Composition for diagnosing follicular thyroid carcinoma using expression level of aminoacyl-tRNA synthetase-related protein and method for detecting diagnostic marker

Abstract

A method for diagnosing follicular thyroid carcinoma is described. The method measures protein expression levels of an aminoacyl-tRNA synthetase (ARS) or an aminoacyl-tRNA synthetase complex-interacting multifunctional protein (AIMP) in a sample, such as a thyroid tissue sample, from a subject suspected of having follicular thyroid carcinoma. The protein expression levels of ARS or AIMP are compared to the measured protein expression levels in a control.

Claims

1. A method for treating a follicular thyroid carcinoma, the method comprising the steps of: (a) obtaining a thyroid tissue sample from a human subject suspected of having follicular thyroid carcinoma; (b) measuring the protein expression level of an aminoacyl-tRNA synthetase (ARS) or an aminoacyl-tRNA synthetase complex-interacting multifunctional protein (AIMP) in the sample; (c) comparing the measured protein expression level of the subject with that of a control; (d) diagnosing the subject with a follicular thyroid carcinoma when the protein expression level of the subject has a change in comparison with that of the control; and (e) treating the diagnosed subject by conducting at least one of a chemotherapy, a surgery; and a radiation therapy.

2. The method of claim 1, herein the control is a patient identified as having thyroid follicular adenoma.

3. The method of claim 1, wherein the change in the protein expression level is an increase in the expression level of at least one protein selected from the group consisting of Aminoacyl tRNA synthase complex-interacting multifunctional protein 1 (AIMP1), Isoleucyl-tRNA synthetase mitochondrial (IARS mitochondrial), seryl-tRNA synthetase mitochondrial (SARS mitochondria), lysyl-tRNA synthetase (KARS); valyl-tRNA synthetase (VARS), and phenylalanyl-tRNA synthetase alpha subunit (FARSA).

4. The method of claim 1, wherein the change in the protein expression level is a decrease in the protein expression level of at least one protein selected from the group consisting of alanyl-tRNA synthetase (AARS), aspartyl-tRNA synthetase (DARS), bifunctional glutamyl-prolyl-tRNA synthetase (EPRS), tryptophanyl-tRNA synthetase (WARS), glycyl-tRNA synthetase (GARS), isoleucyl-tRNA synthetase cytoplasmic (IARS cytoplasmic), tyrosyl-tRNA synthetase (YARS), asparagyl-tRNA synthetase (NARS), glutaminyl-tRNA synthetase (OARS), arginyl-tRNA synthetase (RARS), seryl-tRNA synthetase cytoplasmic (SARS cytoplasmic) and threonyl-tRNA synthetase (TARS).

5. The method of claim 1, wherein the measurement of the protein expression level is performed by a method selected from the group consisting of western blotting, dot blotting, enzyme-linked immunosorbant assay (ELISA), radio immune assay (RIA), radial immunodiffusion assay, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry, immunoprecipitation, complement fixation assay, Flow Cytometry (FRCS), protein chip and mass spectrometry.

6. The method of claim 1, wherein the aminoacyl-tRNA synthetase (ARS) is at least one selected from the group consisting of Isoleucyl-tRNA synthetase mitochondrial (IARS mitochondrial, GenBank GI No. 94730583), seryl-tRNA synthetase mitochondria (SARS mitochondria, GenBank GI No. 23822219), lysyl-tRNA synthetase (KARS, GenBank GI No. 20178333), valyl-tRNA synthetase (VARS, GenBank GI No. 1194845281), phenylalanyl-tRNA synthetase alpha subunit (FARSA, GenBank GI No. 12643946), alanyl-tRNA synthetase (AARS, GenBank GI No. 115502460), aspartyl-tRNA synthetase (GARS, GenBank GI No. 20178330), bifunctional glutamyl-prolyl-tRNA synthetase (EPRS, GenBank GI No. 288558855), tryptophanyl-tRNA synthetase (WARS, GenBank GI No. 135191), glycyl-tRNA synthetase (GARS, GenBank GI No. 313104283), isoleucyl-tRNA synthetase cytoplasmic (IARS cytoplasmic, GenBank GI No. 239938717), tyrosyl-tRNA synthetase (YAKS, GenBank GI No. 13638438), asparagyl-tRNA synthetase (NARS, GenBank GI No. 3915059), glutaminyl-tRNA synthetase (OARS, GenBank GI No. 1351170), arginyl-tRNA synthetase (BARS, GenBank GI No. 20178331), seryl-tRNA synthetase cytoplasmic (SARS cytoplasmic, GenBank GI No. 19860217) and threonyl-tRNA synthetase (TARS, GenBank GI No. 60267755).

7. The method of claim 1, wherein the aminoacyl-tRNA synthetase complex-interacting multifunctional protein (AIMP) is an aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1, GenBank GI No. 215490009).

8. The method of claim 1, wherein the step of measuring is conducted with a composition comprising an agent for measuring the expression level of an aminoacyl-tRNA synthetase (ARS) or an aminoacyl-tRNA synthetase complex-interacting multifunctional protein (AIMP).

9. The method of claim 8, wherein the aminoacyl-tRNA synthetase (ARS) is at least one selected from the group consisting of Isoleucyl-tRNA synthetase mitochondrial (IARS mitochondrial), seryl-tRNA synthetase mitochondria (SARS mitochondria), lysyl-tRNA synthetase (KARS), valyl-tRNA synthetase (VARS), phenylalanyl-tRNA synthetase alpha subunit (FARSA), alanyl-tRNA synthetase (AARS), aspartyl-tRNA synthetase (DARS), bifunctional glutamyl-prolyl-tRNA synthetase (EPRS), tryptophanyl-tRNA synthetase (WARS), glycyl-tRNA synthetase (GARS), isoleucyl-tRNA synthetase cytoplasmic (IARS cytoplasmic), tyrosyl-tRNA synthetase (YARS), asparagyl-tRNA synthetase (MARS), glutaminyl-tRNA synthetase (OARS), arginyl-tRNA synthetase (BARS), seryl-tRNA synthetase cytoplasmic (SARS cytoplasmic) and threonyl-tRNA synthetase (TARS).

10. The method of claim 8, wherein the aminoacyl-tRNA synthetase complex-interacting multifunctional protein (AIMP) is an aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1).

11. The method of claim 1, wherein the follicular thyroid carcinoma is differentially diagnosed from a follicular adenoma.

12. The method of claim 8, wherein the agent is a peptide, antibody, or aptamer comprising a binding domain specific for the ARS or AIMP protein.

13. The method of claim 8, wherein the composition comprising the agent is contained in a kit for diagnosing a follicular thyroid carcinoma.

Description

BRIEF DESCRIPTION OF DRAWINGS/FIGURES

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 shows the number of proteins identified as a result of proteomic analysis of follicular adenoma (FA) and follicular thyroid carcinoma (FTC) tissues.

(3) FIG. 2 shows mass spectrometer (MS) results comparing the protein level of AIMP1, IARS mitochondrial, SARS mitochondrial, KARS, VARS, and FARSA (FARS alpha subunit) in follicular adenoma (FA) and follicular thyroid carcinoma (FTC) tissues, respectively. The y-axis of the graph represents an ion peak area detected by the mass spectrometer for peptides constituting the proteins, which can be used as a quantitative value.

(4) FIG. 3 shows mass spectrometer (MS) results comparing the protein level of AARS, DARS, EPRS, WARS, GARS, and IASRS cytoplasmic in follicular adenoma (FA) and follicular thyroid carcinoma (FTC) tissues, respectively. The y-axis of the graph represents an ion peak area detected by the mass spectrometer for peptides constituting the proteins, which can be used as a quantitative value.

(5) FIG. 4 shows mass spectrometer (MS) results comparing the protein level of YARS, NARS, QARS, RARS, SARS cytoplasmic, and TARS in follicular adenoma (FA) and follicular thyroid carcinoma (FTC) tissues, respectively. The y-axis of the graph represents an ion peak area detected by the mass spectrometer for peptides constituting the proteins, which can be used as a quantitative value.

(6) FIG. 5 shows Western blot results of AIMP1 protein levels in follicular adenoma and follicular thyroid carcinoma tissues, respectively.

(7) FIG. 6 show the result of Area Under Curve (AUC) calculation and Receiver Operating Characteristic (ROC) analysis via deriving the band intensity via western blot after confirming AIMP1 protein level in follicular adenoma and follicular thyroid carcinoma tissue by western blotting.

(8) FIG. 7 shows the results of an Interactive plotting analysis for classification of follicular adenoma and follicular thyroid carcinoma by the measurement of AIMP1 protein level, confirming that AIMP1 was able to distinguish between follicular thyroid carcinoma and follicular adenoma with sensitivity of 90%, and specificity of 70%.

MODE FOR CARRYING OUT INVENTION

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

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

(11) Method

(12) 1. Clinical Sample

(13) Thyroid tissue samples were used which were donated to Bio-Resource Center of the Asan Medical Center upon consent to donation of human tissue for a study. The study protocol was approved by the Asan Medical Center Institutional Review Board (approval number: 2013-0539). In this study, 10 cases of thyroid tissue collected from follicular thyroid carcinoma (FTC) patients and 10 cases of thyroid tissue collected from follicular adenoma (FA) patients were used as tissue samples. Patients were randomly selected regardless of their age and gender to minimize artificial bias.

(14) 2. Protein Analysis

(15) Tissue homogenization was performed on the thyroid tissue collected from FTC and FA patients, respectively, and protein extraction was the performed with RIPA buffer supplemented with 1% SUS and protease/phosphatase inhibitor cocktail. The extracted proteins were quantitated with BCA, and 100 g of each sample was mixed to prepare a pooling set. Peptides were prepared by the filter-aided sample preparation (FASP) method, followed by Nano LC-Q Exactive mass spectrometry and protein analysis. Specific experimental methods are as follows:

(16) Fractionated on a 20 cm C18 capillary column (OD 360 m, ID 75 m) for 120 minutes;

(17) Fractionated on gradient (5-45% acetonitrile and 0.1% formic acid solution) for 80 minutes;

(18) Collecting data in data dependent acquisition (DDA) mode for Top 5 intensity precursor; and

(19) The collected data were compared with the sequence database using the Proteome discoverer 1.4 program and the peak area was analyzed. DAVID gene ontology analysis was also performed.

(20) 3. Western Blot Analysis

(21) 20 ug of thyroid tissue lysate collected from 10 FTC and 10 FA patients, respectively, were subjected to SDS PAGE electrophoresis. After the electrophoresed PAGE gel was transferred to polyvinylidene difluoride (PVDF) membrane (Millipore), and blocking with Bovine serum albumin was performed. The first reaction was a monoclonal mouse anti-human AIMP1 antibody (1:500) and mouse anti-human beta actin (1:1000) antibody reacted for 6 hours at 4 C., followed by blot detection using goat anti-mouse HRP-conjugated secondary antibody (1:4000). Band Intensity was measured using ImageJ (version 1.48), while Receiver Operating Characteristics (ROC) analysis, Area Under Curve (AUC), and intact plot analysis were performed using MedCalc (version 17.6).

Example 1

Identification of Thyroid Tissue Proteins

(22) A total of 2,909 proteins in follicular adenoma tissues and 2,739 proteins in follicular thyroid carcinoma tissues were identified, respectively, while a total of 4,162 proteins were identified (FIG. 1). This is the most extensive thyroid cancer-related protein analysis results that has not been reported in the art so for.

Example 2

Gene Ontology Analysis

(23) As a result of Gene ontology analysis, the functional classification of proteins related to oxidation/reduction, protein localization, and intracellular transport was prominent in follicular adenoma groups (Table 2), whereas the functional classification of proteolysis, macromolecule catabolic process, RNA processing and cell cycle related proteins was differentiated in follicular thyroid carcinoma groups (Table 1).

(24) TABLE-US-00001 TABLE 1 Proteins that are specifically expressed in FTC Functional annotation (FTC Specific proteome) Term RT Count % P-value Benjamin Proteolysis RT 65 8.9 4.9E3 1.9E1 protein localization RT 59 8.0 1.3E3 6.4E2 establishment of protein RT 56 7.6 2.3E4 1.6E2 localization intracellular transport RT 55 75 6.2E6 6.2E4 protein transport RT 54 7.4 5.8E4 3.4E2 macromolecule catabolic process RT 54 7.4 1.0E3 5.3E2 RNA processing RT 50 6.8 1.6E6 2.1E4 cell cycle RT 50 6.8 6.4E3 2.1E1 negative regulation of RT 49 6.7 3.6E3 1.5E1 macromolecule metabolic process positive regulation of RT 49 6.7 4.9E2 5.9E1 macromolecule metabolic process

(25) TABLE-US-00002 TABLE 2 Proteins that are specifically expressed in FA Functional annotation (FA specific proteome) Term RT Count % P-value Benjamin oxidation reduction RT 56 7.1 4.0E6 5.3E3 protein localization RT 54 6.8 2.3E2 6.2E1 intracellular transport RT 52 6.6 1.4E4 3.9E2 protein transport RT 50 6.3 9.1E3 4.5E1 establishment of protein RT 50 6.3 1.1E2 4.7E1 localization phosphorylation RT 48 6.1 4.3E2 7.0E1 translation RT 45 5.7 1.5E10 3.9E7 biological adhesion RT 45 5.7 1.9E2 5.9E1 cell adhesion RT 45 5.7 2.0E2 5.9E1 vesicle-mediated transport RT 44 5.5 1.0E3 1.5E1

(26) In particular, among the proteins specifically expressed in follicular thyroid carcinoma thyroid tissue, the focal adhesion-related proteins were significantly increased compared to follicular adenoma thyroid tissue. Also, spliceosome-related proteins were found to be specifically increased in follicular thyroid carcinoma.

Example 3

Trend Analysis of ARS (Aminoacyl-tRNA Synthetase)-Related Protein Expression

(27) Among the proteins identified from the analysis result, ARS-related proteins were selected and label free semi-quantification was performed based on the peak area. As a result, 20 ARS and AIMP in follicular adenoma, 21 ARS and AIMP in follicular thyroid carcinoma tissues were identified, and quantitative analysis results are shown in the table below (Table 3, Table 4).

(28) TABLE-US-00003 TABLE 3 ARS and AIMP proteins expressed in FA ARSs in Follicular adenoma tissue Gene Description symbol Uniprot ID Peak Area Alanine-tRNA ligase, cytoplasmic AARS SYAC_HUMAN 1125849500 Aminoacyl tRNA synthase AIMP1 AIMP1_HUMAN 85053716 complex-interacting multifunctional protein 1 Aminoacyl tRNA synthase AIMP2 A8MU58_HUMAN 71773240 complex-interacting multifunctional protein 2 Arginine-tRNA ligase, RARS SYRC_HUMAN 661244666 cytoplasmic Asparagine-tRNA ligase, NARS SYNC_HUMAN 943432533 cytoplasmic Aspartyl-tRNA synthetase, DARS D3DP78_HUMAN 796052746 isoform CRA Bifunctional glutamate/proline- EPRS SYEP_HUMAN 1905052478 tRNA ligase Glycine-tRNA liagase GARS SYG_HUMAN 289437841 Isoleucine-tRNA ligase. IARS J3KR24_HUMAN 5433348221 Cytoplasmic Isoleucine-tRNA ligase, IARS2 SYMI_HUMAN 310599507 mitochondrial Lysine-tRNA ligase KARS SYK_HUMAN 171681552 Phenylalanine-tRNA ligase alpha FARSA B4E363_HUMAN 91568703 subunit QARS protein (Fragment) QARS Q96AW5_HUMAN 382807833 Serine-tRNA ligase, cytoplasmic SARS SYSC_HUMAN 13939590732 Serine-tRNA ligase, SARS2 SYSM_HUMAN 54572943 mitochondrial Threonine-tRNA ligase, TARS SYTC_HUMAN 366450168 cytoplasmic Tryptophan-tRNA ligase, WARS SYWC_HUMAN 1005292231 cytoplasmic Tyrosine-tRNA ligase, YARS SYYC_HUMAN 2381826769 cytoplasmic Tyrosine-tRNA ligase, YARS2 SYYM_HUMAN 78858264 mitochondrial Valyl-tRNA synthetase VARS A2BEY0_HUMAN 112446540 (Fragment)

(29) TABLE-US-00004 TABLE 4 ARS and AIMP proteins expressed in FTC ARSs in Follicular thyroid carcinam tissue Description Gene symbol Uniprot ID Peak Area Aspartate-tRNA ligase, DARS SYDC_HUMAN 676501313 cytoplasmic Isoleucine-tRNA, mitochondrial IARS2 SYIM_HUMAN 405032150 Serine-tRNA ligase, cytoplasmic SARS Q5T5C7_HUMAN 6841913593 Alanine-tRNA ligase, cytoplasmic AARS SYAC_HUMAN 259092335 Asparagine-tRNA ligase, NARS SYNC_HUMAN 699339102 cytoplasmic Tryptophan-tRNA ligase, WARS SYWC_HUMAN 488259749 cytoplasmic Tyrosine-tRNA ligase, cytoplasmic YARS SYYC_HUMAN 28939829 Valine-tRNA ligase VARS B0V043_HUMAN 380930687 Phenylalanine-tRNA ligase alpha FARSA SYFA_HUMAN 116709397 subunit Histidine-tRNA ligase, cytoplasmic HARS J3KNE5_HUMAN 244441275 Threonine-tRNA ligase, TARS B5DEG8_HUMAN 226134539 cytoplasmic FARSB protein (Fragment) FARSB Q9BR63_HUMAN 209463210 Cysteine-tRNA ligase, cytoplasmic CARS B4DKY_HUMAN 64458226 Glutamine-tRNA ligase QARS SYQ_HUMAN 293130531 Serine--tRNA ligase, mitochondrial SARS2 SYSM_HUMAN 3864844815 Glycine-tRNA ligase GARS SYG_HUMAN 276263425 Isoleucine-tRNA ligase, IARS J3KR24_HUMAN 229288160 cytoplasmic Aminoacyl tRNA synthase AIMP1 AIMP1_HUMAN 412726062 complex-interacting multifunctional protein 1 Lysine-tRNA ligase KARS SYK_HUMAN 127812747 Bifunctional glutamate/proline- EPRS SYEP_HUMAN 399164615 tRNA ligase Arginine-tRNA ligase, cytoplasmic RARS SYRC_HUMAN 196971392

(30) In particular, AARS is decreased and AIMP1, KARS and VARS are increased in follicular thyroid carcinoma tissue, compared with follicular adenoma tissue (FIG. 2 and FIG. 3). The increase of AIMP1 and KARS in follicular thyroid carcinoma tissues compared to benign tumors of follicular adenomas was confirmed for the first time in this study.

(31) In addition to, AARS, aspartate-tRNA ligase (DARS), bifunctional glutamate/proline-tRNA ligase (EPRS), tryptophan-tRNA ligase (WARS), glycine-tRNA ligase (GARS), isoleucine-tRNA ligase cytoplasmic (EARS cytoplasmic), tyrosine-tRNA ligase (PARS), asparagine-tRNA ligase (MARS), glutamine-tRNA ligase (QARS), arginine-tRNA ligase (RARS), serine-tRNA ligase cytoplasmic (SARS cytoplasmic), threonine-tRNA ligase (TARS) and the like showed decreased protein levels in follicular thyroid carcinomas compared to follicular adenomas (FIG. 3 and FIG. 4). In contrast, AIMP1, isoleucine-tRNA ligase mitochondrial (IARS mitochondrial), serine-tRNA ligase mitochondrial (SARS mitochondrial), lysine-tRNA ligase (KARS), valine-tRNA ligase (VARS), phenylalanine-tRNA ligase alpha subunit (FARS alpha subunit) and the like showed the opposite tendency (FIG. 2). The proteins showing this difference can be used as biomarkers to differentiate follicular thyroid carcinoma from follicular adenomas, and sequence information of the biomarkers discovered in the present invention is provided in Table 5 below.

(32) TABLE-US-00005 TABLE 5 seq. reference Classification Gene symbol (GenBank GI No.) up AIMP1 215490009 IARS mitochondrial 94730583 SARS mitochondria 23822219 KARS 20178333 VARS 1194845281 FARSA 12643946 down AARS 115502460 DARS 20178330 EPRS 288558855 WARS 135191 GARS 313104283 IARS cytoplasmic 239938717 YARS 13638438 NARS 3915059 QARS 1351170 RARS 20178331 SARS cytoplasmic 19860217 TARS 60267755

Example 4

Quantitative Analysis of AIMP1 Protein in FA and FTC Groups

(33) Among the FTC and FA-differentiating biomarkers in Example 3, AIMP1 was used as a representative, to confirm its detection ability described. The amount of AIMP1 protein in 10 follicular adenoma tissues and 10 follicular thyroid carcinoma tissues was analyzed by Western blot, respectively. Representative analysis results are shown in FIG. 5. Receiver Operating Characteristic (ROC) analysis was performed by deriving the band intensities and Area Under Curve (AUC) was calculated. As a result, the AUC was 0.770 and the significance level was 0.0195 (see FIG. 6). As shown in FIG. 7, the result of the interactive plotting analysis showed that the AIMP1 can distinguish the follicular thyroid carcinoma from follicular adenoma with the sensitivity of 90% and the specificity of 70%.

INDUSTRIAL APPLICABILITY

(34) As described above, the ARS or AIMP protein types disclosed in the present invention are diagnostic markers that can differentially detect benign tumors such as follicular adenomas without tissue collection through surgery for follicular thyroid carcinoma in which a simple and definite diagnosis method is not available, thus being able to be used in fields such as an in vitro diagnostic industry.