KR101141190B1 - Biomarker indicative of prostate cancer and diagnosis using the same - Google Patents

Abstract

The present invention relates to a kit for diagnosing or prognostic prostate cancer comprising an antibody or aptamer specifically binding to a resistin protein, a nucleotide sequence encoding a resistin protein, a sequence complementary to the nucleotide sequence, or a fragment of the nucleotide. . The present invention is a marker with greatly improved accuracy and reliability as a marker for prostate cancer. In addition, the present invention can be used to determine the early diagnosis and prognosis of prostate cancer by using a resistin whose expression is specifically increased only in the tissue of a prostate cancer patient.

Resistin, Prostate, Cancer, Diagnosis, Marker

Description

Biomarker for Prostate Cancer and Diagnosis of Prostate Cancer Using the Same {Biomarker Indicative of Prostate Cancer and Diagnosis Using the Same}

The present invention relates to biomarkers specific for prostate cancer and their use.

Prostate cancer has recently gained much attention, especially in an aging society. The incidence and disease-specific mortality rates of prostate cancer are highest in North America and Europe. ^{1 The} incidence of prostate cancer among Asian immigrants in the United States is higher than that of mainland Asians, and the incidence of prostate cancer has increased dramatically in Asian countries over the past 20 years. ² These reports suggest that Western lifestyle and lack of exercise associated with high fat intake are important factors in the development of prostate cancer. Several studies have also reported that dietary and fat intake affect the incidence of prostate cancer, and ³ correlates with the body mass index (BMI). ⁴

Obesity, especially abdominal obesity patients have increased insulin, arguably increased insulin is known to cause insulin resistance and hyperinsulinemia, thereby increasing the incidence and progression of prostate cancer. ⁵ Adipocytes associated with obesity have been previously thought to play a passive role as a reservoir of excess energy in the body, but recent studies have shown that fat cells have a tumor necrosis factor-α (TNF-α). ), Leptin, interleukin-6 (IL-6), plasminogen activator inhibitor-1, adiponectin, resistin, and the like. It has been shown to act as an endocrine organ that produces and secretes biologically active cytokines called adipokines. ⁶

Resistin, named in the sense of resistance to insulin, has a unique and varied action, and blood resist levels are reduced by the diabetic rosiglitazone, which is increased by dietary and obesity genes, which leads to obesity. Have them speculate that this hormone is a link between insulin and insulin resistance. ⁷ Holcomb ⁸ et al. Described for the first time the unique distribution in the registrar gene family and tissues, which are found in various parts of the human body and are known to be closely related to the development of obesity, diabetes and hypertension. In addition, a study has been reported that resistin directly affects cell proliferation in cultured vascular endothelial cells. ^{9 It has} already been reported that resistin is involved in the development and progression of choriocaricnoma and breast cancer. ^10,11

Insulin resistance is associated with the expression and progression of prostate cancer, and adipocaine targets cancer progenitor cells, activating signal transduction cascades, leading to abnormal cell proliferation and transformation into malignant cells. Based on this fact, it can be hypothesized that increased expression of resistin in prostate epithelial cells is involved in the correlation between prostate cancer and insulin resistance. ^{12 In} this study, we investigated the relationship between the expression of resistin in prostate epithelial cells and the differentiation and progression of prostate cancer.

Currently, a method for early diagnosis of endometrial cancer using a multimarker containing resistin (US Patent Publication No. 20080090258) and a resistin protein as a diagnostic marker for cardiovascular disease (US Patent Publication No. 20050244892) Patent literature for is reported.

However, no literature has been reported that increases the expression of resistin in prostate cancer tissues and cells and that it can be used as a diagnostic marker for prostate cancer.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have made diligent research efforts to discover novel biomarkers that can rapidly and accurately molecularly diagnose prostate cancer. As a result, the present invention was completed by identifying that resistin is a marker capable of early diagnosis of prostate cancer and a prognosis.

Accordingly, it is an object of the present invention to provide a kit for diagnosing or prognosticting prostate cancer.

Another object of the present invention is to provide a method for detecting prostate cancer markers in order to provide information necessary for the diagnosis or prognosis of prostate cancer.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention provides a prostate cancer diagnosis comprising an antibody or aptamer specifically binding to a resistin protein, a nucleotide sequence encoding a resistin protein, a sequence complementary to the nucleotide sequence, or a fragment of the nucleotide. Or a kit for prognostic analysis.

According to another aspect of the present invention, the present invention provides a method for detecting prostate cancer through a method of detecting the expression of the nucleotide sequence of the resistin in a biological sample of human in order to provide necessary information for prostate cancer diagnosis or prognosis. do.

The present inventors have made diligent efforts to discover novel molecular markers that can rapidly and accurately molecularly diagnose prostate cancer. As a result, it was found that the above-described molecular markers can be used to diagnose prostate cancer early and determine the prognosis. In particular, the marker of the present invention is a marker with greatly improved accuracy and reliability as a marker for prostate cancer.

The molecular marker of the present invention may be an indicator for the development and development of prostate cancer, and may be used for the diagnosis of the development and development of prostate cancer.

As used herein, the term “diagnosis” refers to determining the susceptibility of an object to a particular disease or condition, determining whether an object currently has a particular disease or condition, or having a particular disease or condition. Determining the prognosis of a subject (eg, identifying a metastatic or metastatic cancer state, determining the stage of the cancer or responsiveness of the cancer to treatment), or therametrics (eg, treating efficacy Monitoring the status of an object to provide information about it).

Resistin proteins specifically express diffuse positivity in cases where prostate cancer cells or tissues differentiate and in cells or tissues of distant metastatic prostate cancer (see Examples of the Invention). As used herein, the expression "subtractive expression" refers to a pattern in which a resistin protein is uniformly and widely expressed in a raw sample (for example, cancer tissue or cancer cells), and more specifically, immune tissue using a resistin-specific antibody. When staining prostate tissue by immunohistochemical staining means that the cells to be stained is more than 70% of the total cells. Thus, the kit of the present invention can not only diagnose prostate cancer, but also can very specifically diagnose prostate cancer or distant metastatic prostate cancer that differentiates. Judging from the diffuse expression of resistin, differentiated prostate cancer or distant metastatic prostate cancer is 1.8-3 times higher than that of other states (e.g., organ-limited prostate cancer and locally advanced prostate cancer). do.

According to an embodiment of the present invention, it can be seen that resistin specifically expresses prostate cancer epithelial cells more than normal prostate epithelial cells or prostatic hypertrophy epithelial cells. More specifically, 5-10-fold, more preferably 7-10-fold and most preferably 9-10-fold resistin high expression in prostate cancer epithelial cells than prostatic hypertrophic epithelial cells based on the diffuse expression pattern of resistin. (See Example Table 1). It is common for prostate hyperplasia and prostate cancer to be indistinguishable using a biomarker. If the resist of the present invention is used, prostate cancer can be diagnosed by clearly distinguishing prostate hyperplasia and prostate cancer.

According to a preferred embodiment of the present invention, the kit of the present invention can selectively diagnose only a prostate cancer sample among prostatic hyperplasia samples and prostate cancer samples. Preferably, the sample is human tissue, cells, blood, serum or plasma.

According to a preferred embodiment of the invention, the invention can be carried out in an immunoassay mode, ie, antigen-antibody response mode. In this case, the antibody or aptamer specifically binds to the prostate cancer marker of the present invention described above.

The antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Antibodies may be commonly used in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) Or phage antibody library methods (Clackson et al, Nature , 352: 624-628 (1991) and Marks et al, J. Mol. Biol. , 222: 58, 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art. Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

When the method of the present invention is carried out using antibodies or aptamers, the present invention can be used to diagnose prostate cancer by performing according to conventional immunoassay methods.

Such immunoassays can be performed according to various quantitative or qualitative immunoassay protocols developed in the prior art. The immunoassay format includes radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or hardwood analysis, sandwich analysis, flow cytometry, and immunoassay. Including but not limited to fluorescent staining and immunoaffinity purification. The immunoassay or method of immunostaining is described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, if the method of the present invention is carried out according to the method radioactive immunoassay, radioactive isotope is an antibody labeled (e.g., C _14, I _125, P ₃₂ and S ₃₅₎ of detecting the marker molecules of the present invention .

When the method of the invention is carried out in an ELISA manner, certain embodiments of the invention comprise the steps of: (i) coating an unknown cell sample lysate to be analyzed on the surface of a solid substrate; (Ii) reacting said cell lysate with an antibody against a marker as a primary antibody; (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iii) measuring the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates.

The enzyme bound to the secondary antibody may include an enzyme catalyzing a chromogenic reaction, a fluorescence reaction, a luminescent reaction, or an infrared reaction, but is not limited thereto. For example, an alkaline phosphatase,? -Galactosidase, Radish peroxidase, luciferase, and cytochrome P450. When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as a substrate Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis) if colorimetric substrates such as -B1-phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase is used -N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS substrates such as phenzaine methosulfate can be used. The.

When the method of the invention is carried out in a capture-ELISA mode, certain embodiments of the invention comprise (i) coating an antibody against a marker of the invention as a capturing antibody on the surface of a solid substrate; (Ii) reacting the capture antibody with the sample; (Iii) reacting the product of step (ii) with a detecting antibody that has a label that generates a signal and that specifically reacts with a resistin protein; And (iv) measuring a signal originating from said label.

The detection antibody carries a label which generates a detectable signal. The labels may include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P450), radioactive substances (eg C14, I125, P32 and S35). ), Fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent and fluorescence resonance energy transfer (FRET), including but not limited to various labels and labeling methods Ed Harlow and David Lane , Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

Measurement of the final enzyme activity or signal in the ELISA method and the capture-ELISA method can be carried out according to various methods known in the art. Detection of these signals allows for qualitative or quantitative analysis of the markers of the invention. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used.

According to another variant of the present invention, an aptamer that specifically binds to the marker of the present invention may be used instead of the antibody. Aptamers are oligonucleic acid or peptide molecules, the general contents of which are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA . 95 (24): 142727 (1998).

By analyzing the final signal intensity by the above-described immunoassay process, prostate cancer can be diagnosed. That is, if the signal for the marker of the present invention in the sample is stronger than the normal sample, it is diagnosed as prostate cancer.

The kit of the present invention may further include other components in addition to the above components. For example, when the kit of the present invention is subjected to a PCR amplification process, the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus). (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs. Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above.

According to a preferred embodiment of the invention, the kit of the invention may be a microarray.

According to a preferred embodiment of the present invention, the kit of the present invention is a gene amplification kit.

When the kit of the present invention is a microarray, the probe is immobilized on the solid surface of the microarray. When the kit of the present invention is a gene amplification kit, it includes a primer.

The probe or primer used in the diagnostic kit of the present invention has a sequence complementary to the resistin nucleotide sequence. As used herein, the term “complementary” means having complementarity enough to selectively hybridize to the above-described nucleotide sequence under certain specific hybridization or annealing conditions. Thus, the term “complementary” has a different meaning from the term perfectly complementary, and the primers or probes of the present invention may be capable of selectively hybridizing to the above-described nucleotide sequence so long as one or more mismatches ( mismatch) may have a nucleotide sequence.

As used herein, the term “primer” refers to a single that can serve as a starting point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in a suitable buffer at a suitable temperature. -Refers to stranded oligonucleotides. The suitable length of the primer is typically 15-30 nucleotides, although it varies with various factors such as temperature and use of the primer. Short primer molecules generally require lower temperatures to form hybrid complexes that are sufficiently stable with the template.

The sequence of the primer does not need to have a sequence that is completely complementary to some sequences of the template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with the template to perform the primer-specific function. Therefore, the primer in the present invention does not need to have a sequence that is perfectly complementary to the above-described nucleotide sequence as a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing to the gene sequence and acting as a primer. The design of such primers can be easily carried out by those skilled in the art with reference to the above-described nucleotide sequence, for example, by using a program for primer design (eg, PRIMER 3 program).

As used herein, the term “probe” refers to a linear oligomer of natural or modified monomers or linkages, includes deoxyribonucleotides and ribonucleotides, and can specifically hybridize to a target nucleotide sequence, naturally Present or artificially synthesized. The probe of the present invention is preferably a single strand, and is an oligodioxyribonucleotide.

The nucleotide sequence of the marker of the present invention, which should be referred to when constructing a primer or probe, can be identified in GenBank using the accession number of the above-described resistin, and the primer or probe can be designed with reference to this sequence.

In the microarray of the present invention, the probe is used as a hybridizable array element and is immobilized on a substrate. Preferred gases include, for example, membranes, filters, chips, slides, wafers, fibers, magnetic beads or non-magnetic beads, gels, tubing, plates, polymers, microparticles and capillaries, as suitable rigid or semi-rigid supports. Said hybridization array element is arranged and immobilized on said gas phase. This immobilization is carried out by chemical bonding methods or by covalent bonding methods such as UV. For example, the hybridization array element can be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and can also be bonded by UV at the polylysine coating surface. In addition, the hybridization array element may be coupled to the gas through a linker (e.g., ethylene glycol oligomer and diamine).

On the other hand, the sample DNA applied to the microarray of the present invention can be labeled and hybridized with array elements on the microarray. Hybridization conditions can vary. The detection and analysis of the hybridization degree can be variously carried out according to the labeling substance.

The prostate cancer diagnostic kit of the present invention can be carried out based on hybridization. In this case, a probe having a sequence complementary to the nucleotide sequence of the marker of the present invention described above is used.

Prostate cancer may be determined by hybridization-based analysis using a probe hybridized to the nucleotide sequence of the marker of the present invention described above.

The label of the probe can provide a signal that allows detection of hybridization, which can be linked to oligonucleotides. Suitable labels include fluorophores (e.g. fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia), chromophores, chemilumines, magnetic particles, radioisotopes Elements (P ³² and S ³⁵ ), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, substrates for enzymes, heavy metals (eg gold) and antibodies, streptabi Hapten with specific binding partners, such as, but not limited to, dine, biotin, digoxigenin and chelating groups. Labeling is carried out in a variety of ways conventionally practiced in the art, such as nick translation methods, random priming methods (Multiprime DNA labeling systems booklet, "Amersham" (1989)) and chination methods (Maxam & Gilbert, Methods). in Enzymology , 65: 499 (1986)). The label provides signals that can be detected by fluorescence, radioactivity, colorimetry, weighing, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, and nanocrystals.

The nucleic acid sample to be analyzed can be prepared using mRNA obtained from various biosamples. The raw sample is preferably a prostate tissue cell. The hybridization reaction-based assay may be performed by labeling the cDNA to be analyzed instead of the probe.

If a probe is used, the probe is hybridized with the cDNA molecule. In the present invention, suitable hybridization conditions can be determined in a series of procedures by an optimization procedure. This procedure is carried out by a person skilled in the art in order to establish a protocol for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and wash times, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. Detailed conditions for hybridization are described by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc .; NY (1999). For example, high stringency conditions were hybridized at 65 ° C in 0.5 M NaHPO ₄ , 7% SDS (sodium dodecyl sulfate) and 1 mM EDTA, followed by addition of 0.1 x SSC / 0.1% SDS Lt; RTI ID = 0.0 > 68 C < / RTI > Alternatively, high stringency conditions means washing at < RTI ID = 0.0 > 48 C < / RTI > in 6 x SSC / 0.05% sodium pyrophosphate. Low stringency conditions mean, for example, washing in 0.2 x SSC / 0.1% SDS at 42 ° C.

After the hybridization reaction, the hybridization signal coming out of the hybridization reaction is detected. The hybridization signal can be performed by various methods, for example, depending on the type of label bound to the probe. For example, if the probe is labeled by an enzyme, the substrate of the enzyme can be reacted with the hybridization product to confirm hybridization. Combinations of enzymes / substrates that can be used include peroxidase (eg horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium). Nitrate), resorphin benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine (TMB), ABTS (2 , 2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine; Alkaline phosphatase with bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF substrates; Glucose oxidase, t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate). When the probe is labeled with gold particles, it can be detected by silver dyeing using silver nitrate. Therefore, when the method for detecting the prostate cancer marker of the present invention is performed based on hybridization, specifically, (i) hybridizing a probe having a sequence complementary to the nucleotide sequence of the marker of the present invention to a nucleic acid sample; (ii) detecting whether the hybridization reaction occurs. By analyzing the strength of the hybridization signal by the hybridization process, it is possible to determine whether prostate cancer. That is, if the hybridization signal for the nucleotide sequence of the marker of the present invention in the sample is stronger than the normal sample (for example, normal prostate tissue cells), it is diagnosed as prostate cancer.

According to a preferred embodiment of the present invention, the human prostate cancer diagnostic kit of the present invention may be a gene amplification kit.

As used herein, the term "amplification" refers to a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the art, which include polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822), ligase chain reaction (LCR) ( 17, 18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA, WO 88/10315), self-maintaining sequence replication (self sustained sequence replication, WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction ( CP-PCR), US Pat. No. 4,437,975), optional print Arbitrarily primed polymerase chain reaction (AP-PCR), US Pat. Nos. 5,413,909 and 5,861,245, Nucleic acid sequence based amplification (NASBA), US Pat. No. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification (21, 22) and loop-mediated isothermal amplification; LAMP) 23, but is not limited thereto. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.

PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (inverse polymerase) chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information on PCR see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.

When the diagnostic kit of the present invention is carried out using a primer, a gene amplification reaction is performed to examine the expression level of the nucleotide sequence of the marker of the present invention. Since the present invention analyzes the expression level of the nucleotide sequence of the marker of the present invention, the mRNA amount of the nucleotide sequence of the marker of the present invention is examined in a sample (for example, prostate tissue, blood or urine) of the analysis target. The degree of expression of the nucleotide sequence of the marker is determined.

Therefore, in principle, the present invention uses a mRNA in a sample as a template and performs a gene amplification reaction using a primer that binds to mRNA or cDNA.

To obtain mRNA, total RNA is isolated from the sample. Isolation of total RNA can be carried out according to conventional methods known in the art. See Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Tesniere. , C. et al., Plant Mol. Biol. Rep. , 9: 242 (1991); Ausubel, FM et al., Current Protocols in Molecular Biology , John Willey & Sons (1987); and Chomczynski, P. et al , Anal.Biochem. 162: 156 (1987)). For example, TRIzol can be used to easily isolate total RNA in a cell. Subsequently, cDNA is synthesized from the isolated mRNA, and this cDNA is amplified. Since the total RNA of the present invention is isolated from human samples, the end of the mRNA has a poly-A tail, and cDNA can be easily synthesized using oligo dT primers and reverse transcriptases using these sequence characteristics. PNAS USA, 85: 8998 (1988); Libert F, et al., Science , 244: 569 (1989); and Sambrook, J. et al., Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001). Next, the synthesized cDNA is amplified through gene amplification reaction.

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Suitable nucleic acid hybridization conditions for forming such a double-chain structure include Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach, IRL Press, Washington, DC (1985).

Various DNA polymerases can be used for amplification of the present invention and include “Clenow” fragments of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from various bacterial species, which include Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). Include.

When carrying out the polymerization reaction, it is preferable to provide an excess amount of the components required for the reaction to the reaction vessel. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. It is desired to provide cofactors such as Mg ²⁺ , dATP, dCTP, dGTP and dTTP to the reaction mixture such that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer ensures that all enzymes are close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.

Annealing in the present invention is carried out under stringent conditions allowing specific binding between the target nucleotide sequence and the primer. Stringent conditions for annealing are sequence-dependent and vary depending on the surrounding environmental variables.

The cDNA of the nucleotide sequence of the marker of the present invention thus amplified is analyzed by a suitable method to investigate the expression level of the nucleotide sequence of the marker of the present invention. For example, the degree of expression of the nucleotide sequence of the marker of the present invention is examined by gel electrophoresis of the amplification reaction product described above, and by observing and analyzing the resulting band. Through this amplification reaction, prostate cancer is diagnosed when the expression of the nucleotide sequence of the marker of the present invention in a raw sample is higher than that of a normal sample (normal cell).

Therefore, when the prostate cancer marker detection method of the present invention is performed based on an amplification reaction using cDNA, specifically (i) performing an amplification reaction using a primer annealed to the nucleotide sequence of the marker of the present invention; And (ii) analyzing the product of the amplification reaction to determine the expression level of the nucleotide sequence of the marker of the present invention.

The marker of the present invention is a biomolecule that is highly expressed in prostate cancer. High expression of such markers can be measured at the mRNA or protein level. The term "high expression" as used herein refers to a case where the expression level of the nucleotide sequence of interest in the sample to be investigated is high compared to the normal sample. For example, expression assay methods commonly used in the art, such as RT-PCR methods or ELISA methods (Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)) In the case of expression analysis according to, it means a case where the expression is analyzed to be many. For example, as a result of analysis according to the above-described analysis method, when the marker of the present invention is expressed 2-10 times higher than normal cells, it is determined as "high expression" in the present invention and is determined as prostate cancer. .

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides novel molecular markers for prostate cancer.

(Ii) Resistin in the present invention is a marker with greatly improved accuracy and reliability as a marker for prostate cancer.

(Iii) In addition, the present invention can be used to determine early diagnosis and prognosis of infectious cancer using resistin whose expression is specifically increased only in the tissue of a prostate cancer patient.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Unless stated otherwise, solids / solids are (weight / weight) parts or%, solids / liquids are (weight / volume) parts or%, and liquids / liquids are (volume / volume) parts or%.

Materials and Methods

1. Selection of subjects and tissues

From January to September 2007, 26 patients (mean age 68.7 ± 8.3 years) diagnosed with prostate hyperplasia were diagnosed as prostate hyperplasia and 67 patients (mean age 68.1 ± 8.1 years old) diagnosed with prostate cancer. Cancer was included. Patients with prostatic hyperplasia have a lower urinary tract symptom, have a prostate-specific antigen (PSA) level of normal (<4 ng / ml) and have no abnormalities on rectal balance examination. Tissues were obtained by prostate cancer, and prostate cancer patients obtained by ultrasound-guided prostate biopsy.

2. Classification of test items and experimental groups

All clinical trial participants had written informed consent and blood sampling (PSA, fasting blood glucose, total cholesterol) and anthropometric measurements (height, weight) were performed. Body mass index was calculated by dividing the weight (kg) by the square of the height (㎡). The prostate cancer group combines PSA levels at diagnosis, pathological grade of radical prostatectomy or prostate biopsy tissue, computed tomography (CT) or magnetic resonance imaging (MRI) findings, and bone scan findings. Classified according to TNM system. The prostate cancer group was classified into three groups according to the degree of progression: organ-confined (n = 37), locally advanced (n = 13), and metastatic disease (n = 17), and Gleason (Gleason). ) Group 3 with low grade (low grade, n = 23), 7 grades with intermediate grade (n = 24), and more than 7 grades with high grade (high grade, n = 20). Classified as

3. Immunohistochemical Staining of Resistin Protein

All tissues were fixed in 10% formalin buffer and paraffin embedded. The sections having a thickness of 5 μm were sliced and deparaffinized, and then washed with distilled water for 5 minutes in anhydrous alcohol, 90%, 75%, and 50% alcohol in order. H & E staining and immunohistochemical staining were performed using ABC (the avidin-biotin-streptavidin complex) technique (Biogenex kit). Resistin was diluted with primary monoclonal antibody (R & D system, Cat. No: MAB1359, clone: 184335, Minneapolis, USA) at 1:70 and treated at room temperature for one day. Microwave irradiation and EDTA-NaOH (TiTriplex, Merck) buffer at pH 8.0 ¹⁴ were used for antigenic recovery. Negative controls were treated with non-immune mouse serum without treatment with primary antibody.

4. Evaluation of immunohistochemical staining

The determination of staining for resistin was evaluated semi-quantitatively on a four-point scale, depending on the degree of red-brown staining of the cytoplasm under a microscope. Negative cells were stained with no staining, less than 30% for 1+ (focal positivity), 30-70% for 2+ (multifocalpositivity), and 70% or more for 3+ (less than diffuse positivity). ) And pathological readings were performed in the same laboratory (FIG. 1).

5. Result Analysis

The expression levels of resistin were compared according to the enlargement of the prostate gland, the prostate cancer group, the progression of the prostate cancer, and the Gleason score. The SPSS software package version 11.0 (Statistical Package for Social SciencesTM, Chicago, USA) was used to analyze using the Mann-Whitney U test, chi-square test and Kruskal-Wallis test. It makes sense.

Experiment result

1. Characteristics of Target Group

Serum PSA was higher in the prostate cancer group (p <0.05), and there was no difference in age, body mass index, fasting blood glucose and total cholesterol levels between the two groups (Table 1). In the prostate cancer group, the age and serum PSA of the prostate cancer group significantly increased in proportion to the progression (p <0.01), but there was no difference in body mass index, fasting blood glucose and total cholesterol (Table 2). ). As the Gleason score increased, the patient's age increased as the Gleason score increased (p <0.05), but there was no difference in plasma PSA, body mass index, fasting glucose and total cholesterol (Table 3).

2. Comparison of Resistin Expression in Prostate Hyperplasia and Prostate Cancer

Diffuse (3+) expression was found in one case (3.8%) in the prostate hyperplasia group, 25 cases (37.3%) in the prostate cancer group, and the total resistivity was significantly higher in the prostate cancer group than in the prostate hypertrophy group. (P <0.001) (Table 1).

Body mass index (BMI): body mass index, prostate-specific antigen (PSA): prostate-specific antigen, benign prostatic hyperplasia (BPH): prostatic hyperplasia, prostate cancer (PCa): prostate cancer, ^a : Mann-Whitney U test, ^b : chi-square test

3. Comparison of the expression level of resistin according to the progression of prostate cancer

Diffuse expression was classified in 10 cases (27.0%) in the localized group, 4 cases (30.8%) in the local invasion group, and 11 cases (64.7%) in the distant metastasis group. As a result, the resistin showed statistically significant expression (p <0.05) (Table 2).

BMI (body mass index): body mass index, PSA (prostate-specific antigen): prostate-specific antigen, PCa (prostate cancer): prostate cancer, ^a : Kruskal-Wallis test, ^b : chi-square test

4. Comparison of Resistin Expression According to Gleason Score

In the classification according to Gleason score of prostate cancer, diffuse expression was found in 4 cases (17.4%) at low grade, 7 cases (29.2%) at middle grade, and 14 cases (70.0%) at high grade. High hand scores showed statistically significant expression (p <0.05) (Table 3 and FIG. 1).

GS (Gleason score): Gleason score, BMI (body mass index): body mass index, PSA (prostate-specific antigen): prostate-specific antigen, PCa (prostate cancer): prostate cancer, ^a : Kruskal-Wallis test, ^b : chi-square test

Review

Obesity is increasing rapidly over the past decade as one of the major health problems in 30% of the American people is has been reported in obese patients, our country is no exception in experiencing a rapid change of the change of the ¹³ diet and social environment not come Bureau of Statistics data in 2001 According to the report, 30.6% of the total population reported obesity, including overweight. ¹⁴ Obesity is associated with the occurrence of various chronic diseases such as coronary artery disease, hypertension, diabetes, and is known to be associated with several cancers, including breast and ¹⁵ colorectal cancer. ^10,11 Overweight and obesity increase mortality associated with various malignancies, including prostate cancer. ⁴ Epidemiologic studies have argued that an increase in body mass index is associated with prostate cancer progression. According to a recent study, the incidence of prostate cancer increased by 9% in patients with a body mass index of 30 kg / m² or higher, but there was no statistical significance. There was a significant difference. ⁵ This suggests that obesity may be associated with prostate cancer rather than prostate cancer. ¹⁶ In addition, the increase in body mass index underwent radical prostatectomy patients rating or is associated with an increase in weapons, it has been reported to be associated with higher biochemical recurrence after surgery, significant mortality by ¹⁷ Prostate Cancer Increase. ⁴ Some reasons for this high relapse rate are suggested. As the BMI increases are also a major cause of increasing differentiation of prostate cancer, obesity caused by bleeding in the ^18,19 and ¹⁸ high difficulty of the surgical technique yangseongil possibility of surgical margins, designed to radiation in the area of radiation therapy ^19,20 Because it is difficult to do so, the effect of treatment may be impaired. In addition, obesity may be delayed, so it may be difficult to expect good results at the time of diagnosis. In addition, as the BMI increases, the prostate cancer mortality rate increases, and cancer prevention studies (CPS) I and II have reported an increased risk of dying from prostate cancer in obesity patients by 21-27% or more. Obesity has been reported to increase the risk of death from prostate cancer by 34% or more than normal individuals. ²¹ Resistin is the most recently described adipocyte derived peptide and was initially suggested to have a role in causing insulin resistance and obesity. Resistin has been reported to antagonize insulin action in both in vivo and ex vivo cells, receptors have not yet been identified and isolated and little signal transduction is known. It has been reported that blood resistin concentrations are increased in obese and diabetic patients, with an average blood serum resistin concentration of 40 ng / ml in diabetic patients (15 ng / ml in dry and non-diabetic patients). ²² Shalev et al. ²³ reported a significant increase in blood mean mean resistin concentration of 30.7 ng / ml in healthy controls and 49.7 ng / ml in diabetic patients.

In this study, the expression of resistin in prostate cancer tissue was higher than that of prostatic hypertrophy tissue, which may explain the hypothesis that resistin may be involved in the development of prostate cancer. For the other adipic myrtle of leptin There are reports this fact, Stattin and ²⁴ is related to the development and progression of the prostate cancer, leptin, and was measured the expression of the receptor, all in serum leptin levels and prostate cancer tissue In our previous studies, leptin was significantly higher in prostate cancer patients than in prostatic hypertrophy tissues. ²⁵ The present study also showed that there was a significant positive correlation between cell differentiation, prostate cancer stage, and resistin expression. This implies that increased expression of resistin in tissues may eventually affect the degree of differentiation and progression of prostate cancer, suggesting that resistin may play a bridge role in the relationship between obesity and prostate cancer.

A recent study reported the correlation between plasma resistin concentration and tumors associated with obesity and insulin resistance. Kang et al ¹¹ reported that plasma levels rejiseutin the higher the higher the incidence of breast cancer, plasma rejiseutin value is not less than the average value, increase the chance of the pathologic grade of a tumor appears highest. These results suggest that high plasma resistin levels are another adipocytokine that increases the risk of developing tumors associated with insulin resistance. In the present study, the expression of resistin increases with the progression of prostate cancer, suggesting that it can induce the differentiation and progression of prostate cancer, but the mechanism is still unknown. Calabro et al. ⁹ found that resistin induces cell proliferation through two pathways in human aortic vascular smooth muscle experiments. Resistin demonstrated cell proliferation through activation of P42 / p44 mitogen activated protein kinase (MAPK) and activation of Akt serine / threonine kinase (PI3K) by phosphatidylinositol 3-kinase (PI3K). These pathways may suggest the potential for involvement in the development of prostate cancer.

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Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Figure 1 is an image showing the immunohistochemical staining of the resistin (resistin) protein. Panel (A) is an enlarged prostate epithelial cell, panel (B) is a low grade prostatic epithelial cell, panel (C) is an intermediate prostate epithelial cell, and panel (D) is a prostate cancer prostate epithelium. Immunohistochemical staining for resistin protein in cells is the result image (anti-resistin antibody, x 100 magnified image). The intensity of prostate epithelial cell resistin expression was significantly higher in the prostate cancer group than in the prostatic hypertrophy group, and was significantly increased in the high grade.

Claims (14)

A kit for diagnosing or prognostic prostate cancer comprising an antibody that specifically binds a resistin protein, a nucleotide sequence encoding a resistin protein, a sequence complementary to the nucleotide sequence, or a fragment of the nucleotide. The kit of claim 1, wherein the prostate cancer is differentiated prostate cancer or distant metastatic prostate cancer. The kit according to claim 1, wherein the kit is a kit capable of selectively diagnosing only prostate cancer among prostatic hyperplasia and prostate cancer. delete The kit for diagnosing or prognosticing prostate cancer according to claim 1, wherein the kit is a kit for immunoassay. The kit for diagnosing or prognosticing prostate cancer according to claim 1, wherein the kit is a microarray. The kit for diagnosing or prognosticing prostate cancer according to claim 1, wherein the kit is a gene amplification kit. A method for detecting prostate cancer diagnosis or prognostic markers by detecting the expression of the resistin protein or the nucleotide sequence of the resistin in a human biological sample to provide information necessary for prostate cancer diagnosis or prognostic analysis. 10. The method of claim 8, wherein the prostate cancer is differentiated prostate cancer or distant metastatic prostate cancer. The method of claim 8, wherein the method is a method capable of selectively diagnosing only a prostate cancer sample from an enlarged prostate sample and a prostate cancer sample. The method of claim 10, wherein the sample is human tissue, cells, blood, serum or plasma. 9. The method of claim 8, wherein the method is carried out in an antigen-antibody reaction mode. 9. The method of claim 8, wherein the method is carried out in a microarray manner. The method of claim 8, wherein the method is performed by gene amplification.

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