Application of lncRNA Meg3 as target spot in anti-addiction drugs

Abstract

The invention relates to the field of biological medicine, and discloses application of lncRNA Meg3 as a target spot in anti-addiction drugs. Through next-generation sequencing, it is found that lncRNA Meg3 in hippocampus tissues of oxycodone addiction mice is highly expressed, and m6A methylation is mediated by METTL3. SiRNA Meg3 is designed aiming at the target spot and is wrapped in lipid nanoparticles, and the oxycodone addiction behavior can be remarkably inhibited through nasal administration. The siRNA lipid particle has no addiction, has no influence on normal behaviors such as sweet water preference and the like, and has specificity. Experiments prove that the compound plays a role by blocking an ERK / MEK signal channel. The invention provides a new strategy for the development of anti-addiction drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of lncRNA Meg3 as a target in anti-addiction drugs. Background Art

[0002] Drug addiction is an ongoing public health issue. In recent years, addiction to the opioid oxycodone has gradually come into the public eye. In 2019, the National Medical Products Administration (NMPA) designated oxycodone-containing compound preparations and other products as psychotropic substances. Research on oxycodone addiction is lagging, and its pathogenesis and treatment have yet to be discovered. Currently, opioid receptor antagonists or agonists, such as methadone, naltrexone, and buprenorphine, are used to treat oxycodone addiction. However, these drugs are inherently addictive and are non-specific treatments. Summary of the Invention

[0003] In response to the problems existing in the prior art, the present invention provides the use of lncRNA Meg3 as a target in anti-addiction drugs. The present invention screened out lncRNA Meg3 by performing second-generation sequencing on the hippocampal tissue of oxycodone-addicted mice, designed and synthesized siRNA Meg3 targeting lncRNA Meg3, and encapsulated siRNA Meg3 by preparing lipid nanoparticles. Subsequent nasal administration can inhibit oxycodone addiction behavior. The administration of siRNA Meg3 lipid nanoparticles alone did not lead to addictive behavior in mice, indicating that they are not addictive in themselves. siRNAMeg3 lipid nanoparticles had no effect on the mice's preference for sugar water, indicating specificity in inhibiting oxycodone addiction.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0005] The present invention discloses the use of a lncRNA Meg3 expression level inhibitor in the preparation of a drug for treating oxycodone addiction.

[0006] Furthermore, the lncRNA Meg3 expression inhibitor includes an siRNA specifically targeting lncRNA Meg3, and the nucleotide sequence of the siRNA is shown in any one of SEQ ID NO.1 to SEQ ID NO.2.

[0007] The present invention also discloses a pharmaceutical composition for resisting oxycodone addiction, comprising an active substance for targeted inhibition of lncRNA Meg3 expression and a pharmaceutical carrier.

[0008] Furthermore, the active substance that inhibits the expression of lncRNA Meg3 is siRNA.

[0009] Furthermore, the siRNA sequences are shown in SEQ ID NO: 1 and NO: 2.

[0010] The present invention also discloses an anti-addiction drug composition, characterized in that it comprises any of the above-mentioned drugs and a pharmaceutical carrier.

[0011] Furthermore, the carrier is a lipid nanoparticle (LNP), and the LNP contains DLin-MC3-DMA, DSPC, cholesterol, and PEG2000-DMG, and the molar ratio thereof is (45±2):(10±1):(43.5±2):(1.5±0.2).

[0012] Furthermore, the mass ratio of total lipid to siRNA in the LNP is (30±5):1.

[0013] Furthermore, the LNP has a Zeta potential of -10 mV to -20 mV and an average particle size of 80 nm to 100 nm.

[0014] The present invention also discloses the use of a lncRNA Meg3 expression detection reagent in the preparation of an oxycodone addiction diagnostic kit, characterized in that the reagent comprises a primer pair or a probe that specifically recognizes lncRNA Meg3.

[0015] Compared with the prior art, the present invention has the following beneficial effects.

[0016] 1. The present invention discovered for the first time that siRNA Meg3 can specifically inhibit oxycodone addiction and is not addictive itself, thus overcoming the difficulty of the drug itself producing addiction.

[0017] 2. The siRNA Meg3 lipid nanoparticles, developed for the first time by this invention, specifically inhibit oxycodone addiction and can be used to treat oxycodone addiction. Nasal administration allows the drug to enter the brain faster and take effect more quickly than traditional oral treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Oxycodone upregulates METTL3 expression in mouse hippocampal neurons. (AB) Mass spectrometry detection of two glutamate channels; (C) Mass spectrometry detection of glutamate fragmentation in samples; (D) Mass spectrometry detected increased glutamate release in the hippocampus of oxycodone-addicted mice; (E) RT-qPCR detected upregulation of METTL3 and METTL14 expression in the hippocampus of oxycodone-addicted mice; (F) Blockade of glutamate receptors downregulated METTL3 expression; (G) Conditioned place preference experiments confirmed that oxycodone addiction worsens with increasing dose; (H) METTL3 expression increases with increasing oxycodone dose (top) or duration of administration (bottom); (I) METTL3 expression in different cell types.

[0019] Figure 2 Oxycodone promotes the binding of METTL3 and METTL14 in mouse hippocampal neurons. (A) METTL14 expression increases with increasing oxycodone dose (top) or duration (bottom); (B) Co-IP assay detects increased binding of METTL3 and METTL14; (C) Immunofluorescence assay detects upregulation of METTL14 in hippocampal neurons of oxycodone-addicted mice; (D) Immunofluorescence assay detects increased colocalization of METTL3 and METTL14 in the hippocampus of oxycodone-addicted mice.

[0020] Figure 3 Oxycodone induces m6A methylation of lncRNA Meg3 via METTL3. (AC) Next-generation sequencing detected upregulated expression of lncRNA Meg3 in the hippocampus; (D) RT-qPCR verified the increased expression of lncRNA Meg3; (E) SRAMP database predicted potential m6A methylation sites on lncRNA Meg3; (FG) Oxycodone promoted methylation of sites #5 and #8 on lncRNA Meg3 via METTL3.

[0021] Figure 4 siRNA Meg3 lipid nanoparticles inhibit oxycodone addiction. (AD) siRNA Meg3 inhibits the ERK / MEK signaling pathway; (EF) siRNA Meg3 inhibits oxycodone addiction; (GL) siRNA Meg3 lipid nanoparticles specifically inhibit oxycodone addiction. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments, as all technologies implemented based on the present invention fall within the scope of the present invention.

[0023] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0024] Example 1: Oxycodone upregulates the expression of METTL3 in mouse hippocampal neurons.

[0025] 1. Experimental Materials .

[0026] 2. Experimental methods

[0027] (1) High performance liquid chromatography-mass spectrometry. An Agilent 1260 high performance liquid chromatograph and a 6420 QQQ mass spectrometer (Agilent Technologies, Inc., USA) with an electrospray ionization source were used for analysis. Data were processed using a MassHunter B.07.00 workstation. Multiple reaction monitoring (MRM) mode was used for component analysis. The ion fragments of glutamic acid were m / z 148→102 and m / z 148→84. An Agilent C18 column was used for separation. The mobile phase consisted of solution A: 0.1% methanol aqueous solution and solution B: acetonitrile (A:B, 30:70, v / v), with a flow rate of 0.4 mL / min. The sample preparation procedure was as follows: 100 μL of hippocampal tissue homogenate was taken, 500 μL of acetonitrile was added for protein precipitation, the mixture was centrifuged at 13,000 r / min for 5 min, and the supernatant was collected for LC-MS / MS analysis.

[0028] (2) Fluorescence real-time quantitative polymerase chain reaction (RT-qPCR). Total RNA extraction from tissues: Fresh mouse hippocampus was placed in a pre-cooled 2 mL EP tube. 1 mL of Trizol solution was added, ultrasonically disrupted on ice for 20 min, and then allowed to stand for 5 min. The supernatant was transferred to an EP tube, 200 µL of chloroform was added, and vortexed for 30 s until the tube became milky. After standing at room temperature for 3 min, the tube was centrifuged at 4°C, 12000 g, for 15 min. 400 µL of the upper aqueous phase was transferred to a new EP tube, 400 µL of isopropanol was added, and the tube was thoroughly mixed by inversion. After standing at room temperature for 10 min, the tube was centrifuged at 4°C, 12000 g, for 10 min, and the supernatant was discarded. 1 mL of ethanol was added, the tube was centrifuged at 4°C, 7500 g, for 5 min, and the supernatant was discarded. 1 mL of ethanol was added, the tube was centrifuged at 4°C, 7500 g, for 5 min, and the supernatant was discarded. 1 mL of ethanol was added, the tube was centrifuged in reverse at 4°C, 7500 g, for 5 min, and the supernatant was discarded. After absorbing excess water, the concentration and A260 / A280 ratio were tested using an RNA concentration detector and then stored at -80°C for a long term.

[0029] RNA Reverse Transcription: Extracted RNA was reverse transcribed into cDNA using a reverse transcriptase instrument. Store the cDNA at -20°C. RNase-free ddH2O, 4× gDNA Wiper Mix, 5× HiScript III qRT SuperMix, and 4× No RT Control Mix were added in the order specified in the manufacturer's instructions to remove genomic DNA and perform reverse transcription to generate the cDNA product.

[0030] Real-time fluorescence quantitative PCR: performed using the Stratagene Mx3000P system (Agilent Technologies, Inc., USA), with each sample tested three times. Each 20µL qPCR reaction system contained 10ng of cDNA template, 10µL of 2×SYBR Green qPCR Master Mix, 10µM of forward and reverse primers, and 0.4 of ROX reference dye. The cycling conditions for qPCR were as follows: (a) initial denaturation: 95°C for 30s; (b) 35 cycles of amplification: denaturation at 95°C for 15s; (c) annealing and extension at 60°C for 1min. GAPDH was used as an internal reference for normalization, and 2 -ΔΔCt The relevant primer sequences are shown in Table 1.

[0031] Table 1: Primer sequences (5'-3') .

[0032] (3) Western Blot. Place the mouse hippocampus sample in a pre-cooled 2 mL EP tube, add RIPA and PMSF in a fixed ratio (tissue:RIPA:PMSF = 1:10:0.1), and ultrasonically disrupt the tissue until it is fully lysed. Then transfer it to a 2 mL EP tube and centrifuge it at 12,000 rpm for 15 min at 4°C. Take the supernatant and measure the protein concentration. Add 5× Loading Buffer, shake it, and heat it in a water bath for 10 min to denature it. Store it at -20°C until ready for use.

[0033] Prepare the separating gel according to the recipe in Table 2. Once the separating gel has solidified, pour the prepared stacking gel on top and insert a comb to allow it to solidify. Once the stacking gel has solidified, remove the comb and place the stacking gel plate in the electrophoresis tank. Add the appropriate amount of electrophoresis buffer and then load 3µL of marker and 5µL of protein sample into each well. Electrophoresis was performed using the stacking gel (70V, 30min) and the separating gel (110V, 1.5h). After electrophoresis, the lower separating gel and a PVDF membrane activated in methanol were sandwiched in sequence. Transfer was performed in an ice bath at a constant current of 260mA for a time consistent with the protein's molecular weight. After transfer, the strips were blocked in 5% skim milk for 120min. After blocking, the PVDF membrane was washed three times with TBST and incubated with the primary antibody overnight at 4°C. The dilution ratio for METTL3 was 1:1000; the dilution ratio for NR2B was 1:1000; and the dilution ratio for GAPDH was 1:5000. The next day, remove the PVDF membrane and place it on a shaker to rewarm for 40 minutes. Wash it three times with TBST solution for 15 minutes each. After washing, incubate it with the secondary antibody at a dilution ratio of 1:5000 for 70 minutes at room temperature on a shaker. Remove the PVDF membrane and wash it three times with TBST solution for 15 minutes each. After washing, prepare the ECL developer. Mix equal volumes of Solution A and Solution B. Remove the PVDF membrane and place it in a developer. Add an appropriate amount of developer solution. Collect the results using a chemiluminescence gel imaging system.

[0034] Table 2. SDS-PAGE separation gel and stacking gel preparation .

[0035] (4) Conditioned place preference (CPP). Adult male ICR mice, 5-8 weeks old, weighing 25-30 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd., with an ethics number of 2023PS692K. Four groups of animals were randomly assigned to the study group: blank group (Sal, n=12), model group 1 (Oxy1.5, 1.5 mg / kg, n=12), model group 2 (Oxy3, 3 mg / kg, n=12), and model group 3 (Oxy6, 6 mg / kg, n=12). The mice were given the drug once a day for 4 consecutive days. The drug volume was 0.1 mL / 10 g and injected intraperitoneally into the mice.

[0036] The CPP enclosure was an SPF-grade mouse cage with horizontal and vertical stripes taped to the walls using electrical tape (18 mm wide). The cage was divided into two rectangular chambers, each measuring 15 cm × 10.5 cm × 15 cm. Black vertical stripes were evenly distributed on the walls of one chamber, with 2 cm between each stripe; the other chamber's walls were evenly distributed with black horizontal stripes, also with 2 cm between each stripe. A small door measuring 5 cm × 5 cm was located in the lower portion of the access panel, and the partition was completely closed. Mice were placed in the CPP enclosure using the access panel as a partition for two days of familiarization. Pre-CPP was then conducted. Mice were allowed to freely move between the two sides for 15 minutes, and the time they spent on each side was recorded to determine whether they had a natural preference for the horizontal and vertical stripes. After the pre-CPP results determined the drug-compensated side, Oxy and Sal were administered starting the next day. The model group received Oxy in the morning and Sal in the afternoon, with a 6-hour interval; the control group received Sal in both the morning and afternoon. Each group received the drug in the morning on the drug-treated side and in the afternoon on the non-drug-treated side for four consecutive days. On the sixth day, a post-CPP test was performed, allowing mice to move freely on both sides for 15 minutes while recording the time they spent on the drug-treated side. Hippocampi were harvested one hour after behavioral testing.

[0037] (5) Immunofluorescence staining (IF). Tissue perfusion and sampling: After anesthetizing the mouse with 2% isoflurane, cut the chest skin, lift the xiphoid process with hemostats, and cut the ribs with scissors to expose the heart. Pass the syringe needle through the left ventricle into the ascending aorta, fix the needle with hemostats, and puncture the right atrial appendage. Quickly inject 20 mL of pre-cooled Sal. After the liver turns light brown, inject 20 mL of pre-cooled 4% paraformaldehyde solution. When the mouse becomes pale and its tail becomes stiff, the perfusion is successful. Then, take the whole mouse brain and soak it in 4% paraformaldehyde.

[0038] Paraffin section preparation: After fixation for 48 hours in 4% paraformaldehyde, the tissue was placed in a gradient of alcohol (50%, 75%, 95%, 100%) for 2 hours each step, followed by immersion in 100% xylene for 1.5 hours. The treated tissue was placed in a 1:1 mixture of paraffin and xylene and oven-dried at 40°C overnight. The temperature of the incubator was raised to 60°C, and the tissue-soaking solution was replaced with liquid paraffin three times, each for 2 hours. The paraffin block box was then placed on a hot plate at 65°C. The tissue was then poured into the box and arranged. The box was then filled with paraffin and gently placed in a basin of cold water, with the bottom touching the cold water. Once solidified, the block was removed and air-dried for sectioning. The paraffin block was trimmed, ensuring that the material was surrounded by paraffin wax. A small amount of paraffin debris was melted with a heated scalpel. The viscosity of the melted paraffin allowed the block to be firmly adhered to the wood board. Paraffin sections of tissue were then cut to a thickness of 2.5 µm. Place the cut tissue slices at room temperature for staining.

[0039] Immunofluorescence staining: Paraffin sections were dewaxed and washed three times with PBS (5 minutes each). Sections were retrievaled with sodium citrate antigen retrieval solution for 8.5 minutes. After cooling to room temperature, sections were transferred to PBS and rinsed three times (5 minutes each). Tissues were circled with an immunohistochemical pen and blocked with 5% goat serum in a humidified chamber for 60 minutes at room temperature. Primary antibodies were diluted in PBS to the following ratios: METTL3 at 1:200, NeuN at 1:500, GFAP at 1:200, and Iba-1 at 1:200. After blocking, the goat serum was removed and METTL3 was added for double labeling with NeuN, GFAP, and Iba-1, respectively. Approximately 50 µL of each solution was added to each circle and incubated overnight at 4°C in a humidified chamber. The following day, sections were removed and rewarmed at room temperature for 30 minutes. The primary antibody was aspirated and the sections were rinsed three times (5 minutes each) with PBS. Add the corresponding fluorescent secondary antibody (1:300 dilution) to each tissue slice, approximately 50 µL per circle. Incubate the slices in a light-proof, humidified chamber at room temperature for 2 hours. Aspirate the secondary antibody and rinse three times with PBS, 5 minutes each. Allow the slices to dry and mount them with anti-fluorescence decay mounting medium to prevent air bubbles. Observe and capture images under a fluorescence microscope.

[0040] 3. Experimental results. Figure 1 As shown, glutamate release in the hippocampus of oxycodone-addicted mice was increased ( Figure 1 AD), RNA methyltransferases METTL3 and METTL14 were upregulated ( Figure 1 E); METTL3 is mediated by glutamate release ( Figure 1 F); Oxycodone addiction is dose-dependent ( Figure 1 G); the upregulation of METTL3 has a dose-effect and time-effect relationship with oxycodone dose ( Figure 1 H); METTL3 is localized in neurons of the mouse hippocampus, but not in microglia and astrocytes ( Figure 1 I). The above results suggest that oxycodone causes increased expression of METTL3 in mouse hippocampal neurons.

[0041] Example 2: Oxycodone promotes the binding of METTL3 and METTL14 in mouse hippocampal neurons.

[0042] 1. Experimental Materials .

[0043] 2. Experimental methods

[0044] (1) Western Blot: Prepare and conduct relevant experiments according to the method described in Example 1 (3).

[0045] (2) Immunofluorescence staining (IF): Prepare / perform the relevant experiments according to the method described in Example 1 (5).

[0046] (3) Co-immunoprecipitation (Co-IP). Place the mouse hippocampal tissue sample in a pre-cooled 2 mL EP tube, add lysis buffer, lyse on ice for 30 min, centrifuge at 12,000 rpm for 15 min at 4°C, and collect the supernatant. Determine the protein concentration using a BCA kit. Add 50 µL Protein A / G magnetic beads to the lysate and incubate at 4°C for 1 h. After incubation, centrifuge at 1000 g for 1 min at 4°C and collect the supernatant. Take 500 µL of the supernatant and add METTL3 (1:50) and METTL14 (1:50) primary antibodies, and shake slowly at 4°C overnight. Add 100 µL Protein A / G magnetic beads to capture the antigen-antibody complex, and shake slowly at 4°C overnight. Centrifuge at 14,000 rpm for 5 s to collect the magnetic bead-antigen-antibody complex, remove the supernatant, and wash three times with 800 µL RIPA buffer. Resuspend the magnetic bead-antigen-antibody complex in 60 µL of 2× loading buffer and mix gently. Heat the loaded sample in a water bath (100°C) for 5 minutes to collect the remaining magnetic beads. Heat and denature again for 5 minutes before electrophoresis. Follow the subsequent Western blotting procedure (1). The dilution ratio of METTL3 for Western blotting is 1:1000; the dilution ratio of METTL14 is 1:1000.

[0047] 3. Experimental results. Figure 2 As shown, the upregulation of METTL14 has a dose-response and time-response relationship with oxycodone dose ( Figure 2 A); Increased binding of METTL3 and METTL14 in the hippocampus of oxycodone-addicted mice ( Figure 2 B); Increased expression of METTL14 in hippocampal neurons of oxycodone-addicted mice ( Figure 2 C), increased colocalization of METTL3 and METTL14 ( Figure 2 D) The above results confirm that oxycodone causes the binding of METTL3 and METTL14 in mouse hippocampal neurons.

[0048] Example 3: Oxycodone induces m6A methylation of lncRNA Meg3 through METTL3.

[0049] 1. Experimental Materials .

[0050] 2. Experimental methods

[0051] (1) RNA-seq detection. The hippocampus tissue of mice was taken, and total RNA was extracted according to the "Tissue Total RNA Extraction" section in Example 1 (2), and the RNA concentration was determined. RNA-seq library preparation and sample sequencing were performed using BGI GENE (Shenzhen, China). The sequencing library was constructed using the MGISEQ-2000RS high-throughput sequencing kit. The QC-qualified library was subjected to SE50 sequencing using MGI-2000. The library was sequenced using high-intensity DNA nanochip technology and combined probe anchor synthesis technology (cPAS). The reference genome version was GCF_000001635.26_GRCm38.p6 of NCBI, and the sequencing data was filtered using SOAPnuke (v1.5.6). Analysis and data mining of Dr Tom's multi-omics data were performed.

[0052] (2) RNA methylation immunoprecipitation-qPCR (MeRIP-qPCR). Sample analysis was performed using the MeRIP kit according to the manufacturer's instructions. Mouse hippocampus was obtained, RNA was extracted by the Trizol method, and its concentration purity, A260 / 280 and A260 / 230 ratios were tested. Then, affinity magnetic beads were mixed for RNA immunocapture and cleavage. 1-2µL (200-400ng) of RNA in the input group was added to 20µL of ICB, and then 1.5µL of NDE and 1µL of CEM were added and incubated at room temperature. After capture, RNA was enriched by mixing proteinase K and PDB in a ratio of 1:10, and then eluting the suspended beads. After removing genomic DNA from the enriched RNA, it was reverse transcribed into cDNA, and the reverse transcribed RNA was subjected to subsequent RT-qPCR analysis. The experimental process was the same as the method described in Example 1 (2) to prepare / conduct related experiments. The primer sequences of the lncRNA MEG3 modification site are shown in Table 3.

[0053] Table 3: Primer sequences (5'-3') .

[0054] (3) RNA immunoprecipitation-qPCR (RIP-qPCR). Use the RIP kit for detection and analysis. Take fresh mouse hippocampus, add 500µL Lysis Buffer, 1µL 0.5M DTT, 1.25µL 40U / µL RNase Inhibitor and 1µL 100mM PMSF, and homogenize at 4℃. Centrifuge the homogenate at 4℃, 14000g, for 10min, and take the supernatant as the prepared tissue sample. Take 30µL of sample as input for subsequent detection. Add 30µL Protein A / G Agarose suspension to the enzyme-free tube, then add 1mL NT2 Wash Buffer and gently pipette to resuspend Protein A / G Agarose. Centrifuge at 4℃, 1000g for 1min, and remove the supernatant. Add 100µL of NT2 Wash Buffer to the washed Protein A / G Agarose solution to resuspend it appropriately. Then add METTL3 antibody (1:50) and negative control IgG (1:50) and incubate on a shaker at 4°C for 4 hours. After pre-binding the Protein A / G Agarose to the antibody, centrifuge at 1000g for 1 minute at 4°C. Discard the supernatant of the pre-bound Protein A / G Agarose. Add 270µL of tissue sample to the antibody-pre-bound Protein A / G Agarose solution, mix thoroughly by inversion, and incubate on a shaker at 4°C for 4 hours. After incubation, centrifuge at 1000g for 1 minute at 4°C and discard the supernatant. Add 1mL of NT2 Wash Buffer, resuspend the Protein A / G Agarose solution by pipetting, centrifuge at 1000g for 1 minute at 4°C, wash four times, and discard the supernatant. Add 100µL of Elution Buffer, resuspend evenly, and incubate at 55°C for 30 minutes. Mix equal volumes of the Elution Buffer and Protein A / G Agarose mixture with the binding buffer provided in the kit and perform column purification of RNA. The extracted RNA was reverse transcribed into cDNA using a reverse transcription instrument. Following the instructions for the RNA reverse transcription kit, RNase-free ddH2O, 4× gDNA wiper mix, 5× HiScript III qRT SuperMix, and 4× No RT Control Mix were added in order to remove genomic DNA and perform reverse transcription to generate the cDNA product.

[0055] RT-qPCR: The expression levels of relevant indicators were detected according to the instructions of the cDNA fluorescence quantitative PCR kit, and GAPDH was used as the internal reference for normalization. In this study, 2 -ΔΔCtThe gene expression level was calculated by the method. Primers #5 and #8 were selected for detection. The experimental process was the same as that described in Example 1 (2) to prepare / perform the relevant experiment. Finally, the relevant bands were detected by 1% agarose gel electrophoresis.

[0056] (4) Agarose gel electrophoresis retardation experiment. Dissolve 0.4 g of low-melting-point agarose powder in 40 mL of 1×TAE buffer and heat the mixture in a microwave for 2 min to ensure that the agarose is completely dissolved. After the solution becomes uniform and clear, let it cool briefly to about 40°C. Then, add 4 µL of GelRed nucleic acid dye to the solution. Mix the dye thoroughly into the agarose-buffer mixture to ensure uniform distribution. After the gel solidifies, use a pipette to carefully add the sample to the gel loading well. Connect the gel apparatus to a power supply, set the voltage to 120 V, and start electrophoresis. Run the electrophoresis for 15 min. After the electrophoresis is completed, carefully remove the gel from the gel tray and place it on the gel imaging system. Use the imaging system software to visualize the siRNA bands.

[0057] (5) Fluorescence real-time quantitative polymerase chain reaction: Prepare / perform relevant experiments according to the method described in Example 1 (2).

[0058] 3. Experimental results. Figure 3 As shown in Figure 2, next-generation sequencing revealed that oxycodone led to upregulation of lncRNA Meg3 expression ( Figure 3 AC); lncRNA Meg3 expression was increased by RT-qPCR ( Figure 3 D); SRAMP database predicts the possible m6A methylation sites of lncRNA Meg3 ( Figure 3 E); 8 pairs of primers were designed and m6A enrichment increase was detected at sites #5 and #8 ( Figure 3 F); RIP experiments confirmed that oxycodone promoted methylation of sites #5 and #8 on lncRNAMeg3 through METTL3 ( Figure 3 G). The above results confirmed that oxycodone promoted m6A methylation of lncRNA Meg3 through METTL3, thereby promoting its own expression.

[0059] Example 4: siRNA Meg3 lipid nanoparticles inhibit oxycodone addiction.

[0060] 1. Experimental Materials .

[0061] 2. Experimental methods

[0062] (1) Western Blot: Prepare and conduct relevant experiments according to the method described in Example 1 (3).

[0063] (2) Conditioned Place Preference: Prepare and conduct relevant experiments according to the method described in Example 1 (4).

[0064] (3) Preparation of lipid nanoparticles (LNP). According to the molar ratio of DLin-MC3-DMA / DSPC / cholesterol / PEG2000-DMG of 45:10:43.5:1.5, the corresponding amount of excipients was dissolved in 0.25 mL of anhydrous ethanol to form an oil phase. At the same time, according to the total lipid:siRNA ratio of 30:1 (w / w / ) (N / P 6), the corresponding amount of siRNA was dissolved in 0.75 mL of citric acid buffer to prepare an aqueous phase. The organic phase was quickly added to the aqueous phase (volume ratio 1:3) and vortexed for 10 minutes. After sample collection, the ethanol concentration was immediately diluted to less than 1% with 30 volumes of PBS. The ethanol was removed by dialyzing with pH 7.4 PBS (10 mM) at 4°C for 1 hour. The LNP nanovaccine was stored at 4°C.

[0065] (4) Synthesis of siRNA Meg3: Completed by Sangon Biotech (Shanghai) Co., Ltd. The positive chain sequence is 5'-CCAGGACCCUCCAACUGUA / dT / / dT / -3', The antisense strand sequence is 5'-UACAGUUGGAGGGUCCUGG / dT / / dT / -3'.

[0066] (5) Characterization of physicochemical properties. The prepared lipid nanoparticles (LNPs) were diluted with deionized water at a dilution ratio of 1:300. Subsequently, the Z-average particle size and Zeta potential of these diluted LNP samples were measured using a Zetasizer Nano ZS instrument (Malvern Panalytical, UK). They were further diluted 100 times with deionized water. A small amount of the diluted sample was dropped onto a 300-mesh copper grid, and after the sample evaporated naturally, it was negatively stained with a 2% sodium phosphotungstate solution. To observe and analyze the morphology of LNPs, a Hitachi H-7650 transmission electron microscope (Hitachi, Japan) was used to obtain high-resolution images of LNPs. This transmission electron microscopy analysis provides detailed information on the size, shape, and distribution of LNPs in the optimal formulation.

[0067] (6) Agarose gel electrophoresis retardation experiment: Prepare / perform the relevant experiment according to the method described in Example 3 (4).

[0068] (7) Nasal administration. After anesthetizing the mouse with 2% isoflurane, place the mouse in a supine position on the experimental table with the head tilted up 30 degrees. Gently pinch the nose skin with the thumb and index finger to keep the nostril open. Use a microsyringe with a maximum range of 100µL to draw 50µL of siRNA Meg3 lipid nanoparticle solution. After the syringe needle enters the mouse nostril 0.5cm, slowly inject the drug solution. The injection time is 5min / unilateral nostril, and the administration volume is 25µL / unilateral nostril. After the administration is completed, keep the mouse in a supine position for 10min to prevent the drug solution from leaking out.

[0069] (8) Immunofluorescence staining (IF): 0.5 h after nasal administration of fluorescently labeled siRNA Meg3 lipid nanoparticles, the relevant experiments were prepared / conducted according to the "tissue perfusion and sampling" and "paraffin section preparation" methods described in Example 1 (5).

[0070] Immunofluorescence staining: Paraffin sections were deparaffinized and washed with PBS three times for 5 minutes each. Sections were retrievaled with sodium citrate antigen retrieval solution for 8.5 minutes. After cooling to room temperature, sections were transferred to PBS and rinsed three times for 5 minutes each. Tissues were circled with an immunohistochemical pen and blocked with 5% goat serum in a humidified chamber for 60 minutes at room temperature. The primary antibody (NeuN) was diluted 1:500 in PBS. After blocking, the goat serum was removed and 50 µL of NeuN primary antibody dilution was added. The sections were incubated overnight at 4°C in a humidified chamber. The next day, sections were removed and rewarmed at room temperature for 30 minutes. The primary antibody was aspirated and the sections were rinsed three times for 5 minutes each with PBS. The corresponding fluorescent secondary antibody (diluted 1:300) was added to each circle, approximately 50 µL per circle. The sections were incubated in a light-proof chamber for 2 hours at room temperature. The secondary antibody was aspirated and the sections were rinsed three times for 5 minutes each with PBS. After the tissue sections are dried, they are mounted with anti-fluorescence attenuation mounting medium to avoid air bubbles. Observe and collect images under a fluorescence microscope.

[0071] 3. Experimental results. Figure 4 As shown, siRNA Meg3 reduces the upregulation of c-Fos caused by oxycodone through the ERK / MEK signaling pathway ( Figure 4 AD), thereby inhibiting oxycodone addiction behavior ( Figure 4 EF); the encapsulation efficiency of siRNA Meg3 lipid nanoparticles is about 95%, and the appearance is a spherical structure ( Figure 4 G), After the lipid nanoparticles that successfully encapsulated siRNA Meg3 were demulsified, a large amount of siRNA was detected ( Figure 4 H), Zeta potential and particle size ( Figure 4 IJ), siRNA Meg3 lipid nanoparticles can inhibit oxycodone addiction, have no effect on sugar water preference, and do not produce place preference after self-administration ( Figure 4 K), siRNA Meg3 lipid nanoparticles colocalize with hippocampal neurons ( Figure 4 L). The above results confirm that siRNA Meg3 lipid nanoparticles can specifically inhibit oxycodone addiction and are not addictive themselves.

[0072] In summary, the present invention screened lncRNA Meg3 by performing second-generation sequencing on the hippocampal tissue of oxycodone-addicted mice, designed and synthesized siRNA Meg3 targeting lncRNA Meg3, and then prepared lipid nanoparticles encapsulating siRNA Meg3. Subsequent nasal administration inhibited oxycodone addiction. siRNA Meg3 lipid nanoparticles administered alone did not induce addictive behavior in mice, suggesting that they are not addictive. siRNA Meg3 lipid nanoparticles also had no effect on the mice's sugar preference, demonstrating their specificity in inhibiting oxycodone addiction.

[0073] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Application of inhibitors of lncRNA Meg3 expression levels in the preparation of drugs for the treatment of oxycodone addiction.

2. The use according to claim 1, characterized in that The lncRNA Meg3 expression inhibitor includes an siRNA specifically targeting lncRNA Meg3, and the nucleotide sequence of the siRNA is shown in any one of SEQ ID NO.1 to SEQ ID NO.

2.

3. A pharmaceutical composition for combating oxycodone addiction, comprising an active substance that targets and inhibits the expression of lncRNA Meg3 and a pharmaceutical carrier.

4. The pharmaceutical composition according to claim 2, characterized in that The active substance that inhibits the expression of lncRNA Meg3 is siRNA.

5. The pharmaceutical composition according to claim 3, characterized in that The siRNA sequences are shown in SEQ ID NO: 1 (5'-CCAGGACCCUCCAACUGUA-3') and NO: 2 (5'-UACAGUUGGAGGGUCCUGG-3').

6. An anti-addiction drug composition, characterized in that: Comprising the drug according to any one of claims 1 to 2 and a pharmaceutical carrier.

7. The pharmaceutical composition according to claim 6, characterized in that The carrier is a lipid nanoparticle (LNP), and the LNP contains DLin-MC3-DMA, DSPC, cholesterol, and PEG2000-DMG, and the molar ratio thereof is (45±2): (10±1): (43.5±2): (1.5±0.2).

8. The composition according to claim 7, characterized in that: The mass ratio of total lipid to siRNA in the LNP is (30±5):

1.

9. The composition according to claim 7, characterized in that: The LNP has a zeta potential of -10 mV to -20 mV and an average particle size of 80 nm to 100 nm.

10. Use of a lncRNA Meg3 expression detection reagent in the preparation of an oxycodone addiction diagnostic kit, characterized in that: The reagents include a primer pair or a probe that specifically recognizes lncRNA Meg3.

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