KR20200113556A - Composition for preventing or treating diabetes and method for screening antidiabetic agents using ST8SIA1 - Google Patents

Abstract

The present invention relates to a composition for preventing or treating diabetes using ST8SIA1 and a method for screening an antidiabetic agent. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating diabetes containing an inhibitor of the expression or activity of a ST8SIA1 protein or insulin-secreting cells in which the ST8SIA1 gene is deleted, and a method for screening an antidiabetic agent. The present invention can be usefully used to develop the antidiabetic agent with a completely novel mechanism of action which inhibits the expression or activity of the ST8SIA1 protein through the fact that the amount of insulin secretion is increased due to the inhibition of ST8SIA1. In addition, in the case of administration, transplantation, or subcutaneous administration of insulin-secreting cells from which the ST8SIA1 gene are knocked-out to an individual, insulin secretion is increased, resulting in a decrease in blood sugar in the individual and excellent ability to regulate blood sugar level, thereby being able to be usefully used as the antidiabetic agent.

Description

Composition for preventing or treating diabetes and method for screening antidiabetic agents using ST8SIA1}

The present invention relates to a composition for preventing or treating diabetes using ST8SIA1 and a method for screening a diabetes therapeutic agent, and more particularly, to a diabetes mellitus comprising as an active ingredient an inhibitor of the expression or activity of the ST8SIA1 protein or insulin-secreting cells in which the ST8SIA1 gene is deleted. Contacting the prophylactic or therapeutic pharmaceutical composition and (a) a test substance to cells expressing the ST8SIA1 gene; (b) measuring the ST8SIA1 gene expression level in the control cells not contacting the cells and the test substance; And (c) selecting a test substance that decreases the ST8SIA1 gene expression level compared to the control cells as a candidate substance for the treatment of diabetes.

Glucose toxicity in diabetes causes apoptosis of β-cells and affects various organ systems, including the pancreas. However, the basic mechanism is not fully known. Impairment of β-cells and impaired insulin production are typical features of diabetes (Xu, G et al., Nat. Med. 19, 1141-1146. 2013), but apoptosis of β-cells is prevented despite the rapid spread of diabetes. The exact molecular mechanism of the resulting glucose toxicity is still unknown (Chen, J et al., Diabetes 57, 938-944. 2008).

Apoptosis is a process that naturally occurs in the body by activating specialized intracellular signal transduction to destroy cells. It is also a homeostatic mechanism for maintaining cell populations in tissues. Inadequate apoptosis is fundamentally common in a variety of pathologies, including metabolic stress such as ischemic injury, autoimmune diseases and many types of cancer, neurodegenerative diseases, and glucose toxicity (Elmore, S et al., Toxicol. Pathol. 35, 495-516. 2007). Such apoptosis is caused by the intrinsic pathway that activates caspase-9 and -3 by cytochrome c released from the mitochondria, and caspase-8 and -10 by death receptors such as TNF-α (tumor nerosis factor-α) and Fas ligand. This activation is divided into two pathways of the extrinsic pathway. In addition, it is known that activation of various proteins independent of caspase is involved in apoptosis signaling.

On the other hand, the ST8SIA1 gene is a gene that encodes an enzyme called alpha-N-acetyl neuraminide alpha-2,8-sialyl transferase, and the enzyme transfers sialic acid from CMP-sialic acid to GM3 to ganglioside ( Ganglioside) is a type II membrane protein that produces GD3 and GT3. Ganglioside GD3 is known to be important for cell adhesion and growth of cultured malignant cells. The ST8SIA1 protein can often be found in the Golgi apparatus and is a member of the glycosyltransferase family.

However, until now, it has not been reported that ST8SIA1 can act as a target of diabetes treatment as in the present invention.

Accordingly, the inventors of the present invention have studied to develop a new diabetes treatment, and as a result, ST8SIA1 has a function of inhibiting apoptosis of pancreatic beta cells, particularly in a high glucose state caused by diabetes, and inhibiting this. The present invention was completed by confirming that the amount of insulin secreted in the body was increased by the insulin secreting cells and blood sugar was normally regulated.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes, comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.

Another object of the present invention

(a) contacting the test substance with cells expressing the ST8SIA1 gene;

(b) measuring the ST8SIA1 gene expression level in the control cells not contacting the cells and the test substance; And

(c) To provide a diabetes treatment screening method comprising the step of selecting a test substance that decreases the ST8SIA1 gene expression level compared to the control cells as a candidate substance for the treatment of diabetes.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes, comprising as an active ingredient insulin secreting cells in which the ST8SIA1 gene is deleted.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.

In order to achieve another object of the present invention, the present invention

(a) contacting the test substance with cells expressing the ST8SIA1 gene;

(b) measuring the ST8SIA1 gene expression level in the control cells not contacting the cells and the test substance; And

(c) To provide a diabetes treatment screening method comprising the step of selecting a test substance that decreases the ST8SIA1 gene expression level compared to the control cells as a candidate substance for the treatment of diabetes.

In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising as an active ingredient insulin secreting cells in which the ST8SIA1 gene is deleted.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.

In the present specification,'treatment' means suppressing the occurrence or recurrence of a disease, alleviating symptoms, reducing direct or indirect pathological consequences of a disease, reducing the rate of disease progression, improving, improving, alleviating, or improving prognosis. do. The term “prevention” used in the present invention means any action that suppresses the onset or delays the progression of a disease.

'Protein' is used interchangeably with'polypeptide' or'peptide', for example, refers to a polymer of amino acid residues as commonly found in proteins in nature. The "fragment" of the FGF12 protein refers to a peptide of a portion of the FGF12 protein.

In the present invention, “polynucleotide” or “nucleic acid” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in the form of single or double strands. Unless otherwise limited, also include known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to those of naturally occurring nucleotides. In general, DNA is composed of four bases: adenine (A), guanine (G), cytosine (C), and thymine (T), and RNA is composed of uracil (U) instead of thymine. Have. In the double-stranded nucleic acid, A forms a hydrogen bond with T or U, and C forms a hydrogen bond with the G base, and the relationship between these bases is called "complementary".

On the other hand,'mRNA (messenger RNA or messenger RNA)' is an RNA that serves as a blueprint for polypeptide synthesis (protein translation, translation) by transferring the genetic information of the nucleotide sequence of a specific gene to the ribosome during protein synthesis. Using the gene as a template, single-stranded mRNA is synthesized through a transcription process.

In the present invention,'diabetes', which is an object of treatment or prevention, is a metabolic disorder syndrome characterized by hyperglycemia caused by a lack of insulin hormone or an abnormality in insulin resistance produced by beta cells of the pancreas and further defects of both. These diabetes can be divided into insulin-dependent diabetes (IDDM, Type 1) and insulin-independent diabetes (NIDDM, Type 2) caused by impaired insulin resistance and insulin secretion. In both type 1 and type 2 diabetes, various complications such as heart disease, bowel disease, eye disease, neurological disease, stroke, etc. occur, which result in chronic neurological disease and cardiovascular disease due to elevated blood sugar and insulin levels for a long time. The short-term hypoglycemic and hyperglycemic reactions cause acute complications. Diabetes, as hyperglycemia persists chronically, causes disorders in lipid and protein metabolism as well as sugar metabolism. The conditions are diverse and are directly caused by hyperglycemia, and include diabetic peripheral nerve disorders, diabetic retinopathy, diabetic nephropathy, diabetic cataract, cornea, diabetic arteriosclerosis, etc. in the retina, kidney, nerve, and cardiovascular system.

One of the important pathologies that cause and deepen diabetes is the death of pancreatic β-cells due to hyperglycemia. In the present inventors, in the glucose toxicity (glucose toxicity or glucotoxicity) that affects various organs including the pancreas in a state of elevated blood sugar, ST8SIA1 modulates the p38MAPK/ERK signaling system in human pancreatic β cells and prevents apoptosis by inhibiting ST8SIA1. It has been identified that it plays a role. Therefore, it can be understood that by inhibiting the expression or activity of the ST8SIA1 protein, it is possible to inhibit the occurrence or progression of diabetes by preventing the death of the pancreas, particularly β cells, due to glucose toxicity.

Accordingly, the diabetes in the present invention may be a target of diabetes caused by glucose toxicity or deep or advanced diabetes, and more specifically, selected from the group consisting of type 1 diabetes, type 2 diabetes, and gestational diabetes. Can be.

The term "ST8SIA1" used in the present invention refers to a gene or a protein thereof encoding alpha-N-acetylneuraminide alpha-2,8-sialyltransferase. It is a type II membrane protein that moves sialic acid from CMP-sialic acid to GM3 to produce ganglioside GD3 and GT3. Ganglioside GD3 is known to be important for cell adhesion and growth of cultured malignant cells.

The ST8SIA1 protein (alpha-N-acetylneuraminide alpha-2,8-sialyltransferase) in the present invention may be derived from mammals, preferably derived from humans. Most preferably, the ST8SIA1 protein in the present invention comprises the amino acid sequence of human ST8SIA1 isoform 1 (NP_003025.1) represented by SEQ ID NO: 1 or human ST8SIA1 isoform 2 (NP_001291379.1) represented by SEQ ID NO: 2 Characterized in that (NCBI Genbank accession number in parentheses).

The ST8SIA1 protein expression inhibitor may be an antisense oligonucleotide, siRNA, shRNA, miRNA, ribozyme, crispr-cas9, DNAzyme, or protein nucleic acid (PNA) that complementarily binds to ST8SIA1 mRNA. In addition, the ST8SIA1 mRNA most preferably contains a nucleotide sequence of human ST8SIA1 mRNA transcript variant 1 (NM_003034.3) represented by SEQ ID NO: 3 or human ST8SIA1 mRNA transcript variant 2 (NM_001304450.1) represented by SEQ ID NO: 4 (NCBI Genbank accession number in parentheses). Features such as the coding region (exon) of the above-described human ST8SIA1 mRNA transcript variants are identified in the sequence information displayed by searching the NCBI database with the Genbank accession number in parentheses.

The'siRNA (small interfering RNA or short interfering RNA or silencing RNA)' is artificially introduced into the cell to induce the degradation of the mRNA of a specific gene, thereby preventing protein translation, thereby inhibiting gene expression. Interference) is a short double-stranded RNA that is composed of 20 to 25 nucleotides complementary to a specific site of the target mRNA. Among the double strands of siRNA in cells, an antisense strand complementary to the target mRNA binds to the RNA-induced silencing complex (RISC) protein complex and binds to the target mRNA, and the argonaute protein in the RISC complex cleaves and degrades the target mRNA. It suppresses the expression of specific genes through mechanisms such as suppressing the binding of proteins and ribosomes important for protein translation with mRNA.

The shRNA according to the present invention is a substance that induces RNA interference, and has a double-stranded structure within a molecule and becomes a hairpin-like structure by partially containing a palindromic nucleotide sequence in a single-stranded RNA. It is more than a base molecule. In addition, when a shRNA expression plasmid is introduced into a cell and expressed, a 21-23 base pair siRNA is generated by RNase III (ribonuclease III) enzyme called dicer in the cell to induce RNAi.

The siRNA or shRNA according to the present invention may be obtained by applying various modifications to improve the stability of oligonucleotides in vivo, impart resistance to nucleases, and reduce non-specific immune responses. In the modification of the oligonucleotide, the OH group at the 2′ carbon position of the sugar structure in one or more nucleotides is -CH3 (methyl), -OCH3 (methoxy), -NH2, -F, -O-2-methoxyethyl, -O- Propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -ON-methylacetamido or -O-dimethylamidooxyethyl Modification by substitution with Modification in which oxygen in the sugar structure in the nucleotide is substituted with sulfur; Alternatively, one or more modifications selected from modifications of nucleotide bonds to phosphorothioate, boranophosphate, and methyl phosphonate bonds may be used in combination, and PNA (peptide nucleic acid), Modification into the form of LNA (locked nucleic acid) or UNA (unlocked nucleic acid) can also be used.

In addition, the ST8SIA1 protein activity inhibitor may be a compound, peptide, peptide mimetics, aptamer, antibody, natural extract or synthetic compound that specifically binds to the ST8SIA1 protein.

The pharmaceutical composition according to the present invention may be variously formulated according to the route of administration by a method known in the art together with a pharmaceutically acceptable carrier for the treatment of diabetes. The carrier includes all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes. Preferably, it is formulated using a transfection reagent, and can be delivered to cells using subcutaneous, blood, bone marrow, or abdominal cavity.

The pharmaceutical composition according to the present invention may be administered to a patient in a pharmaceutically effective amount, that is, in an amount sufficient to prevent diabetes or alleviate and treat symptoms. For example, a typical daily dosage may be administered in the range of about 0.01 to 1000 mg/kg, and preferably, it may be administered in the range of about 1 to 100 mg/kg. The pharmaceutical composition of the present invention can be administered in one or several divided doses within a preferred dosage range. In addition, the dosage of the pharmaceutical composition according to the present invention may be appropriately selected by a person skilled in the art according to the route of administration, the subject of administration, age, sex, weight, individual differences, and disease conditions.

The route of administration may be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, rectal, or pancreatic It may be intravenous administration, but is not limited thereto, and most preferably it is administered directly to the pancreas.

In the case of oral administration of the pharmaceutical composition of the present invention, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to a method known in the art together with a suitable carrier for oral administration. It can be formulated in the form of such as. Examples of suitable carriers include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Fillers such as celluloses including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethylcellulose, gelatin, and polyvinylpyrrolidone may be included. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition may further include an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.

In addition, when administered parenterally, the pharmaceutical composition of the present invention can be formulated according to a method known in the art in the form of injections, transdermal administrations and nasal inhalants together with a suitable parenteral carrier.

In addition, the pharmaceutical composition can be administered by any device capable of moving the active substance to the target cell. Preferred modes of administration and formulations are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections or instillations. Injectables include aqueous solvents such as physiological saline or ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid ethyl, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, or glycerin), and other non-aqueous solvents. Stabilizers for preventing deterioration (e.g., ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, and for inhibiting the growth of microorganisms Pharmaceutical carriers such as preservatives (eg, mercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.

In the case of such injections, they must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include, but are not limited to, water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), a mixture thereof and/or a solvent or dispersion medium containing vegetable oil. I can. More preferably, suitable carriers include isotonic solutions such as Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. Etc. can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, and the like may be additionally included. In addition, the injection may further include an isotonic agent such as sugar or sodium chloride in most cases.

In the case of transdermal administration, ointments, creams, lotions, gels, external solutions, pasta, liniment, air rolls, and the like are included. In the above, "transdermal administration" means that the active ingredient in an effective amount contained in the pharmaceutical composition is delivered into the skin by topically administering the pharmaceutical composition to the skin. For example, the pharmaceutical composition of the present invention may be prepared in an injectable formulation and administered by lightly pricking the skin with a 30 gauge thin injection needle or applying it directly to the skin. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania, a prescription generally known for pharmaceutical chemistry.

In the case of inhalation administration, the compounds used according to the invention can be used in pressurized packs or with suitable propellants, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered from a nebulizer in the form of an aerosol spray. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

Other pharmaceutically acceptable carriers may be referred to as those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

Pharmaceutical compositions according to the present invention include one or more buffers (e.g., saline or PBS), carbohythrate (e.g., glucose, mannose, sucrose or dextran), antioxidants, bacteriostatic agents, chelating agents (e.g. For example, EDTA or glutathione), adjuvants (eg, aluminum hydroxide), suspending agents, thickening agents and/or preservatives may further be included.

In addition, the pharmaceutical compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

In addition, the pharmaceutical composition of the present invention may be administered alone or may be administered in combination with a known compound that protects pancreatic cells and has an effect of treating diabetes.

In addition, the present invention

(a) contacting the test substance with cells expressing the ST8SIA1 gene;

(b) measuring the ST8SIA1 gene expression level in the control cells not contacting the cells and the test substance; And

(c) It provides a diabetes treatment screening method comprising the step of selecting a test substance that reduces the ST8SIA1 gene expression level compared to the control cells as a diabetes treatment candidate substance.

The step (a) is a step of contacting a cell expressing the ST8SIA1 gene in order to confirm whether the test substance to be analyzed has an activity to inhibit the expression of the ST8SIA1 gene.

In the method of the present invention,'contacting' is in a general sense, and combining two or more agents (eg, two polypeptides), or combining an agent and cells (eg, proteins and cells). Say. Contact can occur in vitro. For example, combining two or more agents in a test tube or other container, or combining a test agent with cells or cell lysates and a test agent. Contact can also occur in cells or in situ. For example, two polypeptides are brought into contact in a cell or cell lysate by coexpressing a recombinant polynucleotide encoding two polypeptides in a cell. In addition, a protein chip or a protein array in which the protein to be tested is arranged on the surface of a stationary bed may be used.

In addition, in the method of the present invention, the'test substance' can be used interchangeably with a test agent or agent, and any substance, molecule, element, compound ( Includes a compound, entity, or combination thereof. Examples include proteins, polypeptides, small organic molecules, polysaccharides, and polynucleotides. It may also be a natural product, a synthetic compound or a chemical compound, or a combination of two or more substances.

More specifically, test agents that can be screened by the method of the present invention include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines. ), oligomeric N-substituted glycines, oligocarbamates, saccharides, fatty acids, purines, pyrimidines or derivatives thereof, structural analogues, or combinations thereof. Test formulations can be synthetic or natural. The test formulations can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. Combinatorial libraries can be produced with several types of compounds that can be synthesized in a step-by-step manner. Compounds of multiple combinatorial libraries can be prepared by the encoded synthetic libraries (ESL) method (WO 95/12608, WO93/06121, WO 94/08051, WO 95/395503 and WO 95/30642). Peptide libraries can be prepared by the phage display method (WO91/18980). Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained from commercial sources or collected in the field. Known pharmacological agents can be directed or applied to a random chemical modification, such as acylation, alkylation, esterification, amidification, to prepare structural analogues.

The test agent may be a naturally occurring protein or fragment thereof. Such test formulations can be obtained from natural sources, such as cell or tissue lysates. Libraries of polypeptide preparations can be produced, for example, by conventional methods or obtained from commercially available cDNA libraries. The test agent may be a peptide, such as a peptide having about 5 to 30 amino acids, preferably about 5 to 20, more preferably about 7 to 15 amino acids. The peptide may be a naturally occurring protein, a random peptide, or a cleavage product of a “biased” random peptide. In addition, the test agent may be a nucleic acid. The nucleic acid test agent can be a naturally occurring nucleic acid, a random nucleic acid, or a “biased” random nucleic acid. For example, cuts of prokaryotic or eukaryotic genomes can be used analogously to those described above.

Further, the test agent may be a small molecule (eg, a molecule having a molecular weight of about 1,000 Da or less). A high throughput assay may be preferably applied to a method for screening a small molecule modulating agent. Many assays are useful for this screening (Shultz, Bioorg. Med. Chem. Lett., 8:2409-2414, 1998; Weller, Mol. Drivers., 3:61-70, 1997; Fernandes, Curr. Opin. Chem. Biol., 2:597-603, 1998; and Sittampalam, Curr. Opin.Chem. Biol., 1:384-91, 1997).

In the present specification, "expression" means that a protein or nucleic acid is produced in a cell. The cell expressing ST8SIA1 may be a cell that internally expresses ST8SIA1, or may be a cell transformed with a recombinant expression vector containing a polynucleotide encoding ST8SIA1 to overexpress ST8SIA1. Preferably, the cells expressing the ST8SIA1 gene may be pancreatic beta cells or cells derived from pancreatic beta cells. The present inventors have used the IPCs cell line derived from human pancreatic beta cells as cells that internally express ST8SIA1.

The step (b) is a step of measuring the gene expression level of ST8SIA1 in cells expressing ST8SIA1 contacted with the test substance and cells expressing ST8SIA1 without contact with the test substance.

The method of measuring the expression level of the ST8SIA1 gene may be performed by measuring the mRNA or protein level of ST8SIA1.

For the measurement of mRNA expression, conventional methods for determining expression levels in the art can be used without limitation, and examples of analysis methods include reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR (competitive RT-PCR). ), real-time RT-PCR, RNase protection assay (RPA), northern blotting, DNA microarray chip, RNA sequencing ), but is not limited to these.

In addition, methods for measuring the expression level of proteins can be used without limitation, methods known in the art, such as western blotting, dot blotting, enzyme-linked immunosorbent assay. , Radioactive Immunoassay (RIA), Radioimmune Diffusion Method, Ouchteroni Immunity Diffusion Method, Rocket Immunoelectrophoresis, Immunohistochemical Staining, Immunoprecipitation, Complement Fixation Analysis, Flow Cytometry (FACS) or Protein Chip Method, etc. However, it is not limited to these.

The step (c) is a step of selecting a test substance that reduces the expression level of the ST8SIA1 gene compared to the control cells as a candidate substance for treating diabetes.

As identified by the present inventors, upon inhibition of ST8SIA1, insulin secretion is increased, and it serves to prevent cell death. Therefore, the test substance that reduces the expression level of the ST8SIA1 gene reduces the protein level and the activity level of the protein of ST8SIA1 to prevent apoptosis caused by glucose toxicity in pancreatic β cells, and thus delay or symptomatic diabetes caused by this. It can be seen that it can alleviate.

In addition, the present invention

It provides a pharmaceutical composition for preventing or treating diabetes, comprising as an active ingredient insulin secreting cells in which the ST8SIA1 gene is deleted.

The term "insulin producing cells (IPCs)" of the present invention refers to cells capable of secreting insulin, and the type is not limited as long as it has insulin secretion ability, but generally refers to pancreatic beta cells.

The method of producing the "ST8SIA1 gene-deleted insulin secreting cell" of the present invention is not limited thereto, but may include the following steps:

(a) separating exosomes from insulin-secreting cells;

(b) transfecting the exosomes isolated in step (a) with an antibody that specifically binds to ST8SIA1; And

(c) transfecting the exosomes prepared in step (c) into insulin secreting cells.

In the present invention, the exosome can serve as a carrier for transporting the protein of interest to the target cells or tissues of the target protein, including the protein of interest (antibody in the present invention) inside. The protein of interest can be used to delete a specific gene (suppress the expression or activity of a gene) by acting on a target cell or tissue.

In the case of deletion of ST8SIA1 (inhibiting the expression or activity of ST8SIA1) in insulin secreting cells using exosomes transfected with antibodies according to the above manufacturing method, phagocytosis by macrophages by internalizing the antibody in an unstable state into exosomes Action or decomposition can be avoided and can circulate for a long time in the body. In addition, the antibody-transfected exosomes have the advantage that they can potentially avoid endosome pathways and lysosome degradation, and thus can deliver antibodies directly to targets in insulin-secreting cells (Acta Pharmaceutica Sinica B Volume 6, Issue 4). , July 2016, Pages 287-296, WO2013084000A2, etc.)

In one embodiment of the present invention, the exosomes are isolated from insulin-secreting cells, the ST8SIA1 antibody is transfected into the exosomes, and then the prepared ST8SIA1 antibody-transfected exosomes are transfected into the insulin-secreting cells. Expression or activity of the protein was knocked out. Deleting the ST8SIA1 gene of insulin-secreting cells using exosomes was the first discovery by the present inventors.

In addition, in one embodiment of the present invention, when the insulin-secreting cells from which the ST8SIA1 gene is knocked-out is administered or transplanted to an individual, insulin secretion is increased, so that the blood sugar in the individual decreases and the ability to control blood glucose level is very excellent. there was.

The “insulin secreting cells” may be differentiated from stem cells, but are not limited thereto. The method of differentiating insulin-secreting cells from stem cells is not limited as long as it is known, for example, Soria et al. [Soria B, Roche E, Berna G and Leon-Quinto T (2000) Insulin-secreting cells derived from embryonic stem. cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 49, 157-162], Assady et al. [Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, and Tzukerman M. (2001) Insulin production by human embryonic stem cells. Diabetes 50, 1691-1707], Fujikawa et al. [Fujikawa T, Oh SH, Pi L, Hatch HM, Shupe T, and Petersen BE (2005) Teratoma formation leads to failure of treatment for type I diabetes using embryonic stem cell-derived insulin -producing cells. Am J Pathol 166, 1781-1791, et al.

The stem cells are undifferentiated cells having the ability to differentiate into various body tissues, which are classified into totipotent stem cells, pluripotent stem cells, and multipotent stem cells. Can be.

In the present invention, the stem cells may be adult stem cells, embryonic stem cells, middle lateral stem cells, tumor stem cells, or induced pluripotent stem cells, depending on their origin or type.

The term “mesenchymal stem cell (MSC)” as used herein has the ability to differentiate into various mesodermal cells including bone, cartilage, fat, and muscle cells, or ectodermal cells such as neurons. It is a multipotent stem cell. The mesenchymal stem cells may preferably be derived from those selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, chorionic membrane, deciduous membrane, and placenta. In addition, the mesenchymal stem cells may be derived from humans, fetuses, or mammals other than humans. The mammals other than humans are more preferably canine, feline, monkey, cow, sheep, pig, horse, rat, mouse, guinea pig, and the like, and the origin is not limited.

The active ingredient is a concept including a cell culture medium containing the cells, a concentrate of a cell culture, and the like.

The present invention can prevent apoptosis by controlling the expression of related factors upon inhibition of ST8SIA1 in hyperglycemic conditions, decrease insulin resistance and increase sensitivity through increased tyrosine phosphorylation, and increase insulin secretion due to ST8SIA1 inhibition. It can be usefully used to develop a therapeutic agent for diabetes with a completely new mechanism of action that inhibits the expression or activity of the ST8SIA1 protein. In addition, since ST8SIA1 plays a role in promoting apoptosis in pancreatic beta cells under hyperglycemic conditions, lowering the activity of ST8SIA1 inhibits apoptosis caused by hyperglycemia and has the effect of protecting pancreatic beta cells. Using this, ST8SIA1 expression Alternatively, antidiabetic candidate substances can be selected by screening for agents that inhibit activity. In addition, in the case of administering or transplanting insulin-secreting cells from which the ST8SIA1 gene has been knocked-out to an individual, or when directly administered subcutaneously, insulin secretion is increased, so that blood sugar in the individual decreases and the ability to regulate blood glucose level is very excellent.

1 is a result of investigation of the protein level of cleaved caspase 3, an apoptosis-related protein, by Western blotting when ST8SIA1 is inhibited (ST8SIA1KO-IPCs) in insulin producing cells (IPCs) differentiated from mesenchymal stem cells. β-actin was used as a protein loading control.
2 is a result of investigation of the phosphorylation result of ERK by Western blotting when ST8SIA1 is inhibited in IPCs cells. β-actin was used as a protein loading control.
Figure 3 shows the change in the amount of insulin secretion when ST8SIA1 is inhibited in IPCs cells. PBS was used as a control.
FIG. 4 shows the results of measuring the blood glucose concentration of db/db mice by dividing 9-week-old mice into 8 mice per group and transplanting ST8SIA1KO-IPCs at 1 week of breeding.
5 shows the results of measuring insulin sensitivity by dividing 9-week-old db/db mice into 8 mice per group and transplanting ST8SIA1KO-IPCs at 1 week of breeding.
Figure 6 is a 9-week-old db/db mice divided by 8 per group, and ST8SIA1KO-IPCs were transplanted at 1 week of breeding, and glucose tolerance was measured.
Figure 7 shows the results of measuring the blood glucose concentration of db/db mice by dividing 9-week-old mice into 8 mice per group, using anti-ST8SIA1-Ab as an adjuvant with 0.5% saponin and 100 ul of saponin at 1 week.

Hereinafter, the present invention will be described in detail.

However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

<Experiment method>

Cell culture

Insulin secreting cell line IPCs cells were differentiated from mesenchymal stem cells. Low glucose (5mM glucose) DMEM containing 10% FBS, 1% penicillin-streptomycin, 10uM troglitazone and 16.7 μM zinc sulfate was used, and cultured at 37°C and 5% CO ₂ . Treatment with 33mM glucose for 48 hours induces glucose toxicity. Then, 10 μM ST8SIA1 antibody (ST pharm) was treated for 48 hours.

Western blot analysis

Cells were lysed using RIPA buffer (50mMTris-HCl, 150mMNaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitor cocktail (Roche Diagnostics). The protein composition of the cell lysate was quantified with a BCA protein assay kit (Roche Diagnostics), and then the cell lysate containing the same amount of protein was separated by SDS-PAGE. Then, the protein was transferred to Immobilon NC membranes. Blocking was performed for 1 hour with TBS and Tween 20 solution (0.05% Tween 20) containing 5% skim milk or 5% bovine albumin serum (BSV). Antibodies and probes against ST8SIA1 (Abnova, Taiwan), GAPDH (Santa Cruz Biotechnology), p38MAPK (pMAPK, Santa Cruz), and phosphated ERK (pERK, cell signaling) were reacted overnight at 4°C, and peroxidase bound for 1 hour The resulting secondary antibody was reacted. The membrane was washed 3 times with TBS and Tween 20 for 5 minutes each, and the band was observed with a ChemiDoc MP system (Bio-Rad) using a chemiluminescence system (Thermo Fisher Scientific). The intensity of the band was measured using the Image J program.

ST8SIA1 knockout insulin secreting cell induction

In order to separate bone marrow derived mesenchyma stem cells (BD-MSC) from bone marrow, bone marrow cells obtained from bone marrow of female non-diabetic C57BL/6 mice were used in alpha-MEM (15% fbs) media. Incubate for 12 days. The media was replaced every 3 days, and then, 5% FBS high DMEM was cultured for 15 days to separate MSCs. In the next step, BD-MSC was incubated for 7 days in Low DMEM medium containing 5% FBS and 20 μmol/L nicotinamide, and incubated for 7 more days in Low DMEM medium containing 10 μmol/L Exclusive-4 in the last 3 steps. As a result, insulin producing cells (IPCs) were induced. In the cells, the ST8SIA1 antibody was added to the exosomes, and the exosomes were again transfected into IPCs to remove the ST8SIA1 gene to generate the ST8SIA1KO-IPCs cell line.

9-week-old db/db mice were reared by dividing 8 mice per group. At 1 week of breeding, 3×10 6 ST8SIA1KO-IPCs were transplanted into the mouse pancreas.

Exosome transfection

Isolate exosomes from 10ml of IPCs culture media using ExoQuick kt (SBI bio). The amount of exosomes in 500ul of sterile PBS is approximately 50-300ug. In a sterilized 1.5ml tube, 10ul Exo-Fect solution, 10ul PBS solution, 50ul purified exosomes, and ST8SIA1 antibody (20ul) were put together and mixed well by repeating 3 times. The mixed exosome transfection solution is incubated with a shaker at 37°C for 1 hour and immediately placed the tube on ice. Transfect 30ul of ExoQuickTC reagent into the exosome sample suspension, mix well and mix 6 times. Again, the transfection sample is stabilized on ice or at 4°C for 30 minutes. To obtain the transfected exosomes, the sample was centrifuged for 3 minutes at 13,000-14,000 rpm in a microfuge (maximum speed), the supernatant was removed, and the transfected exosome pellet was released in 300ul 1x PBS. Add the transfected exosomes (300ul) to the IPCs grown to about 1×105 in a 6-well, and incubate at 37°C incubation for 24 hours.

Example 1: Effect of the expression level of ST8SIA1 on apoptosis

The association between ST8SIA1 inhibition and apoptosis was investigated.

IPCs (ST8SIA1KO-IPCs) cells transfected with exosomes were cultured for 2 days under conditions of normal glucose (NG) or high glucose (HG: 33 mM), respectively. β-actin was used as a protein loading control. In order to confirm the effect of ST8SIA1 expression on apoptosis induced by glucose toxicity, the expression of ST8SIA1 was inhibited with the ST8SIA1 antibody under high glucose conditions and the expression level of cleaved caspase 3 was measured.

As a result, as shown in FIG. 1, when ST8SIA1 is inhibited with an antibody (anti-ST8SIA1 antibody lane), cleaved caspase 3 (c-caspase-3), which is important for apoptosis compared to a control lane that does not inhibit it, is Appeared to decrease. Through this, it was confirmed that apoptosis was increased by high glucose concentration, as known, whereas when the expression level of ST8SIA1 decreased, apoptosis could be prevented from apoptosis caused by high glucose concentration.

Example 2: Relationship between tyrosine phosphorylation of insulin receptor

Diabetes is known to be mainly caused by an abnormality in sub-receptor signaling, especially inhibition of IRS-1 (insulin receptor substrate-1)-associated PI3K activity, rather than a defect in the insulin receptor (IR) itself. This IRS-1-associated PI3K activity is regulated by the tyrosine phosphorylation of IRS-1 or the amount of IRS-1 protein itself, and a decrease in the amount of tyrosine phosphorylation protein of IRS-1 is observed in various diabetic animal models or diabetic environments.

Accordingly, it was confirmed whether insulin-induced tyrosine phosphorylation was increased when ST8SIA1 was inhibited in pancreatic beta cells. To this end, ST8SIA1KO-IPCs cells were not treated with or treated with 100 nM insulin for 10 minutes to measure tyrosine phosphorylation by Western blot.

As a result, as shown in Figure 2, compared to the increase in tyrosine phosphorylation in the case of insulin treatment (anti-ST8SIA1 antibody lane), there is no significant effect on tyrosine phosphorylation in the control lane not treated with the antibody. Confirmed. Therefore, it was confirmed that inhibition of ST8SIA1 as in the present invention results in an increase in tyrosine phosphorylation, decreases insulin resistance to insulin, and increases sensitivity.

Example 3: Effect of ST8SIA1KO-IPCs on diabetes

9-week-old db/db mice were reared by dividing 8 mice per group. ST8SIA1KO-IPCs cells transfected with exosomes were transplanted at 1 week of breeding, and each mouse was sacrificed 8 weeks later to measure the blood glucose concentration and insulin concentration of db/db mice (Fig. 3). Blood glucose concentration was measured according to the manufacturer's instructions using a commercial kit (Allmedicus), and insulin concentration was measured according to the manufacturer's instructions using a commercial kit (peprotec) by ELISA method.

As a result, as shown in FIG. 3, it was found that the blood insulin concentration was significantly increased in the ST8SIA1KO-IPCs administration group compared to the control group. These results confirmed that the ST8SIA1KO-IPCs of the present invention had an excellent insulin increase by implantation.

In addition, as shown in FIG. 4, compared to the control group, the ST8SIA1KO-IPCs group showed that blood sugar was rapidly decreased to and maintained at a normal level, whereas in the control group, it was confirmed that the blood sugar was continuously increased or increased.

Example 4: Effect on insulin sensitivity and glucose tolerance

9-week-old db/db mice were reared by dividing 8 mice per group. ST8SIA1KO-IPCs transfected with exosomes at 1 week of breeding were transplanted, and glucose tolerance (FIG. 4) and insulin sensitivity (FIG. 5) of db/db mice were measured.

To this end, an oral glucose tolerance test and an insulin resistance test were performed.

For the oral glucose tolerance test (IPGTT), glucose (dosage: 2g/kg body weight) was administered intraperitoneally (IPGTT) at 10 weeks of age. Glucose levels were monitored at 60 and 120 minutes after testing by the tail bleed method.

For the insulin resistance test (ITT), insulin (0.5U/kg body weight) was administered by intraperitoneal injection at 14 weeks of age. Glucose levels were monitored at 10, 20, 40 and 60 minutes after testing by tail bleed method.

As a result, as shown in FIG. 5, as a result of measuring the change in blood glucose level over time after glucose administration, it was confirmed that blood glucose was significantly controlled in the group administered with ST8SIA1KO-IPCs compared to the db/db mouse control group.

In addition, as shown in FIG. 6, as a result of measuring the blood glucose level after insulin administration, it was confirmed that the ST8SIA1KO-IPCs group was quickly removed from the blood compared to the mouse control group (Obese-no injection).

In addition, as shown in FIG. 7, in the group administered subcutaneously with an anti-ST8SIA1-Ab mixed with 100ul of 0.5% saponin (Sigma) solution as a delivery vehicle, blood sugar rapidly decreased to a normal level and maintained, whereas in the control group It was confirmed that the blood sugar was continuously increased or maintained in an increased state.

The present invention can screen an agent that inhibits the expression or activity of ST8SIA1 to screen for antidiabetic candidate substances, and when the insulin-secreting cells from which the ST8SIA1 gene is knocked-out is administered or transplanted to an individual, insulin secretion is increased. As a result, the blood sugar in the individual decreases and the ability to control blood sugar levels is very good, so there is an industrial applicability.

<110> HWANG, In Hu <120> Composition for preventing or treating diabetes and method for screening antidiabetic agents using ST8SIA1 <130> NP19-0028 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 356 <212> PRT <213> Artificial Sequence <220> <223> human ST8SIA1 isoform 1 <400> 1 Met Ser Pro Cys Gly Arg Ala Arg Arg Gln Thr Ser Arg Gly Ala Met 1 5 10 15 Ala Val Leu Ala Trp Lys Phe Pro Arg Thr Arg Leu Pro Met Gly Ala 20 25 30 Ser Ala Leu Cys Val Val Val Leu Cys Trp Leu Tyr Ile Phe Pro Val 35 40 45 Tyr Arg Leu Pro Asn Glu Lys Glu Ile Val Gln Gly Val Leu Gln Gln 50 55 60 Gly Thr Ala Trp Arg Arg Asn Gln Thr Ala Ala Arg Ala Phe Arg Lys 65 70 75 80 Gln Met Glu Asp Cys Cys Asp Pro Ala His Leu Phe Ala Met Thr Lys 85 90 95 Met Asn Ser Pro Met Gly Lys Ser Met Trp Tyr Asp Gly Glu Phe Leu 100 105 110 Tyr Ser Phe Thr Ile Asp Asn Ser Thr Tyr Ser Leu Phe Pro Gln Ala 115 120 125 Thr Pro Phe Gln Leu Pro Leu Lys Lys Cys Ala Val Val Gly Asn Gly 130 135 140 Gly Ile Leu Lys Lys Ser Gly Cys Gly Arg Gln Ile Asp Glu Ala Asn 145 150 155 160 Phe Val Met Arg Cys Asn Leu Pro Pro Leu Ser Ser Glu Tyr Thr Lys 165 170 175 Asp Val Gly Ser Lys Ser Gln Leu Val Thr Ala Asn Pro Ser Ile Ile 180 185 190 Arg Gln Arg Phe Gln Asn Leu Leu Trp Ser Arg Lys Thr Phe Val Asp 195 200 205 Asn Met Lys Ile Tyr Asn His Ser Tyr Ile Tyr Met Pro Ala Phe Ser 210 215 220 Met Lys Thr Gly Thr Glu Pro Ser Leu Arg Val Tyr Tyr Thr Leu Ser 225 230 235 240 Asp Val Gly Ala Asn Gln Thr Val Leu Phe Ala Asn Pro Asn Phe Leu 245 250 255 Arg Ser Ile Gly Lys Phe Trp Lys Ser Arg Gly Ile His Ala Lys Arg 260 265 270 Leu Ser Thr Gly Leu Phe Leu Val Ser Ala Ala Leu Gly Leu Cys Glu 275 280 285 Glu Val Ala Ile Tyr Gly Phe Trp Pro Phe Ser Val Asn Met His Glu 290 295 300 Gln Pro Ile Ser His His Tyr Tyr Asp Asn Val Leu Pro Phe Ser Gly 305 310 315 320 Phe His Ala Met Pro Glu Glu Phe Leu Gln Leu Trp Tyr Leu His Lys 325 330 335 Ile Gly Ala Leu Arg Met Gln Leu Asp Pro Cys Glu Asp Thr Ser Leu 340 345 350 Gln Pro Thr Ser 355 <210> 2 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> human ST8SIA1 isoform 2 <400> 2 Met Thr Gly Ser Phe Tyr Thr His Ser Pro Leu Thr Ile Gln Leu Thr 1 5 10 15 Leu Ser Ser His Arg Cys Asn Leu Pro Pro Leu Ser Ser Glu Tyr Thr 20 25 30 Lys Asp Val Gly Ser Lys Ser Gln Leu Val Thr Ala Asn Pro Ser Ile 35 40 45 Ile Arg Gln Arg Phe Gln Asn Leu Leu Trp Ser Arg Lys Thr Phe Val 50 55 60 Asp Asn Met Lys Ile Tyr Asn His Ser Tyr Ile Tyr Met Pro Ala Phe 65 70 75 80 Ser Met Lys Thr Gly Thr Glu Pro Ser Leu Arg Val Tyr Tyr Thr Leu 85 90 95 Ser Asp Val Gly Ala Asn Gln Thr Val Leu Phe Ala Asn Pro Asn Phe 100 105 110 Leu Arg Ser Ile Gly Lys Phe Trp Lys Ser Arg Gly Ile His Ala Lys 115 120 125 Arg Leu Ser Thr Gly Leu Phe Leu Val Ser Ala Ala Leu Gly Leu Cys 130 135 140 Glu Glu Val Ala Ile Tyr Gly Phe Trp Pro Phe Ser Val Asn Met His 145 150 155 160 Glu Gln Pro Ile Ser His His Tyr Tyr Asp Asn Val Leu Pro Phe Ser 165 170 175 Gly Phe His Ala Met Pro Glu Glu Phe Leu Gln Leu Trp Tyr Leu His 180 185 190 Lys Ile Gly Ala Leu Arg Met Gln Leu Asp Pro Cys Glu Asp Thr Ser 195 200 205 Leu Gln Pro Thr Ser 210 <210> 3 <211> 9737 <212> DNA <213> Artificial Sequence <220> <223> human ST8SIA1 mRNA transcript variant 1 <400> 3 ggggggggtt gcctggcggc gcagcagcgc gggaggcggc gaagggcgca ggagcatcgc 60 tcggagggga caaggggacg ccacgggcca catgtttagg agggagccga gccttctccc 120 ggaccctcgc cgagggcgac cgtgatgctg cagaaccggc gggagcgact cgccgccgcc 180 gctctctgcg cactcggaga ccccagcgcc cgccttctgc aggggaagcg accatggcca 240 tagatcgtga cttcaccccc agccacttcc cctagaaaga aatccttgga aaagttgcat 300 ttgaaaaaat ccttgcgctg acctttgggg ccgacggggc cgaagaagcg tgcgtgcgtt 360 tgcaagtaag agaaccaaag gtgtgtgtgc atggggggct ggcggtgggg gaccctccgc 420 tgccacttcg cctagctttg tgctgaggcc ccggcccccg cccctgggac gccggggctg 480 cgatgagccc ctgcgggcgg gcccggcgac aaacgtccag aggggccatg gctgtactgg 540 cgtggaagtt cccgcggacc cggctgccca tgggagccag tgccctctgt gtcgtggtcc 600 tctgttggct ctacatcttc cccgtctacc ggctgcccaa cgagaaagag atcgtgcagg 660 gggtgctgca acagggcacg gcgtggagga ggaaccagac cgcggccaga gcgttcagga 720 aacaaatgga agactgctgc gaccctgccc atctctttgc tatgactaaa atgaattccc 780 ctatggggaa gagcatgtgg tatgacgggg agtttttata ctcattcacc attgacaatt 840 caacttactc tctcttccca caggcaaccc cattccagct gccattgaag aaatgcgcgg 900 tggtgggaaa tggtgggatt ctgaagaaga gtggctgtgg ccgtcaaata gatgaagcaa 960 attttgtcat gcgatgcaat ctccctcctt tgtcaagtga atacactaag gatgttggat 1020 ccaaaagtca gttagtgaca gctaatccca gcataattcg gcaaaggttt cagaaccttc 1080 tgtggtccag aaagacattt gtggacaaca tgaaaattta taaccacagt tacatctaca 1140 tgcctgcctt ttctatgaag acaggaacag agccatcttt gagggtttat tatacactgt 1200 cagatgttgg tgccaatcaa acagtgctgt ttgccaaccc caactttctg cgtagcattg 1260 gaaagttctg gaaaagtaga ggaatccatg ccaagcgcct gtccacagga ctttttctgg 1320 tgagcgcagc tctgggtctc tgtgaagagg tggccatcta tggcttctgg cccttctctg 1380 tgaatatgca tgagcagccc atcagccacc actactatga caacgtctta cccttttctg 1440 gcttccatgc catgcccgag gaatttctcc aactctggta tcttcataaa atcggtgcac 1500 tgagaatgca gctggaccca tgtgaagata cctcactcca gcccacttcc taggaacaat 1560 ggaagaagaa aggactgaac cagggtattt ttgttaggtt ttctatgtga ctccaagagg 1620 gaatggtcaa gttgtttcat gagtttgcat gggcccttgg aaaaacagga aaggagcaat 1680 gaagatccaa gcaaaacttt actttcagcg ttggcttgga ggacaaataa gaaatgaaac 1740 atcctatgaa atactttata gcacatggca gatttgcaac tagtaaaatg ctggtgaaat 1800 gctgttggta aagcacatgg ttcaaatcta gaagatgcag ttcaaaaaca agacagactc 1860 gagttgttag ggctgaggaa ccaatcaagg tagaacaaag aaaatgttgg ggtaaaagtg 1920 ttgctgattg tcaacacaaa ctggcttaat aatattaata agaacctgtc ttattaagac 1980 tggctttaga accgtaggtt tttttaaaaa attattattt atttttgccc tctttgggga 2040 agtgggtggg tagatttaaa aaatcccttc ctgagtaata aagatacaaa atgttactgc 2100 tgataattgt gatttgttga gccacgtcta tattaactat agctcccctc tatttttaaa 2160 attttacata agaattgctt cttcctcttt tgtcaagtcc acagagacaa tgtgaagcat 2220 tattgaattt agactgtgtt gcttcatagt ctggataaca ggagccactt tccttcctca 2280 gaatttagga cataaaccca attttaggtc tgcattatgt aagcctgttt gcattgtcat 2340 ttttatttta gtgcaaaaga ctaccaatat gagtgaatta tttgtttgta aattggcata 2400 attgtcttca taatttaatg ttcagaaatg cagtaaagca cgttgatgaa aagcttagtc 2460 tctgtggaag acagaccaga agacaaatcc cttttctatc tactgtggca tattcactgc 2520 aaggtaaagg acatgtatgc atacatgcaa gaatgaattg agcgaaactt tgaatatgtt 2580 tcctcttttg agctattgtt atttagtagt tacctaaaaa agaatacatt tttactagta 2640 taccattaaa aatgtaaaag gcttaacatg tatgcataca tgtagacaca catacacaca 2700 cacaaagtta aacaaagctc attactgtgc aactatgaaa gctgaactca tatgacctgg 2760 tatggcaaaa atttgtaacc agtgttggag aatcacaaat gtgttatgca ctatccatta 2820 catgttgata taagacaacc gagctcactg gttgccgcac ataataccca tagcagcaca 2880 agatgcaagt cttaccccca aatgcttacc cggaatgagc agaattgtct tactcatcct 2940 gattttaccc aaaatctcat gaatgggctt gctgttgagc gtggtcacac aaggtatagt 3000 ttgtacaagt aaagaaatgt gaattagtag ctatttattt ccttagtagc aggtaggact 3060 acttagcttg ttacaacagc tttgatacac agtaagaaaa ctctactgac agattacaag 3120 cagccaaatg tgtttacatt tcagagaagg gtaagggaag cctctcgttg tcttcatact 3180 ttggaatgtc ttaagctaaa atgtgacact agagttcaat ttctttttta ttttaaatgt 3240 cactaaattg taggtaaact gtaccaaaaa gttctagttt aattttaaca gtaaaaactc 3300 agcataccat gttaccacgt aaacaataat ggctggtatt ttctgtgggt taatttaaca 3360 tgacataaac ataggaaagc ccttggcaaa attctacatc attgaagtag accatttcta 3420 aatgcaatta aatagtgagg ttattataaa aattagaatt taaaatatga cttgttgata 3480 gtgcagatta taatgttact tagaaagtta agaattcgag agtaatgatg ctattggaga 3540 actggtttgc tatacggcag caaaaacata atgagaatgt accatgaaga aattgtgtta 3600 aatattcact ttgagtgaaa ctttaaatat gtttcctctt ttgagctact gttatttagc 3660 agttacctaa aaaagaatac atttttacta gtctaccatt aaaaatataa aaggcttaac 3720 aaaaacgcat ataaaagtta ataaaatgtt actcagatta gtactttaat tgttaaaatg 3780 taattaatga gctccataga gcccatacat cacagttcca ttgaaaatat catctggact 3840 agaatctctt aaacccagga agtggaggtt gcagtgagcc aagatggtgc cactgcactc 3900 cagcctgggt gacagggcaa gaccctgtct caaaaaaaaa aaaaagaaaa gaaaaagaaa 3960 gaaaatatta tgtggaaatt tttcatcttt ttatgatgcc tgggatagat ttatgcagta 4020 gatttatata taatgtttag tttacaaact tgtgaaacta tttcatgtgg gttctgaaag 4080 gcaaaacaga aatggccagt gctggtgatg tttgattaga ccagctcttc tcagctgggg 4140 tgattcatta tccctctctg gtgacatttg gcaatgtctg gagaccagag acatttttta 4200 ttttcatgac tcaagggtgc taatagcatg tacttagtag aggttaggga tcctgctaaa 4260 gatcttacaa tgcacaagtc agcctctcac agcaaaataa ttatccaggc tgagatgtca 4320 gtagtatcaa ggttgacatc tcagacctgg attagacctg gattagacca aaatgaaatt 4380 ttgattgttc caccttgtct tcacttaaag atagatatac atgctcacag cattcaaaat 4440 atgactacaa tttaatgaaa atattttctt atggtagatt acaactcact agatacacca 4500 tacatatgcc ttttggacat aattggttag ataggagtgt gtgtgtgtgt gtgtgtgtgt 4560 gtgtgtgtgt gtgtgtgtgt gtagcatgaa gttaggcatc tctatcctta actgtgctat 4620 gtgttcctaa gtaaggacct cgctagaatc atcctgtaga gtgctctgga atactctatt 4680 attaagatct tcaattctaa gctcccatta tctttctttg tttatctgaa attaagacct 4740 tcagaattag gcctggtact tttccagatt tgatcagtta cattttgata taactgactc 4800 ttaaaattca gcttagaaga cattcaataa gaaaatcttt aatcactttt ggtgatgcac 4860 ctgccaagag ggggcatcac tttgtaccat cagactaatt aaaaaaattg ttgtgtagtc 4920 ataaaaatta ggctgatcca taggggcaaa aggaagggtg aagagactaa aaactagtac 4980 cctgatagaa ttttagtttt tattagcctc gatttgagaa ctgcattgat tccaaagatc 5040 atgaaggtag gtgaaggatt aagtcattcc ataattaaga aaagtaatgt ttatatgaaa 5100 gatttttata tggacaatga ctcacagaca catatgcttt gtatagagga ttttgccaaa 5160 agtttataca aaattgcaca tctaaactgc ataattctca tacaagaagt taaggaagtg 5220 ctcactggat tttgcttctg tatagttcac ttttatctgt cttcacaagg gtaggaagta 5280 aatgatctca aatggcacat gttcagacaa tactgtgttt ggctttgtga tgtctcatgt 5340 ctttctcttg gctaaaaaaa attgtttagg ctatatataa cttgtacctg aagcaatctg 5400 gacaacatgg cttaaaatgg aaaataactg aaaaaatcct gggattttaa atttgccaaa 5460 aatacccttg gattagtcaa aggctttaca agagtgttat agatgatcca aaatggaaac 5520 aatattgata acatttcttt tcgtcagcag ctacatattc cagttttata actttattat 5580 atacattttt tgattattaa tagaaagtca gccatccaga gttttaactt ctgtggttga 5640 aatattgact ttgtgaaatt tttcactgac aaatacttgt taggagggac ctaaggggga 5700 ggggaatgtt accagtccac tgaaaacttt aggaaatgtt cattgtgttg tgtaaatact 5760 ttcccaccag caccaggtaa tatattctct atgtttgtag aattccaaag atcaaaagct 5820 gtaaggacgt ttatgtgaac ttgacgcaaa cttgtggagg aataaggact gagagtaatg 5880 ttgtgctttt tctttatctt cccgtcgtat aggtcgactc tcaggttata tgacttttat 5940 tgggcacacg tcaggggtgg aagtggtaga agtgggttaa caatttcaag gcctggcaac 6000 ctaggtcaaa acctctaata aagtcagcag taaacataac tagcaacttc attccactat 6060 gtgctaggag gagggtgagg caagttgaaa ggagatgacc ctatccccac actctcagga 6120 tgtaaaaggg cagagtgctc tctaccctta accttggctt tttagcaccg tcagtctctg 6180 tcctctccca ggagagctgc cttggctcca gagttgagct agtagcattg tgactagaga 6240 ggtagtgtgg aaattcgctg aggacctcaa aattttacac tgccttaaac tgttttgttt 6300 ttgcttaaac tcgtgctgtc taaatgtggg tgttttgtgg cagagtctga tcaccccgac 6360 ctagttatag ccctgccagt gattctaaag tggaactttt ccccctcatt catatggata 6420 gtctgagtcc ttgaagctag ggagatatca aatattctac cagttcctac atagcttctt 6480 taaaagtcct agagactaac tcacaggaag tctgaagccc agagcgtctg tttataaact 6540 cctaaatatg aggccacaat atttaggttg agcactagaa aatttctatt ttgcaagtgg 6600 aaggagactt gaatctataa ttgctgtagc tttttttccc ttctatcata agaaaatgga 6660 taccatctgc tttaccctca gcccagactt gtcttttgaa ttttttacat acatcttagg 6720 ggtgagagat tgaaacaagt gcatgaatga aaagcttatt gaaacataac acagcccctg 6780 ctcaccattt cagtaaggga aaatacatgt ttctgtttcc ctctaatttg ttttctctgg 6840 tgccaacatt tctgatttct tctgtctcta atacataagg aataaatacc atattttaaa 6900 ttgacttaca tgcctgcgag catttgccag ataatcaaag cctatgactc taacttttta 6960 aagcacagaa gaggaattat attttatggt cactcctctg tgtttctatt gatctctata 7020 ttgtgctaaa attctttcag gtaatttttt tcttgttgaa gctgtgggat ataatcaatg 7080 atgccaagct gtctgagaat tttatagaat aatatccaaa tatattgatc ctgcctcttt 7140 ctgcttctat gtaattaaac tttcttttga tgtttatttt taaaatgtat gcttcacaca 7200 acaccaataa acatctaaga gatgtgatat aatccaaggc aaagaaaagt aagaatcagt 7260 taatgtctat tttatctgca cagcattacg aacagaaaat tgtaataaaa aaaggtttaa 7320 ccaaactgaa aaaaagagag acaaaatctt ggttttaatt tgaaaagatg attgggagtg 7380 atctcagaag agagttcagc aggaagcaag gaagaggctg ttgaccttga gtgcagcctc 7440 aagaatggaa cctttttgaa atattttaaa ggaaatatct tgtcatccag ctgttatggt 7500 taataatgaa tagttttctc tgggtgaagc ttatccattg cacacctgat gtgctggccc 7560 ctgcgatttc cccaaatgtt ggaatgttgc ctgcataatt tgtggtaatg ggggactgtg 7620 ttcctgcttt tacctggtta caaaaaacaa aacaaaacaa aacaaaacaa caaacagaaa 7680 aaaaaagaga gaaaagatgc tagaggaagc agtgtgacag acagatcatg agatgctgac 7740 ggagagaaca gagtggtacc tgtttaccca cttggaccat gacagtatca aggctccacc 7800 ccttgctgtc ccttcccaag aggtgctctc ctgggagttt gatcacagtg aaacaacact 7860 cccgttttct ttttcctttc tttctttctt tctttttttt tttttttttt ttttttgttg 7920 agacagagtt ttttgctctt gttgcccagg ctggagtgca atggcgtgat ctcggctcac 7980 cgcaacctcc gcctcccagg ttcaagcgat tctcgtgcct cagcctctgg agtagctggg 8040 attacaggca tgcgccacca cacccggcta attttgtact tttagtagag atggggtttc 8100 tccatattgg tcaggctggt cccgaactcc tgacctcagg tgatctgccc gcctcagcct 8160 cccaaagtgc tgggattaca ggcataagcc accgagccca gccccacact cctgttttca 8220 tagccctttt ttgtctgtac tttggtacat attccccaaa taagattatt atttacatct 8280 gtcatgatga ggcagtgata gcagcaagtg tcactttgac cctcaccctt ggaaagtggg 8340 ctatgcatag ctagagttag aacaggaggt tctaatcctg cttccacaat tacttcagta 8400 acaggctttt gtatccatga ttacttaagc tgacttagat gcattttcta ccctgtattc 8460 tagtgtacac aacacaaaca cacataagcc ctttacctgt ggagaagagg gagatggttg 8520 attcagtggg agtggggagt ggcagagggt gccttaactt tttctggctt gacccctact 8580 tccttatgtg tttccttcat atacctctgt cactttagag atgtaatcat gtctgttcca 8640 agttgtatcg ttattatcaa gaagtcctat aaatgtagca aatgtgacta ttttatagaa 8700 atgcacaata gtttataggc cttggagaag tctgataata gtatgtaaat atccttcatt 8760 gaagtcatag aaccttctag taggaagctc cataggtgat aactgcaaga ggattaatgt 8820 ttgtttctcc agtaaacacc ggaagcaagg aatcaatctg tcactgttta tcattcagtc 8880 ttgagcaccg ttgggtgttg ataggttact atagacactc cccttataat taatatacag 8940 gactcacaat ttaggatgtc tgatggccac tagggtacat catagagaca ctagagagaa 9000 gagaacaaat atagctccat caaaaagcct gcttaatctt taacaagtct gaaatattac 9060 taaaattcca gttaccgtgc agcatagtca gtctaaaaag cactggggaa aaaggtgtct 9120 ttttggtctc tttaattcta ttatggcaca ttttaatatt attccacatg tgtgcaaact 9180 atttctcaag agcaaagttt gaggctggag ttaagaaaat agaaaaatta aaacattatt 9240 ttttctcata taatataatc tgactactag ttcatggata taataattct gggaaaaatt 9300 ttagattaaa ctataaaatt gaaatcacct ttgaagagta tatggaattg cttctttcta 9360 ttaaagccaa cctacattgg caaaaagtgt ggagaaaatc agtgtgctca attctttttg 9420 tagagtttca tctaaaatgt tgcaggtaaa aaagaaaact ttaattggta ctttctacgg 9480 tgtgattaga aaaattagta tctatctctg ttggttctgc tgacttgctt ctttccaccg 9540 aatctaaatg caatgtcata ggaggaggtt tgctgtaaaa acatgtcttt ttctttggtg 9600 tattcattgt aattatggct ggcagtgaga aggttcggta agtctcaatt tctctacctc 9660 ctttacttgg agaaaatttg taaatgtacc ctgtgaataa aatgattttt tataaaaaaa 9720 aaaaaaaaaa aaaaaaa 9737 <210> 4 <211> 9627 <212> DNA <213> Artificial Sequence <220> <223> human ST8SIA1 mRNA transcript variant 2 <400> 4 ggggggggtt gcctggcggc gcagcagcgc gggaggcggc gaagggcgca ggagcatcgc 60 tcggagggga caaggggacg ccacgggcca catgtttagg agggagccga gccttctccc 120 ggaccctcgc cgagggcgac cgtgatgctg cagaaccggc gggagcgact cgccgccgcc 180 gctctctgcg cactcggaga ccccagcgcc cgccttctgc aggggaagcg accatggcca 240 tagatcgtga cttcaccccc agccacttcc cctagaaaga aatccttgga aaagttgcat 300 ttgaaaaaat ccttgcgctg acctttgggg ccgacggggc cgaagaagcg tgcgtgcgtt 360 tgcaagtaag agaaccaaag gtgtgtgtgc atggggggct ggcggtgggg gaccctccgc 420 tgccacttcg cctagctttg tgctgaggcc ccggcccccg cccctgggac gccggggctg 480 cgatgagccc ctgcgggcgg gcccggcgac aaacgtccag aggggccatg gctgtactgg 540 cgtggaagtt cccgcggacc cggctgccca tgggagccag tgccctctgt gtcgtggtcc 600 tctgttggct ctacatcttc cccgtctacc ggctgcccaa cgagaaagag atcgtgcagg 660 gggtgctgca acagggcacg gcgtggagga ggaaccagac cgcggccaga gcgttcagga 720 aacaaatgga agactgctgc gaccctgccc atctctttgc tatgactaaa atgaattccc 780 ctatggggaa gagcatgtgg tatgacgggg agtttttata ctcattcacc attgacaatt 840 caacttactc tctcttccca cagatgcaat ctccctcctt tgtcaagtga atacactaag 900 gatgttggat ccaaaagtca gttagtgaca gctaatccca gcataattcg gcaaaggttt 960 cagaaccttc tgtggtccag aaagacattt gtggacaaca tgaaaattta taaccacagt 1020 tacatctaca tgcctgcctt ttctatgaag acaggaacag agccatcttt gagggtttat 1080 tatacactgt cagatgttgg tgccaatcaa acagtgctgt ttgccaaccc caactttctg 1140 cgtagcattg gaaagttctg gaaaagtaga ggaatccatg ccaagcgcct gtccacagga 1200 ctttttctgg tgagcgcagc tctgggtctc tgtgaagagg tggccatcta tggcttctgg 1260 cccttctctg tgaatatgca tgagcagccc atcagccacc actactatga caacgtctta 1320 cccttttctg gcttccatgc catgcccgag gaatttctcc aactctggta tcttcataaa 1380 atcggtgcac tgagaatgca gctggaccca tgtgaagata cctcactcca gcccacttcc 1440 taggaacaat ggaagaagaa aggactgaac cagggtattt ttgttaggtt ttctatgtga 1500 ctccaagagg gaatggtcaa gttgtttcat gagtttgcat gggcccttgg aaaaacagga 1560 aaggagcaat gaagatccaa gcaaaacttt actttcagcg ttggcttgga ggacaaataa 1620 gaaatgaaac atcctatgaa atactttata gcacatggca gatttgcaac tagtaaaatg 1680 ctggtgaaat gctgttggta aagcacatgg ttcaaatcta gaagatgcag ttcaaaaaca 1740 agacagactc gagttgttag ggctgaggaa ccaatcaagg tagaacaaag aaaatgttgg 1800 ggtaaaagtg ttgctgattg tcaacacaaa ctggcttaat aatattaata agaacctgtc 1860 ttattaagac tggctttaga accgtaggtt tttttaaaaa attattattt atttttgccc 1920 tctttgggga agtgggtggg tagatttaaa aaatcccttc ctgagtaata aagatacaaa 1980 atgttactgc tgataattgt gatttgttga gccacgtcta tattaactat agctcccctc 2040 tatttttaaa attttacata agaattgctt cttcctcttt tgtcaagtcc acagagacaa 2100 tgtgaagcat tattgaattt agactgtgtt gcttcatagt ctggataaca ggagccactt 2160 tccttcctca gaatttagga cataaaccca attttaggtc tgcattatgt aagcctgttt 2220 gcattgtcat ttttatttta gtgcaaaaga ctaccaatat gagtgaatta tttgtttgta 2280 aattggcata attgtcttca taatttaatg ttcagaaatg cagtaaagca cgttgatgaa 2340 aagcttagtc tctgtggaag acagaccaga agacaaatcc cttttctatc tactgtggca 2400 tattcactgc aaggtaaagg acatgtatgc atacatgcaa gaatgaattg agcgaaactt 2460 tgaatatgtt tcctcttttg agctattgtt atttagtagt tacctaaaaa agaatacatt 2520 tttactagta taccattaaa aatgtaaaag gcttaacatg tatgcataca tgtagacaca 2580 catacacaca cacaaagtta aacaaagctc attactgtgc aactatgaaa gctgaactca 2640 tatgacctgg tatggcaaaa atttgtaacc agtgttggag aatcacaaat gtgttatgca 2700 ctatccatta catgttgata taagacaacc gagctcactg gttgccgcac ataataccca 2760 tagcagcaca agatgcaagt cttaccccca aatgcttacc cggaatgagc agaattgtct 2820 tactcatcct gattttaccc aaaatctcat gaatgggctt gctgttgagc gtggtcacac 2880 aaggtatagt ttgtacaagt aaagaaatgt gaattagtag ctatttattt ccttagtagc 2940 aggtaggact acttagcttg ttacaacagc tttgatacac agtaagaaaa ctctactgac 3000 agattacaag cagccaaatg tgtttacatt tcagagaagg gtaagggaag cctctcgttg 3060 tcttcatact ttggaatgtc ttaagctaaa atgtgacact agagttcaat ttctttttta 3120 ttttaaatgt cactaaattg taggtaaact gtaccaaaaa gttctagttt aattttaaca 3180 gtaaaaactc agcataccat gttaccacgt aaacaataat ggctggtatt ttctgtgggt 3240 taatttaaca tgacataaac ataggaaagc ccttggcaaa attctacatc attgaagtag 3300 accatttcta aatgcaatta aatagtgagg ttattataaa aattagaatt taaaatatga 3360 cttgttgata gtgcagatta taatgttact tagaaagtta agaattcgag agtaatgatg 3420 ctattggaga actggtttgc tatacggcag caaaaacata atgagaatgt accatgaaga 3480 aattgtgtta aatattcact ttgagtgaaa ctttaaatat gtttcctctt ttgagctact 3540 gttatttagc agttacctaa aaaagaatac atttttacta gtctaccatt aaaaatataa 3600 aaggcttaac aaaaacgcat ataaaagtta ataaaatgtt actcagatta gtactttaat 3660 tgttaaaatg taattaatga gctccataga gcccatacat cacagttcca ttgaaaatat 3720 catctggact agaatctctt aaacccagga agtggaggtt gcagtgagcc aagatggtgc 3780 cactgcactc cagcctgggt gacagggcaa gaccctgtct caaaaaaaaa aaaaagaaaa 3840 gaaaaagaaa gaaaatatta tgtggaaatt tttcatcttt ttatgatgcc tgggatagat 3900 ttatgcagta gatttatata taatgtttag tttacaaact tgtgaaacta tttcatgtgg 3960 gttctgaaag gcaaaacaga aatggccagt gctggtgatg tttgattaga ccagctcttc 4020 tcagctgggg tgattcatta tccctctctg gtgacatttg gcaatgtctg gagaccagag 4080 acatttttta ttttcatgac tcaagggtgc taatagcatg tacttagtag aggttaggga 4140 tcctgctaaa gatcttacaa tgcacaagtc agcctctcac agcaaaataa ttatccaggc 4200 tgagatgtca gtagtatcaa ggttgacatc tcagacctgg attagacctg gattagacca 4260 aaatgaaatt ttgattgttc caccttgtct tcacttaaag atagatatac atgctcacag 4320 cattcaaaat atgactacaa tttaatgaaa atattttctt atggtagatt acaactcact 4380 agatacacca tacatatgcc ttttggacat aattggttag ataggagtgt gtgtgtgtgt 4440 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtagcatgaa gttaggcatc tctatcctta 4500 actgtgctat gtgttcctaa gtaaggacct cgctagaatc atcctgtaga gtgctctgga 4560 atactctatt attaagatct tcaattctaa gctcccatta tctttctttg tttatctgaa 4620 attaagacct tcagaattag gcctggtact tttccagatt tgatcagtta cattttgata 4680 taactgactc ttaaaattca gcttagaaga cattcaataa gaaaatcttt aatcactttt 4740 ggtgatgcac ctgccaagag ggggcatcac tttgtaccat cagactaatt aaaaaaattg 4800 ttgtgtagtc ataaaaatta ggctgatcca taggggcaaa aggaagggtg aagagactaa 4860 aaactagtac cctgatagaa ttttagtttt tattagcctc gatttgagaa ctgcattgat 4920 tccaaagatc atgaaggtag gtgaaggatt aagtcattcc ataattaaga aaagtaatgt 4980 ttatatgaaa gatttttata tggacaatga ctcacagaca catatgcttt gtatagagga 5040 ttttgccaaa agtttataca aaattgcaca tctaaactgc ataattctca tacaagaagt 5100 taaggaagtg ctcactggat tttgcttctg tatagttcac ttttatctgt cttcacaagg 5160 gtaggaagta aatgatctca aatggcacat gttcagacaa tactgtgttt ggctttgtga 5220 tgtctcatgt ctttctcttg gctaaaaaaa attgtttagg ctatatataa cttgtacctg 5280 aagcaatctg gacaacatgg cttaaaatgg aaaataactg aaaaaatcct gggattttaa 5340 atttgccaaa aatacccttg gattagtcaa aggctttaca agagtgttat agatgatcca 5400 aaatggaaac aatattgata acatttcttt tcgtcagcag ctacatattc cagttttata 5460 actttattat atacattttt tgattattaa tagaaagtca gccatccaga gttttaactt 5520 ctgtggttga aatattgact ttgtgaaatt tttcactgac aaatacttgt taggagggac 5580 ctaaggggga ggggaatgtt accagtccac tgaaaacttt aggaaatgtt cattgtgttg 5640 tgtaaatact ttcccaccag caccaggtaa tatattctct atgtttgtag aattccaaag 5700 atcaaaagct gtaaggacgt ttatgtgaac ttgacgcaaa cttgtggagg aataaggact 5760 gagagtaatg ttgtgctttt tctttatctt cccgtcgtat aggtcgactc tcaggttata 5820 tgacttttat tgggcacacg tcaggggtgg aagtggtaga agtgggttaa caatttcaag 5880 gcctggcaac ctaggtcaaa acctctaata aagtcagcag taaacataac tagcaacttc 5940 attccactat gtgctaggag gagggtgagg caagttgaaa ggagatgacc ctatccccac 6000 actctcagga tgtaaaaggg cagagtgctc tctaccctta accttggctt tttagcaccg 6060 tcagtctctg tcctctccca ggagagctgc cttggctcca gagttgagct agtagcattg 6120 tgactagaga ggtagtgtgg aaattcgctg aggacctcaa aattttacac tgccttaaac 6180 tgttttgttt ttgcttaaac tcgtgctgtc taaatgtggg tgttttgtgg cagagtctga 6240 tcaccccgac ctagttatag ccctgccagt gattctaaag tggaactttt ccccctcatt 6300 catatggata gtctgagtcc ttgaagctag ggagatatca aatattctac cagttcctac 6360 atagcttctt taaaagtcct agagactaac tcacaggaag tctgaagccc agagcgtctg 6420 tttataaact cctaaatatg aggccacaat atttaggttg agcactagaa aatttctatt 6480 ttgcaagtgg aaggagactt gaatctataa ttgctgtagc tttttttccc ttctatcata 6540 agaaaatgga taccatctgc tttaccctca gcccagactt gtcttttgaa ttttttacat 6600 acatcttagg ggtgagagat tgaaacaagt gcatgaatga aaagcttatt gaaacataac 6660 acagcccctg ctcaccattt cagtaaggga aaatacatgt ttctgtttcc ctctaatttg 6720 ttttctctgg tgccaacatt tctgatttct tctgtctcta atacataagg aataaatacc 6780 atattttaaa ttgacttaca tgcctgcgag catttgccag ataatcaaag cctatgactc 6840 taacttttta aagcacagaa gaggaattat attttatggt cactcctctg tgtttctatt 6900 gatctctata ttgtgctaaa attctttcag gtaatttttt tcttgttgaa gctgtgggat 6960 ataatcaatg atgccaagct gtctgagaat tttatagaat aatatccaaa tatattgatc 7020 ctgcctcttt ctgcttctat gtaattaaac tttcttttga tgtttatttt taaaatgtat 7080 gcttcacaca acaccaataa acatctaaga gatgtgatat aatccaaggc aaagaaaagt 7140 aagaatcagt taatgtctat tttatctgca cagcattacg aacagaaaat tgtaataaaa 7200 aaaggtttaa ccaaactgaa aaaaagagag acaaaatctt ggttttaatt tgaaaagatg 7260 attgggagtg atctcagaag agagttcagc aggaagcaag gaagaggctg ttgaccttga 7320 gtgcagcctc aagaatggaa cctttttgaa atattttaaa ggaaatatct tgtcatccag 7380 ctgttatggt taataatgaa tagttttctc tgggtgaagc ttatccattg cacacctgat 7440 gtgctggccc ctgcgatttc cccaaatgtt ggaatgttgc ctgcataatt tgtggtaatg 7500 ggggactgtg ttcctgcttt tacctggtta caaaaaacaa aacaaaacaa aacaaaacaa 7560 caaacagaaa aaaaaagaga gaaaagatgc tagaggaagc agtgtgacag acagatcatg 7620 agatgctgac ggagagaaca gagtggtacc tgtttaccca cttggaccat gacagtatca 7680 aggctccacc ccttgctgtc ccttcccaag aggtgctctc ctgggagttt gatcacagtg 7740 aaacaacact cccgttttct ttttcctttc tttctttctt tctttttttt tttttttttt 7800 ttttttgttg agacagagtt ttttgctctt gttgcccagg ctggagtgca atggcgtgat 7860 ctcggctcac cgcaacctcc gcctcccagg ttcaagcgat tctcgtgcct cagcctctgg 7920 agtagctggg attacaggca tgcgccacca cacccggcta attttgtact tttagtagag 7980 atggggtttc tccatattgg tcaggctggt cccgaactcc tgacctcagg tgatctgccc 8040 gcctcagcct cccaaagtgc tgggattaca ggcataagcc accgagccca gccccacact 8100 cctgttttca tagccctttt ttgtctgtac tttggtacat attccccaaa taagattatt 8160 atttacatct gtcatgatga ggcagtgata gcagcaagtg tcactttgac cctcaccctt 8220 ggaaagtggg ctatgcatag ctagagttag aacaggaggt tctaatcctg cttccacaat 8280 tacttcagta acaggctttt gtatccatga ttacttaagc tgacttagat gcattttcta 8340 ccctgtattc tagtgtacac aacacaaaca cacataagcc ctttacctgt ggagaagagg 8400 gagatggttg attcagtggg agtggggagt ggcagagggt gccttaactt tttctggctt 8460 gacccctact tccttatgtg tttccttcat atacctctgt cactttagag atgtaatcat 8520 gtctgttcca agttgtatcg ttattatcaa gaagtcctat aaatgtagca aatgtgacta 8580 ttttatagaa atgcacaata gtttataggc cttggagaag tctgataata gtatgtaaat 8640 atccttcatt gaagtcatag aaccttctag taggaagctc cataggtgat aactgcaaga 8700 ggattaatgt ttgtttctcc agtaaacacc ggaagcaagg aatcaatctg tcactgttta 8760 tcattcagtc ttgagcaccg ttgggtgttg ataggttact atagacactc cccttataat 8820 taatatacag gactcacaat ttaggatgtc tgatggccac tagggtacat catagagaca 8880 ctagagagaa gagaacaaat atagctccat caaaaagcct gcttaatctt taacaagtct 8940 gaaatattac taaaattcca gttaccgtgc agcatagtca gtctaaaaag cactggggaa 9000 aaaggtgtct ttttggtctc tttaattcta ttatggcaca ttttaatatt attccacatg 9060 tgtgcaaact atttctcaag agcaaagttt gaggctggag ttaagaaaat agaaaaatta 9120 aaacattatt ttttctcata taatataatc tgactactag ttcatggata taataattct 9180 gggaaaaatt ttagattaaa ctataaaatt gaaatcacct ttgaagagta tatggaattg 9240 cttctttcta ttaaagccaa cctacattgg caaaaagtgt ggagaaaatc agtgtgctca 9300 attctttttg tagagtttca tctaaaatgt tgcaggtaaa aaagaaaact ttaattggta 9360 ctttctacgg tgtgattaga aaaattagta tctatctctg ttggttctgc tgacttgctt 9420 ctttccaccg aatctaaatg caatgtcata ggaggaggtt tgctgtaaaa acatgtcttt 9480 ttctttggtg tattcattgt aattatggct ggcagtgaga aggttcggta agtctcaatt 9540 tctctacctc ctttacttgg agaaaatttg taaatgtacc ctgtgaataa aatgattttt 9600 tataaaaaaa aaaaaaaaaa aaaaaaa 9627

Claims (15)

A pharmaceutical composition for preventing or treating diabetes, comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
The composition of claim 1, wherein the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes and gestational diabetes.
The composition of claim 1, wherein the ST8SIA1 protein comprises an amino line sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
The method of claim 1, wherein the ST8SIA1 protein expression inhibitor is composed of an antisense oligonucleotide, siRNA, shRNA, miRNA, ribozyme, crispr-cas9, DNAzyme, and PNA (protein nucleic acid) complementary to ST8SIA1 mRNA Composition, characterized in that any one selected from the group.
The composition of claim 4, wherein the ST8SIA1 mRNA comprises a nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.
The method of claim 1, wherein the ST8SIA1 protein activity inhibitor is any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, natural extracts, and synthetic compounds that specifically bind to ST8SIA1 protein. Composition made by.
(a) contacting the test substance with cells expressing the ST8SIA1 gene;
(b) measuring the ST8SIA1 gene expression level in the control cells not contacting the cells and the test substance; And
(C) a diabetes treatment screening method comprising the step of selecting a test substance that reduces the ST8SIA1 gene expression level compared to the control cells as a diabetes treatment candidate substance.
The method according to claim 7, wherein the cells expressing the ST8SIA1 gene are pancreatic beta cells or cells derived from pancreatic beta cells.
The method of claim 7, wherein the ST8SIA1 gene expression level is characterized in that the ST8SIA1 mRNA or protein expression level is measured.
A pharmaceutical composition for preventing or treating diabetes, comprising as an active ingredient insulin secreting cells in which ST8SIA1 gene expression is suppressed.
The composition of claim 10, wherein the inhibition of the expression of the ST8SIA1 gene is transfected with an exosome containing an antibody that specifically binds to ST8SIA1.
11. The pharmaceutical composition of claim 10, wherein the composition is formulated as a parenteral formulation.
The pharmaceutical composition according to claim 10, wherein the parenteral formulation is an injection or infusion.
The pharmaceutical composition of claim 10, wherein the injection or infusion agent is for injecting the composition into the pancreas.
The method of claim 10, wherein the insulin secreting cells
(a) separating exosomes from insulin-secreting cells;
(b) transfecting the exosomes isolated in step (a) with an antibody that specifically binds to ST8SIA1; And
(c) A composition, characterized in that it is prepared by a method comprising the step of transfecting the exosome prepared in step (c) into insulin-secreting cells.

Images