Disclosure of Invention
It is an object of the present invention to provide a method for diagnosing colon adenocarcinoma by detecting the difference in the expression of VCAN gene or protein.
The invention also aims to provide a method for predicting the prognosis of colon adenocarcinoma by detecting the expression difference of VCAN genes or proteins.
It is a further object of the present invention to provide a method for treating colon adenocarcinoma by activating the VCAN gene or VCAN protein.
The fourth purpose of the invention is to provide a method for screening a medicament for treating colon adenocarcinoma.
The fifth purpose of the invention is to provide a medicine for treating colon adenocarcinoma.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides application of a product for detecting VCAN in preparing a colon adenocarcinoma diagnostic tool.
Further, the product for detecting VCAN comprises a product for detecting the expression level of VCAN genes.
Still further, the product for detecting VCAN includes a product capable of quantifying VCAN gene mRNA, and/or a product capable of quantifying VCAN protein.
The product of the present invention for quantifying VCAN gene mRNA can exert its function based on known methods using nucleic acid molecules: such as PCR, e.g., Southern hybridization, Northern hybridization, dot hybridization, Fluorescence In Situ Hybridization (FISH), DNA microarray, ASO methods, high throughput sequencing platforms, etc. The product can be used to conduct the assay qualitatively, quantitatively, or semi-quantitatively.
The nucleic acid contained in the above-mentioned products can be obtained by chemical synthesis, or by preparing a gene containing a desired nucleic acid from a biological material and then amplifying it using a primer designed to amplify the desired nucleic acid.
Further, the PCR method is a known method, for example, ARMS (Amplification Refractorymutation System) method, RT-PCR (reverse transcriptase-PCR) method, nested PCR method, or the like. The amplified nucleic acid can be detected by using a dot blot hybridization method, a surface plasmon resonance method (SPR method), a PCR-RFLP method, an in situ RT-PCR method, a PCR-SSO (sequence specific oligonucleotide) method, a PCR-SSP method, an AMPFLP (amplifiable fragment length polymorphism) method, an MVR-PCR method, and a PCR-SSCP (single strand conformation polymorphism) method.
The product capable of quantifying VCAN gene mRNA comprises a primer used in real-time quantitative PCR for specifically amplifying VCAN gene, and the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2.
The primers included in the product can be prepared by chemical synthesis, appropriately designed by referring to known information using a method known to those skilled in the art, and prepared by chemical synthesis.
The above-mentioned nucleic acids may further include a probe which can be prepared by chemical synthesis, appropriately designed by referring to known information using a method known to those skilled in the art, and prepared by chemical synthesis, or can be prepared by preparing a gene containing a desired nucleic acid sequence from a biological material and amplifying it using a primer designed for amplifying the desired nucleic acid sequence.
The product of the present invention for quantifying VCAN protein can exert its function based on known methods using antibodies: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blotting, etc. may be included.
The products of the invention for quantifying VCAN protein include antibodies or fragments thereof that specifically bind to VCAN protein. An antibody or fragment thereof of any structure, size, immunoglobulin class, origin, etc., may be used so long as it binds to the target protein. The antibodies or fragments thereof included in the assay products of the invention may be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody (partial fragment) or a peptide containing a portion of an antibody that retains the binding activity of the antibody to an antigen. Antibody fragments may include F (ab')2Fab', Fab, single chain fv (scfv), disulfide-bonded fv (dsfv) or polymers thereof, dimerized V regions (diabodies), or CDR-containing peptides. The products of the invention for quantifying VCAN protein can include an isolated nucleic acid encoding an amino acid sequence of an antibody or encoding a fragment of an antibody, a vector comprising the nucleic acid, and a cell harboring the vector.
Antibodies can be obtained by methods well known to those skilled in the art. For example, mammalian cell expression vectors that retain all or part of the target protein or incorporate polynucleotides encoding them are prepared as antigens. After immunizing an animal with an antigen, immune cells are obtained from the immunized animal and myeloma cells are fused to obtain hybridomas. The antibody is then collected from the hybridoma culture. Finally, a monoclonal antibody against the VCAN protein can be obtained by performing antigen-specific purification of the obtained antibody using the VCAN protein or a portion thereof used as an antigen. Polyclonal antibodies can be prepared as follows: an animal is immunized with the same antigen as above, a blood sample is collected from the immunized animal, serum is separated from the blood, and then antigen-specific purification is performed on the serum using the above antigen. The antibody fragment can be obtained by treating the obtained antibody with an enzyme or by using sequence information of the obtained antibody.
Binding of the label to the antibody or fragment thereof can be carried out by methods generally known in the art. For example, proteins or peptides may be fluorescently labeled as follows: the protein or peptide is washed with phosphate buffer, a dye prepared with DMSO, a buffer, or the like is added, and the solution is mixed and left at room temperature for 10 minutes. In addition, labeling may be carried out using commercially available labeling kits, such as biotin labeling kit, e.g., biotin labeling kit-NH 2, biotin labeling kit-SH (Dojindo laboratories); alkaline phosphatase labeling kits such as alkaline phosphatase labeling kit-NH 2, alkaline phosphatase labeling kit-sh (dojindo laboratories); peroxidase labeling kits such as peroxidase labeling kit-NH 2, peroxidase labeling kit-NH 2(Dojindo Laboratories); phycobiliprotein labeling kits such as phycobiliprotein labeling kit-NH 2, phycobiliprotein labeling kit-SH, B-phycoerythrin labeling kit-NH 2, B-phycoerythrin labeling kit-SH, R-phycoerythrin labeling kit-NH 2, R-phycoerythrin labeling kit SH (dojindo laboratories); fluorescent labeling kits such as fluorescein labeling kit-NH 2, HiLyte Fluor (TM)555 labeling kit-NH 2, HiLyte Fluor (TM)647 labeling kit-NH 2(Dojindo Laboratories); and DyLight 547 and DyLight647(Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody labeling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation), and EZ-marker protein labeling kit (Funakoshi Corporation). For proper labeling, a suitable instrument can be used to detect the labeled antibody or fragment thereof.
As a sample of the detection product according to the present invention, a tissue sample or fluid obtained from a biopsy subject, for example, can be used. The sample is not particularly limited as long as it is suitable for the assay of the present invention; for example, it may comprise tissue, blood, plasma, serum, lymph, urine, serosal cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, saliva, or fractions or treated materials thereof.
In a specific embodiment of the invention, the sample is from a tissue of a subject.
Further, the product for quantifying the VCAN gene or VCAN protein may be a reagent for detecting the VCAN gene or VCAN protein, a kit, a chip, a test paper and the like containing the reagent, and may also be a high-throughput sequencing platform using the reagent.
The invention also provides a tool for diagnosing the colon adenocarcinoma, and the tool can detect the VCAN gene expression level.
Further, the means comprise agents capable of quantifying VCAN gene mRNA, and/or agents capable of quantifying VCAN protein.
Furthermore, the reagent capable of quantifying VCAN gene mRNA is a primer for specifically amplifying VCAN gene used in real-time quantitative PCR, and the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2.
Further, the means for diagnosing colon adenocarcinoma includes, but is not limited to, a chip, a kit, a strip, or a high throughput sequencing platform; the high-throughput sequencing platform is a special tool for diagnosing the colon adenocarcinoma, and the construction of a gene expression profile of a person becomes very convenient and fast work along with the development of a high-throughput sequencing technology. By comparing the gene expression profiles of patients with diseases and normal people, the abnormality of which gene is related to the disease can be easily analyzed. Therefore, the finding that an abnormality in the VCAN gene is associated with colon adenocarcinoma in high-throughput sequencing is also within the scope of VCAN application.
The number of amino acids recognized by the anti-VCAN antibody or a fragment thereof used in the detection product, the diagnostic tool of the present invention is not particularly limited as long as the antibody is capable of binding to VCAN.
The present invention also provides a method for diagnosing colon adenocarcinoma, comprising the steps of:
(1) obtaining a sample from a subject;
(2) detecting the expression level of a VCAN gene or protein in a sample from the subject;
(3) correlating the measured expression level of the VCAN gene or protein with the presence or absence of disease in the subject.
(4) A decrease in the expression level of a VCAN gene or protein, as compared to a control, and the subject is judged to have or be at risk of having colon adenocarcinoma, or a patient with colon adenocarcinoma is judged to have a relapse, or a patient with colon adenocarcinoma is judged to have a poor prognosis.
The invention also provides a method of treating colon adenocarcinoma, said method comprising activating the VCAN gene or VCAN protein.
Further, the method comprises promoting expression of a VCAN gene, or promoting expression of a VCAN protein or enhancing activity of a VCAN protein.
The invention also provides a screening method of the tumor drug, which can measure the effect of the tumor drug on improving the tumor prognosis by measuring the expression level of VCAN gene or VCAN protein after adding the test drug to the cancer cells or at a certain period after applying the test drug to the tumor model animal. More specifically, when the expression level of VCAN gene or VCAN protein is elevated or restored to a normal level after addition or administration of a test drug, the drug may be selected as a therapeutic drug for improving the prognosis of tumor.
The invention also provides a medicament for treating colon adenocarcinoma, which comprises an activator of VCAN.
The inventive VCAN activator is not limited as long as the activator can promote or enhance the expression or activity of VCAN or a substance involved in an upstream or downstream pathway of VCAN, and is a drug effective for treating tumors.
The invention also provides application of the activator in preparing a medicament for treating the colon adenocarcinoma.
Further, the activator comprises a VCAN gene, a VCAN protein, a promoting miRNA, a promoting transcriptional regulator, or a promoting targeted small molecule compound.
The activator of the invention can be used for supplementing the deletion or deficiency of endogenous VCAN protein and treating colon adenocarcinoma caused by VCAN protein deficiency by increasing the expression of VCAN protein. In another aspect, the compounds can be used to enhance the activity of VCAN proteins and thereby treat colon adenocarcinoma.
The medicament of the present invention may be administered alone or together with other medicaments as a medicine. Other drugs that can be administered together with the inventive drug are not limited as long as it does not impair the effect of the inventive therapeutic or prophylactic drug, and preferably, drugs for treating or preventing tumors may include, for example, alkylating agents such as ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, procarbazine, melphalan, and ramustine; antimetabolites such as enocitabine, capecitabine, carmofur, cladribine, gemcitabine, cytarabine octadecylphosphate (cytarabine ocfosfate), tegafur-uracil, tegafur-gimeracil oteracil potassium, doxifluridine, hydroxyurea, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine, and methotrexate; plant alkaloids such as irinotecan, etoposide, sobuzolff, docetaxel, nogitecan, paclitaxel, vinorelbine, vindesine, and vinblastine; anticancer antibiotics such as actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin, netostatin stimalamer, daunorubicin, doxorubicin, pirarubicin, bleomycin, pellomycin, mitomycin C, and mitoxantrone; platinum-based drugs such as oxaliplatin, carboplatin, cisplatin, and nedaplatin; hormonal agents such as anastrozole, exemestane, estramustine, ethinylestradiol, chlormadinone, goserelin, tamoxifen, dexamethasone, toremifene, bicalutamide, flutamide, prednisolone, fosfestrol, mitotane, methyltestosterone, medroxyprogesterone, meindroxane, leuprorelin, and letrozole; biological response modifiers such as interferon alpha, interferon beta, interferon gamma, interleukin, ubenimex, dry BCG, and lentinan; and molecularly targeted drugs such as imatinib (imatinib), gefitinib (gefitinib), gemumab, ozomicin, tamibarotene, trastuzumab, tretinoin, bortezomib (bortezomib), and rituximab, and the like.
The medicine of the present invention may be prepared into various preparation forms. Including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral use.
The route of administration of the drug of the present invention is not limited as long as it exerts the desired therapeutic or prophylactic effect, and includes, but is not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, transdermal, transpleural, topical, inhalation, transmucosal, dermal, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic, intratumoral. In some cases, the administration may be systemic. In some cases topical administration.
The dose of the drug of the present invention is not limited as long as the desired therapeutic effect or prophylactic effect is obtained, and can be appropriately determined depending on the symptoms, sex, age, and the like. The dose of the therapeutic agent or prophylactic agent of the present invention can be determined using, for example, the therapeutic effect or prophylactic effect on a disease as an index.
In the context of the present invention, "diagnosing colon adenocarcinoma" includes determining whether a subject has had colon adenocarcinoma, determining whether a subject is at risk for having colon adenocarcinoma, determining whether a patient with colon adenocarcinoma has relapsed and metastasized, determining the responsiveness of a patient with colon adenocarcinoma to drug treatment, or determining the prognosis of a patient with colon adenocarcinoma.
As used herein, "treatment" encompasses treatment-related diseases or disease states in a mammal, such as a human, having the associated disease or disorder, and includes:
(1) preventing the occurrence of a disease or condition in a mammal, particularly when the mammal is susceptible to said disease condition but has not been diagnosed as having such a disease condition;
(2) inhibiting a disease or disease state, i.e., preventing its occurrence; or
(3) Alleviating the disease or condition, i.e., causing regression of the disease or condition.
The term "treatment" generally refers to the treatment of a human or animal (e.g., as applied by a veterinarian) wherein some desired therapeutic effect is achieved, e.g., inhibiting the progression of a condition (including slowing the progression, stopping the progression), ameliorating the condition, and curing the condition. Treatment as a prophylactic measure (e.g., prophylaxis) is also included. The use of a patient who has not yet developed a condition but who is at risk of developing the condition is also encompassed by the term "treatment".
The invention has the advantages and beneficial effects that:
the invention discloses a molecular marker for diagnosing colon adenocarcinoma, which can be used for judging the early stage of colon adenocarcinoma occurrence and provides the survival rate of patients.
The therapeutic agent of the present invention comprising an activator of VCAN gene or protein can be used as a novel therapeutic agent for colon adenocarcinoma.