JP2008169132A - APPLICATION OF Lin7c TO METASTASIS DIAGNOSIS OF CANCER - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To specify a gene having the relationship with presence or absence and degree of lymph node metastasis and recovery from the lymph node metastasis. <P>SOLUTION: The identification is succeeded in an Lin7c gene in which the relationship with the presence or absence and degree of the lymph node metastasis and recovery from the lymph node metastasis is not suggested as a gene significantly changing the expression level between a cancer patient group developing the lymph node metastasis and a patient group without developing the lymph node metastasis. Furthermore, it is proved that prophylaxis and recovery from the lymph node metastasis can be carried out by raising the expression level of the Lin7c gene. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an agent for improving lymph node metastasis which prevents further metastasis and spread of lymph node metastasis, a prophylactic agent which can effectively prevent lymph node metastasis, and a gene therapy vector which can be used for these, and lymph node metastasis The present invention relates to a diagnostic agent and a lymph node metastasis determination method for the presence or absence of lymph node metastasis applicable to the diagnosis of metastasis, and further to a screening method for a lymph node metastasis inhibitor.

  Metastasis refers to a mode in which a tumor progresses discontinuously, and tumor cells leave the primary lesion and spread to other organs / tissues. Among them, lymph node metastasis is a tumor cell that enters the lymphatic vessel at the primary site, is transported to the lymph fluid, and migrates to the downstream lymph node, forming a mass with the second and third growth sites.

  Methods for diagnosing lymph node metastasis include computed tomography (CT), magnetic resonance imaging (MRI), ultrasonography (US), and scintigraphy as imaging tests, and biopsy tests during surgery There is a method for pathological diagnosis of lymph nodes that receive lymph from cancer lesions. Treatment is surgically lymph node dissection, in addition to chemotherapy and radiation therapy. By the way, as a membrane protein containing one PDZ domain, a membrane protein called Lin7c is known as one of Lin7 discovered in nematodes. Lin7 binds to LET23, a nematode homologue of the EGF receptor via the PDZ domain, contributes to its localization, and is involved in intercellular signals for cell proliferation and differentiation. There is no finding that mammalian Lin7 is involved in the localization of EGF receptors. However, it is involved in the localization of epithelial cells by binding to GABA receptors and suppressing their endocytosis. In addition, the PDZ region is used as a scaffold to bind to proteins containing various PDZ regions. See Non-Patent Documents 1 to 3.

  There is no suggestion of the relationship between such a membrane protein containing one PDZ domain, particularly the Lin7 protein, and the onset of lymph node metastasis as described above.

Butz S, Okamoto M, Sudhof TC.A tripartite protein complex with the potential to couple synaptic vesicle exocytosis to cell adhesion in brain.Cell. 1998 Sep 18; 94 (6): 773-82. Simske JS, Kaech SM, Harp SA, Kim SK. LET-23 receptor localization by the cell junction protein LIN-7 during C. elegans vulval induction. Cell. 1996 Apr 19; 85 (2): 195-204. Perego C, Vanoni C, Villa A, Longhi R, Kaech SM, Frohli E, Hajnal A, Kim SK, Pietrini G. PDZ-mediated interactions retain the epithelial GABA transporter on the basolateral surface of polarized epithelial cells.EMBO J. 1999 May 4; 18 (9): 2384-93

  Therefore, the present invention specifies a gene having a relationship with the presence and extent of lymph node metastasis, an improving agent for preventing further metastasis / spreading of lymph node metastasis, and a prophylactic agent capable of effectively preventing lymph node metastasis And it aims at providing the vector for gene therapy which can be used for these. In addition, the present invention specifies a gene having a relationship with the presence and degree of lymph node metastasis, and can be applied to the diagnosis of lymph node metastasis. An object of the present invention is to provide a screening method for a node metastasis inhibitor.

  As a result of intensive studies by the present inventors in order to achieve the above-described object, as a gene whose expression level was significantly changed between a cancer patient group that developed lymph node metastasis and a cancer patient group that did not develop lymph node metastasis. The present inventors have succeeded in identifying a specific gene that has not been suggested to be associated with the presence and extent of lymph node metastasis. Furthermore, the present inventors succeeded in demonstrating that lymph node metastasis can be prevented by enhancing the expression level of the gene, and completed the present invention.

  That is, the present invention includes the following.

(1) An agent for improving lymph node metastasis comprising, as an active ingredient, a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence substantially identical to the amino acid sequence.
(2) The agent for improving lymph node metastasis according to (1), wherein the polynucleotide is supported on a gene therapy carrier.
(3) The agent for improving lymph node metastasis according to (2), wherein the carrier for gene therapy is a viral vector.
(4) The improving agent according to (1), wherein the lymph node metastasis is lymph node metastasis of squamous cell carcinoma that develops in the oral cavity, pharynx, esophagus or lung.

(5) A preventive agent for lymph node metastasis comprising, as an active ingredient, a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence substantially identical thereto.
(6) The preventive agent for lymph node metastasis according to (5), wherein the polynucleotide is carried on a carrier for gene therapy.
(7) The preventive agent for lymph node metastasis according to (6), wherein the carrier for gene therapy is a viral vector.
(8) The lymph node metastasis preventive agent according to (5), wherein the lymph node metastasis is lymph node metastasis of squamous cell carcinoma that develops in the oral cavity, pharynx, esophagus or lung.
(9) A gene therapy vector having a functioning gene comprising a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 or a substantially identical amino acid sequence thereto.

(10) The gene therapy vector according to (9), which is a viral vector.
(11) The gene therapy vector according to (9), which is used for lymph node metastasis cases.
(12) The gene therapy vector according to (9), wherein the lymph node metastasis is lymph node metastasis of squamous cell carcinoma that develops in the oral cavity, pharynx, esophagus or lung.
(13) A diagnostic agent for the presence or absence of lymph node metastasis, comprising a reagent capable of measuring the amino acid sequence represented by SEQ ID NO: 2 or a protein consisting of the amino acid sequence substantially identical thereto.
(14) The diagnostic agent for the presence or absence of lymph node metastasis according to (13), wherein the reagent contains an antibody against a protein that is an anti-Lin7c antibody.

(15) The diagnostic agent for the presence or absence of lymph node metastasis according to (13), wherein the lymph node metastasis is lymph node metastasis of squamous cell carcinoma that develops in the oral cavity, pharynx, esophagus or lung.
(16) Diagnosis of the presence or absence of lymph node metastasis, including a reagent capable of measuring the expression level of the gene comprising the amino acid sequence represented by SEQ ID NO: 2 or a polynucleotide encoding a substantially identical amino acid sequence thereto medicine.
(17) The reagent according to (16), wherein the reagent contains a single-stranded or double-stranded polynucleotide that specifically hybridizes to the mRNA of the polynucleotide, or a label thereof. A diagnostic agent for the presence or absence of lymph node metastasis.
(18) The diagnostic agent for the presence or absence of lymph node metastasis according to (16), wherein the lymph node metastasis is lymph node metastasis of squamous cell carcinoma that develops in the oral cavity, pharynx, esophagus or lung.
(19) a step of measuring the expression level of a gene containing a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 or substantially the same amino acid sequence in a biological sample derived from a patient to be examined; And determining the presence and / or possibility of lymph node metastasis in the patient to be examined based on the expression level of the lymph node metastasis.

(20) The method for determining lymph node metastasis according to (19), wherein the lymph node metastasis is lymph node metastasis of squamous cell carcinoma that develops in the oral cavity, pharynx, esophagus or lung.
(21) A step of bringing a test substance into contact with a cancer cell-derived cell line, and a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 or substantially the same amino acid sequence in the cell line A lymph node comprising: a step of detecting gene expression; and a step of identifying the test substance as a candidate for a lymph node metastasis inhibitor when the expression level of the gene is promoted by the test substance. A screening method for metastasis inhibitors.
(22) The method for screening a lymph node metastasis inhibitor according to (21), wherein the lymph node metastasis is lymph node metastasis of squamous cell carcinoma that develops in the oral cavity, pharynx, esophagus or lung.

Hereinafter, the present invention will be described in detail.
The present invention is based on the knowledge that lymph node metastasis can be prevented by identifying a gene showing a profile such that the expression level is significantly decreased in a group of cancer patients who have developed lymph node metastasis and enhancing the expression of the gene. Is. In the present invention, metastasis refers to a mode in which a tumor progresses discontinuously, and tumor cells are separated from the primary lesion and spread to other organs / tissues. Among them, lymph node metastasis is a tumor cell that enters the lymphatic vessel at the primary site, is transported to the lymph fluid, and migrates to the downstream lymph node, forming a mass with the second and third growth sites. Due to the increased expression of Lin7c, tumor cells bind firmly to each other in the primary lesion or metastatic lymph node, making it difficult to invade lymphatic vessels or infiltrate surrounding tissues, thereby preventing further metastasis.

  In particular, in the present invention, Lin7c is a major factor of the cell adhesion factor cadherin, and it has been clarified that metastasis, prevention, and treatment of squamous cell carcinoma can be performed by measuring or regulating the expression level. The lymph node metastasis targeted in the present invention has cadherin acting as a main mechanism for cell adhesion in solid cancer, particularly squamous cell carcinoma. For this reason, it is considered that the present invention extends to solid cancers, particularly squamous cell carcinomas that develop in the oral cavity, pharynx, esophagus, lungs and the like.

  According to the present invention, there are provided an improving agent for preventing further metastasis / spreading of lymph node metastasis specified by the above definition, a prophylactic agent capable of effectively preventing lymph node metastasis, and a gene therapy vector usable for them. Can be provided. In addition, according to the present invention, it is possible to provide a diagnostic agent and a method for determining lymph node metastasis that can diagnose the presence and degree of lymph node metastasis specified by the above definition. Furthermore, according to the present invention, it is possible to provide a screening method for a novel lymph node metastasis inhibitor capable of preventing lymph node metastasis specified by the above definition. Hereinafter, these pharmaceutical-related inventions provided by the present invention will be specifically described.

Lymph node metastasis-improving agent and prophylactic agent As shown in the Examples described later, it was revealed that the decrease in Lin7c gene expression is involved in the development of lymph node metastasis. Here, the Lin7c gene encodes a protein having a PDZ domain that is a protein binding site. The nucleotide sequence of the human-derived Lin7c gene is shown in SEQ ID NO: 1, and the amino acid sequence of the human-derived Lin7c protein encoded by the human-derived Lin7c gene is shown in SEQ ID NO: 2. The Lin7c gene is a non-human organism such as Danio rerio (Accession No: NP_001004672), Mus musculus (Accession No: NP_035829), Gallus gallus (Accession No: NP_001026238), Rattus norvegicus (Accession No: NP_068623). ), Macaca mulatta (Accession No: XP_001089490) has also been identified. When expressed as the Lin7c gene in the present invention, it is not limited to the origin, but includes all organism-derived Lin7c genes as a technical scope.

  In particular, as a lymph node metastasis improving agent and preventive agent according to the present invention, it is preferable to use a human-derived Lin7c gene as an active ingredient. In the present invention, the lymph node metastasis improving agent is a drug prescribed for a patient who has developed lymph node metastasis, and prevents the lymph node metastasis from further metastasizing and spreading. In the present invention, the lymph node metastasis preventive agent is a drug prescribed for a patient who has not developed lymph node metastasis (particularly a patient suspected of having lymph node metastasis) and is a drug that prevents the onset of lymph node metastasis. is there. When a gene therapy agent containing the Lin7c gene as an active ingredient is introduced into a patient who develops lymph node metastasis or a patient who prevents the development of lymph node metastasis, the administration form is not particularly limited. Examples of the administration form include a form using a non-viral vector and a form using a viral vector. The outline of the treatment plan using gene therapy agents can be found in publicly known literature (eg, separate experimental medicine, basic technology of gene therapy (Yodosha (1996)), separate experimental medicine, gene transfer & expression analysis experiment method (sheep) Satosha, (1997))).

  In the form using a non-viral vector, a Lin7c gene expression vector incorporating the Lin7c gene is prepared. The obtained gene expression vector is then subjected to a phosphate-calcium coprecipitation method, a method of introducing a DNA molecule using a liposome (liposome method, HVJ-liposome method, cationic liposome method, lipofectin method, lipofectamine method). Incorporation into the cells by DNA direct injection method (named-DNA method), electroporation method, microinjection method, gene gun (Gene Gun) together with carrier (metal particles) and DNA molecules . Examples of the expression vector used here include pCAGGS (Gene 108, 193-200 (1991)), pBK-CMV, pcDNA3.1, pZeoSV (Invitrogen, Stratagene) and the like.

  On the other hand, in the form using a viral vector, a viral vector such as a recombinant adenovirus or a retrovirus can be used. Specifically, for example, a detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, shinbis virus, Sendai virus, SV40, immunodeficiency virus (HIV), etc. A recombinant virus is prepared by introducing a gene expression cassette containing the Lin7c gene into a DNA virus or RNA virus. The Lin7c gene can be introduced into the cell by infecting the cell with the obtained recombinant virus. Among the viral vectors, it is known that adenovirus infection efficiency is much higher than when other viral vectors are used. From this viewpoint, it is preferable to use an adenoviral vector system.

  Examples of methods for introducing the lymph node metastasis ameliorating agent or prophylactic agent of the present invention into patients include an in vivo method in which a gene therapeutic agent is directly introduced into the body, and taking out certain cells from humans and introducing DNA into the cells outside the body. And ex vivo method for returning the cells to the body (Nikkei Science, April 1994, pp. 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23-48 (1994), Experimental Medicine Extra Number, 12 ( 15), (1994)).

  When administered by the in vivo method, it can be administered by an appropriate route according to the disease to be treated, the target organ and the like. For example, it can be administered intravenously, arterially, subcutaneously, intramuscularly, or can be locally administered directly to the tissue where the lesion is observed. Here, in gene therapy with a non-viral vector, it is preferable to guide the gene to the vicinity of the target site by a combination with local administration or a dosage form with enhanced tissue transfer, but in gene therapy with a viral vector, There is no need for local administration, and intravenous administration is also possible.

  As a preparation form, various preparation forms (for example, a liquid agent etc.) suitable for each of the above administration forms can be taken. For example, when an injection containing the Lin7c gene as an active ingredient is used, the injection can be prepared by a conventional method. For example, in an appropriate solvent (buffer solution such as PBS, physiological saline, sterilized water, etc.) After dissolution, it can be prepared by sterilizing by filtration with a filter or the like and then filling into an aseptic container. A conventional carrier or the like may be added to the injection as necessary. Liposomes such as HVJ-liposomes can be in the form of liposome preparations such as a suspension, a freezing agent, and a centrifugal concentrated freezing agent. In addition, in order to facilitate the presence of genes around the diseased site, it is possible to prepare sustained-release preparations (mini-pellet preparations, etc.) and implant them near the affected area, or use an osmotic pump etc. It is also possible to administer gradually and continuously.

  The content of the Lin7c gene in the preparation can be adjusted as appropriate depending on the disease to be treated, the age of the patient, the body weight, etc., and this is preferably administered once every several days to several months. The gene improving agent or prophylactic agent of the present invention as described above can be applied to all lymph node metastasis patients or patients who prevent the onset of lymph node metastasis, but preferably suffers from squamous cell carcinoma and lymph nodes. Effectively used for patients who have developed metastases, patients who have oral squamous cell carcinoma, and who are determined to have a high risk of developing lymph node metastases.

The diagnostic agent for lymph node metastasis and the determination method The diagnostic agent for lymph node metastasis according to the present invention includes a reagent containing a reagent capable of measuring Lin7c protein. Specific examples of the component of “a reagent capable of measuring Lin7c protein” include an anti-Lin7c antibody and a label thereof. The Lin7c protein has a PDZ domain and is known to bind to CASK, PSD-95, Mint1, and the like. Therefore, CASK, PSD-95, Mint1, etc., and these labels can be used as reagents.

  Here, the “anti-Lin7c antibody” can be easily prepared according to known literature, or can be purchased as a marketed product. Known documents for preparing anti-Lin7c antibodies include, for example, Antibodies; A Laboratory Manual, Lane, HD et al., Cold Spring Harbor Laboratory Press (1989), New Cell Engineering Experimental Protocol, Shujunsha (1993) Etc. Specifically, an anti-Lin7c antibody can be produced by appropriately immunizing an animal by a conventional method using Lin7c protein or a part thereof as an immunogen. Here, immunogens for producing anti-Lin7c antibodies include (A) Lin7c protein, (B) fusion protein of Lin7c and GST, and (C) oligopeptide consisting of part of Lin7c protein and KLH, etc. Conjugates, and the like.

  The Lin7c protein shown in (A) is produced by cloning the Lin7c gene, inserting it into an appropriate expression vector, introducing it into Escherichia coli or a cultured cell line, and producing the protein in large quantities from the transformant by a conventional method. It can be obtained by purification. First, when cloning a human-derived Lin7c gene as a Lin7 gene, a cDNA containing a polynucleotide having the base sequence shown in SEQ ID NO: 1 is prepared. The cDNA can be cloned, for example, by PCR reaction using mRNA extracted from human-derived cultured cells as a template. Such cloning can be easily carried out by those skilled in the art based on basic documents such as Molecular cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989). Next, the cloned cDNA is incorporated into expression vectors such as pcDNA3.1 derivatives, pRc / RSV, pRc / CMV, pEF derivatives, pREP9 derivatives (Invitrogen), pIRESneo (Clontech), and introduced into host cells. Thus, a transformant is prepared. The host cell may be any cell that can stably integrate a foreign gene into the host chromosome. For example, CHO cell, L929 cell, C127 cell, BALB / c3T3 cell (deficient in dihydrofolate reductase, thymidine kinase, etc.) These cells can be obtained from ATCC (American Type Culture Collection) or the like. In addition, expression vectors can be introduced into host cells by, for example, the calcium phosphate method (J. Virol., 52, 456-467 (1973)), the method using LT-1 (Panvera), the lipid for gene introduction (Lipofectamine, Lipofectin; Gibco). -BRL) method. After the introduction, the transformant in which the recombinant expression vector is stably integrated into the host chromosome can be selected by culturing in a normal medium containing a selection marker.

  By continuing to culture the transformant obtained as described above under suitable conditions, the Lin7c gene can be highly expressed and the Lin7c protein can be produced. Examples of the method for isolating and purifying the expressed and produced Lin7c protein include the methods described in the Japanese Biochemical Society, New Chemistry Experiment Course 1, Protein I-Separation / Purification / Property- (1990). As described above, Lin7c protein as an immunogen can be prepared.

  In the fusion protein shown in (B), for example, a cDNA containing the above-mentioned Lin7c gene is introduced into a GST fusion protein expression vector such as pGEX-6P-1 (Pharmacia), and this is introduced into Escherichia coli to transform the transformant. Then, the cells can be obtained by disrupting the cells by a conventional method, extracting the fusion protein, and performing purification using glutathione sepharose 4B (Pharmacia) or the like. The conjugate shown in (C) can be obtained by synthesizing an appropriate oligopeptide and then mixing with KLH or BSA protein.

  The animal species to be immunized with the immunogen as described above may be any of rabbit, mouse, rat, chicken, cow, donkey, sheep, horse and the like. Further, in order to obtain a purified antibody from the obtained antiserum, affinity purification may be performed by a conventional method. Either a polyclonal antibody or a monoclonal antibody can be used for diagnosis, but a monoclonal antibody is desirable.

  Various antibody fragments can be prepared based on the antibody obtained as described above. The antibody fragment is, for example, an F (ab ′) 2 fragment that can be generated by pepsin digestion of an antibody, an Fab ′ fragment that can be generated by reducing the disulfide bond of an F (ab ′) 2 fragment, or an antibody containing papain and Examples include 2Fab or Fab fragment that can be produced by treatment with a reducing agent, and these antibody fragments can also be used as the “reagent capable of measuring Lin7c protein” in the present invention.

  Furthermore, a labeled product obtained by labeling these antibodies or antibody fragments with an enzyme or the like can also be a “reagent capable of measuring Lin7c protein” in the present invention. Specific enzyme labeling methods include, for example, the glutaraldehyde method, periodo method, maleimide method, and pyridyl disulfide method. Examples of the enzyme used for labeling include bovine small intestine / alkaline phosphatase and horseradish / peroxidase. These labeled antibodies can be easily prepared by those skilled in the art according to basic books such as enzyme immunoassay, Medical School (1978) and the like.

  As described above, CASK, PSD-95, Mint1, etc., which are known to bind to the Lin7c protein, and labels thereof can also be used as the “reagent capable of measuring the Lin7c protein” in the present invention. Reagents containing the above anti-Lin7c antibody, CASK, PSD-95, Mint1, etc. or a label thereof are present in an appropriate buffer such as a bovine serum albumin-containing phosphate buffer (pH 7.0). Therefore, it can be used as a component of a diagnostic agent for lymph node metastasis. In that case, it can be used in the form of a kit that also contains, for example, an enzyme-labeled secondary antibody, a color former, a color former, a stop solution, and a standard product, depending on the measurement method.

  Examples of methods for diagnosing lymph node metastasis using a diagnostic agent containing a reagent capable of measuring Lin7c protein as described above include the following methods.

  First, the test sample is not particularly limited as long as it is a tissue reflecting the pathological condition of lymph node metastasis onset. For example, as a biopsy tissue, a tumor site from a patient or, if available, a part of a normal site obtained by biopsy can be used. In addition, when there is a possibility of metastasis to lymph nodes, lymph node tissue can be used as a test sample. In addition, blood can be used as the test sample.

  When using an anti-Lin7c antibody or a labeled form thereof as a reagent capable of measuring Lin7c protein, the test sample described above is treated with a fluorescent antibody method, Western blot method, immunoprecipitation method, immunohistochemical staining method, radioimmunoassay method ( The expression level of the Lin7c gene in the test sample can be detected by subjecting it to conventional methods such as RIA) and enzyme immunoassay (ELISA). Detection can be easily carried out based on basic documents such as enzyme immunoassay, Medical School (1978) and the like. In the case of the ELISA method, the plate is coated with a primary antibody (anti-Lin7c antibody), and pyruvate carboxylase present in the sample is bound, then reacted with a secondary antibody (labeled antibody such as alkaline phosphatase), and the Lin7c gene A technique for detecting an antibody bound to an expression product (Lin7c protein) is simple.

  As a result of detection of the expression level of the Lin7c gene as described above, compared to the detection of Lin7c protein in a negative subject (a healthy subject sample or a cancer patient who has not developed lymph node metastasis), Lin7c in a diagnostic subject When the protein expression level is decreased or the expression level is lost, it is determined that the diagnosis target has developed lymph node metastasis or has a high risk of developing lymph node metastasis.

  Next, the diagnostic agent for lymph node metastasis according to the present invention is exemplified by one containing a reagent capable of measuring the expression level of the Lin7c gene. Specifically, examples of the reagent include a polynucleotide capable of specifically detecting Lin7c gene expression (presence of mRNA), or a labeled form thereof. The usable polynucleotide may be single-stranded or double-stranded. Here, as a polynucleotide capable of specifically detecting the mRNA of the Lin7c gene, a polynucleotide that functions as a probe that specifically hybridizes to the mRNA of the Lin7c gene and as a primer in an amplification reaction using the mRNA of the Lin7c gene as a template Mention what works.

  The polynucleotide functioning as the probe is not limited to the position of hybridization as long as it can specifically hybridize to the mRNA of the Lin7c gene. Furthermore, the length of hybridization is not limited as long as it can specifically hybridize to mRNA of the Lin7c gene.

  More specifically, a polynucleotide capable of specifically detecting the mRNA of the Lin7c gene can be appropriately designed based on the base sequence of the human-derived Lin7c gene shown in SEQ ID NO: 1. The designed polynucleotide can be appropriately prepared by synthesizing using a DNA synthesizer if it is short, by PCR if it is long, and using an appropriate restriction enzyme site if it is present. it can.

  A component of the diagnostic agent for lymph node metastasis by allowing the reagent for measuring the expression level of the Lin7c gene containing the polynucleotide as described above to exist in an appropriate buffer that does not interfere with the reaction such as TE buffer. It can be. At that time, depending on the measurement method, it can be used in the form of a kit containing, for example, reverse transcriptase, Taq polymerase, dNTP, reaction stop solution and the like.

  Specifically, the polynucleotide for measuring mRNA of the Lin7c gene is used for diagnosis of lymph node metastasis as a hybridization probe or a PCR primer. Here, a preferable length when used as a PCR primer includes a length of about 15 to 50 bases, and a preferable length when used as a hybridization probe is about 20 to 591 bases. Length.

  Examples of a method for diagnosing lymph node metastasis using the diagnostic polynucleotide of the present invention include the following methods. 1) Total RNA or poly (A) RNA obtained from a test sample or the like collected from a diagnostic target using the above-described diagnostic single-stranded polynucleotide (forward strand and reverse strand) as a primer in a nucleic acid amplification reaction. A method for detecting the expression level of the Lin7c gene by a nucleic acid amplification reaction using a template. 2) The above-mentioned diagnostic single-stranded or double-stranded polynucleotide is labeled and used as a probe in, for example, Northern blot analysis, and the above-mentioned total RNA or poly (A) RNA is used as a hybridized target to produce Lin7c A method for detecting the expression level of a gene.

  The detection of the expression level of the Lin7c gene in the above 1) and 2) can be performed based on a basic document such as Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989). Specific examples are shown below.

  When detecting the expression level of the Lin7c gene by a nucleic acid amplification reaction, first, the above-described primer polynucleotide is synthesized by a conventional method. Next, total RNA or poly (A) RNA is extracted and purified from a test cell or the like by a conventional method, and single-stranded cDNA is prepared by reverse transcriptase using this RNA as a template. The single-stranded cDNA can be easily prepared by using, for example, Superscript pre-amplification system (Life Technologies, Gaithersburg, MD). Then, the previous primer is added and, for example, PCR reaction is performed by a conventional method. The PCR reaction conditions include, for example, conditions of heating at 72 ° C. for 10 minutes after 30 cycles of 95 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 2 minutes. The PCR reaction product is electrophoresed on an agarose gel with an appropriate concentration, whereby the mRNA of the Lin7c gene can be detected and the mRNA can be quantified. As a result, the expression level (mRNA level) of the Lin7c gene is reduced or disappears in the test cell, unlike negative subjects (samples from healthy subjects or patients who have not developed lymph node metastasis). If so, it is determined that the diagnosis subject has developed lymph node metastasis or has a high risk of developing lymph node metastasis.

  Furthermore, as a diagnostic method using a nucleic acid amplification reaction, a method based on the following principle is exemplified. First, RNA is extracted and purified from a biopsy tissue or the like in the same manner as described above, and amplification (PCR reaction) of a target sample using a biotinylated primer is performed by the same method as described above. Thereafter, the amplification product is unified by alkali treatment and hybridized with a solid phase on which a complementary DNA probe is immobilized. Thereafter, the solid phase is washed and reacted with enzyme-labeled avidin. After washing the solid phase, the presence or absence of mRNA of the Lin7c gene can be detected by adding a colorant reaction by adding an enzyme substrate and measuring the absorbance.

  In addition, diagnosis can also be performed by using in situ PCR method (Fernandez et al., Mol. Carcinog, 20,317-326, 1997).

Next, when the expression level of the Lin7c gene is detected by the hybridization reaction of 2), first, a probe is prepared by radiolabeling or biotin labeling the single-stranded or double-stranded polynucleotide described above. The double-stranded polynucleotide is produced, for example, by labeling the PCR reaction product prepared by the technique of 1) with ³² P by the nick translation method or the random prime labeling method. Next, total RNA or poly (A) RNA is extracted and purified from a test cell or the like by a conventional method, and formaldehyde gel electrophoresis and blotting to a nylon membrane are performed by a conventional method. The presence of mRNA expression of the Lin7c gene can be detected by performing hybridization between this membrane and the previous probe. As a hybridization condition, when a double-stranded polynucleotide is used as a probe, for example, 50% (v / v) formamide, 1M NaCl, 10% (w / v) dextran, 1% (w / v) SDS, 100 μg / After hybridization for 16-24 hours at 42 ° C. under the conditions of ml salmon sperm DNA, the plate was washed twice in 2 × SSC at room temperature for 10 minutes, and further washed in 60 ° C., 2 × SSC, 1% SDS for 20 minutes. The conditions for washing twice are listed. As a result of the hybridization, the expression level (mRNA amount) of the Lin7c gene is decreased or not expressed in the test cell, unlike a negative subject (a sample from a healthy subject or a patient who has not developed lymph node metastasis). When it has disappeared, it is determined that the subject to be diagnosed has developed lymph node metastasis or has a high risk of developing lymph node metastasis.

  The detection using the primer and the probe is applied not only to diagnosis but also to in situ hybridization of the tissue.

  Furthermore, the expression level of the Lin7c gene can also be detected using a microarray (DNA chip). In this case, first, the full length of the Lin7c gene or the above-described diagnostic polynucleotide is synthesized on the microarray or immobilized on the surface of the microarray. Examples of the length of the polynucleotide to be immobilized include those of about 15 to 50 bases. Next, total RNA or poly (A) RNA is prepared from test cells or the like, and this is labeled with fluorescence or the like. A solution containing the fluorescently labeled total RNA or poly (A) RNA is brought into contact with the microarray, and the immobilized polynucleotide and the fluorescently labeled total RNA or poly (A) RNA are hybridized. Thereafter, the expression level of the Lin7c gene can be detected, for example, by detecting fluorescence on the microarray.

Screening method for lymph node metastasis inhibitor As described above, improvement of the lymph node metastasis can be expected by enhancing the expression of the Lin7c gene, and the Lin7c gene itself can be used to improve or prevent the lymph node metastasis. However, the substance that increases the Lin7c gene expression can also be used as an active ingredient of an agent for improving / preventing lymph node metastasis. In the present invention, a method for screening for a substance that promotes the expression of the Lin7c gene can be provided.

  That is, as a screening system for a Lin7c gene expression promoting substance provided by the present invention, a plasmid in which a reporter gene is linked to the 3 ′ end of the expression control region (promoter / enhancer) of the Lin7c gene is introduced into a host cell. A system in which a transformed cell is prepared and a test substance is added to the transformed cell is exemplified. Here, the reporter gene is not particularly limited as long as it can detect promoter activity, and for example, luciferase, CAT (chloramphenicol acetyltransferase), ALP (alkaline phosphatase), or GH (growth hormone). And other genes. Reporter vectors incorporating the gene are commercially available, for example, pGL3-Basic Vector (luciferase vector; Promega), and are readily available. Common host cells include COS cells and CHO cells. The transformed cell may be one that transiently expresses the reporter gene or one that can stably express the gene. Determine the ability of the test substance to promote the expression of the Lin7c gene by adding a test substance to the prepared transformed cells, incubating, crushing the cells, and measuring the reporter activity (for example, luciferase activity) Can do.

  A test substance determined to have the ability to promote the expression of the Lin7c gene in this screening system will be subjected to a pharmacological test as a candidate substance as an agent for improving / preventing lymph node metastasis. Here, the test substance is not particularly limited, and may be a substance whose function is unknown or a substance whose function is known. Further, as the test substance, a substance known to have an action of suppressing or preventing lymph node metastasis may be used. In this case, the function of promoting the expression of the Lin7c gene by a substance known to have a lymph node metastasis inhibitory action or preventive action is tested, or the lymph node metastasis inhibitory action is re-evaluated.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is not limitedly interpreted by the following Examples.

[Analysis of genes involved in lymph node metastasis cases]
Cell lines derived from human oral squamous cell carcinoma used in the examples described below are Ca9-22, Ho-1-N-1, Ho-1-u-1, HSC-2, HSC-3, HSC-4 (Human Science Research Resource Bank, Osaka), OK92 (originally cultured from tongue cancer in the inventor's laboratory), Sa3 and H1 (provided by Professor Fujita of Wakayama Medical University) It is. All cell lines were maintained on a 150x20 mm dish (Nunc, Roskilde, Denmark) at 37 ° C (atmosphere maintained at humidity with 5% carbon dioxide and 95% air) and 10% fetal bovine serum And 50 Units / ml penicillin and streptomycin in Dulbecco's modified Eagle's medium F-12 HAM (SIGMA Chemical Co, St. Louis, MO).

  Oral normal keratinocytes collected from 5 patients who underwent oral surgery were used as a control (NOK) throughout the experiment. Informed consent is obtained from these patients prior to the start of the study. The obtained tissue of normal oral keratinocytes was washed in Dulbecco's phosphate buffered saline (PBS) and then immersed in 0.25% trypsin EDTA solution (SIGMA) overnight at 4 ° C. After separating epithelial tissue from connective tissue, they were incubated with 0.25% trypsin EDTA lysis solution for 15 minutes at 37 ° C. while gently pipetting them, and disassembled into cell components. Isolated epithelial cells are seeded in collagen I, coated 60 mm dishes, 0.4% bovine pituitary extract (BPE), 0.1% hydrocortisone adrenal hormone, 0.1% transferrin, 0.1% epinephrine, and 0.1% Of Keratinocyte Basal Medium-2 (Clonetics, MD, USA) containing GA-1000 (Clonetics).

  In this example, the tumor tissue and normal oral tissue (when available) were simultaneously obtained from the patient who obtained informed consent in accordance with the explanation approved by the Ethics Review Committee of Chiba University. Obtained at the time of surgery. The excised tissue was divided into two parts, one frozen immediately and stored at −80 ° C. until use, the other fixed with 10% formalin for pathological diagnosis. Histopathological diagnosis of each cancer tissue was performed according to the international tissue classification of tumors by the pathology department of Chiba University Hospital. The stage of clinical pathology was determined by the TNM classification of International Union against Cancer. All patients were histologically diagnosed with squamous cell carcinoma, and each tumor sample was checked to ensure that over 80% of the specimen was tumor tissue. Genomic DNA was extracted by proteinase K digestion, and total RNA was extracted using Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer's protocol.

Identification of genes whose expression is significantly reduced in patients with lymph node metastasis
Protein solutions extracted from HSC-2, HSC-3, and NOKs were subjected to two-dimensional electrophoresis, and gel images were analyzed to identify spots where expression was significantly reduced in lymph node metastasis cases. Thereafter, the spot was subjected to mass spectrometry, and the protein of the spot was identified by peptide-mass fingerprinting. Details are described below.

  First, cultured cells grown into a complete monolayer were treated with lysis buffer containing 7M urea, 2M thiourea, 4% w / v CHAPS, 10 mM Tris pH 8.0, and sonicated (3x10s pulse on ice). Was dissolved. The sample was centrifuged at 13000 rpm for 20 minutes, and the supernatant containing the protein was removed. Protein concentration was measured by Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA) and adjusted to 1 mg / ml with lysis buffer. The protein sample was adjusted to pH 8.5 with 30 mM Tris HCl. In this study, five normal oral mucosal epithelial cell (NOKs) proteins were independently extracted and mixed in equal amounts after quantification.

  The same amount of protein was taken from each sample before labeling and used as a control sample (pooled standard sample). Approximately 50 μg of each protein extracted into 4 μl was mixed with 1 μl of CyDye Cy2, Cy3 or Cy5 (each 0.2 mM) (Amersham Biosciences, Buckinghamshire, UK) and incubated on ice for 30 seconds. Then, the reaction was stopped by adding 10 mM lysine (1 μl per 400 pmol of reagent), and left on ice for 10 minutes.

  As described above, the control sample labeled with Cy2 and the sample labeled with Cy3 or Cy5 were mixed in a combination according to the experimental design and used for isoelectric focusing (IEF). The same amount of 2x sample buffer (7 M urea, 2 M thiourea, 4% w / v CHAPS, 2% v / v Pharmalytes pH3-10) was added to the combined labeled samples. The total sample volume was adjusted to 450 μl with standard rehydration buffer (7 M urea, 2 M thiourea, 4% w / v CHAPS, 1% v / v Pharmalytes pH 3-10). Immobiline DryStrips (pH 3-10 NL, 24 cm) were rehydrated according to the manufacturer's protocol (Amersham Biosciences) prior to use for IEF. IEF was performed using an IPGphor II IEF unit (Amersham Biosciences) at a maximum current of 50 μA / strip at 20 ° C., 500 Vh for 500 V, 1000 Vh for 1000 V, and 64000 Vh for 8000 V. Second dimension electrophoresis was performed by standard SDS polyacrylamide gel electrophoresis (SDS-PAGE) protocol using Ettan DIGE system (Amersham Biosciences).

  Two-dimensional electrophoresis was performed on a 12% acrylamide isocratic Laemmli gel using an Ettan DALT six instrument (Amersham Biosciences). Electrophoresis was terminated when the bromophenol blue dye reached the bottom of the gel at 20 ° C.

  The labeled proteins on the gel were then visualized using a Typhoon 9400 imager (Amersham Biosciences) for gel image analysis. The Cy2 image was scanned using a 488 nm laser with a fluorescence filter 520 nm BP (band pass) 40. Cy3 images were scanned using a 532 nm laser with a fluorescence filter 580 nm BP30. Cy5 images were scanned using a 633 nm laser with a fluorescence filter 670 nm BP30. All gels were scanned with a resolution of 100 μm.

  Prior to analysis, the image was cut out using ImageQuant V 5.0 (Amersham Biosciences) to remove areas unrelated to the gel image. Gel images were automatically analyzed using a DeCyder Batch Processor and DIA (differential in-gel analysis) software module (Amersham Biosciences). In individual gels, Cy2 labeled images (control samples) are compared to either Cy3 or Cy5 images from NOKs, HSC-2, and HSC-3, respectively, using DeCyder-DIA software. It was. Use DyCyder-BVA (biological variation analysis) software (Amersham Biosciences) to standardize the images and match each gel against the control sample labeled with Cy2 on each gel. Compared. The approximate number for spot detection on the gel was set to 3000. Following statistical analysis of changes in protein content between samples, the reference spot map was superimposed between multiple gels using the DeCyder-BVA software module. Statistical significance of each expression level was calculated using Student's t test with log ratio. This analysis was equivalent to t-testing in pairs and increased accuracy by taking into account pairs of samples in the gel. Only protein spots that showed a statistically significant (p <0.001) increase or decrease in expression levels in all gels used in the study were considered spots with differential expression.

  The result of two-dimensional electrophoresis and the result of gel image analysis are shown in FIG. In FIG. 1, spots indicated by arrows indicate proteins whose expression is significantly reduced in lymph node metastasis cases.

  Spots with significantly reduced expression in lymph node metastasis cases were excised and subjected to mass spectrometry and peptide-mass fingerprinting. For each of HSC-2, HSC-3 and NOKs, separately electrophoresed gels for spot excision were fixed and stained with SYPRO Ruby (Molecular Probes, Inc., Eugene, OR, USA). The gel fluorescence image was superimposed with the CyDye image using DeCyder software. A list of spots to pick up was generated from DeCyder and exported to an automated spot handling system, Ettan spot picker (Amersham Biosciences).

Pixel information was converted into xy coordinates with reference markers from DeCyder software by Spot picker. The target spot was excised as a round plug with a diameter of 2 mm and transferred into a 96-well microtiter plate. Protein spots excised from the gel were digested in gel with an enzyme solution containing 50 mM NH ₄ HCO ₃ , 5 mM CaCl ₂ , and 12.5 ng / μl trypsin. The purified sample was fixed on a stainless steel target mixed with matrix crystals of α-cyano-4-hydroxycinnamic acid and dried in air. Mass determination was performed using an AXIMA-CFR mass spectrometer (Shimadzu Co. Ltd., Kyoto, Japan). Proteins were identified by peptide-mass fingerprinting (PMF) method using Mascot Search on the web (Matrix Science, Ltd., London, UK).

  The results of MALDI-TOF-mass are shown in FIG. As a result, it was revealed that the abundance of Lin7c protein was significantly reduced in lymph node metastasis cases.

Immunohistochemistry Next, in this example, the expression level of Lin7c protein was observed in lymph node metastasis cases and cases without lymph node metastasis by immunohistochemistry (IHC) staining. For IHC staining, paraffin-embedded specimens were sliced at 4 μm thickness, deparaffinized and washed with water, treated with 0.3% H ₂ O ₂ for 30 minutes, then treated with 1.5% blocking for 2 hours at room temperature. Treat with serum (Santa Cruz Biotechnology, Santa Cruz, CA) in PBS, incubate 100-fold diluted Lin7C antibody (Santa Cruz Biotechnology) in a wet box for 30 minutes at room temperature and wash 3 times with PBS buffer I went there.

  Next, treatment with Envision Regent Kit (Dako, Kyoto, Japan) and color development with 3,3'-diaminobenzidine tetrahydrochloride (Dako). Finally, it was counterstained with hematoxylin and mounted. As a negative control, the two sections were immunostained without primary antibody. The results of immunohistochemical staining are shown in FIGS. 3A-D. FIG. 3A is a representative example of normal oral tissue, and shows that this Lin7c protein is expressed in the cell membrane. FIG. 3B is a representative example of a case of oral squamous cell carcinoma tissue without lymph node metastasis, and shows that this protein is expressed in the cell membrane. FIG. 3C is a representative example of a case of oral squamous cell carcinoma tissue with lymph node metastasis, and shows that the expression of Lin7c protein in the cell membrane is decreased. FIG. 3D is a representative example of lymph node metastasis and shows that the expression of this protein in the cell membrane is reduced.

  Next, a scoring method expressed as the average percentage of positive tumor cells was used to measure Lin7c protein expression. The method was a 400x magnification in each section, randomly selecting at least 5 fields and scoring for Lin7c staining intensity as follows. That is, point 1 was added to weak, point 2 was added to moderate, and point 3 was added to intense. This value multiplied by the percentage of positive tumor cells was taken as the IHC score. Cases with a Lin7c-IHC score of less than 56 were considered negative. Two pathologists without any information or knowledge related to the patient's clinical status scored each case separately. Statistically significant differences between Lin7c-IHC score and clinicopathological function were assessed by Fisher's exact test. P <0.05 was considered important.

  The result of comparison with the clinical index is shown in FIG. 4A-C as an IHC score as a scatter diagram. In FIG. 4A, T means a tumor clinical specimen, N means normal oral tissue, pN (−) means a non-lymph node metastasis case, and pN (+) means a lymph node metastasis case . FIG. 4B shows a scatter diagram obtained by dividing the IHC score of the cancer site by the IHC score of the normal tissue, and dividing it into a non-lymph node metastasis case (pN (−)) and a lymph node metastasis case (pN (+)). Yes. Fig. 4C shows the percentage of the expression value of the Lin7c gene divided by normal tissue using the quantitative RT-PCR method using the mRNA of the specimen used in immunohistological staining. These are shown as a scatter diagram by dividing into lymph node non-metastasis cases (pN (−)) and lymph node metastasis cases (pN (+)). As can be seen from FIGS. 4A to 4C, in the case of lymph node metastasis, it was revealed that the expression of Lin7c protein was significantly reduced at the protein level and the mRNA level.

In addition, quantitative RT-PCR for measuring the expression level of the Lin7c gene mRNA was performed as follows. A control without reverse transcription was prepared. Prior to cDNA synthesis, residual genomic DNA was removed from total RNA using DNase I treatment (DNA-free; Ambion, Austin, TX), and Superscript ^TM reverse transcriptase (Life Technologies, Grand Island, NY) and oligod (T) Reverse transcription was performed using primer.

  Designed 5'-ACGCAGGCAACAGACCTAAT-3 '(nucleotides 580-599; SEQ ID NO: 3) and 5'-CCACTACTCCCGTTAAGACA-3' (nucleotides 821-802; SEQ ID NO: 4) as primers for confirming the mRNA expression of Lin7c gene And synthesized. The amplified product was subjected to electrophoresis using a 3% agarose gel to confirm the size and purity of the product. In order to confirm the PCR product, cloning was performed using pCR 2.1vector (Invitrogen). Then, Real-Time PCR was performed using LightCycler FastStart DNA Master SYBR GreenIkit (Roche Diagnostics GmbH, Mannheim, Germany).

To create a standard curve, cDNA was transcribed from 300, 30, 3.0, and 0.3 ng of total RNA extracted from normal oral tissues. In the PCR reaction using LightCycler (Roche), a final reaction volume of 20 μl containing ₂ μl of FirstStart DNA Master SYBR Green Imix (Roche), 3 mM MgCl _{2 and} 0.2 μl of primer was used according to a conventional method. The reaction solution is placed in a glass capillary tube, initial denaturation is 95 ° C for 10 minutes, the number of cycles for amplification is 45 times, denaturation is 95 ° C for 10 seconds, annealing is 56 ° C for 10 seconds, and extension reaction is 72 ° C. The temperature change rate was set to 20 ° C./second. The amount of mRNA of the transcribed Lin7c gene was calculated from each standard curve and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Analysis of Lin7c gene with decreased expression
DNA sequence method and PCR-based methylation assay were performed in patients with lymph node metastasis with significantly decreased Lin7c gene expression, assuming that the decrease in Lin7c gene expression was due to DNA mutation or epigenetic regulation. .

  Changes in the sequence of Lin7c gene were analyzed using the PCR-direct sequencing method. The full length cloning of the Lin-7C gene containing exon 1-5 was performed using 5 oligo primers.

All exons and adjacent intro sequences were referenced from human genome science data. Primers used for PCR were prepared using the Primer 3 program. The primers used for each exon amplification are as follows.
In order to amplify exon 1, 5′-GGTACTCACTTTTCCGGCTTC-3 ′ (SEQ ID NO: 5) and 5′-ATTGGCATTCGGGAGGAGACA-3 ′ (SEQ ID NO: 6) were designed. To amplify exon 2, 5′-TTGCCCTTGAGGAATGAATC-3 ′ (SEQ ID NO: 7) and 5′-GGTCAGGGAGAGTAAAAACC-3 ′ (SEQ ID NO: 8) were designed. In order to amplify exon 3, 5'-TCACAGAGAACTCAAGCATGG-3 '(SEQ ID NO: 9) and 5'-AAGTTCGATGTGGTTTAAAGAGT-3' (SEQ ID NO: 10) were designed. To amplify exon 4, 5′-GGGGGAAACAAAAGATGAAAG-3 ′ (SEQ ID NO: 11) and 5′-TTTACGGAACTCCCTTAGGA-3 ′ (SEQ ID NO: 12) were designed. To amplify exon 5, 5′-TGGTAAGGCAGGATTATTTCTT-3 ′ (SEQ ID NO: 13) and 5′-ATGATTACCGACGTTACGGT-3 ′ (SEQ ID NO: 14) were designed.

  Using the above primer set, 0.01 unit of LA-Taq DNA polymerase (Takara) was added to 100 ng of template DNA, and adjusted to 25 μl with sterile distilled water. PCR was performed in the following cycle. That is, after a denaturation treatment at 94 ° C. for 10 seconds, an annealing reaction at 60 ° C. for 10 seconds and an extension reaction at 72 ° C. for 30 seconds were taken as one cycle, and 45 cycles were performed. Sequencing was performed using Dye Terminator sequencing kit from Perkin Elmer. The ethanol precipitate was run and extracted on a 36 cm long gel containing 5% LongRange / 8 M urea, and then analyzed with Perkin Elmer ABI377.

  The chart obtained as a result of the analysis is shown in FIG. Changes were observed in the DNA sequences of 6 out of 20 cases in which the expression of the Lin7c gene was reduced, and among these 6 cases, mutations with amino acid changes were detected in 4 cases shown in FIG.

The methylation around the CpG island of the Lin7c gene was analyzed by PCR-based methylation assay. The DNA sample was precipitated in ethanol, dissolved in sterilized distilled water, and treated in mCpG-sensitive BstUI (1 μg / unit, New England Biolabs, Beverly, Mass.) At 60 ° C. for 16 hours. The treated DNA was amplified using the following primers (see FIG. 6). That is, forward primer 5′-CCAGGCTTGACTCCTCTACG-3 ′ (SEQ ID NO: 15) and reverse primer 5′-ATCGAAAACACGGGGAGGAT-3 ′ (SEQ ID NO: 16) were used. PCR was performed using 2.5 pmol primer, 50 μM dNTPs, 10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ and 0.5 units of AmpliTaq (Applied Biosystems, Foster City, Calif.) For 1 μl of the treated DNA sample. Was performed in 25 μl of the reaction solution. The amplified PCR product was electrophoresed on a 3% agarose gel, infected with ethylene bromide, and then visualized by irradiation with ultraviolet rays. Peripheral blood DNA treated with SssImethylase (New England Biolabs) was used as a positive control. Untreated peripheral blood was used as a negative control. The result of electrophoresis is shown in FIG.

  In addition, demethylation analysis was performed using 5-aza-2'-deoxycytidine (5-aza-2'-dC) (Sigma Chemical Co., St. Louis, MO), a DNA methyltransferase inhibitor, and the Lin7c gene MRNA restoration of oral squamous cell carcinoma cell lines (Ca9-22, OK92, Ho-1-N-1, Ho-1-u-1, HSC-2, HSC-3, HSC-4, Sa3 And H1). Each cell was treated in a solution of 5-aza-2'-dC agent at a concentration of 0 or 2 μM for 5 days, washed with PBS, and further cultured in an unreacted solution for 10 days. Thereafter, RNA was extracted, and the expression state of the Lin7c gene was confirmed by quantitative real-time PCR. The results are shown in FIG.

  As shown in FIG. 6, when there is a CpG island in which the gene 5 ′ promoter region is rich in guanine and cytosine, the presence or absence of methylation in that region is said to regulate gene expression. . In normal tissue, it is not methylated and the gene expression switch is in an ON state, but in cancer tissue, it is methylated and the expression switch is in OFF state. This is thought to be the cause of the decrease in the expression of an important tumor suppressor gene that has now been recognized. As shown in FIG. 7, in oral squamous cell carcinoma-derived cell lines, methylation of the CpG island region was detected in 18 out of 20 cases of 9 out of 9 lines and the expression of the Lin7c gene decreased. Moreover, as shown in FIG. 8, in all oral squamous cell carcinoma-derived cell lines in which methylation was observed, the expression of the Lin7c gene mRNA was restored when the demethylating agent was placed in the culture medium and cultured.

[Functional analysis of Lin7c gene]
Invasion Assay
First, an expression vector (pME18SFL3) into which cDNA of human Lin7c was introduced was transiently introduced into nine oral squamous cell carcinoma-derived cell lines using FuGENE-6 as a transfection reagent. After the introduction, it was left for 48 hours. A cell line in which only a vector (pME18SFL3) was similarly introduced as a Mock cell was used as a control. After transfection, the gene expression state was confirmed by real-time PCR. Real-time PCR is as described above. The results are shown in FIG.

  Next, after transfection, each cell line was cultured on a glass slide and fixed with 4% paraformaldehyde for 5 minutes. Thereafter, the solution was permeated for 10 minutes in 0.01% Triton X-100 PBS solution (pH 7.4), and non-specific antibody blocking treatment was performed in 5% skim milk PBS solution (pH 7.4) for 1 hour. Then, incubate in primary antibody (goat anti-Lin7c (Santa Cruz Biotechnology, 1: 100 dilution) for 2 hours, washed with PBS, then secondary antibody (rabbit anti-goat secondary antibody labeled with Alexa Fluor 488 (Molecular Probes, Leiden, The Netherlands)) was incubated for 1 hour and then observed with a fluorescence microscope, which was a Leica DMIRBE inverted stand equipped with a Leica TCS2-MP confocal system (Leica Laserteknik, Mannheim, Germany) and Coherent Mira tuneable pulsed titanium. A sapphire laser (Coherent Laser Group, Santa Clara, CA, USA) was used and the results are shown in FIG.

Next, the invasion ability of the obtained nine transfected cells was analyzed using QCM ^™ 96-Well Cell Invasion Assay Kit (Chemicon, Temecula, Calif.). 0.1 ml of a serum-free medium (Dulbecco's modified Eagle's medium F-12 HAM) was added to one well of a 96-well chamber made of a membrane coated with ECMatrix ^{™ so} that the cell concentration was 5 × 10 ⁵ cells / ml. The chamber was inserted into a 96-well tray containing 150 μl of serum-containing medium per well and cultured for 16 hours in an incubator at 37 ° C., 5% CO ₂ . Thereafter, in order to detach cells having invasive ability that passed through the membrane, the cells were inserted into a 96-well tray in which 150 μl of detachment solution was placed in one well and treated for 30 minutes. Cells detached from the membrane were developed with CyQuant GR dye and detected at 480/520 nm. The results are shown in FIG.

  As shown in FIG. 9, the Lin7c gene whose expression had been reduced was successfully forcibly expressed by the gene introduction method. In FIG. 9, “Mock” represents an oral squamous cell carcinoma-derived cell line into which only an expression vector has been introduced, and “Lin7c gene” represents an oral squamous cell carcinoma-derived cell line into which an expression vector having incorporated the Lin7c gene has been introduced. As shown in FIG. 9, the mRNA of Lin7c gene could be forcibly expressed in the range of 50 to 3000 times.

  In addition, as shown in FIG. 10, it was confirmed that the forcedly expressed Lin7c gene was present in the cell membrane as a Lin7c protein. Furthermore, as shown in FIG. 11, it was revealed that cell migration and invasion ability were suppressed in oral squamous cell carcinoma-derived cell lines expressing Lin7c protein in the cell membrane.

Pathway analysis Next, a target gene group encoding a protein that interacts with the Lin7c protein was subjected to a pathway analysis using Ingenuity Pathway Analysis software (Ingenuity Systems, Mountain View, CA). As a result, 20 genes were selected (FIG. 12). The expression level of mRNA of each selected gene was analyzed using real-time PCR. Primers used for real-time PCR were prepared using the above-mentioned Primer 3 program. Each primer used is as shown in Table 1.

  The result of real-time PCR is shown in FIG. As shown in FIG. 13, it was revealed that the expression level changed in all 20-gene mRNAs selected by pathway analysis. That is, it became clear that Lin7c protein interacts directly or indirectly with the protein group encoded by these 20 genes. These 20 genes include a group of genes involved in the cell adhesion mechanism cadherin-catenin system, which has been reported to be associated with cell adhesion and invasion / metastasis in oral squamous cell carcinoma.

Among genes listed by immunochemical staining and Western blot analysis pathway analysis, genes involved in the cadherin-catenin system, a cell adhesion mechanism that has been reported to be associated with cell adhesion and invasion / metastasis in oral squamous cell carcinoma Attention was paid to CTNNB1 (No. 1 in Table 1) and CASK (No. 2 in Table 1). From the results shown in FIG. 13, the mRNA level of each of these genes is lower in oral squamous cell carcinoma cells than in normal oral mucosa-derived epidermal keratinocytes, but when the Lin7c gene is forcibly expressed, Along with this, strong expression was observed. According to pathway analysis, the Lin7c gene is associated with the CTNNB1 gene via the CASK gene. The expression status of CASK gene and CTNNB1 gene and the expression status of Lin7c gene show similar profiles, suggesting that the expression status of Lin7c gene and cell adhesion mechanism are closely related. . That is, it was considered that the cancer cell migration was allowed as a result of the collapse of the cell expression system due to the decreased expression of the Lin7c gene. In order to support this, immunochemical staining and Western blot analysis were performed using Lin7c antibody, CASK antibody and CTNNB1 antibody.

  In immunochemical staining, cell line HSC3 was first cultured on a glass slide, washed with PBS, fixed with 4% paraformaldehyde for 5 minutes, permeated with PBS containing 0.01% Triton X-100 for 10 minutes, and contained 5% skim milk. Nonspecific antibody blocking treatment with PBS was performed for 1 hour. After washing with PBS, antibodies of Lin7c, CASK and CTNNB1 were incubated for 2 hours. After washing with PBS, Alexa Fluor 488 (Molecular Probes, Leiden, the Netherlands) was incubated for 1 hour. Furthermore, it was stained with DAPI (4,6-diamidino-2-phenylindole dihydrochloride) for nuclear staining. A cover glass was put on and observed with a microscope (Leica DMIRBE) equipped with a fluorescent device, and a fluorescent image was imaged with a Leica TCS2-MP confocal system (Leica Laserteknik, Mannheim, Germany). The results are shown in FIG.

  In the Western blot analysis, protein components were extracted from the cell line introduced with Lin7c, the cell line introduced with MOCK, and the non-introduced cell line, and Western blotting was performed using Lin7c antibody, CASK antibody, and CTNNB1 antibody. .

  Specifically, the protein extraction solution was electrophoresed on a 10% polyacrylamide gel and transferred to a PVDF membrane (Bio Lad). After washing with 0.1% Tween-20-containing TBS (T-TBST), blocking was performed with 5% skim milk-containing T-TBST for 30 minutes, and Lin7c antibody, CASK antibody, or CTNNB1 antibody was incubated for 16 hours. After washing with T-TBST, a histofine biotin-labeled anti-goat IgG antibody (Nichirei) secondary antibody was incubated for 2 hours, and luminescence was visualized using ECL + Kit (Amersham). The results are shown in FIG.

  As shown in FIGS. 14 and 15, it was revealed at the protein level that the expression levels of the CASK gene and the CTNNB1 gene are restored by forcibly expressing the Lin7c gene. As a result, it was clarified that by restoring the expression level of the Lin7c gene, the cell adhesion cell adhesion system can be restored, and the invasion ability and metastasis ability once acquired by the cancer cells can be suppressed.

Analysis of cell migration ability Next, as part of the functional analysis of the Lin7c gene, cell migration ability was analyzed by a wound healing assay. First, human full-length Lin7c construct was purchased from Toyobo, inserted into pcDNA4 / TO vectors (Invitrogen), and then introduced into Ca9-22 cell line using FuGENE6 kit (Roche). Cloning using Zeocin (Sigma) produced Ca9-22Lin-7C 5-5 and Ca9-22Lin-7C a-1 which are Lin7c stable expression strains. The expression of Lin7c gene was confirmed by Western blotting using a monoclonal antibody (Santa Cruz).

  A wound healing assay (scratch test) was performed with the prepared Ca9-22Lin-7C 5-5, Ca9-22Lin-7C a-1, and Ca9-22Lin-7C Mock (a strain in which only the vector was introduced). Specifically, cells were seeded in a dish, and the cells were scratched in a straight line using a micropipette tip in a monolayer confluent state. Thereafter, the state of the cells was observed 4 hours later and 8 hours later with a microscope (Leica DMIRBE) at 200 magnification. The results are shown in FIG.

  As shown in FIG. 16, when the cell migration ability was compared between two oral squamous cell carcinoma cell lines in which the Lin7c gene was forcibly expressed and a cell line into which only the expression vector had been introduced, the confluent cell monolayer was scratched. Eight hours later, the cell migration distance was clearly different between the two gene expression strains and the vector-only cells. From this result, it was recognized that the cell migration ability in oral squamous cell carcinoma cells was significantly suppressed by the expression of Lin7c gene.

Analysis of metastatic potential Next, as part of the functional analysis of the Lin7c gene, we created an experimental animal transplanted with a human oral squamous cell carcinoma-derived cell line on the tongue, and observed the cancer metastasis in the animal, thereby compelling the Lin7c gene. The effect of inhibiting metastasis by expression was verified.

First, 2 × 10 ⁵ cells were prepared from each clone and directly injected into the tongue of the BALB / cAnNcrj-nu / nu mice (Charles River Japan) at 6 weeks of age. Thereafter, the submandibular gland, liver, and lung were collected together with the tongue of the sample mouse, and DNA was extracted. The expression of the sample was confirmed by RT-PCR using primers of a human-specific Alu sequence. Further, as a reference for comparison of the expression level, detection of ARG RICH, which is a mouse-specific gene, was similarly performed by PCR for each sample.

  All mice were kept under specific pathogen-free conditions. All animal experiments were conducted in accordance with Chiba University laboratory animal center guidelines. The results of RT-PCR are shown in FIG. As a result, metastasis to the lung was observed in 4 out of 5 cases transplanted with vector-only cells, whereas no metastasis was observed in all 5 cases of transplantation of the cell line forcibly expressing the Lin7c gene. . From this result, it was confirmed in a system using animal experiments that metastasis was significantly suppressed by the expression of the Lin7c gene.

It is a photograph which shows the result of two-dimensional electrophoresis and gel image analysis. It is a spectrum figure which shows the result of MALDI-TOF-mass. It is a photograph showing the result of immunohistochemical staining. It is a dispersion | distribution figure which shows the result of a comparison with a clinical parameter | index with an IHC score. It is a chart figure obtained as a result of sequence analysis. It is a schematic diagram which shows CpG island in the promoter region of Lin7c gene. It is a photograph which shows the result of the electrophoresis in PCR-based methylation assay. It is a characteristic view which shows the result of having confirmed the expression state of Lin7c gene by quantitative real-time PCR. It is a characteristic view which shows the result of real-time PCR in Invasion Assay. It is a photograph which shows the result of the fluorescence microscope observation in Invasion Assay. It is a characteristic view which shows the invasion ability in Invasion Assay. It is a schematic diagram which shows the network of 20 genes selected by the pathway analysis. It is a characteristic view which shows the result of real-time PCR which shows the expression state of 20 genes selected by the pathway analysis. It is a photograph which shows the result of the immunochemical staining using CTNNB1 antibody and CASK antibody. It is a photograph which shows the result of the western blotting using Lin7c antibody, CTNNB1 antibody, and CASK antibody. It is a photograph which shows the result of wound healing assay. It is a photograph which shows the result of RT-PCR in the various tissues extract | collected from the experimental animal which transplanted the human oral squamous cell carcinoma origin cell line to the tongue.

Claims (22)

  An agent for improving lymph node metastasis, comprising, as an active ingredient, a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence substantially the same as the amino acid sequence.   The agent for improving lymph node metastasis according to claim 1, wherein the polynucleotide is supported on a carrier for gene therapy.   The agent for improving lymph node metastasis according to claim 2, wherein the carrier for gene therapy is a viral vector.   The improving agent according to claim 1, wherein the lymph node metastasis is lymph node metastasis of squamous cell carcinoma that develops in the oral cavity, pharynx, esophagus or lung.   An agent for preventing lymph node metastasis, comprising as an active ingredient a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence substantially identical to the amino acid sequence.   6. The agent for preventing lymph node metastasis according to claim 5, wherein the polynucleotide is carried on a carrier for gene therapy.   The agent for preventing lymph node metastasis according to claim 6, wherein the carrier for gene therapy is a viral vector.   6. The agent for preventing lymph node metastasis according to claim 5, wherein the lymph node metastasis is lymph node metastasis of squamous cell carcinoma that develops in the oral cavity, pharynx, esophagus or lung.   A gene therapy vector having a functioning gene comprising an amino acid sequence represented by SEQ ID NO: 2 or a polynucleotide encoding a substantially identical amino acid sequence thereto.   The vector for gene therapy according to claim 9, which is a viral vector.   10. The gene therapy vector according to claim 9, which is used for lymph node metastasis cases.   The gene therapy vector according to claim 9, wherein the lymph node metastasis is lymph node metastasis of squamous cell carcinoma that develops in the oral cavity, pharynx, esophagus or lung.   A diagnostic agent for the presence or absence of lymph node metastasis, comprising a reagent capable of measuring a protein comprising the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence substantially the same as the amino acid sequence.   14. The diagnostic agent for the presence or absence of lymph node metastasis according to claim 13, wherein the reagent contains an antibody against a protein that is an anti-Lin7c antibody.   The diagnostic agent for the presence or absence of lymph node metastasis according to claim 13, wherein the lymph node metastasis is lymph node metastasis of squamous cell carcinoma that develops in the oral cavity, pharynx, esophagus or lung.   A diagnostic agent for the presence or absence of lymph node metastasis, comprising a reagent capable of measuring the expression level of a gene comprising the amino acid sequence represented by SEQ ID NO: 2 or a polynucleotide encoding a substantially identical amino acid sequence thereto.   The lymph node metastasis according to claim 16, wherein the reagent contains a single-stranded or double-stranded polynucleotide that specifically hybridizes to the mRNA of the polynucleotide, or a label thereof. The presence or absence of diagnosing agent.   The diagnostic agent for the presence or absence of lymph node metastasis according to claim 16, wherein the lymph node metastasis is lymph node metastasis of squamous cell carcinoma that develops in the oral cavity, pharynx, esophagus or lung. Measuring the expression level of a gene containing a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 or substantially the same amino acid sequence in a biological sample derived from a patient to be tested;
Determining the presence and / or possibility of lymph node metastasis in the patient to be examined based on the expression level of the gene.   20. The method for determining lymph node metastasis according to claim 19, wherein the lymph node metastasis is lymph node metastasis of squamous cell carcinoma that develops in the oral cavity, pharynx, esophagus or lung. A step of bringing a test substance into contact with a cancer cell-derived cell line; and expression of a gene containing a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence substantially identical thereto in the cell line Detecting step;
A method for screening a lymph node metastasis inhibitor, comprising the step of identifying the test substance as a candidate substance for a lymph node metastasis inhibitor when the expression level of the gene is promoted by the test substance.   22. The method for screening a lymph node metastasis inhibitor according to claim 21, wherein the lymph node metastasis is lymph node metastasis of squamous cell carcinoma that develops in the oral cavity, pharynx, esophagus or lung.

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