RNAi-mediated Down-Regulation Of DREAM Gene Expression In Hippocampus And Nucleus Accumbens Inhibited The Conditioned Placed Preference And Withdrawal Syndromes In Morphine-addicted Rats

Addiction to opiates is a chronic, relapsing brain disease induced by long-term use of morphine repeated and uncontrolled. If left untreated, it can cause major medical, social, and economic problems. The clinical manifestations included physiological dependence and psychological dependence. Physiological dependence, which is also called physical, is defined by the appearance of characteristic withdrawal symptoms when opiates are suddenly discontinued. Psychological dependence, which is also called psychic dependence, refers to a sence of well-being and contentment cheerfulness induced by repeated use of opiates. This pleasure effects produce an overwhelming desire to continue with the drug experience followed by a craving or compulsive to use the drug. At present, various effective methods have been devised to improve physiological dependent status. However, it is difficult to get rid of psychological dependence induced by opiates, which tends to relapse.Dynorphin and the kappa opioid receptor (KOPr) are localized in several areas of the dopaminergic nigrostriatal and mesolimbic-mesocortical systems. Dynorphin peptides and pro- dynorphin mRNA are particularly abundant in the nucleus accumbens, caudate, amygdala, hippocampus, and hypothalamus. Dynophin and KOPr play an important role in a modulation of opioid, cocaine, and other rewarding stimuli, presumably through modulation of basal and drug-induced dopaminergic tone. In contrast to the ligand of mu opioid receptor, dynorphin peptides, the ligand of kappa opioid receptor, decrease basal and drug-induced dopamine levels in several areas of the dopaminergic nigrostriatal and mesolimbic-mesocortical system. The KOPr-dynorphin system may therefore be considered to be a part of the countermodulatory mechanisms of the brain after direct or indirect drug-induced dopaminergic stimulation. Earlier studies showed that pretreatment with KOPr agonists decreases the psychostimulant and conditioned rewarding effects of cocaine in rats and decreases the rate of intravenous cocaine self-administration. Thus it has been hypothesized that ligands acting at KOPr may be potential pharmacotherapeutic agents for specific stages in the treatment of addictive diseases.Expression of the human PDYN gene is regulated by the calcium-binding protein downstream regulatory element (DRE) antagonist modulator (DREAM). DREAM, a multifunctional protein, binds as a tetramer downstream to the promoter via an intragenic sequence termed the downstream regulatory element (DRE), blocking transcription. DREAM as a PPD gene transcriptional inhibitor is commonly agreed upon now. If point mutations occur in the DRE site, PPD gene expression will be increased.RNA interference is a gene silencing phenomenon, induced by double-stranded RNA(dsRNA) efficient and special blocking or down-regulating homologous gene expression in various biological cells. Gene silencing induced by RNAi is also known as post-transcriptional gene silencing (PTGS). For mammal animals, transduction of long dsRNA above 30bp into cells often induces unexpected antiviral response. So the common way for mammal animals is to prepare siRNAs (19-23bp) and then transduct it into cells; or make DNA expression vector containing shot hairpin RNAs(shRNAs) and then transduct it into cells to express shRNA, which is cleaved into siRNAs by an enzyme called Dicer. The siRNAs in cells recruit additional components to form an RNA-induced Silencing Complex (RISC). siRNA strands guide RISCs to target mRNA molecules homologous with siRNAs by base pairing, then cleave the target mRNA at the midpoint and result in degradation of it. Finally the corresponding proteins synthesis reduced and thus the target gene expression silencing. siRNAs are characterized in high specificity and can inhibit target gene expression effectively without prejudice to irrelevant genes. This conclusion makes siRNA a powerful tool identifying gene function.The key step in siRNA therapy is using a suitable delivery system to facilitate siRNA access to its intracellular site of action. Delivery system roughly divided into two types:viral gene vectors delivery systems and nonviral gene delivery systems. Nonviral gene delivery systems included bare siRNA, covalent modificated siRNAs, liposomes and lipid complex, nanoparticle, proteins and polypeptide and antibody conjugate. Viral gene vectors included adenovirus vector, adeno-associated virus vectors, retroviral vectors and lentiviral vectors and other viral vectors. Virus vector, is characterized by high transfection efficiency and high expression efficiency, is a powerful tool for gene transfer on the whole and cells level. Adeno-associated virus(AAV), a virus no evidence exist to suggest be related to human disease until now, have great advantages over other viral vectors. It is high safety due to very low cytotoxicity. It has a boarder host range due to its ability of infecting dividing and nondividing cells. It has low immunogenicity because it cannot induce the intensive immunologic reaction. Furthermore, it can integrate into chromosome of host cell and stably express foreign gene in vivo for a long time. These features made AAV a most hopeful gene delivery vector, which is widely used for gene therapy and Vaccines Research.On the basis of the above theory, we hypothesized that if the DREAM in central nervous system silenced by RNAi, the craving related to increased dopamine level will be improved through removing the inhibition of PPD gene expression and thus decreasing the release of dopamine in mesolimbic-mesocortical system. We designed two parts experiments to evaluate the inhibition effect of RNAi-mediated down-regulation of DREAM gene expression in hippocampus and nucleus accumbens on the conditioned placed preference and withdrawl syndromes in morphine-addicted rats.1. To construct recombined adeno-associated viral vector containing DREAM short hairpin RNA and then infect PC 12 cells in vitro. To observe efficiency of rAAV infecting PC 12 cells. Western blotting was used to detect the DREAM protein expression in order to evaluate the RNA interfering effect.2. To transduct DREAM shRNA structure enconding siRNA into hippocampus and nucleus accumbens of rats by means of rAAV using brain stereotaxic technology. After DREAM siRNA stabilize its expression in vivo, we build morphine addict model in rats and to evaluate the inhibition effect of gene therapy on the conditioned rewarding effect and withdrawl syndromes by observing behavior and conditioned placed preference. Methods and results 1. Construction of recombined adeno-associated viral vectors containing DREAM shRNA and identification of its interfering efficiency.Methods:We designed siRNA targeting the rat DREAM coding sequence based on recommendations described by previous literature. The targets of the siRNA comprised of 19 nucleotides of the rat DREAM gene. We designed and synthesized two DNA model-strands encoding DREAM shRNA. After annealing a double strand was resembled and BamHⅠand HindⅢsites was added at both ends simultaneously. Then we ligated the double strand into the plasmid pDC316-EGFP-U6 had been digested by the restriction enzyme BamHⅠand HindⅢ. The recombinant plasmid pDC316- U6-Dream shRNA-EGFP was construct successfully. We designed two primers, one of which complement with 5'(five prime) end of reading frame of EGFP gene on plasmid pDC316- U6-Dream shRNA-EGFP in order to induce EcoRI site, the other of which complement with 5'end of U6 promoter in order to induce SalI site. 华中科技大学博士学位论文Then polymerase chain reaction(PCR) amplification taking pDC316-U6-Dream shRNA-EGFP as model was carried using the two primers. PCR products and plamid pSNAV2.0 were digested by the restriction enzyme EcoRI and SalI and set up ligation reaction to get recombinant vector plasmid pSNAV2.0-EGFP-DREAM-shRNA-U6. Had been identified by enzyme cutting and sequencing, pSNAV2.0 vector carrying foreign gene transfected BHK-21 cells and AAV vector cell strains were developed. Using helper virus HSV1-rc/ΔUL2 carrying rep and cap gene of AAV virus to infect the AAV vector cell strains, we get rAAV2/1 vector rAAV2/1-DREAM-shRNA-EGFP containing foreign gene by a series of steps of cell lysis and purification. Finally, rAAV2/1 vector containing DREAM shRNA infected PC 12 cells in vitro and western blot was used to detect DREAM protein expression in order to evaluate interfering efficiency of rAAV2/1-DREAM shRNA-EGFP. rAAV2/1-EGFP not containing DREAM shRNA were regarded as negative control.Results:PCR identification and sequencing confirmed the successful construction of recombinant adeno-associate viral vector containing DREAM gene shRNA, which can inhibit DREAM protein expression markedly after infected PC 12 cell in vitro.2. stereotaxic injection of rAAV2/1 vector containing DREAM shRNA into hippocampus and nucleus accumbens inhibit the conditioned placed preference and withdrawl syndromes in morphine-addicted rats.Methods:36 SD rats confirm to a certain standard were randomly divided into four groups:rAAV2/1-DREAM group, rAAV2/1 group, PBS group and control group. Bilateral stereotaxic injection of rAAV2/1-DREAM shRNA-EGFP into hippocampus and nucleus accumbens of rats was carry out in rAAV2/1-DREAM group, rAAV2/1-EGFP in rAAV2/1 group and PBS in PBS group. Control group had no injection. Two weeks later morphine-addicted model and conditioned placed preference model of rats were established. The rats in rAAV2/1-DREAM group, rAAV2/1 group and PBS group were administered intraperitoneally with gradually increasing doses of morphine twice daily(interval between two dose is 12 hours) for 10 consecutive days(day1:5 and 5mg/kg; day 2:10 and 10mg/kg; day 3:20 and 20mg/kg; day4:30 and 30mg/kg; day 5:40 and 40mg/kg; day 6:50 and 50 mg/kg; day 7:60 and 60mg/kg; day 8:70 and 70mg/kg; day 9:80 and 80mg/kg; day 10:90 and 90mg/kg). The animals were confined for 1 h to the drug-paired chamber when received the injection of morphine and then move freely. The rats were injected equivalent volume physiological saline intraperitoneally 3h before or after every dose of morphine and then confined for 1h to the non-drug-paired chamber. The rats in control group received physiological saline instead of morphine and the same injection procedure. 3h after the last dose of morphine was given, the rats in every group were administered with nalxone (2mg/kg, i.p.) to induce withdrawal behaviors. The behavioral signs of withdrawal syndrome were measured by the numbers of jumping, wet-dog shaking, mastication and the body weight loss within 30min.3d after the last dose of morphine was given, conditioned placed preference test was carry on. Time spent in each compartment was recorded separately for each animal for a total of 15min. Animals were sacrificed immediately following behavioral testing and their brains rapidly extracted and different brain regions dissected out. Immunohistochemical and western blot were used to detect the DREAM protein expression in hippocampus and nucleus accumbens of rats.Results:1. morphine-addicted rats that had received naloxone injection showed obvious signs of withdrawal. The rAAV2/1 and PBS groups showed marked body weight loss and dramatically increased signs of withdrawal, which were manifested by jumping, wet-dog shaking and mastication, compared to the rAAV2/1-DREAM group (P<0.05).2. The time in drug-paired chamber in rAAV2/1 and PBS group (575.17±66.69,555.50±49.79) are significantly longer than that in the control group (315.33±31.95) (P<0.01). There was no difference in the time change in the drug-paired side in rAAV2/1-DREAM group (366.00±26.38) and control group (315.33± 31.95) (P>0.05).3. Immunohistochemistry and western blotting results show that the DREAM protein expression in hippocampus and nucleus accumbens of rats significantly decreased in rAAV2/1-DREAM group, compared with PBS and rAAV2/1 group(P<0.01).3. Statistical analysisAll of the Measurement data were shown as mean±standard deviation. Statistical analyses were performed with SPSS11.0. Comparison among groups was made using a One-way ANOVA. Values of P<0.05 was considered statistically significant.ConclusionIn our study we successfully constructed Recombinant adeno-associated viral vector containing DREAM gene shRAN, which would targetedly reduce dream protein expression after it infected cells with endogenous expression of DREAM. When DREAM siRNA stabilized its expression in vivo after we transducted DREAM shRNA structure enconding siRNA into hippocampus and nucleus accumbens of rats by means of rAAV delivery system under the guidance of steric orientation technique, it can significantly reduced the DREAM protein expression in hippocampus and nucleus accumbens of rats and dramatically inhibit morphine induced withdrawal symptoms and the formation of conditioned placed preference. These results indicate that siRNA-mediated DREAM gene silencing can inhibit effectively morphine-induced conditioned rewarding effect.SummaryWe successfully constructed recombinant adeno-associated viral silence expression vector for DREAM and obtained stable expression in cultured cells in vitro and in vivo. The interfering effect of DREAM siRNA was confirmed by corresponding protein expression level. The recombinant adeno-associated viral silence expression vector overcome defects of transient expression in previous research on studying gene function using RNAi. The recombinant adeno-associated viral silence expression vector transferred into brain of rats can dramatically inhibit morphine induced withdrawal symptoms and the formation of conditioned placed preference. It's a effective genetically modified therapy methods to treat the syndrome of addiction to opiates with great feasibility and a pilot-study for the therapy of addiction. Furthermore, we indicated that DREAM may be a potential therapy target to alleviate rewarding effect induced by opiates.