CN114214396A - Application of GABRD methylation as heroin relapse resisting target - Google Patents
Application of GABRD methylation as heroin relapse resisting target Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
The invention relates to an application of GABRD methylation as an anti-heroin relapse target spot, and compared with the prior art, the invention has the advantages that: according to the invention, research finds that the heroin relapse behavior can be effectively reduced by reducing the GABRD gene methylation level, and the research and development values and the development significance of the drug for treating heroin relapse by taking the GABRD gene methylation as a target point are important.
Description
Technical Field
The invention relates to a GABRD methylation target spot, in particular to an application of GABRD methylation as an anti-heroin relapse target spot.
Background
Opioid addiction, particularly heroin addiction, causes significant medical and economic hazards. According to investigation data of drugs and crime problem offices (UNODC) of the United nations, 10.96 thousands of drug breaking and acquiring crime cases all the year round in 2018 are shown in the world, 13.74 thousands of criminal suspects are caught, 67.9 tons of various drugs are paid, the heroin addicts still occupy a large proportion, and the heroin abuse situation is severe. Heroin long-term activation of opioid receptors allows compensatory adaptation of both opioid and non-opioid receptor systems, leading to addiction. The chronic use of heroin for a long period of time leads to strong physical and mental dependence, which leads to a strong craving for re-use, which is the main cause of a high relapse rate. The midbrain Ventral Tegmental Area (VTA) to nucleus accumbens (NAc) brain region dopaminergic system is the major pathway for drug reward that is currently recognized. NAc is an important nucleus in the reward circuit of the brain, the mesospiny neuron in the nucleus accumbens, and produces gamma-aminobutyric acid (GABA), a major central nervous system inhibitory neurotransmitter. The A type GABA receptor delta subtype (GABRD) gene is proved to be related to diseases such as epilepsy and convulsion, and the methylation of the gene is found to be possibly involved in addiction and relapse of drugs such as ***e. Therefore, the research on the heroin addiction intervention mechanism and the screening of drug treatment targets are beneficial to promoting the prevention and treatment of heroin addiction and relapse.
Disclosure of Invention
The invention aims to solve the technical problem of providing an application of GABRD methylation as an anti-heroin relapse target point aiming at the current state of the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows: the application of GABRD methylation as a drug target in screening drugs for preventing relapse after heroin withdrawal is characterized in that: the DMR sequence of the differentially methylated region of GABRD is:
CGGACAGCACCCGGGAGGCGGGAGAGAGGTCAGAGGGGTCCTCAGAACTGGAATAGCAGCACGCG。
further, the amplification primer and the sequencing primer for amplifying the DMR sequence of the GABRD differential methylation region are respectively as follows:
methylation specific forward primers: 5 '-Biotin-GTGGTTAAGGGTAAGAATGAAGAGA-3';
methylation specific downstream primers, 5'-CCCATTAAATCCACCCAATTTTACTT-3';
methylation specific sequencing primers, 5'-CCACCCAATTTTACTTT-3'.
Further, the drug for preventing relapse is a GABRD methyltransferase inhibitor.
5 'AZA is a DNA methyltransferase inhibitor, the medicament has an inhibitory action on GABRD methylation, the invention evaluates the inhibitory action effect of 5' AZA on the relapse prevention after the withdrawal of heroin by peripherally injecting 5 'AZA on the relapse behavior of a rat which is self-administered with heroin and centrally injecting 5' AZA on the relapse behavior of a rat which is self-administered with heroin; and the DNA methylation and the protein expression change of the GABRD gene in the NAc brain area of the rat are reabsorbed after the heroin self administration is subsided by central injection of 5' AZA, and the possibility that the GABRD methylation is taken as a drug-dropping treatment target is observed.
The results show that: the peripheral injection of 5 'AZA can effectively inhibit the recovery of the drug-seeking behavior of heroin, and the central injection of 5' AZA can effectively inhibit the recovery of the drug-seeking behavior of heroin, and the results show that: GABRD methylation plays an important role in drug addiction, and drug design aiming at GABRD methylation is expected to give up drug addiction.
Compared with the prior art, the invention has the advantages that: according to the invention, research finds that the heroin relapse behavior can be effectively reduced by reducing the GABRD gene methylation level, and the research and development values and the development significance of the drug for treating heroin relapse by taking the GABRD gene methylation as a target point are important.
Drawings
FIG. 1A is the record of effective nasal triggers and ineffective nasal triggers in the construction of Heroin model of the present invention (Heroin self-administered group Heroin; physiological saline passive administered group Control group);
FIG. 1B is a graph showing the number of injection needles recorded in the construction of the Heroin model of the present invention (Heroin self administration group: Heroin self-administration group; Heroin passive administration group: Yoke Heroin group; physiological saline passive administration group: Control group);
FIG. 2 is a diagram showing the GABRD candidate fragment and the position information of the to-be-detected site according to the present invention;
FIG. 3 shows the changes of methylation of the GABRD gene of the present invention in the Heroin self-administration group (Heroin self-administration group), the Heroin passive administration group (Yoke Heroin group) and the Control group (Control group);
FIG. 4 is a graph showing the results of significant reduction in methylation level of CG3 test site in the heroin self-administered group in the GABRD gene of the present invention;
FIG. 5 is a graph showing the results of significant reduction in methylation level of CG4 test site in the heroin self-administered group in the GABRD gene of the present invention;
FIG. 6 is a graph showing the results of significant reduction in methylation level of CG5 test site in the heroin self-administered group in the GABRD gene of the present invention;
FIG. 7 shows the expression of mRNA in the Heroin self-administration group (Heroin self-administration group), the Heroin passive administration group (Yoke Heroin group) and the Control group (Control group) by the GABRD of the present invention;
FIG. 8 shows the protein expression of the GABRD of the present invention in the Heroin self-administration group (Heroin self-administration group), the Heroin passive administration group (Yoke Heroin group) and the Control group (Control group);
FIG. 9 shows the results of methylation function validation of candidate fragments of the GABRD gene and fragments (Pro is pGL3-promoter plasmid, F-Luc is Firefly luciferase, R-Luc is Renilla luciferase, pGL3-promoter No-load control group, Pro-GABRD is GABRD insert group pGL3-promoter-GABRD, Me-Pro-GABRD is methylated GABRD insert group, pGL3-promoter-GABRD methylated group;
FIG. 10 is a graph showing the effect of peripherally injected methyltransferase inhibitor 5 ' AZA of the present invention on the resuscitative behavior of Heroin ignition after the extinction of Heroin self-administration in rats (Active: number of Active nasal triggers; Inactive: number of Inactive nasal triggers; Heroin: Heroin; SAL: normal saline; 5 ' AZA: 5-AZA-2 ' -deoxycytidine, 5-AZA-2-deoxycytidine);
FIG. 11 is a graph of the effect of peripherally injected methyltransferase inhibitor 5 ' AZA of the present invention on conditioned cue-induced relapse behavior after the self-administration of Heroin in rat bars has subsided (Active: number of effective nasal triggers; Inactive: number of ineffective nasal triggers; Heroin: Heroin; SAL: normal saline; 5 ' AZA: 5-AZA-2 ' -deoxycytidine, 5-AZA-2-deoxycytidine);
FIG. 12 is a graph showing the effect of microinjection of 5 ' AZA, a methyltransferase inhibitor, into the brain region of NAc according to the present invention on the resuscitative behavior of Heroin elicitation in rats (Active: number of Active nasal contacts; Inactive: number of Inactive nasal contacts; Her: Heroin, Heroin; Sal: physiological Saline, Saline; aCSF: vehicle agent; 5 ' AZA: 5-AZA-2 ' -deoxycytidine, 5-AZA-2-deoxycytidine);
FIG. 13 is a graph showing the effect of microinjection of methyltransferase inhibitor 5 ' AZA of the present invention on the methylation of the GABRD gene in the brain region of NAc (Her: Heroin, Heroin; Sal: Saline, Saline; aCSF: vehicle; 5 ' AZA: 5-AZA-2 ' -deoxycytidine, 5-AZA-2-deoxycytidine);
FIG. 14 is a graph of the effect of NAc brain region injection of the methyltransferase inhibitor 5 ' AZA of the present invention on GABRD protein expression (Her: Heroin, Heroin; Sal: Saline, Saline; aCSF: vehicle; 5 ' AZA: 5-AZA-2 ' -deoxycytidine, 5-AZA-2-deoxycytidine).
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
The purity of the heroin used in the embodiment of the invention is 98%, and the heroin purity is provided by a material evidence identification center of the ministry of public security.
Example 1 construction of a model for intravenous self-administration of heroin
Intravenous Self-Administration (Intravenous Drug Self-Administration) is one of the most classical animal models for studying Drug addiction, and the Intravenous Self-Administration model is a classical animal model reflecting the active Drug seeking and Administration behaviors of a user, and the Drug Administration motivation and the active compulsive Administration behaviors can be examined by using experimental animals.
The classical rat heroin self-administration model can well simulate the heroin ingestion behavior of a patient who takes drug clinically, the rat can generate self-administration to the heroin after training, and meanwhile, the continuously rising behavior reaction rate and the dosage to the heroin in the training process can reflect the compulsive drug seeking behavior of the drug-taking patient from the beginning of contacting with the heroin to the addiction of a large amount of heroin.
We used the following method to establish a model for rat self-administration: firstly, SD male rats are subjected to jugular vein cannulation operation, a section of PE tube is inserted into the right jugular vein and penetrates out of the body through the back, and the SD male rats are subjected to antibiosis and recovery for more than one week after the operation. Subsequently, adopt with external sound insulation light-proof self dosing cage, be equipped with in the cage that the nose touches the lamp and is used for writing down effective nose and touch with invalid nose, the syringe pump is connected to top cage lamp, forms nose and touches lamp, cage lamp and syringe pump integrated device. Before self-administration training, a syringe on a syringe pump is filled with 5mL of heroin solution, and after a rat is placed in a cage, a connecting pipe on the cage is connected with a back cannula of the rat. The training procedure is FR-1 intensive training, the procedure is started, a yellow signal lamp in an effective nose touch is lightened at the moment, after a rat touches the effective nose, the signal lamp is extinguished, a cage lamp is lightened, a needle of heroin is obtained along with the peristaltic sound of the injection pump, the cage lamp is continuously lightened for 5s every time, and the dosage of the heroin is as follows: 0.1mg/kg on days 1-2, 0.05mg/kg on days 3-6, and 0.025mg/kg on days 7-14. In the next 20s, the effective nasal contact is in the refractory period (Time out), namely, the rats touch the effective nasal contact only during the period to record data on a computer, but no heroin can be really obtained, the next cycle is carried out after the refractory period, while training 4h by using the heroin self-administered rats, the rats in the passive heroin administered group are also injected with the same amount of heroin, and the rats in the passive saline group are simultaneously injected with the same amount of saline as a normal control group. The self-administration training is carried out for 1 time and 4 hours every day, the effective nasal contact reaction rate and the injection amount are obviously increased for 14 continuous days, and the effective nasal contact number is not more than 10% after the last 3 days (see figure 1A). As can be seen from FIG. 1B, after 14 days of self-administration training, the number of the heroin injection needles of rats in the heroin self-administration group and the passive administration group is significantly higher than that in the normal saline group and is maintained above 40 needles, which indicates that a stable heroin self-administration model is successfully established and meets the requirements of subsequent experiments.
Example 2Illumina HiSeq sequencing
After stable establishment of the heroin self-administration model for 12h, rats were administered intraperitoneally with pentobarbital sodium and the brain area of NAc was sacrificed and isolated after deep anesthesia. A Heroin self-administration group (Heroin self-administration), a Heroin passive administration group (Yoke Heroin group) and a passive saline Control group (Control) were established (n is 3 in each group). The genome DNA of the tissue sample is extracted by a PerkinElmer full-automatic nucleic acid extractor (Shanghai Limited product of Perkin Elmer medical diagnosis), and the concentration of the obtained DNA is detected by a Qubit4.0 nucleic acid protein determinator. And (3) carrying out end repair and adding a sequencing linker after DNA fragmentation and purification, then carrying out Bisulfit treatment, carrying out library construction and quality inspection after amplification, and then carrying out Illumina HiSeq whole genome methylation sequencing on a computer. Finally, differential methylation sites and regions (DMR) are analyzed and screened, and the changes of methylation levels of each group are compared, so that DMR exists on the GABRD gene and is screened and found in chr 5: 172809066 and 172809129 regions were also located on the CpG52 island (see table I and fig. 2).
And the DMR sequence is:
CGGACAGCACCCGGGAGGCGGGAGAGAGGTCAGAGGGGTCCTCAGAACTGGAATAGCAGCACGCG；
table I: sequencing and detecting the DMR region information of the GABRD gene
>rn6_cpgIslandExt_CpG:52 range＝chr5:172809013-172809608 5'pad＝0 3'pad＝0 strand＝+repeatMasking＝none
CGCCTGGCGACTCCAATCCCTCCCCAAAGCTCAGCGTTCAGCACCGAGGACGGCGGACAGCACCCGGGAGGCGGGAGAGAGGTCAGAGGGGTCCTCAGAACTGGAATAGCAGCACGCGCAAAGCAAAACTGGGTGGACCTAATGGGTGAAGACTCGGGGTTCTGGCAACTCGGAACTCTGCCCCACCGAGGGTGCCCGCCAAAGGACGAGTCCCTCCCGCCTCATATCGTCCCCGGCCCAGGAGCTGCTCACCTGGCGCCATGGTGCGGCTGCGTGCACAGCAGAAGGAGCGGCAGCAGCAGCCAGCCCAGAACGTCCATGGCCTTGCGGGCTGTGCGCGGGACAGGTCCGGCTAGCCCCAGCGCAAGCAAAGTTGCTTCGCCTGCGGCCGCGCGGCGGCACCAGAAGGGCGGGAGCGCGAAGGAGAGTGGAACGGGCGCGGGGGGAGGACGGAACGTCGAGCCTGCCCCGCCCCGCCCCGCCCGGCGCTCCAGTCCCACCCCCGCTCGCGCGCCCCACTGCACGCGGGAGGCTTCTGCGCACCGCATAGCACCGGGTGGTAGCTCCACCGCGCTCCCAGAGCCTCGCACACCGCG
Meanwhile, the nucleotide sequence can be correspondingly referred to SEQ ID NO.1 of the sequence table.
EXAMPLE 3 amplification of samples to detect changes in the DNA methylation level of the GABRD Gene
The rat Heroin self-administration model was re-established as in example 1, and a Heroin self-administration group (Heroin self-administration), a Heroin passive administration group (Yoke Heroin group) and a passive saline Control group (Control) were established. Taking each group of NAc brain tissues, and extracting tissue DNA. DNA methylation level detection is carried out on 5 CpG sites in DMR of the CpG island region of the GABRD gene by adopting a bisulfite pyrosequencing technology. The basic principle of this technique: after a DNA sample is treated by bisulfite in the kit, a Polymerase Chain Reaction (PCR) is applied to amplify an upstream primer and a downstream primer by methylation specificity amplification, and then pyrophosphoric acid sequencing is carried out on an amplified product by a specificity sequencing primer, so that the methylation degree of each site is calculated. In the research, PyroMark Assay 2.0 software is adopted for primer Design, and PCR amplification primers and sequencing primers used for experiments are as follows:
(1) methylation specific Forward primer (Forward primer)
5’-Biotin-GTGGTTAAGGGTAAGAATGAAGAGA-3’；
(2) Methylation specific downstream primer (Reverse primer)
5’-CCCATTAAATCCACCCAATTTTACTT-3’；
(3) Methylation specific Sequencing primer (Sequencing primer)
5’-CCACCCAATTTTACTTT-3’。
The specific steps of the amplification are as follows:
a. the genomic DNA of both the addicts and controls was Bisulfite converted using the Qiagen EpiTect bisufite Kit (Qiagen, # 59104).
b. And B, taking 10-20ng of the DNA sample transformed in the step A, adding the DNA sample into a Pyromark PCR Kit (Pyromark PCR Kit; Qiagen; #978703), adding the pair of GABRD gene CpG52 region methylation specific amplification primers, and carrying out PCR amplification under the conditions: firstly, denaturation at 95 ℃ for 3 min; then carrying out 40 cycles of reactions at 95 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1 min; then the extension reaction was carried out at 72 ℃ for 7 min.
c. Pyrosequencing detection, designing a program to be run in PyroMark Q48 software, importing the program into a U disk, and inserting the program into a USB interface of PyroMark Q48. And clicking the 'Sequence' displayed on the touch screen of the instrument, and loading the running program in the USB flash disk. Putting a cotton sliver absorbing waste liquid, and ensuring that the head and tail connection part is in the right left (9 o' clock) direction. And pushing a cover of the instrument open, opening the cover of each clip, selecting the clip to be poured, displaying a water drop icon on the selected clip, and selecting whether the current run is the last run. The reagents are added to each clip in sequence, depending on the volume of each reagent displayed on the touch screen. When the reagent is added into the card holder, please use the sample adding gun to adhere to the wall and add slowly, avoid generating bubbles. After adding the reagent, the lid of the clip is closed and locked. Clicking "Start" starts to test whether the cardholder functions properly, and a green arrow appears behind the passing cardholder. The loading disc is placed in the machine, care is taken to align the hole in the disc in a defined position, otherwise the machine will alarm. And screwing a fixing screw for fixing the sample loading disc. The beads were shaken and then applied to the loading disc using a continuous application gun fed with a random machine, 3. mu.l per well, 3 wells at a time. Mu.l of PCR product containing biotin label is added to the well of the loading disc, and if the amount of PCR product is less than 10. mu.l, 10. mu.l is supplemented with pure water to ensure that the magnetic beads are completely covered with the PCR product liquid. The Q48 program is run, clicking "Start". And when the operation is finished, the result can be automatically led into the USB flash disk, the USB flash disk is pulled out, the instrument is closed from the software, and then the instrument is closed. Finally, the methylation status of each site was analyzed using Pyro Q CpG software available from a pyrosequencer. Data from the results of the analysis obtained from the pyrosequencer (see FIG. 3 for example). The horizontal axis represents the order of dDNTP added, and the vertical axis represents the intensity of the fluorescence signal. Among these, blue was the best quality sequenced, yellow second, and red was the worst quality. However, if there is blue in one reaction, it is shown that the overall quality is not problematic, and the red and yellow results are normal. Analysis of the data using SPSS 16.0 found: significant correlation (R >0.5, P <0.01, see R values in the table of fig. 2) was found between the 5 sites tested (CpG1, CpG2, CpG3, CpG4 and CpG5), and thus the total methylation of CpG1-CpG5 was analyzed. The difference in methylation level of CpG52 region of GABRD gene between the passive group and the control group was found in the heroin-self-administered group, and as a result (see fig. 3, the methylation level of GABRD in the heroin-self-administered group was found to be significantly lower than that in the control group (P ═ 0.012) —, each site was compared and analyzed, and it was shown that the methylation level was significantly lower in all three sites CG3, CG4, and CG5 in the heroin-self-administered group relative to the control group (see fig. 4, 5, and 6).
Example 4 variation of mRNA expression levels of GABRD Gene in NAc brain region
The GBRD gene mRNA expression is detected by reverse transcription real-time quantitative PCR (RT-qPCR) of a Roche LightCycler 480 instrument. Extracting naloxone self-administered group, passive administered group and control group rat NAc brain tissue RNA, and using total RNA
(Invitrogen; Thermo Fisher Scientific, Inc.) kit extraction. The total RNA was reverse transcribed to synthesize cDNA using the MiScript II RT kit (Qiagen gmbH). The qPCR primer nucleotide sequences are shown in table II, GAPDH gene as internal reference. The reaction system contained 5. mu.l of SYBR Green premix,mu.l of primers (10. mu.M), 1.5. mu.l of cDNA and 1.5. mu.l of water. The amplification conditions were: 95 ℃, 10min, 40 cycles: 95 ℃, 10S, 55 ℃, 15S and 72 ℃, 15S. Use 2-ΔΔCqMethods analysis RT-qPCR data was analyzed for relative changes in GABRD mRNA expression levels. The analysis of the results showed that the level of mRNA expression of the GABRD gene in the heroin-self-administered group was significantly increased as compared with the control group and the heroin-passively administered group, respectively (F)(3,24)＝9.065，P＝3.42E-4；F(2,24)10.238, P0.001, see fig. 7).
TABLE II qPCR primer sequence information
Example 5 changes in protein expression levels of the GABRD Gene in the NAc brain region
Meanwhile, the expression level of GABRD protein in NAc brain region in the Heroin self-administration group (Heroin self-administration), the Heroin passive administration group (Yoke Heroin group) and the passive saline Control group (Control) was measured by Western Blot method. The detection method comprises the following steps: dissolve RIPA lysate (petun yunnan, P0013B) and mix well. An appropriate amount of lysate was taken, PMSF was added several minutes before use to give a PMSF final concentration of 1mM, 150 μ l of lysate was added to the tissue, homogenized using a homogenizer, and then repeatedly blown with a pipette to disrupt the tissue and lyse it sufficiently. Placing on ice for 30min, vortexing once every 10min to fully crack the tissue, centrifuging at 4 ℃ of 10000-; mixing the protein solution and loading buffer, placing in 100 deg.C water bath for 5min, centrifuging, placing on ice, and waiting for sample loading. SDS-PAGE separation gel is prepared, added into two gel-making glass plates and sealed by isopropanol. After the lower layer gel solidified, the isopropanol was poured off and ddH was used2And (4) after O washing, sucking dry and starting to prepare concentrated glue. Adding the concentrated glue into two glue-making glass plates, immediately inserting the comb without bubbles, and pulling out the comb after solidification. After a gel making groove is made, the gel making groove is placed on ice, and after the electrophoresis liquid is poured in, the sample loading is started, and 10-35 mu l of sample is loaded in each hole. Recording the sample loading sequence in advanceRunning buffer (Tris 3.028g (25mM), Gly 14.413g (192mM), 10% SDS 10mL (0.1% SDS) was prepared with ddH2And (4) metering the volume of O to 1000mL, and pre-cooling at 4 ℃. Transfer buffer (Tris 3.028g) (25mM), Gly 14.413g (192mM), Methanol (Methanol)200mL with ddH2O is metered to 1000mL), and the solution is pre-cooled at 4 ℃. The upper layer glue is 60v for 30min, and the lower layer glue is 110v for 120 min. After electrophoresis, the gel needs to be rinsed in PBS for 2min, and then put into a membrane-transferring buffer solution for soaking and balancing. Soaking the sheared PVDF membrane in methanol for 2min, and then in ddH2Soaking in O for 2min, and soaking in transfer buffer for 15 min. Placement of the cushion, film and glue: red, (+) white shelf, pad, filter paper, membrane, glue, filter paper, pad, black shelf (-), black. The trans-film strips were from-to +. Transfer on ice, 100v, 40min/70 min. And after the transfer is finished, putting the membrane into PBS for soaking for 3-5min, and shearing the membrane. Blocking with 5% skim milk (0.75g milk powder +15mL PBS) at 37 ℃ for 1-2 h. The membranes were washed three times with 1 XPBS-T (1000mL of 1 XPBS +1mL of Tween-20) for 5-10 min. Incubate primary antibody, internal control (1: 5000), GABRD protein (both diluted at 1: 1000), shake at 4 deg.C overnight (freezer). Taking out the shaking table from the ice house, washing the membrane, and carrying out 1 XPBST for 5-10min for three times. Add secondary antibody (1:10000), at 37 deg.C for 2h, shake the membrane with 1 XPBST for 5min, wash the membrane five times. And recording and storing the result by using the chemiluminescence developing ice, and performing gray scale analysis. The results showed that there was a significant difference in the expression level of proteins between groups (F)(2,3)21.942, P ═ 0.016), multiple comparisons showed a significant increase in the level of GABRD gene mRNA expression in the heroin self-administered group relative to the control group and the heroin passive-administered group, respectively (P ═ 0.016, P ═ 0.009, see fig. 8).
Example 6 Dual luciferase reporter Gene experiments
The dual-luciferase reporter gene vector system contains two luciferase genes which can be expressed in the same cell at the same time, and comprises a firefly luciferase reporter gene and a Renilla luciferase reporter gene. The regulatory sequence of the target gene to be researched is cloned into an expression plasmid containing a reporter gene, then the recombinant plasmid is introduced into an appropriate cell line, and the induction effect of the target sequence on the gene expression is indirectly evaluated by measuring the expression level of the reporter gene.
In order to verify the biological function of a target sequence of the GABRD gene, test whether the target sequence has enhancer activity and detect the influence of sequence methylation on the activity, a dual-luciferase reporter gene experiment is carried out, 293T cell amplification culture is firstly carried out, and then a rat GABRD target DMR sequence is synthesized: CGGACAGCACCCGGGAGGCGGGAGAGAGGTCAGAGGGGTCCTCAGAACTGGAATAGCAGCACGCG, the synthetic sequence was inserted into the PGL3-Promoter to construct a plasmid. The constructed product was methylated in vitro using the SssI kit (Thermo, EM 0821). Methylation identification is carried out by using an EpiJET DNA Methylation Analysis Kit, the Methylation effect is verified by electrophoresis, then the plasmid which is successfully constructed and successfully methylated is transfected into 293T cells, and dual-luciferase reporter gene detection is carried out 48h later, and the grouping is as follows: pGL3-promoter No-load control (Pro); GABRD insertion set pGL3-promoter-GABRD (Pro-GABRD); methylated GABRD insert set pGL3-promoter-GABRD methylation set (Me-Pro-GABRD). Before detection, the culture medium is discarded for rinsing, 100 mul of diluted 1 XPLB is added into each hole, and the cells are cracked by shaking for 15min at the normal temperature of a shaking table. The lysate was collected into a 1.5mL Ep tube, centrifuged at 10000g for 5min, and the supernatant was collected for use. Opening a multifunctional microplate reader (TECAN Infinite M1000 Pro)), opening software, adding 10mL of the pre-mixed luciferase test reagent LAR II into a 15mL centrifuge tube, and putting the centrifuge tube into an A channel suction tube; 200. mu.l of 50X
Substrate was added to 10mL
In Buffer, configured as Stop&Glo Reagent, and Stop&The Glo Reagent was added to a 15mL centrifuge tube, the B channel pipette was placed, the software was turned on, and the parameters were set. Adding 50 μ l of the supernatant into each well of a white opaque 96-well enzyme-labeled plate, placing into a multifunctional enzyme-labeled instrument, starting detection, adding 50 μ l of the pre-mixed luciferase test reagent LARII into each well, measuring data after 2s, and adding 50 μ l of Stop into each well&Glo Reagent, after resting for 2s, data were measured and the differences compared according to the obtained ratioThe degree of activation of the reporter gene of interest between samples; then analyzed using Graphpad prism (Graphpad Software, San Diego, Calif.) Software, statistically tested for One-way anova followed by SNK for differences between groups, and P for differences<0.05 is the screening criterion for significant differences. As shown in fig. 9, compared with the Pro group, the fluorescence ratio of the Pro-GABRD group is significantly increased, and the fluorescence ratio of the methylation group Me-Pro-GABRD is significantly decreased, which suggests that the DMR sequence of the GABRD gene has a significant enhancer-like function, and the enhancer-like activity of the fragment is significantly inhibited after methylation. The results show that the target DMR sequence of GABRD acts as an enhancer, while the methylation of GABRD reduces its gene expression promoter function.
Example 75' Effect of AZA on the Retention conditioned cue-induced or heroin-induced relapse behavior in rat treated with heroin self-administration
The re-inhalation is the difficulty for clinically treating the heroin addiction, and after the heroin patient is detoxified and abstained, the heroin patient is induced by contacting with the related environment of the previous drug taking, the drug itself or the drive of the poison friends, so that the impulsion of taking the heroin is generated, and the re-inhalation of the heroin is finally caused. The rat relapse model can better reflect the behavior of relapse of heroin (re-addiction) caused by the fact that a patient in the heroin withdrawal period contacts the environment related to previous heroin ingestion or the heroin per se again.
(1) Effect of peripheral injection of 5' AZA on the Return to suckback behavior after Adenosine self-administered rats regressed
To evaluate whether 5' AZA has an intervention effect on the relapse behavior of heroin addicted rats, a conditioned cue or a heroin ignition induced rat relapse model is established respectively, and the relapse behavior is tested. First, rats were trained to receive 14 days of Heroin self-administration training in self-administration operating cages, all experimental rats were trained to receive 9 days of Heroin or saline self-administration training using the FR1 procedure (4 h/day), and then, on day 10, the rats with Heroin self-administration were randomly divided into Heroin +5-Aza-dc administration group (Heroin/5 'AZA) and Heroin + saline group (Heroin/SAL), and the Heroin/5' AZA group rats were divided half an hour before daily Heroin self-administration trainingRats in the Heroin/SAL group were given physiological saline instead of 5 'AZA half an hour prior to daily self-administration training for 8 consecutive days, 5' AZA (5mg/kg, s.c.). Statistical analysis shows that peripheral injection of DNA methyltransferase inhibitor 5' AZA can significantly reduce the number of re-inhalation effective nasal-tactile responses (F) of heroin-induced ignition in self-administered rats(2,6)＝9.249，P<0.05, see fig. 10), whereas peripheral injection of 5' AZA tended to decrease the effective nasal contact number of the heroin-self-administered rats on conditioned cue-induced relapse, but was not statistically significant (F)(2,6)0.012, P0.915, see fig. 11). The results suggest that peripheral injection of 5' AZA can effectively inhibit the recovery of heroin foraging behavior.
(2) Effect of Central injection of 5' AZA on the Return to suckback behavior in heroin-dosed rats
To further evaluate whether the effect of 5' AZA on the relapse behavior of heroin self-dosed rats was centrally acting, we re-established a conditioned cue or heroin elicitation induced rat relapse model and performed a test of relapse behavior. First, all experimental rats were trained for 14 days of heroin or saline self-administration using the FR1 procedure (4h/days) and then regressed, starting on the 10 th day of the regression training, by bilaterally microinjecting 5 ' AZA (1 ug/side, 1mg/ml concentration) or aCSF in the NAc brain area half an hour prior to the training, for 14 days of regression, and the rats that were self-administered with heroin and saline were randomly divided into a heroin +5-AZA-dc administration group (Her/5 ' AZA), a heroin + saline + physiological saline group (Her/aCSF), a physiological saline +5-AZA-dc administration group (Sal/5 ' AZA) and a physiological saline + vehicle (Sal/aCSF). Statistical analysis showed that 5-Aza-dC (F (1,19) ═ 14.402, P ═ 0.001), Heroin (F (1,19) ═ 28.475, P ═ 5.46E-5) and interaction (Heroin and 5-Aza-dC) (F (1,19) ═ 16.367, P ═ 8.40E-4) all resulted in a change in the number of effective rhinobyes as the main effect, and multiple comparisons showed a significant decrease in the number of effective rhinobyes due to Heroin-induced re-inhalation in the Heroin/5' Aza group compared to the Heroin/aCSF group (P ═ 2.45E-5, see fig. 12). The results suggest that central injection of 5' AZA can effectively inhibit the recovery of the foraging behavior of heroin.
Example 8 Effect of Central injection of 5' AZA on DNA methylation and protein expression of the GABRD Gene in the brain region of NAc in rats that repopulated the regression of heroin self-administration
All rats in example 7 were sacrificed immediately after the relapse behavior test, and the NAc brain region was taken to extract DNA and protein, respectively, and the DNA was assayed for methylation level of the target fragment of the GABRD gene by the pyrosequencing method. The results showed a significant reduction in methylation levels of the fragments of interest of the Heroin/5' AZA group GABRD genes compared to the Heroin/aCSF group (results are shown in fig. 13: 44.71 ± 1.19, 32.68 ± 5.29, P ═ 0.038). Also the methylation level of the target fragment of the Sal/5' AZA group GABRD gene was significantly reduced compared to Sal/aCSF (36.99 + -1.17, 11.36 + -2.56, P-4.64E-4). Furthermore, there was a tendency for elevated methylation in Heroin/SAL versus Control/SAL groups, but no statistical difference, as shown in the following Table:
in addition, the protein expression level of the extracted protein is detected by WB, and the expression level of the Heroin/5' AZA group GABRD protein is found to be remarkably increased compared with the Heroin/aCSF group (the result is shown in figure 14: P ═ 0.018). At the same time, the methylation level of the target fragment of the Sal/5' AZA group GABRD gene was also significantly increased compared to Sal/aCSF (P0.010). The results suggest that central injection of 5 'AZA effectively inhibited the recovery of heroin's foraging behavior, likely acting in part by decreasing methylation of the GABRD gene to increase its protein expression level.
Sequence listing
<110> Ningbo City Kangning Hospital (Ningbo City center for mental disease prevention and control, Ningbo City microcirculation and hyoscyami research institute)
Application of <120> GABRD methylation as anti-heroin relapse target
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 596
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgcctggcga ctccaatccc tccccaaagc tcagcgttca gcaccgagga cggcggacag 60
cacccgggag gcgggagaga ggtcagaggg gtcctcagaa ctggaatagc agcacgcgca 120
aagcaaaact gggtggacct aatgggtgaa gactcggggt tctggcaact cggaactctg 180
ccccaccgag ggtgcccgcc aaaggacgag tccctcccgc ctcatatcgt ccccggccca 240
ggagctgctc acctggcgcc atggtgcggc tgcgtgcaca gcagaaggag cggcagcagc 300
agccagccca gaacgtccat ggccttgcgg gctgtgcgcg ggacaggtcc ggctagcccc 360
agcgcaagca aagttgcttc gcctgcggcc gcgcggcggc accagaaggg cgggagcgcg 420
aaggagagtg gaacgggcgc ggggggagga cggaacgtcg agcctgcccc gccccgcccc 480
gcccggcgct ccagtcccac ccccgctcgc gcgccccact gcacgcggga ggcttctgcg 540
caccgcatag caccgggtgg tagctccacc gcgctcccag agcctcgcac accgcg 596
Claims (3)
1. The application of GABRD methylation as a drug target in screening drugs for preventing relapse after heroin withdrawal is characterized in that: the DMR sequence of the differentially methylated region of GABRD is:
CGGACAGCACCCGGGAGGCGGGAGAGAGGTCAGAGGGGTCCTCAGAACTGGAATAGCAGCACGCG。
2. use according to claim 1, characterized in that: the amplification primer and the sequencing primer for amplifying the DMR sequence of the GABRD differential methylation region are respectively as follows:
methylation specific forward primers: 5 '-Biotin-GTGGTTAAGGGTAAGAATGAAGAGA-3';
methylation specific downstream primers, 5'-CCCATTAAATCCACCCAATTTTACTT-3';
methylation specific sequencing primers, 5'-CCACCCAATTTTACTTT-3'.
3. Use according to claim 1, characterized in that: the drug for preventing relapse is a GABRD methyltransferase inhibitor.
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