KR101844845B1 - Composition for predicting susceptibility to drugs for the therapy of biliary tract cancer - Google Patents

Abstract

The present invention provides a composition comprising an agent for measuring the expression level of a gene, a protein and / or a microRNA capable of predicting susceptibility to a drug for treating a bile duct cancer, and a kit comprising the composition. The present invention also provides a method for predicting the sensitivity of a drug for treating biliary cancer by measuring the expression level of genes, proteins and / or microRNAs.

Description

Technical Field [0001] The present invention relates to a composition for predicting drug susceptibility for the treatment of biliary cancer,

The present invention relates to a composition for predicting susceptibility to a bile duct cancer therapeutic agent, and more particularly, to a composition for predicting susceptibility to a bile duct cancer therapeutic agent, which comprises a gene, protein or microRNA (microRNA, miRNA) To a composition capable of predicting the susceptibility to the biliary cancer therapeutic agent.

Cholangiocarcinoma is a cancer that occurs in the liver bile ducts and is known to occur in stem cells such as liver cancer (Sell and Dunsford Am. J. Pathol. 134: 1347-1363, 1989). Worldwide, the incidence of biliary cancer is much lower than that of liver cancer, but the incidence is higher in Southeast Asia than in Europe or North America. Biliary cancer is characterized by a high recurrence rate, which is not effective, and general chemotherapy or radiotherapy is not effective (Pederson et al. Cancer Res. 4325-4332, 1997).

The treatment of surgical operations for biliary cancer, radiation therapy, chemotherapy or a combination of these is generally used. In particular, gemcitabine, cisplatin, oxaliplatin and 5FU have been used for the treatment of the above-mentioned bile duct cancer, but the therapeutic effect is insufficient due to the resistance of the therapeutic agent due to tolerance . Thus, although GP treatment for treating cholangiocarcinoma with cisplatin in combination with gemcitabine has been used to treat biliary cancer, the cure rate is remarkably low.

On the other hand, in general, in the case of chemotherapy, the reactivity of the organism when the anticancer drug is administered is highly dependent on the sensitivity of the cancer cell to the anticancer drug. Such sensitivity varies depending on the kind of cancer, and the difference is due to the quantitative or qualitative difference of the target molecule of the anticancer drug or the related factors, or the acquisition of drug resistance. Although research on the therapeutic reactivity of individual anticancer agents according to specific genes has been continuously developed, there is a problem that there is no biomarker capable of predicting anticancer drug susceptibility due to the complex action of the related factors and the therapeutic agent diversity.

Based on this background, if the target cancer cell is susceptible to the drug, it is possible to identify the genetic change of the cancer cell specifically, and it is possible to determine the effect of the drug early and select a new treatment method, Which may contribute to higher survival rates.

It is an object of the present invention to provide a composition for predicting susceptibility, which comprises a preparation capable of measuring gene, protein and / or microRNA, which is a biomarker capable of predicting susceptibility to a drug for the treatment of bile duct cancer, And a kit comprising the composition.

It is another object of the present invention to provide a method for providing information for predicting the susceptibility of a drug for the treatment of bile duct cancer using the biomarker according to the present invention.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The inventors of the present invention have found that when a bile duct cancer cell line and a resistant cholangiocarcinoma cell line are treated with a bile duct cancer treatment or a drug for overcoming resistance, the expression of specific genes and proteins decreases, and the expression of microRNAs (miRNAs) And found the present invention.

That is, the present invention provides a therapeutic effect on a drug for treating biliary cancer by measuring a change in the expression of a specific gene, microRNA (miRNA) and protein after administration of a drug for treating bile duct cancer in a patient with biliary cancer, And to overcome resistance to

In the present invention, the "susceptibility prediction" means prediction of the degree of therapeutic effect when a specific drug is administered to individual cancer of a patient or resistance to tolerance in a patient suffering from resistance cancer. If the sensitivity to the specific drug is high, an excellent therapeutic effect can be expected, or an effect of overcoming tolerance of tolerant cancer patients can be expected. On the other hand, if the susceptibility is low, the desired therapeutic effect can not be obtained or the effect of failing to overcome tolerance can be expected. Through the sensitivity prediction, it can be predicted whether the patient will respond favorably to administration of the drug, surgical intervention and chemotherapy, or whether the patient can survive for a long time after the therapeutic treatment.

According to one embodiment of the present invention, there is provided a biomarker for predicting susceptibility to a drug for the treatment of bile duct cancer comprising the following genes, proteins and / or microRNAs (miRNAs) The susceptibility to drugs for cancer treatment can be predicted. In the above sensitivity-sensitive prediction, the biliary cancer cells are separated from the bile duct cancer tissues obtained by the biopsy or the like before the actual treatment, and the bile duct cancer treatment drug is administered. Thereafter, the change in the expression amount of the biomarker The measurement can give clinical usefulness because it can predict the efficacy of the treatment and overcome the drug resistance in advance.

In the present invention, the term "biomarker" refers to a molecule that can be detected and evaluated as an indicator of a normal biological process, an onset process, or a pharmacological response to therapeutic intervention, and specifically includes a protein or a poly- , MicroRNA, and the like. Such biomarkers are characterized in that they can be measured by reflecting the physiological, pharmacological or disease process or condition.

Specifically, the gene of the present invention may be selected from the group consisting of YAP1, TAZ, TEAD1, TEAD2, TEAD3, TEAD4, TGF-beta, SMAD2, SMAD3, SMAD4, CEACAM5, CEACAM6, EGFR, MET, AKT1, AKT2, AKT3, NOTCH3, , HES5, GAS6, and AXL. In the present invention, when the biliary cancer cancer drug is administered to a patient with biliary cancer, the expression of the gene is down-regulated compared to before administration of the drug, , But is not limited thereto.

The protein of the present invention may be at least one selected from the group consisting of YAP1, TEAD4, TGF-beta, SMAD4, EGFR, C-MET, NOTCH3, HES5 and AXL. The TGF-beta may be present in the cell or secreted outside the cell in the cell. In the present invention, when a biliary cancer cancer drug is administered to a patient suffering from biliary cancer, the expression of the protein is down-regulated compared to before administration of the drug, , But is not limited thereto.

In addition, the microRNA of the present invention can be used for the production of a microarray of hsa-miR-92a-1-5p, hsa-miR-135b-3p, hsa-miR-31, hsa- Hsa-miR-196b-3p, hsa-miR-18a-3p, hsa-miR-149-5p, hsa-miR-99b-3p, hsa-miR-4778-5p, HSA-miR-421, hsa-miR-1301-3p, hsa-miR-93-3p, hsa-miR-100-5p, hsa-miR-4304, hsa-miR-1273g-3p, HSA-miR-4669, hsa-miR-98-5p, hsa-miR-378e, hsa-miR-4444, hsa-miR-6734-5p, hsa-miR-193b-5p, hs-miR-6740-5p, hsa-miR-629-3p, hsa-miR-4684-3p, hsa-miR-6746-5p, hsa-miR-99b, hsa-miR-422a, 5p, hsa-miR-423-5p, hsa-miR-6777-5p, hsa-miR-4442, hsa-let-7i-5p, hsa-miR-3911, Hs-miR-675-5p, hsa-miR-7107-5p, hsa-miR- hs-miR-146a-3p, hsa-miR-3459, hsa-miR-455-3p, hsa-miR-146a-3p, hsa-miR- hs-miR-368g, hsa-miR-106b-3p, hsa-miR-320c, hsa-miR-3621, hsa-miR-3613-3p, -mR-1469, hs HSA-miR-1443-5p, hsa-miR-4467, hsa-miR-146b-5p, hsa-miR-210-3p, hsa-miR-8063, Hs-miR-4660-5p, hsa-miR-1237-5p, hsa-miR-4486, hsa-miR-2977, hsa-miR- Hs-miR-3693-3p, hsa-miR-7114-5p, hsa-miR-3149, hsa-miR-4502, hsa-miR-509-3p, hsa-miR-192-5p, hsa- HSA-miR-194-3p, hsa-miR-8075, hsa-miR-27a-3p, hsa-miR-200a-3p, hsa-miR-6723-5p, hsa-miR-7108, hsa-miR-6786-5p, hsa-miR-6776-5p, hsa-miR-6722-3p, hsa-miR- miR-690, hsa-miR-4632-5p, hsa-miR-6848-5p, hsa-miR-4651, hsa-miR- HSA-miR-1915-3p, hsa-miR-22-3p, hsa-miR-638, hsa-miR-4763-3p, hsa-miR-141-3p, hsa-miR-6727-5p, hsa-miR-4466, hsa-miR-6791-5p, hsa-miR-2861, hsa-miR-762, hsa-miR-6724-5p, hsa- one species selected from the group consisting of miR-3185, hsa-miR-4745-5p, hsa-miR-8069, hsa-miR-6850-5p, hsa-miR-6789-5p and hsa- Can be.

In one embodiment of the present invention, the microRNA is selected from the group consisting of hsa-miR-92a-1-5p, hsa-miR-135b-3p, hsa-miR-31, hsa- HSA-miR-18a-5p, hsa-miR-196b-3p, hsa-miR-18a-3p, hsa-miR-149-5p, miR-805, hsa-miR-421, hsa-miR-1301-3p, hsa-miR-93-3p, hsa-miR- miR-1469, hsa-miR-3671, hsa-miR-3613-3p, hsa-miR-1968-5p, hsa-miR-4669, hsa-miR- HSA-miR-1343-5p, hsa-miR-4467, hsa-miR-146b-5p, hsa-miR-210-3p, hsa-miR- hs-miR-1237-5p, hsa-miR-4486, hsa-miR-297, hsa-miR-4690-5p, hsa-miR-6860, miR-365-3p, hsa-miR-7114-5p hsa-miR-22-5p and hsa-miR- -mR-194-3p. < / RTI > In the present invention, when the microRNA is down-regulated, it is highly susceptible to a drug for treating biliary cancer, and it can be predicted that the drug will exhibit a therapeutic effect on biliary cancer cells. However, no.

In another embodiment of the present invention, the microRNA is selected from the group consisting of hsa-miR-98-5p, hsa-miR-378e, hsa-miR-4444, hsa-miR-6734-5p, hsa- HSA-miR-6740-5p, hsa-miR-629-3p, hsa-miR-4684-3p, hsa-miR-6746-5p, hsa-miR-99b, miR-391, hsa-let-7e-5p, hs-miR-423-5p, hsa-miR-6777-5p, hsa-miR-4442, hsa-let- HSA-miR-6831-5p, hsa-miR-5006-5p, hsa-miR-99b-5p, hsa-miR-601, hsa-miR- Hs-miR-146a-3p, hsa-miR-3459, hsa-miR-183-5p, hsa-miR-151a-3p, hsa- miR-4298, hsa-miR-378g, hsa-miR-106b-3p and hsa-miR-320c. In the present invention, when the microRNA is down-regulated, it is highly susceptible to the drug for treating biliary cancer, and the tolerance-overcoming effect of the drug can be predicted.

In another embodiment of the present invention, the microRNA is selected from the group consisting of hsa-miR-3621, hsa-miR-3613-3p, hsa-miR-1908-5p, hsa-miR- HSA-miR-1343-5p, hsa-miR-4467, hsa-miR-146b-5p, hsa-miR-210-3p, hsa-miR-8063, hsa-miR-1228-5p, hsa hi-miR-1237-5p, hsa-miR-4486, hsa-miR-297, hsa-miR-4690-5p, hsa-miR-6860, hsa-miR-194-5p, hsa miR-3693-3p, hsa-miR-7114-5p, hsa-miR-22-5p, hsa-miR- HSA-miR-194-3p, hsa-miR-8075, hsa-miR-27a-3p, hsa-miR-200a-3p, hsa-miR-6723-5p, hsa-miR-92b-5p, -7108, hsa-miR-6786-5p, hsa-miR-6776-5p, hsa-miR-6722-3p, hsa-miR-6125, hsa-miR-7704, Hs-miR-4616, hsa-miR-4632-5p, hsa-miR-6848-5p, hsa-miR-4651, hsa-miR-6089, hsa-miR-6800, hsa-miR-7108-5p , hsa-miR-1915-3p, hsa-miR-22-3p, hsa-miR-638, hsa-miR-4763-3p, hsa-miR-141-3p, hsa-miR-6727-5p, -4466, hsa-miR-6791-5p, hsa-miR-2861, hsa-miR-762, hsa-miR-6724-5p, hsa-miR-3940-5p, 1, hsa-miR-3185, hsa-miR-4745-5p, hsa-miR-8069, hsa-miR-6850-5p, hsa-miR-6789-5p and hsa- Or more. In the present invention, when the microRNA is up-regulated, the drug is highly susceptible to a drug for treating biliary cancer, and the drug may exhibit a therapeutic effect on biliary cancer cells, but is not limited thereto .

In another embodiment of the present invention, the microRNA of the present invention is selected from the group consisting of hsa-miR-8075, hsa-miR-27a-3p, hsa-miR-200a-3p, hsa- Hs-miR-7704, hsa-miR-6776-5p, hsa-miR-6776-5p, hsa-miR-6722-3p, hsa-miR-7108, hs-miR-6905, hsa-miR-6325p, hsa-miR-6748-5p, hsa-miR- -mR-7158-5p, hsa-miR-1915-3p, hsa-miR-22-3p, hsa-miR-638, hsa-miR-4763-3p, hsa-miR-141-3p, HSA-miR-4466, hsa-miR-6791-5p, hsa-miR-2861, hsa-miR-762, hsa-miR-6724-5p, hsa-miR-6789-5p, hsa-miR-4745-5p, hsa-miR-8069, hsa-miR-6750-5p and hsa-miR-4734. . In the present invention, when the microRNA is up-regulated, it is highly susceptible to the drug for treating biliary cancer, and the tolerance-overcoming effect of the drug can be predicted.

According to another embodiment of the present invention, there is provided a composition for predicting susceptibility comprising an agent for measuring an expression level of a gene, a protein and / or a microRNA according to the present invention. In the present invention, by using a composition capable of measuring a change in the expression level of the biomarker described above, it is possible to anticipate the effectiveness of the treatment and / or overcome the drug resistance before administration of the drug to the biliary cancer patient, It is possible to impart useful utility.

The term " composition for predicting sensitivity "of the present invention means a preparation for measuring expression levels of genes, proteins and microRNAs capable of predicting susceptibility to drugs for treating bile duct cancer.

In one embodiment of the present invention, the agent capable of measuring the expression level of the gene may be a primer pair or a probe capable of specifically binding to a gene contained in the biomarker of the present invention, but is not limited thereto .

In another embodiment of the present invention, the agent capable of measuring the expression level of the protein may be an antibody, a peptide or a nucleotide that specifically binds to a protein contained in the biomarker of the present invention, but is not limited thereto.

In another embodiment of the present invention, the agent capable of measuring the expression level of the microRNA may be a primer pair or a probe capable of specifically binding to the microRNA contained in the biomarker of the present invention, It is not.

Another embodiment of the present invention relates to a kit for predicting susceptibility to drugs for treating biliary cancer, which comprises the composition for predicting susceptibility according to the present invention. The kit provided by the present invention can measure the expression levels of genes, proteins and / or microRNAs according to the present invention, so that the effectiveness of the drug for the treatment of bile duct cancer and / And thus can be given clinical usefulness.

In the present invention, the term "kit " means a set of a composition and accessories necessary for a specific purpose. For the purpose of the present invention, the kit of the present invention can estimate the sensitivity of the drug for the treatment of biliary cancer by measuring the expression levels of genes, proteins and microRNAs capable of predicting susceptibility to drugs for treating bile duct cancer.

Specifically, the kit includes not only an agent for measuring the expression level of a gene, protein or microRNA corresponding to the biomarker according to the present invention, but also an agent for measuring the expression level, Tools, reagents, and the like. Examples of such tools or reagents include, but are not limited to, suitable carriers, markers capable of generating detectable signals, chromophores, solubilizers, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Examples of the insoluble carrier include polystyrene, polyethylene, polypropylene, polyester, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, Acrylonitrile, fluorine resin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose and combinations thereof.

Since the kit for predicting susceptibility to drugs for treating biliary cancer of the present invention contains the aforementioned composition for predicting susceptibility capable of measuring genes, proteins and / or microRNAs according to the present invention as an effective ingredient, The description thereof is omitted in order to avoid the excessive complexity of the present specification.

According to another embodiment of the present invention, there is provided a method for providing information on the susceptibility of a drug for the treatment of bile duct cancer using gene, protein and / or microRNA according to the present invention (hereinafter referred to as 'information providing method'). . Specifically, (a) measuring the expression level of a gene, protein and / or microRNA in a biological sample separated from a desired individual, (b) administering a drug for the treatment of bile duct cancer to the biological sample, (c) (D) measuring the level of expression of the gene, protein and / or microRNA in the biological sample after administration of the drug for the treatment of bile duct cancer; and (d) measuring the expression level measured in step (a) RTI ID = 0.0 > expression level < / RTI >

The information providing method of the present invention is a method for providing information to a biliary cancer patient by measuring changes in the expression levels of genes, proteins and / or microRNAs according to the present invention before and after administration of a drug for treating bile duct cancer to a biological sample obtained from a biliary cancer patient It is possible to predict the therapeutic effect of the patient by predicting the susceptibility to the bile duct cancer treatment drug before the direct administration of the drug for treating the bile duct cancer and further to predict whether the resistance against the drug is overcome.

However, the above-mentioned "intended subject " of the present invention means an individual, preferably a biliary cancer patient, before or after treatment with a drug for treating bile duct cancer. The above-mentioned biliary cancer patients may include patients who have undergone chemotherapy, radiotherapy, chemotherapy or the like, patients who are not treated or scheduled by the therapies, and patients who are resistant to drugs.

The term "biological sample" obtained from the subject in the present invention means a tissue, cell, blood, serum, plasma, saliva, sputum, cerebrospinal fluid, feces or the like in which the level of the gene, protein and / Or urine, and the like, but are not limited thereto.

In the present invention, the process of separating a gene, protein or microRNA (miRNA) from a biological sample obtained from the desired subject can be performed through a known process.

In the present invention, the gene whose expression level is measured in the steps (a) and (c) is selected from the group consisting of YAP1, TAZ, TEAD1, TEAD2, TEAD3, TEAD4, TGF-beta, SMAD2, SMAD3, SMAD4, CEACAM5, CEACAM6, EGFR, MET, AKT1, AKT2, AKT3, NOTCH3, DLL1, JAG1, HES5, GAS6 and AXL. In the present invention, when the expression level of the gene measured in step (c) is down-regulated from the expression level of the gene measured in step (a), sensitivity to the drug for treating biliary cancer is high, It can be predicted that the drug will have a therapeutic effect.

In the present invention, the protein whose expression level is measured in steps (a) and (c) may be one selected from the group consisting of YAP1, TEAD4, TGF-beta, SMAD4, EGFR, C-MET, NOTCH3, HES5 and AXL It may be at least one selected. However, the TGF-beta may be present in the cell or secreted outside the cell in the cell. In the present invention, when the expression level of the protein measured in the step (c) is down-regulated from the expression level of the protein measured in the step (a), the sensitivity to the drug for treating biliary cancer is high, It can be predicted that the drug will have a therapeutic effect.

In the present invention, the microRNAs whose expression levels are measured in the steps (a) and (c) are hsa-miR-92a-1-5p, hsa-miR-135b-3p, hsa-miR- HSA-miR-188-3p, hsa-miR-149b-5p, hsa-miR-196b-3p, hsa-miR- Hs-miR-159p, hsa-miR-1580p, hsa-miR- HSA-miR-4444, hsa-miR-6744, hsa-miR-375e, hsa-miR- Hs-miR-6746-5p, hsa-miR-6743-5p, hsa-miR-1943b-5p, hsa-miR- hs-miR-422a, hsa-miR-378a-5p, hsa-miR-423-5p, hsa-miR-6777-5p, hsa-miR-4442, hsa-let-7i-5p, hsa hs-miR-125b-5p, hsa-miR-931-5p, hsa-miR-6831-5p, hsa-miR- hs-miR-6785-5p, hsa-miR-7107-5p, hsa-miR-455-3p, hsa-miR-146a-3p, hsa-miR-345, hsa-miR-4459, HSA-miR-369, hsa-miR-106b-3p, hsa-miR-320c, hsa-miR-3698 hsa-miR-3643-3p, hsa-miR-1843-5p, hsa-miR-4674, hsa-miR-1469, hsa-miR-6805-5p, hs-miR-146b-5p, hsa-miR-210-3p, hsa-miR-8063, hsa-miR-1228-5p, hsa-miR-3065-5p, -4486, hsa-miR-297, hsa-miR-4690-5p, hsa-miR-6860, hsa-miR-194-5p, hsa-miR-3149, hs-miR-192-5p, hsa-miR-3663-3p, hsa-miR-7114-5p, hsa-miR-22-5p, hsa-miR-194-3p, Hs-miR-67a-3p, hsa-miR-67a-5p, hsa-miR-67a-5p, hsa-miR- hs-miR-6325, hsa-miR-6125, hsa-miR-7704, hsa-miR-2278, hsa-miR-6722-5p, hsa-miR-4516, HSA-miR-4651, hsa-miR-6089, hsa-miR-6800, hsa-miR-7108-5p, hsa-miR-1915-3p, miR-6781-5p, hsa-miR-1461-3p, hsa-miR-6727-5p, hsa-miR-6366, hsa-miR-4763-3p, hs-miR-6794-5p, hsa-miR-3940-5p, hsa-miR-4741, hsa-miR-3185, hsa-miR-4745-5p, -6850-5p, hsa-miR-6789-5p, hsa-miR-4734 < / RTI >

In one embodiment of the present invention, the microRNAs for which expression levels are measured in steps (a) and (c) are hsa-miR-92a-a1-5p, hsa-miR-135b-3p, hsa-miR-31 hsa-miR-184a-5p, hsa-miR-196b-3p, hsa-miR-18a-3p, hsa-miR- Hsa-miR-1005p, hsa-miR-475-5p, hsa-miR-8060, hsa-miR-421, hsa-miR-1371-3p, hsa-miR- hsa-miR-3613-3p, hsa-miR-1783-3p, hsa-miR-7162-3p, hsa-miR-4669, hsa-miR- HSA-miR-1469, hsa-miR-6805-5p, hsa-miR-1343-5p, hsa-miR-4467, hsa-miR-146b-5p, hs-miR-1286-5p, hsa-miR-1236-5p, hsa-miR-1237-5p, hsa-miR-8063, hsa-miR-1228-5p, hsa-miR- hs-miR-365-3p, hsa-miR-365-3p, hsa-miR- -mR-7114-5p hsa-miR-22-5p and hsa-miR-194-3p. In the present invention, when the expression level of the microRNA measured in the step (c) is down-regulated below the expression level of the microRNA measured in the step (a), the sensitivity to the drug for treating biliary cancer It can be predicted that the drug can exhibit a therapeutic effect on the drug.

In another embodiment of the present invention, the microRNAs for which expression level is measured in steps (a) and (c) are hsa-miR-98-5p, hsa-miR-378e, hsa-miR- HSA-miR-6743-5p, hsa-miR-1943b-5p, hsa-miR-4499, hsa-miR-6740-5p, hsa-miR-629-3p, hs-miR-442, hsa-miR-378a-5p, hsa-miR-423-5p, hsa-miR-677-5p, hsa-miR-4442, hs-miR-391, hsa-let-7e-5p, hsa-miR-6831-5p, hsa-miR-5006-5p, hsa-miR-99b-5p, Hs-miR-6785-5p, hsa-miR-7107-5p, hsa-miR-455-3p, hsa-miR-146a-3p, hsa-miR-345, hsa-miR-4459, Hsa-miR-106b-3p, and hsa-miR-320c, hsa-miR-151a-3p, hsa-miR-4298, hsa-miR-378g, hsa-miR-106b-3p and hsa-miR-320c. In the present invention, when the expression level of the microRNA measured in the step (c) is down-regulated below the expression level of the microRNA measured in the step (a), the sensitivity to the drug for treating biliary cancer And it can be predicted that the drug has overcome tolerance to the drug.

In another embodiment of the present invention, the microRNAs whose expression level is measured in steps (a) and (c) are selected from the group consisting of hsa-miR-3621, hsa-miR-3613-3p, hsa-miR- HSA-miR-4674, hsa-miR-1469, hsa-miR-6805-5p, hsa-miR-1343-5p, hsa-miR- HSA-miR-8063, hsa-miR-1228-5p, hsa-miR-3065-5p, hsa-miR-1237-5p, hsa-miR-4486, hsa- hs-miR-1960-p, hsa-miR-1960-p, hsa-miR- HSA-miR-7114-5p, hsa-miR-22-5p, hsa-miR-194-3p, hsa-miR-8075, hsa-miR-27a-3p, 6723-5p, hsa-miR-6722-3p, hsa-miR-6125, hsa-miR-6786-5p, hsa-miR- miR-4651-5, hsa-miR-4651, hsa-miR-4682-5p, hsa-miR- HSA-miR-6800, hsa-miR-7108-5p, hsa-miR-1915-3p, hsa-miR-22-3p, hsa-miR-638, hsa-miR-4763-3p, hsa-miR -141-3p, hsa-miR-6727-5p, hsa-miR-4466, hsa-miR-6791-5p, hsa-miR-2 HSA-miR-7125p, hsa-miR-9705p, hsa-miR-7141, hsa-miR- hsa-miR-6750-5p, hsa-miR-6789-5p and hsa-miR-4734hsa-miR-92b-5p, hsa-miR- HSA-miR-6125, hsa-miR-7704, hsa-miR-2278, hsa-miR-6729-5p, hsa-miR- HSA-miR-6800, hsa-miR-7108-5p, hsa-miR-1915-3p, hsa-miR-22-3p, hsa-miR-638, HSA-miR-4466, hsa-miR-4466, hsa-miR-6727-5p, hsa-miR-4466, hsa-miR-6791-5p, hsa- Hs-miR-6750-5p, hsa-miR-6789-5, hsa-miR-3940-5p, hsa-miR-4741, hsa-miR-3185, hsa- 5p and hsa-miR-4734. In the present invention, when the expression level of the microRNA measured in the step (c) is up-regulated above the expression level of the microRNA measured in the step (a), the sensitivity to the drug for treating the biliary cancer And is expected to show a therapeutic effect on the drug, but is not limited thereto.

In another embodiment of the present invention, the microRNAs whose expression level is measured in steps (a) and (c) are hsa-miR-8075, hsa-miR-27a-3p, hsa-miR- hsa-miR-6723-5p, hsa-miR-92b-5p, hsa-miR-7108, hsa-miR-6786-5p, hsa-miR- MiR-4648-5p, hsa-miR-4651, hsa-miR-7124, hsa-miR-7704, hsa-miR-2278, hsa-miR-6729-5p, hsa- HSA-miR-6089, hsa-miR-6800, hsa-miR-7108-5p, hsa-miR-1915-3p, hsa-miR-22-3p, hsa- HSA-miR-672-5p, hsa-miR-4466, hsa-miR-6791-5p, hsa-miR-141-3p, hsa-miR- hsa-miR-6740-5p, hsa-miR-6741-5p, hsa-miR-4741-5, hsa-miR- hsa-miR-4734 < / RTI > In the present invention, when the expression level of the microRNA measured in the step (c) is up-regulated above the expression level of the microRNA measured in the step (a), the sensitivity to the drug for treating biliary cancer And it can be predicted that the drug has overcome tolerance to the drug.

In the present invention, the method for measuring the change in the expression level of the gene is a method for measuring the amount of the complementary complex of the gene to be synthesized by using a primer or a probe specifically binding to the gene. The specific method is not particularly limited, (RT-PCR), competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), and reverse transcription-polymerase chain reaction Northern blot assay, DNA chip or the like.

In the present invention, the method for measuring the change in the expression level of the protein is to measure the amount of the antigen-antibody complex formed using an antibody that specifically binds to the protein. Specific methods are not particularly limited, Western blot assay, ELISA, 2D, and the like.

In addition, in the present invention, the method for measuring the change in expression of the microRNA is to measure the formation amount of a complementary complex of a gene using a primer or a probe specifically binding to the microRNA, For example, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blot analysis (Northern blot analysis) assay or DNA chip analysis.

However, the "antigen-antibody complex" of the present invention means a conjugate of the protein and the antibody specific thereto, and the amount of the antigen-antibody complex formed is quantitatively determined through the intensity of the signal of the detection label It is measurable. The term "gene complementary binding complex" of the present invention refers to a combination of the gene and a primer or probe specific thereto, and the formation amount of the gene complementary binding complex is determined by the intensity of a signal of a detection label Lt; RTI ID = 0.0 > quantitative < / RTI >

Specifically, the detection label may be selected from the group consisting of an enzyme, a minerals, a ligand, a luminescent material, a microparticle, a redox molecule, and a radioisotope, but is not limited thereto. More specifically, enzymes that can be used as detection labels include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholine esterase A glucosoxidase, a hexokinase and a GDPase, an RNase, a glucose oxidase and a luciferase, a phosphofructokinase, a phosphoenolpyruvate carboxylase, an aspartate aminotransferase, a phosphoenolpyruvate decarboxylase Lase, < / RTI > But is not limited thereto. Examples of the above-mentioned minerals that can be used include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, phycocyanin, allophycocyanin, o-phthaldehyde, . The ligand may be a biotin derivative or the like, but is not limited thereto. Examples of the luminescent material include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. The fine particles include, but are not limited to, colloidal gold and colored latex. In addition, the redox molecules include ferrocene, ruthenium complex compounds, Biology hydrogen, quinone, Ti ions, Cs ions, diimide, 1,4-benzoquinone, _{hydroquinone, K 4 W (CN) 8} , [Os (bpy) 3] ^{2 +} , [RU (bpy) ₃ ] ^{2 +} , [MO (CN) ₈ ] ^{4 -} , and the like. In addition, the radioisotope includes a ^{3 H, 14 C, 32 P} , 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re, In It is not limited.

The "biliary cancer" of the present invention is a carcinoma arising in the epithelial cells surrounding the gallbladder, and cholangiocarcinoma and gallbladder cancer are collectively referred to as bile duct cancer. For the purpose of the present invention, the cholangiocarcinoma comprises a bile duct cancer resistant to a bile duct cancer therapeutic drug.

The "drug-resistant biliary cancer" of the present invention is extremely sensitive to the treatment of drugs for treatment of bile duct cancer, in particular, anticancer drugs, and the symptoms of the cancer are improved, relieved, alleviated or treated by treatment with anti- Biliary cancer does not mean. The drug-resistant biliary cancer may be resistant to certain anticancer agents from the beginning and may not develop resistance to the same therapeutic agent due to gene mutation in cancer cells due to long-term drug treatment, It is possible. Preferably, the resistance-inducing drug may be, but is not limited to, gemcitabine.

The term "primer" of the present invention means a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming a base pair complementary to a template, Refers to a short nucleic acid sequence that serves as a starting point for replication. The primers can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. Preferably, in the invention, PCR amplification is performed using a sense and antisense primer of a microRNA polynucleotide to predict susceptibility through production of a product. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art.

The "probe" of the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or several hundreds of nucleotides which can be specifically bound to the gene or the microRNA. The oligonucleotide oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, and RNA probes. For easier detection, they can be labeled with fluorescent molecules, biotin, isotopes, and the like. But is not limited thereto.

In addition, the "antibody" of the present invention includes functional forms of antibody molecules as well as complete forms having polyclonal antibodies, monoclonal antibodies, recombinant antibodies and two full-length light chains and two full- , F (ab '), F (ab') 2, and Fv.

In the present invention, the drug for treating bile duct cancer to be subjected to the susceptibility prediction in the present invention is an anticancer agent capable of inhibiting the growth of biliary cancer cells and inducing apoptosis, and the kind thereof is not particularly limited. For example, , Imatinib, oxaliplatin, rituximab, elotinib, neratinib, lapatinib, zetitib, vandetanib, nrotinib, semathanib, conservatib, acitinib, cediranib, lestauritinib, trastuzumab Cefotaxime, biscuit alum, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramer, cefotaxime, cefotaxime, cisplatin, Amlodipine, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxifluridine, gemcitabine, But are not limited to, dexamethasone, deamin, femetrexed, tegafur, capecitabine, gimeracin, atheracyl, azacytidine, methotrexate, uracil, cytarabine, fluorouracil, , Capecitabine, mercaptopurine, thioguanine, cladribine, calmophor, ralitriptycide, docetaxel, paclitaxel, irinotecan, velotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, tenny But are not limited to, but are not limited to, fosiod, doxorubicin, dirubicin, epirubicin, mitoxantrone, mitomycin, blormomycin, daunorubicin, dactinomycin, pyrabicin, aclarubicin, But are not limited to, thiourea, zolomide, laver, iospasmide, cyclophosphamide, melphalan, altretin, docavazine, thiotepa, nimustine, chlorambucil, mitolactol, reucoborin, Glutethimide, anagrelide, (E) -N1- (3), < / RTI > < RTI ID = 0.0 > (Dimethylamino) propyl) -N8-hydroxy-2 - ((naphthalen-1-yloxy) methyl) oct- N 8 -hydroxy-2 - ((naphthalen-1-yloxy) methyl) oct-2-enediamide and carmustine, preferably a histone deacetylation inhibitor, Lt; / RTI > More preferably at least one selected from the group consisting of vorinostat, romidepsin, valproic acid, panobinostat, entinostat, belinostat, ((naphthalen-1-yloxy) methyl) oct-2-ene-2- Consisting of (E) -N1- (3- (dimethylamino) propyl) -N8-hydroxy-2- ((naphthalen-1-yloxy) methyl) oct-2-enediamide and resminostat (E) -N1- (3- (dimethylamino) propyl) -N8-hydroxy-2 - ((naphthalen-1-yloxy) methyl) oct- (E) -N1- (3- (dimethylamino) propyl) -N8-hydroxy-2- ((naphthalen-1-yloxy) methyl) oct-2-enediamide.

The biomarker of the present invention predicts the sensitivity of a bile duct cancer cell to a drug for treatment of bile duct cancer and effectively predicts the therapeutic effect of the bile duct cancer cell against the drug for treating a specific bile duct cancer and whether the survival of the bile duct cancer cell is overcome.

In addition, the present invention measures the expression level of a biomarker for predicting susceptibility to a drug for the treatment of bile duct cancer in a sample of an objective individual, and determines the therapeutic effect of the drug for the treatment of biliary cancer and whether the drug is resistant to the drug Can be provided.

FIG. 1 shows the results of IC ₅₀ by growth inhibition of a bile duct cancer cell line according to an embodiment of the present invention.
FIG. 2 shows a gene array result of a cholangiocarcinoma cell line according to an embodiment of the present invention.
3 (a) to 3 (e) show results of Western blot analysis according to an embodiment of the present invention.
FIG. 4 shows a result of a microRNA (microarray) microarray analysis according to an embodiment of the present invention in which expression is commonly decreased in biliary tumor cells and resistant cell lines.
Figure 5 shows the results of microRNA (microarray) microarray analysis according to an embodiment of the present invention showing decreased expression in resistant cell lines.
FIG. 6 is a microarray analysis of a microRNA (miRNA) according to an embodiment of the present invention, showing a common expression increase in biliary tumor cells and resistant cell lines.
FIG. 7 shows the results of microRNA (microarray) microarray analysis according to an embodiment of the present invention showing an increase in expression in the resistant cell line.
FIG. 8 shows the results of quantitative reverse transcription PCR (qRT-PCR) of microRNA according to an embodiment of the present invention.
FIG. 9 is a Western blot analysis result of a cell signal transduction pathway according to transformation of microRNA according to an embodiment of the present invention.
FIG. 10 shows the results of in situ hybridization and immunohistochemical staining according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

[ Example ]

[ Example  One] Resistant biliary cancer cell line ( SNU1196 / GR )

A resistant biliary cancer cell line (SNU1196 / GR) with drug resistance was constructed by using the bile duct cancer cell line SNU1196 as a parent cell (SNU1196 / P).

Specifically, the gemcitabine drug was administered to the cholangiocarcinoma cell line SNU1196 for 3 days at a concentration of 0.075 μM corresponding to the IC ₅₀ estimated by MTT assay. Thereafter, the volume of RPMI 1640 without drug is cultured in a nutrient medium containing 10% fetal bovine serum (FBS; hyclone, logan, UT), and when cells are recovered, the gemcitabine drug is administered at a concentration twice the IC ₅₀ And the restoration process was repeated three days later. The above procedure was repeated for one year to construct a resistant biliary cancer cell line (SNU1196 / GR) with therapeutic resistance.

[ Example  2] Cholangiocarcinoma cell line ( SNU1196 / P and SNU1196 / GR ) Culture

(E) -2- (naphthalen-1-yl-oxymethyl) -oct-2-enedioic acid-1 - [(3- dimethylamino- propyl) -amide] 8-hydroxy To overcome the tolerance of resistant biliary cancer cells by amide (CG200745), bile duct cancer cell lines SNU1196 / P and SNU1196 / GR were subcultured. SNU1196 / P cell line was purchased from Korean Cell Line Bank (KCLB, Korean Cell Line Bank, Seoul, Korea). The cell line was cultured in a 5% CO ₂ incubator at 37 ° C in a nutrient medium containing 10% fetal bovine serum (FBS; hyclone, logan, UT) in a volume of RPMI 1640 according to the protocol presented by the Korean Cell Line Bank Respectively.

[ Example  3] Overcome the resistance and tolerance of CG200745 in resistant biliary cancer cell line

MTT experiments were performed to confirm resistance and therapeutic efficacy of CG200745 in biliary cancer cell lines SNU1196 / P and SNU1196 / GR.

The MTT assay was performed by attaching the cholangiocarcinoma cell line to a 96-well plate at 3 × 10 ³ cells / well, followed by gemcitabine and CG200745 diluted to 1/10 of the following day, and cultured for 72 hours. After the incubation, MTT (3- [4,5-dimethylthiazol-2-yl] -2,5 diphenyl tetrazolium bromide , Sigma-Aldrich))) was added at a rate of 100 μl / well and reacted for 3 hours under light shielding conditions. After 3 hours, the reaction solution was removed, and 100 μl / well of DMSO (dimethyl sulfoxide) was added thereto. The absorbance measured at 570 nm was calculated by ICP ₅₀ using Graphpad Prism 4 software. 1.

As shown in FIG. 1, gemcitabine has an IC ₅₀ of more than 100 times higher than that of SNU1196 / P in SNU1196 / GR. On the other hand, the IC ₅₀ was estimated CG200745 in 1.5x compared to SNU1196 / P in SNU1196 / GR. In addition, it was confirmed that the IC ₅₀ for CG200745 corresponds to a low concentration corresponding to 1 μM or less for each of the above-described cholangiocarcinoma cell lines.

These results indicate that the treatment and drug tolerance can be overcome by the CG200745 drug when resistance to gemcitabine is developed in the bile duct cancer.

[ Example  4] Change of gene expression by CG200745 in biliary cancer cell line

As shown in Example 3 above, microarray analysis was performed for analysis of cDNAs formed by reverse transcription of messenger RNA (mRNA) when resistance to and / or therapeutic effect was achieved.

RNA extraction for the above analysis was performed using a mirVara miRNA isolation kit (Embion, Austin, Tex.), A protocol provided by the manufacturer, to extract total RNA. Agilent 2100 bio-analyzer (Agilent Technologies) and RNA 6000 nanochip (Agilent Technologies) were used for quantitative analysis of total RNA using Nanodrop (ND-1000, Technologies, Inc., DE) The quantitative and qualitative analyzed total RNA was synthesized by cDNA using GeneChip (Human Gene 2.0 ST Array) platform using GeneChip WT (whole transcript) amplification kit. Sense cDNA of the synthesized cDNA was fragmented and labeled with biotin using a CeneChip WT terminal labeling kit. Approximately 5.5 μg of the labeled cDNA was hybridized on an Affymetrix GenecChip Array at 45 ° C. for 16 hours. The hybridized cDNA was washed and stained with GeneChip Fluidic Station 450 and then scanned through a GCS3000 scanner (Affymetix). Analysis of the scanned images was performed using the Affymetrix GeneChip Command Console Software (AGCC), Affymetrix Expression Console Software (http://www.affymetrix.com/estore/catalog/131414/AFFY/Expression-Console-Software#1_1 ) And R 3.0.2 (www.r-project.org), and the results are shown in FIG. 2 and Table 1.

HIPPO, TGF-beta, AXL / GAS6, and Growth Factor Receptor, respectively, when the cholangiocarcinoma cell line had a resistance to and / or therapeutic effect by the CG200745 drug, as shown in FIG. 2 and Table 1. [ factor receptor gene expression was markedly decreased.

Inhibition of the expression of the gene can be utilized as a biomarker capable of predicting the therapeutic effect and / or overcoming tolerance by the CG200745 drug.

[ Example  5] Changes in protein expression by CG200745 in cholangiocarcinoma cell line

Western blot analysis was performed in order to confirm the cell signaling mechanism in the case of having resistance to and / or therapeutic effect from the cholangiocarcinoma cell line according to CG200745.

The preparation method of the protein used in Western blot analysis is as follows. The cholangiocarcinoma cell line was harvested by treatment with Accutase (Sigma-Aldrich), washed with PBS, and then lysed using a cell lysis buffer containing a protein degradation inhibitor. The whole solution was fractionated using a centrifuge, and only the protein-containing solution was extracted. In order to obtain secreted proteins from the cell culture medium, the cells were cultured in serum-free RPMI medium without fetal bovine serum (FBS) for 24 hours, and then the cell by-products were removed by centrifugation using a filter. The protein from which the cell byproduct was removed was extracted with the medium through concentration of the protein by acetone precipitation. The extracted proteins were each quantified by the Bradford method. Proteins at the same concentration obtained by quantification were separated by performing SDS-polyacrylamide gel electrophoresis and then transferred to a PVDF membrane. The membranes from which the proteins were transferred were blocked with TBS-T (Tris-buffered saline / 0.05% Tween-20) solution containing 5% non-fat milk for 1 hour at room temperature in order to reduce non-specific binding. After the reaction, the primary antibody (1: 000 dilution) was reacted overnight at 4 ° C, and the secondary antibody (1: 000 dilution) was reacted at room temperature for 1 hour. For visualization, an enhanced chemiluminescence system of the PIERCE product was used.

Western blot analysis revealed that GAPDH, a housekeeping gene for the quantitative comparison of YAP, TEAD4, TGF-beta2, secreted TGF-beta2, SMAD3, AXL, C-MET, EGFR, Notch3, HES5 and protein expression And the results are shown in Figs. 3 (a) to 3 (e).

As shown in FIGS. 3 (a) to 3 (e), YAP / HIPPO, Growth factor receptor, Notch, AXL / GAS6 and the like, known as cancer cell reproduction and maintenance, Although the degree of TGF-beta protein is different, the expression of CG200745 in all of the other proteins except EGFR is decreased in the bile duct cancer cell line and the resistant bile duct cancer cell line.

Through the above results, when CG200745 according to the present invention was administered to a bile duct cancer cell line and drug-resistant cholangiocarcinoma cell line, the expression of the protein associated with the cell signal transduction was remarkably reduced, thereby overcoming tolerance to and / Furthermore, the protein of Table 1 can be utilized as a biomarker capable of predicting the tolerance and / or therapeutic efficacy.

[ Example  6] In cholangiocarcinoma cell line by CG200745 miRNA  Identification of expression changes

Microarray was performed to confirm the change of microRNA expression when the resistance against CG200745 and / or therapeutic effect occurred from the cholangiocarcinoma cell line.

The microarray was a platform of Affymetrix Gene Chip miRNA 4.0 Array (Homo sapiens), and the procedure is as follows. 1 μg of the total RNA obtained by the method of Example 4 was labeled with the FlashTag ™ Biotin HSR RNA Labeling Kit and hybridized to Affymetrix miRNA microarray in a GeneChip® Hybridization Oven. The hybrid Affymetrix miRNA microarray was scanned using the GeneChip® Scanner and analyzed using the Affymetrix® GeneChip Command Console® Software (AGCC). After this analysis, further analysis was performed using the Affymetrix® Expression Console ™ Software (http://www.affymetrix.com/estore/catalog/131414/AFFY/Expression-Console- Software # 1_1) and R 2.15.1 (www.r- project.org) was used. The procedure was performed twice for each cholangiocarcinoma cell line to improve accuracy, and the results are shown in FIGS. 4 to 7. FIG.

As shown in FIGS. 4 to 7, when the effect of treatment and immunity overcome by CG200745 according to the present invention occurs, 36 microRNAs whose expression is remarkably reduced in biliary cancer-resistant cell lines and 36 microRNAs whose expression is remarkably increased Twenty-six miRNAs with significantly increased expression in the bile duct cancer cell lines and 20 miRNAs with decreased expression in the bile duct cancer cell line were identified.

[ Example  7] Microcirculation by CG200745 in cholangiocarcinoma cell line RNA  Quantitative confirmation of expression

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed on microRNAs whose expression levels were significantly increased in bile duct cancer cell lines among the microRNAs of Example 6 above.

Quantitative reverse transcription PCR (qRT-PCR) was performed using the TaqMan reverse transcription kit (Applied Biosystems, Foster City, Calif.), TaqMan miRNA analyzer (Applied Biosystems) and TaqMan gene expression master mix And proceeded according to the manufacturer's protocol. The PCR amplification conditions consisted of 40 cycles consisting of the initiation step of 95 10 minutes, 95 30 seconds, 56 30 seconds and 72 15 seconds. The PCR amplification conditions consisted of mir-22-3p, mir-22-5p, mir- MicroRNAs and endogenous microRNAs of mir-194-5p, mir-210-3p, mir-3663-3p, mir-6860 and mir-331-3p were purchased from Applied Biosystems Quantitative reverse transcription PCR (qRT-PCR) was performed on an ABI Prism 7300 Sequence Detection System (Applied Biosystems) and the results are shown in FIG.

As shown in FIG. 8, when normalized with miR-331-3p in the cholangiocarcinoma cell line or drug-resistant cholangiocarcinoma cell line treated with CG200745 compared to the cholangiocarcinoma cell line without CG200745, miR-22 The expression of miR-22-5p, miR-192-5p, miR-194-3p, miR-194-5p, miR-210-3p and miR-509-3p was increased 1.5-fold there was.

The increase in expression of miR-22-3p, miR-22-5p, miR-192-5p, miR-194-3p, miR-194-5p, miR-210-3p and miR- , And as a biomarker for predicting susceptibility to treatment and / or tolerance overcome by the administration of the CG200745 drug according to the present invention.

[ Example  8] Microalgae in Biliary Cancer Cell Lines To RNA  by Mechanism of cell signaling  Confirm change

When the therapeutic effect of CG200745 was observed in Example 6, microRNAs with altered expression were transfected into SNU1196 and 1196 / GR using lifofectamin according to the protocol provided by the manufacturer After that, the cell signal transduction protein having a change in expression was identified in Example 5 above.

Western blot analysis was performed in the same manner as in Example 5, and changes in YAP, C-MET and AXL protein expression were measured. The results are shown in FIG.

As shown in FIG. 9, the expression of YAP protein was detected in the case of transfection of miR-22-5p, miR-141-3p and miR-509-3p in SNU1196 and miR-141-3p and miR194- 3p, miR-194-5p and miR-509-3p. In C-MET, miR-192, miR-194-3p and mir-509-3p were transfected with SNU1196 and miR-141-3p, miR-192-5p and mir-194- 3p. ≪ / RTI > In addition, AXL was decreased when transfected with miR-192-5p and miR-509-3p in SNU1196 and when transfected with miR-141-3p and miR-509-3p in SNU1196 / GR.

From these results, it can be seen that the expression of microRNAs affects the cell signal transduction pathway, which is changed by CG200745 treatment.

[ Example  9] Microcirculation by CG200745 in biliary cancer-induced rats RNA  Expression and YAP  Identification of cell signaling mechanism protein

After the tumor cells were transplanted into rats by the conventional method, GC200745 was periodically injected into the abdominal cavity, and the tumor tissues were extracted to examine the expression of microRNA and YAP protein by immunohistochemical staining Respectively.

In situ hybridization and immunohistochemical staining are as follows. After the extraction, paraffin was removed from the tissue slide fixed to paraffin using xylene, and hydration was performed using alcohol. The tissues that underwent the hydration process were treated with Proteinase K to dissolve the membrane proteins so that the probes could bind. The membrane protein-immobilized tissue was fixed with 4% PFA and prehybridized with Roche DIG Easy Hyb reagent (Roche Roche). After prehybridization, AC-HH3, miR-192-5p, miR-194-3p And miR-509-3p probe and Roche DIG Easy Hyb reagent were placed on a slide and hybridization was carried out at 53 DEG C for 16 hours. Subsequently, unhybridized probe was removed using various concentration of saline-sodium citrate (SSC) buffer and incubated with anti-DIG-AP antibody recognizing DIG of the hybridized probe. NBT / BCIP reagent recognizing the AP was used for the color development of the antibody bound to the probe. To check the overall shape of the tissue, counter staining was performed using a neutral red reagent, The results are shown in Figs. 10 (a) and (b).

As shown in FIG. 10 (a), the expression of AC-HH3 was increased in the region treated with CG200745, and it was confirmed that the HDAC inhibitor was normally operated. The site of treatment with miR-192-5p, miR-194-3p and miR-509-3p were expressed.

In addition, as shown in Fig. 10 (b), it was confirmed that the expression of YAP was decreased in the region where miR-509-3p expression was present.

These results indicate that the expression of CG200745 by microRNAs in vivo suppresses the expression of YAP protein and regulates the signal transduction pathway.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the scope of the present invention is not limited to the disclosed exemplary embodiments, but various changes and modifications may be made without departing from the scope of the invention. It will be obvious to those who have knowledge of

Claims (37)

delete delete delete delete delete A composition for predicting susceptibility to a drug for the treatment of bile duct cancer, which comprises an agent for measuring an expression level of a protein of YAP1, TAZ, TEAD4, C-MET and AXL,
A drug for the treatment of biliary cancer, which is predicted to be highly susceptible to drugs for treating biliary cancer when the level of expression of the protein is down-regulated after treatment of the biliary cancer cell line with a drug for treating bile duct cancer, Composition for predicting susceptibility to. The method according to claim 6,
Wherein the agent for measuring the expression level comprises an antibody, a peptide or a nucleotide that specifically binds to the protein. The method according to claim 6,
The composition for predicting susceptibility is used for predicting whether or not to overcome tolerance to a drug for treating biliary cancer, the composition for predicting susceptibility to a drug for treating biliary cancer. The method according to claim 6,
Hs-miR-368-3p, hsa-miR-18b-3p, hsa-miR-314, hsa-miR- HSA-miR-196b-3p, hsa-miR-18a-3p, hsa-miR-149-5p, hsa-miR-99b-3p, hsa-miR- HSA-miR-1301-3p, hsa-miR-93-3p, hsa-miR-100-5p, hsa-miR-4301, hsa-miR-1273g-3p, hsa- HSA-miR-4669, hsa-miR-98-5p, hsa-miR-378e, hsa-miR-4444, hsa-miR-6734-5p, hsa-miR-193b-5p, hs-miR-6740-5p, hsa-miR-629-3p, hsa-miR-4684-3p, hsa-miR-6746-5p, hsa-miR-99b, hsa-miR-422a, hsa-miR-378a-5p hs-miR-423-5p, hsa-miR-6777-5p, hsa-miR-4442, hsa-let-7i-5p, hsa-miR-3911, 5p, hsa-miR-5006-5p, hsa-miR-99b-5p, hsa-miR-601, hsa-miR-125b-5p, hsa-miR-6785-5p, HSA-miR-146a-3p, hsa-miR-345, hsa-miR-183-5p, hsa-miR-151a-3p, miR-378g, hsa-miR-106b-3p, hsa-miR-320c, hsa-miR-3621, hsa-miR-3613-3p, hsa-miR- -1469, hsa-miR- HSA-miR-1343-5p, hsa-miR-4467, hsa-miR-146b-5p, hsa-miR-210-3p, hsa-miR- hs-miR-1237-5p, hsa-miR-4486, hsa-miR-297, hsa-miR-4690-5p, hsa-miR-6860, miR-369-3p, hsa-miR-7114-5p, hsa-miR-22-5p, hs-miR- HSA-miR-194-3p, hsa-miR-8075, hsa-miR-27a-3p, hsa-miR-200a-3p, hsa-miR-6723-5p, 7208, hsa-miR-6776-5p, hsa-miR-6776-5p, hsa-miR-6722-3p, hsa-miR-6125, hsa-miR- Hs-miR-6125p, hsa-miR-6848-5p, hsa-miR-4651, hsa-miR-6089, hsa-miR- HSA-miR-1915-3p, hsa-miR-22-3p, hsa-miR-638, hsa-miR-4763-3p, hsa-miR-141-3p, HSA-miR-4761, hsa-miR-2861, hsa-miR-6724-5p, hsa-miR-3940-5p, hsa-miR- hsa-miR-4745-5p, hsa-miR-8069, hsa-miR-6850-5p, hsa-miR-6789-5p and hsa-miR- A composition for predicting susceptibility to a drug for the treatment of bile duct cancer, which further comprises an agent for measuring the expression level of microRNA. 10. The method of claim 9,
Wherein the agent for measuring the expression level of the microRNA comprises a primer or a probe specifically binding to the microRNA. 10. The method of claim 9,
The microRNA may be selected from the group consisting of hsa-miR-92a-1-5p, hsa-miR-135b-3p, hsa-miR-31, hsa- HSA-miR-196b-3p, hsa-miR-18a-3p, hsa-miR-149-5p, hsa-miR-99b-3p, hsa-miR-4778-5p, hsa- HSA-miR-1301-3p, hsa-miR-93-3p, hsa-miR-100-5p, hsa-miR-4304, hsa-miR-1273g-3p, hsa- miR-4669, hsa-miR-3621, hsa-miR-3613-3p, hsa-miR-1908-5p, hsa-miR-4674, hsa-miR- HSA-miR-4457, hsa-miR-146b-5p, hsa-miR-210-3p, hsa-miR-8063, hsa-miR- HSA-miR-4486, hsa-miR-2977, hsa-miR-4690-5p, hsa-miR-6860, hsa-miR-194-5p, Hs-miR-229p, and hsa-miR-194-3p, and hsa-miR-369-3p, hsa-miR- A composition for predicting susceptibility to a drug for the treatment of bile duct cancer, the composition being at least one selected from the group consisting of. 10. The method of claim 9,
The microRNA may be selected from the group consisting of hsa-miR-98-5p, hsa-miR-378e, hsa-miR-4444, hsa-miR-6734-5p, hsa- HSA-miR-629-3p, hsa-miR-4684-3p, hsa-miR-6746-5p, hsa-miR-99b, hsa-miR-422a, hsa- miR-423-5p, hsa-miR-6777-5p, hsa-miR-4442, hsa-let-7i-5p, hsa-miR- hs-miR-7107-5p, hsa-miR-5006-5p, hsa-miR-99b-5p, hsa-miR-601, hsa-miR- HSA-miR-429, hsa-miR-3459, hsa-miR-183-5p, hsa-miR-146a-3p, hsa-miR- MiR-200a-3p, hsa-miR-6723 = 5p, hsa-miR-80b, hsa-miR-106a-3p, hsa-miR- HSA-miR-7808, hsa-miR-6786-5p, hsa-miR-6776-5p, hsa-miR-6125, hsa-miR- Hs-miR-4616-5p, hsa-miR-4651, hsa-miR-6089, hsa-miR-6800, hsa-miR-7108-5p, hsa-miR-1915-3p, hsa-miR-223p, hsa-miR-638, hsa-miR-4763-3p, hsa-miR-141-3p, hsa-miR- hsa-miR-6791-5 hS-miR-2961, hsa-miR-4745-5p, hsa-miR-2861, hsa-miR-762, hsa-miR-6724-5p, hsa-miR-3940-5p, hsa- A composition for predicting susceptibility to a drug for the treatment of bile duct cancer, wherein the composition is at least one selected from the group consisting of hsa-miR-8069, hsa-miR-6850-5p, hsa-miR-6789-5p and hsa-miR-4734. 12. The method of claim 11,
The microRNA may be selected from the group consisting of hsa-miR-3621, hsa-miR-3613-3p, hsa-miR-1908-5p, hsa-miR-4674, hsa- HSA-miR-4457, hsa-miR-146b-5p, hsa-miR-210-3p, hsa-miR-8063, hsa-miR- HSA-miR-4486, hsa-miR-2977, hsa-miR-4690-5p, hsa-miR-6860, hsa-miR-194-5p, Hs-miR-229p, hsa-miR-229p, hsa-miR-2293p, hsa-miR- Wherein the compound is at least one selected from the group consisting of the compounds of the present invention. 13. The method of claim 12,
The microRNA may be selected from the group consisting of hsa-miR-8075, hsa-miR-27a-3p, hsa-miR-200a-3p, hsa-miR-6723-5p, hsa- miR-6786-5p, hsa-miR-6776-5p, hsa-miR-6722-3p, hsa-miR-6125, hsa-miR-7704, hsa- HSA-miR-4616-5p, hsa-miR-6848-5p, hsa-miR-4651, hsa-miR-6089, hsa-miR- HSA-miR-223p, hsa-miR-638, hsa-miR-4763-3p, hsa-miR-141-3p, hsa-miR-6727-5p, miR-3781-5p, hsa-miR-4741, hsa-miR-3185, hsa-miR-3661, hsa-miR- Predicted susceptibility to drugs for the treatment of bile duct cancer, which is selected from the group consisting of 4745-5p, hsa-miR-8069, hsa-miR-6850-5p, hsa-miR-6789-5p and hsa-miR-4734 / RTI > delete delete A kit for predicting susceptibility to drugs for treating bile duct cancer, comprising the composition of claim 6. 18. The method of claim 17,
Wherein said kit is used for predicting whether or not to overcome tolerance to a drug for the treatment of bile duct cancer. delete delete delete delete delete delete delete (a) measuring the level of expression of the protein in a biological sample separated from the subject;
(b) administering to said biological sample a drug for treating bile duct cancer;
(c) measuring the expression level of the protein in the biological sample after administering the drug for treating bile duct cancer; And
(d) comparing the expression level measured in step (a) with the expression level measured in step (c)
These proteins are YAP1, TAZ, TEAD4, C-MET and AXL,
In the step (d), when the expression level of the protein measured in the step (c) is down-regulated from the expression level of the protein measured in the step (a) A method of providing information for predicting susceptibility of a drug for the treatment of biliary cancer, which is predicted to be highly susceptible to cancer. delete 27. The method of claim 26,
In the step (d), when the expression level of the protein measured in the step (c) is down-regulated from the expression level of the protein measured in the step (a) A method of providing information that predicts that tolerance will be overcome. 27. The method of claim 26,
Further comprising the step of measuring the expression level of the microRNA in the steps (a) and (c)
The microRNA may be selected from the group consisting of hsa-miR-92a-1-5p, hsa-miR-135b-3p, hsa-miR-31, hsa- HSA-miR-196b-3p, hsa-miR-18a-3p, hsa-miR-149-5p, hsa-miR-99b-3p, hsa-miR-4778-5p, hsa- HSA-miR-1301-3p, hsa-miR-93-3p, hsa-miR-100-5p, hsa-miR-4304, hsa-miR-1273g-3p, hsa- hsa-miR-449, hsa-miR-4644, hsa-miR-4653-5p, hsa-miR-193b-5p, hsa-miR- Hsa-miR-624-3p, hsa-miR-4684-3p, hsa-miR-6746-5p, hsa-miR-99b, hsa-miR-422a, hsa-miR-378a-5p, hsa hs-miR-671-5p, hsa-miR-4442, hsa-let-7i-5p, hsa-miR-3911, hsa-let-7e-5p, hsa-miR-6831-5p hs-miR-675-5p, hsa-miR-7107-5p, hsa-miR-580b-5p, hsa-miR- Hs-miR-146a-3p, hsa-miR-345, hsa-miR-4459, hsa-miR-183-5p, hsa-miR-151a-3p, HSA-miR-1464, hsa-miR-3671, hsa-miR-106b-3p, hsa-miR-320c, hsa-miR-3621, hsa-miR- , hsa-miR-6805-5p, hsa hi-miR-1343-5p, hsa-miR-4467, hsa-miR-146b-5p, hsa-miR-210-3p, hsa-miR-8063, hsa-miR-1228-5p, hs-miR-1237-5p, hsa-miR-4486, hsa-miR-297, hsa-miR-4690-5p, hsa-miR-6860, hsa-miR-194-5p, hs-miR-369-3p, hsa-miR-7114-5p, hsa-miR-22-5p, hsa-miR-194 HSA-miR-7108, hsa-miR-17a-3p, hsa-miR-70a-3p, hsa-miR- Hsa-miR-6776-5p, hsa-miR-6722-5p, hsa-miR-6722-5p, hsa-miR-6722-5p, hsa-miR-6722-5p, hsa-miR- HSA-miR-4657, hsa-miR-4632-5p, hsa-miR-6848-5p, hsa-miR-4651, hsa-miR-6089, hsa-miR- HSA-miR-223p, hsa-miR-638, hsa-miR-4763-3p, hsa-miR-141-3p, hsa-miR-6727-5p, HSA-miR-4761, hsa-miR-3161, hsa-miR-4745, hsa-miR-4761, hsa-miR- Hsa-miR-6789-5p, hsa-miR-8069, hsa-miR-6850-5p, hsa-miR- To provide information for predicting susceptibility of therapeutic drugs. 30. The method of claim 29,
The microRNA may be selected from the group consisting of hsa-miR-92a-1-5p, hsa-miR-135b-3p, hsa-miR-31, hsa- HSA-miR-196b-3p, hsa-miR-18a-3p, hsa-miR-149-5p, hsa-miR-99b-3p, hsa-miR-4778-5p, hsa- HSA-miR-1301-3p, hsa-miR-93-3p, hsa-miR-100-5p, hsa-miR-4304, hsa-miR-1273g-3p, hsa- miR-4669, hsa-miR-98-5p, hsa-miR-378e, hsa-miR-4444, hsa-miR-6734-5p, hsa-miR-193b-5p, hsa- HSA-miR-629-3p, hsa-miR-4684-3p, hsa-miR-6746-5p, hsa-miR-99b, hsa-miR-422a, hsa- miR-423-5p, hsa-miR-6777-5p, hsa-miR-4442, hsa-let-7i-5p, hsa-miR- hs-miR-7107-5p, hsa-miR-5006-5p, hsa-miR-99b-5p, hsa-miR-601, hsa-miR- HSA-miR-429, hsa-miR-3459, hsa-miR-183-5p, hsa-miR-146a-3p, hsa-miR- 378g, hsa-miR-106b-3p and hsa-miR-320c. 31. The method of claim 30,
The step (d) may further comprise the step of: when the expression level of the microRNA measured in step (c) is down-regulated from the expression level of the microRNA measured in step (a) A method of providing information that predicts a high susceptibility to a drug. 31. The method of claim 30,
The microRNA may be selected from the group consisting of hsa-miR-98-5p, hsa-miR-378e, hsa-miR-4444, hsa-miR-6734-5p, hsa- HSA-miR-629-3p, hsa-miR-4684-3p, hsa-miR-6746-5p, hsa-miR-99b, hsa-miR-422a, hsa- miR-423-5p, hsa-miR-6777-5p, hsa-miR-4442, hsa-let-7i-5p, hsa-miR- hs-miR-7107-5p, hsa-miR-5006-5p, hsa-miR-99b-5p, hsa-miR-601, hsa-miR- HSA-miR-429, hsa-miR-3459, hsa-miR-183-5p, hsa-miR-146a-3p, hsa-miR- 378g, hsa-miR-106b-3p and hsa-miR-320c. 33. The method of claim 32,
The step (d) may further comprise: when the expression level of the microRNA measured in step (c) is down-regulated from the expression level of the microRNA measured in step (a) A method of providing information that predicts that resistance to a drug is overcome. 30. The method of claim 29,
The microRNA may be selected from the group consisting of hsa-miR-3621, hsa-miR-3613-3p, hsa-miR-1908-5p, hsa-miR-4674, hsa- HSA-miR-4457, hsa-miR-146b-5p, hsa-miR-210-3p, hsa-miR-8063, hsa-miR- HSA-miR-4486, hsa-miR-2977, hsa-miR-4690-5p, hsa-miR-6860, hsa-miR-194-5p, Hs-miR-369-3p, hsa-miR-509-3p, hsa-miR-192-5p, hsa-miR- hs-miR-67a-5p, hsa-miR-92b-5p, hsa-miR-7108, hsa-miR-67a-3p, hsa-miR- Hs-miR-6776-5p, hsa-miR-6125, hsa-miR-7704, hsa-miR-2278, hsa-miR-6729-5p, HSA-miR-4651, hsa-miR-6089, hsa-miR-6800, hsa-miR-7108-5p, hsa-miR-1915-3p, hsa- miR-4761-5p, hsa-miR-2861, hsa-miR-4463, hs-miR- hS-miR-7125p, hsa-miR-3940-5p, hsa-miR-4741, hsa-miR-3185, hsa-miR-4745-5p, 6850-5p , hsa-miR-6789-5p, and hsa-miR-4734. 35. The method of claim 34,
The step (d) may further comprise the step of: when the expression level of the microRNA measured in the step (c) is up-regulated above the expression level of the microRNA measured in the step (a) A method of providing information that predicts a high susceptibility to a drug. 35. The method of claim 34,
The microRNA may be selected from the group consisting of hsa-miR-8075, hsa-miR-27a-3p, hsa-miR-200a-3p, hsa-miR-6723-5p, hsa- miR-6786-5p, hsa-miR-6776-5p, hsa-miR-6722-3p, hsa-miR-6125, hsa-miR-7704, hsa- HSA-miR-4616-5p, hsa-miR-6848-5p, hsa-miR-4651, hsa-miR-6089, hsa-miR- HSA-miR-223p, hsa-miR-638, hsa-miR-4763-3p, hsa-miR-141-3p, hsa-miR-6727-5p, miR-3781-5p, hsa-miR-4741, hsa-miR-3185, hsa-miR-3661, hsa-miR- 4744-5p, hsa-miR-8069, hsa-miR-6850-5p, hsa-miR-6789-5p and hsa-miR-4734. 37. The method of claim 36,
In the step (d), when the expression level of the microRNA measured in the step (c) is up-regulated above the expression level of the microRNA measured in the step (a) A method of providing information that predicts that resistance to a drug is overcome.

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