KR100992239B1 - Novel use of MIG12 gene - Google Patents

Abstract

The present invention is a composition for diagnosing cancer or screening cancer comprising a MIG12 gene or a protein expressed from the MIG12 gene, a composition for treating cancer comprising an inhibitor of the gene or an inhibitor of the protein expressed from the gene and a pharmaceutically acceptable carrier. And it provides a kit for diagnosing cancer comprising a MIG12 gene, or a protein expressed from the MIG12 gene.

Since the MIG12 gene of the present invention is expressed in large amounts in cancer cells and increases the proliferation rate of normal cells, inhibiting the expression of this gene has an effect of inhibiting the growth of cancer cells. Therefore, the MIG12 gene can be used as a target gene for diagnosis or treatment of various cancers.

MIG12 gene, protein, cancer

Description

Novel use of MIG12 gene

The present invention is a composition for diagnosing cancer or screening cancer comprising MIG12 gene or a protein expressed from MIG12 gene, a composition for diagnosing cancer or screening cancer comprising an OIP5 gene or a protein expressed from OIP5 gene, an inhibitor of the gene or the gene It relates to a composition for treating cancer comprising an inhibitor of a protein expressed therefrom and a pharmaceutically acceptable carrier, and a kit for diagnosing cancer comprising at least one of a MIG12 gene, an OIP5 gene or a protein expressed therefrom.

As a result of research on the human genome project, a new era of bio-biotechnology is opening up to understand human diseases at the molecular level, to discover new disease-related targets, and to develop new drugs using them. Accordingly, the era of customized medicine for diagnosing, treating, and predicting various diseases according to individual patients with different genetic environments is coming to reality. Custom medicine includes the discovery of genomic diagnostic biomarkers, targeted therapeutic technology, discovery of disease-specific drug action points, new drugs for targeted therapy, accurate clinical information, patient genome information, genomic epidemiologic results, and bioinformatics that can integrate them. The development of drug genome technology that complements Matics should be accompanied. In particular, in order to be competitive in personalized medicine, it is important to develop a drug prediction system for a disease and an individual, to discover a diagnostic biomarker, to discover new target genes and proteins, and to develop a therapeutic agent using the same.

Recently, there has been a fierce competition for discovering therapeutic targets and using them in diagnosis and development of therapeutics through the study of genes related to the generation and treatment of cancers, which are incurable diseases worldwide. As human genome DNA chip or proteome analysis studies are actively conducted along with the activation of genomic research and a large number of genes related to cancer have been discovered, a list of many genes and related databases have been established, but most of these genes are specific within cells. Biological function and cancer relevance have not yet been studied or uncertain and there are significant difficulties in using them as actual cancer relevance or diagnosis and target genes.

Meanwhile, as the necessity of research on the functions of numerous human genes is highlighted, the utilization and interest of model organisms capable of simple function and morphology studies are increasing. In order to understand the basic and evolutionarily well-preserved mechanisms occurring in cells, researches using model organisms, which have been used in various fields for a long time, for discovering targets for diagnosing or treating cancer are being conducted.

Division, which is used in one of these model organisms yeast S. pombe genes are the genes of the 10,043% 4,824 fewer pieces contain introns (intron) (Wood V. et al , Nature 45:.. 871-880, 2002). In S. pombe , important related genes in eukaryotic cells, such as cell cycle regulation, proteolysis, protein phosphorylation and RNA splicing, are very well preserved. Yeasts , including S. pombe , are the simplest model cells of eukaryotic cells, which can be cultured at low cost, are easy to genetically research and manipulate, and have a well-established biochemical and physiological function analysis system. It has been used as a model cell for the study of the function of disease-causing genes and drug development (Lum, PK et al . , Cell ., 116: 121-137, 2004). In particular, in fission yeast, when genes related to cell cycle and signal transduction are deleted or expressed in cells, abnormality in regulation of signaling system is caused. Thus, growth rate inhibition and morphological changes of cells due to abnormal cell cycle regulation or metabolic abnormalities are caused. Caused. Thus, by exploring these phenotype changes, we can infer the function of the gene.

On the other hand, the use of RNAi (RNA interference) experimental techniques for genes expressed in cancer cells can confirm the possibility of use as a therapeutic cancer target. RNAi is a technique that induces the degradation of mRNA of the corresponding nucleotide sequence specifically in a cell using various types of oligonucleotides (miRNA, expressed dsRNA, synthetic siRNA) of various types, thereby preventing the function of genes from appearing. As a result of observing the expression of cells whose gene expression is inhibited, it is possible to study the function of the gene and to analyze the signaling pathways, thereby developing new drugs through target validation.

Among the oligo double ribonucleic acids available for RNAi technique, siRNA (small interfering RNA), which has recently been in the spotlight, is a breakthrough method that was selected as a "breakthrough of the year" in the 2002 Science Journal. siRNA is a short 19-30 double strand RNA duplex, when introduced intracellularly has the effect of inhibiting the expression of a specific gene only without non-specific inhibition (non-specific inhibition). The mechanism of siRNA is combined with RISC (RNA-induced silencing complex) in the cell to cut off the sense strand of the gene corresponding to mRNA, and the antisense stand complementary to the sense strand is complexed with RISC and has a complementary base mRNA In other words, it is known to inhibit the expression of genes by binding to and degradation of the mRNA of the target gene. Therefore, siRNA for a gene that promotes the production of cancer and inhibits apoptosis has the effect of inhibiting the action of the gene in the cell to kill cancer cells, and can use this to verify a therapeutic target gene. Therefore, siRNA research for the discovery of target genes for target diseases, validation of target genes, and development of therapeutic agents is very active.

siRNAs have short hairpin RNAs (shRNAs) that induce gene expression inhibition by transient transfection and appropriate delivery systems and have a transient effect, whereas they have a relatively long-term RNAi effect. shRNA synthesizes oligo DNA linking 3-10 base linkers between sense of the target gene siRNA sequence and complementary nonsense and then clones into a plasmid vector or shRNAs are retroviruses lentivirus and adenovirus. When inserted into (adenovirus) and expressed, a short hairpin RNA (shRNA) with a looped hairpin structure is created and converted into siRNA by Dicer in the cell, showing RNAi effect. shRNA has the advantage of showing a relatively long RNAi effect and is widely used in in vivo experiments and is being studied for clinical application as a new drug.

Meanwhile, MIG12 (GenBank Accession No .: NM_021242) is a gene related to Opitz G / BBB syndrome and was discovered as a gene that interacts with MID1, which is associated with microtubules (BMC Cell Biol. 2004; 5: 9). . MIG12, along with MID1, is involved in the stabilization of microtubules and is thought to be involved in intracellular processes that require stabilization of microtubules such as cell division and migration. When MID1 and MIG12 are introduced into the cell at the same time, MIG12 recruits a filament composed of tubulin, and the microtubule bundle composed of acetylated tubulin is depolymerizing agent. Resistance to). There have been no reported reports of cancer related to MIG12.

OIP5 (GenBank Registry Number: NM_007280) was first discovered as a protein that interacts with OpaP, a membrane protein associated with the invasion of human cells, related to the opacity of Neisseria gonorrhoeae (John M. Gi-ChungLiZhu & Richard F. Molecular Microbiology 27171- January 1998). OIP is a protein that interacts with the Raf kinase isoform (Genomics Volume 81, 112-125) and a PH domain (pleckstrin homology domain) that binds to phosphatidylinositol 4-phosphate. Although it has been identified as a protein that interacts with FAPP1 (S. Dowler et al., Biochem. J. 351 (2000), pp. 19-31), the specific physiological function of OIP has not been reported yet. none.

The present invention aims to provide a novel use of the MIG12 (MID1 interacting protein 1) gene and the OIP5 gene.

More specifically, the present invention is a composition for diagnosing cancer or screening cancer comprising the MIG12 gene or a protein expressed from the MIG12 gene, a composition for diagnosing cancer or screening an anticancer agent comprising a protein expressed from the OIP5 gene or OIP5 gene, of the gene It is intended to provide a cancer diagnostic composition comprising an inhibitor or an inhibitor of a protein expressed from the gene and a pharmaceutically acceptable carrier, and a kit for cancer diagnosis comprising at least one of a MIG12 gene, an OIP5 gene or a protein expressed therefrom. .

The present invention also provides a method for diagnosing or screening a cancer comprising a MIG12 gene or a protein expressed from a MIG12 gene, and a method for diagnosing or screening a cancer, or for screening a cancer comprising an OIP5 gene or a protein expressed from an OIP5 gene. Use of the composition and methods of diagnosis or screening, inhibitors of the genes or inhibitors of proteins expressed from the genes and uses of the composition for the treatment of cancer comprising a pharmaceutically acceptable carrier and methods of treatment.

The present invention provides a composition for diagnosing cancer comprising the MIG12 gene and a composition for diagnosing cancer comprising the OIP5 gene.

The 'MIG12 gene' or 'OIP5 gene' of the present invention refers to DNA or mRNA of these genes, and is a concept that includes all or part of DNA or mRNA.

The composition of the present invention may include distilled water or a buffer to keep the structure of the nucleic acid stable in addition to the gene.

As can be seen from Examples 2 to 3 below, the MIG12 or OIP5 gene is specifically increased in cancer cell lines and cancer cells. Therefore, cancer can be diagnosed by investigating whether the MIG12 or OIP5 gene is overexpressed using the gene of the present invention.

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

Confirmation of the reaction between the sample and the composition comprising the gene in the diagnostic method of the present invention is a conventional method used to determine whether the reaction between DNA-DNA, DNA-RNA, DNA-protein, such as DNA chips, protein chips, Polymerase chain reaction (PCR), Northern blotting, Southern blotting, Enzyme Linked Immunosorbent assay (ELISA), yeast two-hybrid, 2-D gel analysis and in vitro binding assay ) And the like can be used. For example, all or part of the gene is used as a probe to hybridize with nucleic acid isolated from the body fluid of the subject, and then various methods known in the art, such as reverse transcription polymerases chain reaction and southern blotting ( By detecting this by southern blotting, Northern blotting, or the like, it is possible to determine whether cancer occurs by examining whether the gene is highly expressed in the subject. Labeling the probe with a radioisotope or enzyme can easily confirm the presence of the gene.

The present invention also provides a cancer diagnostic composition comprising a protein expressed from the MIG12 gene and a cancer diagnostic composition comprising a protein expressed from the OIP gene.

In addition to the protein, the composition of the present invention may include distilled water or a buffer to keep the structure of the protein stable.

As mentioned above, since MIG12 or OIP5 gene is specifically increased in cancer cells, cancer can be diagnosed by investigating whether MIG12 or OIP5 gene or protein is overexpressed using the protein expressed from the genes.

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

Confirmation of the reaction between the composition comprising the protein and the sample in the diagnostic method of the present invention is a common method used to confirm the reaction between the DNA-protein, RNA-protein, protein-protein, such as DNA chips, protein chips, Polymerase chain reaction (PCR), Northern blotting, Southern blotting, Western blotting, Enzyme Linked Immunosorbent assay (ELISA), specific immunostaining, yeast two-hybrid, 2-D Gel assays and in vitro binding assays can be used. For example, all or part of a protein expressed from the genes as a probe may be hybridized with a nucleic acid or protein isolated from a subject's body fluid, and then various methods known in the art, such as reverse transcription polymerases chain. By detecting this by reaction, western blotting, or the like, it is possible to determine whether the cancer has occurred by examining whether the gene is highly expressed in the subject. Labeling the probe with a radioisotope or enzyme can easily confirm the presence of the gene.

Alternatively, the composition of the present invention may include a specific antibody to the protein instead of the protein. In cancer cells, the MIG12 or OIP5 gene is overexpressed, resulting in an increase in the amount of protein expressed from the gene. Therefore, when using an antibody against a protein expressed from the gene as in Example 1, it is possible to diagnose the cancer by detecting the protein through an antigen-antibody reaction.

The monoclonal antibody to the protein may be prepared and used through a monoclonal antibody manufacturing method customary in the art, or may be used commercially. Monoclonal antibodies to these proteins generally use secondary antibodies conjugated with enzymes such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) and their substrates. It may be quantitatively analyzed by color reaction, or may be directly quantitatively analyzed by conjugating AP or HRP enzyme to a monoclonal antibody to the protein. In addition, a polyclonal antibody that recognizes the protein may be used instead of the monoclonal antibody, which may be manufactured and used through an antiserum production method conventional in the art.

The present invention also provides an anticancer agent screening composition comprising the MIG12 gene and an anticancer agent screening composition comprising the OIP5 gene. In another aspect, the present invention uses the anticancer screening of the composition and using the composition as a target material in contact with the test substance, confirming the reaction between the target material and the test substance, the test substance to improve the expression of the gene It provides a method for screening an anticancer agent comprising the step of determining whether it exhibits activity or inhibitory activity.

In the screening method of the present invention, confirming the reaction between the composition containing the gene and the test substance may use conventional methods used to confirm the reaction between DNA-DNA, DNA-RNA, DNA-protein, and DNA-compound. have. For example, hybridization tests to confirm the binding between the gene and the test substance in vitro, reaction of mammalian cells with the test substance, and then Northern analysis, quantitative PCR, quantitative real-time PCR, etc. Method for measuring the expression rate of the gene, or a method of connecting the reporter gene to the gene introduced into the cell and then reacted with the test substance and measuring the expression rate of the reporter protein. In such a case, the composition of the present invention may include distilled water or a buffer solution to keep the structure of the nucleic acid stable, in addition to the gene.

In another aspect, the present invention provides an anticancer screening method comprising the step of determining whether the composition exhibits the activity of inhibiting the expression of the gene by inducing phenotypic changes of the model cells or animal cells using the composition as a target material. .

In the anti-cancer drug screening method of the present invention, cells through phenotype investigation such as growth rate, morphological changes, temperature sensitivity, etc., using model organisms and animal cells overexpressing the genes -based analysis can be performed to screen substances that directly inhibit the expression of the gene and the action of the protein.

The present invention also provides a composition for screening an anticancer agent comprising a protein expressed from the MIG12 gene and a composition for screening an anticancer agent comprising a protein expressed from the OIP5 gene. The present invention also provides a method for screening an anticancer agent of the composition and using the composition as a target material to contact a test substance and confirming a reaction between the target substance and the test substance, thereby improving the function of the protein. It provides a method for screening an anticancer agent comprising the step of determining whether it exhibits activity or inhibitory activity.

In the screening method of the present invention, the reaction between the composition comprising the protein and the test substance may use conventional methods used to confirm the reaction between the protein and the protein and the protein. For example, a method of measuring activity after reacting the protein with a test substance, yeast two-hybrid, phage-displayed peptide clone binding to the protein, High throughput screening (HTS) using natural products and chemical libraries, drug hit HTS, cell-based screening, or DNA array (DNA array) Can be used. In such a case, the composition of the present invention may include, in addition to the protein, a buffer or a reaction solution that stably maintains the structure or physiological activity of the protein. In addition, the composition of the present invention may include a cell expressing the protein, or a cell containing the plasmid expressing the protein under a promoter capable of controlling transcription rate for in vivo experiments.

In the screening method of the present invention, the test substance may be individual nucleic acids, proteins, other extracts or natural products, compounds, or the like, which are estimated to have potential as cancer metastasis inhibitors according to a conventional selection method or randomly selected.

The test substance obtained through the screening method of the present invention, which exhibits the activity of enhancing gene expression or enhancing the function of the protein, and the test substance exhibiting activity of inhibiting gene expression or inhibiting the function of the protein, In this case, it can be an anticancer drug candidate by developing an inhibitor for the test substance, and in the latter case, it can be an anticancer drug candidate. Such an anticancer drug candidate acts as a leading compound in a future anticancer drug development process, and by modifying and optimizing its structure so that the leading material can exhibit the inhibitory effect of the gene or the protein expressed therefrom, Can develop anticancer drugs.

The present invention also provides siRNA of MIG12 gene or siRNA of OIP5 gene.

siRNA is introduced into the cell by synthesizing the forward strand (sense strand) of the gene and the reverse sequence (anti-sense strand) showing the actual gene suppression effect in the cell together and then combined with a double ribonucleic acid chain.

The siRNA sequences of the present invention refer to the sense strand of a gene for convenience, unless otherwise stated.

The candidate siRNA sequence region of the MIG12 gene (NM_021242) may be a sequence of 19-30 sequences belonging to the nucleotide sequence of SEQ ID NO: 14, 15, 16, 17, or 18, wherein the 14, 15, 16, 17 , Or 18 is a partial sequence of the MIG12 gene (NM_021242) of SEQ ID NO: 1, 845 to 950 base sequence, 970 to 1030 base sequence, 1080 to 1160 base sequence, 1220 to 1290 base sequence, Corresponding to the 1320-1400th base sequence, the sequence consists of 61-106 bases. That is, the base sequence complementary to the base sequence may be a sequence for efficiently inducing RNAi.

The siRNA of the MIG12 gene may include a nucleotide sequence corresponding to an mRNA sequence derived from 19 to 30 sequential sequences in one or more sequences selected from SEQ ID NOs: 14, 15, 16, 17, and 18.

The base sequence of siRNA of the MIG12 gene may be SEQ ID NO: 3 or 4. SEQ ID NO: 3 is a sequence in which a single chain of two bases (TT) is suspended in 19 nucleotide sequences corresponding to mRNA sequences derived from 1331 to 1349 base sequences of the MIG12 gene of SEQ ID NO: 1, and SEQ ID NO: 4 is SEQ ID NO: A single-chain binucleotide (TT) is hung on 19 nucleotide sequences corresponding to mRNA sequences derived from the 1123-1141 nucleotide sequences of the MIG12 gene of 1, and has an anti-sense strand complementary to the sequence. Genetic inhibitory effect (RNAi effect) can be excellent.

The candidate siRNA nucleotide sequence region of the OIP5 gene (NM_007280) may be a sequence of 19-30 nucleotide sequences belonging to the nucleotide sequence of SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, or 26, wherein the sequence SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, or 26 is a subsequence of the OIP5 gene (NM_007280) of SEQ ID NO: 2, the 1st to 60th nucleotide sequences, the 120th to 180th nucleotide sequences, respectively Corresponds to 380 to 460 base sequence, 518 to 580 base sequence, 720 to 780 base sequence, 850 to 890 base sequence, 920 to 980 base sequence, or 1040 to 1100 base sequence, and has a base number of 41-81. A sequence of dogs. That is, the base sequence complementary to the base sequence may be a sequence for efficiently inducing RNAi.

The siRNA of the OIP5 gene is a nucleotide sequence corresponding to an mRNA sequence derived from 19 to 30 sequential sequences in one or more sequences selected from SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, and 26. It may include.

The base sequence of the siRNA of the OIP5 gene may be SEQ ID NO: 5 or 6. SEQ ID NO: 5 is a sequence in which a single chain of two bases (TT) is suspended in 19 nucleotide sequences corresponding to mRNA sequences derived from 426-444 base sequences of the OIP5 gene of SEQ ID NO: 2, and SEQ ID NO: 6 is SEQ ID NO: A single chain of two bases (TT) is suspended in 19 nucleotide sequences corresponding to mRNA sequences derived from the 417-435 base sequences of the OIP5 gene of 2, and an anti-sense strand complementary to the sequence is shown. Genetic inhibitory effect (RNAi effect) can be excellent.

In addition, the siRNA of the present invention may be in a chemically modified form to prevent rapid degradation by nucleic acid enzymes in vivo. The siRNA has a double helix structure, which is relatively more stable than single or helix ribonucleic acid or antisense oligonucleotide, but is degraded rapidly by in vivo nucleic acid enzymes, thereby reducing the rate of degradation through chemical modification. Can be. Methods of chemically modifying siRNAs to make them stable and resistant so that they are not readily degraded are well known to those skilled in the art. The most commonly used method of chemical modification of siRNA is boranophosphate or phosphorothioate modification. These substances stably form the linkage between the nucleosides of the siRNA, consequently conferring resistance to nucleic acid degradation. Ribonucleic acid modified with boranophosphate is characterized by poor nucleic acid degradation, but is not synthesized by chemical reactions, and is synthesized only by boranophosphate entering ribonucleic acid by in vitro transcription. Boranophosphate modification method belongs to a relatively easy method, but has a disadvantage that it is difficult to modify in a specific position. On the other hand, phosphorothioate modification method has the advantage of introducing elemental sulfur to the desired part, but severe phosphothioation reduces the efficacy, toxicity, nonspecific RNA-induced silencing complex (RISC) Problems such as formation may appear. Therefore, in recent years, in addition to the above two methods, a method of chemically modifying only the end position of ribonucleic acid (above the 3 'end) to impart resistance to the nuclease is more preferred. In addition, chemical modification of the ribose ring (ribose ring) is known to increase the resistance to nucleases, in particular the variation in the ribose 2'-position of the original cells stabilizes the siRNA. However, there is a problem in that the stability is increased only when the methyl group is precisely inserted in this position, and too many methyl groups lose the ribonucleic acid-mediated interference phenomenon. The reason for such chemical modification is also to increase the efficacy and increase the pharmacokinetic residence time in vivo (Mark et. Al., Molecular Therapy, 13: 644-670, 2006).

In addition to chemical modification methods, a safe and efficient delivery system is required to increase the intracellular delivery efficiency of siRNA. To this end, the siRNA of the present invention may be included in a pharmaceutical composition for treating cancer in the form of a complex with a nucleic acid delivery system.

Nucleic acid carriers for delivering nucleic acid materials into cells can be largely divided into viral and non-viral vectors. The most widely used is a viral vector because of its high delivery efficiency and long duration. Among various viral vectors, retroviral vectors, adenovirus vectors, and adeno-associated viral vectors are mainly used. Such a viral vector is efficient in intracellular delivery of ribonucleic acid, but has various problems in terms of safety, such as recombination into a virus having in vivo activity, inducing an immune response, and random insertion into a host chromosome. In contrast, nonviral vectors have advantages over viral vectors, which have a low toxicity and immune response, can be repeatedly administered, easily form complexes with ribonucleic acids, and facilitate mass production. In addition, ligands specific to diseased cells or tissues are conjugated to non-viral vectors to enable long-term cell-selective nucleic acid delivery. As the non-viral vector, various formulations such as liposomes, cationic polymers, micelles, emulsions, nanoparticles and the like can be used. Nucleic acid carriers can significantly enhance the transport efficiency of a desired nucleic acid in an animal cell and can be delivered to any animal cell depending on the purpose of use of the nucleic acid to be delivered.

SiRNA of the present invention, when introduced into the short 19-30 double ribonucleic acid chain shows the effect of inhibiting the expression of MIG12 gene or OIP5 gene without non-specific inhibition, specifically anticancer activity that kills cancer cells It can also be used to study the function of related genes.

The invention also provides shRNAs of the MIG12 gene or shRNAs of the OIP5 gene.

The shRNA (small hairpin RNA) synthesizes oligo DNAs linking 3-10 base linkers between the sense of the target gene siRNA and complementary nonsense, and then clones into a plasmid vector or shRNA is a retrovirus lentiviral When inserted into and expressed in lentiviruses and adenoviruses, short hairpin RNAs (shRNAs) with a looped hairpin structure are produced and converted into siRNAs by Dicer in the cell to show RNAi effects. The shRNA has a relatively long RNAi effect compared to siRNA.

The shRNA of the MIG12 gene also corresponds to an mRNA sequence derived from consecutive 19 to 30 base sequences in one or more sequences selected from SEQ ID NOs: 14, 15, 16, 17, and 18 of the MIG12 gene, similarly to the siRNA of the MIG12 gene. It may include a nucleotide sequence.

The shRNA of the MIG12 gene may include an RNA base sequence of SEQ ID NO: 4 and an RNA base sequence complementary to SEQ ID NO: 4 connected to a loop region of 3-10 base. The RNA base sequence of SEQ ID NO: 4 is 19 base sequences corresponding to mRNA sequences derived from the 1123-1141 base sequences of the MIG12 gene of SEQ ID NO: 1, and the anti-sense strand of the sequence complementary to the sequence (anti-sense strand) Genetic inhibitory effect (RNAi effect) can be excellent.

The 'RNA base sequence' refers to an RNA base sequence portion excluding the double base (TT) of the short chain from the base sequence of SEQ ID NO.

The top strand for producing an expression vector for expressing the shRNA of the MIG12 gene may be SEQ ID NO: 27.

The shRNA of the OIP5 gene is also an mRNA derived from 19 to 30 base sequences in one or more sequences selected from SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, and 26, similarly to the siRNA of the OIP5 gene. It may include a base sequence corresponding to the sequence.

The shRNA of the OIP5 gene may include an RNA base sequence of SEQ ID NO: 5 and an RNA base sequence complementary to SEQ ID NO: 5 connected to a loop region of 3-10 base. The RNA base sequence of SEQ ID NO: 5 is 19 base sequences corresponding to the mRNA sequence derived from the 426-444 base sequences of the OIP5 gene of SEQ ID NO: 2, and the sequence of anti-sense strand complementary to the sequence Genetic inhibitory effect (RNAi effect) can be excellent.

The 'RNA base sequence' refers to an RNA base sequence portion excluding the double base (TT) of the short chain from the base sequence of SEQ ID NO.

The top strand for producing an expression vector for expressing the shRNA of the OIP5 gene may be SEQ ID NO: 28.

Expression constructs / vectors comprising small hairpin RNAs (shRNAs) including hairpin structures to increase the intracellular and transfection effects of siRNAs are known to those skilled in the art. For example, US applications 2004/106567 and 2004/0086884, in addition to viral vectors comprising shRNAs, nonviral vectors, as well as delivery mechanisms including liposome delivery vehicles, plasmid injection systems, artificial viral envelopes and polylysine conjugates, Many expression constructs / vectors are provided.

The present invention also provides a composition for treating cancer comprising an inhibitor for the MIG12 gene and a composition for treating cancer comprising an inhibitor for the OIP5 gene.

'Inhibitors for genes' of the present invention encompass the concept of inhibitors of expression of genes.

The composition may further comprise a pharmaceutically acceptable carrier.

As can be seen in the following examples, the gene of the present invention is expressed in large amounts in cancer cells, the inhibitor of the gene can be inhibited by inhibiting the expression of the gene can inhibit cancer.

Accordingly, the present invention also provides a method of treating cancer comprising the use of an inhibitor of the gene for cancer treatment and administering to the patient an effective amount of the inhibitor of the gene. Cancer treatment in the present invention includes the prevention and suppression 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 successfully used to achieve gene-specific inhibition 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 prevent the expression of a protein encoded by a DNA or RNA target by binding to the target and stopping expression at the stage of transcription, translation or splicing. Antisense oligonucleotides have been successfully used in cell culture and animal models of disease (Hogrefe, 1999). Other modifications of antisense oligonucleotides to make them more stable and resistant to degradation of oligonucleotides are known and understood by those skilled in the art. Antisense oligonucleotides as used herein include oligonucleotides having double or single stranded DNA, double or single stranded RNA, DNA / RNA hybrids, DNA and RNA analogs and base, sugar or backbone modifications. Oligonucleotides are modified by methods known in the art to increase stability and to increase resistance to nuclease degradation. These modifications are known in the art and include, but are not limited to, modifications to oligonucleotide backbones, modifications of sugar moieties, or modifications of bases.

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

siRNA is introduced into the cell by synthesizing the forward strand (sense strand) of the gene and the reverse sequence (anti-sense strand) showing the actual gene suppression effect in the cell together and then combined with a double ribonucleic acid chain.

The siRNA sequences of the present invention refer to the sense strand of a gene for convenience, unless otherwise stated.

The candidate siRNA sequence region of the MIG12 gene (NM_021242) may be a sequence of 19-30 sequences belonging to the nucleotide sequence of SEQ ID NO: 14, 15, 16, 17, or 18, wherein the 14, 15, 16, 17 , Or 18 is a partial sequence of the MIG12 gene (NM_021242) of SEQ ID NO: 1, 845 to 950 base sequence, 970 to 1030 base sequence, 1080 to 1160 base sequence, 1220 to 1290 base sequence, Corresponding to the 1320-1400th base sequence, the sequence consists of 61-106 bases. That is, the base sequence complementary to the base sequence may be a sequence for efficiently inducing RNAi.

The siRNA of the MIG12 gene may include a nucleotide sequence corresponding to an mRNA sequence derived from 19 to 30 sequential sequences in one or more sequences selected from SEQ ID NOs: 14, 15, 16, 17, and 18.

The base sequence of siRNA of the MIG12 gene may be SEQ ID NO: 3 or 4. Said SEQ ID NO: 3 is a sequence in which a single chain of two bases (TT) is suspended in 19 nucleotide sequences corresponding to mRNA sequences derived from the 1331 to 1349 nucleotide sequences of the MIG12 gene of SEQ ID NO: 1, and SEQ ID NO: 4 is a sequence An anti-sense strand having a single chain of two bases (TT) suspended in 19 nucleotide sequences corresponding to an mRNA sequence derived from nucleotides 1123 to 1141 of MIG12 gene of No. 1, and complementary to the sequence. Gene inhibition effect (RNAi effect) of the can be excellent.

The candidate siRNA nucleotide sequence region of the OIP5 gene (NM_007280) may be a sequence of 19-30 nucleotide sequences belonging to the nucleotide sequence of SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, or 26, wherein the sequence SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, or 26 is a subsequence of the OIP5 gene (NM_007280) of SEQ ID NO: 2, the 1st to 60th nucleotide sequences, the 120th to 180th nucleotide sequences, respectively Corresponds to 380 to 460 base sequence, 518 to 580 base sequence, 720 to 780 base sequence, 850 to 890 base sequence, 920 to 980 base sequence, or 1040 to 1100 base sequence, and has a base number of 41-81. A sequence of dogs. That is, the base sequence complementary to the base sequence may be a sequence for efficiently inducing RNAi.

The siRNA of the OIP5 gene is a nucleotide sequence corresponding to an mRNA sequence derived from 19 to 30 sequential sequences in one or more sequences selected from SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, and 26. It may include.

The base sequence of the siRNA of the OIP5 gene may be SEQ ID NO: 5 or 6. SEQ ID NO: 5 is a sequence in which a single chain of two bases (TT) is suspended in 19 base sequences corresponding to an mRNA sequence derived from the 426-444 base sequences of the OIP5 gene of SEQ ID NO: 2, wherein SEQ ID NO: 6 is SEQ ID NO: A single chain of two bases (TT) is suspended in 19 nucleotide sequences corresponding to mRNA sequences derived from the 417-435 base sequences of the OIP5 gene of 2, and an anti-sense strand complementary to the sequence is shown. Genetic inhibitory effect (RNAi effect) can be excellent.

In addition, the siRNA of the present invention may be in a chemically modified form to prevent rapid degradation by nucleic acid enzymes in vivo. Since siRNA has a double helix structure, it is relatively more stable than single-stranded ribonucleic acid or antisense oligonucleotide, but since it is rapidly decomposed by nucleic acid degrading enzymes in vivo, it can be reduced by chemical modification. have. Methods of chemically modifying siRNAs to make them stable and resistant so that they are not readily degraded are well known to those skilled in the art. The most commonly used method of chemical modification of siRNA is boranophosphate or phosphorothioate modification. These substances stably form the linkage between the nucleosides of the siRNA, consequently conferring resistance to nucleic acid degradation. Ribonucleic acid modified with boranophosphate is characterized by poor nucleic acid degradation, but is not synthesized by chemical reactions, and is synthesized only by boranophosphate entering ribonucleic acid by in vitro transcription. Boranophosphate modification method belongs to a relatively easy method, but has a disadvantage that it is difficult to modify in a specific position. Phosphorothioate modifications, on the other hand, have the advantage of introducing elemental sulfur into the desired portion, but severe phosphothioation reduces the efficacy, toxicity, and nonspecific RNA-induced silencing complex (RISC). Problems such as formation may appear. Therefore, in recent years, in addition to the above two methods, a method of chemically modifying only the end position of ribonucleic acid (above the 3 'end) to impart resistance to the nuclease is more preferred. In addition, chemically modifying the ribose ring (ribose ring) is known to increase the resistance to nucleases, in particular, the variation in the ribose 2'-position inherent in the cell stabilizes the siRNA. However, when the methyl group is precisely entered at this position, the stability is increased, and too many methyl groups have the problem that the ribonucleic acid mediated interference phenomenon is lost. The reason for such chemical modification is also to increase the efficacy and increase the pharmacokinetic residence time in vivo (Mark et. Al., Molecular Therapy, 13: 644-670, 2006).

In addition to chemical modification methods, a safe and efficient delivery system is required to increase the intracellular delivery efficiency of siRNA. To this end, the siRNA of the present invention may be included in a pharmaceutical composition for treating cancer in the form of a complex with a nucleic acid delivery system.

Nucleic acid carriers for delivering nucleic acid materials into cells can be largely divided into viral and non-viral vectors. The most widely used is a viral vector because of its high delivery efficiency and long duration. Among various viral vectors, retroviral vectors, adenovirus vectors, and adeno-associated viral vectors are mainly used. Such a viral vector is efficient in intracellular delivery of ribonucleic acid, but has various problems in terms of safety, such as recombination into a virus having in vivo activity, inducing an immune response, and random insertion into a host chromosome. In contrast, nonviral vectors have advantages over viral vectors, which have a low toxicity and immune response, can be repeatedly administered, easily form complexes with ribonucleic acids, and facilitate mass production. Further, ligands specific to diseased cells or tissues are conjugated to non-viral vectors to enable long-term cell-selective nucleic acid delivery. As the non-viral vector, various formulations such as liposomes, cationic polymers, micelles, emulsions, nanoparticles and the like can be used. Nucleic acid carriers can significantly enhance the transport efficiency of a desired nucleic acid in an animal cell and can be delivered to any animal cell depending on the purpose of use of the nucleic acid to be delivered.

SiRNA of the present invention, when introduced into the short 19-30 double ribonucleic acid chain shows the effect of inhibiting the expression of MIG12 gene or OIP5 gene without non-specific inhibition, specifically anticancer activity that kills cancer cells It can also be used to study the function of related genes.

In addition, the inhibitor for the gene may be shRNA of the MIG12 gene or shRNA of the OIP5 gene.

The shRNA (small hairpin RNA) synthesizes oligo DNAs linking 3-10 base linkers between the sense of the target gene siRNA and complementary nonsense, and then clones into a plasmid vector or shRNA is a retrovirus lentiviral When inserted into and expressed in lentiviruses and adenoviruses, short hairpin RNAs (shRNAs) with a looped hairpin structure are produced and converted into siRNAs by Dicer in the cell to show RNAi effects. The shRNA has a relatively long RNAi effect compared to siRNA.

The shRNA of the MIG12 gene also corresponds to an mRNA sequence derived from 19 to 30 base sequences in one or more sequences selected from SEQ ID NOs: 14, 15, 16, 17, and 18 of the MIG12 gene in the same manner as the siRNA of the MIG12 gene. It may include a nucleotide sequence.

The shRNA of the MIG12 gene may include an RNA base sequence of SEQ ID NO: 4 and an RNA base sequence complementary to SEQ ID NO: 4 connected to a loop region of 3-10 base. The RNA base sequence of SEQ ID NO: 4 is 19 base sequences corresponding to mRNA sequences derived from the 1123-1141 base sequences of the MIG12 gene of SEQ ID NO: 1, and the anti-sense strand of the sequence complementary to the sequence (anti-sense strand) Genetic inhibitory effect (RNAi effect) can be excellent.

The 'RNA base sequence' refers to an RNA base sequence portion excluding the double base (TT) of the short chain from the base sequence of SEQ ID NO.

The top strand for producing an expression vector for expressing the shRNA of the MIG12 gene may be SEQ ID NO: 27.

The shRNA of the OIP5 gene is also an mRNA derived from 19 to 30 base sequences in one or more sequences selected from SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, and 26, similarly to the siRNA of the OIP5 gene. It may include a base sequence corresponding to the sequence.

The shRNA of the OIP5 gene may include an RNA base sequence of SEQ ID NO: 5 and an RNA base sequence complementary to SEQ ID NO: 5 connected to a loop region of 3-10 base. The RNA base sequence of SEQ ID NO: 5 is 19 base sequences corresponding to the mRNA sequence derived from the 426-444 base sequences of the OIP5 gene of SEQ ID NO: 2, and the sequence of anti-sense strand complementary to the sequence Genetic inhibitory effect (RNAi effect) can be excellent.

The 'RNA base sequence' refers to an RNA base sequence portion excluding the double base (TT) of the short chain from the base sequence of SEQ ID NO.

The top strand for producing an expression vector for expressing the shRNA of the OIP5 gene may be SEQ ID NO: 28.

Expression constructs / vectors comprising small hairpin RNAs (shRNAs) including hairpin structures to increase the intracellular and transfection effects of siRNAs are known to those skilled in the art. For example, US applications 2004/106567 and 2004/0086884, in addition to viral vectors comprising shRNAs, nonviral vectors, as well as delivery mechanisms including liposome delivery vehicles, plasmid injection systems, artificial viral envelopes and polylysine conjugates, Many expression constructs / vectors are provided.

siRNA has the potential to be a very strong drug against inhibition of expression of specific genes in vivo in terms of cell culture and long lasting effects, ability to transfect cells in vivo and resistance to serum degradation. (Bertrand et al., 2002).

siRNAs have been proposed as alternatives to antisense oligonucleotides because they can achieve an effect equal to or higher than that obtained by antisense oligonucleotides even at relatively low concentrations (Thompson, 2002). The use of siRNAs has shown popularity in inhibiting gene expression in animal models of disease. One skilled in the art can synthesize and modify the antisense oligonucleotides and siRNAs in any desired manner using methods known in the art (eg, Andreas Henschel, Frank Buchholz1 and Bianca Habermann (2004) DEQOR: a web-based tool for the design and quality control of siRNAs.Nucleic Acids Research 32 (Web Server Issue): W113-W120. Reference). Those skilled in the art also understand well regulatory sequences (eg, constitutive promoters, inducible promoters, tissue-specific promoters or combinations thereof) useful for antisense oligonucleotides, siRNAs, or shRNA expression constructs / vectors.

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

Inhibitors of the gene may be any of low molecular weight compounds, natural products, bioavailable proteins, proteins or novel proteins in vivo, synthetic and natural compounds that inhibit the expression of the gene in addition to the antisense oligonucleotide, siRNA, or shRNA. Therefore, compounds known as inhibitors of the genes in the art and materials discovered through screening using the genes are also available.

The present invention also provides a composition for treating cancer comprising an inhibitor for a protein expressed from the MIG12 gene and a composition for treating cancer comprising an inhibitor for a protein expressed from the OIP5 gene.

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

Accordingly, the present invention provides a method for treating cancer comprising the use of an inhibitor of the protein for cancer treatment and administering to the patient an effective amount of the inhibitor of the protein. Cancer treatment in the present invention includes the prevention and suppression of cancer.

Inhibitors against the protein may be an antibody against a protein expressed from a gene of the present invention. The monoclonal antibody to the protein may be prepared and used through a monoclonal antibody manufacturing method customary in the art, or may be used commercially. In addition, a polyclonal antibody that recognizes the protein may be used instead of the monoclonal antibody, which may be manufactured and used through an antiserum production method conventional in the art.

The composition of the present invention may be preferably formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers in addition to the active ingredient described above for administration. If the inhibitor for a protein of the invention is an antibody, the pharmaceutically acceptable carrier may consist of a minimum amount of auxiliary material, such as a wetting or emulsifying agent, preservative or buffer, which increases the shelf life or effectiveness of the binding protein.

In addition, the composition for treating cancer of the present invention may be used with one or more anticancer agents. Compositions for treating cancer of the present invention are, for example, chemotherapeutic agents well known to those skilled in the art, such as cyclophosphamide, aziridine, alkylalconsulfonate, nitrosourea, dacarbazine, carboplatin, Alkylating agents such as cisplatin, antibiotics such as mitomycin C, anthracycline, doxorubicin (adriamycin), plants such as antimetabolitic agents such as methotrexate, 5-fluorouracin, cytarabine, and vinca alkaloids Derived drugs, hormones, and the like.

The composition for treating cancer of the present invention may include a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and such adjuvant may include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants. Agent, solubilizer, and the like.

In addition, the composition of the present invention may be preferably formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers in addition to the active ingredient described above for administration.

Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. 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. Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component according to the appropriate method in the art.

Pharmaceutical formulation forms of the compositions of the invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release formulations of the active compounds, and the like. have.

Compositions of the present invention may be administered in conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, sternum, transdermal, nasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. May be administered.

In the method of treatment of the present invention, "effective amount" means the amount required to achieve the effect of inhibiting cancer. Thus, the "effective amount" of the active ingredient of the present invention may include the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the age, body weight, general state of health, sex and It can be adjusted according to various factors including the diet, the time of administration, the route of administration and the rate of secretion of the composition, the duration of treatment, and the drugs used simultaneously. In adults, when the inhibitor of the gene or protein is administered once to several times a day, 0.01ng / kg ~ 10mg / kg for siRNA, 0.01ng / kg ~ 10mg / kg for shRNA, mRNA of the gene 0.01ng / kg ~ 10mg / kg for antisense oligonucleotides, 0.1ng / kg ~ 10mg / kg for compounds, 0.1ng / kg ~ 10mg / kg for monoclonal antibodies to the protein, In the case of low molecular weight compounds, 0.1ng / kg ~ 10mg / kg, in the case of natural products, 0.1ng / kg ~ 10mg / kg, in the case of bioavailable protein, it is preferable to administer at a dose of 0.1ng / kg ~ 10mg / kg.

In addition, the present invention provides a kit for diagnosing cancer comprising at least one of a MIG12 gene, an OIP5 gene, or a protein expressed therefrom.

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

In the present invention, the MIG12 gene may have a nucleotide sequence of SEQ ID NO: 1 or one or more bases of one or more bases of the nucleotide sequence is deleted, substituted or inserted.

In addition, in the present invention, the OIP5 gene may have a nucleotide sequence of SEQ ID NO: 2 or one or more bases of one or more bases of the nucleotide sequence is deleted, substituted or inserted.

It is apparent to those skilled in the art that the above-described MIG12 gene, OIP5 gene, and siRNA or shRNA sequences thereof are exemplary but not limited thereto. Sequences having substantial sequence identity or substantial sequence homology to those sequences are also within the scope of the present invention. As used herein, the term "substantial sequence identity" or "substantial sequence homology" is used to express that the sequence represents substantial structural or functional identity with another sequence. This difference is due, for example, to inherent variations in codon usage between different species. If there is a significant amount of sequence overlap or similarity between two or more different sequences, the structural differences are negligible if they have similar physical properties, even if their lengths or structures differ.

Matters related to genetic engineering in the present invention are described in Sambrook et al. (Sambrook, et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY (2001)) and Frederick et al. M. Ausubel et al., Current protocols in molecular biology volumes 1, 2, 3, John Wiley & Sons, Inc. (1994)).

The MIG12 and OIP5 genes of the present invention are associated with cell cycle regulation and growth factors as a result of overexpression in model cells, are expressed in large amounts in cancer cell lines, and increase the rate of proliferation of cells. It has a depressing effect. Therefore, the MIG12 and OIP5 genes can be used as target genes for diagnosis or treatment of various cancers. Therefore, the present invention provides bio-new drugs such as siRNA / shRNA new drugs, antibody new drugs, and protein new drugs, which are customized anti-cancer drugs with low side effects such as therapeutic agents specific to cancer target genes, and low-molecular synthetic drugs, natural drug new drugs targeting MIG12 and OIP5 proteins, etc. It will be used for the development of new drugs such as target anticancer drugs. It will also contribute to the development of original technologies that lay the foundation for the study of new cancer-related mechanism pathways.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various forms. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

<Example 1> MIG12 or OIP5 gene expression and its effect confirmed

Fission yeast is caused by a lack of genes related to cell cycle and signal transduction or overexpression in the cell, resulting in abnormality in the regulation of the signaling system. Overexpression of the human gene MIG12 or OIP5 in cleavage yeast confirmed the cancer-related effects of gene overexpression, including inhibition of cell growth and morphological changes.

1-1. Manufacture of MIG12 or OIP5 Gene Expression Yeast

The Gateway ^TM system (Invitrogen, USA) was used to easily express large amounts of MIG12 or OIP5 genes in cleavage yeast, and a gene expression vector with an nmt1 promoter was used to regulate expression of the gene depending on the presence or absence of thiamin. In addition, each of the human genes is S. pombe The p173DES vector (Accession No .: KCTC 10757BP) with a conversion cassette was used to reduce the need to clone to the expression vector system. An entry vector cloned with a human gene MIG12 or OIP5 (provided by Frontier Human Genome Function Corp., Korea) was fused with the p173DES vector in vitro to prepare a cleavage yeast gene expression vector expressing human genes (FIG. One).

Plasmids cloned with the human genes MIG12 and OIP5, respectively, were used to examine the cellular phenotypic and phenotypic changes in the overexpression of genes cloned into the p173DES vector, ED665 (h-) (Fanters, a haploid strain of S. pombe ). , University of Glasgow, UK) was transformed by a lithium acetate transformation method.

Selection medium of the transformants was added to adenine and leucine in EMM medium (Edinburgh Minimal Media) in consideration of the nutritional requirements of uracil, a marker of the cleavage yeast plasmid pDES173, and the strain. A medium (hereinafter referred to as EMM + AL) was used.

1-2. Cell growth inhibitory effect

In order to express cancer-related human genes in the ED665 strain, EMM + AL + Thia with 2 μM of thiamine (hereinafter referred to as Thia), which acts as a repressor of the promoter, and EMM + AL without addition were used, respectively. . As a control, pDES173 without human genes was expressed together. After incubation at 30 ° C. for 3 days, colonies grown in EMM + AL solid medium and EMM + AL + Thia solid medium were compared to observe whether cell growth was inhibited. As a result, it was confirmed that colony formation of the strain in which the MIG12 or OIP5 gene was cultured in the EMM + AL solid medium was suppressed, so that excessive expression of MIG12 or OIP5 caused the growth inhibition of cells (FIG. 2A and 3A).

1-3. Cytoplasmic effect

In addition, the cells cultured in EMM + AL + Thia solid medium were washed three times with sterilized distilled water to completely remove thiamin, and then EMM + AL liquid medium and EMM + AL + Thia (2μM) liquid were used to confirm the cell transformation. Each 10 ml of the medium was inoculated again with an OD ₆₀₀ of 0.3. After incubation at 30 ° C. for 6-8 hours, the OD ₆₀₀ was diluted to 0.1 in the same medium, and then incubated for 16 hours. The obtained cells were observed under an optical microscope to compare the cell trait changes that occur when human genes were overexpressed with those of normal cells. When human genes were overexpressed in S. pombe , morphological changes occurred in long cells with multiple diaphragms. It was confirmed (Fig. 2B, 3B).

1-4. Cell cycle abnormal effects

In addition, FACScan ^TM that analyzes the DNA content after staining the chromosomal DNA by treating PI (propidium iodide) to the cells of Example 1-3 (Becton, Dickinson and company) was confirmed that the over-expression of human genes cause abnormalities in the cell cycle (Fig. 2C, 3C).

1-5. Human gene expression

Whether the human gene was properly expressed was confirmed by Western blot using a monoclonal antibody (Cell signaling, USA) that recognizes the HA tag at the N- terminal portion of the expressed protein ( Fig. 2D, 3D ).

From the results of 1-2 to 1-4, it can be seen that overexpression of the MIG12 or OIP5 gene according to the present invention results in cell growth, cell type change, and cell cycle abnormalities characteristic of cancer. Therefore, diagnosis of cancer and drug screening are possible by measuring the overexpression of MIG12 or OIP5 gene. For example, as shown in the results of 1-5, by using an antibody against a protein expressed by the MIG12 or OIP5 gene, the expression of the MIG12 or OIP5 gene is measured to diagnose cancer and screen the drug.

Example 2 MIG12 or OIP5 Gene Expression in Cancer Cell Lines

A simple way of estimating the cancer's relevance to a gene is to compare changes in the amount of expression of that gene in cancer tissues or cancer cell lines. Investigate the relationship between the expression and the amount of cancer generation and progression by comparing whether the cancer cells are expressed or reduced in a large amount compared to normal cells. To this end, extracting the total RNA from the cancer cell line to see how the actual expression of the gene is expressed in the cancer cell line, and performing the RT-PCR using the oligonucleotide of the gene to compare the amount of each gene with the normal cell line in the cell of the gene Check the expression level.

The expression level of MIG12 or OIP5 gene was analyzed by RT-PCR in human gastric cancer cell line, liver cancer cell line and normal cell. Sun620, Sun216, Sun484, Sun601, and Sun638 (Seoul National University Cell Line Bank) as gastric cancer cell lines, Huh7, SH-J1, and Ck-1a (prepaid from Dr. Kim Dae-gon, Chonnam National University), Chang (ATCC # CCL-62), Snu790 and Snu354 (Seoul National University Cell Line Bank) were used. In addition, IMR90 (human fetal lung fibroblasts, ATCC # CCL-186) was used as a normal cell line. RT-PCR of this experiment was performed using a one-step RT-PCR PreMix kit (iNtRON Biotechnology, INC). This kit is designed to synthesize and amplify cDNA at once. 1μg / reaction of RNA template of each cancer cell, specific primer, tertiary in 8μl PreMix (OptiScriptTM RT, i-StarTaqTM DNA polymerase) RT-PCR was performed by adding distilled water to a total volume of 20 μl. Specific primers used in the RT-PCR are as follows (Table 1).

N-terminal C-terminal MIG12 SEQ ID NO: 10
5'-ATGATGCAAATCTGCGACACC-3 ' SEQ ID NO: 11
5'-TCAGTGGCCCCAATTGCCGAA-3 ' OIP5 SEQ ID NO: 12
5'-GGCTGGGGGCTGAGGAGCCA-3 ' SEQ ID NO: 13
5'-CACTTCACTCAGAATCTTCA-3 '

As a result of the RT-PCR, MIG12 and OIP5 was confirmed that the RNA level is increased in most human gastric cancer and liver cancer cell line compared to the normal cell line ( Fig. 4 ).

Therefore, by measuring the mRNA level of MIG12 or OIP5 using RT-PCR or the like, it can be seen that diagnosis, drug screening and the like of cancer associated with MIG12 or OIP5 is possible.

Example 3 Expression of OIP5 Gene in Colorectal Cancer Cell Lines

3-1. Expression of OIP5 in Cancerous and Normal Tissues of Colorectal Cancer Patients

Colorectal cancer and normal tissue from 66 patients with colorectal cancer were obtained from Samsung Medical Center (Seoul, South Korea). 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 quantified using Experion RNA StdSens (Bio-Rad) chip. First, the colon and normal tissues of the colon cancer were cut to an appropriate size, and then dissolved in 15 ml of kit digestion buffer containing 150ul of beta mercapto ethanol. 15 ml of 70% ethanol was added thereto, mixed well, and centrifuged at 3000 g for 5 minutes to attach total RNA to the membrane. After washing twice, total RNA was isolated by adding 1.2 ml of RNase-free water. Attached cell lines were recovered using trypsin, EDTA, and lysed in 1 ml of digestion buffer in the kit.

The extracted total RNA was hybridized using an Illumina TotalPrep RNA Amplification Kit (Ambion). T7 Oligo (dT) to synthesize cDNA using a primer, and using the biotin-UTP in In vitro transcription was performed to prepare biotin-labeled cRNA. The prepared cRNA was quantified using NanoDrop. CRNAs prepared from normal colon epithelial cells and colon cancer cells were hybridized to a Human-6 V2 (Illumina) chip. In order to remove nonspecific hybridization after hybridization, DNA chips were washed using Illumina Gene Expression System wash (Illumina), and the washed DNA chips were labeled with streptavidin-Cy3 (Amersham) fluorescent dye. Fluorescently labeled DNA chips were scanned using a confocal laser scanner (Illumina) to obtain data of fluorescence present in each spot and stored as image files in TIFF format. TIFF image files were quantified with BeadStudio version 3 (Illumina) to quantify the fluorescence values of each spot. Quantified results were corrected using the 'quantile' function with Avadis Prophetic version 3.3 (Strand Genomics) program.

The results are shown in Fig. 5 is an OIP5 gene expression profile measured after mRNA extraction from cancerous and normal tissues of colorectal cancer patients, and is highly expressed (when the amount of mRNA in cancer tissues relative to normal tissues is relatively increased). In case of low expression (relative amount of mRNA in cancer tissue relative to normal tissue is relatively reduced), it is shown in green. In Figure 5, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and ACTB (Homo sapiens actin, beta) refers to the standard gene for determining the difference in gene expression.

As a result, it was confirmed that the expression level of the OIP5 gene was more than doubled in 58 patients out of a total of 66 colon cancer patients (FIG. 5), and the average expression of the OIP5 gene in the colon cancer cells was increased by 3.7 times. In Figure 5, DF is a patient who did not relapse after surgical treatment, RC is a comparison of the degree of OIP5 expression in the patient group relapsed even after surgical treatment, there seems to be no difference in the amount of OIP5 expression in the two patients.

3-2. Comparison of mRNA Expression of OIP5 in Cancer Tissues of Colorectal Cancer Patients

RT-PCR was performed in the same manner as in Example 2, except that cancer tissues of 10 colorectal cancer patients randomly selected from the cancer tissues of 66 colon cancer patients of 3-1 above were used instead of the cancer cell line.

The results are shown in FIG. 6 is a result of measuring the RNA level of OIP5 using RT-PCR in 10 cancer tissues of colorectal cancer patients, T means cancer tissue, NT means normal tissue used as a control. As a result, it was confirmed that OIP5 gene was highly expressed in 8 patients colorectal cancer tissues out of 10 patients colorectal cancer tissues.

The results of 3-1 and 3-2 show a high correlation between cell lines obtained from actual colorectal cancer patients and the OIP5 gene, indicating that the OIP5 gene can be used as a diagnostic, drug screening, and therapeutic target.

Example 4 Increasing Cell Proliferation Rate upon Expression of MIG12 or OIP5 Gene

Taking advantage of the fact that the gene affects the rate of cell proliferation when the gene is involved in cancer production, overexpression of the gene using the CMV promoter in NIH3T3, a non-transformed mammalian cell line, and measuring the cell number for 72 hours, By examining the effect of the gene on the cell proliferation by comparing the rate of cell proliferation with the control group, cancer relatedness of the MIG12 or OIP5 gene was confirmed.

First, vector DNA containing MIG12 or OIP5 was purified through a column on a large scale, and the transformation was performed in 6-well cell culture plates. NIH3T3 (mouse embryonicfibroblast cell line) cell line for transformation (ATCC CRL-1658) was incubated with 60-70% confluency in DMEM medium supplemented with 10% calf serum.

10 μl of DNA (1 μg / μl), 10 μl of Lipofectamine in reagent (GIBCO / BRL), and 500 μl of serum-free DMEM medium (Dulbelcco's Modified Eagle's Medium) were mixed and reacted for 45 minutes at room temperature. Incubated at 37 ℃ for 48 hours. Four to six hours after the addition of the DNA mixture solution was replaced with fresh medium, the cell growth rate was examined by measuring the number of cells. The results are shown in FIG. 7 is a graph showing the results of proliferation assay after the expression of MIG12 or OIP5 gene in animal cells (NIH3T3) as a test for predicting cancer-related, white is not overexpressed as a control The results are for cell lines, and black is the result for cell lines with overexpressed genes. As a result, when the gene MIG12 or OIP5 was expressed in the NIH3T3 cell line, which is a normal cell line of mice, it was confirmed that the cell growth rate was increased ( FIG. 7 ).

These results support the cancer-related by the expression of MIG12 or OIP5 gene to increase the growth rate of cells, and by measuring the expression level of MIG12 or OIP5, diagnosis of cancer, drug screening, etc. related to MIG12 or OIP5 gene It can be seen that this is possible.

Example 5 Design and Synthesis of siRNA

For genes that are expressed in large amounts in cancer cell tissues or cancer cell lines, the siRNA technique can be used to observe the change in cell proliferation rate by inhibiting gene expression. Therefore, each siRNA for MIG12 and OIP5 can be designed and applied to various cancer cell lines. After the introduction, the growth rate of the cancer cell line was observed according to the decrease of mRNA expression.

The siRNAs of the MIG12 gene and the OIP5 gene were designed in a structure in which two bases (TT) of 3'- in a single chain were suspended in a double ribonucleic acid chain of 19 oligonucleotides.

The thermodynamic binding energy between siRNA and target mRNA designed to design siRNAs of MIG12 and OIP5 genes was calculated to select the base sequence with the most suitable energy distribution for intracellular RISC (RNA-induced silencing complex) protein binding. For this purpose, the energy profile analysis of mRNA was performed to find the most efficient target region of the gene (FIG. 8), and siRNA was designed by ranking the selected siRNA.

Sites that can be designed as siRNA sequences by energy profile analysis of the MIG12 gene (NM_021242) mRNA are complementary to contiguous 19-30 sequences belonging to SEQ ID NOs: 14, 15, 16, 17, or 18 Nucleotide sequence. The base sequence of SEQ ID NO: 14, 15, 16, 17, or 18 is a partial sequence of the MIG12 gene (NM_021242) of SEQ ID NO: 1, 845 to 950 base sequence, 970 to 1030 base sequence, 1080 to 1160 Corresponds to the base sequence, the 1220 to 1290 base sequence, and the base sequence complementary to the 1320 to 1400 base sequence. That is, the base sequence of the siRNA that can be designed is a base sequence corresponding to an mRNA sequence derived from 19 to 30 consecutive nucleotide sequences as part of each of the base sequences of SEQ ID NO: 14, 15, 16, 17, or 18 to be.

Sites that can be designed as siRNA sequences by energy profile analysis of the OIP5 gene (NM_021242) mRNA are sequenced 19- belonging to the nucleotide sequences of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, or 26. 30 base sequences. The nucleotide sequence of SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, or 26 is a partial sequence of the OIP5 gene of SEQ ID NO: 2, 1 to 60 nucleotide sequences, 120 to 180 nucleotide sequences, 380 Corresponds to the 460th base sequence, 518-580 base sequence, 720-780 base sequence, 850-890 base sequence, 920-980 base sequence, 1040-1100 base sequence. That is, the base sequence of the siRNA that can be designed is mRNA derived from 19-30 consecutive base sequences as part of each of the base sequences of SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, or 26. The base sequence corresponding to the sequence.

Using the energy profile analysis results, siMIG12 was designed to have the nucleotide sequence of SEQ ID NO: 3 or 4, siOIP5 was designed to have the nucleotide sequence of SEQ ID NO: 5 or 6. The nucleotide sequence of SEQ ID NO: 3 is a structure in which a single base of a two-chain (TT) is suspended in a nucleotide sequence corresponding to the mRNA sequence derived from the 1331-1349 th nucleotide sequence of SEQ ID NO: 1, and the nucleotide sequence of SEQ ID NO: 4 is 1123-. It is a structure in which a short chain of two bases (TT) is suspended in the base sequence corresponding to the mRNA sequence derived from the 1141st base sequence. In addition, SEQ ID NO: 5 is a structure in which a double-chain (TT) of a single chain is suspended in the base sequence corresponding to the mRNA sequence derived from the 426-444 base sequence of the OIP5 gene of SEQ ID NO: 2, SEQ ID NO: 6 It is a structure in which a single chain of two bases (TT) is suspended in the nucleotide sequence corresponding to the mRNA sequence derived from the 417th to 435th nucleotide sequences of the OIP5 gene of 2.

In addition, RABGTTA (Rab geranylgeranyl transferase alpha-subunit gene) was used as a gene showing cancer-related properties, and the siRNA of RABGTTA was designed to have a nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 8.

In addition, GFP (Green Flourescence Protein) having no effect on cells was used as a negative control of the siRNA experiment, and siGFP was designed to have a nucleotide sequence of SEQ ID NO: 9.

The siRNAs of all genes were designed by synthesizing the forward strand (sense strand) of the gene and the reverse sequence (anti-sense strand) showing the actual gene suppression effect in the cell, and then incorporated into the double ribonucleic acid chain.

The designed siRNA was synthesized by Samchully Pharmaceutical (Korea).

Example 6 Growth Inhibition Effect of Cancer Cell Line by Effect of MIG12 or OIP5 Gene siRNA

In the case of genes that are expressed in large amounts in cancer cell tissues or cancer cell lines, it is possible to observe whether there is a change in cell proliferation rate by inhibiting gene expression through siRNA technique, so that the siRNA prepared in Example 5 is grown in various cancer cell lines. Inhibition, apoptosis effect was confirmed.

6-1. Growth Inhibitory Effects of Cancer Cell Lines

Gastric cancer cell line Snu638 (Seoul National University Cell Line Bank), liver cancer cell line HCC (Seoul National University Cell Line Bank), Huh7 (pre-distribution from Chonnam National University), Snu387 (Seoul National University Cell Line Bank), CK-1a (pre-distribution from Chonnam National University) Lung cancer cell line A549 (Seoul National University Cell Line Bank), colon cancer cell line colo205 (Seoul National University Cell Line Bank), sw620 (Seoul National University Cell Line Bank), DLD-1 (Seoul National University Cell Line Bank), KM120 (Seoul National University Cell Line Bank) were used. After incubation with 30% confluency, the siMIG12 (SEQ ID NO: 3 and SEQ ID NO: 4), siOIP5 (SEQ ID NO: 5 and SEQ ID NO: 6), siRABGGTA (SEQ ID NO: 7 and SEQ ID NO: 8) were determined by the oligofectamine method (Invitrogen Co.). ), siGFP (SEQ ID NO: 9) was introduced into the cells, transformed, and after 5 hours, the cells were washed with PBS and replaced with fresh medium, and cultured for 72 hours.

Inhibition of growth of cancer cells by siRNA and staining with SRB (Sulfur rhodamine, Sigma Co.) was investigated through microscopic and changes by the number of cells stained with SRB (FIG. 9, FIG. 10). 9 is a graph showing the degree of inhibition of cell growth in various gastric cancer, liver cancer, lung cancer and colorectal cancer cell lines as a result of siRNA experiment to confirm the cancer target gene potential of MIG12 and OIP5 gene, Figure 10 is a cancer of the MIG12 and OIP5 gene As a result of siRNA experiments to confirm the target gene potential, it is a cell photograph showing cell growth inhibition in various gastric, liver, lung and colorectal cancer cell lines. As a result, GFP siRNA was used as a control (1.0), indicating the degree of growth inhibition by the RNAi effect of each gene in various cancer cell lines. Therefore, siMIG12 and siOIP5 according to the present invention showed a different cell line specificity, showing a different degree of inhibition, and it can be seen that there is an anticancer effect because it has a cell growth inhibitory effect of gastric cancer cell line, lung cancer cell line, liver cancer cell line and colon cancer cell line. ( FIG. 9 ).

In addition, in the HCC, Hur7, Snu638, Snu387 and CK-K1 cell line experiments, there was no significant change in the cell line treated with control GFP siRNA, whereas gastric cancer cell line Snu638 treated with siRNA of MIG12 or OIP5, HCC, Huh7, and Snu387 is shown to inhibit the growth of cells ( FIG. 10 ).

6-2. Apoptosis effect

Fluorescence Activated Cell Sorter (FACS) analysis was performed to measure the degree of apoptosis by inhibition of expression of the gene after RNAi experiment in Snu638 cell line ( FIG. 11 ). When the siRNA of RABGTTA was treated, the growth inhibitory effect of the cell line was not observed. In the CK-1a cell line, the growth inhibitory effect of the cell line was not observed even though the expression of three genes was inhibited. It was observed that apoptosis was induced by FACS analysis of Snu638 cell line in which siOIP was introduced ( FIG. 11 ). In the cancer cell line containing the control siGFP, DNA fragments, which are the main phenomena of apoptosis, were seen after 72 hours, but the presence of relatively large amounts of DNA fragments was observed when siMIG12 or siOIP5 was treated.

In addition, RNAi experiments using siRNAs of MIG12 and OIP5 confirmed that apoptosis of cells occurred when MIG12 or OIP5 expression was reduced through FACS analysis. RT-PCR was performed to determine the expression changes of any genes involved in apoptosis. After incubation for 72 hours after siRNA treatment, cells (Snu638) were collected and total RNA was isolated. Synthesis of cDNA from RNA and RT-PCR using Gene Expression Profiling kit (GeneXp ™) (Seegene, Seoul, Korea) to treat aRNA and induce apoptosis in apoptosis cells The expression levels of related genes were examined and compared (FIG. 12). As a result, the expression levels of caspase 2, caspase 7, caspase 8, caspase 9, caspase 10, BAK1 and granzyme, which are known to be directly involved in apoptosis, were increased in the case of siMIG12. In the case of siOIP5, the expression levels of caspase 2, caspase 8, caspase 9, caspase 10, BAK1 and granzyme, which are known to be directly involved in the induction of apoptosis, were increased. In addition, Mcl-1, which counteracts apoptosis induction, is reduced. It was confirmed that the expression of Bcl2 was increased and the expression of P53 was increased when the expression of OIP5 gene was decreased when the two gene siRNA treatments were induced. This shows that apoptosis is induced when the expression of MIG12 and OIP5 genes are decreased, and the mechanism of apoptosis is different. have.

As a result, it can be seen that the siRNA of the present invention induces apoptosis effect of cancer cell line and shows anticancer effect.

Example 7 shRNA Design and Synthesis of MIG12 and OIP5 Genes

In the case of genes that are expressed in large amounts in cancer cell tissues or cancer cell lines, it is possible to observe whether there is a change in the proliferation rate of cancer cells by inhibiting gene expression through shRNA technique. Therefore, each shRNA for MIG12 and OIP5 is designed and intracellularly. After cloning into a vector for introduction into the cancer cell line, the growth rate of the cancer cell line was observed according to mRNA expression degradation.

First, shRNAs of MIG12, OIP5 and GFP were designed and synthesized in Bioneer (South Korea). Green Flourescence Protein (GFP) was used as a negative control for shRNA experiments that had no effect on cells. To prepare shRNAs, a pair of 67 nucleotides (top and bottom strands) including DNA (forward and reverse) and loop sequences corresponding to siRNA sequences were synthesized, and a pENTR-U6-BHRNX vector having a U6 promoter Clone). The structure of 67 nucleotides consists of gatcc-siRNA forward sequence-loop -siRNA reverse sequence-TTTTTTACGCGTg and forms a stem-loop structure in vivo (FIG. 13A). SEQ ID NO: 27 used siMIG12 ′ SEQ ID NO: 4 as a top strand for shRNA of the MIG12 gene. SEQ ID NO: 28 was siOIP5 SEQ ID NO: 5 as a top strand for shRNA of the OIP5 gene. The sequence of the synthesized 67mer nucleotide was cloned into pENTR-U6-BHRNX vector (Nurogenex) and the sequence was confirmed. GFP shRNAs were designed and synthesized in the same manner as shRNAs of MIG12 and OIP5 genes except that the nucleotide sequence of SEQ ID NO: 29 was used as the top strand.

Example 8 Growth Inhibition Effect of Cancer Cell Line by shRNA of MIG12 or OIP5 Gene

After culturing the liver cancer cell line Huh7 in 60% confluency, it was introduced into cells to express the shRNA of Example 7 from the vector by the lipofectamine method (Invitrogen Co.), and then transformed into a fresh medium after 5 hours, 72 Incubated for hours. Cell growth inhibition was observed under a microscope and stained with SRB (Sulfur rhodamine, Sigma Co.). While there was no significant change in the cell line treated with GFP shRNA as a control, the growth inhibition of cells was shown in the liver cancer cell line Huh7 treated with shRNAs of MIG12 and OIP5 (FIG. 13B) .

Example 9 Drug Screening Using Human Gene MIG12 or OIP5 Overexpression Effect

The promoter of the gene expression vector for expressing cancer-related human genes in the cleavage yeast ED665 strain of Example 1 is regulated by thiamin. That is, the ED665 strain into which the MIG12 or OIP5 gene was introduced was cultured in a medium (EMM + AL + Thia) to which 15 μM of thiamine (thiamine, hereinafter referred to as Thia) was added, but the medium without expressing the MIG12 or OIP5 gene (EMM) was added. + AL) expresses large amounts of MIG12 or OIP5 genes in culture. Using this property, it was confirmed that anti-cancer screening was possible using thiamin as a test substance and MIG12 or OIP5 gene as a target substance.

9-1. Cell Growth Inhibition Effect of Cleavage Yeast by MIG12 or OIP5 Overexpression

In order to confirm cell growth inhibition by MIG12 or OIP5 gene expression, ED665 strain cultured in EMM + AL + Thia solid medium was washed three times with sterile distilled water to completely remove thiamin, and then EMM + AL liquid medium and EMM +. Cell growth was observed by inoculating 10 ml of AL + Thia (15 uM) liquid medium so that the OD ₆₀₀ value was 0.1 again. As a control (control), a strain in which only an expression vector was introduced without gene cloning was used. Figure 14 shows the difference in cell growth after 23 hours. In FIG. 14, (+) means the result in the medium containing thiamin, and (-) means the result in the medium not containing thiamin. As a result, MIG12 and OIP5 protein expression began to be induced 12 hours after thiamin was removed, and as shown in FIG. 14, the difference in cell growth according to protein expression induction was significantly increased ( FIG. 14 ). That is, it was confirmed that cell growth was decreased by expression of MIG12 or OIP5, and cell growth inhibition did not occur when MIG12 or OIP5 gene expression was inhibited by thiamine.

9-2. Quantitative Measurement of MIG12 or OIP5 Overexpression Effects in 96-well Plates

The extent of cell growth during overexpression of genes cloned into the p173DES vector was observed on 96well plates. Cells inoculated in EMM + AL + Thia solid medium were washed three times with sterile distilled water to completely remove thiamin, and then the OD value was added to EMM + AL liquid medium and 200ul of EMM + AL + Thia (15uM) liquid medium. After inoculation to 0.05, the cells were aliquoted into 96well plates, diluted in 1/5 ratios, and incubated for 24 hours. As a control (control), a strain in which only an expression vector was introduced without gene cloning was used. In the control group containing no human gene, there was little effect on cell growth with or without thiamin. However, in the group in which the MIG12 or OIP5 gene was introduced, the cell growth was significantly inhibited when the MIG12 or OIP5 gene was overexpressed because thiamin was not added. Was confirmed ( FIG. 15A ). In addition, cells grown on 96-well plates were diluted four-fold in EMM + AL solid medium and EMM + AL + Thia (15uM) solid medium, respectively. The degree of growth in EMM + AL + Thia (15uM) solid medium was not overexpressed. All grew well without any difference, but it was confirmed that the growth inhibition of cells was shown in EMM + AL solid medium induced overexpression of MIG12 or OIP5 gene ( FIG. 15B ).

From the results of 9-1 to 9-2, the cell growth was restored to normal through drug (test substance) treatment for the cells whose growth was inhibited due to overexpression of the MIG12 and OIP5 genes (target material) according to the present invention. By culturing in 96-well plate and solid medium, the growth inhibition effect can be easily identified quantitatively and can be used as a drug screening system because it can search for anticancer drugs targeting MIG12 or OIP5.

1 is a human gene from the plasmid (Entry clone) in which the human gene is inserted S. pombe It is a schematic diagram cloned into a destination vector.

Figure 2 shows the change in phenotype when the human gene MIG12 is overexpressed in S. pombe , Figure 2A shows cell growth inhibition, Figure 2B shows the morphological changes of the cells, Figure 2C shows the change in the amount of DNA in the cell. 2D shows the results of confirming the expression of human genes.

Figure 3 shows the change in phenotype when the human gene OIP5 is overexpressed in S. pombe , Figure 3A shows cell growth inhibition, Figure 3B shows the morphological changes of the cells, Figure 3C shows the change in the amount of DNA in the cell. 3D shows the results of confirming human gene expression.

Figure 4 shows the results of measuring the mRNA amount of MIG12 or OIP5 gene in liver and gastric cancer cell lines using RT-PCR.

5 is a graph comparing the expression of OIP5 gene in cancerous tissues and normal tissues of colon cancer patients.

Figure 6 is a comparison of the mRNA expression of OIP5 using RT-PCR in cancer tissue of colorectal cancer patients.

FIG. 7 is a graph showing the results of proliferation assay after the expression of MIG12 or OIP5 gene in animal cells (NIH3T3) as an experiment for predicting cancer relatedness.

8 is a diagram showing the energy profile of the MIG12 or OIP5 gene.

9 is a siRNA experiment for confirming the possibility of cancer target gene of the MIG12 or OIP5 gene, a graph showing the degree of cell growth inhibition in various gastric, liver, lung and colorectal cancer cell lines.

FIG. 10 is a cell photograph showing cell growth inhibition in various gastric and liver cancer cell lines as a result of siRNA experiments to confirm the possibility of cancer target genes of MIG12 or OIP5 genes.

11 shows the results of FACS analysis for measuring the degree of cell death by inhibition of expression of the gene after siRNA experiment.

12 shows expression changes of genes related to apoptosis through RNAi experiments using siMIG12 or siOIP5.

Figure 13 shows shRNA design (A) and experimental results (B).

14 is a graph showing the growth rate inhibition and degree of growth of cells after overexpression of MIG12 or OIP5 gene in S. pombe of model cell division yeast.

Figure 15 shows the results of investigating the possibility of HTS screening cystel development targeting MIG12 and OIP5 gene using 96-well plate.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel use of MIG12 gene <130> P07-067-KRI <150> KR1020060124438 <151> 2006-12-08 <160> 29 <170> KopatentIn 1.71 <210> 1 <211> 2454 <212> DNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (2454) <223> MIG12, Homo sapiens MID1 interacting protein 1 (gastrulation          specific G12 homolog (zebrafish)) (MID1IP1), transcript variant          1, mRNA. <400> 1 ggctcactct gcaaccaagg cacgtgcatt ctggtcatcc cacgcgggga gcgcgcgcaa 60 ggcccgccca gcccccacat gccagcccca ccctccagtc ggtccggacg ccgacgcctt 120 tttgaccctc gctgtgcccg gccctcctca tctggcctgc ccagggcttg gtgctggcgg 180 ggtccagctg ctccaatccc tcctcctctg ctctgccctg ccctgccctg gcctgccccg 240 gcgccctccc tcagcccggg tatcaggcga gaggcggagc tggcccggcg cgccccgccc 300 ccgctgtaga aagggccggg cgagtgttac tcgcggtcat cccggcctgg gccttttatc 360 tcggtgctgc cgggggaggc gggaggagga gacaccaggg gtggccctga gcgccggcga 420 cacctttcct ggactataaa ttgagcacct gggatgggta gggggccaac gcagtcaccg 480 ccgtccgcag tcacagtcca gccactgacc gcagcagcgc ccttgcgtag cagccgcttg 540 cagcgagaac actgaattgc caacgagcag gagagtctca aggcgcaaga ggaggccagg 600 gctcgaccca cagagcaccc tcagccatcg cgagtttccg ggcgccaaag ccaggagaag 660 ccgcccatcc cgcagggccg gtctgccagc gagacgagag ttggcgaggg cggaggagtg 720 ccgggaatcc cgccacaccg gctatagcca ggcccccagc gcgggccttg gagagcgcgt 780 gaaggcgggc atccccttga cccggccgac catccccgtg cccctgcgtc cctgcgctcc 840 aacgtccgcg cggccaccat gatgcaaatc tgcgacacct acaaccagaa gcactcgctc 900 tttaacgcca tgaatcgctt cattggcgcc gtgaacaaca tggaccagac ggtgatggtg 960 cccagcttgc tgcgcgacgt gcccctggct gaccccgggt tagacaacga tgttggcgtg 1020 gaggtaggcg gcagtggcgg ctgcctggag gagcgcacgc ccccagtccc cgactcggga 1080 agcgccaatg gcagcttttt cgcgccctct cgggacatgt acagccacta cgtgcttctc 1140 aagtccatcc gcaacgacat cgagtggggg gtcctgcacc agccgcctcc accggctggg 1200 agcgaggagg gcagtgcctg gaagtccaag gacatcctgg tggacctggg ccacttggag 1260 ggtgcggacg ccggcgaaga agacctggaa cagcagttcc actaccacct gcgcgggctg 1320 cacactgtgc tctcgaaact cacgcgcaaa gccaacatcc tcactaacag atacaagcag 1380 gagatcggct tcggcaattg gggccactga ggcgtggcgc ccgtggctgc ccagcacctt 1440 cttcgaccca tctcaccctc tctcattcct caaagctttt tttttttttc ctggctgggg 1500 ggcgggaagg gcagactgca aactgggggg ctgcgtacgt gcaggaggcg cggtggggct 1560 gcgtggagga gggggccacg tgtgagagag aagaaaatgg tggccggaga tgggagggcc 1620 caaggaacct cctgggaggg ggcctgcatt ctatgttggt gggaatggga ctgggctgac 1680 gccctgcatt cagcctgtgc ctttcctggg gtttcttttc tgttcttttc ggaggagagg 1740 gcccgagaag gggccatacc agggcgcggc gctgggttgc cacacttggg aaagcagccc 1800 ggagctgggt gctggggaag gcggggcgcg tagcctcccg ccgccctgcg gttgggccgg 1860 tggaggccca ggcgttgcta ggattgcatc agttttcctg tttgcactat ttctttttgt 1920 aacattggcc ctgtgtgaag tatttcgaat ctcctccttg ctctgaaact tcagcgattc 1980 cattgtgata agcgcacaaa cagcactgtc tgtcggtaat cggtactact ttattaatga 2040 ttttctgtta cactgtatag tagtcctatg gcacccccac cccatccctt tcgtgccact 2100 cccgtcccca cccccacccc agtgtgtata agctggcatt tcgccagctt gtacgtagct 2160 tgccactcag tgaaaataat aacattatta tgagaaagtg gacttaaccg aaatggaacc 2220 aactgacatt ctatcgtgtt gtacatagaa tgatgaaggg ttccactgtt gttgtatgtc 2280 ttaaatttat ttaaaacttt ttttaatcca gatgtagact atattctaaa aaataaaaaa 2340 gcaaatgtgt caactaaatt ggacaagcgt ctggtcctca ttaatctgcc aatgaatggt 2400 ttcgtcatta aataaaaatc aatttaattg atttactagc aaaaaaaaaa aaaa 2454 <210> 2 <211> 1249 <212> DNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (1249) Homo sapiens Opa interacting protein 5 (OIP5), mRNA. <400> 2 gagctgtgct tggggcgggg cctggcgtgt attcgaaagg aaggcgccgg ctgcgggaag 60 atggcggctc agccgctgcg gcatcgctca cgttgtgcaa cgccgccccg gggggacttt 120 tgtggtggca ctgagagggc gattgaccaa gcttctttta cgacctccat ggagtgggat 180 acgcaggtgg tgaaggggtc ctcgccgctc ggccccgcag ggctgggggc tgaggagcca 240 gccgccggcc cgcagctgcc gtcttggctg cagcctgaga ggtgcgctgt gttccagtgc 300 gcacagtgtc acgcagtgct cgccgactcg gtgcacctcg cctgggacct gtcgcggtcc 360 ctcggggccg tggtcttctc cagagttaca aataacgtcg ttttggaagc gcccttccta 420 gttggcattg aaggttcact caaaggcagt acttacaacc ttttattctg tggttcttgt 480 gggattcccg ttggtttcca tctgtattct acccatgctg ccctggctgc cttgagaggt 540 cacttctgcc tttccagtga caaaatggtg tgctatctct taaaaacaaa agccatagta 600 aatgcatcag agatggatat tcaaaatgtt cctctatcag aaaagattgc agagctgaaa 660 gagaagatag tgctaacgca caatcgctta aaatcactaa tgaagattct gagtgaagtg 720 actcctgacc agtccaagcc agaaaactga tcctgtacca aagcttgagt gtcaggttca 780 ggctttattg ctgtcttcaa caacaggtgc tgcttagtca tttcttgaaa aagattggct 840 tcaagaatgg aggggaaatg cagtttctat ttacctttag gctgattttc caaattattt 900 gtgaagctgt ttttagaaga tgagagacta aggattcttc tcttttatag ctatttgcct 960 taagaactta ctttagattc ttattgaatt cataatactt atctctgaaa atgtctttga 1020 ctgtaaattt aggaattaag atgcagagtc ccatgtgtcc tctgatctaa agttgcatgg 1080 ttggtctgaa aatagagttg ggcttaatgt tgacttctat tactcctgca tggagcagtt 1140 gttatgaata ctaatacatc actttttaac ttctgtaaaa tacagatcat aatattctat 1200 aggtaatgtt taataaattg cctgaataat atacaaaaaa aaaaaaaaa 1249 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA for MIG12 (NM_021242: 1331-1349) <220> <223> siRNA <400> 3 ucucgaaacu cacgcgcaat t 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA for MIG12 (NM_021242: 1123-1141) <220> <223> siRNA <400> 4 agccacuacg ugcuucucat t 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA for OIP5 (NM_007280: 426-444) <220> <223> siRNA <400> 5 cauugaaggu ucacucaaat t 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA for OIP5 (NM_007280: 417-435) <220> <223> siRNA <400> 6 ccuaguuggc auugaaggut t 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA for RABGGTA (NM_182836: 1110-1128) <220> <223> siRNA <400> 7 acauacauuu cgcgucauut t 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA for RABGGTA (NM_182836: 1509-1527) <220> <223> siRNA <400> 8 ggcucacaag gaucugacat t 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA for GFP <220> <223> siRNA <400> 9 guucagcgug uccggcgagt t 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> N-terminal primer for PCR of MIG 12 <400> 10 atgatgcaaa tctgcgacac c 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> C-terminal primer for PCR of MIG 12 <400> 11 tcagtggccc caattgccga a 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> N-terminal primer for PCR of OIP 5 <400> 12 ggctgggggc tgaggagcca 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> C-terminal primer for PCR of OIP 5 <400> 13 cacttcactc agaatcttca 20 <210> 14 <211> 106 <212> DNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (106) <223> partial sequence of MIG12 (NM_021242: 845-950) <400> 14 tccgcgcggc caccatgatg caaatctgcg acacctacaa ccagaagcac tcgctcttta 60 acgccatgaa tcgcttcatt ggcgccgtga acaacatgga ccagac 106 <210> 15 <211> 61 <212> DNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (61) <223> partial sequence of MIG12 (NM-021242: 970-1030) <400> 15 ctgcgcgacg tgcccctggc tgaccccggg ttagacaacg atgttggcgt ggaggtaggc 60 g 61 <210> 16 <211> 81 <212> DNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (81) <223> partial sequence of MIG12 (NM_021242: 1080-1160) <400> 16 aagcgccaat ggcagctttt tcgcgccctc tcgggacatg tacagccact acgtgcttct 60 caagtccatc cgcaacgaca t 81 <210> 17 <211> 71 <212> DNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (71) <223> partial sequence of MIG12 (NM_021242: 1220-1290) <400> 17 ggaagtccaa ggacatcctg gtggacctgg gccacttgga gggtgcggac gccggcgaag 60 aagacctgga a 71 <210> 18 <211> 81 <212> DNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (81) <223> partial sequence of MIG12 (NM_021242: 1320-1400) <400> 18 gcacactgtg ctctcgaaac tcacgcgcaa agccaacatc ctcactaaca gatacaagca 60 ggagatcggc ttcggcaatt g 81 <210> 19 <211> 60 <212> DNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (60) <223> partial sequence of OIP5 (NM_007280: 1-60) <400> 19 gagctgtgct tggggcgggg cctggcgtgt attcgaaagg aaggcgccgg ctgcgggaag 60                                                                           60 <210> 20 <211> 61 <212> DNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (61) <223> partial sequence of OIP5 (NM_007280: 120-180) <400> 20 ttgtggtggc actgagaggg cgattgacca agcttctttt acgacctcca tggagtggga 60 t 61 <210> 21 <211> 81 <212> DNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (81) <223> partial sequence of OIP5 (NM_007280: 380-460) <400> 21 ccagagttac aaataacgtc gttttggaag cgcccttcct agttggcatt gaaggttcac 60 tcaaaggcag tacttacaac c 81 <210> 22 <211> 63 <212> DNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (63) <223> partial sequence of OIP5 (NM_007280: 518-580) <400> 22 ctgccctggc tgccttgaga ggtcacttct gcctttccag tgacaaaatg gtgtgctatc 60 tct 63 <210> 23 <211> 61 <212> DNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (61) <223> partial sequence of OIP5 (NM_007280: 720-780) <400> 23 gactcctgac cagtccaagc cagaaaactg atcctgtacc aaagcttgag tgtcaggttc 60 a 61 <210> 24 <211> 41 <212> DNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (41) <223> partial sequence of OIP5 (NM_007280: 850-890) <400> 24 gaggggaaat gcagtttcta tttaccttta ggctgatttt c 41 <210> 25 <211> 61 <212> DNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (61) <223> partial sequence of OIP5 (NM_007280: 920-980) <400> 25 atgagagact aaggattctt ctcttttata gctatttgcc ttaagaactt actttagatt 60 c 61 <210> 26 <211> 61 <212> DNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (61) <223> partial sequence of OIP5 (NM_007280: 1040-1100) <400> 26 gatgcagagt cccatgtgtc ctctgatcta aagttgcatg gttggtctga aaatagagtt 60 g 61 <210> 27 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> top strand for shMIG12 <400> 27 gatccgagcc actacgtgct tctcattcaa gagatgagaa gcacgtagtg gctctttttt 60 acgcgtg 67 <210> 28 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> top strand for shOIP 5 <400> 28 gatccgcatt gaaggttcac tcaaattcaa gagatttgag tgaaccttca atgctttttt 60 acgcgtg 67 <210> 29 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> top strand for shGFP <400> 29 gatccggttc agcgtgtccg gcgagttcaa gagactcgcc ggacacgctg aacctttttt 60 acgcgtg 67  

Claims (28)

A cancer diagnostic composition comprising a MIG12 (MID1 interacting protein 1) gene, wherein the cancer is at least one selected from liver cancer or gastric cancer. A cancer diagnostic composition comprising a protein expressed from a MIG12 (MID1 interacting protein 1) gene, wherein the cancer is at least one selected from liver cancer or gastric cancer. A cancer diagnostic composition comprising an antibody against a protein expressed from a MIG12 (MID1 interacting protein 1) gene, wherein the cancer is at least one selected from liver cancer or gastric cancer. A composition for screening an anticancer agent comprising a MIG12 (MID1 interaction protein 1) gene, wherein the cancer is at least one selected from liver cancer, gastric cancer, or colorectal cancer. A composition for screening an anticancer agent comprising a protein expressed from a MIG12 (MID1 interacting protein 1) gene, wherein the anticancer agent is at least one selected from an anticancer agent or an anticancer agent. By using the composition of claim 4 as a target substance, the test substance is contacted and the reaction between the target substance and the test substance is checked to determine whether the test substance exhibits an activity for enhancing or inhibiting expression of the gene. A method for screening an anticancer agent comprising the step, wherein the anticancer agent is at least one selected from an anticancer agent, an anticancer agent, or an anticolon cancer agent. A method for screening an anticancer agent comprising the step of determining whether the composition of claim 4 exhibits the activity of inhibiting the expression of the gene by inducing phenotypic changes of model cells or animal cells using the composition of claim 4, wherein the anticancer agent At least one selected from anti-hepatic cancer, anti-gastric, or anti-colon cancer. Using the composition of claim 5 as a target material to contact a test substance and confirming a reaction between the target substance and the test substance, to determine whether the test substance exhibits an activity to enhance or inhibit the function of the protein. A method for screening an anticancer agent comprising the step, wherein the anticancer agent is at least one selected from an anticancer agent or an anticancer agent. delete delete delete delete delete delete delete delete An inhibitor of the MIG12 (MID1 interacting protein 1) gene, which corresponds to an mRNA sequence derived from 19 to 30 consecutive sequences in one or more sequences selected from SEQ ID NOs: 14, 15, 16, 17, and 18 SiRNA comprising a nucleotide sequence, or shRNA comprising a nucleotide sequence corresponding to an mRNA sequence derived from a sequence of 19 to 30 base sequences in one or more sequences selected from SEQ ID NOs: 14, 15, 16, 17 and 18 A composition for treating cancer, wherein the cancer is at least one selected from liver cancer, stomach cancer, or colorectal cancer. delete delete The composition of claim 17, wherein the base sequence of the siRNA is SEQ ID NO: 3 or 4. 18. The composition of claim 17, wherein the siRNA is in a chemically modified form. delete 18. The composition of claim 17, wherein the shRNA comprises an RNA base sequence of SEQ ID NO: 4 and an RNA base sequence complementary to SEQ ID NO: 4 linked to a loop region of 3 to 10 bases. The composition for treating cancer according to claim 23, wherein the top strand for preparing an expression vector for expressing the shRNA is SEQ ID NO: 27. delete delete delete A kit for diagnosing cancer comprising a protein expressed from a MIG12 (MID1 interacting protein 1) gene or a MIG12 (MID1 interacting protein 1) gene, wherein the cancer is at least one selected from liver cancer or gastric cancer.

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