KR102565608B1 - Marker for diagnosis of prostate cancer - Google Patents

Abstract

The present invention relates to a marker for diagnosis and prognosis of prostate cancer. According to the present invention, since the amount of mRNA expression and detection of PSA in blood cancer cells isolated from blood are specific to the stage of prostate cancer, the marker of the present invention The PSA gene in blood cancer cells can be usefully used for diagnosis and prognosis of prostate cancer.

Description

Marker for diagnosis of prostate cancer {MARKER FOR DIAGNOSIS OF PROSTATE CANCER}

The present invention relates to markers for diagnosis and prognosis of prostate cancer.

Isolation of cell types or intracellular components is required as a preparatory tool for diagnosis and treatment in medicine, and for end-purpose or other analysis in research. There are currently many methods of sorting cell types or intracellular components used in laboratories and clinical laboratories. BACKGROUND OF THE INVENTION Rapid isolation of different types of particles, such as viruses, bacteria, cells and multicellular organisms, is a central step in a variety of applications in the areas of pharmaceutical research, clinical diagnostics and environmental analysis. Rapidly growing knowledge in drug discovery and protein research is enabling researchers to rapidly gain a better understanding of protein-protein interactions, cell signaling pathways, and markers of metabolic processes. Such information is difficult or impossible to obtain using single protein detection methods, eg traditional methods such as ELISA or Western blotting. Therefore, separation of cell types or intracellular components is increasingly required.

As a method for separating a specific target biomolecule from a biological sample such as blood plasma, a method using silica, glass fiber, anion exchange resin or magnetic beads is known. Among them, according to the method using magnetic beads, magnetic beads having a probe capable of binding to a target biomolecule are put into a sample solution to capture the target biomolecule, and then the magnetic beads are separated from the sample solution to separate the target biomolecule. extract molecules. A bead-based separation method for separating target biomolecules using magnetic beads has already been commercialized and is widely used to separate cells, proteins, nucleic acids, or other biomolecules. For example, US Patent US 6,893,881 suggests a method for isolating specific target cells using antibody-coated paramagnetic beads.

The magnetophoresis separation technology using high gradient magnetic separation (HGMS) to separate these magnetic beads has a simple structure, high efficiency, ease of use, and hydrolysis compared to dielectrophoresis. It is a field that has been continuously researched for a long time because it has advantages that do not have characteristics. Another advantage of such magnetophoresis is that biological specificity is maintained by a biocompatible bond between magnetic particles and a bioanalyte, and magnetophoresis is not affected by media.

A conventional magnetophoresis method has a magnetic energy source for applying a magnetic field for magnetophoretic separation of a sample having magnetism to be separated, and a magnetic microstructure region for amplifying the magnetic field gradient applied by the external magnetic energy source. A method of applying a magnetic field from a magnetic energy source to a sample with a magnetic field and separating it according to a magnetic density gradient is used.

Cancer has a high mortality rate worldwide and is the second most common cause of death in Western societies after cardiovascular disease. In particular, lung cancer is on the rise due to the aging of the population, an increase in the smoking population, and air pollution. Dietary habits have become westernized and high-fat diets have become common, and colorectal and breast cancers have rapidly increased in environmental pollutants and increased alcohol consumption. , prostate cancer, etc. are continuously increasing. In this situation, there is an urgent need for the creation of anti-cancer substances that can contribute to the improvement of human health, the improvement of healthy quality of life, and the promotion of human health by enabling early prevention and treatment of cancer. Innovative cancer research focused on a deeper understanding of the fundamental mechanisms of DNA damage/repair, apoptosis, tumorigenesis, and treatment in molecular terms is strongly needed to conquer cancer (Varmus, 2006; Alberts, 2008, 2011). ), which could eventually become a breakthrough in cancer therapy. In most cases, a malignant tumor develops in one organ (lung, liver, kidney, stomach, large intestine, rectum, etc.) and then spreads from the organ, which is the primary site, to other tissues. It is called metastasis. Metastasis is a phenomenon accompanying the progression of malignant tumors. As malignant tumor cells proliferate and cancer progresses, they acquire new genetic traits necessary for metastasis, infiltrate into blood vessels and lymph glands, circulate along blood and lymph vessels, and settle in other tissues. After that, it proliferates.

Despite the recent increase in early cancer diagnosis and development of treatments such as targeted therapy, cancer still accounts for a large portion of the causes of death, and most cancers can still be cured only through surgical removal. However, it has been confirmed that even after the primary tumor is removed, local relapse and metastasis of cancer occur due to the involvement of many factors. Therefore, one of the most important factors determining cancer treatment outcomes and prognosis is determining whether metastasized cancer cells are present at the time of initial diagnosis or during treatment. The metastasis of cancer cells to lymph nodes or bone marrow is defined as “disseminated tumor cell (DTC)”, and cancer cells circulating in the peripheral blood of cancer patients are defined as “circulating tumor cell (CTC)”. These are cells shed from primary or metastatic lesions. Circulating tumor cells (CTC) are not only expected to be a potent biomarker in cancer diagnosis, treatment prognosis analysis, and micrometastasis analysis, but also are noninvasive in analysis methods compared to existing cancer diagnosis methods. Because of its advantages, it is very promising as a future cancer diagnosis method. However, since the distribution rate of cancer cells in the blood is very low at the level of one cancer cell per billion cells or one cancer cell per 10 ⁶ to 10 ⁷ leukocytes, it is very difficult to accurately analyze it and requires a very sophisticated analysis method.

Prostate cancer is one of the most common urinary tract tumors worldwide, and is the third most frequently occurring tumor after breast cancer and lung cancer. Prostate cancer is an uncommon disease before the age of 50, but it increases rapidly after the age of 50. With the recent extension of life expectancy, the number of elderly men is rapidly increasing in Korea, and prostate cancer is diagnosed at an early stage and continuous management is required to prevent it from getting worse. In particular, human prostate cancer seems to have a propensity to metastasize to the bone, and it is known that it inevitably progresses from an androgen-dependent state to an androgen-resistant state, increasing the patient's mortality. Most prostate cancers are initially androgen dependent (AD) (Feldman BJ etal., Nat Rev Cancer 20011:34-45. and Han M etal., J Urol 2001166:416-9.). Prostate cancer cells initially require androgens for continued proliferation. In response to surgical (orchiectomy) or medical (GnRH agonists or estrogens) removal of testosterone through androgen deprivation therapy (ADT), or blockade of androgen action with anti-androgens, the growth of susceptible prostate cancer cells Rapid apoptosis is induced. The positive response rate is about 86% based on reduction of prostate specific antigen (PSA) and stabilization or reduction of tumor volume. Apoptosis usually occurs during the first few days to a week. However, cells remaining after positive reaction undergo a period of proliferation arrest in which they tend not to die. 18-36 months after hormone removal and blockade, hyperplasia relapses in 90% of cases. Surviving cancer cells consistently become androgen-independent or unresponsive, followed by androgen-independent (AI) cell proliferation, resulting in hormone-refractory prostate cancer (HRPC) or castration resistant prostate cancer. prostate cancer, CRPC) (Scher HI, Sawyers CL. J Clin Oncol 2006 23:8253-61.). There is no currently developed treatment for this CRPC, so the patient will eventually die. Androgen independence resulting in conversion to CRPC appears to be achieved by a variety of mechanisms. Moreover, about 25% of men treated for prostate cancer have a recurrence of the disease and require additional treatment, and it is currently the second leading cause of cancer death in men in the United States. Early diagnosis and treatment for this disease is in need.

An object of the present invention is to develop a prostate cancer-specific marker detectable in CTCs, in particular, a diagnostic marker capable of diagnosing the onset and progression of prostate cancer.

In order to achieve the above object, the present invention provides a composition for diagnosing prostate cancer.

In addition, the present invention provides a prostate cancer diagnosis kit.

In addition, the present invention is a kit for screening prostate cancer inhibitors

In addition, the present invention provides a method for providing information necessary for diagnosis or prognosis of prostate cancer.

According to the present invention, since the amount of mRNA expression and detection of PSA in CTCs isolated from blood is specific to the stage of prostate cancer, it can be usefully used for diagnosis and prognosis of prostate cancer.

1 is a schematic view of the entire experimental sequence for the isolation and genetic analysis of cerebral cancer cells (CTC).
2 shows a process of manufacturing a device for separating fine particles by controlling the direction of magnetophoresis.
3 shows a device for separating fine particles by controlling the direction of the magnetophore.
4 is a diagram showing the detection rate of the PSA gene detected from prostate cancer patients and the amount of PSA expression detected.
5 is a diagram confirming the correlation between the mRNA level of cancer cell-derived PSA gene in blood and the PSA level in patient serum.

Hereinafter, a preferred embodiment of the marker for diagnosing prostate cancer according to the present invention will be described with reference to the accompanying drawings.

In one aspect, the present invention relates to a diagnostic marker for prostate cancer comprising the PSA gene in CTCs.

In one aspect, the present invention relates to a marker for prognostic prognosis of prostate cancer comprising the PSA gene in CTCs.

In the present invention, the term “marker for prognosis, biomarker for determining prognosis, or prognosis marker” refers to a substance that can predict/diagnose the risk of prostate cancer recurrence, which is recurred and developed after treatment of prostate cancer. Polypeptides or nucleic acids showing increased or decreased expression in a significant correlation with the possibility of developing hormone-refractory prostate cancer (HRPC) or castration resistant prostate cancer (CRPC) (Example: mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), and the like. For the purpose of the present invention, the marker for predicting the prognosis of prostate cancer is a gene PSA in blood cancer cells, which is a gene whose mRNA expression level is increased specifically for prostate cancer. Preferably, such a marker includes not only a gene but also DNA or mRNA complementary to any one marker.

In one aspect, the present invention relates to a composition for diagnosing prostate cancer, including an agent for measuring the expression level of a prostate specific antigen (PSA) gene in circulating tumor cells (CTC).

In one embodiment, the prostate cancer may be in localized stages or metastatic stages, metastatic hormone-sensitive prostate cancer (mHSPC) or metastatic castrate-resistant prostate cancer (metastatic castrate-resistant prostate cancer). resistant prostate cancer, mCRPC) is more preferable.

In one embodiment, the composition may include an agent for measuring the expression level of the PSA gene at the mRNA level or protein level of the PSA gene, more preferably including an agent for measuring the mRNA level of the PSA gene in CTCs do.

In one embodiment, the agent measured at the mRNA level is a primer pair that specifically recognizes a nucleic acid sequence of the gene, a nucleic acid sequence complementary to the nucleic acid sequence, a fragment of the nucleic acid sequence and complementary sequence, a probe, or It may be a primer pair and a probe, and the measurement is polymerase chain reaction, real-time RT-PCR (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, DNA chip, multiplex PCR or ddPCR may be performed by one or more methods selected from the group consisting of.

In one embodiment, the agent measured at the protein level is an antibody, antibody fragment, aptamer, avidity multimer, or peptidomimetics that specifically recognize the full-length protein of a gene or a fragment thereof. ), and the measurement is Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay , Complement Fixation Assay, FACS, mass spectrometry, or protein microarray.

In one embodiment, the gene is a device (biochip) that separates fine particles by adjusting the direction of magnetophoresis registered by the present applicant ('10-1622342', 'fine particle separation device using magnetophoresis and this In the isolated blood cancer cells, the expression increased specifically for the onset and stage of prostate cancer, and in the localized stages, the PSA mRNA in the serum PSA and CTC of each patient increased. Although no correlation was shown, in the metastatic group, mHSPC and mCRPC, the PSA level in serum and the PSA mRNA level in CTC showed the same increasing pattern of each patient.

As used herein, the terms "detection" or "measurement" mean detecting or quantifying the level (concentration) of a measured object.

As used herein, the term "primer" is a nucleic acid sequence having a short free 3-terminal hydroxyl group, which can form a base pair with a complementary template and serves as a starting point for copying the template strand. Refers to a short nucleic acid sequence that functions as a point. Primers can initiate DNA synthesis in the presence of reagents for polymerization (i.e., DNA polymerate or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases to several hundred bases in length that can form a specific binding with mRNA, and is labeled to determine the presence or absence of a specific mRNA can be checked. 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 the PSA gene, and the degree of expression of the gene in CTC can be diagnosed through hybridization. Selection of an appropriate probe and hybridization conditions may be modified based on those known in the art, so the present invention is not particularly limited thereto.

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, capping, substitution of one or more homologues of a natural nucleotide, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phossotriesters, phosphoro amidates, carbamates, etc.) or to charged linkages (eg phosphorothioates, phosphorodithioates, etc.).

In the present invention, suitable conditions for hybridizing a probe with a cDNA molecule can be determined in a series of steps by an optimization procedure. This procedure is carried out as a series of procedures by those skilled in the art to establish a protocol for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and washing time, 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 in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y. (1999). For example, among the above stringent conditions, high stringency conditions are hybridization at 65 ° C in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS) and 1 mM EDTA, and 68 ° C in 0.1 x standard saline citrate (SSC) / 0.1% SDS. It means washing under conditions. Alternatively, high stringency means washing in 6 x SSC/0.05% sodium pyrophosphate at 48°C. Low stringency means, for example, washing at 42° C. in 0.2 x SSC/0.1% SDS.

As used herein, 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 the PSA gene, which is the marker of the present invention, and the method for producing the antibody can be prepared using a well-known method. This includes partial peptides that can be made from the protein. The form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody, or any antibody having antigen-binding properties is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies.

In one aspect, the present invention relates to a pharmaceutical composition for inhibiting or treating prostate cancer, comprising an inhibitor of PSA expression in CTC as an active ingredient.

The pharmaceutical composition according to the present invention may contain any substance capable of inhibiting the expression of PSA in CTC.

In the present invention, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. In the above, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not cause an allergic reaction such as gastrointestinal disorder, dizziness, or similar reaction when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate and stearic acid, and carriers for parenteral administration such as water, suitable oil, saline, aqueous glucose and glycol, etc. and the like, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. As other pharmaceutically acceptable carriers, reference may be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition according to the present invention may be formulated in a suitable form according to a method known in the art together with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various forms for parenteral or oral administration according to known methods, and a representative formulation for parenteral administration is an isotonic aqueous solution or suspension for injection. Formulations for injection may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, it can be formulated for injection by dissolving each component in saline or buffer. In addition, dosage forms for oral administration include, but are not limited to, powders, granules, tablets, pills, and capsules. The pharmaceutical composition formulated in the above way may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular. It means that the administration route of the substance can be administered through any general route as long as it can reach the target tissue. In the above, "effective amount" refers to an amount that exhibits a preventive or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may vary depending on various factors such as the type and severity of the patient's disease, age, sex, weight, sensitivity to drugs, the type of current treatment, administration method, target cells, etc. can be easily determined by experts in In addition, the pharmaceutical composition of the present invention may be administered in combination with conventional therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Preferably, considering all of the above factors, it is possible to administer an amount that can obtain the maximum effect with the minimum amount without side effects, more preferably 1 to 10000 μg/kg body weight/day, and even more preferably 10 to 1000 It can be administered repeatedly several times a day at an effective dose of mg/kg body weight/day.

In one embodiment, the PSA expression inhibitor may preferably include a chemical substance, nucleotide, antisense, siRNA oligonucleotide or natural product extract as an active ingredient, more preferably a sequence complementary to the nucleotide sequence of the gene Eggplant may contain antisense or small interference RNA (siRNA) oligonucleotides as an active ingredient.

As used herein, the term "expression inhibition" means to cause a reduction in the expression (into mRNA) or translation (into protein) of a target gene, preferably by which the expression of the target gene is undetectable or insignificant. means to exist.

As used herein, the term "small interfering RNA (siRNA)" refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (International Patent Publication Nos. 00/44895, 01/36646, 99/32619, 01 /29058, 99/07409 and 00/44914). Since siRNA can suppress the expression of a target gene, it is provided as an efficient gene knock-down method or a gene therapy method. The siRNA molecule of the present invention may have a structure in which the sense strand (sequence corresponding to the mRNA sequence of PSA) and the antisense strand (sequence complementary to the mRNA sequence of PSA) are positioned opposite each other to form a double-stranded structure. The siRNA molecules of the present invention may have a single-stranded structure with self-complementary sense and antisense strands. Furthermore, siRNA is not limited to complete pairing of double-stranded RNA parts paired with each other, but pairing by mismatch (corresponding bases are not complementary), bulge (no base corresponding to one chain), etc. Unfinished parts may be included. In addition, the siRNA end structure can be either a blunt end or a cohesive end as long as it can suppress the expression of the PSA gene by the RNAi effect, and the cohesive end structure is a 3'-end protruding structure and a 5'-end All extruded structures are possible. In addition, the siRNA molecule of the present invention may have a form in which a short nucleotide sequence is inserted between the self-complementary sense and antisense strands, in which case the siRNA molecule formed by expression of the nucleotide sequence is resistant to intramolecular hybridization. Thus, a hairpin structure is formed, and a stem-and-loop structure is formed as a whole. This stem-and-loop structure can be processed in vitro or in vivo to generate active siRNA molecules capable of mediating RNAi. Methods for producing siRNA include direct synthesis of siRNA in a test tube and introduction into cells through a transformation process, and transfection of siRNA expression vectors or PCR-derived siRNA expression cassettes prepared so that siRNA is expressed in cells into cells. There is a way to convert or infect.

In one embodiment, the composition of the present invention including the gene-specific siRNA may include an agent that promotes the entry of siRNA into cells. Agents that promote siRNA uptake into cells can generally use agents that promote nucleic acid uptake. For example, liposomes are used or one of a number of sterols including cholesterol, cholate, and deoxycholic acid is used. It can be formulated with an oily carrier. In addition, poly-L-lysine, spermine, polysilazane, polyethylenimine (PEI), polydihydroimidazolenium, polyallylamine ), a cationic polymer such as chitosan may be used, and succinylated PLL (succinylated PLL), succinylated PEI (polyglutamic acid), polyaspartic acid Anionic polymers such as polyaspartic acid, polyacrylic acid, polymethacylic acid, dextran sulfate, heparin, and hyaluronic acid available.

In one embodiment, when an antibody specific to the protein is used as a substance that reduces the expression and activity of the PSA protein, the antibody is coupled directly with an existing therapeutic agent or indirectly through a linker (eg, covalent can be combined).

As used herein, the term "antisense oligonucleotide" refers to DNA or RNA or derivatives thereof containing a nucleic acid sequence complementary to a specific mRNA sequence, and binds to a complementary sequence in the mRNA to form a PSA protein. The antisense sequence of the present invention refers to a DNA or RNA sequence that is complementary to PSA mRNA and capable of binding to PSA mRNA, and is responsible for translation, cytoplasmic translocation, and maturation of PSA mRNA. ) or other essential activity for overall biological function In addition, the antisense nucleic acid can be modified at one or more bases, sugars or backbone positions to enhance efficacy (De Mesmaeker et al ., Curr Opin Struct Biol., 5, 3, 343-55, 1995) Nucleic acid backbones include phosphorothioates, phosphotriesters, methyl phosphonates, short-chain alkyls, cycloalkyls, short-chain heteroatomic, heterocyclic can be modified with intersugar linkages, etc. Antisense nucleic acids can also contain one or more substituted sugar moieties Antisense nucleic acids can contain modified bases The modified bases include hypoxanthine , 6-methyladenine, 5-methylpyrimidine (especially 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiocyl HMC, 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine, etc. , The antisense nucleic acid of the present invention can be chemically combined with one or more moieties or conjugates that enhance the activity and cell adhesion of the antisense nucleic acid. lipid-soluble moieties such as acids, thioethers, thiocholesterol, fatty chains, phospholipids, polyamines, polyethylene glycol chains, adamantane acetic acid, palmityl moieties, octadecylamine, hexylaminocarbonyl-oxycholesterol moieties, and the like; and is not limited thereto. Oligonucleotides containing fat-soluble moieties and preparation methods are already well known in the art (U.S. Patent Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acid may increase stability to nucleases and increase binding affinity between the antisense nucleic acid and the target mRNA. The antisense oligonucleotide may be synthesized in vitro by a conventional method and administered into a living body, or the antisense oligonucleotide may be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro is using RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow antisense RNA to be transcribed using a vector in which the origin of the recognition site (MCS) is reversed. Such antisense RNA preferably has a translational stop codon in the sequence so that it is not translated into the peptide sequence.

In one aspect, the present invention relates to a prostate cancer diagnostic kit comprising the composition of the present invention.

In one embodiment, the prostate cancer may be metastatic hormone-positive prostate cancer or metastatic castration-resistant prostate cancer.

In one embodiment, the kit may further comprise tools and/or reagents for collecting a biological sample from a subject or patient as well as tools and/or reagents for preparing genomic DNA, cDNA, RNA or protein from the sample. there is. For example, it may include PCR primers to amplify relevant regions of genomic DNA. The kit may contain probes of genetic factors useful for pharmacogenomic profiling. Also, in the use of such kits, labeled oligonucleotides can be used for easy identification during analysis.

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

In one aspect, the present invention relates to a kit for screening an inhibitor of prostate cancer, including an agent for measuring the expression level of the PSA gene in CTCs at the mRNA or protein level.

In one embodiment, the prostate cancer can be metastatic hormone positive prostate cancer or metastatic castration-resistant prostate cancer.

In one embodiment, the kit can screen inhibitors that inhibit progression or metastasis of prostate cancer.

In one aspect, the present invention comprises the steps of determining the expression level of mRNA or protein of the PSA gene in a sample isolated from the test subject; And it relates to a method for providing information necessary for diagnosis or prognosis of prostate cancer, comprising the step of comparing with the corresponding result of the corresponding gene in a normal control sample.

In one embodiment, the method may further include preparing a sample separated from the test subject by isolating CTCs from the test subject's blood sample using a biochip.

In one embodiment, the sample isolated from the test subject may be CTCs, more preferably CTCs isolated from blood separated from the test subject using a biochip, and the biochip is the dielectrophoresis and magnetophoresis of the present invention. A device for separating fine particles by adjusting the direction, that is, '10-1622342' previously registered by the present applicant, 'a device for separating fine particles using magnetophoresis and a method for separating fine particles using the same' is preferred.

In one embodiment, the prostate cancer can be metastatic hormone positive prostate cancer or metastatic castration-resistant prostate cancer.

In one embodiment, the step of distinguishing the stage of prostate cancer according to the mRNA expression level of the PSA gene may be further included, and the stage of prostate cancer is T2, T3 and T4 stages of localized stages, mHSPC and mCRPC. can be

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

manufacturing example. Manufacture of device for separating fine particles by controlling the direction of magnetophoresis

To form a microfluidic channel, a Ti/Cu/Cr seed layer is deposited on a bottom glass substrate (BorofloatTM, Howard Glass Co, Worchester, MA) with a thickness of 0.7 mm, a pattern is formed through photoresist, and a plating process is performed. Through this, a ferromagnetic nickel wire having a thickness of 30 μm was formed.

Two ferromagnetic nickel wires were included in the lower glass substrate of the microfluidic channel part for separation, one of which was included at an inclination angle of 57 ° with respect to the flow direction of the sample, and the other wire proceeded at 71 ° and then separated After changing the inclination angle to 113 ° at the distal end of the microfluidic channel, the pattern was formed by changing the inclination angle to 90 ° again.

In order to reduce cell adhesion to the microfluidic channel surface, the ferromagnetic nickel wire was configured to be 100 μm apart from the channel surface of the microfluidic channel part for separation. A lower substrate including a ferromagnetic nickel wire as a magnetic structure was prepared by removing the photoresist, applying an epoxy adhesive, and flattening the surface.

Subsequently, SU-8 was formed as a microfluidic channel pattern on the upper glass substrate, and a sample inlet was made using Nitril rubber O-rings (size 001-1/2, McMaster-Carr, IL, USA) to form an upper glass substrate. The fine particle separator using magnetic flow was finally completed by manufacturing and bonding the prepared lower glass substrate with a UV adhesive (1187-M, DYMAX Co, Torrington, CT).

Example 1. Prostate cancer patient classification, clinical data and sample collection

Five samples collected from 5 control patients without cancer and 64 samples collected from 58 patients with prostate cancer were classified into localized disease according to the summary stage classification of the SEER program of the National Cancer Institute (USA). Localized stages (T2, T3 and T4) and metastatic stages belonging to progressive disease due to distant metastasis (metastatic hormone-sensitive prostate cancer (mHSPC) and metastatic castrate-resistant prostate cancer) -resistant prostate cancer, mCRPC)), and clinical data were collected and analyzed (Table 1).

Example 2. Isolation of CTCs from Samples

Whole blood collected from the patients was diluted with PBS solution (Invitrogen, USA) at a ratio of 1 to 5 and treated with anticoagulation (Heparin-Agarose, H-1027, Sigma Diagnostics, USA) to prepare a blood sample , This was pretreated by injecting EpCAM-specific antigen-reactive immunomagnetic nanobeads. The prepared sample was injected at a rate of 8 μl/h into the sample inlet of the device for separating microparticles by controlling the direction of dielectrophoresis and magnetophoresis prepared in Preparation Example above to separate CTCs.

Example 3. Correlation analysis of PSA in CTC and PCA in serum

After aggregating and disrupting the CBC cells isolated in Example 2, the cell lysate and magnetic beads having OligodT ₂₅ attached to the surface were mixed to obtain the poly-A tail of the mRNA in the cell lysate and the beads. T ₂₅ was combined. Thereafter, the mRNA-bound magnetic beads were washed twice with washing buffer A type (removal of suspended cells) and twice with wash buffer B type (removal of other suspended cell matter attached between mRNAs and stabilization of mRNA). The washed mRNA-magnetic beads were desorbed with Tris-HCL buffer, and the desorbed mRNA was synthesized into cDNA (complementary DNA). Multiplex PCR was performed using the synthesized cDNA, and the PSA gene was detected by the ddPCR method under the conditions shown in Table 2 below. Here, since the ddPCR method measures absolute values rather than relative values, a threshold value is set when measuring gene expression (PSA threshold value 0.01), and samples (patients) corresponding to or above the threshold value are measured for each gene. was regarded as As the reference value, a value equal to or greater than the average measured value of the control sample was used. The correlation between the level of PSA mRNA in the detected CTCs and the level of PSA in serum among the clinical data identified in Table 1 was analyzed.

The amount of CTC-derived mRNA PSA gene expression of the patient was derived by liner fitting in order from least to greatest, followed by liner fitting of Serum PSA protein value for each patient.

As a result of confirming the PSA gene detection rate and expression level through the mRNA level isolated from CTC, the PSA mRNA level of CTC increased as the clinical stage increased. In particular, the PSA gene expression level and detection rate increased in mHSPC (38.47). The expression level increased 3428 times with the localized stage (0.07462 copies/ul) and mHSPC (255.833 copies/ul). In addition, as a result of confirming the correlation between the PSA level in serum (Serum PSA) and the level of PSA mRNA in CTC, there was no correlation between serum PSA and PSA mRNA in CTC in each patient in the localized stage, but metastatic group mHSPC and In mCRPC, the PSA level in serum and the PSA mRNA level in CTC of each patient showed the same increasing pattern (Fig. 5).

Claims (16)

a) confirming the expression level of mRNA or protein of a prostate specific antigen (PSA) gene in circulating tumor cells isolated from the blood of a test subject using a biochip that separates circulating tumor cells (CTC);
b) comparing with the corresponding result of the gene of interest in a normal control sample; and
c) 0.07462 copies/ul as the reference value for prostate cancer in localized stages according to the mRNA expression level and detection level of the gene,
Metastatic hormone-sensitive prostate cancer (mHSPC) of metastatic stages was set at 255.833 copies/ul as the reference value
Distinguishing mHSPC or metastatic castrate-resistant prostate cancer (mCRPC) of the localized stage, metastatic stage; including,
The biochip for isolating CTCs in step a) is a device for separating microparticles by adjusting the direction of dielectrophoresis and magnetophoresis, comprising: an upper glass substrate; a lower glass substrate including a patterned ferromagnetic nickel wire; and a microfluidic channel through which a sample containing fine particles passes, wherein the microfluidic channel includes a microfluidic channel for separation through which the fine particles included in the injected sample pass while being separated by magnetophoresis, wherein the The microfluidic channel for separation includes a plurality of ferromagnetic nickel wires, one of which is included at an inclination angle of 57° with respect to the flow direction of the sample, and the other wire runs at an angle of 71° to the microfluidic channel for separation. Characterized in that the pattern is formed by changing the inclination angle to 113 ° at the distal end and then changing the inclination angle to 90 ° again,
A method for providing information necessary for the diagnosis or prognosis of prostate cancer. The method of claim 1, wherein the limited-stage prostate cancer is T2, T3, or T4 stage prostate cancer. delete The method of claim 1, wherein the step of determining the expression level of step a) is to determine the expression level of the PSA gene using an agent that measures the expression level of the PSA gene at the mRNA or protein level of the PSA gene. A method of providing information necessary for diagnosis or prognosis. The method of claim 4, wherein the agent measured at the mRNA level comprises a nucleic acid sequence of the gene, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair that specifically recognizes a fragment of the nucleic acid sequence and a complementary sequence, a probe, or primer pairs and probes, a method for providing information necessary for diagnosis or prognosis of prostate cancer. The method of claim 5, wherein the measurement is polymerase chain reaction, real-time RT-PCR (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, DNA chip, multiplex PCR, or ddPCR performed by one or more methods selected from the group consisting of, a method for providing information necessary for prostate cancer diagnosis or prognosis. The method of claim 6, wherein the agent measured at the protein level is an antibody, antibody fragment, aptamer, avidity multimer or peptidomimetic that specifically recognizes the full-length protein of a gene or a fragment thereof ( peptidomimetics), a method of providing information necessary for diagnosis or prognosis of prostate cancer. The method of claim 7, wherein the measurement is Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay , Complement Fixation Assay, FACS, mass spectrometry, or protein microarray. delete delete delete delete delete delete delete delete