Colorectal cancer diagnostic composition, and method for detecting diagnostic marker

Abstract

The present invention relates to a colorectal cancer diagnostic composition and method for detecting a diagnostic marker, more specifically to a colorectal cancer diagnostic composition comprising one or more mRNAs selected from the group consisting of lysyl-tRNA synthetase (KRS) and aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1) of a preparation for measuring protein expression levels thereof, and a method for detecting a marker form a sample obtained from a test subject in order to provide information necessary for diagnosing colorectal cancer. The colorectal cancer diagnostic marker comprising KRS and AIMP1, according to the present invention, has raised expression levels of same in the serum of a colorectal cancer patient in comparison to a normal control group. Therefore, whether colorectal cancer is present can be accurately and rapidly determined by measuring the expression levels of one or more markers selected from the group consisting of KRS and AIMP1.

Claims

1. A method for treating a colon cancer in a subject, the method comprising the steps of: (a) obtaining a cell-free sample from a subject suspected of having colon cancer; (b) measuring the level the secreted form of lysyl-tRNA synthetase (KRS) and/or aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1) in the sample; (c) identifying the subject as having an increased level of secreted KRS and/or AIMP1 in the cell-free sample, relative to a normal control sample of a healthy subject; (d) diagnosing the subject with a colon cancer; and (e) treating the diagnosed subject by at least one of (i) administering an effective amount of a therapeutic agent for colon cancer to the diagnosed subject, (ii) conducting a curative surgery, and (iii) conducting a radiation therapy.

2. The method of claim 1, wherein the sample is selected from the group consisting of plasma and serum.

3. The method of claim 1, wherein the protein level is measured by one selected from the group consisting of Western blotting, ELISA, radioimmunoassay, radial immunodiffusion assay, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry, Immunoprecipitation assay, complement fixation assay, FACS and protein chip method.

4. A method for screening an anti-colon cancer agent, the method comprising the steps of: (a) administering an anti-colon cancer agent candidate to a sample obtained from a colon cancer patient; (b) measuring a protein level of at least one secreted protein selected from the group consisting of lysyl-tRNA synthetase (KRS) and aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1) in the sample under the presence or absence of the anti-colon cancer agent candidate; (c) comparing the protein level under the presence of the candidate with the mRNA or protein level under the absence of the candidate; (d) selecting the candidate that decreases the protein level under the presence of the candidate; and (e) determining the anticancer activity of the selected candidate in a cell or an animal.

5. The method of claim 1, wherein the step of measuring the protein level comprises using an antibody specific for the KRS or AIMP1 protein.

6. The method of claim 1, wherein the KRS protein comprises the amino acid sequence of SEQ ID NO: 3 and the AIMP1 protein comprises the amino acid sequence of SEQ ID NO: 4.

7. The method of claim 1, wherein the measuring step uses a kit containing an agent for measuring a protein level of at least one secreted protein selected from the group consisting of lysyl-tRNA synthetase (KRS) and aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1).

8. The method of claim 7, wherein the kit is RT-PCR kit, DNA chip kit or a protein chip kit.

Description

BRIEF DESCRIPTION OF DRAWINGS/FIGURES

(1) FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G and 1H show the serum protein levels of colon cancer patients and normal controls by dot blot, respectively (A: GRS, B: KRS, C: AIMP1, D: HRS, E: WRS, F: CA-19-9, G: TNF-α, H: IL-10).

(2) FIGS. 2A, 2B and 2C are graphs showing an ROC curve of serum protein levels.

MODE FOR CARRYING OUT INVENTION

(3) Hereinafter, the present invention will be described in detail.

(4) However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

(5) Methods

(6) 1. Obtaining of Experimental Samples

(7) Serum from colon cancer patients was obtained from the Samsung Medical Center (Seoul, Republic of Korea) according to the regulations of the Clinical Examination Committee. Samples of 32 normal controls and 164 colon cancer patients were obtained and analyzed.

(8) The clinical information of the subjects participated in the experiment is as shown in Table 1.

(9) TABLE-US-00001 TABLE 1 Clinical and pathologic information of normal controls and colon cancer patients Normal controls Colon cancer patients Number of 32 164 subjcects Gender Male 20 93 Female 12 71 Average age 44.33 ± 9.27 60.2 ± 12.2 Tumor stage Stage I — 14 Stage II — 50 Stage III — 50 Stage IV — 50 Timor size(cm) 5.75 ± 1.89

(10) 2. ELISA Assay

(11) The level of GRS (glycyl-tRNA synthetase), KRS (lysyl-tRNA synthetase), HRS (histidyl-tRNA synthetase), WRS (tryptophanyl-tRNA synthetase), AIMP-1 (aminoacyl-tRNA synthetase complex-interacting multifunctional Protein 1), TNF-α, IL-10 and CA-19-9 secreted in the serum of normal controls and colon cancer patients were analyzed using an enzyme immunoassay kit according to the manufacturer's instructions, respectively. The amount of protein secretion was measured using a microplate reader (TECAN). The manufacturers of each serum protein assay kit are as follows:

(12) GRS, HRS, WRS ELISA kit (Cusabio, China)

(13) AIMP1 ELISA kit (Elab science, China)

(14) KRS ELISA kit (Mybiosource, USA)

(15) TNF-a, IL-10 (BD science, USA)

(16) CA 19-9 (Abnova, Taiwan).

(17) 3. Statistical Analysis

(18) The P value between the proteins secreted in the serum of normal controls and colon cancer patients was analyzed using the Mann-Whitney test/Two-tailed test with XLASTAT software. Dotblot plot, ROC curve, AUC, and standard deviation were analyzed using Graphpad Prism 6 software.

(19) Results

Example 1: Serum Analysis of Normal Controls and Colon Cancer Patients

(20) To search for markers specific for colon cancer, serum proteins were analyzed by enzyme-linked immunosorbent assay (ELISA) for 32 normal controls and 164 colon cancer patients.

(21) The results are shown in Table 2 and FIGS. 1A to 1H, respectively.

(22) TABLE-US-00002 TABLE 2 Serum Protein Analysis of Normal controls and Colon Cancer Patients (pg/ml) Normal controls Colon cancer patients GRS 561.2 ± 137.3 535.5 ± 39.08 KRS 775.6 ± 53.4    507 ± 561.1 WRS 2345 ± 276.2  2628 ± 115.6 HRS 1574 ± 290.1  1302 ± 55.33 AIMP1 1711 ± 143.5  2600 ± 68.32 TNF-α 142.5 ± 20.01 113.6 ± 5.092 IL-10 87.40 ± 13.17 167.9 ± 4.54  CA 19-9 76.63 ± 11.80 284.3 ± 48.03

(23) As shown in Table 2 and FIGS. 1A to 1H, the levels of KRS (lysyl-tRNA synthetase) and AIMP1 (aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1) proteins were significantly increased in the serum of the colon cancer patients compared with those of normal controls. In particular, KRS was found to be significantly superior to CA 19-9, one of the conventional colon cancer diagnostic markers.

Example 2: ROC Curve Assay

(24) As shown in FIGS. 2A to 2C, the AUC of KRS and AIMP1 ROC is greater than 0.6 with p value of less than 0.01, verifying that the KRS and AIMP1 are excellent colon cancer markers since the levels of KRS and AIMP1 in the serum of the colon cancer patients were statistically significantly higher than those of normal controls. In addition, KRS and AIMP1 were found to be better markers than CA-19-9, a conventional biomarker of colon cancer.

INDUSTRIAL AVAILABILITY

(25) The colon cancer diagnostic markers of KRS and AIMP1 according to the present invention are found to have increased expression levels in the serum of colon cancer patients compared with the normal control. Therefore, by measuring the expression levels of at least one markers selected from the group consisting of KRS and AIMP1, the presence or absence of colon cancer can be accurately and rapidly verified. Thus, the colon cancer diagnostic markers according to the present invention are considered to have excellent industrial applicability.