KR101466661B1 - novel use of TMC5 gene - Google Patents

Abstract

The present invention relates to a composition for screening for cancer diagnosis or anticancer drug comprising an antibody against a protein expressed from TMC5 gene or TMC5 gene, an inhibitor of said gene or an inhibitor of protein expressed from said gene and a cancer comprising a pharmaceutically acceptable carrier A therapeutic composition, a TMC5 gene, or a protein expressed from a TMC5 gene. The TMC5 gene of the present invention is abundantly expressed in cancer cells and increases the proliferation rate of normal cells. Therefore, when the expression of these genes is inhibited, the growth of cancer cells is inhibited. Therefore, the TMC5 gene can be used as a target gene of a diagnostic or therapeutic agent for various cancers.

TMC5, cancer, siRNA

Description

Novel use of TMC5 gene {Novel use of TMC5 gene}

The present invention relates to a composition for screening for cancer diagnosis or anticancer drug comprising an antibody against a protein expressed from TMC5 gene or TMC5 gene, an inhibitor of said gene or an inhibitor of protein expressed from said gene and a cancer comprising a pharmaceutically acceptable carrier A therapeutic composition, a TMC5 gene, or a protein expressed from a TMC5 gene.

Human Genome Project Research shows that there is a new era of bio-biotechnology that aims to understand human diseases at the molecular level, to discover new disease-related targets, and to develop new drugs using them. As a result, the era of personalized medicine that diagnoses, treats, and predicts various diseases according to individual patients with different genetic environments is coming to the reality. Customized medicine includes biomarkers for genome diagnosis, targeting treatment technology, discovery of disease-specific drug action points, new drugs for target treatment, accurate clinical information, genome information of patients, genome dynamics results, Matrix and others should be complemented by the development of drug genome technology. In particular, in order to be competitive in customized medicine, it is important to develop a drug prediction system for diseases and individuals, to discover diagnostic biomarkers, to develop new target genes and proteins, and to develop therapeutic agents using them.

In recent years, there has been a fierce competition for exploring therapeutic targets and studying the functions of genes involved in the generation and treatment of cancer, which is a worldwide incurable disease, in order to use them for diagnosis and therapeutic drug development. With the activation of the genome research, studies on human gene DNA chip or proteome analysis have been actively conducted, and a large number of genes related to cancer have been discovered. Therefore, a list of many genes and related databases have been established, but most of these genes Biological function and cancer relevance have not yet been studied or are uncertain, and there are considerable difficulties in actual cancer-related or diagnostic and target genes.

On the other hand, as the need for research on the functions of numerous human genes is highlighted, the utilization and interest of model organisms capable of simple functional and morphological studies are increasing. In order to understand basic and evolutionally well-preserved mechanisms occurring within the cell, studies are underway to utilize the model organism, which has been used in various fields for a long time, in cancer diagnosis or therapeutic target discovery.

On the other hand, the use of RNA interference (RNAi) technique for large-scale gene expression in cancer cells can be used to confirm the possibility of use as a therapeutic cancer target. RNAi is a technique for inducing the degradation of the mRNA of a specific nucleotide sequence in cells by using various types of oligonucleotides (miRNA, expressed dsRNA, synthetic siRNA) to prevent the function of the gene from appearing. By observing the phenomenon of expression of cells in which gene expression has been inhibited, new gene development studies and target pathway analysis can be performed, and new drug development studies are conducted through target validation and the like.

Among the oligo-ribonucleic acids that can be used for the RNAi technique, a technique using a small interfering RNA (siRNA), which is currently in the spotlight, is a breakthrough method of selecting "breakthrough of the year" in the 2002 Science Journal. siRNA is a short 19-23 double ribonucleic acid chain that is introduced into a cell to inhibit the expression of a specific gene without non-specific inhibition. Therefore, the siRNA promotes the production of cancer, siRNA inhibits the action of the corresponding gene in the cell and kills cancer cells, which can be used to verify the therapeutic target gene. Therefore, recent research on siRNAs for target gene discovery, target gene verification, and therapeutic agent development is very active.

On the other hand, the TMC5 gene (GeneBank registration number: NM_024780, FLJ13593, EXPASY registration number: Q6UXY8) belongs to recently discovered transmembrane channel-like protein group and its function is unknown. Not yet reported. In mice, TMC5 has been reported to be expressed in the colon, small intestine, liver, lung, testis and ovary (BMC Genomics. 2003; 4: 24).

The present invention aims at providing a novel use of the TMC5 gene.

More specifically, the present invention relates to a composition for screening cancer-diagnosing or anticancer agents comprising an antibody against a protein expressed from TMC5 gene or TMC5 gene, an inhibitor of the gene or an inhibitor of the protein expressed from the gene and a pharmaceutically acceptable carrier And a cancer diagnostic kit comprising at least one of the TMC5 gene or the protein expressed from the TMC5 gene.

The present invention provides a cancer diagnostic composition comprising a TMC5 gene.

In the present invention, 'TMC5 gene' refers to the DNA or mRNA of the gene and includes all or a part of DNA or mRNA.

In addition to the gene, the composition of the present invention may contain distilled water or a buffer to stably maintain the structure of the nucleic acid.

As can be seen from the following Examples 1 to 4, the TMC5 gene specifically increases expression in cancer cells. Therefore, cancer can be diagnosed by examining the overexpression of the TMC5 gene.

Therefore, the present invention also provides a method of diagnosing cancer by checking the use of said genes for cancer diagnosis and the reaction between said composition and a sample obtained from a subject.

In the diagnostic method of the present invention, the confirmation of the reaction between the composition containing the gene and the sample can be performed by a conventional method used for confirming the reaction between DNA-DNA, DNA-RNA, and DNA- (PCR), Northern blotting, Southern blotting, ELISA (enzyme linked immunosorbent assay), yeast two-hybrid, 2-D gel analysis and in vitro binding assay ) Can be used. For example, the whole or a part of the gene may be used as a probe to be hybridized with a nucleic acid isolated from a body fluid of a subject, followed by various methods known in the art, for example, reverse transcription polymerase chain reaction, Southern blotting southern blotting, northern blotting, or the like to detect whether the gene is highly expressed in a subject, thereby determining whether or not cancer has occurred. When the probe is labeled with a radioactive isotope or an enzyme, the presence of the gene can be easily confirmed.

The present invention also provides a cancer diagnostic composition comprising a protein expressed from the TMC5 gene.

In addition to the protein, the composition of the present invention may contain distilled water or a buffer to stably maintain the structure of the protein.

As mentioned above, since the expression of TMC5 gene is specifically increased in cancer cells, cancer can be diagnosed by examining overexpression of TMC5 gene or protein using protein expressed from the gene.

Therefore, the present invention also provides a method for diagnosing cancer by checking the use of said proteins for cancer diagnosis and the reaction between said composition and a sample obtained from a subject.

In the diagnostic method of the present invention, confirmation of the reaction between the composition containing the protein and the sample can be carried out by a conventional method used for confirming the reaction between DNA-protein, RNA-protein and protein-protein such as DNA chip, (PCR), Northern blotting, Southern blotting, Western blotting, ELISA (enzyme linked immunosorbent assay), specific immuno-staining (histoimmunostaining), yeast two-hybrid method, 2-D Gel analysis and an in vitro binding assay can be used. For example, all or a part of proteins expressed from the genes may be used as a probe to be hybridized with a nucleic acid or a protein separated from a body fluid of a subject, and then subjected to various methods known in the art, for example, reverse transcription polymerases chain reaction, western blotting or the like to detect whether the gene is highly expressed in the subject, thereby determining whether or not cancer has occurred. When the probe is labeled with a radioactive isotope or an enzyme, the presence of the gene can be easily confirmed.

Alternatively, the composition of the present invention may contain a specific antibody for the protein instead of the protein. In cancer cells, the TMC5 gene is overexpressed and the amount of the protein expressed from the gene is also increased. Therefore, when an antibody against a protein expressed from the gene is used, cancer can be diagnosed by detecting the protein through an antigen-antibody reaction.

The monoclonal antibody to the protein may be prepared by using a monoclonal antibody production method customary in the art or may be commercially available. The monoclonal antibody to the protein may be a secondary antibody conjugated with an enzyme, such as alkaline phosphatase (AP) or horseradish peroxidase (HRP), and their substrates Or may be quantitatively analyzed using a conjugate of AP or HRP enzyme or the like directly to the monoclonal antibody against the protein. In addition, a polyclonal antibody recognizing the protein may be used in place of the monoclonal antibody, or it may be prepared and used through an antiserum preparation method customary in the art.

The present invention also provides a composition for screening an anti-cancer agent comprising the TMC5 gene. The present invention also relates to a use of the composition for screening an anticancer agent and a method of contacting the test subject using the composition as a target substance and confirming the reaction between the target substance and the test subject to determine whether the test substance enhances expression of the gene Activity or suppressing activity of the anti-cancer agent.

In the screening method of the present invention, the reaction between the composition containing the gene and the test substance can be confirmed by conventional methods used for confirming whether DNA-DNA, DNA-RNA, DNA-protein or DNA- have. For example, a hybridization test for confirming the binding between the gene and a test substance in vitro, a reaction between a mammalian cell and a test substance, a Northern analysis, a quantitative PCR, and a quantitative real-time PCR A method for measuring the expression rate of the gene or a method for introducing a reporter gene into the gene, introducing the gene into a cell, reacting with a test substance, and measuring the expression rate of a reporter protein. In this case, in addition to the gene, the composition of the present invention may contain distilled water or a buffer to stably maintain the structure of the nucleic acid.

The present invention also provides a composition for screening an anticancer agent comprising a protein expressed from a TMC5 gene. The present invention also relates to a use of the composition for screening an anticancer agent and a method for contacting the test substance using the composition as a target substance and confirming the reaction between the target substance and the test substance, And determining whether the compound exhibits activity or inhibitory activity.

In the screening method of the present invention, the reaction between the composition containing the protein and the test substance can be confirmed by conventional methods used for confirming the reaction between protein-protein and protein-compound. For example, there are a method of measuring the activity after reacting the protein with a test substance, a yeast two-hybrid method, a search of a phage-displayed peptide clone binding to the protein, High throughput screening using natural materials and chemical libraries, drug-hit HTS, cell-based screening, or screening using DNA arrays, etc. Can be used. In this case, in addition to the protein, the composition of the present invention may contain a buffer or a reaction solution that stably maintains the structure or physiological activity of the protein. The composition of the present invention may further comprise a cell expressing the protein, or a cell containing a plasmid expressing the protein under a promoter capable of regulating the transcription rate, for in vivo experiments.

In the screening method of the present invention, the substance to be tested may be individual nucleic acids, proteins, other extracts or natural substances, compounds, etc., which are presumed to have potential as anticancer drugs according to a conventional selection method or randomly selected.

The test substance obtained through the screening method of the present invention exhibiting the activity of promoting gene expression or enhancing the function of protein and the test substance exhibiting the activity of suppressing gene expression or suppressing the function of protein on the contrary, Can be a candidate for an anticancer agent, and in the latter case, it can be an anticancer agent candidate substance by developing an inhibitor against the test substance. Such anticancer drug candidates act as a leading compound in the subsequent development of anticancer drugs. By modifying and optimizing the structure of the drug so that the leading substance exhibits the function of suppressing the function of the gene or the protein expressed therefrom, It is possible to develop an anticancer drug.

The present invention also provides a composition for treating cancer comprising an inhibitor against the TMC5 gene and a pharmaceutically acceptable carrier.

As can be seen in the following examples, the gene of the present invention is expressed in a large amount in cancer cells, so that cancer can be inhibited by inhibiting the expression of the gene by administering an inhibitor of the gene.

Accordingly, the present invention also provides a cancer treatment method comprising administering to a patient an inhibitor of said gene and an effective amount of said inhibitor of said gene. In the present invention, cancer treatment includes prevention and inhibition of cancer.

In the present invention, the inhibitor for the gene may be an antisense oligonucleotide for the mRNA of the gene.

Antisense oligonucleotides have been used successfully to achieve gene-specific inhibition both in vivo as well as in vitro. Antisense nucleotides are short length DNA synthesis strands (or DNA analogs) that are antisense (or complementary) to a particular DNA or RNA target. Antisense oligonucleotides have been proposed to inhibit the expression of proteins encoded by DNA or RNA targets by binding to the target and stopping expression at the transcription, translation or splicing step. Antisense oligonucleotides have also been successfully used in animal models of cell culture and disease (Hogrefe, 1999). Other variations of antisense oligonucleotides to make the oligonucleotides more stable and resistant to degradation are known and understood by those skilled in the art. Antisense oligonucleotides used herein include double stranded or single stranded DNA, double stranded or single stranded RNA, DNA / RNA hybrids, DNA and RNA analogs and oligonucleotides with base, sugar or backbone modifications. Oligonucleotides are modified by methods known in the art to increase stability and increase resistance to nuclease degradation. These modifications are known in the art and include, but are not limited to, modification of the oligonucleotide backbone, modification of the sugar moiety, or modification of the base.

In addition, the inhibitor for the gene may be siRNA (Small Interfering RNA) of the gene.

siRNAs have been proposed as an alternative to antisense oligonucleotides because they can achieve equivalent or even higher effects than those obtained by antisense oligonucleotides at relatively low concentrations (Thompson, 2002). The use of siRNAs has been shown to inhibit gene expression in animal models of disease. siRNA has the potential to become a very potent drug against the inhibition of the expression of specific genes in vivo in terms of long-lasting effects in cell culture and in vivo, the ability to transfect cells in vivo, and resistance to serum degradation (Bertrand et al., 2002). Expression constructs / vectors including siRNA delivery and siRNA are known to those skilled in the art. For example, U.S. Patent Application Nos. 2004/106567 and 2004/0086884 disclose delivery mechanisms including viral vectors, non-viral vectors, liposome delivery vehicles, plasmid injection systems, artificial viral envelopes and polylysine conjugates, / Vector.

One skilled in the art can synthesize and modify the antisense oligonucleotides and siRNAs in any manner desired using methods known in the art. (Web Server Issue: W113-W120.) Those skilled in the art will also appreciate that antisense oligonucleotides or antisense oligonucleotides or antisense oligonucleotides or antisense oligonucleotides (e.g., a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a combination thereof) useful in the expression construct / vector with the siRNA.

The antisense oligonucleotides or siRNA of the present invention used for the treatment of cancer may be administered in the form of a composition further comprising a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrin, glycerol, ethanol as well as combinations thereof. Such compositions may be formulated to provide a rapid release, sustained or delayed release of the active ingredient after administration.

The inhibitor of the gene may be any substance that inhibits the expression of the gene in addition to the antisense oligonucleotide or siRNA. Therefore, compounds known as inhibitors of the genes in the art are also available.

The present invention also provides a composition for treating cancer comprising an inhibitor for a protein expressed from a TMC5 gene and a pharmaceutically acceptable carrier.

When the gene of the present invention is inhibited, expression of the protein is inhibited and cancer is suppressed. Thus, inhibition of the protein of the gene can inhibit cancer.

Accordingly, the present invention provides a method for treating cancer, comprising administering to a patient an inhibitor of said protein and an effective amount of an inhibitor of said protein. In the present invention, cancer treatment includes prevention and inhibition of cancer.

The inhibitor for the protein may be a nucleic acid, a compound, a natural product or a protein. For example, an inhibitor of the protein may be an antibody to a protein expressed from the gene of the present invention. The monoclonal antibody to the protein may be prepared by using a monoclonal antibody production method customary in the art or may be commercially available. In addition, a polyclonal antibody recognizing the protein may be used in place of the monoclonal antibody, or it may be prepared and used through an antiserum preparation method customary in the art.

The composition of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. When the inhibitor for the protein of the present invention is an antibody, the pharmaceutically acceptable carrier may be composed of a minimum amount of auxiliary substance such as a wetting agent or an emulsifying agent, an antiseptic or a buffer to increase the shelf life or effectiveness of the binding protein.

In addition, the composition for treating cancer of the present invention can be used together with one or more anticancer agents. The composition for treating cancer of the present invention may be administered orally or parenterally, for example, by administering a chemotherapeutic agent such as cyclophosphamide, aziridine, alkylalcone sulfonate, nitroso urea, Antibiotics such as alkylating agents such as cisplatin, mitomycin C, anthracycline and doxorubicin (adriamycin), antimetabolitic agents such as methotrexate, 5-fluorouracil and cytarabine, plants such as vinca alkaloids Derived medicines, hormones, and the like.

The composition for treating cancer of the present invention may include pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned effective ingredients. Examples of such adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, And solubilizing agents.

In addition, the composition of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration.

Examples of the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water suitable for the living body such as saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

Pharmaceutical dosage forms of the compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, dripping or suspending agents, have.

The compositions of the present invention may be formulated and administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, percutaneous, intranasal, inhalation, topical, rectal, Lt; / RTI >

In the treatment method of the present invention, "effective amount" means an amount required for achieving the effect of inhibiting cancer. Therefore, the "effective amount" of the active ingredient of the present invention may vary depending on the kind of the disease, the severity of the disease, the kind and amount of the active ingredient and the other ingredients contained in the composition, the type of the formulation and the age, And the diet, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used concurrently, and the like. In the case of an adult, 0.01 ng / kg to 10 mg / kg in the case of siRNA and 0.01 ng / kg to 10 mg / kg in the case of the antisense oligonucleotide against mRNA of the gene when the gene or protein inhibitor is administered once to several times a day, Kg to 10 mg / kg in the case of the compound, and 0.1 ng / kg to 10 mg / kg in the case of the monoclonal antibody against the protein.

In addition, the present invention provides a cancer diagnostic kit comprising at least one of the TMC5 gene or a protein expressed from the TMC5 gene.

The cancer diagnostic kit may include, in addition to the diagnostic composition of the present invention, a material for application such as a DNA chip, a protein chip and the like used for grasping the reaction between the composition and the sample.

In the present invention, the TMC5 gene may have a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence in which at least one base of the nucleotide sequence is deleted, substituted or inserted.

In the present invention, the siRNA of the TMC5 gene may have the sense sequence of SEQ ID NO: 2 and the antisense sequence of SEQ ID NO: 3.

The sequence of the siRNA for the above TMC5 gene is illustrative, but it is obvious to those skilled in the art. Sequences having substantial sequence identity or substantial sequence homology to the sequences are also within the scope of the present invention. The term "substantial sequence identity" or "substantial sequence homology ", as used herein, is used to describe that a sequence represents a substantial structural or functional identity with another sequence. These differences are due, for example, to inherent variations in the codon usage between different species. If there is a significant amount of sequence redundancy or similarity between two or more different sequences, the structural differences will be negligible if they have similar physical properties even if the length or structure of these sequences is different.

(2001)) and Frederick et al. (Frederick et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, M. Ausubel et al., Current Protocols in Molecular Biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994)).

The TMC5 gene of the present invention is abundantly expressed in cancer cells and increases the proliferation rate of normal cells. Therefore, when the expression of these genes is inhibited, the growth of cancer cells is inhibited. Therefore, the TMC5 gene can be used as a target gene of a diagnostic or therapeutic agent for various cancers. Therefore, the present invention will be greatly utilized in the development of biopharmaceuticals and tailor-made anticancer drugs having less side effects such as therapeutic agents specific to cancer target genes. It will also contribute to the development of the underlying technology that will lay the foundation for new cancer pathway research.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

<Example 1> Determination of expression level of TMC5 gene in cancer cell line

A simple way of estimating the cancer association of a gene is to compare changes in the amount of expression of that gene in cancer tissues or cancer cell lines. The relationship between the expression level of cancer cells and the progression of cancer should be investigated by comparing the expression level of cancer cells with that of normal cells. To do this, we extracted the total RNA from the cancer cell line and then RT-PCR using the oligonucleotide of the gene to compare the amount of each gene with that of the normal cell line. And the amount of expression is confirmed.

In the present invention, the degree of expression of TMC5 gene was analyzed by RT-PCR in human gastric cancer cell line, liver cancer cell line and normal cells. (ATCC # CCL13), SNU709, and SNU354 (SNU601, SNU608, SNU638, SNU638, SNU604 and SNU638 as the gastric cancer cell lines, respectively), Huh7, SH-J1 and Ck- (Seoul National University Cell Line Bank). IMR90 (human fetal lung fibroblasts, ATCC # CCL-186) was used as the normal cell line. RT-PCR of this experiment was performed using one-step RT-PCR PreMix kit (iNtRON Biotechnology, INC). This kit was designed so that cDNA can be synthesized and amplified at once. 8 μl of PreMix (OptiScript ™ RT, i-StarTaq ™ DNA polymerase) is mixed with 1 μg / reaction of RNA template of each cancer cell, specific primer, The volume was adjusted to 20 μl total by adding distilled water and RT-PCR was performed. The specific primers used in the RT-PCR are as follows.

N-terminal primer C-terminal primer  TMC5 SEQ ID NO: 6
5'-TGGCGGGCCTCACAGATGATG-3 SEQ ID NO: 7
5'-GGCCCTTGGATTACCTTCTTG-3 '

As a result of RT-PCR, it was confirmed that the degree of mRNA of TMC5 was increased in human gastric cancer and liver cancer cell line as compared with normal cell line (Fig. 1). It is estimated that alternative splicing of mRNA occurs because RT-PCR size is different in normal cell line and cancer cell line.

Example 2 Expression of TMC5 Gene in Cancerous Tissue and Normal Tissue of Colon Cancer Patients

From the Samsung Medical Center (Seoul, Korea), 66 colon cancer patients and normal tissues were obtained, respectively. Each tissue was surgically removed from the patient and stored in liquid nitrogen until analysis.

Total RNA was isolated using QIAGEN kit (RNeasy Maxi kit: cat # 75162) and quantitated using Experion RNA StdSens (Bio-Rad) chip. First, the colon cancer tissue and normal tissue were cut into an appropriate size, and then dissolved in a 15 ml kit of digestion buffer supplemented with 150 ul of beta mercaptoethanol. To this, 15 ml of 70% ethane wool was added and mixed well. The total RNA was then attached to the membrane by centrifugation at 3000 g for 5 minutes. After two washes, total RNA was isolated by adding 1.2 ml of RNase-free water. In the case of the attached cell line, it was recovered using trypsin and EDTA, and then dissolved in 1 ml of the digestion buffer in the kit.

The extracted total RNA was hybridized using Illumina TotalPrep RNA Amplification Kit (Ambion). T7 Oligo (dT) to synthesize cDNA using a primer, and using the biotin-UTP in vitro transcription was performed to produce biotin-labeled cRNA. The prepared cRNA was quantified using NanoDrop. CRNA produced in normal colon epithelial cells and colon cancer cells was hybridized with Human-6 V2 (Illumina) chip. To remove non-specific hybridization after hybridization, the DNA chip was washed with Illumina Gene Expression System washes (Illumina), and the washed DNA chip was labeled with streptavidin-Cy3 (Amersham) fluorescent dye. The fluorescence-labeled DNA chip was scanned using a confocal laser scanner (Illumina) to obtain fluorescence data in each spot and stored as a TIFF image file. TIFF image file was quantified with BeadStudio version 3 (Illumina), and the fluorescence value of each spot was quantified. The quantified results were calibrated using 'quantile' function with Avadis Prophetic version 3.3 (Strand Genomics) program.

The results are shown in Fig. FIG. 2 shows the TMC5 gene expression profile measured in cancer tissues and normal tissues of patients with colorectal cancer, when the tumor was highly expressed, it was red, and when it was low, it was green. The ACTB (Homo sapiens actin, beta) in FIG. 2 means a standard gene for determining the difference in gene expression. TMC5 expression increased in colorectal cancer tissues by an average of 2.8-fold and increased by more than 2-fold in 45 of 66 patients.

<Example 3> Expression of TMC5 gene in colorectal cancer tissue by RT-PCR

A simple way of estimating the cancer association of a gene is to compare changes in the amount of expression of that gene in cancer tissues or cancer cell lines. The relationship between the expression level of cancer cells and the progression of cancer should be investigated by comparing the expression level of cancer cells with that of normal cells. To do this, we extracted the total RNA from the cancer cell line and then RT-PCR using the oligonucleotide of the gene to compare the amount of each gene with that of the normal cell line. And the amount of expression is confirmed.

In the present invention, the expression level of TMC5 gene was analyzed by RT-PCR in colorectal cancer tissues. RT-PCR of this experiment was performed using one-step RT-PCR PreMix kit (iNtRON Biotechnology, INC). This kit was designed so that cDNA can be synthesized and amplified at once. 8 μl of PreMix (OptiScript ™ RT, i-StarTaq ™ DNA polymerase) is mixed with 1 μg / reaction of RNA template of each cancer cell, specific primer, The volume was adjusted to 20 μl total by adding distilled water and RT-PCR was performed. The specific primers used in the above-mentioned RT-PCR were the same as those used in Example 1.

Figure 3 shows the range of expression of the TMC5 gene in cancer patients. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) in FIG. 3 represents a standard gene for determining the difference in gene expression, wherein N represents Normal and T represents Tumor. As can be seen from FIG. 3, when the normal tissues of 20 patients were compared with cancer tissues, it was confirmed that the mRNA of TMC5 was increased in the cancer tissues of the patients as compared with the normal tissues (FIG. 3).

Example 4 Confirmation of Cancer Cell Growth Inhibition by TMC5 siRNA Transfection in Cancer Cells

In the case of genes that are expressed in large amounts in cancer cell lines or cancer cells, it is possible to observe whether there is a change in cell proliferation rate by inhibiting gene expression through siRNA technique. Thus, in the present invention, the siRNA for TMC5 was designed and introduced into the gastric cancer cell line Snu638, and the change of the growth rate of the cancer cell following the decrease of the mRNA expression of the gene was observed.

First, siRNAs of TMC5 and GFP were designed and synthesized (Samchully Pharmaceutical). The synthesized siRNA consists of a double ribonucleotide chain of 19 oligonucleotides designed on the basis of the gene base sequence and a structure in which a short base of two nucleotides is suspended. GFP (Green Flourescence Protein) was used as a negative control for siRNA experiments that had no effect on cell proliferation.

SiGFP (SEQ ID NO: 2 + 3) and siGFP (SEQ ID NO: 4 + 5) were introduced into the cells by the hyperfect method (Qiagen Co.) after culturing the gastric cancer cell line Snu638 (Seoul National University Cell Line Bank) with 70% Lt; / RTI &gt; and transformed. RNA was extracted from each cell and RT-PCR was performed to confirm that the mRNA of the gene was reduced and gene expression was inhibited by siRNA (FIG. 4 top). NT indicates the amount of gene expression in cells not treated with siRNA, and - indicates that there is no target to target siRNA for sequences lacking homology with human genes.

(Seoul National University Cell Line Bank), KM12SM (Seoul National University Cell Line Bank), Colo205 (Seoul National University Cell Line Bank), SW620 (Seoul National University Cell Line Bank), HCT116 (SNU 387, SNU 388), prostate cancer cell line PC-3 (Seoul National University Cell Line Bank), SP2 / 0 cell line (Seoul National University cell line bank) lung cancer cell line A549, liver cancer cell line Huh7 (SEQ ID NO: 2 + 3) and siGFP (SEQ ID NO: 4 + 5) were introduced into the cells using a high-throughput method (Qiagen Co.) Cell growth inhibition was also examined. Each cell was stained with SRB (Sulfur rhodamine, Sigma Co.) and subjected to SRB analysis based on the cell line treated with GFP siRNA, which is a control with no significant change in cell growth via a microscope. (Fig. 4, bottom line). Inhibition of TMC5 gene expression in most colorectal cancer cell lines induced cell growth inhibition. In addition, siRNA of TMC5 gene showed excellent cell growth inhibitory effect on liver cancer cell line SNU387 and lung cancer cell line A549. These results suggest that TMC5 gene can be used as a cancer treatment target because siRNA treatment of TMC5 gene can inhibit the growth of colon cancer cell line, lung cancer and liver cancer cell line.

Each cell treated with siRNA was stained with SRB (Sulfur rhodamine, Sigma Co.) in colorectal cancer cell line Colo205 and stomach cancer cell line Snu638, and the cells were stained with SRB-stained cells treated with GFP siRNA, Observation of the photograph revealed the degree of inhibition of cell growth and morphological changes (Fig. 5).

FIG. 1 shows the results of RT-PCR analysis of changes in the expression level of TMC5 gene in human gastric cancer cell line, liver cancer cell line, and normal cell. GAPDH is a control gene.

FIG. 2 is a graph showing the expression of TMC5 gene in tissues of colon cancer patients by microarray experiment using Illumina 48K chip.

FIG. 3 shows the results of RT-PCR analysis of changes in the expression level of the TMC5 gene in 20 cancer tissues of colon cancer patients. GAPDH is a control gene.

FIG. 4 is a graph showing the degree of inhibition of cell growth by SRB staining when treating siRNA of TMC5 gene in colorectal cancer, lung cancer, liver cancer, stomach cancer, and prostate cancer cell line, to be.

FIG. 5 is a photograph showing that cell growth was inhibited when treating TMC5 gene siRNA in colon cancer and gastric cancer cell line. FIG.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel use of TMC5 gene <130> P07-076-KRI <160> 7 <170> Kopatentin 1.71 <210> 1 <211> 3687 <212> DNA <213> Homo sapiens <400> 1 acagaattct ctgcaggaaa acactccttc tcctgagaaa actcctcagg ggttagatgc 60 agggagttat gttgtgattt gtgtaggggt ggctttggtg agctcatcct ggcattttaa 120 aaataatgtg aatggtgtat ccctttgtga tcatcatcac tttttccctg cgagtcccaa 180 tcaatgcggg tgtgactgct gtatgaagtc tcaggaagcc caccccagtg gagaagaagg 240 cttgcaggag gcaggagatg ctgtccgatg accacgtgaa tgaaatcatc atacaggttg 300 agaatgtttc ctctggggtc caaagccacc catcctcaaa tcagattttt caagaaaagg 360 tgctgctaga ctcaagcatc aacatggttt tgtcaatatc tgacattgat gtgatagact 420 ctcagacagt cagcaaaagg aatgaccaaa agggtaacca ggtgctgcgg ttttcaacat 480 ctttgaatga gtcgatgtct cagacccttc atagcctaga atgcatgggc atagacactc 540 ctggttcttc acatgaaact gttcaaggac agaagttaat cgcatccctt atacccatga 600 catccagaga cagaattaaa gccatcagga accagccaag gaccatggaa gagaaaagga 660 accttaggaa aatagttgac aaagaaaaaa gcaaacagac ccatcgtatc cttcagctca 720 attgctgtat tcagtgtctg aactccattt cccgggctta tcggagatcc aagaacagcc 780 tgtcggaaat tctgaattcc atcagcctgt ggcagaagac gctgaagatc attggaggca 840 agtttggaac cagcgtcctc tcctatttca actttctgag atggcttttg aagttcaaca 900 ttttctcatt catcctgaac ttcagcttca tcataatccc tcagtttacc gtggccaaaa 960 agaacaccct ccagttcact gggctggagt ttttcactgg ggtgggttat tttagggaca 1020 cagtgatgta ctatggcttt tacaccaatt ccaccatcca gcacgggaac agcggggcat 1080 cctacaacat gcagctggcc tacatcttca caatcggagc atgcttgacc acctgcttct 1140 tcagtttgct gttcagcatg gccaagtatt tccggaacaa cttcattaat ccccacattt 1200 actccggagg gatcaccaag ctgatctttt gctgggactt cactgtcact catgaaaaag 1260 ctgtgaagct aaaacagaag aatcttagca ctgagataag ggagaacctg tcagagctcc 1320 gtcaggagaa ttccaagttg acgttcaatc agctgctgac ccgcttctct gcctacatgg 1380 tagcctgggt tgtctctaca ggagtggcca tagcctgctg tgcagccgtt tattacctgg 1440 ctgagtacaa cttagagttc ctgaagacac acagtaaccc tggggcggtg ctgttactgc 1500 ctttcgttgt gtcctgcatt aatctggccg tgccatgcat ctactccatg ttcaggcttg 1560 tggagaggta cgagatgcca cggcacgaag tctacgttct cctgatccga aacatctttt 1620 tgaaaatatc aatcattggc attctttgtt actattggct caacaccgtg gccctgtctg 1680 gtgaagagtg ttgggaaacc ctcattggcc aggacatcta ccggctcctt ctgatggatt 1740 ttgtgttctc tttagtcaat tccttcctgg gggagtttct gaggagaatc attgggatgc 1800 aactgatcac aagtcttggc cttcaggagt ttgacattgc caggaacgtt ctagaactga 1860 tctatgcaca aactctggtg tggattggca tcttcttctg ccccctgctg ccctttatcc 1920 aaatgattat gcttttcatc atgttctact ccaaaaatat cagcctgatg atgaatttcc 1980 agcctccgag caaagcctgg cgggcctcac agatgatgac tttcttcatc ttcttgctct 2040 ttttcccatc cttcaccggg gtcttgtgca ccctggccat caccatctgg agattgaagc 2100 cttcagctga ctgtggccct tttcgaggtc tgcctctctt cattcactcc atctacagct 2160 ggatcgacac cctaagtaca cggcctggct acctgtgggt tgtttggatc tatcggaacc 2220 tcattggaag tgtgcacttc tttttcatcc tcaccctcat tgtgctaatc atcacctatc 2280 tttactggca gatcacagag ggaaggaaga ttatgataag gctgctccat gagcagatca 2340 ttaatgaggg caaagataaa atgttcctga tagaaaaatt gatcaagctg caggatatgg 2400 agaagaaagc aaaccccagc tcacttgttc tggaaaggag agaggtggag caacaaggct 2460 ttttgcattt gggggaacat gatggcagtc ttgacttgcg atctagaaga tcagttcaag 2520 aaggtaatcc aagggcctga tgactctttt ggtaaccaga caccaatcaa ataaggggag 2580 gagacgaaaa tggaatgatt tcttccatgc cacctgtgcc tttaggaact gcccagaaga 2640 aaatccaagg ctttagccag gagcggaaac tgactaccat gtaattatca aagtaaaatt 2700 gggcattcca tgctattttt aatacctgga ttgctgattt ttcaagacaa aatacttggg 2760 gttttccaat aaagattgtt gtaatattga aatgagccta caaaaaccta ggaagagata 2820 actagggaat aatgtatatt atcttcaaga agtgtgtgca ggaatgattg gttcttagaa 2880 atctctcctg ccagacttcc cagacctggc aaaggtttag aaactgttgc taagaaaagt 2940 ggtccatcct gaataaacat gtaatactcc agcagggata tgaagcctct gaattgtaga 3000 acctgcattt atttgtgact ttgaactaaa gacatccccc atgtcccaaa ggtggaatac 3060 aaccagaggt ctcatctctg aactttcttg cgtactgatt acatgagtct ttggagtcgg 3120 ggatggagga ggttctgccc ctgtgaggtg ttatacatga ccatcaaagt cctacgtcaa 3180 gctagctttg cagtggcagt accgtagcca atgagattta tccgagacgc gattattgct 3240 aattggaaat tttcccaata ccccaccgtg atgacttgaa atataatcag cgctggcaat 3300 ttttgacagt ctctacggag actgaataag aaaaaagaaa agaaaagaaa ttagctgggt 3360 gcgatggctt atgcctgtaa tcccggcact ttgggaggct gaggcaagcg gatcacttaa 3420 tgtcaggagt tcaagaccag cctggccaac atggtgaaac cccgtctcta ctaaaaataa 3480 aaaaactagc tgggcgtggt ggtacatgcc tataatccca gctactcggg aggctgaggc 3540 aggagaattg cttgaacctg ggaggcagag gttgcagtga ggcgagattg taccactgca 3600 ttccagcctg ggcaacagtg agactctgcc tcaaaaaaat aaataaataa ataaataaag 3660 taaattaaaa gtcttattca aaccaaa 3687 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA for TMC5, sense <220> <223> siRNA for TMC5, sense <400> 2 guuaaucgca ucccuuauat t 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA for TMC5, antisense <220> <223> siRNA for TMC5, antisense <400> 3 cucaagcauc aacaugguut t 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA for GFP, sense <220> <223> siRNA for GFP, sense <400> 4 guucagcgug uccggcgagt t 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA for GFP, antisense <220> <223> siRNA for GFP, antisense <400> 5 cucgccggac acgcugaact t 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> N-terminal primers for PCR of TMC5 <400> 6 tggcgggcct cacagatgat g 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > C-terminal primers for PCR of TMC5 <400> 7 ggcccttgga ttaccttctt g 21

Claims (23)

A composition for diagnosing cancer comprising TMC5 gene. A composition for diagnosing cancer comprising an antibody to a protein expressed from a TMC5 gene. A composition for screening an anticancer agent comprising the TMC5 gene. delete delete A composition for treating cancer, comprising an antisense oligonucleotide for the mRNA of the TMC5 gene and a pharmaceutically acceptable carrier. A siRNA of the TMC5 gene, and a pharmaceutically acceptable carrier. 8. The composition of claim 7, wherein the siRNA is an siRNA having the sense sequence of SEQ ID NO: 2 and the antisense sequence of SEQ ID NO: 3. delete A composition for treating cancer comprising an antibody to a protein expressed from a TMC5 gene and a pharmaceutically acceptable carrier. The composition for treating cancer according to any one of claims 6, 7, 8, and 10, wherein the TMC5 gene has the nucleotide sequence of SEQ ID NO: 1. delete delete delete delete delete delete delete delete delete delete delete A cancer diagnostic kit comprising the TMC5 gene.

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