WO2020130190A1 - Information providing method for predicting or diagnosing risk of developing renal cancer - Google Patents

Abstract

The present invention relates to a specific protein biomarker that can predict or diagnose the risk of developing renal cancer, and relates to an information providing method, a screening method, a diagnostic composition, and a pharmaceutical composition using same.

Description

Method of providing information to predict or diagnose the risk of developing kidney cancer

The present invention relates to a specific protein biomarker that can predict or diagnose the risk of developing kidney cancer.

SIRTs (Sirtuins) are nicotinamide-dependent deacetylases that are well preserved in most species, from bacteria to humans. In mammals, there are a total of seven types of Sirtuin, from SIRT1 to SIRT7.

Cancer is a disease with a very heterogeneous cause and progression, and is similar in morphology and pathology, but progresses significantly differently at the molecular level. Recent clinical experience also supports this, with ERBB2 and EGFR targeting drugs successfully used in clinical trials such as Herceptin and Gefitinib being mutated or overexpressed in only about 20 to 30% of all cancer patients. .

As described above, since the progress of the cancer cancer process is heterogeneous, it is necessary to acquire and analyze as many clinical samples as possible in order to find important targets. Currently, only a few research groups around the world have secured a large-scale gene expression database and are using it to develop targeting and cancer targeting studies, and many studies in Korea have yet to secure large-scale databases.

Therefore, there is a need to discover a new “cancer” treatment target and to develop a new “cancer therapy” using the discovered target.

The present invention relates to a specific protein biomarker capable of predicting or diagnosing the risk of developing kidney cancer, and providing an information providing method of predicting or diagnosing the risk of developing kidney cancer, comprising measuring the expression level of the protein. There is this.

In addition, the object of the present invention is to provide a screening method for candidates for preventing or treating kidney cancer, comprising measuring the expression level of the protein.

In addition, the present invention is a composition for diagnosing kidney cancer comprising a nucleotide sequence of a gene encoding the protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance specifically binding to a protein encoded by the nucleotide sequence. The purpose is to provide.

In addition, the object of the present invention is to provide a pharmaceutical composition for preventing or treating kidney cancer, which includes a substance that increases the expression of the protein or promotes activity.

1. A method of providing information for predicting or diagnosing the risk of developing kidney cancer, comprising measuring the expression level of SIRT1, SIRT3, or SIRT6 protein in a sample isolated from an individual.

2. In the above 1, when the expression level is lower than the control object, further comprising the step of predicting that the risk of developing kidney cancer is higher.

3. The method of 1 above, wherein the sample is kidney cancer cells.

4. A method of screening for a candidate for prevention or treatment of kidney cancer, comprising the step of measuring the expression level of SIRT1, SIRT3, or SIRT6 protein before and after treatment by treating the sample separated from the kidney cancer individual.

5. In the above 4, when the expression of the protein is increased compared to before the treatment of the test substance, the screening method further comprises the step of selecting the test substance as a candidate for prevention or treatment of kidney cancer.

6. The method of 4 above, wherein the sample is kidney cancer cells.

7. For diagnosis of kidney cancer comprising a nucleotide sequence of a gene encoding SIRT1, SIRT3 or SIRT6 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance specifically binding to a protein encoded by the nucleotide sequence Composition.

8. Kidney cancer diagnostic kit comprising the composition of the above 7.

9. A pharmaceutical composition for preventing or treating kidney cancer comprising a substance that increases the expression of SIRT1, SIRT3 or SIRT6 protein or promotes activity.

The information providing method of the present invention can quickly and accurately provide information for predicting or diagnosing the risk of developing kidney cancer.

The screening method of the present invention can quickly and accurately screen candidates for preventing or treating kidney cancer.

The diagnostic composition of the present invention can quickly and accurately diagnose kidney cancer, and the pharmaceutical composition can exert excellent effects in the prevention or treatment of kidney cancer.

1 and 2 confirm the expression of seven SIRTs in normal and renal cancer tissues by immunohistochemical staining.

3 is a graph of Kaplan-Meier cancer specific survival analysis results, showing that the high SIRT3 expression group has a higher survival rate than the low SIRT3 expression group.

Hereinafter, the present invention will be described in detail.

The present invention relates to the development of kidney cancer comprising the step of measuring the expression of SIRT1 (GenBank: AAH12499.1), SIRT3 (GenBank: AAD40851.1) or SIRT6 (GenBank: CAG33481.1) protein in a sample isolated from an individual. Provides information provision method for risk prediction or diagnosis.

The present invention is based on finding that SIRT1, SIRT3, or SIRT6 is expressed at a specifically low level in a sample obtained from a patient with kidney cancer, and discovering its utility as a novel biomarker or indicator.

The sample of the individual and the sample of the control are biological samples, which means all samples obtained from an individual whose expression of SIRT1, SIRT3 or SIRT6 of the present invention can be detected, wherein the biological samples are biopsy, blood, immune cells , May be any one selected from the group consisting of nerve cells and skin tissue, and preferably kidney cancer cells, but is not particularly limited thereto, and may be prepared by treatment using a method commonly used in the technical field of the present invention. .

In the information providing method, when the expression level is lower than that of a control object, it may further include a step of predicting a higher risk of developing kidney cancer.

As a method for measuring the expression level of the indicator factor, a method of measuring a concentration in a sample of mRNA, a transcript of a gene encoding an indicator factor, or a concentration in a sample of the indicator factor protein may be selected, but is not limited thereto. It can be carried out by selecting a method commonly used in the technical field of the present invention.

As a method for measuring the concentration in the sample of the mRNA, reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), real-time reverse transcriptase polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting and DNA chip, but are not limited thereto.

As a method of measuring the concentration in the sample of the protein, the amount of the protein can be confirmed using an antibody that specifically binds to the protein. As an analysis method for this, immunostaining test, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectric Youngdong, tissue immunostaining (immunohistochemistry), immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), FACS (fluorescence-activated cell sorting) and protein chip (protein chip), but are not limited thereto. .

According to the method for providing information of the present invention, the expression level of an indicator factor is measured from samples of a suspected kidney cancer sample and a control group (unsuspected patient) according to the above method, the measured results are compared with each other, and then the indicator factor When the expression level of is measured in a sample of a suspected patient lower than that of a control group, it is possible to predict or diagnose that the risk of developing kidney cancer is high, and the expression level of the indicator factor is similar or higher than a sample of the control group in a sample of the suspected patient. When measured, it can provide information that can predict or diagnose a low risk of developing kidney cancer.

The present invention provides a method for screening a candidate material for preventing or treating kidney cancer, comprising treating a sample separated from a kidney cancer individual and comparing the expression level of SIRT1, SIRT3, or SIRT6 protein before and after treatment.

The test substance is a newly synthesized or known compound, and may include, without limitation, substances expected to exhibit effects in the prevention or treatment of kidney cancer, for example, nucleic acids, nucleotides, proteins, peptides, amino acids, sugars, lipids And it may be at least one selected from the group consisting of compounds, but is not particularly limited thereto.

The screening method of the present invention measures the expression level of the indicator factor of the present invention by administering the test substance to the sample, and when the expression of the indicator factor increases compared to before the treatment of the test substance, the test substance is renal cancer. When screening as a prophylactic or therapeutic agent, and the expression of the index factor remains unchanged or decreased compared to before the treatment of the test substance, the candidate substance is not screened as a prophylactic or therapeutic agent for renal cancer. can do.

In addition, in the screening method of the present invention, matters related to a sample, measurement of expression level, and the like are as described above.

The present invention is a kidney cancer comprising a material that specifically binds to a nucleotide sequence of a gene encoding SIRT1, SIRT3 or SIRT6 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a protein encoded by the nucleotide sequence. Provided is a diagnostic composition.

The substance that specifically binds to the protein may be an antibody, and the antibody refers to a specific immunoglobulin directed against an antigenic site, wherein the 　 antibody specifically refers to SIRT1, SIRT3 or SIRT6 protein A binding antibody, and a gene encoding SIRT1, SIRT3 or SIRT6 is cloned into an expression vector to obtain a SIRT1, SIRT3 or SIRT6 protein, and an antibody can be prepared according to a conventional method in the art. The form of the “antibody” includes polyclonal “antibody” or monoclonal “antibody” and includes all immunoglobulin “antibodies”. The 　antibody does not have the structure of a complete form intact 　antibody with two light chains and two heavy chains, as well as a complete form with two full length light chains and two heavy chains, but is directed against an antigenic site. Also includes functional fragments of “antibody” molecules that have specific antigen-binding sites (binding domains) and retain antigen-binding functions. In the diagnosis of kidney cancer using the composition of the present invention, kidney cancer is diagnosed by measuring the expression level of SIRT1, SIRT3 or SIRT6 through hybridization using SIRT1, SIRT3 or SIRT6 protein and the antibody. Can. The appropriate 　 antibody selection and hybridization conditions can be appropriately selected according to techniques known in the art.

The nucleotide sequence, a sequence complementary to the nucleotide sequence, and a substance specifically binding to the fragment of the nucleotide may be specifically a probe or a primer.

The probe means a nucleotide fragment such as RNA or DNA corresponding to a few bases to a few hundred bases, which is capable of specifically binding with nucleotides such as mRNA, and is labeled with a radioactive element, etc. Presence or absence (content) can be checked. The probe may be produced in the form of an oligonucleotide (probe), a single-stranded DNA (probe), a double-stranded DNA (probe), or an RNA-probe, and a gene encoding SIRT1, SIRT3 or SIRT6 Kidney cancer can be diagnosed by performing hybridization using the 　 probe complementary to the mRNA of, and measuring the amount of mRNA expression through the degree of hybridization. The appropriate conditions for selection and hybridization of the probe may be appropriately selected according to techniques known in the art.

The primer refers to a short nucleotide sequence that can form a complementary template and base pair with a nucleotide sequence having a short free 3-terminal hydroxyl group and serves as a starting point for template strand copying. The primer is capable of initiating DNA synthesis in the presence of a reagent (i.e., DNA polymerase/polymerase or reverse transcriptase) and four different nucleoside triphosphates for polymerization at an appropriate buffer and temperature, and the SIRT1, Kidney cancer can be diagnosed by measuring the desired expression level of SIRT1, SIRT3 or SIRT6 protein by performing PCR amplification using the mRNA primer of the gene encoding SIRT3 or SIRT6. PCR conditions, and the length of the “primer” set can be appropriately selected according to techniques known in the art.

The nucleotide sequence of the gene encoding the SIRT1, SIRT3 or SIRT6, the sequence complementary to the nucleotide sequence, or the 　probe or 　primer specifically binding to the fragment of the nucleotide is known as the nucleotide sequence of the gene encoding SIRT1, SIRT3 or SIRT6 Therefore, those skilled in the art can design the primer 　 or 　 probe based on the sequence according to conventional methods in the art.

The 　probe or 　primer can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods, and has a length of 10 to 100 nucleotides (hereinafter referred to as'nt'), 10 to 90 nt, 10 to 80 nt, 10 to 70 nt, 10 to 60 nt, 10 to 50 nt, 10 to 40 nt, 10 to 30 nt, 10 to 25 nt, 20 to 100 nt, 30 to 90 nt, 40 to 80 It may be nt, 50 to 70 nt, 20 to 60 nt, 20 to 50 nt, 30 to 40 nt, 20 to 30 nt, or 20 to 25 nt.

The present invention provides a kit for diagnosing kidney cancer comprising the composition.

The kit can diagnose kidney cancer by measuring the expression level of SIRT1, SIRT3 or SIRT6 by measuring the expression level of mRNA or SIRT1, SIRT3 or SIRT6 protein of the gene encoding SIRT1, SIRT3 or SIRT6. The kit not only includes a nucleotide sequence of a gene encoding SIRT1, SIRT3 or SIRT6 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance specifically binding to a protein encoded by the nucleotide sequence, , The kit may include one or more other components　composition, solution, or device suitable for an analytical method for measuring the expression level of SIRT1, SIRT3 or SIRT6 protein used by the kit.

If the kit is a kit for measuring the expression level of mRNA of a gene encoding SIRT1, SIRT3 or SIRT6 protein, it may be a kit containing essential elements necessary for performing RT-PCR. RT-PCR kits include test tubes or other suitable containers, reaction buffers, enzymes such as deoxyribonucleotides (dNTPs), Taq-polymerases and reverse transcriptases, in addition to each “primer” pair specific for mRNA of the marker gene, DNase, RNase Inhibitors, DEPC-water (dEPC water), sterile water, and the like. In addition, it may include a "primer" pair specific to the gene used as a quantitative control.

The kit provides immunological detection of a material that specifically binds to a nucleotide sequence of a gene encoding a SIRT1, SIRT3 or SIRT6 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a protein encoded by the nucleotide sequence. In order to include a substrate, a suitable buffer solution, a secondary antibody labeled with a coloring enzyme or a fluorescent substance, a coloring substrate. The substrate may be a nitrocellulose membrane, a 96 well plate synthesized from polyvinyl resin, a 96 well plate synthesized from polystyrene resin, glass slide glass, and the like, and the chrominase may be peroxidase, alkaline phosphatase ( alkaline phosphatase) can be used, FITC, RITC, etc. can be used for the fluorescent material, and the color development substrate is 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or o- Phenylenediamine (OPD), tetramethyl benzidine (TMB), and the like can be used.

The kit may be a microarray for diagnosing kidney cancer capable of measuring the mRNA expression level of a gene encoding SIRT1, SIRT3 or SIRT6 protein or a protein thereof. The microarray can be easily prepared by a person skilled in the art according to a method known in the art using the index factor, and according to one embodiment, to the mRNA or fragment thereof of the gene encoding the SIRT1, SIRT3 or SIRT6 protein The cDNA of the corresponding sequence may be a microarray attached to the substrate as a #probe.

The present invention provides a composition for preventing or treating kidney cancer comprising a substance that increases the expression of SIRT1, SIRT3 or SIRT6 protein or promotes activity.

"Treatment" means an approach to obtain beneficial or desirable clinical results. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease range, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, disease state Improvement or temporary relief and alleviation (partially or entirely), detectable or not. In addition, "treatment" may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. Treatment refers to both therapeutic treatment and prophylactic or preventative measures. The treatments include treatments that are required for disorders that have already occurred as well as preventive disorders.

"Prevention" means any action that suppresses or delays the onset of a related disease. It will be apparent to those skilled in the art that the compositions herein can prevent related diseases when administered prior to initial symptoms, or onset.

Substances that increase the expression of SIRT1, SIRT3 or SIRT6 protein or promote activity are low molecular weight drugs, gene drugs, protein drugs, extracts, nucleic acids, oligonucleotides, proteins, peptides, antibodies, RNA, DNA, PNA, pressure Tamers, chemicals, enzymes, amino acids, sugars, lipids, compounds (natural compounds and/or synthetic compounds), and at least one selected from the group consisting of components, but may increase SIRT1, SIRT3 or SIRT6 protein expression Or, if the substance has an effect of promoting activity, it is not particularly limited.

Meanwhile, the composition may further include a pharmaceutically acceptable carrier, and may be formulated together with the carrier. The term "pharmaceutically acceptable carrier" in the present invention refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound. As a pharmaceutical carrier that is acceptable in a composition formulated as a liquid solution, as a sterile and biocompatible material, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets.

The composition of the present invention is applicable to any formulation containing an agent that increases the expression of SIRT1, SIRT3 or SIRT6 protein or promotes activity as an active ingredient, and can be prepared as an oral or parenteral formulation. The pharmaceutical formulations of the present invention are oral, rectal, nasal, topical (including cheek and sublingual), subcutaneous, vaginal or parenteral; intramuscular and subcutaneous. And forms suitable for administration by inhalation (including intravenous) or administration by inhalation or insufflation.

The composition of the present invention is administered in a pharmaceutically effective amount. The effective dose level depends on the type of patient's disease, severity, drug activity, sensitivity to the drug, duration of administration, rate of administration and release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field. Can be determined. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the composition of the present invention can vary widely depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and disease severity, and an appropriate dosage is, for example, The amount of drugs accumulated in the body and/or the expression of the SIRT1, SIRT3 or SIRT6 protein to be used may be increased or may vary depending on the specific efficacy level of the substance promoting the activity. In general, it can be calculated based on the EC50 measured as effective in the in vivo animal model and in vitro, for example, may be 0.01 μg to 1 g per 1 kg of body weight, and in a unit period of daily, weekly, monthly or yearly, It may be administered once or several times per unit period, or may be continuously administered for a long period of time using an infusion pump. The number of repeated doses is determined taking into account the time the drug stays in the body and the concentration of the drug in the body. The composition may be administered for relapse even after being treated according to the course of disease treatment.

The composition of the present invention may further contain a compound that maintains/increases the solubility and/or absorption of one or more active ingredients exhibiting the same or similar function in relation to the treatment of kidney cancer. Also optionally, chemotherapeutic agents, anti-inflammatory agents, anti-viral agents and/or immunomodulators may be further included.

In addition, the compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.

Hereinafter, examples will be described in detail to specifically describe the present invention.

Example 1. Experimental method

1. Setting up the experimental group

Between January 2004 and December 2010, clinical, radiological and pathological records of 119 patients who underwent surgical renal cancer surgery were evaluated in accordance with the guidelines of the Institutional Review Board of Gyeongsang National University Hospital. None of the patients received chemotherapy or radiotherapy before surgical cancer surgery. Clinical pathology data, such as gender, age, Karnofsky performance status, Perman grade, and tumor-nodule-metastasis stages, were collected retrospectively from medical records. To assess Cancer-specific survival, survival status and cause of death were investigated using the National Cancer Registration Database and institutional electronic medical records. In the survival analysis, only patients with renal cancer transparent cell types were included, and patients with a positive margin status were excluded.

2. Pathological data analysis

Pathological results were reviewed by two urogenital pathologists. Early kidney cancer tumor-nodule-metastasis stages and grades were established by the American Joint Committee on Cancer (AJCC) stage manual (7 ^th edition) and the Perlman grading system, respectively. Perlman grades 1 and 2 were considered low, and Perlman grades 3 and 4 were considered high.

3. Tissue microarray

The excised cancer samples were fixed overnight in 20% neutral formalin buffer. Samples were thoroughly tested, excised, and inserted into paraffin blocks. Portions representing tumor tissue and adjacent normal tissues (tissues at least 10 mm from the tumor) were selected from each specimen by microscopic examination of H&E (Hematoxylin and Eosin) stained sections. One 2.0 mm tumor tissue core and one 2.0 mm normal tissue core were arrayed per case.

4. Immunohistochemistry analysis

Immunohistochemical staining was performed on tumor tissue and adjacent normal tissue in patients with kidney cancer. Tissue microarray blocks were cut into 4 μm slices for IHC staining. After deparaffinization and rehydration, slides were incubated at 3% hydrogen peroxide for 10 minutes to inhibit the activity of endogenous peroxidase, and non-specific background staining was performed. Sections were heated in a microwave oven (700W) in 10 mM citrate buffer (pH=6.0) for 20 minutes. After inhibiting the activity of the endogenous peroxidase with a peroxidase inhibitor solution, sections were incubated overnight with anti-SIRT antibody at 4°C. Anti-SIRT monoclonal or polyclonal antibodies described below were used: SIRT1 (clone H-95; Santa Cruz Biotechnology), SIRT2 (clone H-300; Santa Cruz Biotechnology), SIRT3 (clone C73E3; Cell Signaling Technology). , SIRT4-7 (ab90485, ab78982, ab118487, and ab78977; Abcam, respectively).

5. Scoring the immune response

The expression levels of SIRT 1 to 7 are the intensity of staining (0, no staining; 1, weak staining; 2, medium staining; and 3, strong staining) and the ratio of staining positive cells (0, <30%; 1,30- 49%; 2,50-69%; and 3, ≥70%) were scored semi-quantitatively. The sum index was obtained by combining the staining intensity and the ratio of cells. Scoring was performed repeatedly from 2 pathologists who did not provide clinical information. The grade of expression was evaluated by calculating the average value of the measured scores, and a final average score of 4 or higher was considered to indicate high expression in the sample. On the other hand, tumors were considered to have a low expression level.

6. Statistical analysis

The correlation between SIRT expression and clinical pathological characteristics according to immunostaining chemistry was analyzed by the Pearson Chi-square test and Fisher's accuracy test. Overall survival was calculated from the day of surgery to the day of death or the last follow-up. The Kaplan-Meier method was used to measure survival, and differences between groups were compared by log rank test. The univariate and multivariate associations of clinical and pathologic variables of cancer-specific survival were measured by Cox regression. The final model was evaluated internally by Bootstrap. All analyzes were performed using SPSS for Windows software (ver. 21.0; SPSS Inc, Chicago, IL). P <0.05 was considered statistically significant.

Example 2. Clinical pathological characteristics of the experimental group

Of the 119 patients, 17 patients were excluded due to lack of expression data. The clinical and pathological parameters of the patients are summarized in Table 1 below. 74 (72.5%) were male and the median age was 59 years (quadrant range: 49.75 ~ 69.25). Among 102 kidney cancer patients, 24 recurrences or metastases were found during the median follow-up period of 73 months (quadrant range: 42.75 to 96.25).

parameter		data
Median age (interquartile range)		59 years old (49.75 ~ 69.25)
gender	male	74 (72.5%)
	female	28 (27.5%)
Karnowski performance index	>80	71 (69.6%)
	≤80	31 (30.4%)
Pathological T stage	One	72 (70.6%)
	2	11 (10.8%)
	3	18 (17.6%)
	4	1 (1.0%)
transition	Yes	24 (23.5%)
	no	78 (76.5%)
Perlman rating	1, 2	81 (79.4%)
	3, 4	21 (3.9%)
Histological form	Transparent cells	81 (79.4%)
	Papillary cells	4 (3.9%)
	Hemoglobin cell	8 (7.8%)
	Etc	9 (8.8%)
Differentiation of breeding		3 (3%)
Positive margin status		2 (2.0%)
Median follow-up period (interquartile range)		73 (42.5 ~ 96.25)

Example 3. Comparison of SIRT expression between normal and renal cancer tissues

To evaluate the role of SIRT protein in kidney cancer, the expression of 7 SIRT proteins was compared between tumor tissue and adjacent normal tissues by immunohistochemical staining for the same person. All 204 samples were analyzed and included 102 malignant tumor samples and 102 adjacent normal tissue samples. As can be seen in Figures 1 and 2, the SIRT family showed different expression in kidney cancer tissue. The chi-square test and Fisher's accuracy test in Tables 2 to 8 below showed that the expression of SIRT1, 3, 6 was significantly lower in tumor tissue compared to normal tissue (P = 0.001, P = 0.006, and P = 0.033, respectively). ), the expression of other SIRT protein did not show a significant difference between the two tissues. SIRT3 expression levels varied according to the histological type of kidney cancer (P = 0.034), 48.1% in clear cells, 100% in papillary cells, and 87.5% in achromatic cells. All types of renal cancer with high SIRT1 expression were classified as tumor stage 1 by AJCC classification, and most cases were of low grade nuclear differentiation except 1 case, although the difference was not statistically significant. Was evaluated. No significant correlation was observed between SIRT expression and metastasis.

		SIRT1
		height	lowness	P
group	normal	88 (86.3%)	14 (13.7%)	0.001
	tumor	16 (15.7%)	86 (84.3%)
Histological form	Transparency	10 (12.3%)	71 (87.7%)	0.067
	Nipples	2 (50.0%)	2 (50.0%)
	An achromatic pigment	1 (12.5%)	7 (87.5%)
	Etc	3 (33.3%)	6 (66.7%)
T stage	One	16 (22.2%)	56 (77.8%)	0.067
	2	0 (0.0%)	11 (100%)
	3	0 (0.0%)	18 (100%)
	4	0 (0.0%)	1 (100%)
Perlman rating	1, 2	15 (18.5%)	66 (81.5%)	0.182
	3, 4	1 (4.8%)	20 (95.2%)
transition	no	14 (17.9%)	64 (82.1%)	0.347
	Yes	2 (8.3%)	22 (91.7%)

		SIRT2
		height	lowness	P
group	normal	87 (85.3%)	15 (14.7%)	0.276
	tumor	80 (78.4%)	22 (21.6%)
Histological form	Transparency	63 (77.8%)	18 (22.2%)	0.913
	Nipples	3 (75.0%)	1 (25.0%)
	An achromatic pigment	6 (75.0%)	2 (25.0%)
	Etc	8 (88.9%)	1 (11.1%)
T stage	One	57 (79.2%)	15 (20.8%)	0.891
	2	8 (72.7%)	3 (27.3%)
	3	14 (77.8%)	4 (22.2%)
	4	1 (100%)	0 (0.0%)
Perlman rating	1, 2	65 (80.2%)	16 (19.8%)	0.381
	3, 4	15 (71.4%)	6 (28.6%)
transition	no	61 (78.2%)	17 (21.8%)	1.000
	Yes	19 (79.2%)	5 (20.8%)

		SIRT3
		height	lowness	P
group	normal	74 (72.5%)	28 (27.5%)	0.006
	tumor	54 (52.9%)	48 (47.1%)
Histological form	Transparency	39 (48.1%)	42 (51.9%)	0.034
	Nipples	4 (100%)	0 (0.0%)
	An achromatic pigment	7 (87.5%)	1 (12.5%)
	Etc	4 (44.4%)	5 (55.6%)
T stage	One	42 (58.3%)	30 (41.7%)	0.266
	2	5 (45.5%)	6 (54.5%)
	3	7 (38.9%)	11 (61.1%)
	4	0 (0.0%)	1 (100%)
Perlman rating	1, 2	42 (51.9%)	39 (48.1%)	0.665
	3, 4	12 (57.1%)	9 (42.9%)
transition	no	45 (57.7%)	33 (42.3%)	0.104
	Yes	9 (37.5%)	15 (62.5%)

		SIRT4
		height	lowness	P
group	normal	11 (10.8%)	91 (89.2%)	0.105
	tumor	4 (3.9%)	98 (96.1%)
Histological form	Transparency	2 (2.5%)	79 (97.5%)	0.077
	Nipples	1 (25.0%)	3 (75.0%)
	An achromatic pigment	1 (12.5%)	7 (75.0%)
	Etc	0 (0.0%)	9 (100%)
T stage	One	3 (4.2%)	69 (95.8%)	1.000
	2	0 (0.0%)	11 (100%)
	3	1 (5.6%)	17 (94.4%)
	4	0 (0.0%)	1 (100%)
Perlman rating	1, 2	3 (3.7%)	78 (96.3%)	1.000
	3, 4	1 (4.8%)	20 (95.2%)
transition	no	4 (5.1%)	74 (94.9%)	0.570
	Yes	0 (0.0%)	24 (100%)

		SIRT5
		height	lowness	P
group	normal	93 (91.2%)	9 (8.8%)	0.593
	tumor	96 (94.1%)	6 (5.9%)
Histological form	Transparency	77 (95.1%)	4 (4.9%)	0.436
	Nipples	4 (100%)	0 (0.0%)
	An achromatic pigment	7 (87.5%)	1 (12.5%)
	Etc	8 (88.9%)	1 (11.1%)
T stage	One	69 (95.8%)	3 (4.2%)	0.314
	2	10 (90.9%)	1 (9.1%)
	3	16 (88.9%)	2 (11.1%)
	4	1 (100%)	0 (0.0%)
Perlman rating	1, 2	76 (93.8%)	5 (6.2%)	1.000
	3, 4	20 (95.2%)	1 (4.8%)
transition	no	75 (96.2%)	3 (3.8%)	0.141
	Yes	21 (87.5%)	3 (12.5%)

		SIRT6
		height	lowness	P
group	normal	67 (65.7%)	35 (34.3%)	0.033
	tumor	51 (50.0%)	51 (50.0%)
Histological form	Transparency	36 (44.4%)	45 (55.6%)	0.180
	Nipples	3 (75.0%)	1 (25.0%)
	An achromatic pigment	6 (75.0%)	2 (25.0%)
	Etc	6 (66.7%)	3 (33.3%)
T stage	One	35 (48.6%)	37 (51.4%)	0.950
	2	6 (54.5%)	5 (45.5%)
	3	9 (50.0%)	9 (50.0%)
	4	1 (100%)	0 (0.0%)
Perlman rating	1, 2	39 (48.1%)	42 (51.9%)	0.463
	3, 4	12 (57.1%)	9 (42.9%)
transition	no	40 (51.3%)	38 (48.7%)	0.816
	Yes	11 (45.8%)	13 (54.2%)

		SIRT7
		height	lowness	P
group	normal	19 (18.6%)	83 (81.4%)	0.183
	tumor	28 (27.5%)	74 (72.5%)
Histological form	Transparency	21 (25.9%)	60 (74.1%)	0.244
	Nipples	3 (75.0%)	1 (25.0%)
	An achromatic pigment	2 (25.0%)	6 (75.0%)
	Etc	2 (22.2%)	7 (77.8%)
T stage	One	23 (31.9%)	49 (68.1%)	0.103
	2	0 (0.0%)	11 (100%)
	3	5 (27.8%)	13 (72.2%)
	4	0 (0.0%)	1 (100%)
Perlman rating	1, 2	22 (27.2%)	59 (72.8%)	0.897
	3, 4	6 (28.6%)	15 (71.4%)
transition	no	23 (29.5%)	55 (70.5%)	0.601
	Yes	5 (20.8%)	19 (79.2%)

Example 4. Correlation between SIRT expression level and survival rate

Of the 102 kidney cancer patients, 81 were transparent cell kidney cancer patients. One patient with positive margin status was excluded, and the survival rate of 80 patients with transparent cell renal cancer was analyzed. During the analysis, 14 (17.5%) patients died of clear cell kidney cancer. To avoid excessive bias, a validation test was performed using the bootstrap method and the Somer's D and Harrell's C values were determined. As a result, the predictive power was found to be sufficient (Somer's D = 0.8313, optimization = 4.4%, C-index = 0.915). The results of the single-variable Cox regression analysis are shown in Table 9 below. Among the clinical pathological features analyzed, age (P = 0.006), T stage (P <0.001), presence of distant metastasis (P <0.001), poor histological grade (P <0.037) and Kartowski performance status index (P <0.001) was associated with significantly poor cancer specific survival. Thus, when SIRT3 was analyzed with age, tumor stage, metastasis, Perman's grade, and Kartovsky performance status index, it was analyzed whether it could be an independent prognostic factor for kidney cancer, and as a result, prognostic factor for long survival It was confirmed that it can be (P = 0.047, Table 10 below). Kaplan-Meier analysis showed a significant difference between the high SIRT3 expression group and the low SIRT3 expression group, and the high SIRT3 expression group was associated with a higher survival rate (P=0.046, FIG. 3).

		HR	Lower 95% CI	Top 95% CI	P value
age		1.083	1.023	1.146	0.006
gender	male	One
	female	0.437	0.057	3.341	0.425
T stage		2.849	1.670	4.861	<0.001
transition		63.156	8.206	486.083	<0.001
Perlman rating	1, 2	One
	3, 4	2.783	1.031	8.321	0.037
Karnowski performance rating index		0.945	0.917	0.974	<0.001
SIRT1		0.370	0.048	2.830	0.338
SIRT2		0.939	0.258	3.418	0.923
SIRT3		0.325	0.102	1.040	0.058
SIRT4		0.046	0.001	6327.766	0.611
SIRT5		0.300	0.067	1.350	0.117
SIRT6		1.190	0.417	3.396	0.744
SIRT7		0.363	0.081	1.627	0.186

	HR	Lower 95% CI	Top 95% CI	P value
Multivariate Cox regression analysis of cancer specific survival (SIRT1)
age	1.023	0.960	1.090	0.490
T stage	1.501	0.743	3.032	0.257
Perlman rating	0.817	0.234	2.853	0.751
transition	32.950	3.494	310.711	0.002
Karnowski performance rating index	0.957	0.920	0.996	0.029
SIRT1	0.343	0.036	3.290	0.354
Multivariate Cox regression analysis of cancer specific survival (SIRT3)
age	1.039	0.975	1.107	0.237
T stage	1.370	0.666	2.819	0.393
Perlman rating	1.858	0.462	7.472	0.383
transition	31.045	3.290	292.916	0.003
Karnowski performance rating index	0.942	0.904	0.983	0.006
SIRT3	0.133	0.018	0.974	0.047
Multivariate Cox regression analysis of cancer specific survival (SIRT6)
age	1.013	0.951	1.078	0.691
T stage	1.719	0.860	3.436	0.125
Perlman rating	0.821	0.243	2.779	0.751
transition	27.157	2.915	288.407	0.004
Karnowski performance rating index	0.951	0.909	0.995	0.030
SIRT6	1.551	0.450	5.346	0.487

Claims (9)

1. Method for providing information for predicting or diagnosing the risk of developing kidney cancer, comprising measuring the expression level of SIRT1, SIRT3, or SIRT6 protein in a sample isolated from an individual.
2. The method according to claim 1, further comprising the step of predicting that the risk of developing kidney cancer is higher when the expression level is lower than that of the control individual.
3. The method according to claim 1, wherein the sample is kidney cancer cells.
4. A method of screening for a candidate for prevention or treatment of kidney cancer, comprising the step of measuring the expression level of SIRT1, SIRT3, or SIRT6 protein before and after treatment by treating a test substance with a sample separated from a kidney cancer individual.
5. The method according to claim 4, When the expression of the protein is increased compared to before the treatment of the test substance, the screening method further comprising the step of selecting the test substance as a candidate for prevention or treatment of kidney cancer.
6. The method according to claim 4, wherein the sample is kidney cancer cells.
7. A composition for diagnosing kidney cancer comprising a nucleotide sequence of a gene encoding SIRT1, SIRT3 or SIRT6 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance specifically binding to a protein encoded by the nucleotide sequence.
8. Kidney cancer diagnostic kit comprising the composition of claim 7.
9. A pharmaceutical composition for preventing or treating kidney cancer comprising a substance that increases the expression of SIRT1, SIRT3 or SIRT6 protein or promotes activity.

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