KR20240043173A - MRPL14 gene marker for diagnosis or prediction of Papillary thyroid carcinoma and uses thereof - Google Patents

Abstract

The present invention relates to the MRPL14 genetic marker for diagnosis or prediction of papillary thyroid cancer and its use. It can be used for diagnosis or prediction of papillary thyroid cancer by analyzing the expression level of the MRPL14 gene or its protein, or is also useful in screening for a therapeutic agent for papillary thyroid cancer. It could be utilized.

Description

MRPL14 genetic marker for diagnosis or prediction of papillary thyroid carcinoma and uses thereof {MRPL14 gene marker for diagnosis or prediction of Papillary thyroid carcinoma and uses thereof}

The present invention relates to the MRPL14 genetic marker for diagnosis or prediction of papillary thyroid cancer and its use.

The thyroid gland is a butterfly-shaped organ located 2 to 3 cm above the protruding part of the front of the neck, i.e., the folds of the neck. Its function is to produce and store thyroid hormones and then export them to necessary organs. Thyroid cancer is a general term for cancer that occurs in the thyroid gland and is the most common malignant tumor of the endocrine system. Factors that increase the incidence of thyroid cancer include radiation exposure, genetic factors, and past history of thyroid disease. In particular, exposure to radiation is the most well-known risk factor for thyroid cancer, and it is known that the risk of developing thyroid cancer increases as the dose of radiation exposed increases. In addition, it is assumed that various reasons such as female hormones, iodine intake, obesity, and genetic factors are related, but the relationship has not been clearly proven.

Thyroid cancer is classified into papillary carcinoma, follicular carcinoma, medullary thyroid carcinoma, and anaplastic carcinoma (undifferentiated thyroid cancer) depending on histological shape, cancer origin cell, and degree of differentiation. , Among these, papillary thyroid cancer is the most common. The prognosis varies depending on the type of thyroid cancer, but papillary thyroid cancer, which accounts for 80-90% of thyroid cancer, is easily treated and has a high cure rate. However, because it is cancer, there is a possibility that it will recur or spread to other organs, and if the cancer has spread to various organs throughout the body, such as the lungs or bones, the prognosis is worse than if it is limited to the neck, so the thyroid gland, organs around the neck, and lymph nodes must be examined in detail. You need to be diagnosed.

Meanwhile, Korean Patent No. 2180982 discloses 'ADM2 genetic marker for diagnosing or predicting prognosis of thyroid cancer and its use', and Korean Patent No. 2321571 discloses 'Thyroid cancer containing an agent capable of detecting mutations in the PLEKHS1 gene. 'Biomarker composition for diagnosis or prediction of prognosis and use thereof' has been disclosed, but there has been no description regarding the MRPL14 genetic marker for diagnosis or prediction of papillary thyroid cancer of the present invention and its use.

The present invention was developed in response to the above-mentioned needs, and the present inventors confirmed that the expression level of MRPL14 (Mitochondrial ribosome protein L14) mRNA was higher in papillary thyroid cancer tissue than in normal thyroid tissue. In addition, when MRPL14 siRNA (small interfering RNA) was transfected into the papillary thyroid cancer cell lines B-CPAP and KTC-1, proliferation, migration, and invasion of B-CPAP and KTC-1 cells occurred. By confirming that this was significantly reduced, the present invention was completed.

In order to solve the above problems, the present invention provides a composition for diagnosing or predicting papillary thyroid cancer, including an agent for measuring the expression level of the mRNA of the MRPL14 (Mitochondrial ribosomal protein L14) gene or its protein.

Additionally, the present invention provides a kit for diagnosing or predicting papillary thyroid cancer comprising the composition.

In addition, the present invention provides the following steps: (1) measuring the expression level of the mRNA of the MRPL14 gene or the protein expressed from the gene from an isolated biological sample from a patient suspected of having papillary thyroid cancer; (2) comparing the expression level with a normal control sample; and (3) determining papillary thyroid cancer when the expression level is higher than that of a normal control sample.

In addition, the present invention provides a screening method for a therapeutic agent for papillary thyroid cancer, comprising measuring the expression level of the MRPL14 gene or the expression level of the protein encoded by the gene after administration of a candidate substance expected to treat papillary thyroid cancer. do.

In addition, the present invention provides a pharmaceutical composition for preventing or treating papillary thyroid cancer, comprising an expression or activity inhibitor of MRPL14 protein as an active ingredient.

The MRPL14 gene or its protein of the present invention is a novel biomarker for diagnosis or prediction of papillary thyroid cancer. It can be used for diagnosis or prediction of papillary thyroid cancer by analyzing the expression level of the MRPL14 gene or its protein, or as a therapeutic agent for papillary thyroid cancer. It is expected that it can be useful for screening as well.

Figure 1 shows the results of confirming the expression of MRPs (mitochondrial ribosome proteins) in tumor samples from papillary thyroid cancer patients and normal thyroid samples from TCGA (The Cancer Genome Atlas). (A) is a schematic diagram showing the process of analyzing MRPs transcripts in tumor samples and normal samples from patients with papillary thyroid cancer, and (B) shows the expression level of MRPs between tumor samples from patients with papillary thyroid cancer and normal thyroid samples. This is the result, (C) is the result of calculating the maximum absolute deviation (MAD) for the 78 MRPs, and (D) is the result of calculating the maximum absolute deviation (MAD) for the 78 MRPs for normal samples (n=57) and tumor samples (n=500). This is a heat map showing the expression level, and (E) is a normal MRP for variable MRPs (n=32) selected according to the application of a cut-off of maximum absolute deviation (MAD)>0.25 among 78 MRPs. Expression levels of up-MRPs (n=9), dn(down)-MRPs (n=3), and other MRPs (n=20) classified through permutation t -test between samples and tumor samples were measured in thyroid cancer patients. The heatmap shown is (left), and (F) is a graph showing the fold change (FC) for the expression of up-MRPs, dn-MRPs, and other MRPs between normal and tumor samples.
Figure 2 shows the results of confirming the expression level of MRPL14 in tumor samples from thyroid cancer patients and normal thyroid samples. (A) shows the results of confirming differentially expressed genes (DEGs) showing differences in expression between tumor samples and normal samples from TCGA thyroid cancer patients, and (B) shows the expression level of MRPL14 in tumor samples and normal samples from TCGA thyroid cancer patients. This is the confirmed result, and (C) is the result of measuring the expression levels of the MRPL14 gene (left) and its protein (right) in tumor samples and normal samples from thyroid cancer patients at Chungnam National University Hospital (CNUH).
Figure 3 shows the expression of MRPL14 mRNA in normal cell lines (N-thy-ori 3.1), papillary thyroid cancer cell lines (B-CPAP, TPC-1, KTC-1), and anaplastic thyroid cancer cell lines (FRO, SW1736, 8505C). These are the results of RT-PCR (Reverse transcription polymerase chain reaction) confirming the level (A), and the results of Western blot (Western bolt) confirming the expression level of MRPL14 protein (B).
Figure 4 shows Western blot (A) and CCK after transfection of MRPL14 siRNA (siMRPL14-#1 or #2 in Table 1) or negative control siRNA (siNC) into papillary thyroid cancer cell lines B-CPAP and KTC-1, respectively. This is the result of performing the -8 assay (B) and cell counting assay (C).
Figure 5 shows cell migration (migration, A) after transfection of MRPL14 siRNA (siMRPL14-#1 or #2 in Table 1) or negative control siRNA (siNC) into papillary thyroid cancer cell lines B-CPAP and KTC-1, respectively. ) and invasion (B).

In order to achieve the object of the present invention, the present invention provides a composition for diagnosing or predicting papillary thyroid cancer, including an agent for measuring the expression level of the mRNA of the MRPL14 (Mitochondrial ribosomal protein L14) gene or its protein.

In the composition of the present invention, the MRPL14 gene may preferably have the base sequence of SEQ ID NO: 1, but is not limited thereto. Additionally, homologs of the above base sequence are included within the scope of the present invention. Specifically, the gene includes a base sequence having sequence homology of at least 70%, preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with the base sequence of SEQ ID NO: 1. can do. The “% sequence homology” for a polynucleotide is determined by comparing a comparison region with two optimally aligned sequences, wherein a portion of the polynucleotide sequence in the comparison region is a reference sequence (additions or deletions) for the optimal alignment of the two sequences. may contain additions or deletions (i.e. gaps) compared to those that do not contain .

SEQ ID NO: 1 is the coding sequence (CDS) of the MRPL14 gene (accession No. NM_001318767.2) registered in NCBI (National Center for Biotechnology Information) GenBank.

Additionally, in the composition of the present invention, the MRPL14 protein may preferably have the amino acid sequence of SEQ ID NO: 2, but is not limited thereto. The scope of the MRPL14 protein according to the present invention includes a protein having the amino acid sequence shown in SEQ ID NO: 2 and functional equivalents of the protein. “Functional equivalent” means at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 90% or more, as a result of addition, substitution or deletion of amino acids, and the amino acid sequence shown in SEQ ID NO: 2. refers to a protein that has more than 95% sequence homology and exhibits substantially the same physiological activity as the protein represented by SEQ ID NO: 2.

In the present invention, the term 'diagnosis' means confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, diagnosis is to determine whether papillary thyroid carcinoma has occurred.

In the present invention, 'mRNA expression level measurement' is a process of confirming the presence and expression level of mRNA of the papillary thyroid cancer marker gene ( MRPL14 ) in a biological sample for diagnosis or prediction of papillary thyroid cancer. Preferably, the amount of mRNA is measured. You can find out by measuring. Analysis methods for this include reverse transcriptase polymerase chain reaction (RT-PCR), competitive RT-PCR (competitive RT-PCR), real time quantitative RT-PCR (RT-PCR), and RNase protection assay (RPA). ), Northern blotting, DNA chip, etc., but are not limited to these.

In the present invention, the agent for measuring the mRNA expression level of the MRPL14 gene is not limited thereto, but preferably may be a primer, probe, or antisense oligonucleotide, and the primer, probe, or antisense oligonucleotide is a nucleotide sequence of the MRPL14 gene. For reference, a person skilled in the art can design it using known methods, programs or tools.

In the present invention, the term 'primer' refers to a nucleic acid sequence with a short free 3' hydroxyl group that can form a base pair with a complementary template and serves as a starting point for copying the template strand. refers to a short nucleic acid sequence that functions as a Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature. In the present invention, papillary epithelial adenocarcinoma can be diagnosed or predicted by performing PCR amplification using sense and antisense primers capable of binding to the base sequence of the MRPL14 gene and determining whether the desired product is produced. PCR conditions and lengths of sense and antisense primers can be modified based on those known in the art.

In the present invention, the term 'probe' refers to a nucleic acid fragment such as RNA or DNA that is as short as a few bases or as long as several hundred bases, capable of forming a specific binding to mRNA, and is labeled to confirm the presence or absence of a specific mRNA. You can. Probes may be manufactured in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, etc. In the present invention, hybridization is performed using a probe complementary to the MRPL14 polynucleotide, and papillary thyroid cancer can be diagnosed or predicted based on hybridization. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art.

In addition, in the present invention, 'measuring protein expression level' is a process of confirming the presence and expression level of a protein expressed in the papillary thyroid cancer marker gene ( MRPL14 ) in a biological sample for the purpose of diagnosing or predicting papillary thyroid cancer, This can usually be done by checking the amount of protein. Analysis methods for this include ELISA (enzyme linked immunosorbent assay), Western blot, radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion method, and rocket. ) Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, FACS (Fluorescence Activated Cell Sorting), and protein chip, etc., but are not limited thereto.

In the present invention, the agent for measuring the protein expression level is not limited thereto, but may preferably be an antibody or aptamer that specifically binds to the MRPL14 protein.

In the present invention, the term 'antibody' is a term known in the art and refers to a specific protein molecule directed to an antigenic site. For the purpose of the present invention, an antibody refers to an antibody that specifically binds to the MRPL14 protein, which is a marker of the present invention. This MRPL14 antibody is produced by cloning the entire length or part of the MRPL14 gene into an expression vector according to a conventional method. It can be produced by a conventional method by obtaining a protein encoded by the full length or part of the MRPL14 gene and using the obtained protein as an immunogen (antigen). The form of the antibody of the present invention is not particularly limited, and polyclonal antibodies, monoclonal antibodies, or functional fragments having antigen binding properties are also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies. The functional fragment of the antibody molecule refers to a fragment that possesses at least an antigen-binding function and includes Fab, F(ab'), F(ab')2, and ScFv.

In the present invention, the term 'aptamer' refers to a nucleic acid that can specifically and strongly bind to a specific molecule while maintaining a stable tertiary structure. Because of its specific binding function, it is compared to antibodies and is evaluated as an alternative technology to antibodies.

The present invention also provides a kit for diagnosing or predicting papillary thyroid cancer, including the composition for diagnosing or predicting papillary thyroid cancer.

The kit of the present invention can diagnose or predict papillary thyroid cancer by checking whether the mRNA of the MRPL14 gene or the protein expressed therefrom is detected. The kit for diagnosing or predicting papillary thyroid cancer of the present invention includes an agent for detecting the expression level of the MRPL14 gene (e.g., a primer or probe) or an agent that can specifically detect a protein (e.g., an antibody or aptamer). In addition, one or more other component compositions, solutions, or devices suitable for the analysis method may be included.

In the present invention, the kit for measuring the mRNA expression level of the MRPL14 gene may be a kit containing the essential elements required to perform RT-PCR. In addition to each primer pair specific for the marker gene, the RT-PCR kit contains test tubes or other suitable containers, reaction buffer (pH and magnesium concentration), deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase. It may contain the same enzyme, DNase, RNase inhibitor, DEPC-water, sterilized water, etc. It may also include a primer pair specific for the gene used as a quantitative control.

Additionally, the kit of the present invention may be a diagnostic or predictive kit containing essential elements required to perform a DNA chip. A DNA chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and reagents, agents, enzymes, etc. for producing a fluorescent label probe. Additionally, the substrate may include cDNA corresponding to a quantitative control gene or a fragment thereof.

In addition, when the kit of the present invention is a kit for measuring the expression level of MRPL14 protein, it includes a substrate, an appropriate buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, etc. for immunological detection of the antibody. can do. In the above, the substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide glass, and the coloring enzyme may be peroxidase or alkaline phosphatase. Fatase (Alkaline Phosphatase) can be used, fluorescent substances such as FITC (Fluorescein isothiocyanate), RITC (Rhodamine B isothiocyanate), etc. can be used, and the coloring substrate solution is ABTS (2,2'-azino-bis (3-ethyl Benzothiazoline-6-sulfonic acid) or OPD (o-phenylenediamine) or TMB (tetramethylbenzidine) can be used.

The present invention also,

(1) measuring the expression level of the mRNA of the MRPL14 gene or the protein expressed from the gene from an isolated biological sample from a patient suspected of having papillary thyroid cancer;

(2) comparing the expression level with a normal control sample; and

(3) determining papillary thyroid cancer when the expression level is higher than that of a normal control sample; providing a method of providing information for diagnosing papillary thyroid cancer, including the step.

In the method of providing information for the diagnosis of papillary thyroid cancer of the present invention, the expression level of the mRNA or protein of the MRPL14 gene from the biological sample can be measured by isolating the mRNA or protein of the gene from the biological sample, Isolation of gene mRNA or protein can be performed using methods known in the art.

In the present invention, the term 'biological sample' includes samples such as tissue, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), paraffin tissue, saliva, sputum, or urine. Without being limited thereto, the biological sample may be used in a manipulated or unmanipulated state.

In the method of providing information for the diagnosis of papillary thyroid cancer according to the present invention, the expression level of the mRNA of the MRPL14 gene or the protein expressed from the gene measured from the separated biological sample of the patient suspected of having papillary thyroid cancer is compared to the normal control sample. If the expression level of the mRNA of the MRPL14 gene or the protein expressed from the gene is higher than that measured in , a patient suspected of having papillary thyroid cancer can be diagnosed as having papillary thyroid cancer.

The analysis method for measuring the mRNA level and the protein level are the same as described above, and the expression level of the marker gene or protein in patients with papillary thyroid cancer is compared with the expression level of the marker gene or protein in the normal control group. can be expressed as an absolute (e.g., μg/ml) or relative (e.g., relative intensity of signal) difference.

The present invention also provides a screening method for a therapeutic agent for papillary thyroid cancer, comprising measuring the expression level of the MRPL14 gene or the expression level of the protein encoded by the gene after administration of a candidate substance expected to treat papillary thyroid cancer. do.

Specifically, a therapeutic agent for papillary thyroid cancer can be screened by comparing the change (increase or decrease) in the expression level of the MRPL14 gene or MRPL14 protein in the presence and absence of a candidate substance for the treatment of papillary thyroid cancer, preferably the MRPL14 gene or Substances that indirectly or directly reduce the expression level of MRPL14 protein can be selected as a treatment for thyroid cancer. That is, the expression level of the MRPL14 gene or MRPL14 protein of the present invention is measured in biological samples of patients with papillary thyroid cancer in the absence of a candidate substance for the treatment of papillary thyroid cancer, and the MRPL14 gene or MRPL14 protein of the present invention is measured in the presence of a candidate substance for the treatment of papillary thyroid cancer. After measuring the expression level of the MRPL14 protein and comparing the two, the expression level of the MRPL14 gene or MRPL14 protein of the present invention in the presence of the papillary thyroid cancer treatment candidate is reduced compared to the expression level in the absence of the papillary thyroid cancer treatment candidate. The substance can be predicted as a treatment for papillary thyroid cancer.

The present invention also provides a pharmaceutical composition for preventing or treating papillary thyroid cancer, comprising an expression or activity inhibitor of MRPL14 protein as an active ingredient.

In the pharmaceutical composition of the present invention, when the expression or activity inhibitor of the MRPL14 protein is transfected into B-CPAP and KTC-1 cells derived from papillary thyroid carcinoma, proliferation of B-CPAP and KTC-1 cells , has the effect of suppressing migration and invasion.

In the pharmaceutical composition of the present invention, the expression or activity inhibitor of the MRPL14 protein is an antibody against the MRPL14 protein or a double stranded RNA (dsRNA), short interfering RNA (siRNA), or shRNA (short) that binds complementary to the mRNA of the MRPL14 gene. hairpin RNA) and antisense nucleotides, preferably siRNA consisting of the base sequences of SEQ ID NOs: 3 to 6 (Table 1), most preferably of SEQ ID NOs: 3 and 4. It may be any one selected from the group consisting of a siRNA duplex consisting of base sequences (AM16708A in Table 1) and a siRNA duplex consisting of base sequences of SEQ ID NOs: 5 and 6 (AM16708B in Table 1), but is not limited thereto.

In the siRNA according to the invention, it is possible for the sense RNA strand and/or the antisense RNA strand to contain at least one chemical modification in its sugar moiety, nucleobase portion or internucleotide structure. This modification may make it possible to inhibit destruction of siRNA by nucleases in vivo. All chemical modifications that can improve the stability and biocompatibility of siRNA according to the present invention in vivo are included within the scope of the present invention. The siRNA according to the invention is “isolated”, meaning that it is not in the natural state, but can be obtained by any means involving human intervention. In particular, siRNA according to the present invention can be obtained by chemical synthesis, amplification of the required nucleotide sequence by polymerase chain reaction (PCR), or purifying already existing siRNA by recombinant synthesis.

In addition, the antibody is capable of binding complementary to the MRPL14 protein of the present invention and includes polyclonal antibodies, monoclonal antibodies, and fragments capable of binding to the epitope. The polyclonal antibody can be produced by a conventional method, purified by any method known in the art, and can be used in any animal species host such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, etc. can be made from The monoclonal antibodies can be produced using any technique that provides for the production of antibody molecules through continuous culture of cell lines. The monoclonal antibodies can be produced using any technique that provides for the production of antibody molecules through continuous culture of cell lines. These technologies include, but are not limited to, hybridoma technology, human B-cell hybridoma technology, and EBV-hybridoma technology.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the above active ingredients. In addition, it can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterilized injection solutions according to conventional methods, but is not limited thereto. . Carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, and methyl. Examples include cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract with at least one excipient, such as starch, calcium carbonate, and sucrose. ) or prepared by mixing lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, Witepsol, Macrogol, Tween 61, cacao, laurin, glycerogeratin, etc. can be used.

The appropriate dosage of the pharmaceutical composition according to the present invention is prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. It can be. The concentration of the active ingredient included in the composition of the present invention can be determined considering the purpose of treatment, patient condition, required period, etc., and is not limited to a specific concentration range.

Hereinafter, the present invention will be described in detail through examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

Materials and Methods

1. Cell lines and reagents

Human papillary thyroid carcinoma (PTC) cell lines ‘B-CPAP, KTC-1 and TPC-1’, normal cell lines ‘N-thy-ori 3.1’ and anaplastic thyroid carcinoma (ATC) The cell line 'FRO, SW1736, 8505C' was provided by Professor Ye-eun Kang of Chungnam National University (Korea).

B-CPAP, N-thy-ori3.1, FRO, SW1736, and 8505c cell lines were cultured in RPMI-1640 medium (Gibco, USA, #LM 011-03), and KTC-1 and TPC-1 cell lines were cultured in high-glucose medium. Cultured in DMEM medium (Gibco, #LM 001-05). The cell lines were cultured in a 10 cm tissue culture dish, and treated with 10% FBS (fetal bovine serum; Gibco, #16000044) and 1% penicillin-streptomycin (Gibco, #15140122). In all experiments, cells were cultured under conditions of 37°C, 5% CO ₂ and 95% air.

2. Data and processing

Reads obtained through RNA-sequencing experiments using the TopHat2 v2.1.5 program were aligned to the Homo sapiens reference genome (UCSC Genome Browser Gateway, GRCh37/hg19) sequence using TopHat2 v2.1.5, with the default TopHat parameter options. This was used. In addition, the Tuxedo protocol was used to analyze the differentially expressed gene (DEG) profile between the normal control group and the papillary thyroid cancer experimental group. Aligned reads were processed through Cufflinks v2.2.1 based on FPKM (Fragment per kilobase of transcript per million mapped reads) values, and unbiased and normalized RNA-sequencing fragment counts were used to analyze relative transcript levels. used. A gene transfer format (GTF) file was created to quantitatively compare the transcript level of each sample with that of the reference GTF file. Next, Cuffdiff was used to calculate the difference in FPKM between each group. FDR (false discovery rate)-adjusted p -values (<0.05) were calculated using the Benjamini-Hochberg method, using TCGA (The Cancer Genome Atlas) and Chungnam National University Hospital (CNUH) databases. The role of MRPL14 (Mitochondrial ribosome protein L14) was investigated.

3. Transcriptome profiling for mitochondrial ribosomal proteins (MRPs)

Transcriptome profiling of 78 MRPs was performed. First, a cut-off of maximum absolute deviation (MAD)>0.25 was applied to select variable MRP among 78 MRPs. To identify signatures related to MRP defects in thyroid cancer, permutation t -test was performed between normal thyroid samples and papillary epithelial adenocarcinoma samples, and the selected MRPs were selected according to false discovery rate (FDR) and fold change (FC). They were classified as up-MRPs, dn-MRPs and other MRPs (FDR<0.05 &FC>0.25,FC<-0.25,-0.25<FC<0.25). The up-MRPs refer to up-regulated MRP, and the dn-MRPs refer to down-regualted MRP. If the fold change of tumor tissue compared to normal tissue is positive, it is 'up', and if it is negative, it is 'down'. ', other-MRPs refer to MRPs with a MAD (maximum absolute deviation) of 0.25 or more, an FDR of 0.05 or less, and a fold change that exists between -0.25 and 0.25.

4. Collection of human-derived samples

To analyze the transcriptome and identify differentially expressed genes (DEGs), RNA was extracted from tumor tissue samples and non-tumor tissue samples obtained from thyroid cancer patients (n=364) in the Chungnam National University Hospital (CNUH) database. An RNA library was constructed using the extracted RNA (1 μg) using the TruSeq stranded mRNA Sample Preparation Kit v2 (Illumina, USA), and the quality of the library was analyzed with an Agilent 2100 Bioanalyzer using the Agilent DNA 1000 Kit. Samples from five patients diagnosed with papillary thyroid cancer (PTC) at CNUH were also included, and tumor tissue samples and normal thyroid tissue were collected from PTC patients. These tissue samples were homogenized in RIPA buffer. The present invention was tested with approval from the Institutional Review Board (IRB) of Chungnam National University Hospital (IRB approval number: 2017-07-005), and the human samples used in the present invention were collected from all patients according to the guidelines of Chungnam National University Hospital. It was used after obtaining prior consent from .

5. Small interfiring RNA transfection

Cells were distributed in a 6-well plate at 1×10 ⁵ cells/well and cultured for 24 hours so that each well was occupied by about 60-70% of cells. The next day, transient transfection was performed using Lipofectamine RNAi MAX reagent (Invitrogen, #56532) according to the manufacturer's protocol, and MRPL14 siRNA duplex (AM-16708; Table 1) and negative control siRNA (Negative Control) were performed according to the manufacturer's protocol. siRNA, #SN-1003) was purchased and used from Bioneer (Korea). After 7 to 8 hours, the medium was replaced, and the transfected cells were cultured at 37°C for 48 hours. Afterwards, immunoblot analysis was performed to evaluate the effect and efficiency of siRNA knockdown. In the present invention, the base sequences of SEQ ID NOs: 3 to 6 refer to the sequences in Table 1 below excluding the bases (tt or tg) corresponding to overhangs.

Sequence information of MRPL14 siRNA duplex siRNA Sequence information (5'→3') (SEQ ID NO) AM16708A sense CGAAUUAAGACACCCAUCCtt (3) anti-sense GGAUGGGUGUCUUAAUUCGtg (4) AM16708B sense GGUGGGCGACCAGAUACUAtt (5) anti-sense UAGUAUCUGGUCGCCCACCtt (6)

tt or tg: overhang (protruding part)

6. Cell proliferation assay

After dispensing and culturing cells at 100 μl (8,000 cells) per well of a 96-well plate, the cultured cells were treated with MRPL14 siRNA and negative control siRNA at a concentration of 50 nM, respectively, and reacted for 48 hours. Cell viability was measured at 450 nm using Cell Counting Kit-8 cell proliferation reagent (DOJINDO Lab., #CK04) and ELISA (enzyme-linked immunosorbent assay) reader. The experiment was repeated 3 times, and statistical analysis was performed using t -test.

7. Western blot analysis

After distributing ^and culturing ^the cells at 1.5 Obtained. The obtained cells were treated with RIPA lysis buffer (Thermo Fisher Scientific, #89900) containing a phosphatase inhibitor (Thermo Fisher Scientific, #78427) and a protease inhibitor cocktail (Roche, #11836170001) to lyse the cells. was dissolved. Total protein concentration was measured using the BCA Protein Assay Kit (Thermo Fisher Scientific, #23228).

For Western blotting, the proteins were denatured by applying heat, separated through SDS-PAGE gel electrophoresis, and then transferred to a PVDF membrane (polyvinylidene fluoride membrane; Merck Millipore, USA). After the transfer was completed, the membrane was placed in 0.1% Tween20 (TBST) containing 5% skim milk and reacted at room temperature for 1 hour, then washed with TBST, and the washed membrane was added to the primary antibody and reacted at 4°C overnight. The next day, the membrane was washed three times with TBST, added to secondary antibody (1:1000; Cell Signaling Technology Inc., USA) and reacted at room temperature for 2 hours, then washed again and subjected to enhanced chemiluminescence (ECL) to detect the protein. . The experiment was repeated 3 times, and statistical analysis was performed using t -test.

8. Analysis of migration and invasion of papillary epithelial adenocarcinoma cells

Migration and invasion of B-CPAP and KTC-1 cells were analyzed through transwell assay. Specifically, before performing the transwell assay, MRPL14 siRNA and negative control siRNA were transfected into cells and cultured for 48 hours, and then the transfected cells were transferred to a matrigel-coated transwell (24-well). ; SPL, Korea) was dispensed into the upper chamfer, and 450 ㎕ of medium containing 10% PBS was dispensed into the lower transwell chamfer and cultured for 24 hours at 37°C and 5% CO ₂ conditions. For cell invasion analysis, transwells coated with Matrigel were used in the upper chamber, and for cell migration analysis, transwells without Matrigel coating were used. After culturing, the cells were stained with 1% crystal violet solution and confirmed using an optical microscope (100× magnification). The experiment was repeated 3 times, and statistical analysis was performed using t -test.

9. Statistical analysis

Statistical analysis was performed using SPSS version 22.0.0 (IBM Corp., USA) and GraphPad Prism 6 (GraphPad Software, USA), and unpaired Student t -test was used. Data from three independent experiments were expressed as mean ± standard deviation, and P -value <0.05 indicates statistical significance ( ^* P <0.05, ^** P <0.01, ^*** P <0.001).

Example 1. Confirmation of abnormal expression of MRP in thyroid cancer patients from TCGA (The Cancer Genome Atlas)

To investigate the role of MRP in thyroid cancer progression, transcriptome analysis was performed on tumor samples (n = 500) and normal thyroid samples (n = 57) from patients with papillary thyroid cancer (PTC) in TCGA ( Figure 1A ). , it was confirmed that the expression levels of all 78 MRPs did not differ between tumor samples and normal samples (Figure 1B). However, as a result of measuring the maximum absolute deviation (MAD) between the two samples, it was confirmed that the expression levels of 78 MRPs were more diverse in tumor samples than in normal samples (Figures 1C and 1D). These results mean that in tumor samples, MRP lost its protein integrity and had expression dysregulation of MRP, while normal samples had a moderate protein integrity.

Next, a permutation t -test between normal and tumor samples was performed on variable MRPs (n=32) selected among the 78 MRPs by applying a cut-off of maximum absolute deviation (MAD)>0.25. The selected MRPs were classified into up-MRPs (n=9), dn-MRPs (n=3), and other MRPs (n=20) according to false discovery rate (FDR) and fold change (FC).

In addition, as a result of measuring the expression levels of up-MRPs, dn-MRPs, and other MRPs through gene set variation analysis, the expression levels of up-MRPs were high in most tumor samples, and the expression levels of dn-MRPs were high. The expression level was confirmed to be low (Figures 1E and 1F). The above results show that dysregulation of MRP expression plays an important role in the development and progression of papillary thyroid cancer.

Example 2. MRPL14 Correlation analysis between genes and thyroid cancer

As a result of checking differentially expressed genes (DEGs) between tumor samples from TCGA papillary thyroid cancer patients and normal thyroid samples, MRPL14 was confirmed to be the most up-regulated gene among 9 up-MRPs with potential as an oncogene ( Figure 2A), it was confirmed that the expression level of MRPL14 was significantly higher in tumor samples compared to normal samples (Figure 2B). In addition, as a result of measuring the expression levels of the MRPL14 gene and its protein in tumor samples and normal thyroid samples from thyroid cancer patients at Chungnam National University Hospital (CNUH), the expression levels of the MRPL14 gene and its protein were significantly higher in tumor samples than in normal samples. was confirmed (Figure 2C).

Additionally, to demonstrate the importance of MRPL14 expression using integrative genomic analysis (IGA), we analyzed the correlation between MRPL14 expression levels and clinicopathological parameters in patients with TCGA papillary thyroid cancer. Patients were separated based on the top 25% and bottom 25% of MRPL14 expression.

As a result, as shown in Table 2, the top 25% with high expression of MRPL14 were age, T stage, extrathyroidal extension, lymph node metastasis, and AJCC (American Joint Committee on Cancer). ) was confirmed to be significantly related to stage classification. The above results show that the expression level of MRPK14 is increased in papillary thyroid cancer samples compared to normal samples, indicating that the MRPK14 gene and its protein are new markers for diagnosing or predicting papillary thyroid cancer.

Example 3. In thyroid cancer cell lines MRPL14 Check the expression level of

Normal cell line (N-thy-ori 3.1), papillary thyroid cancer (PTC) cell line (B-CPAP, TPC-1, KTC-1) and anaplastic thyroid carcinoma (ATC) cell line (FRO, SW1736, 8505C) As a result of measuring the expression level of MRPL14 mRNA, it was confirmed that the expression level of MRPL14 mRNA was significantly increased in all thyroid cancer cell lines except the FRO cell line compared to the normal cell line (Figure 3A).

In addition, as a result of measuring the expression level of MRPL14 protein, it was confirmed that the expression level of MRPL14 was significantly increased in PTC cell lines B-CPAP and KTC-1 and ATC cell lines FRO and SW1736 compared to normal cell lines (Figure 3B).

The MRPL14 gene and its protein showed high expression levels not only in papillary thyroid cancer cell lines but also in anaplastic thyroid cancer. However, as described in Example 2 above, the MRPL14 gene of the present invention has a high expression level between tumor samples from patients with papillary thyroid cancer and normal thyroid samples. Since it was confirmed to have the highest potential as an oncogene among differentially expressed genes (DEGs), B-CPAP and KTC-1 cell lines, which highly expressed both the MRPL14 gene and its protein, were used in subsequent experiments among papillary thyroid cancer cell lines.

Example 4. MRPL14 Confirmation of inhibitory effect on proliferation of papillary thyroid cancer cells due to inhibition of expression

After transfecting MRPL14 siRNA (siMRPL14-#1 or #2 in Table 1) or negative control siRNA (siNC) into the papillary thyroid cancer cell lines B-CPAP and KTC-1, respectively, Western blot was performed. As a result, the negative control group In comparison, the MRPL14 expression level was confirmed to be significantly reduced in the MRPL14 siRNA treatment group (Figure 4A).

In addition, as a result of performing CCK-8 assay and cell counting assay on B-CPAP and KTC-1 transfected with MRPL14 siRNA and negative control siRNA, B-CPAP and KTC- in the MRPL14 siRNA treatment group compared to the negative control group. 1 It was confirmed that cell proliferation was significantly reduced (Figures 4B and 4C).

Example 5. MRPL14 Confirmation of the effect of inhibiting the migration and invasion of papillary thyroid cancer cells by inhibiting expression

To confirm the effect of MRPL14 on papillary thyroid cancer metastasis, MRPL14 siRNA (siMRPL14-#1 or #2 in Table 1) or negative control siRNA (siNC) was applied to papillary thyroid cancer cell lines B-CPAP and KTC-1, respectively. After transfection, the migration and invasion of B-CPAP and KTC-1 cells were analyzed.

As a result, it was confirmed that the migration (Figure 5A) and invasion (Figure 5B) of B-CPAP and KTC-1 cells were significantly reduced in the MRPL14 siRNA treated group compared to the negative control group. This shows that MRPL14 promotes the migration and invasion of papillary thyroid cancer cells.

Claims (9)

A composition for diagnosing or predicting papillary thyroid cancer, comprising an agent for measuring the expression level of the mRNA or protein of the MRPL14 (Mitochondrial ribosomal protein L14) gene. The method of claim 1, wherein the agent for measuring the mRNA expression level of the gene is any one selected from the group consisting of primers, probes, and antisense oligonucleotides that specifically bind to the gene. Composition for prediction. The composition for diagnosing or predicting papillary thyroid cancer according to claim 1, wherein the agent for measuring the protein expression level is an antibody or aptamer specific for the protein. A kit for diagnosing or predicting papillary thyroid cancer, comprising the composition according to any one of claims 1 to 3. The kit for diagnosing or predicting papillary thyroid cancer according to claim 4, wherein the kit is an RT-PCR kit, a DNA chip kit, or a protein chip kit. (1) measuring the expression level of the mRNA of the MRPL14 gene or the protein expressed from the gene from an isolated biological sample from a patient suspected of having papillary thyroid cancer;
(2) comparing the expression level with a normal control sample; and
(3) determining papillary thyroid cancer when the expression level is higher than that of a normal control sample. A method of providing information for diagnosing papillary thyroid cancer, including a step. A screening method for a therapeutic agent for papillary thyroid cancer, comprising measuring the expression level of the MRPL14 gene or the expression level of the protein encoded by the gene after administration of a candidate substance expected to treat papillary thyroid cancer. A pharmaceutical composition for preventing or treating papillary thyroid cancer, comprising an expression or activity inhibitor of the MRPL14 protein as an active ingredient. The method of claim 8, wherein the expression or activity inhibitor of the MRPL14 protein is an antibody against the MRPL14 protein or any one selected from the group consisting of dsRNA, siRNA, shRNA, and antisense nucleotides that bind complementary to the mRNA of the MRPL14 gene. A pharmaceutical composition for preventing or treating papillary thyroid cancer.