KR20170012816A - Compositions for colorectal cancer prognosis prediction comprising a preparation for measuring the level of expression of a gene CIRP - Google Patents

Abstract

The present invention relates to a composition for predicting the prognosis of a colon cancer, which comprises an agent for measuring the mRNA level of the CIRP gene or the level of the protein expressed therefrom, a kit for predicting the prognosis of a colon cancer containing the composition, To a method for providing information for predicting the prognosis of a cancer patient. The use of the composition or kit of the present invention can effectively predict the prognosis of treatment such as recurrence or metastasis in colorectal cancer patients, and thus can be widely used for the effective treatment of colorectal cancer.

Description

[0001] The present invention relates to a composition for predicting colorectal cancer prognosis comprising an agent for measuring the expression level of a CIRP gene,

The present invention relates to a composition for predicting colorectal cancer prognosis using a CIRP gene, and more particularly, to a composition for predicting colorectal cancer prognosis, which comprises a preparation for measuring the mRNA level of a CIRP gene or the level of a protein expressed therefrom, And a method for providing information for predicting a prognosis of a colorectal cancer patient using the level of mRNA of the CIRP gene or the level of the protein expressed therefrom.

The large intestine is a long, tubular digestive organ that extends from the end of the small intestine to the anus, and the cancer that occurs at this site is called colon cancer. Colon cancer is divided into colon cancer and rectal cancer according to each site. Patients with colorectal cancer generally present with symptoms such as changes in bowel habits, stools or stools, thin thickness, weight loss, abdominal discomfort, fatigue and anorexia.

Recent studies have shown that the incidence of colorectal cancer is significantly increasing in Korea due to changes in dietary habits, and the incidence of colorectal cancer has increased by 420% from 1995 to 2002 (Figure 1) Health Insurance Statistical Yearbook, National Health Insurance Corporation). Furthermore, according to data released by the Central Cancer Registry in 2014, colorectal cancer is regarded as a serious problem in society as it is the third most common cause of cancer in Korea in 2012 (12.9%) (National Cancer Information Center) .

The importance of diagnosis and treatment of colorectal cancer is emphasized. Recently, studies on the diagnosis of colorectal cancer using molecular markers of colon cancer have been actively carried out. As a result, WO 2005/015224 discloses a method for diagnosing colorectal cancer using an antibody against 60S acidic ribosomal protein P0 (RLA-0), and WO 2004/079368 discloses that expression of HSP9O is increased in colorectal cancer tissue . WO 2004/071267 discloses a method for early diagnosis of colorectal cancer using NNMT (nicotinamide N-methyltransferase), and WO 2005/015234 discloses that SAHH (S-adenosylhomocysteine hydrolase) . In addition, US Pat. No. 7501243 discloses TTK (Tyrosine threonine kinase) as a colon cancer marker.

However, studies on the diagnosis and treatment of colorectal cancer have been actively conducted, but the prognosis of colorectal cancer has not been sufficiently studied. At present, post-operative complications such as weight loss of the patient, local symptoms such as anorexia, pathologic tumor stage, abdominal and inguinal examination, rectal examination, occult blood test, The prognosis of cancer patients is judged. However, these inspections have disadvantages such that the criterion is not objective, has to go through troublesome procedures, and the result is not correct.

In colorectal cancer, it is known that 20 ~ 50% recurrence after radical resection. Therefore, prediction of colorectal cancer prognosis is as important as diagnosis or treatment of colorectal cancer (National Cancer Information Center). In the case of recurrence, extensive recurrence accompanied with local recurrence and distant metastasis occurs more frequently than local recurrence, and there is a limit to the cure of colorectal cancer by radical resection.

Accordingly, the present inventors have made intensive efforts to develop a composition capable of predicting the prognosis of colorectal cancer. As a result, it has been confirmed that the prognosis of colorectal cancer can be predicted by measuring the mRNA level of CIRP gene or the level of protein expressed therefrom, Thus, the present invention has been completed.

The main object of the present invention is to provide a composition for predicting colorectal cancer prognosis, which comprises an agent for measuring the mRNA level of a CIRP gene or the level of a protein expressed therefrom.

Another object of the present invention is to provide a kit for predicting the prognosis of colon cancer comprising the above composition.

It is another object of the present invention to provide a method for providing information for predicting the prognosis of patients with colorectal cancer by measuring the level of mRNA of CIRP gene or the level of protein expressed therefrom from colorectal cancer tissue of colon cancer patients.

In carrying out various studies to develop a method for predicting the prognosis of colorectal cancer, the present inventors investigated the expression level of RNA-binding protein (RBPs) gene in colon cancer tissue and normal tissue isolated from colorectal cancer patients . In general, an RNA binding protein gene is a gene involved in the expression of various genes responsible for cell growth and proliferation. Such a change in protein gene expression may cause physiological defects of cells and lead to the development of cancer. Thus, the present inventors have found that when the mRNA level of the CIRP gene, which is one of the RNA binding proteins measured from the colorectal cancer tissue of a colorectal cancer patient, is decreased as compared with the mRNA level of the CIRP gene measured from the normal tissue of the colon cancer patient, And the prognosis of cancer patients was bad. As a result, it was analyzed that the gene whose expression level was changed could be used as a gene to predict the prognosis of colon cancer patients. Such a technique for predicting the prognosis of colon cancer using the CIRP gene has not been known at all and has been identified for the first time by the present inventor.

According to one aspect of the present invention, there is provided a composition for predicting colorectal cancer prognosis, which comprises an agent for measuring an mRNA level of a CIRP gene or a level of a protein expressed therefrom.

As used herein, the term "CIRP gene " refers to a gene encoding a cold inducible RNA-binding protein, which is known to play various tissue-specific roles, such as maintaining normal cell function and morphology. The specific base sequence and protein information of the gene is known from NCBI (GenBank: NM - 001080).

The term "agent for measuring mRNA level of a gene " of the present invention means a preparation used for measuring the level of mRNA transcribed from the target gene in order to confirm the expression of the target gene contained in the sample , Preferably RT-PCR, quantitative real time PCR, competitive RT-PCR, real time quantitative RT-PCR, RNase protection (RPA) a primer or a probe capable of specifically binding to a target gene used in a method such as assay, Northern blotting and DNA chip analysis, but is not particularly limited thereto.

The term "primer" of the present invention refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a complementary template and having a starting point for template strand copying ≪ / RTI > Primers can be used to initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.

In the present invention, the primer may be a primer that can be used for amplification of the CIRP gene. In one embodiment of the present invention, the primers of SEQ ID NOS: 1 and 2 are used as primers used for amplification of the CIRP gene , The nucleotide sequence of the primer is not limited as long as it can be complementarily bound to the CIRP gene and amplified by the PCR method.

The term "probe" of the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or several hundreds of nucleotides capable of specifically binding to a gene or mRNA, including oligonucleotide probes, For example, in the form of a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and may be labeled for easier detection.

In the present invention, the probe may be a probe capable of complementarily binding to the CIRP gene, and the nucleotide sequence of the probe is not limited as long as it can complementarily bind to each gene.

The term "agent for measuring the level of protein " of the present invention means a preparation used for measuring the level of a target protein contained in a sample, preferably western blotting, enzyme linked immunosorbent assay, radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay (Immunoprecipitation Assay), Complement Fixation Assay, Immunofluorescence, Immunochromatography, FACS (fluorescence activated cell sorter analysis) and protein chip technology assay Lt; / RTI >

The term "antibody" of the present invention means a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. Such an antibody can be prepared by cloning each gene into an expression vector according to a conventional method, A protein encoded by the marker gene can be obtained and can be produced from the obtained protein by a conventional method. The form of the antibody is not particularly limited, and any polyclonal antibody, monoclonal antibody or antigen-binding antibody thereof may be included in the antibody of the present invention, and any immunoglobulin antibody may be included, Specific antibodies of the invention. In addition, the antibody comprises a functional fragment of an antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and can be Fab, F (ab ') 2, F (ab') 2, Fv and the like.

In the present invention, the antibody may be an antibody capable of specifically binding to a protein expressed from a CIRP gene, preferably a polyclonal antibody capable of specifically binding to each protein, Antibody or a portion thereof.

The term "prognosis" of the present invention is used in the same sense as "prognosis prediction ", which means an act of predicting the progress and result of a disease in advance. More specifically, the prognosis or prognosis prediction means that the progress of the disease after the treatment depends on the physiological or environmental condition of the patient, and it refers to all the actions for predicting the progress of the disease after comprehensive treatment considering the condition of the patient . ≪ / RTI > For the purpose of the present invention, the prognosis can be interpreted as predicting the disease-free survival rate or survival rate of patients with colorectal cancer by predicting the progression and cure of disease after treatment of colorectal cancer. For example, predicting a "good prognosis" indicates a high level of disease-free survival or survival rate after treatment for colorectal cancer, meaning that patients with colorectal cancer are more likely to be treated and predicted "poor prognosis" Is a low level of disease free survival or survival rate after treatment for colorectal cancer, meaning that cancer is likely to recur from patients with colorectal cancer or to die from colon cancer.

The term " disease free survival "of the present invention means the possibility that a patient can survive without recurrence of cancer after treatment of colorectal cancer.

The term "survival rate" of the present invention means the possibility that, after treatment of colorectal cancer, the patient can survive regardless of recurrence of cancer.

Meanwhile, the composition for predicting colorectal cancer prognosis according to the present invention can be used for measuring the mRNA level of the CIRP gene or the level of the protein expressed therefrom, as well as the level of the mRNA of the RBM3 gene or the level of the protein expressed therefrom The formulation may further comprise.

As used herein, the term "RBM3 gene" refers to a gene encoding RNA binding motif protein 3, which is one of glycine-rich RNA binding proteins. The specific base sequence and protein information of the gene is known from NCBI (NM_001017430).

In another aspect of the present invention, there is provided a kit for predicting colorectal cancer prognosis, the composition comprising the composition for predicting colorectal cancer prognosis.

The kit of the present invention can be used for predicting the prognosis of colorectal cancer by measuring the level of mRNA of the CIRP gene or the level of the protein expressed therefrom from a sample of a colon cancer patient, A primer, a probe or an antibody for measuring the level of the protein expressed therefrom, as well as one or more other component compositions, solutions or devices suitable for the assay method. In addition to the above-described composition, in addition to the agent for measuring the mRNA level of the CIRP gene of the present invention or the protein expressed therefrom, the agent for measuring the mRNA level of the RBM3 gene or the level of the protein expressed therefrom May be further included.

The term "sample" of the present invention means a direct object of measuring the expression level of the CIRP gene separated from a colon cancer patient, preferably a tissue sample of a colon cancer patient.

As a specific example, the kit for measuring the mRNA expression level of the CIRP gene of the present invention may be a kit containing necessary elements necessary for performing RT-PCR. The RT-PCR kit can be used in combination with a test tube or other appropriate container, a reaction buffer (pH and magnesium concentration varies), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase , DNase, RNAse inhibitor, DEPC-water, sterile water, and the like. In addition, it may contain a primer pair specific to a gene used as a quantitative control.

As another example, the kit of the present invention may contain the necessary elements necessary for performing DNA chip analysis. The kit for DNA chip analysis may include a substrate on which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and a reagent, a preparation, and an enzyme for producing a fluorescent-labeled probe. In addition, the substrate may contain a cDNA corresponding to a quantitative control gene or a fragment thereof.

As another example, the kit of the present invention may be a kit for analyzing a protein chip for measuring the level of a protein expressed from a CIRP gene. The kit may include, but is not limited to, A suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, and the like. The substrate is not particularly limited, but a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, and a glass slide glass can be used, and the coloring enzyme is not particularly limited The fluorescent substance may be FITC, RITC or the like, and the coloring substrate liquid is not particularly limited, but ABTS (2, 3, 4, 5, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) or OPD (o-phenylenediamine), TMB (tetramethylbenzidine).

In another aspect, the present invention provides a method for predicting the prognosis of a colon cancer patient using the composition or kit for predicting the prognosis of a colorectal cancer. Specifically, the present invention provides a method for predicting the prognosis of a colon cancer patient, Measuring the mRNA level of the gene or the level of the protein expressed therefrom (step 1); And comparing the measured mRNA level or the level of the protein expressed therefrom to the level measured from normal tissue of the colon cancer patient (step 2).

The term "normal tissue" of the present invention means any tissue derived from a patient suffering from a colorectal cancer but not causing pathological symptoms such as onset or metastasis of colon cancer. In one embodiment of the present invention, the mRNA level of the CIRP gene from the colon cancer tissue of the colon cancer patient was compared with the mRNA level of the CIRP gene from the normal tissue of the colorectal cancer patient, Means a tissue in which no cancer has occurred in the region.

Wherein the mRNA level of the CIRP gene measured from the colorectal cancer tissue in step 1 or the level of the protein expressed therefrom is measured from the mRNA level of the CIRP gene measured from the normal tissue in step 2 or from When compared with the level of protein, it is predicted that the prognosis is worse because it is highly likely to recur or migrate to other tissues or organs after treatment of colorectal cancer.

In one experimental example of the present invention, the 5-year disease-free survival rate of the CIRP gene mRNA level was 99.7% when the mRNA level of the CIRP gene was high and 76.3% when the mRNA level of the CIRP gene was low, The overall survival rate over 5 years was also 98.1% for high mRNA level of CIRP gene and 81.1% for low mRNA level of CIRP gene. When the level of mRNA of CIRP gene was low, overall survival and disease free survival And that the prognosis was worse.

In addition to the CIRP gene, the method may further include measuring the mRNA level of the RBM3 gene or the level of the protein expressed therefrom. The level of mRNA of the added RBM3 gene or the level of the protein expressed therefrom can reduce the occurrence of errors in predicting the prognosis of colorectal cancer. For example, if the mRNA level of the RBM3 gene or the level of the protein expressed from the RBM3 gene measured from the colorectal cancer tissue in the step 1 is higher than the mRNA level of the RBM3 gene or the , The prognosis of colorectal cancer may be predicted to be poor.

Meanwhile, the method of measuring the mRNA level of the gene or the level of the protein expressed therefrom is the same as described above.

The use of the composition or kit of the present invention can effectively predict the prognosis of treatment such as recurrence or metastasis after colorectal cancer treatment in patients with colorectal cancer and thus can be widely used for effective treatment of colorectal cancer.

FIG. 1A is a graph showing the disease-free survival rate of colorectal cancer patients according to the expression level of RBM3 gene. FIG.
FIG. 1B is a graph showing the overall survival rate of colorectal cancer patients according to the expression level of RBM3 gene.
2A is a graph showing the disease-free survival rate of patients with colorectal cancer according to the degree of CIRP gene expression.
FIG. 2B is a graph showing the overall survival rate of colon cancer patients according to the degree of CIRP gene expression.

Hereinafter, the constitution and effects of the present invention will be described in more detail with reference to Examples and Experimental Examples. These Examples and Experimental Examples are only for illustrating the present invention, and the scope of the present invention is not limited by these Examples and Experimental Examples.

Example  1: Clinical features of colon cancer patients and collection of colon cancer samples

Samples of colorectal cancer tissues were obtained from September 2009 to September 2011 and were collected immediately after colon cancer surgery and stored at -80 ° C.

The 94 selected patients who provided the samples of the colorectal cancer tissues were the second stage to the fourth stage colorectal cancer patients with healing ability (Gachon University, Gil Cancer Center, Incheon), and the clinical characteristics of the patients were as follows 1.

The consent for the collection of the colon cancer tissue samples was obtained from each patient according to the guidelines issued by the ethics committee of the Gil Cancer Center. All the experimental methods using the colon cancer tissue samples were conducted by the Gil Medical Center .

variable ratio(%) age   Average age 64.0 gender   Male Female 57:37 Cancer antigen, ng / mL Average value (range) 5.9 (1.4-103.2) CA19-9 U / dL Average value (range) 22.9 (3.4-340.5) T armor   T1 + T2 15 16.0   T3 + T2 79 84.0 N armory   No transition 68 72.3   Presence of transition 26 27.7 M armory   Remote metastasis 85 90.4   Remote Transition Presence 9 9.6 Lymph invasion   Absent 57 60.6   existence 37 39.4 Neural infiltration   Absent 90 95.7   existence 4 4.3 Size of tumor    ≤ 5 cm 49 52.1    > 5 cm 45 47.9 Site of tumor   Colon 74 82   Rectum 20 18 Histological differentiation   Well differentiation 5 5.3   Moderate eruption 80 85.1   Almost differentiation X 9 9.6

Example  2: Surveillance of postoperative recurrence of colorectal cancer patients

94 patients selected in Example 1 were monitored for recurrence after colorectal cancer surgery.

Specifically, physical examination, carcino-embryonic antigen (CEA), carbohydrate antigen 19-9 (carbohydrate antigen 19-9, CA19-9), chest radiography, and spiral abdominal computed tomography Three years were conducted every six months, and every year thereafter. Other tests such as endoscopy and abdominal ultrasound were performed every 6 to 12 months depending on the patient's condition.

The mean follow-up period was 51.1 months (range, 3.1 to 57.1 months). All patients were classified according to the 7th edition of American Joint Committee on Cancer (AJCC) staging system.

Analysis of the serum anti-cancer and carbohydrate antigens 19-9 was performed in one laboratory using ADVIA Centaur ™ CEA / CA19-9 Lite Reagent & Solid Phase (Siemens, Germany).

Example  3: Correlation between genes expressed from patients with colorectal cancer and prognosis of colorectal cancer

In order to predict the prognosis for the recurrence of colorectal cancer after colorectal cancer surgery in the 94 patients selected in Example 1, it was confirmed whether the mRNA level of the specific gene is related to the prognosis of colorectal cancer.

Example  3-1: All from the organization of patients with colorectal cancer mRNA  Separation method and mRNA  Quantitative Real-time Polymerase Reaction Method of Binding Protein

MRNA levels of all genes from normal tissues and colon cancer tissues of 94 patients selected in Example 1 were measured by quantitative real-time PCR.

Specifically, tissue flakes were mixed with tubes containing TRIzol reagent (Invitrogen, Carlsbad, Calif.) Using a spatula and the tissues were homogenized on ice. The homogenate was centrifuged at 10,000 g for 15 min at 4 < 0 > C to remove cell debris and insoluble matter present in the homogenized material, and the supernatant of the purified homogenate was collected in a tube without RNase. Thereafter, the same amount of chloroform as that of glycogen (1 μl, 20 mg / ml) was added to each tube in which the supernatant was collected. The tube was vigorously shaken by hand for 30 seconds and left at room temperature for 10 minutes. The tubes were centrifuged at 10,000 g for 20 min. Thereafter, the water layer (top, clean layer) was transferred to a fresh tube without RNase, mixed with an equal amount of isopropanol, and then incubated at 4 ° C for 20 minutes. After centrifuging the cultured tube, the supernatant was removed and the remaining pellet was washed with 70% cold ethanol. All mRNA pellets were obtained and dissolved in RNase-free water, and the dissolved mRNA pellets were used for cDNA synthesis.

All mRNA samples were treated with DNase I and the first strand cDNA was synthesized from a 1 μl aliquot of all mRNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, Calif.). MRNA levels of CIRP, RBM3, and insulin-like growth factor binding protein 2 (IGFBP-2) were measured using SYBR Premix Ex Taq II (Takara, Otsu, Shiga, Japan) and ABI 7900HT Fast Real-time Polymerase Chain Reaction System.

Each of the above samples was subjected to triplicate experiments and the expression of the gene was normalized to the housekeeping gene GAPDH. The sequences of the polymerase reaction primers used for quantification of the genes by real-time PCR are shown in Table 2 below.

Kinds direction SEQ ID NO: The sequence (5'-3 ') of the primer CIRP Forward SEQ ID NO: 1 5'-AGGGCTGAGTTTTGACACCAA-3 ' Reverse SEQ ID NO: 2 5'-ACAAACCCAAATCCCCGAGAT-3 ' RBM3 Forward SEQ ID NO: 3 5'-GAGGGCTCAACTTTAACACCG-3 ' Reverse SEQ ID NO: 4 5'-GACCACCTCAGAGATAGGTCC-3 ' IGFBP-2 Forward SEQ ID NO: 5 5'-AGTGGAATTGCATGGGAAAATCA-3 ' Reverse SEQ ID NO: 6 5'-CAACGGCGGTTTCTGTGTC-3 ' GAPDH Forward SEQ ID NO: 7 5'-AATCCCATCACCATCTTCCA-3 ' Reverse SEQ ID NO: 8 5'-TGGACTCCACGACGTACTCA-3 '

Example  3-2: Quantified mRNA  Analysis of binding protein expression

The level of mRNA of the RNA binding protein gene among all the mRNA quantified by the above Example 3-1 was analyzed.

Specifically, the level of the mRNA of the RNA binding protein gene was normalized based on the mRNA level of the housekeeping gene (GAPDH), and the level of the mRNA of the RNA binding protein gene expressed in the tumor sample was measured using the RNA binding protein gene MRNA levels were compared. A high level in the tumor sample means a high level of mRNA of the RNA binding protein gene and a low level in the tumor sample means a low level of mRNA of the RNA binding protein gene, MRNA levels of binding protein genes were analyzed.

Example  3-3: Correlation between RNA-binding protein and clinicopathological characteristics of patients

In order to confirm whether the prognosis of colorectal cancer can be predicted through the correlation between the RNA-binding protein and the clinical characteristics of the patients, the level of mRNA of the RNA binding protein gene identified in Examples 3-1 and 3-2, The relationship between the clinical characteristics of the selected patients identified in Example 1 was confirmed (Table 3).

RBM3 Low RMB3
High P-value CIRP Low CIRP High P-value IGFBP-2
Low IGFBP-2
High P-value age 0.310 0.461 0.257   ≤60 24 13 30 6 27 9   > 60 42 15 50 8 48 10 south 39 18 0.330 48 9 0.504 31 6 0.307 female 27 10 32 5 44 13 The cancer antigen (ng / mL) 0.038 0.427 0.571   ≤ 5 49 25 63 12 60 15   > 5 17 3 17 2 15 4 CA19-9 (U / dL) 0.238 0.341 0.380   ≤ 20 50 23 64 10 58 16   > 20 16 5 16 4 17 3 T armor 0.371 0.071 0.139   T1 + T2 11 4 15 0 14 One   T3 + T2 55 24 65 14 61 18 N armory 0.503 0.332 0.341   No transition 49 19 59 9 53 15   Presence of transition 17 9 21 5 22 4 M armory 0.719 0.861 0.871 Remote transition
Absent 58 26 71 13 67 17 Remote transition
existence 8 2 9 One 8 2 Lymph invasion 0.099 0.496 0.491   Absent 44 13 49 8 46 11   existence 23 14 31 6 29 8 Histological differentiation 0.275 0.579 0.370   Well differentiation 2 3 5 0 5 0   Moderate differentiation + Almost differentiation X 64 25 75 14 70 19 Size of tumor 0.065 0.562 0.563   ≤ 5 cm 39 10 42 7 38 11   > 5 cm 28 17 38 7 36 9 Site of tumor 0.506 0.614 0.225   Leader 52 22 63 11 61 13   rectal 14 6 17 3 14 6

As shown in Table 3, no correlation was observed between the mRNA levels of the RNA binding proteins RBM3, CIRP, and IGFBP-2 gene and the clinicopathologic characteristics of colon cancer patients.

It was confirmed that the prognosis of colorectal cancer could not be predicted only by the relationship between the RNA binding protein and the clinical pathologic characteristics of the patients.

Example  3-4: RNA binding protein and patients Disease-free survival rate  And The overall survival rate  correlation

To determine whether the prognosis of colorectal cancer can be determined by correlating the levels of RNA-binding protein gene mRNA with the 5-year disease free survival rates (DFS) and overall survival rates (OS) , The correlation between the mRNA level of the RNA binding protein gene identified in Examples 3-1 and 3-2 and the 5-year disease-free survival rate and the total survival rate of the 94 patients selected in Example 1 were confirmed (Table 4) .

N, overall survival rate P-value N, disease free survival rate P-value age 0.870 0.870   ≤60 36 (82.4) 36 (84.4)   > 60 58 (81.8) 58 (83.3) south 57 (78.1) 0.973 57 (84.1) 0.808 female 37 (81.1) 37 (83.7) The cancer antigen (ng / mL) 0.980 0.601   ≤ 5 74 (83.8) 74 (84.2)   > 5 20 (84.2) 20 (78.7) CA19-9 (U / dL) 0.597 0.959   ≤ 20 73 (85.0) 73 (87.4)   > 20 21 (80.0) 21 (81.0) T armor 0.043 0.043   T1 + T2 15 (90.0) 15 (95.0)   T3 + T2 79 (70.7) 79 (75.9) N armory 0.001 0.001   No transition 68 (85.7) 64 (89.7)   Presence of transition 26 (61.0) 27 (63.1) M armory 0.717 0.373   Remote metastasis 84 (84.1) 84 (81.0)   Remote Transition Presence 10 (80.0) 10 (69.9) Lymph invasion 0.071 0.071   Absent 57 (89.2) 57 (89.2)   existence 37 (75.7) 37 (75.7) Histological differentiation 0.933 0.986   Well differentiation 5 (80.0) 5 (80.0) Moderate eruption
+ Almost differentiation X 89 (82.3) 89 (77.9) Size of tumor 0.056 0.007   ≤ 5 cm 49 (89.4) 49 (91.7)   > 5 cm 45 (77.3) 45 (68.9) Site of tumor 0.540 0.970   Leader 74 (84.9) 74 (84.9)   rectal 20 (80.0) 20 (79.7) Expression of RBM3 0.135 0.049   lowness 67 (80.0) 67 (74.2)   height 27 (92.6). 27 (92.6). Expression of CIRP 0.087 0.048   lowness 80 (81.1) 80 (76.3)   height 14 (98.1) 14 (99.7) IGFBP-2 0.172 0.087   lowness 75 (81.1) 75 (76.0)   height 19 (94.7) 19 (94.7)

As a result, a poor prognosis was observed in patients with colorectal cancer when the mRNA levels of RBM3 and CIRP genes were low, and a good prognosis was observed in high cases (Table 4, Figs. 1A to 2B).

Specifically, the 5-year disease-free survival rates of patients with RBM3 gene mRNA levels were 92.6% and 74.2%, respectively, when the mRNA levels of the RBM3 gene were high and 74.2%, respectively Overall survival rate was 92.6% for high mRNA levels of RBM3 gene and 80% for low mRNA levels of RBM3 gene. Overall survival rate and 5-year overall survival rate of high RBM3 gene mRNA levels (Fig. 1 (a) and (b)).

In particular, the 5-year disease-free survival rate and the 5-year overall survival rate of patients according to the level of the mRNA of the CIRP gene show a more significant difference than the level of the mRNA of the RBM3 gene. In conclusion, the 5-year disease-free survival rates of patients with CIRP gene mRNA levels were 99.7% and 76.3%, respectively, when the mRNA level of the CIRP gene was high and the mRNA level of the CIRP gene was low The overall survival rate of the CIRP gene was 98.1% for the high mRNA level and 81.1% for the low mRNA level of the CIRP gene. When the CIRP gene mRNA level was high, And disease-free survival rate were high (Figs. 2a and 2b).

Based on the above results, it was confirmed that the prognosis of patients with colorectal cancer based on mRNA levels of RNA binding proteins, particularly RBM3 and CIRP genes, can be predicted.

Example  3-5: Analysis of prognostic factors of colorectal cancer

Multivariate models were designed to test the independent prognostic factors of colorectal cancer patients and mRNA levels of RNA-binding protein genes (Table 5). In Model A, only the relative risk of pathologic prognostic factors and 95% confidence intervals were identified. In Model B, not only pathological prognostic factors but also mRNA levels of RNA binding protein genes were included, and their relative risk and 95% confidence interval Respectively.

variable P- value Relative risk 95% confidence interval, low 95% confidence interval, high Model A T armor 0.043 1.41 1.17 4.42 N armory 0.002 5.87 2.30 14.9 Size of tumor 0.073 0.91 1.34 5.73 Model B T armor 0.163 1.76 0.73 6.44 N armory 0.001 6.81 3.99 17.4 RBM3 0.024 1.49 1.64 3.55 CIRP 0.020 1.10 1.41 4.51

As a result, we found that the N stage of the pathologic prognostic factors in Model A is the strongest prognostic factor. Furthermore, in Model B, not only N stage but also RBM3 and CIRP could be independent prognostic factors in colorectal cancer.

These results suggest that mRNA levels of RBM3 and CIRP genes are useful prognostic factors for the prognosis of colorectal cancer patients.

<110> GIL MEDICAL CENTER          Gachon University of Industry-Academic cooperation Foundation <120> Compositions for colorectal cancer prognosis prediction          a preparation for measuring the level of expression of          a gene CIRP <130> KPA150525-KR <160> 8 <170> KoPatentin 3.0 <210> 1 <211> 21 <212> DNA <213> Unknown <220> <223> CIRP forward <400> 1 agggctgagt tttgacacca a 21 <210> 2 <211> 21 <212> DNA <213> Unknown <220> <223> CIRP reverse <400> 2 acaaacccaa atccccgaga t 21 <210> 3 <211> 21 <212> DNA <213> Unknown <220> <223> RBM3 Forward <400> 3 gagggctcaa ctttaacacc g 21 <210> 4 <211> 21 <212> DNA <213> Unknown <220> <223> RBM3 Reverse primer <400> 4 gaccacctca gagataggtc c 21 <210> 5 <211> 23 <212> DNA <213> Unknown <220> <223> IGFBP-2 Forward primer <400> 5 agtggaattg catgggaaaa tca 23 <210> 6 <211> 19 <212> DNA <213> Unknown <220> <223> primer <400> 6 caacggcggt ttctgtgtc 19 <210> 7 <211> 20 <212> DNA <213> Unknown <220> <223> primer <400> 7 aatcccatca ccatcttcca 20 <210> 8 <211> 20 <212> DNA <213> Unknown <220> <223> primer <400> 8 tggactccac gacgtactca 20

Claims (12)

A composition for predicting colorectal cancer prognosis, comprising an agent that measures the mRNA level of a cold inducible RNA-binding protein (CIRP) gene or the level of a protein expressed therefrom.
The composition for predicting colorectal cancer prognosis according to claim 1, wherein the agent for measuring the mRNA level of the CIRP gene comprises the primers of SEQ ID NOs: 1 and 2.
The composition for predicting colorectal cancer prognosis according to claim 1, wherein the agent for measuring the level of the CIRP protein comprises an antibody specific to the protein.
The composition for predicting colorectal cancer prognosis according to claim 1, further comprising an agent for measuring an mRNA level of an RNA-binding motif protein 3 (RBM3) gene or a level of a protein expressed therefrom.
A kit for predicting colorectal cancer prognosis, comprising the composition according to any one of claims 1 to 4.
6. The kit for predicting colorectal cancer prognosis according to claim 5, wherein the kit is an RT-polymerase reaction kit, a DNA chip kit or a protein chip kit.
Measuring the mRNA level of the CIRP gene or the level of the protein expressed therefrom from colorectal cancer tissue of a colon cancer patient (step 1); And
Comparing the measured mRNA level or the level of the protein expressed therefrom to the level measured from the normal tissue of the colon cancer patient (step 2)
The method comprising the steps of: (a) providing information for predicting a prognosis of a colon cancer patient.
8. The method according to claim 7, wherein the mRNA level of the gene is determined by quantitative real-time PCR, RT-PCR, competitive RT-PCR, And one or more selected from the group consisting of real time quantitative RT-PCR, RNase protection method, Northern blotting or DNA chip technology assay is used &Lt; / RTI &gt;
8. The method of claim 7, wherein the level of the protein is selected from the group consisting of western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchteroni immunodiffusion Immunohistochemical staining, immunoprecipitation assays, complement fixation assays, immunofluorescence, immunochromatography (immunofluorescence), immunohistochemical staining, immunohistochemical staining, immunohistochemical staining, immunohistochemical staining, immunochromatography, FACS (fluorescence activated cell sorter analysis), and protein chip technology assay.
8. The method according to claim 7, wherein the mRNA level of the CIRP gene measured from the colorectal cancer tissue in step 1 or the level of the protein expressed therefrom is higher than the mRNA level of the CIRP gene measured from the normal tissue in step 2, Wherein the prognosis of colorectal cancer is predicted to be poor when compared to the level of the protein being treated.
8. The method of claim 7, wherein the level of mRNA of the RBM3 gene or the level of the protein expressed therefrom is further measured in steps 1 and 2, respectively.
12. The method according to claim 11, wherein the mRNA level of the RBM3 gene or the level of the protein expressed therefrom further measured from the colorectal cancer tissue in the step 1 is higher than the mRNA level of the RBM3 gene further measured from the normal tissue in the step 2 Or the level of protein expressed therefrom is reduced, the prognosis of colorectal cancer is predicted to be poor.

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