KR20190121578A - Biomarkers for diagnosis of prostate cancer and uses thereof - Google Patents

Abstract

The present invention relates to a biomarker for diagnosing prostate cancer and a usage thereof, and more specifically, to a composition for diagnosing prostate cancer comprising a specific miRNA or an agent for measuring expression level of a fragment of the same, to a kit for diagnosing prostate cancer containing the composition, and to a method for providing information for prostate cancer diagnosis including a step for measuring an expression level of the biomarker. The present invention identified a novel miRNA abnormally expressed in prostate cancer compared with normal tissues for the first time, and uses the same as a biomarker, thereby rapidly diagnosing prostate cancer in a non-invasive method without surgical procedure. Particularly, miRNA for diagnosing prostate cancer of the present invention has relatively large expression amount compared with other genes in a urine of an entity, thereby being able to be stably analyzed through qRT-PCR.

Description

Biomarkers for diagnosis of prostate cancer and uses thereof

The present invention relates to a biomarker for diagnosing prostate cancer and its use, and more particularly to a composition for diagnosing prostate cancer comprising an agent capable of measuring the expression level of a particular miRNA or fragment thereof; Prostate cancer diagnostic kit comprising the composition; And it relates to a method for providing information for diagnosing prostate cancer comprising the step of measuring the expression level of the biomarker.

The prostate is a chestnut-sized male reproductive organ just below the bladder and in front of the rectum that produces and stores part of the semen. The upper part of the bladder is fixed to the anterior phalanx prosthetic ligament, which is adjacent to the urethra from the bladder to the urethra, and the lower part is fixed by the urogenital septum. Most of the cancers that occur in the prostate gland are adenocarcinomas (cancerous cancers) that occur in prostate cells. The type can be classified according to the degree of differentiation of tumor tissue and the characteristics of cells.

Prostate cancer is one of the world's most common urinary tract tumors, with 180,890 newly diagnosed prostate cancer in 2016 alone, accounting for 10.7% of all new tumor diagnoses in the United States, and the third after breast and lung cancer. It is a frequently developing tumor. Based on 2009-2013 statistics, 129.4 out of 100,000 men worldwide have prostate cancer, and prostate cancer mortality rates in 2016 are 26,120. In addition, prostate cancer is a rare disease before age 50, but increases rapidly after age 50. With the recent increase in life expectancy, the number of elderly men in Korea is increasing rapidly, and prolonged diagnosis of prostate cancer requires continuous management so as not to worsen with metastasis. Human prostate cancer, in particular, appears to have a propensity to metastasize to bone and is known to inevitably progress from an androgen-dependent state to an androgen-resistant state, increasing patient mortality. Moreover, about 25% of men treated with prostate cancer are recurrent illnesses that require further treatment, which is currently the second leading cause of cancer deaths in men in the United States. Is needed.

Conventional techniques for diagnosing prostate cancer include prostate specific antigen (PSA) measurements and biopsi. PSA is the most commonly used method of diagnosing prostate cancer, and is a method of determining prostate cancer risk by measuring specific antigen levels produced by prostate cells. Malignant prostate cancer has higher PSA levels, and most men without prostate cancer have levels below 4 ng / mL. However, if the measured PSA levels are very high in the absence of prostate cancer, or if they are in the PSA gray zone (more than 4 ng / mL PSA but less than 10 ng / mL), biops are required. Biopsi is a method of diagnosing malignant tumors such as cancer and sarcoma in a patient's organ through pathological histological examination of the tissue of a suspected cancerous organ. Such a bioptic test is very painful for patients, and the accuracy of prostate cancer diagnosis, especially in the PSA gray zone, is less than 30%. In other words, about 70% of patients suffer from costly and painful rebiopsis due to the limitation of PSA.

Therefore, the development of prostate cancer specific diagnostics with higher accuracy is required to overcome and replace such unnecessary rebiopsis and PSA limitations.

Against this background, the present invention has identified specific miRNAs as prostate cancer specific biomarkers based on urine samples and non-tumor control urine samples from patients diagnosed with prostate cancer, and these miRNA biomarkers are used for advanced prostate cancer and focal prostate cancer. The present invention has been completed by confirming that it is useful for diagnosis of all.

Korean Patent Publication No. 10-2017-0008729 United States Patent No. 8,597,892

Accordingly, an object of the present invention is to provide a composition that can effectively diagnose (differentiate) prostate cancer.

Another object of the present invention is to provide a kit for diagnosing (differentiating) prostate cancer comprising the composition.

Still another object of the present invention is to provide a method for providing information for effectively diagnosing (differentiating) prostate cancer using a biomarker.

In order to achieve the object of the present invention as described above,

The present invention provides a composition for diagnosing prostate cancer, comprising an agent capable of measuring miRNA expression levels of hsa-miR-3659 or hsa-miR-3679-5p.

In one embodiment of the present invention, the hsa-miR-3659 may be composed of the nucleotide sequence of SEQ ID NO: 1, the hsa-miR-3679-5p may be composed of the nucleotide sequence of SEQ ID NO: 2.

In one embodiment of the invention, the agent may be a primer, probe or antibody that specifically binds to the miRNA.

The present invention also provides a diagnostic kit for prostate cancer comprising the composition.

In addition, the present invention comprises the steps of measuring the miRNA expression level of hsa-miR-3659 or hsa-miR-3679-5p in a biological sample isolated from the individual; And comparing the miRNA expression level with a corresponding miRNA expression level in a normal control sample.

In one embodiment of the present invention, the hsa-miR-3659 may be composed of the nucleotide sequence of SEQ ID NO: 1, the hsa-miR-3679-5p may be composed of the nucleotide sequence of SEQ ID NO: 2.

In one embodiment of the invention, the sample may be urine.

In one embodiment of the present invention, the step of measuring the expression level of miRNA is reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time reverse transcriptase polymerase reaction ( It can be measured by real time quantitative RTPCR, RNase protection method, Northern blotting or gene chip.

According to the present invention for the first time to identify a new miRNA that is abnormally expressed in prostate cancer compared to normal tissue, by using it as a biomarker can be quickly and quickly diagnose (differentiate) prostate cancer in a non-invasive way without surgical procedures. In particular, the prostate cancer diagnostic miRNA has a relatively large amount of expression compared to other genes in the urine of the individual has the advantage that can be stably analyzed through qRT-PCR.

1 is a flow chart showing the overall experimental design process of the present invention.
FIG. 2A shows a box plot showing the expression level of miR-3659 in the urine of BPH control and total prostate cancer patients (Total PCa).
FIG. 2B shows a box plot showing the expression level of miR-3679-5p in the urine of patients with prostatic hypertrophy (BPH control) and total prostate cancer (Total PCa).
FIG. 3A shows a box plot showing the expression level of miR-3659 in the urine of patients with BPH control, Localized PCa and Advanced PCa.
FIG. 3B shows a box plot showing the expression level of miR-3679-5p in the urine of patients with BPH control, Localized PCa and Advanced PCa.

The present invention relates to a bioker for diagnosing prostate cancer.

The term "prostate cancer" used in the present invention is a malignant tumor occurring in the prostate, and includes "localized prostate cancer" and "advanced prostate cancer."

As used herein, the term "diagnosis" refers to confirming the presence or characteristic of a pathological condition. In the present invention, the diagnosis may be interpreted as confirming the progression or onset of prostate cancer.

More specifically, the present invention relates to a biomarker for diagnosing prostate cancer of hsa-miR-3659 or hsa-miR-3679-5p.

As another aspect, the present invention provides a diagnostic composition for prostate cancer, comprising an agent capable of measuring the miRNA expression level of hsa-miR-3659 or hsa-miR-3679-5p.

In an embodiment of the invention, the hsa-miR-3659 may be composed of the nucleotide sequence of SEQ ID NO: 1, the hsa-miR-3679-5p may be composed of the nucleotide sequence of SEQ ID NO: 2.

The base sequence described in SEQ ID NO: 1 or 2 may be used as a diagnostic marker for diagnosing the progression or onset of prostate cancer, and the sequence may be modified to some extent in diagnosing the progression or invention of prostate cancer. Do. Those skilled in the art will appreciate that base sequences in which at least 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 98% homology are maintained by such artificial modifications are desired in the present invention. As long as it is possible to significantly compare the expression level between normal individuals and individuals with prostate cancer disease as prognostic cancer diagnostic markers, it will be readily understood that they are equivalent to the nucleotide sequences of the present invention.

As used herein, the term "marker or diagnostic marker" refers to a substance that can diagnose an individual with prostate cancer from a normal cell or a normal individual, and is a cell that has advanced or developed prostate cancer as compared to normal cells. Or organic biomolecules such as polypeptides, proteins or nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that exhibit an increase or decrease in an individual. For the purposes of the present invention, the prostate cancer diagnostic markers of the present invention are miRNAs or fragments of miRNAs that show a difference in expression levels specifically in prostate cancer cells as compared to cells of normal cells or tissues.

As used herein, the term "agent for measuring the level of miRNA or fragment thereof" refers to an agent used in a method for confirming the expression of a corresponding miRNA or fragment thereof contained in a sample, preferably RT-PCR. Methods such as competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting, and gene chip analysis It may be a primer, a probe or an antibody that can specifically bind to a target gene used for, but is not particularly limited thereto.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3 'hydroxyl group, which can form complementary templates and base pairs and starts for template strand copying. By a short nucleic acid sequence that functions as a point. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures.

As used herein, the term “probe” refers to a nucleic acid fragment such as RNA or DNA, which may correspond to a short base of several to long bases capable of specific binding with a gene or mRNA, and oligonucleotide. Probes, single stranded DNA (single stranded DNA) probes, double stranded DNA (double stranded DNA) probes, RNA probes and the like can be produced in the form of, and can be labeled for easier detection.

Primers or probes of the invention can be synthesized chemically using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphotriester, phosph Modifications to poroamidates, carbamates, etc.) or charged linkers (eg, phosphorothioates, phosphorodithioates, etc.).

Specifically, in one embodiment of the present invention derived miRNA that can be used as a diagnostic marker for prostate cancer. The miRNA was analyzed urine samples of 14 patients histologically diagnosed with prostate cancer and 5 non-tumor controls using a microarray technique and a total of 60 genes were selected. Subsequently, we selected gene candidate groups that could be stably analyzed by qRT-PCR because the expression levels of both tumor and non-tumor control urine samples were relatively higher than those of other genes. Two miRNAs (hsa-miR-3659 and hsa-miR-3679-5p) were final selected.

The final two miRNAs of the present invention (hsa-miR-3659 and hsa-miR-3679-5p) were then obtained in a total of 147 urine samples obtained from patients with prostate cancer (95) and prostatic hyperplasia (52). The utility as a detection marker was evaluated. The results showed that the expression levels of hsa-miR-3659 and hsa-miR-3679-5p were markedly down-regulated in the urine samples of all prostate cancer patients, including focal and metastatic prostate cancer. . In particular, it was confirmed that hsa-miR-3659 was expressed downward in the urine samples of patients with local prostate cancer and advanced prostate cancer compared to the control of the enlarged prostate.

Through these results, it was confirmed that the expression level of hsa-miR-3659 and hsa-miR-3679-5p is reduced in the presence of prostate cancer, two new miRNAs of the present invention (hsa-miR-3659 and hsa- It was found that the use of an agent measuring the expression level of miR-3679-5p) can effectively diagnose prostate cancer.

Therefore, the present invention can provide a composition for diagnosing prostate cancer, which comprises an agent capable of measuring miRNA expression levels of hsa-miR-3659 and hsa-miR-3679-5p.

As another aspect, the present invention relates to a kit for diagnosing prostate cancer comprising the composition.

The kit of the present invention is to measure prostate cancer by measuring (identifying) miRNA expression levels of one or more of the two miRNAs (hsa-miR-3659 and hsa-miR-3679-5p), which are diagnostic markers for prostate cancer. It can be used to diagnose. The kit for diagnosing prostate cancer of the present invention is suitable for polynucleotides, primers, probes or antibodies as well as analytical methods for identifying one or more miRNAs from the total of two miRNAs (hsa-miR-3659 and hsa-miR-3679-5p). One or more other component compositions, solutions or devices may be included.

As a specific example, a diagnostic kit for measuring the expression level of one or more miRNAs or fragments thereof of two miRNAs of the present invention (hsa-miR-3659 and hsa-miR-3679-5p) is required to perform RT-PCR. It may be a kit containing essential elements. The RT-PCR kit includes a test tube or other appropriate container, reaction buffer (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, in addition to each primer pair specific for the gene. Such as enzymes, DNase, RNAse inhibitors, DEPC-water (DEPC-water), may include sterile water and the like. It may also comprise primer pairs specific for the genes used as quantitative controls.

As another example, a kit of the present invention may contain the necessary elements necessary to perform a gene chip assay. The gene chip analysis kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and a reagent, an agent, an enzyme, or the like for preparing a fluorescence-labeled probe. In addition, the substrate may comprise cDNA corresponding to the quantitative control gene or fragment thereof.

The kit of the present invention may further comprise an agent in addition to the two new miRNAs, which measure the expression level of a conventionally known prostate cancer diagnostic biomarker gene.

As another aspect, the present invention relates to a microarray for diagnosing prostate cancer comprising the composition.

The microarray may be made of a conventional microarray except for including the polynucleotide, primer or probe of the present invention.

Hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art. The detection involves, for example, labeling a nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent substance, for example, a substance such as Cy3 and Cy5, and then hybridizing onto a microarray and the labeling substance. The hybridization result can be detected by detecting a signal generated from the.

As another aspect, the present invention provides a method for providing information for diagnosing prostate cancer using the diagnostic composition or kit, specifically (a) hsa-miR-3659, hsa in a biological sample isolated from the subject measuring the expression level of miR-3679-5p or a fragment thereof; And (b) comparing the at least one miRNA expression level with a corresponding miRNA expression level in a normal control sample.

In detail, after measuring the expression level of the miRNA or fragment thereof in step (a), and comparing the miRNA expression level in the step (b) with the corresponding miRNA expression level of the normal control sample, hsa-miR-3659 or hsa If the expression level of -miR-3679-5p is significantly reduced, it may be determined that prostate cancer has progressed or developed.

At this time, the step of measuring the expression level of the miRNA or fragment thereof is reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time quantitative RTPCR (real time quantitative RTPCR) ), RNase protection method (RNase protection method), Northern blotting (Northern blotting) or a gene chip can be measured, but the type is not particularly limited.

In one embodiment of the present invention, the hsa-miR-3659 is composed of the nucleotide sequence of SEQ ID NO: 1, the hsa-miR-3679-5p may be composed of the nucleotide sequence of SEQ ID NO: 2.

In another embodiment of the invention, the biological sample isolated from the subject is urine (urine) obtained from a subject.

In another embodiment, the present invention is directed to administering a candidate agent that is expected to be able to prevent or treat the progression or onset of prostate cancer, and wherein the expression level of hsa-miR-3659, hsa-miR-3679-5p or a fragment thereof is administered. It provides a method for screening an agent for preventing or treating prostate cancer comprising the step of measuring.

Specifically, the method for screening an agent for preventing or treating prostate cancer of the present invention includes (i) hsa-miR-3659, hsa-miR-3679-5p, or a fragment thereof, from a sample of an experimental animal having prostate cancer as a control group. Measuring the expression level of the; (Ii) administering to the experimental animal a candidate substance expected to be able to treat prostate cancer; (c) measuring the expression level of hsa-miR-3659, hsa-miR-3679-5p or fragments thereof from a sample of the experimental animal administered with the candidate substance in the experimental group; And, (d) comparing the levels of hsa-miR-3659, hsa-miR-3679-5p, or fragments thereof, measured in the control group with those measured in the experimental group, to a level higher than that measured in the control group. And if present, determining that the candidate material can be used as a prophylactic or therapeutic agent that prevents the progression or onset of prostate cancer.

Measuring the expression level of hsa-miR-3659, hsa-miR-3679-5p or fragments thereof in cells in the absence of a candidate agent capable of preventing or treating prostate cancer, and also in the presence of the candidate agent After measuring the levels of the miRNA or fragments thereof and comparing the two, a substance whose expression level of the miRNA or fragment thereof of the present invention in the presence of the candidate substance increases above the level in the absence of the candidate substance is determined by It can be predicted as a prophylactic or therapeutic agent.

As another aspect, the present invention provides a composition for preventing or treating prostate cancer, comprising an active ingredient targeted to hsa-miR-3659, hsa-miR-3679-5p or a fragment thereof as an active ingredient.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

< Example >

1. Materials and Methods

Urine and Serum Sample Preparation

In the present study, 52 urine samples from prostatic hyperplasia and 95 urine samples (32 prostate cancers and 63 advanced prostate cancers) provided by Chungbuk National University Hospital were used. The first urine was taken in the morning before surgery in patients with prostate cancer and enlarged prostate. Urine samples were centrifuged at 2,500 rpm for 15 minutes and the supernatant was stored at -80 ° C. Serum samples were taken the morning of surgery and stored at -80 ° C.

Microarray

Total RNA was extracted from urine samples using RecoverAll Total Nucleic Acid Isolation Kit (Life Technologies, Carlsbad, Calif., USA). The quantity and integrity of RNA was examined with the RNA 6000 Pico Chip Kit (Agilent Technologies, Santa Clara, Calif., USA) and Agilent 2100 Bioanalyzer. MiRNA profiling was performed using the Agilent Human miRNA Microarray Release 16.0 platform, which examines 1,205 human miRNAs and 144 viral miRNAs.

From urine samples miRNA  extraction

Genolution urine miRNA purification kit (Genolution Pharmaceuticals Inc., Seoul, Korea) was used to extract miRNAs from urine samples. Genomelution 500 μL of the supernatant of each urine sample was added to a tube containing proprietary miRNA isolation solution and vortexed for 20 seconds. Thereafter, 200 μL of chloroform was added, followed by boxing for 10 seconds, followed by centrifugation for 10 minutes at 13,000 rpm at 4 ° C. 600 μL of the aqueous solution of the upper layer was obtained without white precipitate, which was transferred to a 1.5 ml fresh tube, followed by addition of 800 μL of isopropanol, followed by centrifugation at 15,000 rpm for 20 minutes at 4 ° C. The solution after centrifugation was left still to pour into anticipated reverse RNA pellets. Then, 1 ml of 70% ethanol was added and centrifuged at 15,000 rpm for 20 minutes at 4 ° C. After removing the remaining ethanol, the pellet was dissolved in 40 μL of RNase-free water and stored at -80 ° C until used in the experiment.

Extracted from urine samples miRNA  cDNA synthesis

The Mir-XTM miRNA First Strand cDNA Synthesis Kit (Clontech, TAKARA, Otsu, Japan) was used to synthesize cDNA from miRNA extracted from urine samples. 5 μL of mRQ Buffer (2 ×), 1.25 μL of mRQ enzyme and 3.75 μL of miRNA sample were added to each RNase-free 0.2 ml tube and the reagents were mixed well in the thermocycler. The mixture was allowed to react at 37 ° C. for 1 hour and then terminated at 85 ° C. for 5 minutes for enzyme inactivation and left on ice. 90 μl ddH ₂ O was added to bring the total volume to 100 μl.

Real-time PCR

Real-time polymerase chain reaction (RT-PCR) was performed using the Rotor Gene 6000 instrument (Corbett Research, Mortlake, Australia) to amplify miRNAs extracted from urine samples. Micro-reaction tubes (Corbett Research) containing SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules, Calif.) Were used for RT-PCR. A forward primer designed as follows for miRNA amplification was used. MiR-3659: 5′-UGA GUG UUG UCU ACG AGG GCA-3 ′ (21 base pairs). MiR-3679-5p: 5'- UGA GGA UAU GGC AGG GAA GGG GA -3 '(23 base pairs). After thawing the reagent and template cDNA, a final volume of 10 μL containing 5 μL 2X QuantiTect SYBR Green PCR master mix, 0.2 μL 10X miScript universal primer, 0.2 μL 10 pmol forward primer, 2 μL template cDNA and 2.6 μL RNase-free water PCR reaction was performed. RT-PCR conditions are as follows. 45 cycles of 5 seconds at 95 ° C. and 20 seconds at 60 ° C., followed by 60 cycles at 95 ° C., 30 seconds at 55 ° C., and 30 seconds at 95 ° C. were performed once. Melting programs were carried out at a heating rate of 1 ° C. per 5 seconds from 70 ° C. to 99 ° C. Spectral data were captured and analyzed using Rotor-Gene Q software 2.3.1.49. All samples were subjected to 3 replicates.

Statistical analysis

Mann-Whitney U test was used for sample urine miRNA expression in prostate cancer. Statistical analysis is performed by IBM SPSS Statistics ver. 20.0 (IBM Co., Armonk, NY). P <0.05 was considered significant.

2. Results

Research Design Outline

Flow chart and overall experimental design are shown in FIG. 1. To identify candidate urine miRNAs specific for prostate cancer, a discovery cohort consisting of 14 urine samples and 5 prostatic hypertrophy control urine samples from prostate cancer patients was used for miRNA microarray analysis. First, a total of 60 genes were selected through a microarray (see Table 1 below). Final candidate gene selection was performed through initial pilot test cohort (36 prostate cancers (18 local prostate cancers, 18 advanced prostate cancers), 18 prostatic hypertrophy controls). For the final candidate gene selection, ① genes that were stable in qRT-PCR were selected because of their relatively high expression levels in both tumor and non-tumor control urine samples, and ② target candidate groups that satisfy the above conditions. As compared to the non-tumor control group, the genes expressed in the tumor group were expressed downward.

The final two miRNAs derived from the microarray analysis (hsa-miR-3659 and hsa-miR-3679-5p) were then totally 147 urine obtained from patients with prostate cancer (95) and prostatic hyperplasia (52). The usefulness of the sample as a detection marker was evaluated.

60 genes selected via microarray turn gene turn gene One bkv-mir-B1-5p 31 hsa-mir-3115 2 hsa-mir-3162-3p 32 hsa-let-7d-5p 3 hsa-mir-4299 33 hsa-let-7d-3p 4 hsa-mir-1268a 34 hsa-mir-885-3p 5 hsa-mir-320a 35 hsa-mir-99a-5p 6 hsa-mir-671-5p 36 hsa-mir-99a-3p 7 hsa-mir-1268b 37 hsa-mir-767-5p 8 hsa-mir-3605-5p 38 hsa-mir-26a-5p 9 hsa-mir-549a 39 hsa-mir-26a-2-3p 10 hsa-mir-3162-5p 40 hsa-mir-1264 11 hsa-mir-4257 41 hsa-mir-3924 12 hsa-mir-1237d 42 hsa-let-7i-5p 13 hsa-mir-4270 43 hsa-let-7i-3p 14 hsa-mir-3194-3p 44 hsa-mir-3919 15 hsa-mir-3194-5p 45 hsa-mir-1277-5p 16 hsa-mir-3659 46 hsa-mir-1277-3p 17 hsa-mir-638 47 hsa-mir-32-5p 18 hsa-mir-601 48 hsa-mir-32-3p 19 hsa-mir-3679-5p 49 hsa-mir-4320 20 hbv-mir-B2RC 50 hsa-mir-519b-3p 21 hsa-mir-4271 51 hsa-mir-624-5p 22 hsa-mir-3161 52 hsa-let-7a-5p 23 hsa-mir-3195 53 hsa-mir-519c-3p 24 hsa-mir-517c-3p 54 hsa-mir-517c-5p 25 hsa-mir-762 55 hsa-let-7a-3p 26 hsa-mir-769-3p 56 hsa-mir-325 27 hsa-mir-3115 57 hsa-mir-105-5p 28 hsa-mir-612 58 hsa-let-7a-2-3p 29 hsa-mir-3920 59 hsa-mir-624-3p 30 hsa-mir-760 60 hsa-mir-105-3p

Basic characteristics of the study subject

The basic characteristics of the patients and controls are shown in Table 2 below. A total of 147 urine samples (prostate cancer-95, prostatic hyperplasia-52) were used in this experiment. Prostate cancer samples, on the other hand, are classified into 32 focal prostate cancers and 63 advanced prostates. The average age of patients with prostate cancer was 66.86 ± 5.50 years; The mean age of the prostatic hypertrophy control group was 69.13 ± 8.95. In patients with prostate cancer, the average serum prostate-specific antigen (PSA) level was 86.23 ± 465.33 ng / ml; The mean PSA level of the prostatic hypertrophy control group was 43.85 ± 276.73 ng / ml.

Basic characteristics of the study subject (n = 147) Parameters No. of patients (%) PCa BPH p-value No. 95 52 PSA ± SD (ng / ml) 86.23 ± 465.33 43.84 ± 276.73 0.000 Mean ± SD age 66.86 ± 5.50 69.13 ± 8.95 0.153 Gleason score (%) 7 (3 + 4) 30 (31.58) - - 7 (4 + 3) 31 (32.63) - - 8≥ 34 (35.79) - - Stage (%) Localized 32 (33.68) - - Advanced 63 (66.32) - - PCa, prostate cancer; BPH, benign prostate hyperplasia; PSA, prostate specific antigen; SD, standard deviation
Mann-Whitney U test was used to compare the expression levels to clinical variables.
2 patients of PCa has only T and N and without clinical information of M (metastasis)

In prostate cancer and enlarged prostate control urine hsa - miR -3659 and hsa - miR -3679-5p Expression amount  Measure

To verify the usefulness of the prostate cancer miRNA markers of the present invention, miRNA expression was compared in prostatic hypertrophy controls and prostate cancer. As a result, as shown in FIG. 2, the box plot shows that miR-3659 and miR-3679-5p expression levels were significantly lower in the prostate cancer urine samples (including topical prostate cancer and metastatic prostate cancer) compared to the prostatic hypertrophy control. (Each P <0.001, P = 0.007). In addition, as shown in FIG. 3, RT-PCR results showed that miR-3659 expression was down-expressed in urine samples of both prostate cancer and advanced prostate cancer compared to the prostatic hypertrophy urine sample (each P <0.001, P <0.001) and miR-3679-5p were found to be downwardly expressed in advanced prostate cancer compared to prostatic hyperplasia.

Although many studies have been conducted on prostate cancer-specific biomarkers, the sensitivity and specificity of detection of biomarkers in body fluids have been reported to be very low. In addition, PSA (prostate-specific antigen) is widely used to detect prostate cancer, but because it is not a cancer-specific marker, often misdiagnosis occurs, and when a prostate tissue biopsy is performed for accurate examination, the patient suffers and costs are high. It also requires a lot of non-invasive biomarkers for early detection of prostate cancer.

In this experiment, we demonstrated the usefulness of hsa-miR-3659 and hsa-miR-3679-5p as biomarkers to diagnose prostate cancer in urine.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

miRNA: MicroRNA
PCa: prostate cancer
BPH: benign prostate hyperplasia
PSA: prostate-specific antigen

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Biomarkers for diagnosis of prostate cancer and uses <130> NPDC-68276 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-3659 <400> 1 ugaguguugu cuacgagggc a 21 <210> 2 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-3679-5p <400> 2 ugaggauaug gcagggaagg gga 23

Claims (8)

A composition for diagnosing prostate cancer, comprising an agent capable of measuring miRNA expression levels of hsa-miR-3659 or hsa-miR-3679-5p. The method of claim 1,
The hsa-miR-3659 is composed of the nucleotide sequence of SEQ ID NO: 1, the hsa-miR-3679-5p is composed of the nucleotide sequence of SEQ ID NO: 2 diagnostic composition for prostate cancer. The method of claim 1,
The agent is a diagnostic composition for prostate cancer, characterized in that the primer, probe or antibody that specifically binds to the miRNA. A diagnostic kit for prostate cancer comprising the composition according to any one of claims 1 to 3. Measuring miRNA expression levels of hsa-miR-3659 or hsa-miR-3679-5p in a biological sample isolated from the individual; And
Comparing the miRNA expression level with a corresponding miRNA expression level in a normal control sample. The method of claim 5,
The hsa-miR-3659 consists of the nucleotide sequence of SEQ ID NO: 1, wherein the hsa-miR-3679-5p is provided with a base sequence of SEQ ID NO: 2 providing information for diagnosing prostate cancer. The method of claim 5,
The sample is urine, the method of providing information for diagnosing prostate cancer. The method of claim 5,
The step of measuring the expression level of miRNA is reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time quantitative RTPCR, RNase protection assay (RNase protection method), Northern blotting (Northern blotting) or a method for providing information for diagnosing prostate cancer, characterized in that the gene chip.

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