JP2012170335A - Diagnostic method and therapeutic composition for oral squamous cell carcinoma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To newly discover a gene in an amount of expression in an oral squamous cell carcinoma (OSCC) and provide a method for detecting the OSCC using the gene.SOLUTION: The method for detecting the oral squamous cell carcinoma in a biological sample to be inspected includes following steps: (1) a step for detecting at least one gene expression level selected from the group consisting of AIM2 and IFI16 in the biological sample to be inspected and (2) a step for deciding the presence of the oral squamous cell carcinoma when the expression level has been enhanced.

Description

  The present invention relates to a method for detecting oral squamous cell carcinoma, a diagnostic agent, and a diagnostic kit. Furthermore, this invention relates to the screening method of the compound for treating or preventing oral squamous cell carcinoma, and the composition for treatment or prevention.

  Oral cancer accounts for 1-2% of all cancers (35% of head and neck cancers), and oral squamous cell carcinoma (OSCC) represents 80% of oral cancers. The prognosis of OSCC is relatively good compared to other cancers, but the prognosis is poor for advanced cancer and high-grade cancer. In other words, early detection and early treatment are important factors for determining the prognosis. However, although the technology has evolved year by year with respect to diagnosis, there is still no high accuracy, and visual inspection, palpation, and histopathological diagnosis are the main. In terms of treatment, both techniques and procedures have evolved, but many are based on surgery, and resection of oral tissues significantly lowers the patient's quality of life after surgery. If the tumor is wide, radiation therapy and chemotherapy may be combined, but adjuvant therapy does not lead to complete cure.

  Therefore, finding the causative gene of OSCC and elucidating its function are desired because it can be a pioneer for new diagnosis and treatment methods. Patent Documents 1 to 4 report the following methods as diagnostic methods using OSCC genes.

  In Patent Document 1, the expression profile of 33 marker genes with a clear difference in expression level between OSCC and white plate symptom (LP) which is a precancerous symptom of OSCC is an index for more accurate diagnosis of oral cancer. It has been reported to be useful.

  In Patent Document 2, a genetic abnormality on the genome involved in canceration and malignant transformation of cancer is analyzed, and a cancer-related gene that promotes canceration of cells derived from the oral cavity, that is, a Phosphoribosyl transferase domain containing 1 (PRTFDC1) gene, It has been reported that deletion, inactivation or inactivation of PRTFDC1 gene, that is, reduction of PRTFDC1 protein significantly promotes the growth of oral squamous cell carcinoma. And it has been proposed to detect canceration including malignancy of a specimen by detecting deletion or inactivation of the PRTFDC1 gene.

  Patent Document 3 reports that a cancer-related microRNA gene that promotes canceration of oral mucosal epithelial cells has been identified, and that the decrease in the expression level of these genes promotes the proliferation of oral mucosal epithelial cancer. And a method for detecting cancer has been proposed which includes detecting canceration of a specimen using a decrease in expression of a specific microRNA gene as an index.

  In Patent Document 4, after screening for genes frequently deleted in cancer, COBRA (combined bisulfite restriction analysis) method and RT-PCR method are combined to promote canceration of cells derived from the oral cavity. It has been reported that the identification of the gene, that is, the Melatonin Receptor 1A (MTNR1A) gene, and that the decrease in TNR1A protein significantly promotes the growth of oral squamous cell carcinoma. A method for detecting cancer by detecting the amount of MTNR1A protein has been proposed.

JP 2008-259426 A JP 2008-295327 A JP 2009-171876 JP 2010-35525 A

  However, for a diagnostic method that enables early detection of OSCC, it was necessary to investigate more genes, find the causative gene of OSCC, and elucidate its function.

  Accordingly, an object of the present invention is to newly find a gene whose expression level is changed in OSCC, and to provide a method for detecting oral squamous cell carcinoma, a diagnostic agent and a diagnostic kit using the gene. Furthermore, an object of this invention is to provide the screening method of the compound for treating or preventing oral squamous cell carcinoma, and the composition for treatment or prevention.

  The present inventors performed SNP array analysis using OSCC patient specimens and cell lines, detected DNA copy number abnormal regions, genes that were located in the abnormal regions, and genes that showed abnormal expression by DNA microarray analysis And AIM2 and IFI16 located in the 1q23 region were found. And AIM2 and IFI16 were identified as OSCC causative gene candidates from the results of expression analysis using RT-PCR and realtime PCR. The present invention has been completed based on these findings, and has been completed. The following methods for detecting oral squamous cell carcinoma, diagnostic agents, diagnostic kits, screening methods, and therapeutic or preventive compositions are provided. It is to provide.

(I) Detection method for oral squamous cell carcinoma
(I-1) A method for detecting oral squamous cell carcinoma in a biological sample to be tested, comprising the following steps:
(1) a step of detecting an expression level of at least one gene selected from the group consisting of AIM2 and IFI16 in the biological sample; and (2) when the expression level is increased, oral squamous cell carcinoma is The step of determining that it exists.
(I-2) Detection of the expression level of the gene in the step (1)
MRNA of at least one gene selected from the group consisting of AIM2 and IFI16, or
The method according to (I-1), which is performed by detecting a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16.
(I-3) It is determined that oral squamous cell carcinoma is present when the expression level is increased by 100% or more in comparison with the normal sample in the step (2), (I-1) or (I- The method described in 2).

(II) Diagnostic agents and diagnostic kits
(II-1) a nucleic acid that binds to at least one gene selected from the group consisting of AIM2 and IFI16, or
A diagnostic agent for oral squamous cell carcinoma comprising an antibody or antibody fragment that binds to a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16.
(II-2) a nucleic acid that binds to at least one gene selected from the group consisting of AIM2 and IFI16, or
A diagnostic kit for oral squamous cell carcinoma comprising an antibody or antibody fragment that binds to a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16.

(III) Screening method
(III-1) A method for screening a compound for treating or preventing oral squamous cell carcinoma comprising the following steps:
(1) contacting a candidate compound with a cell that expresses at least one gene selected from the group consisting of AIM2 and IFI16, and (2) comparing the gene of said gene with that in the absence of the candidate compound. Selecting a compound that reduces the expression level.
(III-2) A method for screening a compound for treating or preventing oral squamous cell carcinoma comprising the following steps:
(1) contacting a candidate compound with a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16;
(2) detecting the binding activity between the protein and the compound, and (3) selecting a compound that binds to the protein.
(III-3) A method for screening a compound for treating or preventing oral squamous cell carcinoma comprising the following steps:
(1) contacting a candidate compound with a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16;
(2) a step of detecting the cell proliferation activity of the protein obtained in step (1), and (3) a compound that suppresses the cell proliferation activity of the protein as compared with the case where no candidate compound is present. Process.

(IV) Therapeutic or prophylactic composition
(IV-1) A composition for the treatment or prevention of oral squamous cell carcinoma comprising siRNA, shRNA, dsRNA or antisense polynucleotide for at least one gene selected from the group consisting of AIM2 and IFI16.
(IV-2) A composition for the treatment or prevention of oral squamous cell carcinoma comprising an antibody or antibody fragment that binds to a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16.

  According to the present invention, new causal genes (AIM2 and IFI16) of OSCC can be found, and oral squamous cell carcinoma can be detected by using the increased expression of the gene as an index. In addition, by using AIM2 and IFI16 genes, it becomes possible to screen for compounds for treating or preventing oral squamous cell carcinoma. Furthermore, by using a substance (such as shRNA) that inhibits protein production from AIM2 and IFI16 genes, oral squamous cell carcinoma can be treated or prevented.

A: The figure which shows the result of having performed SNP array using high molecular weight genomic DNA, and analyzing the obtained data using the CNAG system, B: The figure which shows the gene map of 1q23. 2 is a graph showing the results of confirming the expression levels of AIM and IFI16 mRNA by Realtime PCR. A: AIM2 (patient sample), B: IFI16 (patient sample), C: AIM2 (cell line), D: IFI16 (cell line) A: graph showing the results of cell proliferation assay, B: diagram showing the results of agarose gel electrophoresis of cDNA, C: graph showing the results of cell cycle evaluation, D, E: cells undergoing apoptosis by Annexin V staining It is a graph which shows the detection result of. A: Diagram showing the results of detection of Iκβα by Western blotting, B: Graph showing the results of cell proliferation assay using NFκβ inhibitor, C: Diagram showing the results of detection of Iκβα by Western blotting when using NFκβ inhibitor , D: Diagram showing the detection result of Iκβα by Western blotting when shAIM2 or shIFI16 is added to SAS, E: Graph showing the result of luciferase assay for examining the transcriptional activity of NFκβ (left: LPS (−), right) : LPS (+)).

  Hereinafter, the present invention will be described in detail.

  In the present invention, “gene” includes double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof unless otherwise specified. In the present invention, “gene” refers to a regulatory region, a coding region, an exon, and an intron without distinction unless otherwise specified.

  In the present invention, “nucleic acid”, “nucleotide” and “polynucleotide” are synonymous and include both DNA and RNA, and may be double-stranded or single-stranded.

  AIM2 and IFI16 genes belong to the p200 family from previous reports. The p200 family is an interferon-inducible gene group, and in particular, AIM2 is an important gene that acts as a defense mechanism against bacterial infection that forms Inflammasome.

  Examples of the base sequence (cDNA) of the AIM2 gene include those represented by SEQ ID NO: 1, and the amino acid sequence encoded by the gene is represented by SEQ ID NO: 2 (GenBank Accession No. NM_004833).

  Examples of the nucleotide sequence (cDNA) of the IFI16 gene include those represented by SEQ ID NO: 3, and the amino acid sequence encoded by the gene is represented by SEQ ID NO: 4 (GenBank Accession No. NM_005531).

  In the present invention, the AIM2 gene and the IFI16 gene have the same biological activity as that of the protein consisting of the amino acid sequence represented by SEQ ID NOs: 2 and 4 above, respectively, as long as the encoded proteins are SEQ ID NO: 1 3 may be a mutant of the gene consisting of the base sequence represented by 3.

  Examples of the mutant include (a) 1 or 2 or more, for example, 1 to 50, 1 to 25, 1 to 12, 1 to 9 in the amino acid sequence represented by SEQ ID NOs: 2 and 4, A gene encoding a protein comprising an amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added; (b) a nucleotide sequence represented by SEQ ID NOs: 1 and 3 and 70% or more, 80% or more, 90% As mentioned above, the gene etc. which consist of a base sequence which has 95% or more identity are mentioned.

  In the present specification, proteins encoded by the AIM2 gene and the IFI16 gene are referred to as “AIM protein” and “IFI16 protein”, respectively.

Method for detecting oral squamous cell carcinoma The method for detecting oral squamous cell carcinoma in the biological sample to be tested of the present invention comprises the following steps:
(1) a step of detecting an expression level of at least one gene selected from the group consisting of AIM2 and IFI16 in the biological sample; and (2) when the expression level is increased, oral squamous cell carcinoma is A step of determining that it exists.

The present invention is based on the following discovery.
[1] High expression of AIM2 and IFI16 in OSCC
As a result of comparing the expression of AIM2 and IFI16 in OSCC specimens and cell lines with normal oral tissues by RT-PCR and realtime PCR, the expression was significantly higher.
[2] Increase in apoptosis due to knockdown of AIM2 and IFI16
AIM2 and IFI16 were knocked down by RNA interference, and cell proliferation was compared with control. As a result, cell growth was suppressed regardless of which gene was knocked down. Therefore, we examined changes in the cell cycle and discovered that the ratio of the subG1 phase increased, and as a result of examining the ratio of apoptotic cells by AnnexinV staining, it was found that either gene was knocked down significantly. We found that apoptosis was occurring.
[3] Suppression of NFκβ activation by AIM2 knockdown
It has been reported that AIM2 is originally reported as an intermediary involved in biological defense, and at that time, NFκβ is activated to induce and mature various cytokines. Previous reports have shown that NFκβ is highly activated in various cancers and contributes to anti-apoptosis of cancer cells. In OSCC, NFκβ was constitutively activated, and when NFκβ inhibitor was introduced, apoptosis was induced. A reporter assay revealed that knockdown of AIM2 in OSCC suppressed activation of NFκβ, demonstrating that apoptosis was induced.

  The test subject in the present invention is a mammal (human, monkey, dog, cat, horse, cow, mouse, rat, etc.), particularly a human. The biological sample is a cell, tissue, body fluid or the like isolated from the test subject, preferably a cell, tissue, body fluid or the like isolated from the oral cavity.

  A method for detecting the expression level of a gene is performed by detecting mRNA of at least one gene selected from the group consisting of AIM2 and IFI16, or a protein encoded by the gene. Extraction of mRNA or protein from a biological sample can be performed according to a conventional method.

  The method for detecting mRNA is not particularly limited as long as it is a method capable of quantifying mRNA, and examples thereof include DNA chip method, Northern blot method, realtime PCR method and the like. The method for detecting mRNA here also includes detecting cDNA corresponding to mRNA.

  The method for detecting the protein is not particularly limited as long as it is a method capable of quantifying the protein, and examples thereof include an ELISA method, a Western blotting method, and an analysis method using a protein chip.

  In the present invention, the expression levels of AIM2 and IFI16 genes are increased by comparison with the expression level of normal samples. The expression level of the normal sample is the expression level of the AIM2 and IFI16 genes (preferably cells isolated from the oral cavity) of an individual who does not suffer from oral squamous cell carcinoma. The expression level of the normal sample may be standardized from data obtained in the past.

  In the present invention, the expression level of at least one gene selected from the group consisting of AIM2 and IFI16 is preferably 100% or more, more preferably 150% or more, particularly preferably 200, as compared to the expression level of a normal sample. If it has increased by more than%, it is determined that oral squamous cell carcinoma is present. If the expression level of at least one of AIM2 and IFI16 genes meets the above criteria, it may be determined that oral squamous cell carcinoma is present, but the expression levels of both AIM2 and IFI16 genes meet the above criteria. It is desirable to satisfy.

Diagnostic Agent and Diagnostic Kit The diagnostic agent or diagnostic kit for oral squamous cell carcinoma of the present invention is selected from a nucleic acid that binds to at least one gene selected from the group consisting of AIM2 and IFI16, or a group consisting of AIM2 and IFI16 An antibody or an antibody fragment that binds to a protein encoded by at least one of the above-mentioned genes.

  A nucleic acid that binds to at least one gene selected from the group consisting of AIM2 and IFI16 is a polynucleotide that can hybridize to the gene, and is used for DNA chip method, Northern blot method, realtime PCR method, etc. By detecting mRNA, the expression level of the gene can be detected.

Antibodies or antibody fragments that bind to AIM2 and IFI16 proteins can be obtained according to known methods, and the antibody may be either a monoclonal antibody or a polyclonal antibody, or may be a humanized chimeric antibody. Examples of the antibody fragment include Fab, Fab ′, F (ab ′) ₂ , Fv, scFv and the like, and the antibody or antibody fragment may be modified with a fluorescent dye or the like. The antibody or antibody fragment can be used in an ELISA method, Western blotting method, analysis method using a protein chip, or the like to detect a protein and detect the expression level of the gene.

  In the present invention, the level of expression of at least one gene selected from the group consisting of AIM2 and IFI16 in a biological sample to be tested is measured in the same manner as described above using the diagnostic agent or diagnostic kit. When the expression level is detected and compared with a normal sample and the expression level is increased, it can be determined that oral squamous cell carcinoma is present.

Screening Method One embodiment of the method for screening a compound for treating or preventing oral squamous cell carcinoma of the present invention is characterized by comprising the following steps:
(1) contacting a candidate compound with a cell that expresses at least one gene selected from the group consisting of AIM2 and IFI16, and (2) comparing the gene of said gene with that in the absence of the candidate compound. Selecting a compound that reduces the expression level.

  A compound selected by the above method and capable of suppressing the expression of at least one gene selected from the group consisting of AIM2 and IFI16 may be effective for the treatment and prevention of oral squamous cell carcinoma.

  The cells are not particularly limited as long as they express at least one gene selected from the group consisting of AIM2 and IFI16, but cells collected from patients suffering from oral squamous cell carcinoma, oral squamous epithelium Examples include cancer cell lines and cells into which AIM2 and IFI16 genes have been introduced.

  Candidate compounds can also be used without particular limitation, and examples include low molecular compounds, high molecular compounds, biopolymers (proteins, nucleic acids, polysaccharides, etc.), and various compound libraries containing such compounds. Can be used.

  The gene expression level when the candidate compound is brought into contact with the cell is 10% or more, preferably 25% or more, more preferably 50% or more, compared with the case where the candidate compound is not present. Preferably, when it is reduced by 75% or more, the compound can be selected as effective for the treatment and prevention of oral squamous cell carcinoma. The gene expression level can be detected by the method described above.

Another embodiment of the method for screening a compound for treating or preventing oral squamous cell carcinoma of the present invention comprises the following steps:
(1) contacting a candidate compound with a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16;
(2) detecting the binding activity between the protein and the compound, and (3) selecting a compound that binds to the protein.

  The compound that binds to AIM2 and IFI16 proteins selected by the above method can inhibit the activity of the proteins and can be effective in the treatment and prevention of oral squamous cell carcinoma.

  The method for detecting the binding activity between the protein and the compound is not particularly limited as long as it can analyze the binding activity between substances, and examples thereof include surface plasmon analysis (SPR) and phage display.

  The compound can be selected by selecting a compound having a high binding activity on the basis of the binding constant or the like as effective for the treatment and prevention of oral squamous cell carcinoma.

Another embodiment of the method for screening a compound for treating or preventing oral squamous cell carcinoma of the present invention comprises the following steps.
(1) contacting a candidate compound with a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16;
(2) a step of detecting the cell proliferation activity of the protein obtained in step (1), and (3) a compound that suppresses the cell proliferation activity of the protein as compared with the case where no candidate compound is present. Process.

  A compound that suppresses cell proliferation activity of a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16, selected by the above method, can suppress the growth of oral squamous cell carcinoma, and oral squamous cell carcinoma It may be effective in the treatment and prevention.

  The method for detecting the cell proliferation activity of a protein that has come into contact with a candidate compound is not particularly limited as long as it can measure the degree of cell proliferation. For example, the protein is added to a culture solution of normal cells and fixed. The degree of cell proliferation can be determined by counting the number of cells after the day.

  The cell proliferation activity when the candidate compound and the protein are brought into contact with the cell is 10% or more, preferably 25%, compared with the case where the candidate compound is not present and only the protein is brought into contact with the cell. As described above, when it is reduced by 50% or more, more preferably 75% or more, the compound can be selected as effective for the treatment and prevention of oral squamous cell carcinoma.

Composition for treatment or prevention of oral squamous cell carcinoma The composition for treatment or prevention of oral squamous cell carcinoma of the present invention is an siRNA, shRNA, dsRNA or at least one gene selected from the group consisting of AIM2 and IFI16. It is a composition for treatment or prevention of oral squamous cell carcinoma comprising an antisense polynucleotide.

  Another embodiment of the composition for treating or preventing oral squamous cell carcinoma of the present invention is an antibody or antibody that binds to a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16 It is characterized by including a fragment.

  siRNA (short interfering RNA) is a double-stranded RNA having a length of about 20 bases or less, and can be introduced into cells to suppress the expression of a target gene. A specific protein binds to siRNA to form RISC, and this complex recognizes and binds to mRNA having the same sequence as siRNA, and cleaves mRNA at the center of siRNA. The siRNA in the present invention is not particularly limited as long as it can cause RNAi and inhibit the production of proteins from AIM2 and IFI16 genes. For example, it is artificially synthesized or biochemically synthesized. Or a short double-stranded RNA of 10 base pairs or more produced by decomposing a double-stranded RNA of about 40 bases or more in the body. The siRNA sequence and the mRNA sequence to be cleaved as a target are preferably 100% identical, but may be 100% identical as long as the cleavage activity remains.

  shRNA (short hairpin RNA) is a single-stranded RNA that contains a partially palindromic base sequence, so it has a double-stranded structure within the molecule, resulting in a hairpin-like structure of about 20 base pairs or more. It is a molecule. Such shRNA, after being introduced into the cell, is degraded to a length of about 20 bases in the cell and can cause RNAi as well as siRNA. The shRNA in the present invention is not particularly limited as long as it can cause RNAi and inhibit the production of proteins from AIM2 and IFI16 genes, but preferably has a 3 ′ overhanging end, and has a double-stranded portion. Is preferably about 10 nucleotides or more, more preferably about 20 nucleotides or more.

  dsRNA (double-stranded RNA) refers to a molecule that is converted within a cell to produce siRNA. Therefore, in the present invention, dsRNA that generates the siRNA may be used. The dsRNA in the present invention is not particularly limited as long as it can cause RNAi and inhibit the production of proteins from the AIM2 and IFI16 genes, but the dsRNA sequence and the mRNA sequence that is cleaved as a target are 100% identical It is preferable to do. However, as long as the cleavage activity by RNAi remains, it may not necessarily match 100%.

  The antisense polynucleotide used in the present invention only needs to be capable of inhibiting the production of proteins from AIM2 and IFI16 genes. For example, 5 to 100 consecutive nucleotide sequences in the DNA sequences of AIM2 and IFI16 genes It can hybridize to. Further, the antisense polynucleotide used in the present invention may be either DNA or RNA, and may be modified. The number of bases of the antisense polynucleotide is preferably 5 to 50, more preferably 9 to 25.

  In addition, it is considered that the growth of oral squamous cell carcinoma can be suppressed by neutralizing proteins encoded by the AIM2 and IFI16 genes by using the antibody or antibody fragment. The antibody or antibody fragment is the same as described above.

  The composition for treating or preventing oral squamous cell carcinoma of the present invention is administered to mammals including humans, and comprises the above siRNA, shRNA, dsRNA antisense polynucleotide, or antibody or antibody fragment as an active ingredient. Include as. The composition for treatment or prevention of oral squamous cell carcinoma of the present invention can optionally contain biologically acceptable carriers, excipients and the like depending on the use form. The composition for treating or preventing oral squamous cell carcinoma of the present invention can be produced according to conventional means. For example, orally as tablets, capsules, elixirs, microcapsules, etc. with sugar coating or enteric coating as needed, or as an external agent such as an ointment or plaster, transdermally as a spray, inhalant, etc. It can be used nasally or tracheally, parenterally in the form of injections such as sterile solutions and suspensions with water or other pharmaceutically acceptable fluids.

  In addition, the composition for treating or preventing oral squamous cell carcinoma of the present invention can express these in cells or tissues in addition to those containing the siRNA, shRNA, dsRNA or antisense polynucleotide itself. It may contain a viral vector or a viral vector. Examples of the administration method using a non-viral vector include a method of introducing a nucleic acid molecule using a liposome, a microinjection method, a method of transferring a nucleic acid molecule together with a carrier with a gene gun, and the like. Examples of methods of administration using viral vectors include methods using viral vectors such as recombinant adenoviruses and retroviruses, and adenoviruses that have been rendered detoxified by vectors expressing siRNA, shRNA, dsRNA, or antisense polynucleotides. A gene can be introduced into a cell or tissue by introducing it into a retrovirus or the like and infecting the cell or tissue with this virus.

  The therapeutic effect can typically be confirmed by a mouse animal model. Specifically, cell lines obtained by introducing shAIM2, shIFI16, shAIM2 / shIFI16 into oral squamous cell carcinoma cell lines are transplanted into immunodeficient NOG mice and treated by examining tumorigenicity, life expectancy, organ invasion ability, etc. It is possible to confirm the effect.

  The dose of the composition for treating or preventing oral squamous cell carcinoma of the present invention varies depending on the age, sex and other conditions of the patient, the degree of symptoms of the patient, etc. The amount is about 0.1 ng to 100 mg / kg / day, preferably about 1 ng to 100 mg / kg / day, more preferably about 1 ng to 10 mg / kg / day. This dose is the same for other animals.

  Examples are given below to illustrate the present invention in more detail. However, the present invention is not limited to these examples and experimental examples.

Experimental Example 1 Identification of cancer-related gene candidates for oral squamous cell carcinoma (OSCC)
(Method)
・ Genomic DNA preparation
10 ⁶ OSCC cell lines were collected, and the patient specimen was only about 1 cm ^{3 in the} tumor area, and lysis buffer (1M Tris-HCl (pH 7.5), 0.5M EDTA (pH 8.0), 5M NaCl, 10 % SDS) was added, proteinase K was added to a final concentration of 150 mg / ml, and the mixture was slowly stirred for 16 hours at 55 ° C. Thereafter, 2 ml of Tris-HCl saturated phenol was added and stirred with a rotator for 30 minutes, followed by centrifugation at 10,000 rpm for 3 minutes. Only the supernatant was transferred to a new tube, and the same operation was performed in the order of Tris-HCl saturated phenol / chloroform and chloroform. 2 ml of isopropanol was added to the final supernatant and mixed slowly, followed by precipitation at 4 ° C. After precipitation, the mixture was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was discarded and washed with 70% ethanol. Ethanol was completely removed and dried, and dissolved in TE to a concentration of 50 ng / μl, and genomic DNA was extracted. Further, it was confirmed by electrophoresis on an agarose gel whether the extracted genomic DNA was cleaved, and a sample was prepared.

-Microarray analysis The microarray data used was Reference Series: GSE3524 (microarray data for 4 normal oral tissues and 16 OSCC) published in NCBI's GEO (gene expresslon omnibus). Calculate the average gene expression level of 4 normal tissues and 16 OSCC cases. Genes with low expression were selected, and genes with p <0.01 were selected in the t-test.

・ Cell culture method
Ca9-22, HSC2, HSC3, HSQ89, and Sa-3 were cultured in DMEM (Wako Pure Chemical Industries) supplemented with 10% fetal bovine blood (FBS) (manufactured by Nichirei Co., Ltd.). Ho1-u-1, HSC4, and SAS were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) (manufactured by Nichirei Co., Ltd.). All cell lines were purchased from the cell bank of RIKEN.

(result)
Prepared from 20 OSCC tumors and 8 OSCC cell lines (Ca9-22, Ho1-u-1, HSC2, HSC3, HSC4, HSQB9, SAS, Sa-3) to identify OSCC cancer-related gene candidates The SNP array (GeneChip Mapping 250K Array, Affymetrix) was performed using the high molecular weight genomic DNA, and the obtained array data was analyzed using the CNAG system (FIG. 1A). Analyzes selected regions with high frequency of DNA copy number abnormalities in OSCC. Regions in which abnormalities were observed in 14/28 cases or more were identified as 78 in the amplification region and 4 in the deletion region, respectively (Table 1).

  Furthermore, microarray analysis (Gene Expresion Omnibus, Affymetrix) was performed to identify OSCC cancer-related gene candidates from the gene group contained in this abnormal region. In 4 normal oral tissues, only genes with an average expression level of 16 times or more or 1/2 or less and p <0.01 (t-test is unequal variance) were selected for 16 cases of oral squamous cell carcinoma Among them, only genes included in the abnormal region identified by SNP array analysis were further selected. As a result, 26 cancer-related gene candidates (PPT1, PSMB2, IFI16, AIM2, ABHD1O, LPP, HLA-C, LY6E, ASS1, P4HA1, RARRES3, CCNB2, KIAA0101, CTSH, LITAF, KRT19, TOP2A, IFI35, CBX1, SLC16A3, BST2, C3, FTL, SUMO3, LGALS1) were identified (Table 1).

Experimental Example 2. High expression of AMI2 mRNA and IFI16 mRNA in OSCC
(Method)
-RNA preparation Stabilly cultured cell lines were collected by centrifugation, the medium was removed, and then 0.8 mL of TRIZOL (registered trademark) (manufactured by Invitrogen) was added. Regarding the patient specimen, the amount of the tumor was only about 1 cm ³ , 0.8 mL of TRIZOL (registered trademark) (manufactured by Invitrogen) was added, and homogenized with a homogenizer. Next, 200 μL of chloroform was added and centrifuged at 14,000 rpm for 15 minutes, and then the upper layer was transferred to another tube. 2-Propanol 500 μL was added, centrifuged at 15,000 rpm for 10 minutes, and washed with 80% ethanol. The obtained RNA was dissolved in RNase-free water, and the amount of RNA was measured with a spectrophotometer at a wavelength of 260 nm.

・ Realtime PCR
1 μg of the RNA obtained above was converted into cDNA using reverse transcriptase (TaKaRa RNA PCR ^™ kit (AMV) Ver.3.0 (manufactured by Takara Bio Inc.)). Each of the obtained cDNAs was used as a template, ABI PRISM 7000 Sequence Detection System (Applied Biosystems) was used as a measuring instrument, and SYBR Green PCR Master Mix (Applied Biosystems) was used according to the protocol. ABI PRISM 7000 SDS software was used to quantify the expression level. Each cDNA was corrected with β-actin and the data were compared. The primers used are shown below.
β-actin: (forward) GACAGGATGCAGAAGGAGATTACT <SEQ ID NO: 5>, (reverse) TGATCCACATCTGCTGGAAGGT <SEQ ID NO: 6>
IFI16: (forward) AGACTCAGCCTCCCTCTCCT <SEQ ID NO: 7>, (reverse) GCCACTGTTTTCGGGTTCT <SEQ ID NO: 8>
AIM2: (forward) GGCCACCATCTGTTTCTGTT <SEQ ID NO: 9>, (reverse) GCCACTAAGTCAAGCTGAAATG <SEQ ID NO: 10>
(result)
Five of the 26 OSCC cancer-related gene candidates identified were interferon-inducible genes. Abnormalities in these gene groups have been reported in various cancers. Of these, 2 genes (AIM2, IFI16) were located in the 1.3 Mb amplification region of 1q23.2 → 1q23.3, so we decided to focus on these 2 genes (FIG. 1B).

  First, the expression levels of AIM2 and IFI16 mRNA were confirmed by realtime PCR using the above 8 cell lines and 11 patient samples, and some of the cell lines and patient samples were excluded from each gene. It was highly expressed (FIGS. 2A, B, C, D). Two normal oral tissues were used as controls.

Experimental Example 3. Inhibition of cell proliferation and apoptosis induction by knockdown of AIM2 and IFI16 in OSCC
(Method)
-Construction of shRNA expression vector and cell introduction method
RNAi Ready pSIREN-RetroQ-ZsGreen (Clontech) pSIREN-RetroQ-ZsGreen vector BamH1-EcoR1 site, shAIM2 (sense strand: GATCCGGCAAACTACATACTGCAAACATTCAAGAGATGTTTGCAGTATGTAGTTTGCCTTTTTTACGCGTG <SEQATGTT ACT CG shIFI16 (sense strand: GATCCGGCTCCACCCAACAGTTCTTCATTCAAGAGATGAAGAACTGTTGGGTGGAGCCTTTTTTACGCGTG <SEQ ID NO: 13> antisense strand: AATTCACGCGTAAAAAAGGCTCCACCCAACAGTTCTTCATCTCTTGAATGAAGAACTGTTGGGTGGAGCCG <SEQ ID No. 14>, IM, vector sh; Each of these vectors was introduced into SAS cells, which are OSCC cell lines, using a gene introduction system nucleofector (manufactured by Amaxa Biosystems). After culturing for 48 hours, collect only GFP-introduced cells (cells with sh vector introduced) using JSAN Desktop Cell Sorter (Bay Bioscience IV), and culture to prepare SAS / shAIM2 and SAS / shIFI16 did. As a control, a control vector (sh Luc) attached to RNAi Ready pSIREN-RetroQ-ZsGreen (manufactured by Clontech) was introduced by the same technique (hereinafter referred to as SAS / shLuc).

・ RT-PCR
Each cDNA was prepared by the same method as described above, amplified by PCR using this as a template, and the expression level of the gene was confirmed by agarose gel electrophoresis. Specifically, TaKaRaExTaq (manufactured by Takara Bio Inc.) was used as the polymerase, and PCR Thermal Cycler DICE (manufactured by Takara Bio Inc.) was used as the PCR device. In addition, each cDNA was corrected with β-actin before amplification by PCR. The same primer as that used for Realtime PCR was used as the base sequence of the primer used for PCR.

・ Cell proliferation experiment
This was performed by using ce11 counting kit8 (manufactured by Dojindo). Inoculate 100 μl of each 1 × 10 ³ pieces of SAS / shAIM2, SAS / shIFI16, and SAS / shLuc in the growth phase into each well of a 96-well microplate (4 wells per day, 4 days each), 24 Incubate for hours. Thereafter, 10 μl of Cell Counting Kit-8 solution was added to each well, and after 2 hours of color reaction in an incubator, absorbance at 450 nm was measured using a microplate reader. This was done 4 times every 24 hours (4days).

・ Cell cycle analysis
After preparing SAS / shAIM2, SAS / shIFI16, and SAS / shLuc cells, culture for 5 days (until the difference in cell growth begins), collect 1 × 10 ⁶ cells, and wash the cells according to the protocol. Then, 5 ml of ice-cold 70% ethanol was added and stirred. Thereafter, it was incubated at −30 ° C. for 30 minutes. Wash cells, add 200 μl RNase (2.5 μg / ml) / PBS, incubate at 37 ° C for 20 minutes, collect cells, add 500 μl PI (10 μg / ml) PBS solution, and incubate at 4 ° C for 10 minutes in the dark. The analysis was performed with FACScan (Becton Dickinson).

・ Apoptosis detection method
Apoptosis Detection kit (manufactured by MBL) was used. After preparing SAS / shAIM2, SAS / shIFI16, and SAS / shLuc cells, culture for 5 days (until the difference in cell growth begins), collect 1 × 10 ⁶ cells, and wash the cells according to the protocol. Then, 0.5 mL of the attached binding buffer and 5 μ1 of Annexin V-PI were added and incubated in a dark room for 5 minutes. FACScan (Becton Dickinson) counted the number of FL-1 (GFP) -positive and apoptotic AnnexinV-positive cells into which sh vector was introduced
(result)
As described above, high expression of AIM2 and IFI16 in OSCC was proved. Next, to examine the effects of these genes on OSCC proliferation and immortalization, short hairpin RNAs that have the activity of cleaving AIM2 and IFI16 gene mRNAs in AIM2 and IFI16 highly expressed OSCC cell lines SAS cells (shRNA), shAIM2, and shIFI16 were introduced to prepare AIM2 expression-suppressed SAS cell lines (hereinafter referred to as “SAS / shAIM2”) and IFI16 expression-suppressed SAS cell lines (hereinafter referred to as “SAS / shIFI16”). As an evaluation that SAS / shAIM2 suppressed AIM2 expression and SAS / shIFI16 suppressed IFI16 expression, RT-PCR was performed to confirm that expression was suppressed (FIG. 3B). A cell proliferation assay was performed on these cells to evaluate their proliferation ability. It was found that the proliferation was significantly suppressed in SAS / AIM2 and SAS / IFI16 cells compared to control cells (FIG. 3A). Therefore, in order to search for the cause of suppression of cell proliferation, changes in the cell cycle were suspected and evaluated. As a result, in both cases, a significant increase in the subGl phase was observed (FIG. 3C). An increase in apoptotic cells was considered from the increase in the subGl phase. Then, next, apoptosis was examined by AnnexinV staining. Compared with control cells, it was found that SAS / AIM2 and SAS / IFI16 cells were significantly apoptotic (FIG. 3D, E).

  These results suggest that AIM2 and IFI16 genes are involved in cell proliferation and anti-apoptosis in OSCC.

Experimental Example 4 Activation of NFκβ in OSCC dependent on the expression of AIM2 and IFI16
(Method)
・ Western blotting
SDS sample buffer (0.05M Tris-HCl , pH6.8,10% glycerol, 2% SDS, 0.01% bromophenol blue, 0.6% 2-mercaptoethanol) was dissolved cell line ¹⁰⁷ cells, as a template, a polyacrylamide gel Electrophoresis. After transferring it to PVDF membrane, the membrane was blocked with 1% BSA, anti-Iκβα antibody (manufactured by Santa Cruz Biotechnology), and anti-β-actin antibody (manufactured by Sigma Aldrich) as a control at 4 ° C for 16 hours 1 Next antibody reaction was carried out. After washing, secondary antibody reaction was performed with anti-Rabbit antibody and anti-mouse antibody for 2 hours. Lumi-Light Plus (Roche Diagnostics) was used for chemiluminescence detection of the antigen on the membrane, and LAS-3000 (Fuji Film) was used for band detection. Each template was corrected based on the amount of ant-β-actin protein before detecting the amount of Iκβα protein.

・ Cell proliferation experiment using NFκβ inhibitor BAY11-7082 (Calbiochem)
Cell proliferation experiments using the NFκβ inhibitor BAY11-7082 (Calbiochem) were cultured for 48 hours in each culture medium, and the above cell counting kit 8 was used to measure the absorbance of each cell at each concentration in 4 wells. Was measured. A graph was prepared so that the number of cells at each concentration was expressed in% when the cells without BAY11-7082 were taken as 100%. In addition, using HSC4, the amount of Iκβα protein at each concentration when BAY11-7082 was added was measured using the same method as the above Western plot.

  Furthermore, the amount of Iκβα protein in SAS / shAIM2 and SAS / shIFI16 was determined using the same method as in the above Western plot. LPS stimulation was performed by stimulating the culture solution at a concentration of 10 μg / ml, collecting cells 10 minutes later, and detecting the amount of Iκβα protein by Western plotting by the above-described method.

・ Luciferase assay
A luciferase assay was performed to examine the transcriptional activity of NFκβ. SAS / shAIM2, SAS / shIFI16, SAS / shLuc were cultured in a 24-well plate, NFκβ luciferase vector (hereinafter referred to as NFκβ-1uc) (provided by University of the Ryukyus) 0.5 μg and pRL-tk 50 ng as internal control Hi1y It was introduced using a MAX liposome reagent (manufactured by Dojin Research Institute). Cells were cultured for 24 hours after the introduction, and fluorescence intensity was detected according to the protocol using Dual-Luciferase (registered trademark) Reporter Assay System (Promega). A 20 / 20n Luminometer was used for detection. For stimulation with LPS, NFκβ-luc and pRL-tk were introduced in the same manner as described above, and after 12 hours, the cells were stimulated at a concentration of 10 μg / ml. After 24 hours, the cells were collected and subjected to luciferase assay. .

(result)
Since activation of AIM2 / IFI16 acts as a defense mechanism accompanying infection, activation of NFκβ and activation of apoptotic pathway can be considered. Therefore, I examined the activation of NFκβ.

  First, the amount of Iκβα protein was compared between OSCC cell lines and normal oral tissues by Western plotting. In general, the amount of Iκβα protein is decreased particularly in OSCC cells (FIG. 4A), and activation of NFκβ is considered. Next, the effect of NFκβ activation on OSCC was examined. Looking at the cellular response to the NFκβ inhibitor BAY11-7082, HSC4 and SAS suppress cell growth in a dose-dependent manner. However, no change was observed in HSQ89 in which the expression of AIM2 and IFI16 was not observed (FIG. 4B). Using HSC4, the amount of Iκβα protein when BAY11-7082 was added was examined at each concentration to examine whether NFκβ activity was suppressed. As a result, Iκβα increased in a dose-dependent manner (FIG. 4C).

  From the above, it was suggested that activation of NFκβ functions in anti-apoptosis in OSCC. Furthermore, when shAIM2 and shIFI16 were added to SAS, the amount of Iκβα protein increased compared to shLuc (FIG. 4D). In order to confirm this, when the transcriptional activation was examined, the activation of NFκβ was dependent on AIM2 and IFI16 (FIG. 4E). In addition, when LPS stimulation was applied to promote NFκβ activity, there were marked differences in both protein content and transcriptional activity. Thus, it was suggested that activation of NFκβ depends on AIM2 and IFI16 in OSCC.

Summary・ AIM2 and IFI16 were frequently expressed in OSCC, indicating the possibility of new diagnostic methods. When the expression was suppressed, the activity of NFκβ was suppressed, and the activity of NFκβ was dependent on AIM2 and IFI16.
-In addition, cell growth suppression was caused by administration of NFκβ activity inhibitor in OSCC, suggesting that NFκβ activity contributes to anti-apoptosis in OSCC. In other words, suppression of AIM2 and IFI16 expression and suppression of NFκβ binding in OSCC leads to treatment of OSCC.

Claims (9)

A method for detecting oral squamous cell carcinoma in a biological sample to be tested, comprising the following steps:
(1) a step of detecting an expression level of at least one gene selected from the group consisting of AIM2 and IFI16 in the biological sample; and (2) when the expression level is increased, oral squamous cell carcinoma is A step of determining that it exists. Detection of the expression level of the gene in the step (1),
MRNA of at least one gene selected from the group consisting of AIM2 and IFI16, or
The method according to claim 1, which is carried out by detecting a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16. A nucleic acid that binds to at least one gene selected from the group consisting of AIM2 and IFI16, or
A diagnostic agent for oral squamous cell carcinoma comprising an antibody or antibody fragment that binds to a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16. A nucleic acid that binds to at least one gene selected from the group consisting of AIM2 and IFI16, or
A diagnostic kit for oral squamous cell carcinoma comprising an antibody or antibody fragment that binds to a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16. A method for screening a compound for treating or preventing oral squamous cell carcinoma comprising the following steps:
(1) contacting a candidate compound with a cell that expresses at least one gene selected from the group consisting of AIM2 and IFI16, and (2) comparing the gene of said gene with that in the absence of the candidate compound. Selecting a compound that reduces the expression level. A method for screening a compound for treating or preventing oral squamous cell carcinoma comprising the following steps:
(1) contacting a candidate compound with a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16;
(2) detecting the binding activity between the protein and the compound, and (3) selecting a compound that binds to the protein. A method for screening a compound for treating or preventing oral squamous cell carcinoma comprising the following steps:
(1) contacting a candidate compound with a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16;
(2) a step of detecting the cell proliferation activity of the protein obtained in step (1), and (3) a compound that suppresses the cell proliferation activity of the protein as compared with the case where no candidate compound is present. Process.   A composition for treating or preventing oral squamous cell carcinoma comprising siRNA, shRNA, dsRNA or antisense polynucleotide for at least one gene selected from the group consisting of AIM2 and IFI16.   A composition for treating or preventing oral squamous cell carcinoma comprising an antibody or an antibody fragment that binds to a protein encoded by at least one gene selected from the group consisting of AIM2 and IFI16.

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