KR102208012B1 - Markers for cocaine addiction - Google Patents

Abstract

The present invention relates to biomarkers whose expression is significantly changed upon acute or chronic administration of cocaine, the biomarkers of the present invention ARHGEF7, PTOV1, GTF2I, ADAM23, CDH2, GRIA1 (GRIA1X3, GRIA1X4), GRIA2 (GRIA2X6 , GRIA2X7), HDAC5, HDAC10, NCAM1, NPY, SNCA, CCNG2, CDC7, CDH8, DLG3, DOCK3, NEUROD6, NEUROG2, SPEN, KDM2B (KDM2BX10, KDM2BX13), KIFAP3, RBM10, MCM311, WNTBM14, TP5311, WNTBD14 , DRD2, DRD3, GNAL, GNG7, LOC101865389, LOC101926461, PPP2R2B, ACHE, ADCY5, AKT3, KCNJ2, LOC101865389, LOC101926461, PLCB1, SLC18A3, SLC5A7, CAMK2A, EGLC1866, B179, LOCL101, BP, ECL101, and BP, LOC101865389 And TNFRSF1A significantly increases or decreases the expression levels of mRNA and protein during acute or chronic administration of cocaine, so cocaine addiction and dependence due to acute or chronic exposure of cocaine can be confirmed through their expression patterns.

Description

Biomarkers specific to cocaine addiction {MARKERS FOR COCAINE ADDICTION}

The present invention relates to biomarkers specific for cocaine addiction, and specifically, to biomarkers whose expression is significantly changed upon acute or chronic administration of cocaine.

Substance abuse refers to the intentional use of drugs for other purposes (non-medical purposes), and substance abuse is distinguished from substance abuse according to the intention of the user. Many drugs, such as opium, hemp, cocaine, and methamphetamine, are being abused for pleasure, anxiety relief, and relaxation. Recently, not only these types, but also various substances such as sedatives, stimulants, inhalants, and hormones are abused. The repeated use of these substances not only causes serious diseases that lead to long-term changes in the brain, but also harms both individuals and society, and such substance abuse is not limited to specific social and economic groups, but widely affects society as a whole. The use of these drugs is regulated nationally because drugs of abuse induce drug-seeking behavior, which is primarily a mental dependence due to strong rewards related to drug intake. (psychological dependence) is formed, and continued abuse leads to tolerance and chronic dependence. When drug use is stopped, physical dependence appears, and this phenomenon occurs due to the physiological function to keep the body in normal condition, and as a result, withdrawal, abstinence syndrome, which is difficult to tolerate physiologically, appears. It depends on the drug.

Psychostimulants, such as cocaine or amphetamine, increase alertness and increase physical capacity in humans. These substances initially increase dopamine permeation, but long-term drug use leads to dysregulation and mental anxiety of the brain reward system, resulting in decreased dopamine activity. Chronic cocaine abuse causes major health problems. Cocaine users may experience acute cardiovascular or cerebrovascular emergencies that can lead to sudden death, such as a heart attack or stroke through vasoconstriction, dilated pupils, and increased body temperature, heart rate and blood pressure, and hyperstimulation, tachycardia. (tachycardia), hypertension, mydriasis, muscle twitching, sleeplessness, extreme nervousness, hallucinations, paranoia, aggressive behavior, and depression The symptoms occur. Other complications associated with cocaine use include heart rhythm disorders, chest pain and respiratory failure, seizures, headaches, and gastrointestinal complications such as abdominal pain and nausea. Also, because cocaine tends to reduce appetite, many long-term users can become malnourished. Furthermore, it is well known that simultaneous abuse of nicotine, cocaine and alcohol is common. The combination of cocaine and alcohol has been shown to exert more cardiovascular toxicity in humans than either drug alone.

Historically, chemical-dependent treatment has primarily involved attempts to persuade patients to voluntarily stop using the substance (behavioral therapy). However, cocaine, morphine, amphetamine, nicotine and alcohol, and other types of dopamine-producing agents are highly addictive substances, and dependence on these drugs can be more difficult to break, and than dependence on most other addictive substances. The damage is significantly greater. In particular, alcohol, cocaine and heroin dependence are usually chronic recurrent disorders. Mental dependence almost always precedes physical dependence, but does not necessarily develop into physical dependence. Tolerance refers to a decrease in response to drug effects requiring administration of higher doses to obtain the same effect. Is metabolic tolerance in which the metabolism of the drug increases after chronic use of the drug, behavioral tolerance (behavioral sensitization) that compensates for the effect of the drug, and compensatory changes in the action of drug receptors, enzymes, or biological membranes. There is a functional tolerance.

For the study of drug addiction and dependence, there have been studies on the mechanism of expression of the dependence of central nervous system stimulants. Desipramine, amantadine and bromocriptine reduce cocaine withdrawal symptoms, and disulfiram It has recently been shown that it can be used in the treatment of cocaine dependence (see, e.g., Bonet et al., Journal of Substance Abuse Treatment, 26 (2004), 225-232). In addition, recent research to discover molecular biological markers or biomarkers of the central nervous system due to drug abuse by applying microarray technology is being conducted.

In the present invention, it is an object of the present invention to be used to diagnose acute or chronic cocaine addiction (or exposure) by discovering genes and protein markers that can serve as major evidence for determining drug addiction by showing changes in expression when cocaine is used.

In order to achieve the above object, the present invention provides a biomarker for confirming cocaine addiction and dependence.

In addition, the present invention provides a biomarker for distinguishing acute and chronic cocaine addiction and dependence.

In addition, the present invention provides a biomarker protein for confirming cocaine addiction and dependence.

In addition, the present invention provides a composition for determining cocaine addiction and dependence.

In addition, the present invention provides a kit for checking cocaine addiction and dependence.

In addition, the present invention provides a method of providing information necessary for cocaine exposure diagnosis.

According to the present invention, ARHGEF7, PTOV1, GTF2I, ADAM23, CDH2, GRIA1 (GRIA1X3, GRIA1X4), GRIA2 (GRIA2X6, GRIA2X7), HDAC5, HDAC10, NCAM1, NPY, SNCA, CCNG2, CDC7, CDH8, DLG3, DLG3 , NEUROG2, SPEN, KDM2B (KDM2BX10, KDM2BX13), KIFAP3, RBM10, RBM14, TP53I11, WNT10B, MCM3AP, DRD1, DRD2, DRD3, GNAL, GNG7, LOC101865389, LOC5J2926461, ACHEC3101, ADC5389, ACHEC3, PPP289 LOC101926461, PLCB1, SLC18A3, SLC5A7, CAMK2A, EGLN2, ENO2, LOC101866179, LOC101866246, BAD, BCL2L1, CASP9, LOC101925175, and TNFRSF1A significantly up or down the expression of mRNA and protein when cocaine is administered acute or chronic. It can be usefully used to confirm cocaine addiction and dependence by acute or chronic exposure of cocaine.

1 is a schematic diagram of the overall experimental protocol of the present invention.
2 is a diagram showing a process of discovering genes that change expression according to acute/chronic cocaine poisoning in the hippocampus through Venn diagram analysis.
Figure 3 shows genes ARHGEF7, PTOV1, GTF2I, ADAM23, CDH2, GRIA1 (GRIA1X3, GRIA1X4), GRIA2 (GRIA2X6, GRIA2X7), HDAC5, HDAC10, NCAM1, NPY, SNCA genes whose expression was changed in the hippocampus according to acute/chronic cocaine poisoning. , CCNG2, CDC7, CDH8, DLG3, DOCK3, NEUROD6, NEUROG2, SPEN, KDM2B (KDM2BX10, KDM2BX13), KIFAP3, RBM10, RBM14, TP53I11, MCM3AP, and WNT10B expression levels:
Con: cocaine untreated group;
CA: acute cocaine administration group;
CC: chronic cocaine administration group;
Acute Cocaine: acute cocaine administration group; And
Chronic Cocaine: Chronic cocaine administration group.
4 is a diagram illustrating a process of discovering genes that change expression according to acute/chronic cocaine poisoning in striatum through Venn diagram analysis.
5 shows genes DRD1, DRD2, DRD3, GNAL, GNG7, LOC101865389, LOC101926461, PPP2R2B, ACHE, ADCY5, AKT3, KCNJ2, LOC101865389, LOC101926461, PLCB1, and SLC5A7926461, PLCB1, and SLC5. , CAMK2A, EGLN2, ENO2, LOC101866179, LOC101866246, BAD, BCL2L1, CASP9, LOC101925175 and TNFRSF1A is a diagram showing the expression levels of:
Con: cocaine untreated group;
CA: acute cocaine administration group; And
CC: chronic cocaine administration group.
6 shows genes whose expression in striatum was changed according to acute/chronic cocaine poisoning: DRD1, DRD2, DRD3, GNAL, GNG7, LOC101865389, LOC101926461, PPP2R2B, ACHE, ADCY5, AKT3, KCNJ2, LOC101865389, LOC101926461, PLCB1, SLC5A7926461, PLCB1, SLC5. , CAMK2A, EGLN2, ENO2, LOC101866179, LOC101866246, BAD, BCL2L1, CASP9, LOC101925175, and TNFRSF1A Pearson correlation analysis results.
7 is a diagram illustrating changes in mRNA expression and protein expression levels of genes CAMK2A and CASP9 whose expression was changed in striatum according to acute/chronic cocaine poisoning:
a: mRNA expression levels of CAMK2A and CASP9;
b: protein expression levels of phosphorylated CAMK2A, CAMK2A and truncated CASP9;
c: quantification of pCAMK2A/CAMK2A;
d: quantification of truncated CASP9;
Con: cocaine untreated group;
CA: acute cocaine administration group; And
CC: chronic cocaine administration group.
8 is a diagram illustrating the regulation of CAMK2A expression in human brain tissue.
9 is a diagram illustrating a functional protein network for CAMK2A.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention. However, the following embodiments are presented as examples of the present invention, whereby the present invention is not limited, and the present invention can be variously modified and applied within the scope of equality interpreted from the description of the claims to be described later. .

Unless otherwise indicated, nucleic acids are written in a 5'→3' orientation from left to right. Numerical ranges recited within the specification include the numbers defining the range, and include each integer or any non-integer fraction within the defined range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in practice to test the present invention, preferred materials and methods are described herein.

In the present invention, the term "subject" or "patient" refers to any single individual in need of treatment, including humans, apes, monkeys, cows, dogs, guinea pigs, rabbits, chickens, insects, and the like. In addition, any subject who participated in a clinical study trial showing no clinical manifestation of any disease, or a subject who participated in an epidemiological study or a subject used as a control group is included. In one embodiment of the present invention, monkeys and humans were targeted.

In the present invention, the term "tissue" or "sample (sample)" refers to a tissue or cell obtained from a subject or patient. The source of the tissue or cell sample may be a fresh, frozen and/or preserved organ or tissue sample or solid tissue from a biopsy or aspirate; Blood or any blood component; It may be a cell at any point in the subject's pregnancy or development. The tissue sample can also be a primary or cultured cell or cell line.

In the present invention, the term "cocaine addiction" refers to the abuse of cocaine, which is an addictive drug, resulting in dependence on the drug, and tolerance occurs when the amount is increased to produce an effect, and if not used It is a phenomenon of withdrawal symptoms that cause abnormalities that are difficult to endure in the whole body, and includes "chronic addiction" exposed to cocaine for a long time and "acute addiction" exposed for a short period of time.

All technical terms used in the present invention, unless otherwise defined, are used in the same sense as those of ordinary skill in the art generally understand in the related field of the present invention. In addition, although preferred methods or samples are described in the present specification, those similar or equivalent are included in the scope of the present invention. The contents of all publications referred to herein by reference are incorporated into the present invention.

In one aspect, the present invention represents a change in expression upon exposure to cocaine ARHGEF7, PTOV1, GTF2I, ADAM23, CDH2, GRIA1 (GRIA1X3, GRIA1X4), GRIA2 (GRIA2X6, GRIA2X7), HDAC5, HDAC10, NCAM1, NPY, SNCA, CCNG2 CDC7, CDH8, DLG3, DOCK3, NEUROD6, NEUROG2, SPEN, KDM2B (KDM2BX10, KDM2BX13), KIFAP3, RBM10, RBM14, TP53I11, WNT10B, MCM3AP, DRD1, DRD2, DRD2101, GNAL, and PCHE2 , ADCY5, AKT3, KCNJ2, LOC101865389, LOC101926461, PLCB1, SLC18A3, SLC5A7, CAMK2A, EGLN2, ENO2, LOC101866179, LOC101866246, BAD, BCL2L1, CASP9, LOC101925175 and TNFRSF1A encoding one or more genes selected from the group or genes selected from the group. It relates to a biomarker for confirming cocaine addiction and dependence, including a protein, and the genes are defined as Accession no. shown in Tables 1 and 2. In one embodiment, in order to identify genes whose expression levels change according to acute/chronic cocaine administration, as a result of performing an analysis as shown in FIG. 1, it was confirmed that the expression levels of the gene groups were significantly increased or down-regulated. .

In one embodiment, upon acute cocaine exposure PTOV1, HDAC5, HDAC10, NPY, SNCA, KDM2BX10, KDM2BX13, KIFAP3, WNT10B, DRD1, DRD2, DRD3, GNAL, PPP2R2B, GNG7, ACHE, SLC5A7, CASP9, LOC1015, KCN175 and KCN175 Expression of one or more genes selected from the group consisting of is upregulated, MCM3AP, GTF2I, ADAM23, CDH2, GRIA1X3, GRIA1X4, GRIA2X6, GRIA2X7, CCNG2, CDC7, CDH8, DLG3, NEUROD6, NEUROG2, SPEN, CAMK2A, EGLN2 And expression of one or more genes selected from the group consisting of BCL2L1 may be down-regulated.

In one embodiment, upon chronic cocaine exposure, expression of at least one gene selected from the group consisting of ARHGEF7, DRD1, DRD2, DRD3, GNAL, PPP2R2B, SLC18A3, ACHE, SLC5A7, ADCY5, KCNJ2, TNFRSF1A, BCL2L1, and BAD is upregulated and , MCM3AP, PTOV1, GTF2I, AKT3, CAMK2A and LOC101866246 expression of one or more genes selected from the group consisting of may be down-regulated.

In one aspect, the present invention relates to a biomarker for acute and chronic cocaine addiction and dependence discrimination, including the ARHGEF7, PTOV1 and GTF2I genes or the protein encoded by the gene, and the expression control pattern of the ARHGEF7, PTOV1 and GTF2I genes Can distinguish between acute and chronic cocaine addiction and dependence. In one embodiment, the expression of ARHGEF7 was increased in the chronic cocaine addicted group, the expression of PTOV1 increased in the acute cocaine addicted group and decreased in the chronic cocaine addicted group, and the expression of GTF2I decreased in the acute and chronic cocaine addicted group. However, it was found that the decrease in expression in the acute cocaine addicted group was remarkable compared to the chronic cocaine addicted group (see the upper graph of Fig. 3a).

In one aspect, the present invention relates to a biomarker protein for confirming cocaine addiction and dependence, including CAMK2A protein, GRIN1 protein, and CASP9 protein whose protein expression level changes upon exposure to cocaine. In one embodiment, it may further include a DLG1 marker protein. In one embodiment, the expression of CAMK2A gene decreased when exposed to cocaine, and the expression of CASP9 increased, but at the protein level, phosphorylation of CAMK2A protein decreased when exposed to acute and chronic cocaine, and the expression level of CAMK2A protein was appreciated during chronic cocaine exposure. , While the cleaved CASP9 increased upon acute cocaine exposure, the cleaved CASP9 was not observed upon chronic cocaine exposure (see FIG. 7).

In one aspect, the present invention represents a change in expression upon exposure to cocaine ARHGEF7, PTOV1, GTF2I, ADAM23, CDH2, GRIA1 (GRIA1X3, GRIA1X4), GRIA2 (GRIA2X6, GRIA2X7), HDAC5, HDAC10, NCAM1, NPY, SNCA, CCNG2 CDC7, CDH8, DLG3, DOCK3, NEUROD6, NEUROG2, SPEN, KDM2B (KDM2BX10, KDM2BX13), KIFAP3, RBM10, RBM14, TP53I11, WNT10B, MCM3AP, DRD1, DRD2, DRD2101, GNAL, and PCHE2 , ADCY5, AKT3, KCNJ2, LOC101865389, LOC101926461, PLCB1, SLC18A3, SLC5A7, CAMK2A, EGLN2, ENO2, LOC101866179, LOC101866246, BAD, BCL2L1, CASP9, LOC101925175 and TNFRSF1A reagent selected from the group of one or more biomarkers for detection It relates to a composition for determining cocaine addiction and dependence, including.

In one embodiment, the detection reagent may be a reagent capable of detecting a marker at the protein or nucleic acid level.

In one embodiment, the reagent for detecting the nucleic acid level of the marker is a primer pair or probe that specifically recognizes the nucleic acid sequence of the marker, a nucleic acid sequence complementary to the nucleic acid sequence, the nucleic acid sequence and a fragment of the complementary sequence, or Primer pairs and probes may be included, which are polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive polymerase chain reaction (Competitive RT-PCR), and Nuclease protection analysis. (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, Northern blot, can be used for gene expression analysis methods including DNA chips, but is not limited thereto.

In one embodiment, the primer pair for the genetic markers of the present invention is a primer pair of SEQ ID NO: 1 and 2, a primer pair of SEQ ID NO: 3 and 4, a primer pair of SEQ ID NO: 5 and 6, a primer of SEQ ID NO: 7 and 8 A pair, a primer pair of SEQ ID NOs: 9 and 10, a primer pair of SEQ ID NOs: 11 and 12, a primer pair of SEQ ID NOs: 13 and 14, a primer pair of SEQ ID NOs: 15 and 16, a primer pair of SEQ ID NOs: 17 and 18, SEQ ID NO: 19 And a primer pair of 20, a primer pair of SEQ ID NOs: 21 and 22, a primer pair of SEQ ID NOs: 23 and 24, a primer pair of SEQ ID NOs: 25 and 26, a primer pair of SEQ ID NOs: 27 and 28, a primer pair of SEQ ID NOs: 29 and 30. , A primer pair of SEQ ID NOs: 31 and 32, a primer pair of SEQ ID NOs: 33 and 34, a primer pair of SEQ ID NOs: 35 and 36, a primer pair of SEQ ID NOs: 37 and 38, a primer pair of SEQ ID NOs: 39 and 40, and SEQ ID NO: 41 and A primer pair of 42, a primer pair of SEQ ID NOs: 43 and 44, a primer pair of SEQ ID NOs: 45 and 46, a primer pair of SEQ ID NOs: 47 and 48, a primer pair of SEQ ID NOs: 49 and 50, a primer pair of SEQ ID NOs: 51 and 52, A primer pair of SEQ ID NOs: 53 and 54, a primer pair of SEQ ID NOs: 55 and 56, a primer pair of SEQ ID NOs: 57 and 58, a primer pair of SEQ ID NOs: 61 and 62, a primer pair of SEQ ID NOs: 63 and 64, and SEQ ID NOs: 65 and 66 A primer pair of, a primer pair of SEQ ID NOs: 67 and 68, a primer pair of SEQ ID NOs: 69 and 70, a primer pair of SEQ ID NOs: 71 and 72, a primer pair of SEQ ID NOs: 73 and 74, a primer pair of SEQ ID NOs: 75 and 76, sequence The primer pair of SEQ ID NO: 77 and 78, the primer pair of SEQ ID NO: 79 and 80, the primer pair of SEQ ID NO: 81 and 82, the primer pair of SEQ ID NO: 83 and 84, the primer pair of SEQ ID NO: 85 and 86, of SEQ ID NO: 87 and 88 Primer pair, primer pair of SEQ ID NO: 89 and 90, SEQ ID NO: 9 Primer pairs of 1 and 92, primer pairs of SEQ ID NOs: 93 and 94, primer pairs of SEQ ID NOs: 95 and 96, primer pairs of SEQ ID NOs: 97 and 98, primer pairs of SEQ ID NOs: 99 and 100, primers of SEQ ID NOs: 101 and 102 A pair, a primer pair of SEQ ID NOs: 103 and 104, a primer pair of SEQ ID NOs: 105 and 106, a primer pair of SEQ ID NOs: 107 and 108, a primer pair of SEQ ID NOs: 109 and 110, a primer pair of SEQ ID NOs: 111 and 112, and SEQ ID NO: It may be selected from the group consisting of primer pairs of 113 and 114.

In one embodiment, the reagent for detecting the protein level of the marker is an antibody, antibody fragment, an aptamer, an avidity multimer, or a peptidomimetics that specifically recognizes the entire protein length of the marker or a fragment thereof. ), which may include Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, and immunoprecipitation assay (Immunoprecipitation assay). , Complement Fixation Assay, FACS, mass spectrometry, or protein quantity analysis methods including protein microarrays, but are not limited thereto.

The term "detection" or "measurement" as used in the present invention means to quantify the concentration of a detected or measured object.

In the present invention, the term "primer" is a nucleic acid sequence having a short free 3 hydroxyl group, which can form a complementary template and a base pair, and is used as a starting point for template strand copying. It refers to a short functional nucleic acid sequence. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerate or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature. In the present invention, PCR amplification using sense and antisense primers specific to genes whose expression changes due to acute or chronic poisoning (exposure) of cocaine can be performed, and the level of mRMA expression can be determined by measuring the level of a desired product. The PCR conditions, the length of the sense and antisense primers can be modified based on those known in the art, but in the present invention, the expression level of each gene was confirmed using a primer pair of SEQ ID NOs: 1 to 114.

In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases to a few hundred bases for a specific binding to mRNA, and is labeled to confirm the presence or absence of a specific mRNA. I can. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. In the present invention, hybridization is performed using a probe complementary to a gene whose expression changes due to acute or chronic poisoning (exposure) of cocaine, and the degree of expression of each gene can be diagnosed through hybridization. Selection and hybridization conditions for suitable probes may be modified based on those known in the art, and thus are not particularly limited in the present invention.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well known methods. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, e.g., uncharged linkers (e.g. methyl phosphonate, phosphotriester, phosphoro Amidates, carbamates, etc.) or to charged linkers (eg phosphorothioate, phosphorodithioate, etc.).

In the present invention, the term "antibody" is a term known in the art and refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a protein expressed in genes that are markers whose expression changes due to acute or chronic poisoning (exposure) of cocaine of the present invention, and the preparation of the antibody The method can be prepared using well known methods. This includes partial peptides that can be made from these proteins. The form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, monoclonal antibody, or any one having antigen-binding properties is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes special antibodies such as humanized antibodies.

The composition for confirming cocaine addiction and dependence of the present invention is a biomarker that appears when cocaine is administered or treated to a subject not only for confirming the cocaine addiction and dependence of the subject, but also to develop a drug for preventing or treating cocaine addiction and dependence. The degree of "addiction and dependence" to confirm the change of the children or the identification of the change.

In one aspect, the present invention ARHGEF7, PTOV1, GTF2I, ADAM23, CDH2, GRIA1 (GRIA1X3, GRIA1X4), GRIA2 (GRIA2X6, GRIA2X7), HDAC5, HDAC10, NCAM1, NPY, SNCA, CCNG2, CDC7, CDH8, CDH8 , NEUROD6, NEUROG2, SPEN, KDM2B (KDM2BX10, KDM2BX13), KIFAP3, RBM10, RBM14, TP53I11, WNT10B, MCM3AP, DRD1, DRD2, DRD3, GNAL, GNG7, LOC101865389, PPPY, LOC2926, ACN, ADC101865389, KJ2, and AKT LOC101865389, LOC101926461, PLCB1, SLC18A3, SLC5A7, CAMK2A, EGLN2, ENO2, LOC101866179, LOC101866246, BAD, BCL2L1, CASP9, LOC101925175 and TNFRSF1A detection reagents for detection of one or more biomarkers selected from the group consisting of poisoning and dependence of cocaine. It is about the dragon kit.

In one embodiment, the kit may further include tools and/or reagents for collecting biological samples from a subject or patient, as well as tools and/or reagents for preparing genomic DNA, cDNA, RNA, or proteins from the samples. have. For example, it may include PCR primers for amplifying the relevant region of genomic DNA. The kit may contain probes of genetic factors useful for pharmacogenomic profiling. In addition, in the use of such a kit, a labeled oligonucleotide can be used to easily identify it during analysis.

In one embodiment, the kit may further contain a labeling material such as DNA polymerase and dNTP (dGTP, dCTP, dATP and dTTP), and a fluorescent material.

In one aspect, the present invention from a biological sample derived from a test subject ARHGEF7, PTOV1, GTF2I, ADAM23, CDH2, GRIA1 (GRIA1X3, GRIA1X4), GRIA2 (GRIA2X6, GRIA2X7), HDAC5, HDAC10, NCAM1, NPY, SNCA, CCNG2, CDC7, CDH8, DLG3, DOCK3, NEUROD6, NEUROG2, SPEN, KDM2B (KDM2BX10, KDM2BX13), KIFAP3, RBM10, RBM14, TP53I11, WNT10B, MCM3AP, DRD1, DRD2, DRD2101, GNAL, and PCHE2 , ADCY5, AKT3, KCNJ2, LOC101865389, LOC101926461, PLCB1, SLC18A3, SLC5A7, CAMK2A, EGLN2, ENO2, LOC101866179, LOC101866246, BAD, BCL2L1, CASP9, LOC101925175 and TNFRSF1A consisting of one or more biomarkers selected from the group of nucleic acids or proteins Detecting the concentration of; Comparing the detection result of the concentration of the nucleic acid or protein with the corresponding result of the corresponding marker of the normal control sample; And compared with the normal control sample, compared with the normal control sample in the subject-derived sample, PTOV1, HDAC5, HDAC10, NPY, SNCA, KDM2BX10, KDM2BX13, KIFAP3, WNT10B, DRD1, DRD2, DRD3, GNAL in the subject-derived sample. , PPP2R2B, GNG7, ACHE, SLC5A7, ADCY5, KCNJ2, LOC101925175, CASP9, ARHGEF7, SLC18A3, TNFRSF1A, BCL2L1, and one or more genes or proteins selected from the group consisting of BAD are upregulated, MCM3AP, GTF2I, ADAM23, and GRIA1X3, GRIA1X4, GRIA2X6, GRIA2X7, CCNG2, CDC7, CDH8, DLG3, NEUROD6, NEUROG2, SPEN, CAMK2A, EGLN2, TNFRSF1A, BCL2L1, PTOV1, AKT3, and LOC101866246 When one or more genes or proteins are downregulated from the group consisting of one or more genes or proteins selected from the group consisting of downregulation It relates to a method of providing information necessary for diagnosing cocaine exposure, comprising the step of determining as cocaine exposure.

In one embodiment, the method is compared with a normal control sample, PTOV1, HDAC5, HDAC10, NPY, SNCA, KDM2BX10, KDM2BX13, KIFAP3, WNT10B, DRD1, DRD2, DRD3, GNAL, PPP2R2B, GNG7, ACHE, SLC5A7, ADCY5 , One or more genes or proteins selected from the group consisting of KCNJ2, LOC101925175 and CASP9 are upregulated; And one or more genes or proteins selected from the group consisting of MCM3AP, GTF2I, ADAM23, CDH2, GRIA1X3, GRIA1X4, GRIA2X6, GRIA2X7, CCNG2, CDC7, CDH8, DLG3, NEUROD6, NEUROG2, SPEN, CAMK2A, EGLN2, TNFRSF1A and BCL2L1. If downregulated, it may further comprise determining that there is an acute cocaine exposure.

In one embodiment, the method is compared to a normal control sample, ARHGEF7, DRD1, DRD2, DRD3, GNAL, PPP2R2B, SLC18A3, ACHE, SLC5A7, ADCY5, KCNJ2, TNFRSF1A, at least one selected from the group consisting of BCL2L1 and BAD. The gene or protein is upregulated; And when one or more genes or proteins selected from the group consisting of MCM3AP, PTOV1, GTF2I, AKT3, CAMK2A, and LOC101866246 are down-regulated, it may further include determining that it is chronic cocaine exposure.

The “biological sample derived from a test subject” includes, but is not limited to, a sample such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine. In addition, a specific method of measuring the expression level of the mRNA or protein thereof of the gene can detect the expression of the gene at the mRNA level or protein level, and the isolation of mRNA or protein from a biological sample is performed using a known process. can do. In one embodiment of the present invention, the expression level of the gene or protein was performed using tissue isolated from the hippocampus or striatum of a monkey.

The present invention will be described in more detail through the following examples. However, the following examples are only for embodiing the contents of the present invention and the present invention is not limited thereto.

Example 1. Acute/chronic cocaine poisoning specific biomarkers in the hippocampus

1-1. Identification of genes that change expression according to acute/chronic cocaine addiction

Female crab monkeys (crab-eating macaque, Macaca fascicularis) (Suzhou, China) of about 5 to 6 years old with an average weight of 3.1 ± 0.1 kg in control (n = 2), acute cocaine administration group (CA, n = 2 to 4) ) And chronic cocaine administration group (CC, n = 2~3). Cocaine hydrochloride (Johnson Matthey Macfarlan Smith, Edinburgh, Scotland) was completely dissolved in 0.9% saline immediately before administration, and then to monkeys 1 to 3 mg/kg (body weight)/once for 5 to 8 weeks. It was injected intramuscularly at 4:00 pm at the daily dose. After cocaine administration, monkeys were sacrificed and hippocampus was obtained. All procedures were performed under the supervision of the KRIBB Institutional Animal Care and Use Committee (IACUC) (Approval No. KRIBB-AEC-16070 and KRIBB-AEC-17079). After separating total RNA from the hippocampus using TRIzol (Invitrogen, Carlsbad, California, USA), confirm that the RIN (RNA integrity number) value is 7 or more using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). I did. For RNA-Seq (RNA sequencing), an RNA library was prepared using a TruSeq RNA library preparation kit (Illumina, San Diego, CA, USA), and then mRNA was purified using magnetic beads attached to oligo-dT. The purified mRNA was cut and reverse transcribed into cDNA using random primers and reverse transcriptase, and a complementary sequence thereof was synthesized after removing the RNA template. After performing end repair, 3'adenylation, and adapter ligation, the purified cDNA library was concentrated by PCR. The prepared library was sequenced with an Illumina HiSeq 2000 sequencer (Macrogen, Seoul, Korea). After confirming the sequences obtained from RNA-Seq with FastQC (version 0.10.0, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), low quality including adapter sequences, contaminated DNA and PCR copies The reading results and artifacts of Trimmomatic ver. Removed with 0.32 (Bolger, Lohse & Usadel, 2014). The reading results were mapped to the reference genome using TopHat (version 2.0.13) software. The transcripts of each sample were assembled into Cufflinks (version 2.2.1) (Trapnell et al., 2010) based on the FPKM (fragments per kilobase of transcript per million mapped reads) method. Genes showing a difference in expression between samples were selected based on the FPKM value of the transcript, and the similarity between the samples was represented by a multidimensional scaling (MDS) plot. RNA-seq data was entrusted to GEO (Gene Expression Omnibus) (accession number GSE104332). As a result, in the control group, acute cocaine addiction and chronic cocaine addiction group, |fold change| 954 genes (transcripts) satisfying ≥ 2 and P <0.05 were identified, hierarchical clustering analysis was performed between the groups (Fig. 2a), and two of the groups (two pair -wise groups) were counted for the number of genes that were up and down regulated (FIG. 2b ). Between the acute cocaine addiction (exposure) group and the chronic cocaine addiction (exposure) group, 408 genes were up-regulated and 381 genes were down-regulated, which was significantly higher than that of the control vs. CA or control vs. CC. In Venn diagram analysis, three duplicated genes ARHGEF7 (Rho guanine nucleotide exchange factor 7), PTOV1 (prostate tumor overexpressed 1), and GTF2I (general transcription factor Iii) were selected through comparison of the three pairs (Fig. 2c). .

In addition, in addition to the above three genes, ADAM23, CDH2, GRIA1 (including GRIA1X3 and GRIA1X4), GRIA2 (including GRIA2X6 GRIA2X7), HDAC10, HDAC5, which are known as genes related to neurogenesis or addiction in NCBI (National Center for Biotechnology Information database). , Genes CCNG2, CDC7, CDH8, DLG3, DOCK3, NEUROD6, NEUROG2, SPEN, KDM2B (including KDM2BX10 and KDM2BX13), KIFAP3, RBM10, RBM14, TP53I11, WNT10B and WNT10B of genes of unknown association with NCAM1, NPY and SNCA were selected. .

1-2. Comparison of mRNA expression patterns of genes according to acute/chronic cocaine poisoning

1 μg of total RNA was reverse transcribed into cDNA using a kit (Thermo Fisher Scientific, Carlsbad, USA). The prepared cDNA, a primer set for the genes selected in Example 1-1 (Table 1) and qRT-PCR (Real time quantitative PCR) using SYBR Green Core Reagents (SYBR Premix Ex Taq II, Takara, Japan) Was performed. For reference, the primer sets of the present invention were confirmed by BLAST against the NCBI cynomolgus monkey genome sequence database to confirm specificity. Specifically, the sample was denatured at 95°C for 3 minutes (1 cycle), annealed at 90°C for 10 seconds and 65°C for 30 seconds for amplification, and then denatured at 95°C for 10 seconds as a final melting curve and 60 Treated at °C for 30 seconds. The data were analyzed by the 2-ΔΔCT method of Applied Biosystems (Carlsbad, CA, USA), a ViiA7 Real-Time PCR system. The final product of the analysis was electrophoresed on a 2% agarose gel and the band was confirmed by UV.

Gene Accession No primer Sequence (5'-3') Sequence number ARHGEF7 XM_005586258.2 Forward GACATTAAAACTCTGGGCAACG One Reverse CCAACGACAGGAATGACAATC 2 PTOV1 XM_005589964.1 Forward CATCCCCTACGACCAGAGC 3 Reverse GAGCCCAACAGTCGGTCCA 4 GTF2I XM_005549400.1 Forward GTATGGAATCCCGAGGCT 5 Reverse CGGAAGCAAGTGGAAGAG 6 ADAM23 XM_005574061.1 Forward CATCAACCAAGACTCGGAAAG 7 Reverse CTCCACATAATCCGAAGACAAC 8 CDH2 XM_005586980.1 Forward GACATTATCACAGTGGCAGC 9 Reverse GCAGTAAACTCTGGAGGATTG 10 GRIA1X3 XM_005558333.1 Forward GGTGGTGGTGGACTGTGAATC 11 Reverse GCTGGGATGGTGTCTGTGTAG 12 GRIA1X4 XM_005558334.1 Forward CAGAACGCCTCAATGCTATC 13 Reverse CACTATTCTTCCACTGCTGC 14 GRIA2X6 XM_005556165.1 Forward CTGGGTTTCAGATAGTGGAC 15 Reverse CTCTGCTTCCTTAGGTTGC 16 GRIA2X7 XM_005556166.1 Forward GGAGACTGTCTGGCAAACC 17 Reverse CTCTGCTTCCTTAGGTTGCG 18 HDAC10 XM_005566924.1 Forward GATGGGAAACGCTGACTAC 19 Reverse CAGCAGATGTGTGAGGTGG 20 HDAC5 XM_005584399.1 Forward CAGTGACACCGTCTGGAATG 21 Reverse CATGGCTGTGGATTCCTCG 22 NCAM1 XM_005579649.1 Forward CACTCCCTCTTCACCATCCATC 23 Reverse CTGGCTTCCTTGGCATCATAC 24 NPY XM_005549975.1 Forward GACTGACCCTCGCCCTGT 25 Reverse CTTGCCATACCTCTGCCTG 26 SNCA XM_005555423.1 Forward GAGTTGTGGCTGCTGCTG 27 Reverse CACCACCGCTCCTCCAAC 28 CCNG2 XM_005554984.1 Forward GGGCTGAGTTTGATTGAGGC 29 Reverse GAACAGACTCCAATGCAAGAC 30 CDC7 XM_005542756.1 Forward CTGACCTGTGACTGCTATG 31 Reverse GTATCGTCCACTAAGCAAAG 32 CDH8 XM_005592128.1 Forward GCTATAAAAAGACTTGACCGG 33 Reverse GCGTTGTCATTGATGTCTTG 34 DLG3 XM_005593877.1 Forward CAGGACGGGGATGATTGAGT 35 Reverse GAACTGCTTTCGCTGTCACTG 36 DOCK3 XM_005547258.1 Forward CTTTGGCTTTGCATTCTCAC 37 Reverse TATTAGGGCAGCCATTGTAG 38 NEUROD6 XM_005549850.1 Forward CCTCAACGACGCTCTGGAC 39 Reverse CTGGTCTCTTGCCGATTCTC 40 NEUROG2 XM_005555708.1 Forward CAAGAAGACCCGTAGACTGAAGG 41 Reverse AGATGTAGTTGTGGGCGAAGC 42 SPEN XM_005544696.1 Forward CAATCAGCAGCAGCAGTAGT 43 Reverse ATCTGTAGAGCGTACTGGAAG 44 KDM2BX10 XM_005572454.1 Forward GTCCCTGTGGTGACTTGGC 45 Reverse AGGCAGGCCTCGCACTTG 46 KDM2BX13 XM_005572457.1 Forward GAGGAGGAGGAGAAGGACGAG 47 Reverse CTTCACGCTCTCCTCTGACTCG 48 KIFAP3 XM_005539960.1 Forward GCTTATCTGTGAAGGAAATGG 49 Reverse AACTCCTCTTCCTCATCATTAG 50 RBM10 XM_005593402.1 Forward GGACGCTACACGATGGATGG 51 Reverse CTGCTCTGCCTCTGACTTGG 52 RBM14 XM_005577130.1 Forward CTACGCCGCACAAGCCAC 53 Reverse GAGTGCGGTAAGGAGCTGC 54 TP53I11 XM_005578119.1 Forward CAGTTCGTCTCTGCTGTGCTC 55 Reverse CCAAGAACTGGACCCCGAAG 56 WNT10B XM_005570737.1 Forward GCTGGCTGCTGGGGTCAT 57 Reverse CAGGGCTGGGCAGAGAGTG 58 MCM3AP XM_005548481.2 Forward CTGAAGTCACAGCCATCC 59 Reverse GGCTATCTTTTGGCACAG 60 GAPDH XM_015430282.1 Forward ACAACAGCCTCAAGATCGTCAG 115 Reverse ACTGTGGTCATGAGTCCTTCC 116

As a result, as shown in Figure 3, the expression of PTOV1, HDAC5, HDAC10, NPY, SNCA, KDM2BX10, KDM2BX13, KIFAP3 and WNT10B was significantly upregulated in the CA group, GTF2I, ADAM23, CDH2, GRIA1X3, GRIA1X4, Expression of GRIA2X6, GRIA2X7, CCNG2, CDC7, CDH8, DLG3, NEUROD6, NEUROG2, SPEN and MCM3AP was significantly downregulated. In addition, the expression of ARHGEF7 was significantly upregulated in the CC group, and the expressions of PTOV1, MCM3AP and GTF2I were significantly downregulated (FIGS. In particular, the expression of ARHGEF7 significantly increased only in the CC group, and the expression of PTOV1 was significantly increased in the CA group and decreased significantly in the CC group. In addition, GTF2I decreased in both CA and CC groups, and the decrease in CA group was found to be significantly decreased compared to CC group. In addition, KDM2BX13 and KIFAP3 were found to increase only in the CA group.

Through this, it was found that CA and CC can be distinguished through the regulation pattern of the markers and the expression regulation of ARHGEF7, PTOV1 and GTF2I (CA: increased PTOV1 expression, decreased GTF2I/CC: increased ARHGEF7, decreased PTOV1, GRF2I) Significantly less than CA)

Example 2. Acute/chronic cocaine poisoning specific biomarkers in striatum

2-1. Selection of genes through Venn diagram analysis

In order to identify genes whose expression significantly changes in striatum when cocaine was administered acutely/chronicly, female crab-eating macaque (crab-eating macaque, about 5 to 6 years old) with an average weight of 3.1 ± 0.1 kg. Macaca fascicularis) (Suzhou, China) was divided into a control group (n = 2), acute cocaine administration group (CA, n = 2), and chronic cocaine administration group (CC, n = 3). All procedures were performed under the supervision of KRIBB Institutional Animal Care and Use Committee (IACUC) (Approval No. KRIBB-AEC-16070). Cocaine hydrochloride (Johnson Matthey Macfarlan Smith, Edinburgh, Scotland) was completely dissolved in 0.9% saline immediately before administration, and then to monkeys 1 to 3 mg/kg (body weight)/once for 5 to 8 weeks. It was injected intramuscularly at 4:00 pm at the daily dose. After cocaine administration, monkeys were sacrificed and striatums were obtained. Total RNA was isolated from the striatum of each group using TRIzol (Invitrogen, Carlsbad, California, USA), and then the RIN (RNA integrity number) value was 7 using 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). It was confirmed that it was abnormal. For RNA-Seq (RNA sequencing), an RNA library was prepared using a TruSeq RNA library preparation kit (Illumina, San Diego, CA, USA), and then mRNA was purified using magnetic beads attached to oligo-dT. The purified mRNA was cut and reverse transcribed into cDNA using random primers and reverse transcriptase, and a complementary sequence thereof was synthesized after removing the RNA template. After performing end repair, 3'adenylation, and adapter ligation, the purified cDNA library was concentrated by PCR. The prepared library was sequenced with an Illumina HiSeq 2000 sequencer (Macrogen, Seoul, Korea). After confirming the sequences obtained from RNA-Seq with FastQC (version 0.10.0, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), low quality including adapter sequences, contaminated DNA and PCR copies The reading results and artifacts of Trimmomatic ver. Removed with 0.32 (Bolger, Lohse & Usadel, 2014). The reading results were mapped to the reference genome Macaca_fascicularis_5.0 (BioSample: SAMN00811240) using TopHat (version 2.0.13) software. The transcripts of each sample were assembled into Cufflinks (version 2.2.1) (Trapnell et al., 2010) based on the FPKM (fragments per kilobase of transcript per million mapped reads) method. Genes showing a difference in expression between samples were selected based on the FPKM value of the transcript, and the similarity between the samples was represented by a multidimensional scaling (MDS) plot. RNA-seq data was entrusted to GEO (Gene Expression Omnibus) (accession number GSE103419). The control group, acute cocaine addiction, and chronic cocaine addiction group did not show a significant difference from each other in MRI analysis and hematoxylin and eosin staining (data not shown), but hierarchical clustering analysis between the groups was performed. As a result, 1613 genes satisfying the expression level 2 times or more and P <0.05 were identified (Figs. 4a and b). In addition, among 1613 genes with fold change of 2.0 or more and a P value of less than 0.05, a total of 263 were differentially expressed in striatum exposed to acute cocaine, and a total of 178 were differentially expressed in striatum exposed to chronic cocaine, and a total of 1413 genes. The gene expression levels were changed differently between the acute and chronic cocaine addiction (exposed) striatum (Fig. 4c). In addition, Venn diagrams (Control vs. CA, Control vs. CC, CA vs. CC) were analyzed between groups using BioVenn (http://www.biovenn.nl/) (Hulsen, de Vlieg & Alkema, 2008). As a result, 13 genes SNAP25, CAMK2A, LOC101867149, KCNAB2, LOC102137553, RBM14, ADD3, PTPRZ1, LOC101865389, ARFGEF2, KCNJ10, LOC101925503, and GOPC could be selected (Figs. 4d and e).

2-2. Selection of genes through functional annotation and pathway analysis

As a result of confirming the sequences of the 13 genes using the human BLAST database, the Macaca fascicularis genome showed 92.8% identity with Homo sapiens (Ebeling et al., 2011). In addition, KEGG pathway (http://www.genome.jp/kegg/pathway.html) analysis was used to analyze biological responses and various standard pathways related to the genes, and cocaine was used in neuronal development and signal transduction. Since it has been shown to affect the pathway, 27 genes related to neurological pathways such as dopaminergic, cholinergic, HIF and apoptosis-related pathways were selected among the KEGG pathways (dopaminergic: DRD1, DRD2, DRD3, GNAL, GNG7. , LOC101865389, LOC101926461, PPP2R2B; Cholinergic: ACHE, ADCY5, AKT3, KCNJ2, LOC101865389, LOC101926461, PLCB1, SLC18A3, SLC5A7; HIF1 alpha pathway: CAMK2A, EGLN2, ENO1, CAS101, BCL9866 and cell death: BCL9866; , LOC101925175, TNFRSF1A) qRT-PCR was performed using the primer pairs in Table 2 below. In addition, to determine the gene expression relationship between acute and chronic cocaine addiction, a Pearson correlation analysis was performed on the expression level of these transcription factors in the qRT-PCR analysis results.

Gene Accession No primer Sequence (5'-3') Amplicon size (bp) Sequence number Dopaminergic synapse DRD1 XM_005579675.1 Forward CCCTCTGATGGGAATGCC 216 61 Reverse CTGGCAATTCTTGGCATGG 62 DRD2 XM_005548104.1 Forward GAGCATTCTGAACTTGTGTG 199 63 Reverse GTTGGCAATGATGCACTC 64 DRD3 XM_005587198.1 Forward CAGCCAGCATCCTGAACCTC 169 65 Reverse CCAAACAGAAGAGGGCAGGAC 66 GNAL XM_005587472.1 Forward CCAAGTGGACAAAGTGAAC 171 67 Reverse GACTCTCTCAGCCTGTTGG 68 GNG7 XM_005547666.1 Forward CGTATCAAGGTCTCCAAAGCGG 106 69 Reverse TAAAGGGGTTCTCGGAGGCAGG 70 LOC101865389 XM_005542907.1 Forward CGAAGTAGGTTGCGGAATGC 161 71 Reverse CATAGGTGTCTGAGCCGAAC 72 LOC101926461 XM_005558151.1 Forward CAGCAAGGGCTACAATAAGG 185 73 Reverse GCAGGTTCCGTAGAAGGTCC 74 PPP2R2B XM_005558151.1 Forward GACCACAGAAGGCTCGGAC 159 75 Reverse GGGTATCAATGTCCTCCTCC 76 Cholinergic synapse ACHE XM_005549276.1 Forward CCTGTGGTAGATGGAGACTTC 150 77 Reverse GTTGTCTTTGCTGAAGCCTGG 78 ADCY5 XM_005547964.1 Forward GACAGGCTTTCCAGGAGA 152 79 Reverse CATATCCTCCTGCTTGGCG 80 AKT3 XM_005539657.1 Forward CAGAACGACCAAAGCCAAACAC 150 81 Reverse CTTCTTGCCTCTGCAGTCTGTC 82 KCNJ2 XM_005584811.1 Forward GAAAGATGGTCACTGTAACG 172 83 Reverse TATCAACCAAAACACACAGC 84 LOC101865389 XM_005547668.1 Forward GAACAAATCGATTCCAGT 204 85 Reverse CGAATCTCTTTGTTGCTGTCC 86 LOC101926461 XM_005542906.1 Forward CTGAAGCAAATAGAGCATAC 219 87 Reverse GAATGTTAGCACTATCTGAGC 88 PLCB1 XM_005568372.1 Forward CTTACCGTTGACCAGATGATG 229 89 Reverse CATTCAAATCCAGTTTCTCAGG 90 SLC18A3 XM_005565137.1 Forward CCTACGGAGAGCGAAGACG 193 91 Reverse CGAACAGCGTGGCGTAGTC 92 SLC5A7 XM_005575201.1 Forward CCTTGCTGACGAAGACTGTG 214 93 Reverse GGGTAATAGCCAGGGTAGAAG 94 HIF-1 signaling pathway CAMK2A XM_005558248.1 Forward GCTGCTGAAGCCCTTAAG 232 95 Reverse CGATGGTGGTGTTGGTGC 96 EGLN2 XM_005589313.1 Forward GCTACAGCCACCTCTACCAC 234 97 Reverse CTCCTGGTTCTCTTGCCTGG 98 ENO2 XM_005569984.1 Forward AAGGTCTTTTCAGGGCTGCAG 138 99 Reverse CGATGGTGGAGTTGATGTGGT 100 LOC101866179 XM_005551953.1 Forward GGAGGAGAAGCAAATCATC 211 101 Reverse CACTGGAATCACTAACTGG 102 LOC101866246 XM_005557063.1 Forward CGTGAGGGCAATGAGAAAG 194 103 Reverse CATCAAGTATTGGTCTCTCG 104 Apoptosis-related genes BAD XM_005577428.1 Forward GGGACAGGCCCTCAGACTC 185 105 Reverse CGAAGGGATGGAGGAGGAG 106 BCL2L1 XM_005568631.1 Forward GGAGACCCCCAGTGCCAT 200 107 Reverse GCTGGGATGTCAGGTCAC 108 CASP9 XM_005544724.1 Forward CCTTCGTTCATCTCCTGCTTAG 204 109 Reverse ATCACCGAATCCTCCAGAACC 110 LOC101925175 XM_005580378.1 Forward GAGAACCTGGAGAAGAAGACGG 204 111 Reverse CTGCTCCAGGTCAGCCATCG 112 TNFRSF1A XM_005569893.1 Forward CACCTGAAAAAGAGGGGGAG 188 113 Reverse CATCTCTCTGCGGGAAGCC 114 GAPDH XM_015430282.1 Forward ACAACAGCCTCAAGATCGTCAG 112 115 Reverse ACTGTGGTCATGAGTCCTTCC 116

As a result, as shown in Figure 5, in the CA group, the expression of DRD1, DRD2, DRD3, GNAL, PPP2R2B, GNG7, ACHE, SLC5A7, ADCY5, KCNJ2, LOC101925175 and CASP9 was upregulated, CAMK2A, EGLN2, TNFRSF1A and BCL2L1. It was downregulated. Of these, GNG7, LOC101925175 and CASP9 were specifically upregulated in CA only, and EGLN2 and BCL2L1 were specifically downregulated only in CA. In addition, in the CC group, DRD1, DRD2, DRD3, GNAL, PPP2R2B, SLC18A3, ACHE, SLC5A7, ADCY5, KCNJ2, TNFRSF1A, BCL2L1 and BAD were upregulated, and AKT3, CAMK2A and LOC101866246 were downregulated. Among these, the expression of BAD was upregulated specifically in the CC group. In addition, the genes whose expression levels were significantly changed according to the control group, CA and CC were DRD1, DRD3, PPP2R2B, GNG7, ACHE, CAMK2A, and BCL2L1. In addition, as a result of Pearson correlation analysis, the genes were found to be correlated after acute and chronic cocaine administration (R = 0.587, P = 0.0013) (Fig. 6).

2-3. Confirmation of the regulation of CAMK2A expression according to cocaine administration

2-3-1. Confirmation of changes in mRNA expression of CAMK2A and CASP9

CAMK2A included in both the independent Venn diagram analysis results (FIG. 4e) and HIF alpha pathway analysis (FIG. 5c), and CASP9 related to apoptosis pathway important for cell homeostasis among the KEGG pathways were selected, and their acute/chronic cocaine administration By confirming the gene expression and protein expression pattern. Specifically, as in Example 2-1, after the administration of acute/chronic cocaine, the final product of RT-PCR analysis using RNA isolated from the brain striatum of monkeys was electrophoresed on an agarose gel to determine their mRNA levels. Confirmed. As a result, similar to the RNA-seq and qRT-PCR data, the expression of CAMK2A decreased by cocaine administration, whereas the expression of CASP9 was increased (Fig. 7a).

2-3-2. Confirmation of changes in protein level of CAMK2A and CASP9

In order to confirm the expression of CAMK2A and CASP9 at the protein level, as in Example 2-1, after the administration of acute/chronic cocaine, total RNA was prepared in the brain striatum of monkeys, and the remaining tissues were dissolved in 1% SDS buffer. . The same amount of protein was electrophoresed on the gel by 10% SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and transferred to a nitrocellulose membrane (Whatman, dassel, Germany). After blocking with 1% skim milk (TBST, 20mM Tris, 137mM NaCl and 0.1% Tween 20) for 1 hour, then anti-pCAMK2A, anti-CAMK2A, anti-cleaved CASP9 (Abcam, Cambridge, UK) and anti-GAPDH (Cell signal, Danvers, Massachusetts, USA) Incubated with antibody. Then, after incubation with horseradish peroxidase-conjugated anti-rabbit IgG (Thermo scientific) diluted 1: 10,000 with a secondary antibody, the band was visualized with PXi4 Chemiluminscent and Fluorescent Imaging System (Syngene, Boston, MA, USA). . Quantification of the intensity of each band, averaging to GAPDH, and relative ratio to control were calculated using Image J software (National Institutes of Health, Bethesda, MD, USA). As a result, the protein level of CAMK2A and its phosphorylation decreased by cocaine administration, and in particular, it was found to be rapidly decreased when chronic cocaine administration (Figs. 7b and c), and CASP9 cleaved in the striatum to which acute cocaine administration was administered increased. (Fig. 7b and d).

2-3-3. Verification of CAMK2A expression regulation pattern in human brain tissue

MRNA of CAMK2A using NCBI (National Center for Biotechnology Information) Gene Expression Omnibus (GEO) database portal (http://www.ncbi.nlm.nih.gov/geo/, accession number: GSE54839, GSE7762, and GSE44456) It was confirmed whether the level was changed by the administration of cocaine even in human brain tissue. Specifically, changes in mRNA expression of CAMK2A in human brain when chronic cocaine administration (GSE54839, GDS5047, probe ID: ILMN_1666445), change in mRNA expression of CAMK2A in human brain when chronic alcohol administration (GSE44456, GDS4879, control n=19, Alcoholic n=20) and chronic morphine administration showed changes in mRNA expression of CAMK2A in mouse striatum (GSE7762, GDS2815, n=36). As a result, it was found that the relative mRNA expression of CAMK2A in the midbrain of chronic cocaine abusers (n=10, triplicate) was significantly reduced compared to that in the control tissue (** P <0.01), but expressed by alcohol and morphine. There was no change in the (Fig. 8). Thus, it was confirmed that the expression of CAMK2A is down-regulated by chronic cocaine administration in the human midbrain.

2-4. Identification of functional protein network for CAMK2A

In order to confirm whether genes functionally related to CAMK2A were changed by cocaine administration, a functional protein network for CAMK2A was identified using the STRING database (Szklarczyk et al., 2017). As a result, it was confirmed that GRIN1 and DLG1 were functionally linked to CAMK2A (FIG. 9A). Accordingly, as a result of confirming the expression levels of CAMK2A, GRIN1 and DLG1 by the method described in the above examples, GRIN1 was down-regulated when administered acute/chronic cocaine similar to CAMK2A, but DLG1 was somewhat down-regulated and chronically It was found that cocaine administration was rather upregulated (Fig. 9b). In addition, as a result of confirming the expression levels of GRIN1 and DLG1 in the GEO data as in Example 2-3-3, unlike that DLG1 did not show a significant change (FIG. 9D ), GRIN1 was also in chronic cocaine-addicted human brain tissue. It showed a downregulated expression pattern similar to CAMK2A (Figs. 9c-f). Through this, it was found that CAMK2A and GRIN1 were related to cocaine addiction.

Example 3. Statistical Analysis

Gene expression levels are expressed as mean ± standard deviation, and the difference in gene expression levels between the two groups was compared using Student's t-test. Differences between groups were analyzed through a one-way variance analysis using SPSS 21 software (SPSS Inc., Chicago, IL, USA). The expression values of each CA and CC group with significant differences through qRT-PCR were analyzed using Pearson's correlation coefficients using SPSS 21 software and GraphPad PRISM 5 (CA, USA), and P values Results below 0.05 (q-values <0.2) were considered significant.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> MARKERS FOR COCAINE ADDICTION <130> PN1712-461 <160> 116 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ARHGEF7 forward primer <400> 1 gacattaaaa ctctgggcaa cg 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ARHGEF7 reverse primer <400> 2 ccaacgacag gaatgacaat c 21 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PTOV1 forward primer <400> 3 catcccctac gaccagagc 19 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PTOV1 reverse primer <400> 4 gagcccaaca gtcggtcca 19 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GTF2I forward primer <400> 5 gtatggaatc ccgaggct 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GTF2I reverse primer <400> 6 cggaagcaag tggaagag 18 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ADAM23 forward primer <400> 7 catcaaccaa gactcggaaa g 21 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ADAM23 reverse primer <400> 8 ctccacataa tccgaagaca ac 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CDH2 forward primer <400> 9 gacattatca cagtggcagc 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CDH2 reverse primer <400> 10 gcagtaaact ctggaggatt g 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GRIA1X3 forward primer <400> 11 ggtggtggtg gactgtgaat c 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GRIA1X3 reverse primer <400> 12 gctgggatgg tgtctgtgta g 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GRIA1X4 forward primer <400> 13 cagaacgcct caatgctatc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GRIA1X4 reverse primer <400> 14 cactattctt ccactgctgc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GRIA2X6 forward primer <400> 15 ctgggtttca gatagtggac 20 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GRIA2X6 reverse primer <400> 16 ctctgcttcc ttaggttgc 19 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GRIA2X7 forward primer <400> 17 ggagactgtc tggcaaacc 19 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GRIA2X7 reverse primer <400> 18 ctctgcttcc ttaggttgcg 20 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HDAC10 forward primer <400> 19 gatgggaaac gctgactac 19 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HDAC10 reverse primer <400> 20 cagcagatgt gtgaggtgg 19 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HDAC5 forward primer <400> 21 cagtgacacc gtctggaatg 20 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HDAC5 reverse primer <400> 22 catggctgtg gattcctcg 19 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NCAM1 forward primer <400> 23 cactccctct tcaccatcca tc 22 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NCAM1 reverse primer <400> 24 ctggcttcct tggcatcata c 21 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NPY forward primer <400> 25 gactgaccct cgccctgt 18 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> NPY reverse primer <400> 26 cttgccatac ctctgcctg 19 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> SNCA forward primer <400> 27 gagttgtggc tgctgctg 18 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> SNCA reverse primer <400> 28 caccaccgct cctccaac 18 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCNG2 forward primer <400> 29 gggctgagtt tgattgaggc 20 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CCNG2 reverse primer <400> 30 gaacagactc caatgcaaga c 21 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CDC7 forward primer <400> 31 ctgacctgtg actgctatg 19 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CDC7 reverse primer <400> 32 gtatcgtcca ctaagcaaag 20 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CDH8 forward primer <400> 33 gctataaaaa gacttgaccg g 21 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CDH8 reverse primer <400> 34 gcgttgtcat tgatgtcttg 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DLG3 forward primer <400> 35 caggacgggg atgattgagt 20 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DLG3 reverse primer <400> 36 gaactgcttt cgctgtcact g 21 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DOCK3 forward primer <400> 37 ctttggcttt gcattctcac 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DOCK3 reverse primer <400> 38 tattagggca gccattgtag 20 <210> 39 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> NEUROD6 forward primer <400> 39 cctcaacgac gctctggac 19 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NEUROD6 reverse primer <400> 40 ctggtctctt gccgattctc 20 <210> 41 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NEUROG2 forward primer <400> 41 caagaagacc cgtagactga agg 23 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NEUROG2 reverse primer <400> 42 agatgtagtt gtgggcgaag c 21 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SPEN forward primer <400> 43 caatcagcag cagcagtagt 20 <210> 44 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SPEN reverse primer <400> 44 atctgtagag cgtactggaa g 21 <210> 45 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> KDM2BX10 forward primer <400> 45 gtccctgtgg tgacttggc 19 <210> 46 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> KDM2BX10 reverse primer <400> 46 aggcaggcct cgcacttg 18 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KDM2BX13 forward primer <400> 47 gaggaggagg agaaggacga g 21 <210> 48 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> KDM2BX13 reverse primer <400> 48 cttcacgctc tcctctgact cg 22 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KIFAP3 forward primer <400> 49 gcttatctgt gaaggaaatg g 21 <210> 50 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> KIFAP3 reverse primer <400> 50 aactcctctt cctcatcatt ag 22 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RBM10 forward primer <400> 51 ggacgctaca cgatggatgg 20 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RBM10 reverse primer <400> 52 ctgctctgcc tctgacttgg 20 <210> 53 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> RBM14 forward primer <400> 53 ctacgccgca caagccac 18 <210> 54 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RBM14 reverse primer <400> 54 gagtgcggta aggagctgc 19 <210> 55 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TP53I11 forward primer <400> 55 cagttcgtct ctgctgtgct c 21 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TP53I11 reverse primer <400> 56 ccaagaactg gaccccgaag 20 <210> 57 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> WNT10B forward primer <400> 57 gctggctgct ggggtcat 18 <210> 58 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> WNT10B reverse primer <400> 58 cagggctggg cagagagtg 19 <210> 59 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> MCM3AP forward primer <400> 59 ctgaagtcac agccatcc 18 <210> 60 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> MCM3AP reverse primer <400> 60 ggctatcttt tggcacag 18 <210> 61 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DRD1 forward primer <400> 61 ccctctgatg ggaatgcc 18 <210> 62 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> DRD1 reverse primer <400> 62 ctggcaattc ttggcatgg 19 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DRD2 forward primer <400> 63 gagcattctg aacttgtgtg 20 <210> 64 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DRD2 reverse primer <400> 64 gttggcaatg atgcactc 18 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DRD3 forward primer <400> 65 cagccagcat cctgaacctc 20 <210> 66 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DRD3 reverse primer <400> 66 ccaaacagaa gagggcagga c 21 <210> 67 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GNAL forward primer <400> 67 ccaagtggac aaagtgaac 19 <210> 68 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GNAL reverse primer <400> 68 gactctctca gcctgttgg 19 <210> 69 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GNG7 forward primer <400> 69 cgtatcaagg tctccaaagc gg 22 <210> 70 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GNG7 reverse primer <400> 70 taaaggggtt ctcggaggca gg 22 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LOC101865389 forward primer <400> 71 cgaagtaggt tgcggaatgc 20 <210> 72 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LOC101865389 reverse primer <400> 72 cataggtgtc tgagccgaac 20 <210> 73 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LOC101926461 forward primer <400> 73 cagcaagggc tacaataagg 20 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LOC101926461 reverse primer <400> 74 gcaggttccg tagaaggtcc 20 <210> 75 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PPP2R2B forward primer <400> 75 gaccacagaa ggctcggac 19 <210> 76 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPP2R2B reverse primer <400> 76 gggtatcaat gtcctcctcc 20 <210> 77 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ACHE forward primer <400> 77 cctgtggtag atggagactt c 21 <210> 78 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ACHE reverse primer <400> 78 gttgtctttg ctgaagcctg g 21 <210> 79 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> ADCY5 forward primer <400> 79 gacaggcttt ccaggaga 18 <210> 80 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ADCY5 reverse primer <400> 80 catatcctcc tgcttggcg 19 <210> 81 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> AKT3 forward primer <400> 81 cagaacgacc aaagccaaac ac 22 <210> 82 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> AKT3 reverse primer <400> 82 cttcttgcct ctgcagtctg tc 22 <210> 83 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KCNJ2 forward primer <400> 83 gaaagatggt cactgtaacg 20 <210> 84 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KCNJ2 reverse primer <400> 84 tatcaaccaa aacacacagc 20 <210> 85 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LOC101865389 forward primer <400> 85 gaacaaatcg attccagt 18 <210> 86 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LOC101865389 reverse primer <400> 86 cgaatctctt tgttgctgtc c 21 <210> 87 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LOC101926461 forward primer <400> 87 ctgaagcaaa tagagcatac 20 <210> 88 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LOC101926461 reverse primer <400> 88 gaatgttagc actatctgag c 21 <210> 89 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PLCB1 forward primer <400> 89 cttaccgttg accagatgat g 21 <210> 90 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PLCB1 reverse primer <400> 90 cattcaaatc cagtttctca gg 22 <210> 91 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SLC18A3 forward primer <400> 91 cctacggaga gcgaagacg 19 <210> 92 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SLC18A3 reverse primer <400> 92 cgaacagcgt ggcgtagtc 19 <210> 93 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SLC5A7 forward primer <400> 93 ccttgctgac gaagactgtg 20 <210> 94 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SLC5A7 reverse primer <400> 94 gggtaatagc cagggtagaa g 21 <210> 95 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CAMK2A forward primer <400> 95 gctgctgaag cccttaag 18 <210> 96 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CAMK2A reverse primer <400> 96 cgatggtggt gttggtgc 18 <210> 97 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EGLN2 forward primer <400> 97 gctacagcca cctctaccac 20 <210> 98 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EGLN2 reverse primer <400> 98 ctcctggttc tcttgcctgg 20 <210> 99 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ENO2 forward primer <400> 99 aaggtctttt cagggctgca g 21 <210> 100 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ENO2 reverse primer <400> 100 cgatggtgga gttgatgtgg t 21 <210> 101 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LOC101866179 forward primer <400> 101 ggaggagaag caaatcatc 19 <210> 102 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LOC101866179 reverse primer <400> 102 cactggaatc actaactgg 19 <210> 103 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LOC101866246 forward primer <400> 103 cgtgagggca atgagaaag 19 <210> 104 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LOC101866246 reverse primer <400> 104 catcaagtat tggtctctcg 20 <210> 105 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> BAD forward primer <400> 105 gggacaggcc ctcagactc 19 <210> 106 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> BAD reverse primer <400> 106 cgaagggatg gaggaggag 19 <210> 107 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> BCL2L1 forward primer <400> 107 ggagaccccc agtgccat 18 <210> 108 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> BCL2L1 reverse primer <400> 108 gctgggatgt caggtcac 18 <210> 109 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CASP9 forward primer <400> 109 ccttcgttca tctcctgctt ag 22 <210> 110 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CASP9 reverse primer <400> 110 atcaccgaat cctccagaac c 21 <210> 111 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LOC101925175 forward primer <400> 111 gagaacctgg agaagaagac gg 22 <210> 112 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LOC101925175 reverse primer <400> 112 ctgctccagg tcagccatcg 20 <210> 113 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNFRSF1A forward primer <400> 113 cacctgaaaa agagggggag 20 <210> 114 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TNFRSF1A reverse primer <400> 114 catctctctg cgggaagcc 19 <210> 115 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 115 acaacagcct caagatcgtc ag 22 <210> 116 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 116 actgtggtca tgagtccttc c 21

Claims (18)

ARHGEF7, PTOV1, GTF2I, ADAM23, CDH2, GRIA1X3, GRIA1X4, GRIA2X6, GRIA2X7, HDAC5, HDAC10, NPY, SNCA, CCNG2, CDC7, CDH8, DLG3, NEUROD6, KDMBX13, which indicate changes in cocaine exposure Genes including KIFAP3, WNT10B, MCM3AP, DRD1, DRD2, DRD3, GNAL, GNG7, PPP2R2B, ACHE, ADCY5, AKT3, KCNJ2, SLC18A3, SLC5A7, CAMK2A, EGLN2, LOC101866246, CASP9RS, LOC101866246, BAD, BAD, BAD, BCL2L1175, and genes including LOC101866246 A biomarker composition for confirming cocaine addiction and dependence comprising a protein encoded by the gene. The method of claim 1, comprising PTOV1, HDAC5, HDAC10, NPY, SNCA, KDM2BX10, KDM2BX13, KIFAP3, WNT10B, DRD1, DRD2, DRD3, GNAL, PPP2R2B, GNG7, ACHE, SLC5A7, ADCY5, KCNJ2, and LOC101925 CAS9, and LOC101175 Biomarker composition for determining cocaine addiction and dependence, which is determined to be acute cocaine exposure when the expression of a gene or a protein encoded by the gene is upregulated. The gene according to claim 1, wherein MCM3AP, GTF2I, ADAM23, CDH2, GRIA1X3, GRIA1X4, GRIA2X6, GRIA2X7, CCNG2, CDC7, CDH8, DLG3, NEUROD6, NEUROG2, SPEN, CAMK2A, EGLN2, TNFRSF1A and BCL gene containing the gene Biomarker composition for determining cocaine addiction and dependence that is determined to be acute cocaine exposure when the expression of the protein encoded by is down-regulated. According to claim 1, ARHGEF7, DRD1, DRD2, DRD3, GNAL, PPP2R2B, SLC18A3, ACHE, SLC5A7, ADCY5, KCNJ2, TNFRSF1A, BCL2L1 and when the expression of the gene or the protein encoded by the BAD is upregulated. Biomarker composition for determining cocaine addiction and dependence, which is determined to be chronic cocaine exposure. The biomarker composition for confirming cocaine addiction and dependence according to claim 1, which is determined to be chronic cocaine exposure when the expression of a gene including MCM3AP, PTOV1, GTF2I, AKT3, CAMK2A and LOC101866246 or a protein encoded by the gene is downregulated . delete delete ARHGEF7, PTOV1, GTF2I, ADAM23, CDH2, GRIA1X3, GRIA1X4, GRIA2X6, GRIA2X7, HDAC5, HDAC10, NPY, SNCA, CCNG2, CDC7, CDH8, DLG3, NEURO, KDM2B, and NEURBX10, indicating changes in expression upon exposure to cocaine Genes including, KIFAP3, WNT10B, MCM3AP, DRD1, DRD2, DRD3, GNAL, GNG7, PPP2R2B, ACHE, ADCY5, AKT3, KCNJ2, SLC18A3, SLC5A7, CAMK2A, EGLN2, LOC101866246, BAD, BAD, BAD9, LOC101866246, CASP9RSF2L1, and LOC101866246 genes Or a composition for confirming cocaine addiction and dependence, comprising a reagent for detecting a biomarker containing a protein encoded by the gene. The composition of claim 8, wherein the detection reagent is a reagent capable of detecting a marker at the protein or nucleic acid level. The method of claim 9, wherein the reagent for detecting the nucleic acid level of the marker is a primer pair or probe that specifically recognizes the nucleic acid sequence of the marker, a nucleic acid sequence complementary to the nucleic acid sequence, the nucleic acid sequence and a fragment of the complementary sequence, or A composition for determining cocaine addiction and dependence, including a primer pair and a probe. The method of claim 10, wherein the detection reagent is polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive polymerase chain reaction (Competitive RT-PCR), Nuclease protection assay (RNase , S1 nuclease assay), in situ hybridization, nucleic acid microarray, Northern blot or used in DNA chips, cocaine addiction and a composition for determining dependence. The method of claim 10, wherein the primer pair is a primer pair of SEQ ID NOs: 1 and 2, a primer pair of SEQ ID NOs: 3 and 4, a primer pair of SEQ ID NOs: 5 and 6, a primer pair of SEQ ID NOs: 7 and 8, and SEQ ID NOs: 9 and 10 A primer pair of, a primer pair of SEQ ID NOs: 11 and 12, a primer pair of SEQ ID NOs: 13 and 14, a primer pair of SEQ ID NOs: 15 and 16, a primer pair of SEQ ID NOs: 17 and 18, a primer pair of SEQ ID NOs: 19 and 20, sequence The primer pairs of SEQ ID NOs: 21 and 22, the primer pairs of SEQ ID NOs: 25 and 26, the primer pairs of SEQ ID NOs: 27 and 28, the primer pairs of SEQ ID NOs: 29 and 30, the primer pairs of SEQ ID NOs: 31 and 32, of SEQ ID NOs: 33 and 34 A primer pair, a primer pair of SEQ ID NOs: 35 and 36, a primer pair of SEQ ID NOs: 39 and 40, a primer pair of SEQ ID NOs: 41 and 42, a primer pair of SEQ ID NOs: 43 and 44, a primer pair of SEQ ID NOs: 45 and 46, SEQ ID NO A primer pair of 47 and 48, a primer pair of SEQ ID NO: 49 and 50, a primer pair of SEQ ID NO: 55 and 56, a primer pair of SEQ ID NO: 57 and 58, a primer pair of SEQ ID NO: 61 and 62, a primer of SEQ ID NO: 63 and 64 A pair, a primer pair of SEQ ID NOs: 65 and 66, a primer pair of SEQ ID NOs: 67 and 68, a primer pair of SEQ ID NOs: 69 and 70, a primer pair of SEQ ID NOs: 75 and 76, a primer pair of SEQ ID NOs: 77 and 78, SEQ ID NO: 79 And a primer pair of 80, a primer pair of SEQ ID NOs: 81 and 82, a primer pair of SEQ ID NOs: 83 and 84, a primer pair of SEQ ID NOs: 91 and 92, a primer pair of SEQ ID NOs: 93 and 94, a primer pair of SEQ ID NOs: 95 and 96. , A primer pair of SEQ ID NOs: 97 and 98, a primer pair of SEQ ID NOs: 103 and 104, a primer pair of SEQ ID NOs: 105 and 106, a primer pair of SEQ ID NOs: 107 and 108, a primer pair of SEQ ID NOs: 109 and 110, and SEQ ID NO: 111 and A primer pair of 112, and SEQ ID NO: 113 And a composition for confirming cocaine addiction and dependence comprising a primer pair of 114. The method of claim 9, wherein the reagent for detecting the protein level of the marker is an antibody, antibody fragment, an aptamer, an avidity multimer, or a peptidomimetic that specifically recognizes the entire protein length of the marker or a fragment thereof. peptidomimetics) containing, cocaine addiction and dependence identification composition. The method of claim 13, wherein the detection reagent is Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay (Immunoprecipitation assay). ), complement fixation assay (Complement Fixation Assay), FACS, used in mass spectrometry or protein microarray, cocaine addiction and a composition for determining dependence. ARHGEF7, PTOV1, GTF2I, ADAM23, CDH2, GRIA1X3, GRIA1X4, GRIA2X6, GRIA2X7, HDAC5, HDAC10, NPY, SNCA, CCNG2, CDC7, CDH8, DLG3, NEUROD6, NEUROG2, SP10B, MAP3, KDMBX3, WDMBX DRD1, DRD2, DRD3, GNAL, GNG7, PPP2R2B, ACHE, ADCY5, AKT3, KCNJ2, SLC18A3, SLC5A7, CAMK2A, EGLN2, LOC101866246, BAD, BCL2L1, CASP9, LOC101925175 and the gene encoding the gene encoding TNFRSF1A, CASP9, LOC101925175 and the gene encoding the gene A kit for confirming cocaine addiction and dependence, including a reagent for detecting a biomarker comprising a. 1) ARHGEF7, PTOV1, GTF2I, ADAM23, CDH2, GRIA1X3, GRIA1X4, GRIA2X6, GRIA2X7, HDAC5, HDAC10, NPY, SNCA, CCNG2, CDC7, CDH8, DLG3, NEUROD6, KEURBX10, NEURBX10, NEUROG2 Including, KDM2BX13, KIFAP3, WNT10B, MCM3AP, DRD1, DRD2, DRD3, GNAL, GNG7, PPP2R2B, ACHE, ADCY5, AKT3, KCNJ2, SLC18A3, SLC5A7, CAMK2A, EGLN2, CAS BRS, LOC101A925, and B175AD CAS, LOC101A925, B175 Detecting a concentration of a biomarker including a gene or a protein encoded by the gene;
2) comparing the detection result of the gene or protein concentration with the corresponding result of the corresponding marker of the normal control sample to which cocaine was not administered;
3) Genes or proteins including PTOV1, HDAC5, HDAC10, NPY, SNCA, KDM2BX10, KDM2BX13, KIFAP3, WNT10B, DRD1, DRD2, DRD3, GNAL, PPP2R2B, GNG7, ACHE, SLC5A7, ADCY5, KCNJ2, LOC101925, and CASP9925175 and proteins Upregulated and downregulated genes or proteins including MCM3AP, GTF2I, ADAM23, CDH2, GRIA1X3, GRIA1X4, GRIA2X6, GRIA2X7, CCNG2, CDC7, CDH8, DLG3, NEUROD6, NEUROG2, SPEN, CAMK2A, EGLN2, TNFRSF1A and BCL2L Determining that it is an acute cocaine exposure; And
4) Compared with the normal control sample, in a sample derived from a subject, ARHGEF7, DRD1, DRD2, DRD3, GNAL, PPP2R2B, SLC18A3, ACHE, SLC5A7, ADCY5, KCNJ2, TNFRSF1A, BCL2L1, and genes containing BAD or the gene are encoded. If the protein is upregulated and the gene or the protein encoded by the gene is down-regulated, including MCM3AP, PTOV1, GTF2I, AKT3, CAMK2A, and LOC101866246, information necessary for the diagnosis of cocaine exposure, including determining that it is chronic cocaine exposure, is provided. How to provide. delete delete

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