KR20170004638A - Composition for Diagnosing of Pheochromocytoma - Google Patents

Abstract

The present invention relates to a composition for diagnosing pheochromocytoma, containing a formulation for measuring the level of mRNA encoding a mu-opioid receptor (OPRM1) gene or a protein thereof. The present invention further relates to a kit including the composition, and a method for providing information required for the diagnosis. By providing an OPRM1 marker enabling an early diagnosis of pheochromocytoma, it is possible to make an early diagnosis on pheochromocytoma and also to predict and grasp the degree of the disease in a meaningful way, by measuring and comparing the level of expression of the OPRM1 mRNA or the protein thereof, showing an increase in the expression in a patient with pheochromocytoma.

Description

{Composition for Diagnosis of Pheochromocytoma}

The present invention relates to a composition for diagnosing pheochromocytoma comprising an agent for measuring mRNA of OPRM1 (mu-opioid receptor) gene or its protein level, a kit containing the same, and a method for providing information for diagnosis.

Pain is the first symptom of cancer, with almost 50% of cancer patients experiencing varying degrees of pain during the course of the cancer. Therefore, analgesic therapy is needed to alleviate such pain in cancer treatment.

Morphine is a representative active opioid alkaloid and has been used as an analgesic for the treatment of various degrees of cancer pain. It has also been reported that morphine is also involved in the recurrence of cancer, and the morphine receptor OPRM1 (μ- opioid receptor signaling mechanisms have been increasingly reported in relation to carcinogenic activity. However, it is not known how OPRM1 affects the proliferation of cancer cells and how the expression pattern of OPRM1 in cancer is specifically known.

On the other hand, the OPRM1 (mu-opioid receptor) encoding the mu-opioid receptor is a receptor that binds opioid drugs (analgesics) such as morphine, heroin and fentanyl, Neuroendocrine hormones, such as endorphin β-endorphin (β-endorphin) and enkephalin (enkephalin), such as receptors, widely expressed in the brain. The OPRM1 protein belongs to the gamma subfamily of class A (rhodopsin-like) GPCRs (G-protein-coupled receptors) and activates the inhibitory G _{i / o} protein when the opiate drug or endogenous opioid receptor binds to adenylate cyclase -cAMP-protein kinase A (PKA) pathway and phospholipase C (PLC) activation, resulting in voltage-dependent Ca2 ⁺ channel inhibition and K ⁺ channel activation. This series of signal transmissions all inhibit the release of neurotransmitters.

In addition to the involvement of OPRM1 in G protein-related signaling in differentiated neurons, various signaling pathways, such as mitogen-activated protein kinases (MAPKs), extracellular-signaling pathways -regulated kinases 1 and 2), SAPK (stress-activated protein kinases), JNK (c-JUN N-terminal kinase), P38, STAT5 (signal transducer and activator of transcription 5), PKB / AKT (protein kinase B) OPRM1 is also reported to activate biologically important signals such as epidermal growth factor receptor (EGFR), which suggests that OPRM1 is associated with tumorigenesis, There is little known about relevance.

Neuroendocrine cells secrete neurotransmitter, neuromodultor and neuropeptide hormone and respond to external stimuli to produce a concentrated granule that secretes hormones by exocytosis And refers to cells without axons and synapses.

Neuroendocrine tumors are a very heterogeneous group of tumors that arise from neuroendocrine cells and include carcinoid, insulinoma, gastrinoma, VIP, pheochromocytoma, Paraganglioma, medullary carcinoma of thyroid, and small cell carcinoma of the lung. Neuroendocrine tumors are not only very rare, but they are also characterized by a wide variety of histologic features and names, ranging from well differentiated carcinoid tumors to undifferentiated small cell carcinomas of the lung or other organs, The biological characteristics, the therapeutic response, and the prognosis are very rare.

Pheochromocytoma, a typical endocrine hypertensive disorder, is a neuroendocrine tumor that secretes catecholamines, most of which originate in chromatin-afflicted cells of the adrenal gland, but some of them occur in the abdominal cavity, abdominal cavity and thoracic cavity after adrenal gland. When derived from extra-adrenal chromaffin cells, it is called adrenal pheochromocytoma or paraganglioma. The paranasal sinuses of the head and neck are derived from parasympathetic nervous system, and most do not produce catecholamines. Although pheochromocytoma is a rare disease, it is mostly a benign tumor. It is a clinically important disease because it is the cause of typical endocrine hypertension and it can be cured by accurate diagnosis and proper medical and surgical treatment. Pheochromocytoma is estimated to account for 0.1-0.6% of patients with hypertension and about 0.05-0.1% of patients with persistent hypertension. However, pheochromocytoma has a high incidence of post-diagnosis pheochromocytomas (0.05%), It is presumed that it contributed to premature death.

Therefore, accurate diagnosis of pheochromocytomas is very important, because resection of the tumor can completely heal hypertension and, if left untreated, can be fatal. Currently, biochemical confirmation of the production of excess catecholamines is a prerequisite for diagnosis, but it is very difficult to reliably eliminate or confirm pheochromocytoma at all times with biochemical tests. Furthermore, there is currently no method for molecular genetic diagnosis of patients suspected of having pheochromocytoma.

Molecular diagnosis is also called genetic diagnosis. It is used to diagnose diseases and diseases by quantitative / qualitative analysis of the genes causing the disease. PCR method accounts for 70% of the whole market, and DNA sequencing and DNA chip analysis The rest of it. However, the conventional PCR method has a tendency to be replaced by a real-time PCR method due to the impossibility of quantitative analysis and false positives caused by cross-contamination. Real-time PCR has a detection limit of over one million times and a high specificity compared with the diagnostic method using antigen-antibody reaction targeting existing proteins, and has been used as a confirmation method for new influenza vaccine tests. However, compared to the immunoassay method based on protein, it is not fully automated and the inspection price is high, so it is mainly used in large hospitals or specialized examination centers.

The inventors of the present invention solved the above problem by solving the problem of molecular diagnosis of pheochromocytoma using isoform detection by alternative splicing of Oprm1 1 gene and by performing multiplexing of isoform detection, Respectively.

Accordingly, the present invention provides a composition for the diagnosis of pheochromocytoma comprising an agent for measuring mRNA of OPRM1 (mu-opioid receptor) gene or its protein level, a kit containing the same, and a method for providing information for diagnosis .

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a composition for diagnosing pheochromocytoma comprising an agent for measuring the mRNA of OPRM1 (μ-opioid receptor) gene or its protein level.

In one embodiment of the present invention, the OPRM1 gene is characterized by comprising the nucleotide sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the agent for measuring the mRNA level of the gene is a primer pair, a probe, or an antisense nucleotide that specifically binds to the gene.

In another embodiment of the present invention, the agent for measuring the protein level is an antibody or an aptamer that specifically binds to the protein.

In addition, the present invention provides a kit for the diagnosis of pheochromocytoma comprising the above composition.

The present invention also relates to a method for detecting an OPRM1 (mu-opioid receptor) gene mRNA expression level or protein expression level from a biological sample; And comparing the expression level of the mRNA or the expression level of the protein with a control sample. The present invention also provides a method for providing information for diagnosis of pheochromocytoma.

In one embodiment of the present invention, the mRNA expression level is measured using a reverse transcriptase polymerase, a competitive reverse transcriptase polymerase, a real-time reverse transcriptase polymerase, an RNase protection assay, or Northern blotting .

In another embodiment of the present invention, the protein expression level is measured using a protein chip, immunoassay, Western blot, or enzyme linked immunosorbent assay (ELISA).

As described above, the present invention provides an OPRM1 marker capable of early diagnosis of pheochromocytomas, thereby measuring and comparing the expression level of OPRM1 mRNA or its protein expressed in a pheochromocytoma patient, And disease severity can be significantly predicted or understood.

1 is a schematic diagram showing the ORPM1 gene structure and isoform (splice variants).
FIG. 2 is a graph showing expression patterns of OPRM1 gene isoform (splice variants) in pheochromocytoma (PCPG) using a TCGA RNA-seq database.
FIG. 3 shows the result of performing an RT-PCR on ORPM1 mRNA in PC12, which is a pancreatic cell (PCPG) cell line, and electrophoresis.

The present invention provides a composition for diagnosing pheochromocytoma comprising an agent for measuring mRNA of OPRM1 (mu-opioid receptor) gene or a protein level thereof.

The genomic structure of the human OPRM1 (hOPRM1) gene has two promoters, which are similar to the promoters of the housekeeping gene due to lack of TATA box or multiple Sp1 binding sites. In addition, OPRM1 is a multi-exon gene consisting of 12 exons. It exhibits a complex expression mechanism. Since OPRM1 causes site or cell-specific RNA processing due to alternative splicing or alternative promoter, OPRM1 (P35372-1, accession number NM_00914) To generate at least 22 splice variants. These various OPRM1 isoforms have different and even opposite effects on opioid action, but the specific splicing regulatory mechanism has not been elucidated.

In order to better understand the expression and the RNA processing of OPRM1 in cancer, the present inventor has found that from the global RNA-seq data of the TCGA (NIH database), OPRM1 　 We have developed an analytical tool for evaluating mRNA levels and isoforms. As a result, the expression of OPRM1 was significantly increased in pheochromocytoma and paraganglioma (PCPG).

The term "diagnosis" as used herein means to identify the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to confirm the onset of pheochromocytoma.

As used herein, the term "diagnostic marker" refers to a polypeptide or nucleic acid that exhibits a significant increase or decrease in the level of gene expression or protein expression in a subject having a pheochromocytoma compared to a normal control (an individual that is not a pheochromocytoma) mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like. For the purpose of the present invention, the marker for the diagnosis of pheochromocytoma is OPRM1 (mu-opioid receptor).

Pheochromocytoma (chromaffinocytoma) is composed of chromaffin cells and is a tumor that synthesizes and liberates catecholamines and some peptide hormones. The disease is characterized by headache, sweating, palpitations, and hypertension. Clinically suspected cases have been diagnosed by measuring catecholamine or its metabolites in plasma or urine. However, the present inventors have found that, in addition to the above-mentioned conventional diagnosis, OPRM1 can be used as a marker for the diagnosis of pheochromocytoma.

As used herein, the term " measurement of mRNA expression level "is used to measure the amount of mRNA in a biological sample by confirming the presence or absence of mRNA and the expression level of the gene for diagnosis of pheochromocytoma in a biological sample. RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection (RPA), and reverse transcriptase-polymerase chain reaction assay, Northern blotting, DNA chip, and the like.

The agent for measuring the level of mRNA expression of the OPRM1 gene is preferably a primer pair or a probe. Since the nucleic acid information of the genes is known by GeneBank et al., A person skilled in the art can use a primer or a primer specifically amplifying a specific region of these genes Probes can be designed.

As used herein, the term "measurement of protein expression level" is a process for confirming the presence and expression level of a protein expressed from a gene for the diagnosis of pheochromocytoma in a biological sample in order to diagnose pheochromocytoma. An amount of the protein can be confirmed by using an antibody, an interaction protein, a ligand, a nanoparticle or an aptamer that specifically binds to the protein or peptide fragment of the gene. However, And may comprise any detection means having an affinity for the antibody, preferably an antibody or an aptamer.

As a method for measuring or comparing the protein expression level, protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI- of Flight Mass Spectrometry analysis, radioimmunoassay, radial immunodiffusion, Oucheroton immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation, two-dimensional electrophoresis analysis, liquid chromatography (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting, and enzyme linked immunosorbent assay (ELISA).

Preferably, the agent that measures the mRNA level may comprise a primer pair, probe or antisense nucleotide that specifically binds to the OPRMl gene.

As used herein, the term "primer pair" is a primer pair that contains all combinations of primer pairs consisting of forward and reverse primers that recognize the target gene sequence, but preferably provides specific and sensitive assay results . The nucleic acid sequence of the primer is inconsistent with the non-target sequence present in the sample and can be given high specificity when it is a primer that amplifies only the target gene sequence containing a complementary primer binding site and does not induce nonspecific amplification .

As used herein, the term "probe" refers to a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically confirming the presence of a target substance in the sample through the binding do. The probe molecule may be a substance commonly used in the art without limitation, but preferably it may be a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a peptide, a polypeptide, a protein, an RNA or a DNA, It is a PNA. More specifically, the probe may be a biomolecule derived from or derived from an organism, or prepared in vitro, such as an enzyme, a protein, an antibody, a microorganism, an animal or plant cell and an organ, a nerve cell, DNA, and RNA DNA includes cDNA, genomic DNA, oligonucleotide, RNA includes genomic RNA, mRNA, oligonucleotide, and examples of the protein include an antibody, an antigen, an enzyme, a peptide, and the like.

The term "antisense" as used in the present invention refers to an antisense oligomer that hybridizes with a target sequence in an RNA by Watson-Crick base pairing to allow the formation of an RNA: oligomer heterodimer, typically in the target sequence, Refers to an oligomer having a sequence of bases and a backbone between subunits. Oligomers may have an exact sequence complement or approximate complementarity to the target sequence.

Preferably, the agent that measures the protein level may comprise an antibody or an aptamer that specifically binds to the OPRM1 protein or peptide fragment.

The term "antibody" as used herein refers to a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to the OPRM1 protein, and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. The production of the antibody can be easily carried out using techniques well known in the art. In addition, the antibodies of the present invention include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

As used herein, the term "aptamer" is a biopolymer substance that inhibits protein interaction through three-dimensional binding with a specific target protein in the form of single or double stranded DNA or RNA. As shown in Fig. Typically, the aptamer may be a small nucleic acid of 15-50 bases in length that folds into defined secondary and tertiary structures, such as a stem-loop structure. Aptamers ^{10-6, 10-8, 10-10,} or ^10- and targets with less than ¹² kd is desirable to combine with the expressed protein or that express the protein. The aptamer can bind a highly expressed protein or a low expression protein with a very high specificity and the aptamer can be composed of a mixture of multiple ribonucleotide units, deoxyribonucleotide units, or two types of nucleotide residues. The aptamers of the present invention may further comprise one or more modified base, sugar or phosphate backbone units.

In another aspect, the present invention provides a kit for the diagnosis of pheochromocytoma comprising the composition for diagnosing pheochromocytoma. Preferably, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit. In addition, preferably, the pheochromocytomic diagnostic kit may further comprise one or more other component compositions, solutions or devices suitable for the assay method.

Preferably, the diagnostic kit comprises a diagnostic kit comprising essential elements necessary for performing a reverse transcription polymerase reaction. The RT-PCR kit contains the respective primer pairs specific for the marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each of the above genes, and has a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also contain a primer specific for the nucleic acid sequence of the control gene. Other reverse transcription polymerase reaction kits include enzymes such as test tubes or other appropriate containers, reaction buffer (pH and magnesium concentration), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC- Water (DEPC-water), sterile water, and the like.

Preferably a diagnostic kit comprising essential elements necessary for carrying out a DNA chip. The DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, preparations, enzymes, and the like for producing a fluorescent-labeled probe. The substrate may also comprise a cDNA or oligonucleotide corresponding to a control gene or fragment thereof.

Also preferably, it may be a diagnostic kit characterized by comprising essential elements necessary for performing ELISA. ELISA kits include antibodies, interacting proteins, ligands, nanoparticles or aptamers that specifically bind to the protein or peptide fragments. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for each marker protein and little cross reactivity to other proteins. The ELISA kit may also include antibodies specific for interacting proteins, ligands, nanoparticles, aptamers or control proteins that specifically bind to the protein or peptide fragment. Other ELISA kits can be used to detect antibodies that can bind a reagent capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e. G., Conjugated to an antibody) Other materials, and the like.

In another aspect, the present invention provides a method for providing information for diagnosis of pheochromocytoma using the composition for diagnosing pheochromocytoma or the kit for diagnosing pheochromocytoma.

Preferably, the information providing method comprises the steps of: measuring the mRNA expression level or protein expression level of the OPRM1 gene from the biological sample separated from the suspected patient of pheochromocytoma; And comparing the expression level of the mRNA or the expression level of the protein with a normal control sample.

The term "biological sample " as used herein includes, but is not limited to, tissues, cells, whole blood, serum, plasma, saliva, cerebrospinal fluid or urine.

The OPRM1 has a characteristic that the expression level is specifically increased in the pheochromocytoma compared with the normal control, and thus, when the level increases, the pheochromocytoma can be diagnosed and information can be provided.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  1: Experimental method

1-1. OPRM1 isoform  denomination

The OPRM1 isoforms are named by referring to the online database spliceMiner (http://projects.insilico.us/SpliceMiner/intro.jsp).

1-2. NGS (next generation sequencing) data analysis

Tumor type descriptions are shown in Table 1 below.

[Table 1]

Level 3-processed RNASeq TCGA data (version 2) was obtained from the TCGA Data Portal 45 (matrix) (http://tcga-data.nci.nih.gov/tcga/) for 31 cancers. RNASeq TCGA data (version 2) contains RNA-seq by Expectation-Maximization (RSEM) of the splice variant. This RNA-seq data was first applied to the statistical program R (http://www.r-project.org/) to group tumors with similar OPRM1 isoforms. Mann-Whitney U test was used to compare gene expression and tumor expression values from normal RNA-seq and determine their significance.

1-3. Cell culture

PC12 cells, the PCPG cell line, were cultured in RPMI 1640 medium containing 10% horse serum and 5% FBS in a 5% CO ₂ incubator maintained at 37 ° C.

1-4. RNA sequencing

Total RNA was isolated by Trizol (Invitrogen) and quantitatively analyzed by Nano-Drop 1000 spectrometer (Thermo Scientific) and electrophoresis (Bioanalyzer 2100; Agilent Technologies). The library was prepared using the TruSeq RNA library kit (Illumina) and sequenced with the HiSeq ^TM 2000 platform (Illumina).

Expression level assessments mapped RNA-Seq read to the human genome using TopHat (version 1.3.3). The reference genome sequence (hg19, Genome Reference Consortium GRCh37) was downloaded from the UCSC website (http://genome.uscs.edu). The number of transcripts was calculated at the gene level, and the relative transcript levels were measured by the FPKM (fragments per kilobase of transcript per million mapped reads) method using Cufflinks software (version 1.2.1).

Using this approach, expression levels were measured for 37,396 Ref-Seq genes arranged based on RNA sequence analyzes reads. Raw data were extracted with FPKM values for all samples and samples with zero values were excluded for more than 50% of the sequences. Statistical significance of changes in drainage was determined by paired t-test.

1-5. RT- PCR

RT-PCR was performed using a QIAGEN OneStep RT-PCR kit. The same amount (0.2 mg) of total RNA was used for each sample. The PCR conditions were: 30 minutes at 50 DEG C, 15 minutes at 95 DEG C; 1 minute at 94 ° C 33 cycles; 72 < 0 > C for 1 minute. The PCR product was electrophoresed on a 2% agarose gel and the sequencing of the PCR products was performed on a 3700 ABI Prism sequencer (Applied Biosystems).

Example  2: Intertumoral OPRM1  Expression analysis

To examine the expression pattern of OPRM1 in various cancers, the OPRM1 expression profile was analyzed for 31 tumor types in the TCGA RNA-seq database. Since OPRM1 pre-mRNA causes alternative splicing to generate multiple protein isoforms, data were analyzed from TCGA level 3 (version 2) to confirm this.

Specifically, OPRM1 was compiled to be compatible with the TCGA database and the normal OPRM1 nomenclature was matched with the OPRM1 nomenclature based on the NCBI and Swiss-Prot databases. For this purpose, the ORPM1 gene structure and isoform were compiled according to the UniProt (http://www.uniprot.org) guidelines using Genbank accession number NM_00914. At this time, P35372-1 (accession number NM_00914) is a reference splice variant (see FIG. 1).

Next, the expression of OPRM1 was analyzed by focusing on the read count frequency of each isoform rather than each read value, because OPRM1 is expressed at a low level in the TCGA database. Since the TCGA database (Version 2) represents RSEM as an expression level, the RSEM values of each isoform were plotted.

The percentage of samples expressing OPRM1 in each tumor was also calculated. As a result, BLCA (bladder cancer), HNSC (head and neck cancer), KIRC (kidney cancer), LUAD (lung cancer) and UCEC (uterine cancer) showed statistically significant OPRM1 overexpression compared to normal tissue (N <10) P < 0.001).

Conversely, COAD (colorectal cancer) and THCA (thyroid cancer) (normal tissue N> 10) showed a statistically significant decrease in OPRM1 expression, from 65% to 9.9% and 16% to 4.8%, respectively &Lt; 0.001).

In addition, PCPG (pheochromocytomas) showed statistically significant OPRM1 overexpression (P <0.001) as compared to normal tissue (N <10) (Cancer tissue).

[Table 2]

These results show that OPRM1 expresses a wide variety of expression patterns in a cancer-specific manner in a wide range of tumor types.

Example  3: OPRM1 isoform  Analysis of expression patterns

To further investigate which of the isoforms of OPRM1 is expressed in cancer, the expression spectrum of isoform was analyzed to confirm that various kinds of OPRM1 isoforms were expressed specifically in cancer.

Specifically, several cancers, including PCPG (pheochromocytoma), were markedly multisoluble. In particular, p35372-10, p35372-12 and p35372-13 isoforms were most expressed in non-neuroendocrine cancers such as HNSC (head and neck cancer) and LUAD (lung cancer).

On the other hand, p35372-1 isoform was most expressed in nerve-derived cancers such as PCPG (pheochromocytoma) (see Fig. 2)

Considering that p35372-10, p35372-12, and p35372-13 isoforms are produced in the exon 1 promoter while the remaining isoforms are produced in the exon 2 promoter, alternative splicing is cell or tissue specific.

To further confirm these results, RT-PCR was performed on PC12, a PCPG (pheochromocytoma) cell line, and the primer set used was as follows.

OPRM1 primer  set

OPRM1 Sense primer 1 (SP1): 5'-TATGTGATTGTAAGATACACCAAAATG-3 '(SEQ ID NO: 2)

OPRM1 Anti-sense primer 1 (AP1): 5'-CAATCTATGGACCCCTGCCTGTA-3 '(SEQ ID NO: 3)

OPRM1 sense primer 2 (SP2): 5'-GGATGGGATCTGGTCCAATGCTATACACCAAAATG-3 '(SEQ ID NO: 4)

OPRM1 Anti-sense primer 1 (AP1): 5'-CAATCTATGGACCCCTGCCTGTA-3 '(SEQ ID NO: 3)

At this time, SP1 anneals exon1 and exon2 of NM_013071.2 reference mu opioid receptor cDNA sequence, SP2 anneals exon 11a and exon2 of NM_013071.2 reference mu opioid receptor cDNA sequence, and AP1 anneals to NM_013071.2 reference mu opioid receptor cDNA Anneals exon2 and exon3 of the sequence.

The control (? Actin ) primer  set

β-actin sense primer: 5'-CTGGGAGGCAGTGACCCTCAACATTACCAG-3 '(SEQ ID NO: 5)

β-actin Anti-sense primer: 5'-GAGAGCCAGTATGCACAGGTCATCGTTCCT-3 '(SEQ ID NO: 6)

As shown in FIG. 3, after the RT-PCR using the two different 5 'primers and the 3' primer in the cavity, the PC12 cells from the bands amplified by each primer set were at least Two OPRM1 isoforms were expressed.

These results strongly suggest that the actual pheochromocytoma cell line (PC12), as well as the results of the TCGA RNA-seq database, has multiple OPRM1 isoforms.

<110> Research and Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Composition for Diagnosing of Pheochromocytoma <130> MP15-031 <160> 6 <170> KoPatentin 3.0 <210> 1 <211> 1203 <212> DNA <213> Homo sapiens <220> <221> gene &Lt; 222 > (1) <223> Oprm1 P35372-1 cDNA <400> 1 atggacagca gcgctgcccc cacgaacgcc agcaattgca ctgatgcctt ggcgtactca 60 agttgctccc cagcacccag ccccggttcc tgggtcaact tgtcccactt agatggcaac 120 ctgtccgacc catgcggtcc gaaccgcacc gacctgggcg ggagagacag cctgtgccct 180 ccgaccggca gtccctccat gatcacggcc atcacgatca tggccctcta ctccatcgtg 240 tgcgtggtgg ggctcttcgg aaacttcctg gtcatgtatg tgattgtcag atacaccaag 300 atgaagactg ccaccaacat ctacattttc aaccttgctc tggcagatgc cttagccacc 360 agtaccctgc ccttccagag tgtgaattac ctaatgggaa catggccatt tggaaccatc 420 ctttgcaaga tagtgatctc catagattac tataacatgt tcaccagcat attcaccctc 480 tgcaccatga gtgttgatcg atacattgca gtctgccacc ctgtcaaggc cttagatttc 540 cgtactcccc gaaatgccaa aattatcaat gtctgcaact ggatcctctc ttcagccatt 600 ggtcttcctg taatgttcat ggctacaaca aaatacaggc aaggttccat agattgtaca 660 ctaacattct ctcatccaac ctggtactgg gaaaacctgc tgaagatctg tgttttcatc 720 ttcgccttca ttatgccagt gctcatcatt accgtgtgct atggactgat gatcttgcgc 780 ctcaagagtg tccgcatgct ctctggctcc aaagaaaagg acaggaatct tcgaaggatc 840 accaggatgg tgctggtggt ggtggctgtg ttcatcgtct gctggactcc cattcacatt 900 tacgtcatca ttaaagcctt ggttacaatc ccagaaacta cgttccagac tgtttcttgg 960 cacttctgca ttgctctagg ttacacaaac agctgcctca acccagtcct ttatgcattt 1020 ctggatgaaa acttcaaacg atgcttcaga gagttctgta tcccaacctc ttccaacatt 1080 gagcaacaaa actccactcg aattcgtcag aacactagag accacccctc cacggccaat 1140 acagtggata gaactaatca tcagctagaa aatctggaag cagaaactgc tccgttgccc 1200 taa 1203 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> OPRM1 Sense primer 1 (SP1) <400> 2 tatgtgattg taagatacac caaaatg 27 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OPRM1 Anti-sense primer 1 (AP1) <400> 3 caatctatgg acccctgcct gta 23 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> OPRM1 Sense primer 2 (SP2) <400> 4 ggatgggatc tggtccaatg ctatacacca aaatg 35 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> b-actin Sense primer <400> 5 ctgggaggca gtgaccctca acattaccag 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> b-actin Anti-sense primer <400> 6 gagagccagt atgcacaggt catcgttcct 30

Claims (8)

A composition for diagnosing pheochromocytoma, comprising an agent for measuring the mRNA of OPRM1 (mu-opioid receptor) gene or a protein level thereof. 2. The composition of claim 1, wherein the OPRM1 gene comprises the nucleotide sequence of SEQ ID NO: 1. The composition according to claim 1, wherein the agent for measuring the mRNA level of the gene is a primer pair, a probe, or an antisense nucleotide that specifically binds to the gene. The composition of claim 1, wherein the agent that measures the protein level is an antibody or an aptamer that specifically binds to the protein. A kit for the diagnosis of pheochromocytoma, comprising the composition according to claim 1. Measuring the mRNA expression level or protein expression level of the OPRMl (mu-opioid receptor) gene from the biological sample; And comparing the expression level of the mRNA or the expression level of the protein with a control sample. &Lt; Desc / Clms Page number 19 &gt; The method according to claim 6, wherein the mRNA expression level is measured using a reverse transcriptase polymerase, a competitive reverse transcriptase polymerase, a real-time reverse transcriptase polymerase, an RNase protection assay, or Northern blotting. Delivery method. The method according to claim 6, wherein the protein expression level is measured using a protein chip, immunoassay, Western blot, or enzyme linked immunosorbent assay (ELISA).

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