdnf mRNA expression levels (P<0.05).</p><p><b>CONCLUSION</b>H3K9 hyperacetylation induces increased Egr-1 binding to gdnf gene promoter II, which might be the reason for the high transcription level of gdnf gene in rat C6 glioma cells.</p>

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.

Comparison of the expression profiles of cell death factors in articular cartilage between Kashin-Beck disease and osteoarthritis / 南方医科大学学报

<p><b>OBJECTIVE</b>To compare the expressions of programmed cell death 5 (PDCD5) and early growth response protein-1 (EGR-1) in the articular cartilage between Kashin-Beck disease (KBD) and primary osteoarthritis and the roles of these factors in KBD cartilage.</p><p><b>METHODS</b>Cartilage specimens were collected from 10 confirmed KBD patients, 15 osteoarthritic patients and 6 healthy subjects. The expression levels of PDCD5 and EGR-1 in the cartilage were detected by immunohistochemistry staining, and the positive chondrocyte counts were recorded in the different layers of KBD and OA cartilages.</p><p><b>RESULTS</b>The KBD cartilages contained a significantly higher percentage of PDCD5-positive chondrocytes in the middle layer [(41.35 ± 2.97)%] than OA cartilages [(26.48 ± 2.04)%, P=0.001] and normal cartilages [(19.02 ± 1.88)%, P=0.000] with also obvious PDCD5 over-expression in the deeper layer compared to OA (P=0.000) and normal cartilages (P=0.029), but PDCD5 expression in the superficial layer of the cartilages showed no significant difference among the 3 groups(P>0.05). The average EGR-1 positivity rate in the superficial layer of the cartilage was significantly higher in KBD patients than in OA patients (P=0.000) and healthy controls (P=0.000), but in the middle layer, its positivity rate in KBD patients was higher than that in the normal control (P=0.017) but lower than that of OA cartilage (P=0.002); EGR-1 expression in the deeper layer was comparable in KBD and OA cartilages but both was higher than that in normal cartilages. PDCD5 and EGR-1 expressions were not correlated in either KBD or normal cartilages, but were positively correlated in the superficial layer of OA cartilages.</p><p><b>CONCLUSIONS</b>KBD cartilages show a significantly increased PDCD5 expression in the deeper layer and enhanced EGR-1 expression in both superficial and deeper layers, suggesting the involvement of PDCD5 and EGR-1 in the pathogenesis of KBD.</p>

Gene-targeted radiation therapy mediated by radiation-sensitive promoter in lung adenocarcinoma and the feasibility of micro-PET/CT in evaluation of therapeutic effectiveness in small animals / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the combined anti-tumor effect of radiation therapy and gene-targeted suppression of tumor neovasculature in lung adenocarcinoma in vivo, and to explore the feasibility of micro-PET/CT in dynamic evaluation of treatment effectiveness.</p><p><b>METHODS</b>Thirty 5-6-week old male BALB/c nude mice were used in this study. The mouse models of xenotransplanted human lung adenocarcinoma were divided into 5 groups at random, six mice in each group: the control group, radiation treatment alone group and three groups of recombinant baculovirus plus radiation treatment (intratumoral injection, tail vein injection, and intramuscular injection). The tumor volume was measured every 2 days. Growth delay time (GD) and growth inhibition rate was calculated. FDG metabolism was evaluated by micro-PET-CT before and after treatment. The expressions of VEGF, CD31 and Ki-67 were detected by immunohistochemistry (IHC).</p><p><b>RESULTS</b>The tumor growth delay was >12 days, and the tumor inhibition rate was >45% in the recombinant baculovirus combined with radiotherapy groups, significantly higher than that of the radiotherapy alone group (P < 0.05). Immunohistochemical analysis showed that the expressions of VEGF, CD31 and Ki-67 were significantly lower than that in other groups (P < 0.05). The micro-PET-CT assessment showed that the FDG-metabolism in the recombinant baculovirus combined with radiotherapy groups was significantly reduced (P < 0.05), and the SUVmax (FDG metabolism) of transplanted tumors after treatment was also markedly decreased in comparison with that of the control group. The tumor volume after treatment was significantly correlated with SUVmax in the recombinant baculovirus intratumoral injection + radiotherapy group(r = 0.976), recombinant baculovirus intravenous injection + radiotherapy group (r = 0.954), recombinant baculovirus intramuscular injection + radiotherapy group (r = 0.929), and radiotherapy alone group (r = 0.871, P < 0.05).</p><p><b>CONCLUSIONS</b>The recombinant baculovirus containing Egr1 promoter and K5 gene combined with radiotherapy enhances the suppressing effect on the growth of lung adenocarcinoma in the tumor-bearing nude mice. The inducibility of Egr1 promoter by radiation allows the targeting and controllability of treatment. Micro-PET-CT results have a good correlation with the treatment effectiveness. Therefore, it can be used in real-time evaluation of tumor metabolic function in vivo.</p>

Neurons in NAc core and BLA are activated during cocaine context-associated reward memory retrieval in mice / 生理学报

The intense associative memories that develop between cocaine-paired contexts and rewarding stimuli make addiction hard to cure by contributing to cocaine seeking and relapse. So it's of great importance to examine the neurobiological basis of addiction memory. Cocaine conditioned place preference (CPP) used in this study is a form of Pavlovian conditioning which can establish associations between drug and contextual factors. c-Fos and Zif268 are commonly used immediate early gene (IEG) makers to identify neurons that are activated after a stimulus or behavioral conditioning. This study was designed to reveal neuronal c-Fos, Zif268 expression pattern in 10 brain regions following cocaine context-associated reward memory retrieval in mice, combining animal behavioral study and immunofluorescence method. C57BL/6 mice were randomly divided into 3 groups: Saline retrieval, Cocaine retrieval, and No retrieval of cocaine groups. Cocaine retrieval and No retrieval of cocaine underwent CPP training (one side paired with cocaine, and the other side with saline) except that No retrieval of cocaine group didn't undergo CPP test. Saline retrieval group received saline injections (i.p) on both sides. The results showed that: Neuronal c-Fos, Zif268 protein expression levels in nucleus accumbens (NAc) core both were elevated in Cocaine retrieval group compared with those in Saline retrieval (Control) group during cocaine context-associated reward memory retrieval. Zif268 protein expression level in basolateral amygdala (BLA) was also elevated in Cocaine retrieval group compared with that in control mice. Elevation was not seen in other regions such as hippocampus, prefrontal cortex (PFC). Thus, NAc core and BLA were activated during cocaine context-associated reward memory retrieval. The results suggest that neurons that are activated in NAc core and BLA are crucial basis of cocaine context-associated reward memory.

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.

Induction of Zenk protein expression within the nucleus taeniae of the amygdala of pigeons following tone and shock stimulation

In this study, we evaluated the expression of the Zenk protein within the nucleus taeniae of the pigeon’s amygdala (TnA) after training in a classical aversive conditioning, in order to improve our understanding of its functional role in birds. Thirty-two 18-month-old adult male pigeons (Columba livia), weighing on average 350 g, were trained under different conditions: with tone-shock associations (experimental group; EG); with shock-alone presentations (shock group; SG); with tone-alone presentations (tone group; TG); with exposure to the training chamber without stimulation (context group; CG), and with daily handling (naive group; NG). The number of immunoreactive nuclei was counted in the whole TnA region and is reported as density of Zenk-positive nuclei. This density of Zenk-positive cells in the TnA was significantly greater for the EG, SG and TG than for the CG and NG (P < 0.05). The data indicate an expression of Zenk in the TnA that was driven by experience, supporting the role of this brain area as a critical element for neural processing of aversive stimuli as well as meaningful novel stimuli.

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.