Egr-1 SiRNA Inhibits Egr-1 Gene Expression And Apoptosis In PC12 Cells After Morphine Withdrawal

BackgroundChronic opiate applications lead to long-term impacts on many functions of the brain and induce tolerance, dependence and addiction. Addiction continues to exact enormous human and financial costs on society, but available treatments remain inadequate for most people. By analogy with other medical disorders, an improved understanding of the biological basis of addiction will lead to more effective treatments and eventually to cures and preventive measures. But it is regrettable that mechanisms of dependence and addiction are still not understood.Previous research suggested that the abnormal gene expression in brain, impairment of neuronal cells and changes in the plasticity of neuron may play important roles in the development of opiate addiction. And chronic morphine treatment and morphine withdrawal cause apoptosis and injury of neuronal cells and other cells. So the neuronal injuries and impairments and changes in neuroplasticity in certain brain regions following chronic opiate treatment and opiate withdrawal could caused by apoptosis and have been implicated in the development of opioid dependence and addiction.Egr-1, also called zif268, nerve growth factor-induced gene A (NGFI-A), belongs to the category of immediate early genes (IEG). It codes for a transcription factor protein, named EGR-1. Acting as a transcription factor, EGR-1 directly controls expression of other genes, which makes this protein an important object of studies aimed at understanding the orchestration of neuronal responses to a variety of stimuli. Specifically expressed in brain, Egr-1 can mediate cellular apoptosis and is involved in several drugs addiction. But it is unclear that Egr-1 is implicated in apoptosis resulted from chronic opiate treatment and opiate withdrawal, and the development of opioid addiction. The present study investigated the effect of acute morphine, glutamic acid, and midazolam administration, and chronic opiate treatment and opiate withdrawal on expression of Egr-1 in PC12 cells and C6 cells. Moreover, we design three Egr-1 siRNAs, and determine their functionality in PC12 cells and C6 cells. At last, selected potent Egr-1 siRNAs were transfected into PC12 cells. We study whether Egr-1siRNAs can inhibit cellular apoptosis and injuries following chronic opiate treatment and opiate withdrawal.In this paper, the study was separated into three parts.Part one The effect of different drugs on the expression of Egr-1. Aim To explore the effects of different drugs on levels of Egr-1 gene expression in PC12 cells or C6 cells.Methods PC12 cells and Rat C6 glioma cells were seeded onto a 60-mm culture dish at a density of 1.5–2×106 cells/dish, and maintained in 5 ml of DMEM supplemented with 10% heat-inactivated fetal calf serum and 5% horse serum, 50 units/ml of penicillin, 50μg/ml of streptomycin at 37°C for 48 h in a humidified incubator containing 95% air–5% CO2 atmosphere. In the experiments testing whether glutamine acid, morphine, naloxone, and midazolam can induce EGR-1 protein expression, we treated the cells with 0-10000μM of glutamine acid, and 0-1000μM of morphine, naloxone, and midazolam. In the experiments testing the effects of pretreatment with morphine, naloxone, and midazolam were also present during treatment of the cells with glutamine acid. Immunocytochemical stain and western blot was used to detect the expression of Egr-1 gene.Results EGR-1 protein staining were observed at 1-10000μM glutamine acid, and the optimal inducing concentration was 100μM. Furthermore, midazalam and morphine can induce expression of Egr-1 gene at a concentration more than 100μM and 1000μM respectively. Pretreatment of PC12 cells with 10μM midazolam, 100μM propofol, 100μM ketamine and 100μM morphine, the expression level of EGR-1 protein induced by glutamine acid significantly declined.Conclusion Glutamine acid can induce expression of Egr-1 gene, but the level of Egr-1 gene expression induced by midazalam and morphine is determined by the concentrations of them. Moreover, midazolam, propofol, ketamine and morphine also could inhibit glutamine acid induced -EGR-1 protein expression.Part two Inhibition of Egr-1 gene expression by Egr-1 siRNA. Aim To design Egr-1 siRNAs for selecting potent siRNAs and facilitating functional gene knockdown studies.Methods Three Egr-1 siRNAs were designed and synthesized, siRNA-Ⅰ: sense, (5'-3') CCAACAGUGGCAACACUUUdTdT, antisense, (3'-5')dTdTGGUUGUCAC -CGU UG UGAAA; siRNA-Ⅱ:sense, (5'-3') GACUUAAAGGCUCUUAAUA dTdT; antisense, (3'-5')dTdT CUGAAUUUCCGAGAAUUAU; siRNA-Ⅲ: sense, (5'-3') GGAC AAGAAA GCAGACAAA dTdT, antisense, (3'-5')dTdTCCUGUUCUUUCGU–CUGUUU. In order to enhance serum and intracellular stability of siRNA, end modification of three terminal by chemical modifications were performed, such as 2'-OMe substitutions. Transfections of different concentrations of FAM-siRNA were performed with Lipofectamine TM 2000.Threir transfection efficiency were determined by immunocytochemistry. In addition, 0-100μM EGFP–siRNAs were used to optimized transfection and test procedures in 6-well plates. Following transfection of three designed siRNA into PC12 cells and C6 cells at a concentration of 50nM, the level of glutamine acid induced-Egr-1 mRNA and EGR-1 protein were observed 48 later by RT-PCR and western-blot respectively.Results Transfection efficiencies of FAM- siRNAs were 80%-90% at a concentration of 50nM, and increasing the concentration did not improve transfection effiencies. Two hours after transfection of EGFP–siRNAs into PC12 cells with pEGFP-C1, the suppressing expression of green fluorescence protein was observed. In addition, EGFP–siRNA had the most potent efficiency of inhibition at 50nM. 48 houes after siRNAⅠ,Ⅱ,Ⅲwere deliveried into PC12 cells and C6 cells, the levels of Egr-1 mRNA and EGR-1 protein induced by glutamine acid had reduced 89%, 47%, 86% and 85%, 30%, 78% in PC12 cells repectively. And in C6 cells, the reducing levels were 92%, 50%, 85% and 90%, 47%, 85% repectively.Conclusion siRNAⅠ,Ⅲare able to down-regulate the expression of Egr-1 gene, and can be used to facilitate functional gene knockdown studies. In addition, EGFP-siRNA and FAM- siRNA can be used to determine the transfection efficiencies and optimized experimental procedures.Part three The effect of Egr-1 siRNAs on cellular injuries and apoptosis following morphine withdrawal.Aim To study the effect of Egr-1 siRNAs on cellular injuries and apoptosis following opiate withdrawal, and explore the mechanism of morphine addiction.Methods After the cells were treated with 100μmol/ L morphine for 48 h, 10μmol/ L naloxone was used to produce acute withdrawal. The invert microscope was applied to observe morphology of the cells, and EGR-1 protein was detected by immunocytochemistry. Following transfection of siRNA, PC12 cells were treated with morphine for 48 h. After acute withdrawal with naloxone, thiazolyl blue tetrazolium bromide (MTT) assays were used to detect the proliferation of the cells and cells apoptosis were determined by AnnexinⅤ/PI flow cytometry.Results Under invert microscope, the forms of cells were irregular and spindle, the chromatin concentrated into masses, the apoptosis bodies were formed 24h after morphine withdral. EGR-1 protein expression was appeared at 1h after withdrawal, reached peak at 8h and was detected even at 24h. MTT assays founded that mortality declined significantly (p<0.05 ), and apoptosis of PC12 cells who transfected siRNAⅠ,Ⅲreduced 26.5% and 20.3% respectively.Conclusion Egr-1 gene is involved in apoptosis after chronic morphine treatment and acute morphine withidrawal. Down-regluation of Egr-1 gene can reduce apoptosis of cells induced by opiate withdrawal.Summary1. Glutamine acid could induce expression of Egr-1 gene, and midazalam, morphine, propofol and ketamine could inhibit its expression.2. The designed siRNAⅠ,Ⅲcan down-regulate the expression of Egr-1 gene in PC12cells and C6 cells.3. Egr-1 gene is involved in apoptosis after chronic morphine treatment and morphine withidrawal.4. Down-regulation of Egr-1 gene can reduce apoptosis of cells induced by morphine withdrawal.5. Apoptosis following chronic opiate treatment and opiate withdrawal could be caused by Egr-1 gene and could be implicated in the development of opioid dependence and addiction.