KR102328654B1 - A composition for preventing or treating cancer - Google Patents

Abstract

The present invention relates to a novel use of the YME1L1 gene and the YME1L1 protein as novel targets that can be used for the prevention or treatment of cancer.

Description

Pharmaceutical composition for preventing or treating cancer

The present invention relates to a pharmaceutical composition for preventing or treating cancer.

In modern society, the incidence rate of cancer is gradually increasing, and various approaches to prevent or treat cancer have been made to try to lower the mortality rate. The statistics are high enough to occupy the first place for a while. Methods for treating cancer include surgery, radiation therapy, chemotherapy, and biological therapy. Therefore, the market size of cancer treatment is expected to reach about 100,000 dollars worldwide and record a high growth rate to exceed 170,000 dollars by 2025.

Cancer is a disease characterized by infinite proliferation and formation of tumors as the regulation related to cell division, growth, and apoptosis is not normally achieved. get affected. Therefore, it is used as a method for preventing or treating cancer by inhibiting the development or progression of cancer when it promotes continuous division of cancer cells or suppresses the expression or protein activity of genes that inhibit cancer cell death. can be

On the other hand, YME1L1 is an ATP-dependent proteolytic enzyme present in the inner membrane of mitochondria, and is known to play a role in decomposing proteins related to lipid transfer. In addition, there have been reports confirming that it has an activity to inhibit nuclear mitochondrial DNA (mtDNA) transfer and that the gene of YME1L1 is mutated with high frequency in colorectal adenocarcinoma cells to increase mtDNA transfer in the nuclear genome. There has been no study or report on the fact that the expression level of the cancer cells can be induced by increasing the expression level or suppressing the expression of YME1L1.

In general, in the process of converting normal cells under normal control into cancer cells, genomic instability increases, and most cells die during the conversion process and do not develop into cancer cells. However, in some cells, by increasing the expression of a protein having an activity to reduce the instability of the genome as described above, cell death is prevented and survival is prevented, and thus cancer cells can be formed. Therefore, when inhibiting the activity of a protein capable of reducing genomic instability or suppressing the expression of a gene encoding the protein, the efficacy of preventing or treating cancer can be expected by inducing apoptosis of cancer cells, thereby lowering the survival rate. Therefore, there is a need to discover new genes or proteins that are specifically expressed in large amounts in cancer cells and play a role in helping the development and progression of cancer, and research and development of new cancer prevention or treatment methods targeting them.

An object of the present invention is to provide a pharmaceutical composition comprising an agent that can be used for preventing or treating cancer by inhibiting cell growth or inducing apoptosis of cancer such as liver cancer, fibrosarcoma, cervical cancer, and the like.

Another object of the present invention is to provide a pharmaceutical composition comprising a polynucleotide encoding the agent that can be used for the prevention or treatment of cancer as described above.

Another object of the present invention is to provide a pharmaceutical composition comprising a cell transduced with the polynucleotide that can be used for the prevention or treatment of cancer as described above.

Another object of the present invention is to provide a method for providing information necessary for diagnosis of cancer by analyzing a sample isolated from a subject.

Another object of the present invention is to provide a method for screening a candidate substance that can be used as a therapeutic agent for cancer by analyzing a test substance.

In order to achieve the above object, one aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising an inhibitor of YME1L1 gene expression or YME1L1 protein activity as an active ingredient.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a polynucleotide encoding a YME1L1 gene expression inhibitor or YME1L1 protein activity inhibitor.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a cell transduced with a polynucleotide encoding a YME1L1 gene expression inhibitor or YME1L1 protein activity inhibitor.

Another aspect of the present invention provides a method for providing information necessary for diagnosing cancer, comprising measuring the expression level of the YME1L1 gene or the activity level of the YME1L1 protein in a sample isolated from a subject.

Another aspect of the present invention provides a method for screening a cancer drug candidate, comprising the step of treating a test substance to a YME1L1 protein-expressing cell line.

The pharmaceutical composition provided in the present invention suppresses the expression of the YME1L1 gene or inhibits the activity of the YME1L1 protein, so that various types of cancer derived from various tissues such as liver cancer, glioma, cervical cancer, lung cancer, colorectal cancer, breast cancer, glioma, etc. It has the effect of causing damage to DNA in cells. Therefore, the pharmaceutical composition of the present invention has an effect of inhibiting the growth of cancer cells or inducing the death of cancer cells, thereby inhibiting the occurrence or progression of cancer, and thus can be usefully used as a pharmaceutical composition for preventing or treating cancer.

In addition, by measuring the YME1L1 gene expression level or YME1L1 protein activity level, which is high in various cancer cell lines, it can be used to provide information for diagnosing the risk of cancer, and can be usefully used for discovering or developing new therapeutic agents. .

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

1 is BM-MSC, Adipo-MSC, Retina, WI38, BJ used as normal cell lines, Huh7, HepG, Hep3B, SNU387, SNU398, SNU449 used as liver cancer cell lines, LN428, U87, Calu1, A549, H460, H1299 used as lung cancer cell lines, SAOS2 used as osteosarcoma cell lines, HT29, HCT116 used as colon cancer cell lines, MDAMB231, MCF7 used as breast cancer cell lines, HT1080 used as fibrosarcoma cell lines, It is a graph comparing the relative amount of YME1L1 gene mRNA measured by real-time RT-PCR.
Figure 2 is a Huh7 liver cancer cell line (A), HT1080 fibrosarcoma cell line (B), and HeLa cervical cancer cell line (C), targeting the three types of siRNA si-YME1L1#1, si-YME1L1 suppressing the expression of YME1L1 #2, si-YME1L1#3 and control-siRNA (si-Con) were treated, respectively, and the cells were stained with crystal violet to confirm the formation of colonies (left figure) and counted the number of formed colonies. These are the comparison graph (middle figure) and the comparison graph (right figure) by measuring the relative amount of YME1L1 mRNA.
FIG. 3 shows additional four types of siRNA that inhibit YME1L1 expression in Huh7 liver cancer cell line (A) and HeLa cervical cancer cell line (B), si-YME1L1#4, si-YME1L1#5, si-YME1L1# 6, si-YME1L1#7 and control-siRNA (si-Con) were treated, respectively, and the cells were stained with crystal violet to confirm the formation of colonies, and a graph comparing the number of colonies formed by counting (left) Figure) and a graph comparing the relative amount of YME1L1 mRNA (Figure on the right).
4 is si-YME1L1#3, two types of siRNAs that inhibit the expression of YME1L1, in HT1080 fibrosarcoma cell line, SNU475 liver cancer cell line, MDAMB231 breast cancer cell line, LN428 glioma cell line, SW480 colorectal cancer cell line and H460 lung cancer cell line. , si-YME1L1#7 and control-siRNA (si-Con) were treated, respectively, and the cells were stained with crystal violet to confirm the formation of colonies (left figure) and counted and compared the number of formed colonies. The graph (right figure) and Western blotting results (middle figure) confirming the protein amount of YME1L1.
5 is a HeLa cervical cancer cell line, two types of siRNA that inhibit the expression of YME1L1, si-YME1L1#3, si-YME1L1#7, and control-siRNA (si-Con), respectively, apoptosis It is a FACS histogram (B) confirming the percentage of cells stained with Annexin V-FITC that can measure the degree of (apoptosis) (A) and stained with a flow cytometer (B).
6 is a HeLa cell line, si-YME1L1#1, si-YME1L1#3, which are siRNAs that inhibit YME1L1 expression, and a control-siRNA (si-Control), respectively, using an anti-γ-H2AX antibody Thus, γ-H2AX, a DNA damage marker, was stained and observed under a fluorescence microscope.

First, the terms used in the present invention are defined.

In the present invention, "cancer" is a disease in which an abnormality occurs in the regulation of cell growth or death, and when the balance of normal cell growth or death is disrupted, it refers to a disease caused by excessive cell proliferation. These abnormal hyperproliferative cells, in some cases, invade surrounding tissues and organs, form a mass, and destroy or deform the normal structure of the body. A tumor refers to a cell mass that has grown abnormally due to the autonomous overgrowth of body tissues, and can be divided into benign tumors and malignant tumors. Malignant tumors have a very rapid growth rate compared to benign tumors, and metastasis occurs while infiltrating surrounding tissues, threatening life, and such malignant tumors are commonly referred to as cancer.

In the present invention, "prevention" means reducing the risk of contracting a disease or disorder, and one or more clinical signs of a disease in a subject who is exposed to or prone to the disease but does not yet have the disease or exhibit symptoms of the disease It means any action that inhibits or delays the onset of a disease by preventing it from progressing.

In the present invention, "treatment" means alleviating a disease or disorder, and refers to any action that improves or beneficially alters the symptoms of a disease by stopping or reducing the progression of the disease or one or more clinical symptoms thereof.

As used herein, “diagnosis” refers to a subject based on observation, testing, or circumstances to identify a subject having a disease, disorder, or condition based on the presence of one or more indicators, such as signs or symptoms of the disease, disorder, or condition. means a clinical or other evaluation of the condition of

In the present invention, "sample" means a collection of similar fluids, cells, or tissues isolated from a subject. The term “sample” refers to fluids of the body (e.g., urine, serum, blood fluids, lymph, gallbladder fluid, ascite fluid, ocular fluids, and fluids collected by bronchial lavage and/or peritoneal rinsing). , ascites, tissue samples or cells from a subject. Also includes teardrops, serum, cerebrospinal fluid, feces, sputum, and cell extracts.

In the present invention, "test substance" means any substance expected to indirectly or directly induce a change in gene expression level or protein activity level that occurs significantly in a cell.

Hereinafter, the present invention will be described in detail.

1. Pharmaceutical composition for preventing or treating cancer

One aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer.

The pharmaceutical composition for preventing or treating cancer includes an inhibitor of YME1L1 gene expression or YME1L1 protein activity inhibitor as an active ingredient.

The YME1L1 gene is a gene encoding a YME1L1 protein, which is an ATP-dependent metalloprotease. The YME1L1 protein expressed from the YME1L1 gene may include an AAA family, an ATP/GTP binding motif, and a HEXXH motif, and the YME1L1 protein has an activity of decomposing proteins such as lipid transport proteins in the inner membrane of mitochondria and has a mitochondrial form It may be involved in maintenance, apoptosis resistance, and cell division. The nucleotide sequence of the YME1L1 gene and the amino acid sequence of the YME1L1 protein can be obtained from public databases such as GenBank of NCBI or the literature.

The YME1L1 gene expression inhibitor includes any agent capable of inhibiting the genetic flow process from the synthesis of mRNA transcribed from the YME1L1 gene to the synthesis of the YME1L1 protein translated from the mRNA. The YME1L1 gene expression inhibitor may be an agent that inhibits transcription of the YME1L1 gene, promotes degradation of transcribed mRNA, inhibits protein synthesis from mRNA, or promotes degradation of synthesized protein. Suppressing the transcription of the YME1L1 gene includes interfering with binding of RNA polymerase, transcriptional regulators, etc. to promoters or regulatory regions on DNA, and inhibiting the process of protein synthesis refers to binding of ribosomes to mRNA. including interfering. The agent promoting the degradation of the protein includes inducing ubiquitination of the YME1L1 protein.

The YME1L1 gene expression inhibitor may be an oligonucleotide complementary to DNA or mRNA of the YME1L1 gene. The oligonucleotide may be any that inhibits gene expression by complementary binding to a part or all of the DNA or mRNA sequence of the YME1L1 gene.

The oligonucleotide may be an antisense nucleotide complementary to DNA or mRNA of the YME1L1 gene, short interfering RNA (siRNA), micro RNA, short hairpin RNA, or an aptamer. , any agent that inhibits the expression of genes known to those skilled in the art, such as inhibiting transcription by binding to a promoter of DNA, degrading transcribed mRNA, or interfering with ribosome binding to mRNA is possible.

The antisense nucleotide has a nucleotide sequence complementary to the DNA, immature-mRNA or mature mRNA of the YME1L1 gene as defined in Watson-Crick base pairing. It may be a nucleotide that interferes with the flow of genetic information to the protein.

The small interfering RNA may be an independent sense RNA homologous to a partial nucleotide target sequence in the mRNA of the YME1L1 gene, and an antisense RNA complementary to the sense RNA to form a double-stranded RNA molecule. The double-stranded region may include an unpaired portion due to mismatch (the corresponding bases are not complementary), bulge (there is no base corresponding to one chain), and the like. The overall length may be 10 to 80 bases, such as 15 to 60 bases or 20 to 40 bases. It is possible that the sense RNA and/or antisense RNA strand contains at least one chemical modification in its sugar moiety, base moiety or internucleotide structure. Such modifications may result in inhibition of destruction of small interfering RNAs by nucleases in vivo. In addition, the small interfering RNA is 'isolated', which means that it is not in its natural state, but can be obtained by any means related to human intervention, in particular the required nucleotides by chemical synthesis, PCR It can be obtained by purifying an already existing small interfering RNA by amplification of the sequence or by recombinant synthesis.

The micro RNA is a single-stranded RNA molecule with a length of 17 to 27 bases, RNA that does not encode a gene, and inhibits protein translation from mRNA by complementarily acting/binding to a specific mRNA or degrading the mRNA to grow, It plays an important role in various regulatory processes in vivo, such as differentiation, proliferation, and death. MicroRNAs are made from long (about 70-80 bases) hairpin-like precursors called pre-miRNAs by Dicer, an RNAse III enzyme. The structure in which the microRNA is formed together with the ribonucleoprotein complex is called miRNP, and the microRNA mediates the down-regulated expression of the target gene by recognizing the target site by antisense complementarity. When the complementarity between the microRNA and the target site on the mRNA is high, for example, 100% or close to that, the target mRNA is cleaved, but if the complementarity between the microRNA and the target site is low, translation of the target gene (expression into protein) can be suppressed. Thousands of microRNAs have been identified so far, and related information can be checked through the miRBase Sequence Database operated and managed by the University of Manchester.

The short hairpin RNA is a single RNA strand of a hairpin structure forming a stem-loop structure in which an independent sense RNA homologous to a partial nucleotide target sequence in the YME1L1 gene mRNA and an antisense RNA complementary to the sense RNA are connected by a loop. can The stem region may include a non-pairing portion due to mismatch (the corresponding bases are not complementary), bulge (there is no base corresponding to one chain), and the like. The overall length may be 10 to 80 bases, such as 15 to 60 bases or 20 to 40 bases. In addition, the loop region has no particular meaning in the sequence, and it is sufficient if it has a length of 3 to 10 bases in order to connect the sense RNA sequence and the antisense RNA sequence at an appropriate interval.

The antisense nucleotides, short interfering RNA (siRNA), micro RNA (micro RNA) or short hairpin RNA (short hairpin RNA) may be included in the form surrounded by LNP (lipid nanoparticles), oligonucleotide form preparation It may be included in any form used for delivery.

The YME1L1 gene expression inhibitor may be a compound, peptide, peptide mimetics, aptamer or antibody that binds to the YME1L1 protein or binds to the YME1L1 binding protein, and the YME1L1 protein by binding to the YME1L1 protein or its binding protein. Any agent that inhibits the expression of a gene known to those skilled in the art, such as inhibiting the expression of the YME1L1 gene as a feedback mechanism by interfering with the interaction between the protein and other proteins is possible.

The YME1L1 protein activity inhibitor may be a compound, peptide, peptide mimetics, aptamer, and antibody that specifically binds to the YME1L1 protein, and prevents the YME1L1 protein from binding to a substrate, from binding to a coenzyme, or YME1L1 Any agent that inhibits the activity of a protein known to those skilled in the art, such as inducing degradation of the protein, is possible.

The peptide mimetics binding to the YME1L1 protein may be a peptide or non-peptide that inhibits the domain site of the YME1L1 protein binding to a substrate or catalyzing a reaction.

The aptamer binding to the YME1L1 protein refers to a single-stranded nucleic acid molecule having a characteristic that can form a specific binding by targeting the protein and has a stable tertiary structure, and is DNA, RNA or modified It may be a nucleic acid. An aptamer having high specificity and affinity for the protein may be synthesized using a SELEX (Systematic Evolution of Ligands by Exponential enrichment) technique or the like.

The antibody binding to the YME1L1 protein refers to an immunoglobulin molecule having reactivity by immunologically binding specifically to an epitope of the protein. All are available. In addition, the antibody may be produced using any technology capable of producing antibody molecules through continuous cell line culture, or may be produced using hybridoma technology. The antibody may include both a monoclonal antibody, a polyclonal antibody, an antibody having a full-length chain structure, an antibody of at least a functional fragment having an antigen-binding function, and a recombinant antibody, and subsequent steps such as washing or separation of the complex It can be used in combination with a solid substrate to facilitate the. The solid substrate may be a synthetic resin, nitrocellulose, a glass substrate, a metal substrate, glass fiber, microspheres or microbeads, and the synthetic resin may be polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF or nylon.

The YME1L1 gene expression inhibitor or YME1L1 protein activity inhibitor has the effect of inducing DNA damage in cancer cells to inhibit cancer cell growth and promote apoptosis, so the YME1L1 gene expression inhibitor or YME1L1 protein activity inhibitor A composition comprising as an active ingredient can be used for preventing or treating cancer.

The cancer is liver cancer, fibrosarcoma, cervical cancer, lung cancer, osteosarcoma, colorectal cancer, breast cancer, glioma, brain tumor, kidney cancer, stomach cancer, prostate cancer, uterine cancer, colon cancer, bladder cancer, pancreatic cancer, spinal cord tumor, head and neck cancer, thymoma, mesothelioma , esophageal cancer, bronchoma, biliary tract cancer, testicular cancer, germcytoma, ovarian cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma or skin cancer, but is not limited thereto, Any type of cancer may be included as long as it is a disease in which growth or death is not normally controlled and thus forms a tumor.

The pharmaceutical composition for preventing or treating cancer of the present invention may include the YME1L1 gene expression inhibitor or YME1L1 protein activity inhibitor itself as it is, and a poly encoding the YME1L1 gene expression inhibitor or YME1L1 protein activity inhibitor. It may also contain nucleotides.

The polynucleotide encoding the YME1L1 gene expression inhibitor or YME1L1 protein activity inhibitor may be inserted into a vector.

The vector may be a linear DNA, a plasmid vector, a recombinant non-viral expression vector or a recombinant viral expression vector.

When the polynucleotide encoding the YME1L1 gene expression inhibitor or the YME1L1 protein activity inhibitor is inserted into a vector, and is included in the pharmaceutical composition for the prevention or treatment of cancer of the present invention, the vector is a YME1L1 gene expression inhibitor or It may further include a polynucleotide encoding an activity inhibitor of the YME1L1 protein and a regulatory sequence for its transcription or translation. Among the above regulatory sequences, particularly important regulatory sequences are those that regulate transcription initiation, such as promoters and enhancers. In addition, it may include a regulatory sequence consisting of a start codon, a stop codon, a polyadenylation signal, Kozak, an enhancer, a signal sequence for membrane targeting and secretion, an IRES (Internal Ribosome Entry Site), and the like. A polynucleotide encoding such a regulatory sequence and an inhibitor of YME1L1 gene expression or YME1L1 protein activity may be operably linked. The 'operably linked' means that when a gene is linked downstream of a promoter sequence, it is linked in a form capable of expression of the gene.

Promoters introduced for specific purposes of recombinant viruses include virus-derived CMV promoters, RSV promoters, and SV promoters that induce continuous gene expression. A promoter containing this induced ERE, a promoter operated by the insect hormone ecdysone, a promoter induced by the antibiotic tetracycline, and the like are included. These promoters can be used by inserting them into the viral genome as appropriate for the purpose of the target genetic material.

The recombinant viral expression vector may be a recombinant baculovirus vector, a recombinant vanisha virus vector, a recombinant retroviral vector, a recombinant adenovirus vector, a recombinant adeno-associated viral vector, a recombinant herpes simplex virus vector, or a recombinant lentivirus vector. can

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a cell transduced with a polynucleotide encoding a YME1L1 gene expression inhibitor or YME1L1 protein activity inhibitor.

Vectors can be introduced into host cells by methods well known to those of ordinary skill in the art. The introduction method includes, but is not limited to, electroporation and lipofection, and a method known in the art may be selected.

The transduction may be stable or transient. One example of transient transduction involves expression of the vector in a particular cell where the vector is not integrated into the genome of the host cell. On the other hand, stable transduction may involve expression of the vector in a particular cell, in which the vector is integrated into the genome of the host cell.

The pharmaceutical composition of the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes. Said 'administration' includes a method of delivering a pharmaceutical composition or medicament into a subject's system or to a specific area in or on the subject. Administration can be administered, for example, enterally, parenterally, intravenously, intramuscularly, subcutaneously, intradermally, intranasally, orally, transdermally, intrauterine dura mater, intracerebroventricular or mucosal. have. It may also be administered locally or systemically.

The pharmaceutical composition of the present invention may be administered together with one or more active ingredients exhibiting the same or similar function. For administration, according to a method that can be easily performed by a person of ordinary skill in the art to which the present invention pertains, one or more pharmaceutically acceptable carriers may be additionally included. The meaning of 'pharmaceutically acceptable' means that it does not inhibit the activity of the active ingredient and does not have more toxicity than the application (prescription) target can adapt to. The 'carrier' is defined as a compound that facilitates the addition of the compound into a cell or tissue.

The pharmaceutical composition of the present invention may be prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and/or excipient, or may be prepared by being introduced into a multi-dose container, and may additionally include a dispersing agent or a stabilizer. .

In addition, the active ingredient included in the pharmaceutical composition may be delivered in a pharmaceutically acceptable carrier such as a colloidal suspension, powder, saline, lipid, liposome, microspheres, or nano-spherical particles. They may form complexes with, or be associated with, a vehicle and are known in the art such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids. It can be delivered in vivo using known delivery systems.

In addition, pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, which are commonly used in the formulation. polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition, it may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components. , and other conventional additives such as a bacteriostatic agent may be added.

The pharmaceutical composition according to the present invention may be administered in various oral and parenteral dosage forms during actual clinical administration. When formulated, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, etc. is prepared using Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, for example, starch, calcium carbonate, sucrose or It is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. The powder may be prepared by simply mixing the active ingredient of the present invention with a suitable pharmaceutically acceptable carrier, such as lactose, starch, microcrystalline cellulose. The granules are prepared by mixing the active ingredient of the present invention, a suitable pharmaceutically acceptable carrier, and a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone and hydroxypropylcellulose, and then adding a solvent such as water, ethanol, isopropanol, etc. It may be manufactured using a wet granulation method using a wet granulation method or a dry granulation method using a compressive force. In addition, tablets may be prepared by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate, and then tableting the granules using a tablet press. Liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerol, gelatin, etc. may be used.

The active ingredient of the present invention is an oral agent, injection (eg, intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection, implant), inhalant, nasal administration depending on the disease to be treated and the condition of the individual It may be administered as a preparation, vaginal preparation, rectal administration preparation, sublingual preparation, transdermal preparation, topical preparation, etc., but is not limited thereto. Depending on the route of administration, it may be formulated into a suitable dosage unit formulation comprising a commonly used, non-toxic, pharmaceutically acceptable carrier, excipient, and vehicle.

Polynucleotides encoding YME1L1 gene expression inhibitors or YME1L1 protein activity inhibitors, YME1L1 gene expression inhibitors or YME1L1 protein activity inhibitors and polynucleotides encoding YME1L1 gene expression inhibitors or YME1L1 protein activity inhibitors, which are active ingredients upon administration The amount of any one component selected from the group consisting of nucleotide-transduced cells may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, target site and severity of disease, etc. Varies.

The active ingredient in the pharmaceutical composition of the present invention may be included in a concentration of 30 μM or more, specifically 35 μM or more, more specifically 40 μM or more.

When a vector containing a polynucleotide encoding a YME1L1 gene expression inhibitor or YME1L1 protein activity inhibitor is included as an active ingredient in the pharmaceutical composition of the present invention, it is preferably contained in an amount of 0.05 to 500 mg, and 0.1 to 300 mg It is more preferable to contain, and in the case of a recombinant virus comprising a polynucleotide encoding a YME1L1 ^{gene expression inhibitor or YME1L1 protein activity inhibitor, 10 3} to 10 ¹² IU (10 to 10 ¹⁰ PFU) is preferably contained and , 10 ⁵ to 10 ¹⁰ IU is more preferably contained.

In addition, the effective dose of a composition containing a vector comprising a polynucleotide encoding a YME1L1 gene expression inhibitor or YME1L1 protein activity inhibitor of the present invention as an active ingredient is 0.05 to 12.5 mg/kg of the vector per 1 kg of body weight. , 10 ⁷ to 10 ¹¹ viral particles (10 ⁵ to 10 ⁹ IU)/kg in the case of a recombinant virus, preferably 0.1 to 10 mg/kg in the case of a vector, and 10 ⁸ to 10 ¹⁰ particles in the case of a recombinant virus (10 ⁶ to 10 ⁸ IU)/kg, and may be administered 2 to 3 times a day. The composition as described above is not necessarily limited thereto, and may vary depending on the condition of the patient and the degree of onset of the disease.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, a 'pharmaceutically effective amount' means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, activity of the drug, Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs, and other factors well known in the medical field may be determined. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent, or may be administered in combination with a therapeutic agent for a disease caused by other contaminants or a therapeutic agent for improving skin aging, and is administered simultaneously, separately, or sequentially with a conventional therapeutic agent may be administered, single or multiple administration. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient into the body, inactivation rate, excretion rate, disease type, and drugs used in combination, the route of administration, It can increase or decrease according to the severity of obesity, sex, weight, age, etc., and can vary according to the severity of the condition being treated. If necessary, for convenience, the total daily dose may be divided and administered several times during the day. For example, the daily dose may be about 0.0001 mg/kg to about 10 g/kg daily, and preferably, about 0.001 mg/kg to about 1 g/kg may be administered once a day. In addition, the administration period may be 1 day to 2 months, but may be administered without limitation until the prevention or treatment effect of the disease appears. In addition, according to the judgment of the doctor or pharmacist, it may be administered several times a day at regular time intervals, for example, divided into 2 to 3 times a day.

2. Methods of diagnosis of cancer

Another aspect of the present invention provides a method for diagnosing cancer, specifically, a method for providing information necessary for diagnosing cancer.

The method of providing information necessary for diagnosis of cancer of the present invention includes measuring the expression level of the YME1L1 gene or the activity level of the YME1L1 protein in a sample isolated from a subject.

The sample isolated from the subject may be cells, tissues, blood, serum, saliva, or urine.

Measuring the expression level of the gene YME1L1 may be a step of measuring, using a formulation that can be amplified, or the amount to be recognized by specific binding to an mRNA of the gene YME1L1.

As a specific example, the mRNA or mRNA may be measured using a primer pair or probe that specifically binds to the nucleotide sequence of cDNA made by reverse transcription, polymerase chain reaction (PCR), RT-PCR (Reverse transcription-PCR) ), Competitive RT-PCR, Real-time RT-PCR, Northern blot, Microarray, RNase protection assay, sequencing The measurement may be performed by a method such as sequencing, but the present invention is not limited thereto, and any method known to those skilled in the art may be used.

Further, the step of measuring the expression level of the gene YME1L1 may be measuring using an agent that can recognize and specifically bind to the protein expressed from the YME1L1 YME1L1 gene.

As a specific example, it may be measured using an antibody or aptamer that specifically binds to the protein, Western blotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining method (immunohistochemical staining, IHC), immunoprecipitation, and immunofluorescence may be measured by methods such as, but not limited to, any method known to those skilled in the art.

The step of measuring the activity level of the YME1L1 protein can be measured by methods such as SDS-PAGE, immunofluorescence, enzyme immunoassay (ELISA), mass spectrometry and protein chip, and if it is a method capable of measuring the activity level of the protein It can be applied without limitation to the present invention.

Meanwhile, the method of providing information required for diagnosis of the cancer of this invention, the activity level of the expression level or YME1L1 protein measured YME1L1 gene measured in the step of expression of YME1L1 gene of normal individuals derived from the sample amount or YME1L1 protein comparing with the activity level of And when the comparison result of the comparing step shows that the expression level of the YME1L1 gene or the activity level of the YME1L1 protein in the sample derived from the subject is higher than that of the sample derived from a normal subject, determining the subject as having a high risk of cancer; further comprising; can do.

When the expression level of the YME1L1 gene in the sample isolated from the subject is compared with the expression level of the YME1L1 gene of a normal individual, the amount of mRNA or protein expressed from the YME1L1 gene will be higher than the amount of mRNA or protein of a normal individual. In this case, it is possible to provide the subject with information that determines that cancer is highly likely to develop in the future or that cancer is currently in progress. In addition, even when the activity level of the YME1L1 protein present in the subject-derived sample is shown to show higher activity than that of the YME1L1 protein of a normal subject, the possibility of future cancer or current cancer is high. It is possible to provide the subject with information that is determined to be true.

3. Screening method for cancer therapeutic candidates

Another aspect of the present invention provides a method for screening a candidate for a therapeutic agent for cancer.

The screening method for the cancer drug candidate includes the step of treating the test substance to a YME1L1 expressing cell line.

On the other hand, the screening method for a cancer therapeutic agent of the present invention comprises the steps of: measuring the expression level of the YME1L1 gene or the activity level of the YME1L1 protein in the cell line treated with the test substance in the step of treating the test substance; and selecting a test substance in which the expression level of the YME1L1 gene or the activity level of the YME1L1 protein in the measuring step is reduced compared to a control group not treated with the test substance; may further include.

The test substance may be a natural compound, a synthetic compound, RNA, DNA, a polypeptide, an enzyme, a protein, a ligand, an antibody, an antigen, a bacterial metabolite, a fungal metabolite, or a bioactive molecule.

How to a method of measuring the expression level of the YME1L1 gene and determine the activity level of YME1L1 protein activity of the method and YME1L1 protein to measure the expression level of the YME1L1 gene described in the diagnosis of cancer through the expression level measured in the YME1L1 gene It is the same as the method of measuring the level.

When the YME1L1 expression cell line was treated with the test substance, the amount of mRNA or protein expressed from the YME1L1 gene was further reduced compared to the control not treated with the test substance, or the activity level of the YME1L1 protein was lower. , the treated test substance may be a candidate substance for a therapeutic agent for cancer that inhibits the YME1L1 protein from advancing or exacerbating cancer.

Hereinafter, the present invention will be described in detail by way of Examples.

However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[Example 1] Confirmation of expression level of YME1L1 in various types of cancer cell lines

cell culture

In order to confirm the relationship between the expression of YME1L1 and cancer, various types of cancer cell lines and normal cell lines were cultured and used in the experiment.

Normal cell lines include BM-MSC, Adipo-MSC, Retina, WI38, BJ, liver cancer cell lines Huh7, HepG, Hep3B, SNU387, SNU398, SNU449, brain tumor-related cell lines LN428, U87, lung cancer cell lines Calu1, A549, H460, H1299, SAOS2 as osteosarcoma cell lines, HT29 and HCT116 as colon cancer cell lines, MDAMB231 and MCF7 as breast cancer cell lines, and HT1080 as fibrosarcoma cell lines were used.

First, Huh7, SNU387, SNU398, SNU449, A549, H460, HCT116, H1299 and MDAMB231 cells were cultured in RPMI medium containing 10% fetal bovine serum (FBS). Hep3B, HepG2, WI38, BJ, MCF7, U87, HeLa and HT1080 cells in MEM medium with 10% FBS, LN428 cells in DMEM medium with 10% FBS Calu1, SAOS2 and HT29 cells in 10% FBS It was cultured in McCoy'5A medium. BM-MSC (Human Mesenchymal Stem Cells) purchased from Lonza is MS-CGM Human Mesenchymal Stem Cell Growth BulletKit?? Cultured in a medium, Adipo-MSC (adipose tissue-drived MSC) cells were cultured in a dedicated medium purchased from Cell Bio. Retina cells were cultured in DMEM/F12 medium containing 10%.

Isolation of RNA and Real-Time RT-RCR

In order to confirm the expression level of the YME1L1 gene from each cell line cultured as described above, RNA was isolated, cDNA was synthesized, and the amount was confirmed by Real-Time RT-PCR. RNA was extracted using Qiagen RNeasy Mini Kit, and the extracted RNA concentration was measured using an ND-1000 spectrophotometer (spectrophotometer, NanoDrop Technologies, Inc. Wilmington, DE, USA). To synthesize cDNA (complementary DNA), 0.5 μg of RNA was used in iScript?? cDNA Synthesis Kit (Biorad) was used, and Real-Time PCR was performed with Hipi Real-time PCR 2X Master Mix (LPS Bio) using SYBR Green. Primers used for Real-Time RT-PCR were used as having the sequence shown in Table 1 below.

SEQ ID NO: sequence name base sequence One YME1L1 forward 5'-TATAGCGCCATCATTCGTGA -3' 2 YME1L1 reverse 5'-TTCGCCTTAGGGAATCATTG -3'

Confirmation of expression level of YME1L1

Among the cell lines cultured as described above, 5 types of normal cell lines (WI38 fibroblast, BJ fibroblast, retinal epithelial cells, BM-MSC (bone marrow-derived stem cells), Adipo-MSC (adipocyte-derived stem cells)) RNA was isolated in the same way and the expression level of YME1L1 was measured through Real-Time RT-PCR. Based on the average value of the YME1L1 expression level measured from the normal cell line, the YME1L1 expression level of each cancer cell line measured by the same method was compared.

As a result, compared with normal cell lines, liver cancer cell lines such as Huh7, HepG2, Hep3B and SNU398, lung cancer cell lines such as Calu1, A549, H460 and H1299, osteosarcoma cell lines such as SAOS2, colon cancer cell lines such as HCT116 and HT29, MDAMB231 and MCF7 It was confirmed that the mRNA of YME1L1 was significantly increased in cancer cell lines such as breast cancer cell lines such as HT1299 and fibrosarcoma cell lines such as HT1299 ( FIG. 1 ). By confirming that a large amount of YME1L1 was expressed in various types of cancer cell lines, it was confirmed that the expression of YME1L1 is related to cancer growth and progression.

[Example 2] Confirmation of the effect of inhibiting the growth of cancer cell lines through inhibition of YME1L1 expression

Confirmation of effect on liver cancer, fibrosarcoma and cervical cancer cell lines

As confirmed in Example 1, since the expression of YME1L1 is related to the growth of cancer, in order to determine whether the growth of cancer cells is inhibited when the expression of YME1L1 is suppressed, the expression of YME1L1 was suppressed using siRNA. . Three different YME1L1-siRNAs (si-YME1L1#1, #2, #3, respectively) were used for inhibition of YME1L1 expression. The si-YME1L1#1, si-YME1L1#2, and si-YME1L1#3 were produced and used by Mbiotech (IDT), and the control-siRNA (si-control) was purchased from Mbiotech and used.

Huh7 liver cancer cell line, HT1080 fibrosarcoma cell line and HeLa cervical cancer cell line were each seeded in 6 well plates, and the next day, the si-YME1L1#1, #2, #3 and control-siRNA were added to RNA iMAX reaction solution (Invitrogen). Each cancer cell line was transfected using After 7 to 10 days, colonies formed by cancer cells were stained with crystal violet to measure the growth level of each cancer cell line. It was checked whether it was properly suppressed by The primers for YME1L1 were used as having the sequence of Table 1, and the primers for β-actin used as controls for the YME1L1-siRNA #1, #2, #3 and Real-Time PCR were the sequences of Table 2 below. was used as having

SEQ ID NO: sequence name base sequence 3 si-YME1L1#1 5'-GCUGCUAUUCGGCAUUUAU -3' 4 si-YME1L1#2 5'-GGUCAUGCCAUUAUUGCAU -3' 5 si-YME1L1#3 5'-GGAGGAAGCUAAACAAGAA -3' 6 control-siRNA 5'-CCUCGUGCCGUUCCAUCAGGUAGUU -3' 7 β-actin forward 5'-GCACCACACCTTCTACAATGA -3' 8 β-actin reverse 5'-TAGCCAGCCTGGATAGCAAC -3'

As a result of real-time PCR, it was confirmed that the expression of YME1L1 mRNA was reduced by reducing the amount of YME1L1 mRNA when treated with YME1L1-siRNA. Compared with , it was confirmed that colony formation was significantly inhibited in all of the Huh7 liver cancer cell line, the HT1080 fibrosarcoma cell line and the HeLa cervical cancer cell line. The result of counting the number of colonies was also measured to decrease when the expression of the YME1L1 gene was suppressed (FIG. 2). Therefore, it was confirmed that by decreasing the expression level of YME1L1, it was possible to induce the effect of inhibiting the growth of the liver cancer cell line.

Confirmation of the effect of using other types of siRNA that inhibits YME1L1 expression - Confirmation of the cancer cell growth inhibitory effect of a total of 7 types of siRNA

In order to confirm that the experimental results using the YME1L1-siRNA #1, #2, and #3 are not the effects caused only by the siRNA recognizing the sequence of some specific region among the mRNA sequence of YME1L1, 4 different types of siRNAs were used. An additional confirmation experiment was performed using

Using YME1L1-siRNA #4, #5, #6, #7 and control-siRNA, which are siRNAs that inhibit the expression of YME1L1 as shown in Table 3 below, Huh7 liver cancer cell line and HeLa cervical cancer cell line After transfection in the same manner as in the experiment, the amount of mRNA of YME1L1 was measured, and the number of colonies was confirmed. The si-YME1L1#4, si-YME1L1#5, si-YME1L1#6 and si-YME1L1#7 were manufactured and used by Mbiotech (IDT), and the control-siRNA (si-control) was purchased from Mbiotech. was used.

SEQ ID NO: sequence name base sequence 9 si-YME1L1#4 5'-GGAACCGACCAUAUUACAA -3' 10 si-YME1L1#5 5'- GCGGAUGGUUACCAAAUUU -3' 11 si-YME1L1#6 5'-CCUUCUAAGGGACUCAUAU -3' 12 si-YME1L1#7 5'-GGAUCAGAUGUUGAGAGUU -3'

As a result, like the results of the experiment in which YME1L1-siRNA #1, #2, and #3 were transfected, even when YME1L1-siRNA #4, #5, #6, #7 was used, the Huh7 liver cancer cell line and HeLa cervical cancer It was determined that the number of colony formations in the cell line decreased ( FIG. 3 ).

Therefore, the effect of inhibiting the growth of cancer cells is not caused only by some siRNA binding to a specific sequence, but inhibiting the growth of cancer cells regardless of the type of siRNA when YME1L1 gene expression is inhibited, thereby preventing or treating cancer. could be confirmed to occur.

Confirmation of growth inhibitory effect on various types of cancer cell lines

Even when targeting cancer cells other than liver cancer cell lines and cervical cancer cell lines, HT1080 fibrosarcoma cell line, SNU475 liver cancer cell line, MDAMB231 breast cancer cell line, LN428 glioma cell line , the experiment was performed on the SW480 colorectal cancer cell line and the H460 lung cancer cell line.

In order to suppress the expression of YME1L1, YME1L1-siRNA #3 and #7 among the siRNAs were transfected with RNA iMAX reaction solution (Invitrogen), and after 24 hours, 1,000 cells were seeded, and again after 7 to 10 days, crystal violet reagent. The number of colonies was observed by staining with . This time, in order to confirm the amount of expressed YME1L1, the protein was isolated from each cell line and Western blotting was performed.

Each harvested cell line was treated with lysis buffer (lysis buffer, 120 mM NaCl, 40 mM Tris-HCl, pH 8.0, 0.5% (w/v) NP40, 1 mM PMSF, protease inhibitor cocktail (100X, GenDEPOT)) for 30 minutes 4 After lysing the cells at °C, centrifugation was performed at 12,000 rpm for 20 minutes. Only the supernatant was separated, quantified with Bradford (BIORAD), electrophoresed on 12% SDS-PAGE gel, transferred to NC membrane (Whatman) at 78V, 2 hours, and then anti-YME1L1 antibody (Protentech) and anti-β - The amount of protein was measured using an actin antibody (Santacruz).

As a result of Western blotting, it was measured that the amount of YME1L1 protein was decreased in all cell lines treated with siRNA for YME1L1. It was confirmed that there is a growth inhibitory effect (FIG. 4). Therefore, it was confirmed that the inhibition of YME1L1 was effective in various types of carcinomas in addition to liver and cervical cancer.

By combining the experimental results of the above examples, it can be confirmed that by inhibiting the expression of YME1L1 (reduction of the mRNA or protein of YME1L1), the growth of cancer cells is inhibited and the effect of preventing or treating cancer can be achieved. there was.

[Example 3] Confirmation of apoptosis-inducing effect of cancer cell lines through inhibition of YME1L1 expression

As confirmed in the experimental results of the above examples, in order to determine whether the growth inhibitory effect of cancer cells according to the inhibition of YME1L1 expression is related to apoptosis, the same HeLa cervical cancer cell line as used in the above example was targeted. Annexin-V staining, an apoptosis marker, was performed.

HeLa cells were seeded in a 60 mm dish, and si-YME1L1#3, #7 and control-siRNA were treated the next day. After 96 hours, cells were collected and stained with Annexin V-FITC for 15 minutes, followed by FACS analysis using a flow cytometer (Cell QuestPro, BD-SCIENCE).

As a result, in the case of cancer cell lines treated with si-YME1L1#3, #7, compared to the case of treatment with control siRNA, it was measured that the staining intensity of Annexin-V was increased on the FACS histogram (FIG. 5). A), the ratio of the number of cells stained with Annexin-V was increased (FIG. 5B).

Through the above results, it can be seen that the growth inhibitory effect of cancer cells that occurs when the expression of YME1L1 is suppressed is due to apoptosis of cancer cells. or could be treated.

[Example 4] Confirmation of the relationship between apoptosis and DNA damage caused by a decrease in YME1L1 expression

To determine whether the effect of inhibiting the expression of YME1L1 to induce cancer cell growth and apoptosis is related to DNA damage in cancer cells, foci stained with γ-H2AX used as a marker of DNA damage was measured. After seeding the same HeLa cell line as used in the above example in a 6-well plate with a cover glass, si-YME1L1#1, si-YME1L1#3 and control-siRNA were treated, respectively. After 72 hours, the cover glass was removed and fixed, and then stained using an anti-γ-H2AX antibody (Cell Siganaling Technology), and observed through a fluorescence microscope.

As a result, it was confirmed that γ-H2AX foci were significantly increased in cells treated with two different siRNAs, si-YME1L1#1 and si-YME1L1#3, compared to cells treated with control-siRNA. (Fig. 6). Therefore, through the above experimental results that the foci of γ-H2AX, a marker of DNA damage, increase, when YME1L1 expression is suppressed, cancer cell death is induced and growth is inhibited as DNA damage increases. was able to confirm

In the above, the present invention has been described in detail only with respect to the described embodiments, but it is obvious to those skilled in the art that various changes and modifications are possible within the scope of the technical spirit of the present invention, and it is natural that such variations and modifications belong to the appended claims.

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Claims (12)

YME1L1 containing oligonucleotides (oligonucleotides) that complementarily bind to DNA or mRNA of the gene as an active ingredient,
As a pharmaceutical composition for the prevention or treatment of cancer,
The cancer is at least one selected from the group consisting of liver cancer, fibrosarcoma, cervical cancer, glioma and colorectal cancer, a pharmaceutical composition for preventing or treating cancer. delete The method according to claim 1,
The oligonucleotide is selected from the group consisting of antisense nucleotides complementary to DNA or mRNA of the YME1L1 gene, short interfering RNA, micro RNA, short hairpin RNA, and an aptamer. A pharmaceutical composition for the prevention or treatment of cancer, which is any one. delete delete delete delete delete delete delete delete delete

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