KR20170112476A - Composition for regulating ecdysis and method for regulating ecdysis - Google Patents

Abstract

The present invention relates to a composition for controlling dissection and a method for controlling dissection. Since the exocrine hormone and excretion can be regulated by controlling the microRNA-8 or v-ATPase gene of the present invention, it can be used in a composition for the control of exfoliation hormone, the prevention or treatment of growth diseases, and the expression of microRNA- Or v-ATPase expression, thereby preparing a macro larva model and using the same to be usefully used for a screening method of a therapeutic agent for a growth disease.

Description

[0001] The present invention relates to a composition for regulating moulting and a method for regulating moulting,

The present invention relates to a composition for controlling dissection and a method for controlling dissection.

MicroRNA (hereinafter referred to as 'miRNA') is a nucleic acid having a size of about 22 nucleotides. It is expressed in most eukaryotes and degrades or mRNAs of target mRNA complementary to the nucleotide sequence. To control gene expression. miRNAs are primarily transfected into primary miRNA transcripts (pri-miRNAs) by RNA polymerase II in the nucleus and then primarily processed into precursor miRNAs (pre-miRNAs). The pre-miRNA then migrates to the cytoplasm and is transformed into a mature miRNA by secondary transformation by Dicer. These miRNAs directly inhibit expression of the gene by directly binding to the Argonaute (Ago) protein, a key element of the RNA-induced silencing complex (RISC), and the target mRNA complementary to the nucleotide sequence. Currently, studies on miRNA production and its mechanism of action are actively underway in Korea and abroad.

Since miRNAs have been reported, the number of related scientific papers has been steadily increasing every year. miRNAs have received great interest both locally and externally, and studies are actively underway.

More than 60% of human genes can be regulated by miRNAs, so the importance of miRNA research has been highlighted both domestically and externally. Actual miRNAs have been found to be involved in almost all biological processes such as biological development and physiology, cell differentiation, proliferation, death, and genetic stability, and are also closely related to virus resistance and human diseases. Therefore, miRNA is being applied by various domestic and international researchers in diverse research fields such as stem cells, cancer, immunity, neurotransmission, but currently, only a part of various biological processes involved in each miRNA are known.

On the other hand, "growth" implies a quantitative change such as an increase in body weight or an increase in body size, while "maturation" refers to a qualitative change that reveals a characteristic of a living body and has a breeding ability . Quantitative growth and qualitative maturation in the development of the living body are partially overlapping, but the balance of these two phases is very important as they have opposite functions. In general, quantitative growth first occurs, and then gradually the rate of qualitative maturity increases. In humans, when this growth-maturation balance is broken, various developmental diseases such as growth disorder, sexual maturity are caused. Understanding the regulatory mechanism of growth-maturation transformation, which is very important in the development of life, is very poor.

Studies on genes related to growth or maturation of living organisms have been carried out at home and abroad, and some studies have also been carried out with respect to miRNA. There have been reports on the role of miRNAs in growth hormone and signal transduction, and some of the functions of miRNAs related to growth promotion and inhibition have been identified to suggest the possibility of diagnosis and treatment of growth diseases. However, And only one target gene. There is no overall study at the level of the full-length genome for miRNAs and their target genes associated with growth, maturation, or growth-maturation transformation.

During development, hormones play an important role in regulating the balance of growth and maturation, both of which are largely classified as protein hormones and steroid hormones. There are many studies on the regulation of growth-maturation transformation by protein hormones such as insulin, but studies on regulation of growth-maturation transformation by steroid hormones are relatively inadequate.

On the other hand, it is known that steroid hormones bind to the corresponding receptors of target cells to form complexes and then move to the nucleus to regulate the expression of various transcription factors at the transcription level. Thus, the transcription factors that are induced by steroid hormones influence the various biological processes in the cell by regulating the expression of numerous genes. Transcription of miRNA genes is also directly or indirectly affected by steroid hormones.

Normal growth and maturation are important not only for the rearing and cultivation of common fauna and flora, but also for humans. In recent years, people have become very sensitive to social disturbances, as well as to growth disorders and some short stature compared with normal levels. In addition, growth disorders and mature-related disorders such as gynecomastia are becoming social problems. According to statistics of gender gait at the Health Insurance Review and Assessment Service, the number of patients treated with gonococcal disease increased from 6,400 in 2006 to 28,000 in 2010, 4.4 times in 5 years, which corresponds to an annual average growth rate of 44.9%. The cost of medical treatment increased by 7.8 times over five years from 2.3 billion won to 17.9 billion won. Despite the growing trend in the number of treatments and therapies associated with growth and maturation-related disorders each year, there is a lack of comprehensive source studies on the regulation of growth-maturation processes.

Thus, steroid hormones are closely related to growth and maturation and are well known to regulate the expression of various genes at transcriptional level. Expression of miRNA genes is also directly or indirectly influenced by steroid hormones, To gain a general understanding of the in vivo function of miRNAs regulated by steroid hormones, it is important to identify all of the miRNAs involved and to identify the actual target genes of each miRNA at the full-length genome level.

Accordingly, the inventors of the present invention have conducted studies on microRNAs involved in growth-maturation transformation of Drosophila and its target genes, and found that it has an effect of regulating the elimination hormone and elimination by regulating microRNA-8 or v-APTase gene Thereby completing the present invention.

It is an object of the present invention to provide a dissolution-inhibiting composition comprising a microRNA-8 (miR-8) or v-ATPase inhibitor or a composition for accelerating dissolution including a microRNA-8 (miR-8) inhibitor or v-ATPase .

It is yet another object of the present invention to provide a method for regulating epithelial migration comprising the step of regulating the expression of microRNA-8 (miR-8) or v-ATPase.

Another object of the present invention is to provide a macro larva model in which the expression of microRNA-8 (miR-8) is increased or the expression of v-ATPase is inhibited.

In order to achieve the above object, the present invention provides a composition for inhibiting dissection, which comprises microRNA-8 (miR-8) or v-ATPase inhibitor.

In addition, the present invention provides a composition for stimulating dissolution, which comprises a microRNA-8 (miR-8) inhibitor or v-ATPase.

The present invention also relates to a method for promoting expression of microRNA-8 (miR-8); Or v-ATPase in the presence of the compound of the present invention.

The present invention also relates to a method for inhibiting the expression of microRNA-8 (miR-8); Or promoting the expression of v-ATPase.

In addition, the present invention provides a macro larva model in which the expression of microRNA-8 (miR-8) is increased or the expression of v-ATPase is inhibited.

Since the exocrine hormone and excretion can be regulated by controlling the microRNA-8 or v-ATPase gene of the present invention, it can be used in a composition for the control of exfoliation hormone, the prevention or treatment of growth diseases, and the expression of microRNA- Or v-ATPase expression, thereby preparing a macro larva model and using the same to be usefully used for a screening method of a therapeutic agent for a growth disease.

FIG. 1 is a graph showing the results of confirming miRNAs showing the difference in binding level with Ago1 in the growth-maturation transformation stage of Drosophila through HITS-CLIP.
FIG. 2 is a graph showing the results of Northern blot analysis of miRNAs showing differences in binding level between Ago1 and Ago1 in the growth-maturation transformation stage of Drosophila.
Fig. 3 shows the result of analysis of a target gene of miR-8. Fig.
FIG. 4 is a diagram illustrating the outline of performing the luciferase reporter assay to confirm whether miR-8 regulates the target gene v-ATPase.
5 shows the result of performing a luciferase reporter assay to confirm whether miR-8 regulates the target gene v-ATPase (*, p <0.05; **, p <0.01; ***, P < 0.001).
FIG. 6 is a graph showing the results of determination of the level of expression of the target genes Vha26 and Vha55 in the liposome upon conversion from the growth stage (WL3) to the maturation stage (PP).
FIG. 7 shows the results of confirming expression levels of target genes Vha26 and Vha55 in the miR-8 mutation at the growth stage (WL3).
FIG. 8 is a graph showing the results of confirming expression levels of Vha26 and Vha55 when miR-8 was overexpressed in S2 cells of Drosophila.
FIG. 9 shows the results of confirming the effect of miR-8 overexpression or v-ATPase expression reduction on intracellular acidification.
FIG. 10 is a graph showing the results of comparing tissue expression levels of miR-8 in the larval stage. FIG.
Fig. 11 shows the result of confirming the production of a larval larva by miR-8 or v-ATPase control.
FIG. 12 is a diagram showing the result of analyzing a mouth claw of a larva controlling miR-8 or v-ATPase.
Fig. 13 shows the results of confirming the expression of BR-C by miR-8 or v-ATPase.
FIG. 14 shows the results of confirming the change of exfoliation hormone by miR-8 or v-ATPase (**, p <0.01; ***, P <0.001).
Fig. 15 is a graph showing the results of confirming the expression level of neverland and shroud, genes related to the exfoliation hormone synthesis by miR-8 or v-ATPase.
Fig. 16 shows the result of confirming whether or not the change of the phenotype due to miR-8 or v-ATPase is restored due to ingestion of exfoliation hormone.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for inhibiting dissection, which comprises microRNA-8 (miR-8) or v-ATPase inhibitor (Vacuolar-type H + -ATPase).

In the present invention, microRNA (miRNA, miRNA, miR) is a single strand RNA molecule of 21-25 nucleotides (nt), which is a regulatory substance that controls gene expression of eukaryotic cells and consists of two stages of processing. The first miRNA transcript is made in the nucleus by the RNase III type enzyme called Drosha, which is made into a stem-loop structure of about 70-90 nt, that is, pre-miRNA, which is then transferred to the cytoplasm and transferred to an enzyme called Dicer To produce 21-25 nt of mature miRNA. Mature miRNAs are processed from longer transcripts and have a length of ~ 22 nucleotides (nt).

MicroRNAs can target specific messenger RNAs (mRNAs) and induce their degradation or translation inhibition, thereby controlling gene expression negatively.

In the present invention, microRNA-8 (miR-8) may be contained in a miR-8 clone form or a recombinant expression vector containing miR-8, but is not limited thereto.

In the present invention, a method of promoting the production of intracellular microRNA-8 can be used as a method of overexpressing microRNA-8. Methods for promoting the production of microRNA-8 include, but are not limited to, known methods for increasing transcription, translation, and mRNA stability. More specifically, a recombinant vector or virus capable of promoting the transcription of microRNA-8, increasing the stability of microRNA-8 producing mRNA, or externally increasing the expression of microRNA-8, . &Lt; / RTI &gt; It is also possible to use modified oligonucleotides which have an effect similar to microRNA-8.

In the present invention, v-ATPase (Vacuolar-type H + -ATPase) plays various functions in eukaryotes and is a gene highly evolutionarily conserved. In particular, it is located in the plasma membrane and functions as a proton pump and plays a role in the acidification of intracellular organelles. It is mainly found in membranes of endothelial cells such as endosomes, lysosomes, secretory vesicles, and is responsible for their acidification. These v-ATPases are composed of various subunits, and in the fruit fly, 33 genes are known to be responsible for each subunit.

The term "v-ATPase inhibitor" in the present invention includes without limitation substances which inhibit the expression of v-ATPase and includes RNA interference (RNAi), siRNA (small interfering RNA), antisense RNA, hairpin ribonucleic acid).

Specifically, the v-ATPase inhibitor may include RNAi, and RNAi is double-stranded RNA, which is introduced into cells to destroy mRNA complementary to the sequence contained in the RNAi molecule. RNAi molecules contain two complementary RNA strands (sense strand and antisense strand) that anneal to each other to form double stranded RNA molecules. Typically, RNAi molecules are derived from exon sequences or coding sequences of the gene to be removed. Preferably, the RNAi molecule is derived from a nucleic acid molecule, wherein the nucleic acid molecule can be composed of the sequence shown in FIG. 4 and is a nucleic acid sequence shown in FIG. 4 or a fragment thereof.

In the present invention, the term "siRNA" refers to a structurally modified siRNA to prevent rapid degradation by a nucleic acid degrading enzyme in vivo. Those skilled in the art can synthesize and modify the siRNAs in any desired manner using methods known in the art (Andreas Henschel, Frank Buchholzl and Bianca Habermann (2004) DEQOR: a web-based tool for the design and quality control of siRNAs. Nucleic Acids Research 32 (Web Server Issue): W113-W120). Since siRNA has a double helix structure, it is a relatively stable structure rather than a single-stranded ribonucleic acid or an antisense oligonucleotide, but is rapidly degraded by a nucleic acid degrading enzyme in vivo, have. Structural modifications of siRNAs to make the siRNAs stable and resistant so that they are not easily degraded are well known to those skilled in the art.

In the present invention, the term "antisense RNA" refers to single-stranded RNA consisting of an antisense sequence, wherein the antisense oligonucleotide binds to the target and is expressed by a DNA or RNA target by stopping expression at the stage of transcription, translation or splicing. It was proposed to prevent the expression of coded proteins. And includes structurally modified antisense RNA to prevent rapid degradation by in vivo nucleic acid degrading enzymes. Those skilled in the art can synthesize and modify the antisense RNA in any desired manner using methods known in the art (European Journal of Biochemistry 2003; 270: 1628-1644 Methods Enzymol. 2000: 313, 3-45. Reference).

In the present invention, the term "shRNA" refers to single-stranded RNA having a short hairpin length of 45 to 70 nucleotides in a cell, and is converted into siRNA by intracellular Dicer to exhibit an RNAi effect. The DNA of the structure in which the sense sequence of the siRNA base sequence and the antisense sequence are linked by 3-10 base linkers (loop sequence) is designed and synthesized and then cloned into the plasmid vector or the shRNA is transferred to the retroviruses lentivirus and adenovirus (adenovirus).

In addition to structural modifications, a safe and efficient delivery system is required to increase the intracellular delivery efficiency of nucleic acid materials such as siRNA. To this end, the siRNA and / or antisense RNA of the present invention may be included in a composition with a nucleic acid delivery system.

The expression constructs / vectors containing RNAi, siRNA, antisense RNA or shRNA (small hairpin RNA) can be prepared by methods known in the art.

In the present invention, the term "ecdysis" refers to a phenomenon in which an animal having a solid cuticle layer on its body surface, such as arthropods such as insects, crustaceans, and linear animals, takes off the old cuticle during the growing process, Snake, frog, and onchidium, a mollusk, are called skin exfoliation, and the molting of mammals and birds can also be seen as a kind of exfoliation.

The molting is controlled by the molting hormone. Ecdysone refers to hormones that induce transplantation and transformation of insects, crustaceans, and the like. These ecdysteroids are collectively referred to as ecdysteroids, and several ecdysteroids with high hormone activity have been found in plants. The exfoliation hormone may include a protein hormone, a steroid hormone, and preferably a steroid hormone.

In the present invention, the peeling is carried out by using let-7-5p, miR-1-3p, miR-10-5p, miR-100-5p, miR-1005-3p, miR-1008-3p, miR- 13p, -3p, miR-125-5p, miR-133-3p, miR-13b-1-3p, miR-13b-2-3p, miR-184-3p, miR-193-3p, miR- 275a-3p, miR-276a-5p, miR-276b-3p, miR-276b-5p, miR-263b-5p, miR- -2p-5p, miR-2c-3p, miR-281-1-5p, miR-281-2-5p, miR-285-3p, miR-2b-2-5p, , miR-315-5p, miR-317-3p, miR-375-3p, miR-8-3p, miR-927-5p, miR-929-5p, miR-92a-3p, -932-5p, miR-956-3p, miR-957-3p, miR-958-3p, miR-960-5p, miR-965-3p, miR-988-3p, miR-9a-5p, -5p, miR-iab-8-5p, and can be regulated by the target gene of the microRNA.

The term "modulation" in the present invention means perturbation of function or activity. In one embodiment of the invention, modulation refers to an increase in gene expression. In one embodiment of the invention, modulation refers to a decrease in gene expression.

The term "Expression" in the present invention means any function and steps by which the encoded information of a gene is converted into and existing in structures existing in the cell.

In the present invention, the microRNA-8 may be a nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

In the present invention, the microRNA-8 can target the v-ATPAse gene and inhibit the expression of the v-ATPAse gene.

The v-ATPase gene is a gene confirmed to be regulated by miR-8 in the present invention, and is a part of v-ATPase subunits (33), VhaM8.9 (Vacuolar H + ATPase M8.9 accessory subunit, FlyBase ID: (Vacuolar H + ATPase 55 kD subunit, FlyBase ID: FBgn0005671), and Vha100-2 (FBhn0037671), Vha26 (Vacuolar H + ATPase 26 kD subunit, FlyBase ID: FBgn0015324), VhaAC45 (Vacuolar H + ATPase AC45 accessory subunit, FlyBase ID: FBgn0262515) Vacuolar H + ATPase 100 kD subunit 2, FlyBase ID: FBgn0028670).

In the present invention, the microRNA-8 targets the v-ATPase gene. In a specific embodiment of the present invention, the microRNA-8 targeting the v-ATPase gene is overexpressed to control the elimination hormone, It is confirmed that the conversion step can be controlled.

In addition, the present invention provides a composition for stimulating dissolution, which comprises a microRNA-8 (miR-8) inhibitor or v-ATPase.

In the present invention, "microRNA-8 (miR-8) inhibitor" includes, without limitation, a substance that inhibits the expression of microRNA-8 and includes RNA interference (RNAi), siRNA (small interfering RNA), antisense RNA ), shRNA (short hairpin ribonucleic acid). A detailed description thereof is as described above.

The present invention also relates to a method for promoting expression of microRNA-8 (miR-8); Or v-ATPase in the presence of the compound of the present invention.

In the present invention, the step of promoting the expression of microRNA-8 can be performed by a method of promoting the production of microRNA-8 in micro cells. Methods for promoting the production of microRNA-8 include, but are not limited to, known methods for increasing transcription, translation, and mRNA stability. More specifically, the present invention provides a recombinant vector capable of promoting the transcription of microRNA-8, increasing the stability of microRNA-8 producing mRNA, or externally increasing the expression of microRNA-8, Or by injecting a virus. It is also possible to use modified oligonucleotides which have an effect similar to microRNA-8.

In the present invention, the step of inhibiting the expression of v-ATPase can be carried out by RNA interference (RNA interference), siRNA (small interfering RNA), antisense RNA, shRNA (short hairpin ribonucleic acid) .

The present invention also relates to a method for inhibiting the expression of microRNA-8 (miR-8); Or promoting the expression of v-ATPase.

In the present invention, the step of inhibiting the expression of microRNA-8 may be performed by RNA interference (RNA interference), siRNA (small interfering RNA), antisense RNA, shRNA (short hairpin ribonucleic acid) have.

In the present invention, the step of promoting the expression of v-ATPase can be carried out by introducing a recombinant vector containing the v-ATPase gene or a virus.

The present invention also provides a giant larva model in which the expression of microRNA-8 (miR-8) is increased or the expression of v-ATPase is inhibited.

In the present invention, the macro larva model may be a fruit fly, and when the animal is a fruit fly, it is not converted from the larval stage to the pupa stage by the overexpression of microRNA-8, .

In addition, the present invention provides a composition for preventing or treating growth disease comprising microRNA-8 (miR-8).

In the present invention, the growth disorder may be selected from the group consisting of familial short stature, constitutional growth retardation, idiopathic short stature, osteochondral dysplasia, short stature due to chromosome abnormality (Down syndrome or Turner syndrome), unfair lightweight Short stature due to chronic systemic disease, short stature due to growth hormone deficiency, short stature due to hypothyroidism, short stature due to sexual maturity, short stature due to Cushing's syndrome, Dysmenorrhea, and psychosocial dwarfism. However, the present invention is not limited thereto, and includes, without limitation, diseases that may be caused by exfoliation hormone.

The term "prophylactic" in the present invention means slowing down or preventing growth disease, initiation, development, or progression of molting during a period including weeks, months or years.

As used herein, the term "treatment" refers to the application of one or more specific procedures used for the treatment or amelioration of a growth disorder. In one embodiment of the invention, the particular procedure comprises administering one or more pharmaceutical agents.

The composition for preventing or treating growth disorders of the present invention may optionally further comprise pharmaceutically acceptable carriers, adjuvants, or additives such as salts, isomers, solvates, hydrates and prodrugs thereof.

The composition may be provided in a physiologically acceptable formulation, such as contained in a pharmaceutically acceptable carrier, using known techniques.

The composition for preventing or treating growth disease of the present invention may be formulated in the form of solid, liquid or aerosol suitable for the route to be administered. Examples of solid compositions include tablets, creams, and implantable dosage units. Tablets can be administered orally. Therapeutic creams may be used topically. Implantable dosage units can be used, for example, locally at a particular site, for example, for the purpose of systematically delivering compositions for the prevention or treatment of subcutaneous growth disorders. Examples of liquid compositions include formulations for subcutaneous, intravenous, intraarterial, topical, and intraocular administration. Examples of aerosol compositions include inhalation formulations for administration through the lung.

The composition may be administered in standard routes. In general, the compositions can be administered by topical, oral, rectal, vaginal, nasal, or parenteral routes (e.g., intravenous, subcutaneous, intramuscular). In addition, the composition may comprise a biodegradable polymer, for example a continuous emission matrix, such as a polymer that is grafted to the target site. Also biodegradable polymers and other polymers can be implanted to enable systemic delivery. Methods of administration include single dose administration, repeated dose administration at predetermined time intervals, and continuous administration at predetermined time intervals.

As used herein, a continuous emission matrix is a matrix made of a material, usually a polymer degradable by enzymatic or acid base hydrolysis or dissolution. Once inserted into the body, the matrix is activated by enzymes and body fluids. Preferably, the sustained release matrix is selected from the group consisting of liposomes, polylactide acid, a polymer of glycolic acid, polylactide co-glycolide (lactose and glycolic acid, Polymers, copolymers of lactic acid and glycolic acid, polyanhydrides, poly (ortho esters), polypeptides, hyaluronic acid, collagen, Chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, phenylalanine, ), Amino acids such as tyrosine and isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone, and silicone a biocompatible material such as silicone can be selected. A preferred biodegradation matrix is any one of polylactide, polyglycolide or co-polymers of lactic acid and glycolic acid.

The amount of the composition to be administered depends on the treatment situation, the particular composition used and other clinical factors such as body weight and the condition of the patient, and the dosage form. The composition can be administered in combination with other components and in the course of treatment of the disease.

Examples of routes of administration include intravenous, intramuscular, subcutaneous, inhaled, topical, mucosal and rectal, as well as parenteral or oral administration. Solutions or suspensions used for oral administration such as blood or subcutaneous administration include solutions such as sterile diluents such as injecting solution, saline solution, fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, benzyl alcohol or methylparaben Bacterial agents; Antioxidants such as ascorbic acid or sodium sulfite; Chelating agents such as ethylenediaminetetraacetic acid; A buffer such as acetate, citrate or phosphate, and an extendability modifier such as sodium chloride or dextrose. Suitable carriers for the manufacture of suppositories are natural and hardened oils, waxes, fats, semi-liquid polyols and the like. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be sealed with ampules, disposable syringes, multi-dose vials made of glass or plastic. Suitable therapeutic agents for injectable use include sterile aqueous solutions or dispersions and sterile powders for sterile preparations of sterile injectable solutions or dispersions. Suitable carriers for intravenous administration include physiological saline, mucilage, CremophorEL (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the therapeutic agent must be sterile and fluid to the extent that it can be easily injected. It must be stable under the conditions of manufacture and storage and should be preserved in the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or a jetting medium containing water, ethanol, a polyol (such as glycerol, propylene glycol and liquid polyetylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a by-pass liquid, and by using a surfactant. Microbial action can be prevented by various antibacterial and antimicrobial agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Polyhydric alcohols such as sugar, mannitol, sorbitol, and isotonic agents such as sodium chloride are preferably included in the therapeutic agent for diseases. Prolonged absorption of the injectable composition can be achieved, for example, by including an absorption delaying agent such as aluminum monostearate and gelatin in the therapeutic agent for disease.

Oral compositions generally include an inert diluent or an edible carrier. For oral therapeutic administration, the active ingredient is incorporated into excipients and used as tablets, troches, or gelatin-type capsules. Oral preparations may also be prepared using a fluid carrier for use as a mouth wash. Examples of suitable carriers for soft gelatine capsules are vegetable oils, waxes, fats, semi-solid and liquid polyols and the like. Examples of carriers suitable for the production of liquid solutions and syrups include water, polyols, saccharose, phosgene, glucose and the like.

Pharmaceutically compatible binders and / or additives may be included as part of the disease treatment agent. The pharmaceutical preparations may also contain preservatives, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavors, salts for controlling osmotic pressure, buffers, coatings or antioxidants. Tablets, pills, capsules, troches and the like may contain any one of the following ingredients or a mixture of similar properties: binders such as microcrystalline cellulose, gum tragacanth or gelatin; Excipients such as starch or lactose, disintegrants such as alginic acid, Primogel TM or corn starch; Lubricants such as magnesium stearate or Sterotes TM; Gildants such as colloidal silicon dioxide; Sweeteners such as sucrose or saccharin; Or a flavoring agent such as peppermint, methyl salicylate, or orange flavor. Oral or parenteral therapeutic agents are advantageously formulated in dosage unit form to facilitate dosage administration and uniformity. The miRNAs can be formulated as medicaments in a conventional manner using one or more pharmaceutically acceptable carriers. Lactose, corn starch or derivatives thereof, talc, stearic acid or its salts can be used, for example, as carriers for tablets and hard gelatine capsules.

The dosage for preventing or treating growth disorders in the present invention can vary widely and, of course, can be adjusted according to individual requirements in each particular case.

Terms not otherwise defined herein have meanings as commonly used in the art to which the present invention belongs.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Example  1: Drosophila growth-maturation transformation Ago1  Binding to protein miRNA  Level change analysis

miRNAs inhibit the expression of genes by directly binding to Ago (Argonaute) proteins and target mRNA, which are key elements of RISC (RNA-induced silencing complex). In order to identify miRNAs showing the difference in binding level with Ago1 in the growth-maturation transformation step, the following procedure was performed.

Ago1 HITS-CLIP was used to identify the RNA base sequence bound to Ago1 by using bioinformatics technique. Among them, the sequence corresponding to the Drosophila miRNA was isolated from the wandering 3rd instar larva (WL3, growth stage) 179 miRNAs were found to bind to 243 miRNAs in Ago1 in the mid-pupa (MP, mature stage). Based on the readings obtained through HITS-CLIP, the readings of Ago1-binding miRNAs obtained from each sample were standardized using DESEQ, and the binding levels of the miRNAs were statistically compared and analyzed. The graphs for the mean (Basemean) expression level of miRNAs identified in the WL3-MP conversion step and the fold change in the expression level between two steps (Fold change) are shown in FIG. miRNAs are represented by dots and triangles, and miRNAs showing a statistically significant difference among miRNAs are indicated in red. MiRNAs represented by triangles are miRNAs identified at only one stage of WL3 or MP.

As shown in Fig. 1 and Table 1, a total of 49 miRNAs having a statistical ( P < 0.05 ) significant difference in the level of binding to Ago1 at the transition stage of maturation were identified: let-7-5p, miR-10-5p, miR-100-5p, miR-1005-3p, miR-1008-3p, miR-1012-3p, miR-125-3p, 133-3p, miR-13b-1-3p, miR-13b-2-3p, miR-184-3p, miR-193-3p, miR-210-3p, miR- 276b-5p, miR-279-3p, miR-276a-3p, miR-276a-5p, miR-273b-5p, miR- 281-1-5p, miR-281-2-5p, miR-285-3p, miR-2b-2-5p, miR-2c-3p, miR- 3p, miR-92b-3p, miR-932-5p, miR-927-5p, miR-929-5p, miR-92a-3p, miR- miR-9a-5p, miR-iab-8-5p, miR-9a-5p, miR-95b-3p, , 31 of these 49 miRNAs were increased and 18 of them were decreased by the transformation of mature stage.

Example 2: Screening and validation of miRNAs involved in the growth-maturation transformation of Drosophila

Based on the results of the mass sequencing analysis of Example 1 above, in order to re-verify the miRNA showing a difference in binding level from Ago1 during WL3-MP conversion, the Ago1 protein was precipitated again in the WL3 and MP stages, 9 kinds of 49 miRNAs which were different in the growth-maturation stage in Example 1 were selected and their binding levels were compared and analyzed by the known Northern blot method. The nine miRNAs were selected from the group consisting of let-7-5p, miR-274-5p, miR-252-5p, miR-279-3p, miR-281-2-5p, miR- -317-3p, and miR-8-3p. The results of the comparative analysis are shown in FIG. 2, and the amount of the precipitated Ago1 protein was confirmed by Western blotting (WB).

As shown in FIG. 2, it was confirmed that the nine kinds of miRNAs identified showed a difference in the level of miRNA bound to Ago1 as in the case of the mass base sequence analysis of Example 1 above.

Example 3: Target gene analysis of miR-8

For the target gene analysis for miR-8 among the miRNAs that showed differences in growth-maturation stages in the above example, based on the data obtained from Ago1 HITS-CLIP, the total target of miRNAs showing different expression levels at the time of WL3- The genes were analyzed. The control RNA between miR-8 and v-ATPase genes can be analyzed by Ago1 CLIP, which can extract not only Ago1-bound miRNAs but also RNAs of target genes bound to miRNAs. After mass sequencing, This technique was used to analyze genes that have important seed sequences for binding to miRNAs. As a result, five genes of v-ATPase subunit identified as target genes of miR-8 are shown in FIG.

miRNA is red, target gene of miR-8 identified by Ago1 HITS-CLIP is green, and target gene regulated by miRNA other than miR-8 is blue. As shown in Fig. 3, it was confirmed that the target gene of miR-8, v-ATPase, was VhaM8.9, Vha26, VhaAC45, Vha55, Vha100-2.

Example 4: Verification of v-ATPase gene regulation of miR-8

Luciferase reporter assay was performed to confirm that the expression of genes identified as target genes of miR-8 identified in Example 3 was directly regulated by miR-8.

As shown in Figure 4, in the RL sub-region of the psiCHECK2 vector capable of simultaneously expressing Renilla Luciferase (RL) and firefly luciferase (FL), 5 ' To prepare a replicator. In addition, a mutant reporter was designed to inhibit the binding of miR-8 by substituting a nucleotide sequence in the important region of the binding region. Fig. 4 shows the binding sites of miR-8 located in the 3'UTR of v-ATPase genes, black indicates normal sequence, and red indicates substitution and deletion of base sequence. The results of the luciferase reporter assay are shown in FIG. Statistical analysis was performed by Dunnett test with ANOVA and multiple tests.

As shown in FIG. 5, in the case of reporter (WT + mir-8) in which the 3'UTR of the gene identified as a target gene is compared with the control (WT + Empty), when miR- Mutation reporter (Mut + mir-8), which replaces some of the nucleotide sequences in the seed region, is not inhibited by miRNA-8 and is increased in its level again .

Thus, it was confirmed that miR-8 target genes were regulated by miR-8.

Western blotting (WB) was also performed to confirm at the protein level whether expression of genes identified as target genes of miR-8 was directly regulated by miR-8.

Specifically, antibodies against Vha26 and Vha55 genes were obtained from five miR-8 target genes, and the expression control relationship between miR-8 and target genes Vha26 and Vha55 was confirmed. Expression levels of miR-8 and its target genes Vha26 and Vha55 in WL3 and prepupa stages were confirmed by western blotting of known methods and the results are shown in Fig. val-tRNA, and beta -Tubulin were used as a loading control.

As shown in FIG. 6, the expression levels of Vha26 and Vha55 were significantly increased as the expression of miR-8 decreased in the conversion from WL3 to PP.

In addition, as shown in FIG. 7, the expression level of the target genes Vha26 and Vha55 in the mutation of miR-8 was analyzed and it was confirmed that the level was increased. The miR-8 mutation (miR-8 ^Δ2 ) is a mutation produced by the Stephen M, Cohen group (Cell, v131, p136), imprecise excision of P-element EP2269, The 1.8 kb portion of the mutation is removed.

In addition, as shown in FIG. 8, it was confirmed that the expression of Vha26 and Vha55 in the case of overexpression of miR-8 (mir-8) in Drosophila S2 cells was significantly decreased at the protein level.

Thus, it was confirmed that the expression of the target gene was negatively regulated by miRNA-8.

Example  5: miR -8 increase or v- ATPase  Identification of regulation of acidification by decreased expression

(Vha26, VhaAC45, Vha55, Vha100-2) in the lipid body of Drosophila larvae using hsFLP in order to confirm whether or not v-ATPase acts as a complex and is associated with intracellular acidification. Clones (target gene knockdown cells) were prepared. In RNAi clones, the expression of each target gene is reduced. Lysotracker staining was performed on the knockdown cells, and the result of staining was shown in FIG. Lysotracker staining can stain the acidic part of the cell. At the same time, a clone (miR-8) overexpressing miR-8 and a miR-8 overexpressing clone were treated with concanamycin A (CMA), which is known as a v-ATPase inhibitor, at a concentration of 150 nM for 10 minutes, CMA) was stained with lysotracker, and the result of staining was shown in FIG.

As shown in FIG. 9, the lysotracker staining portion indicated by the dotted line in the tissue clone in which the expression of the target gene was reduced was decreased, so that it was confirmed that the portion showing acidity was significantly reduced. In addition, a simultaneous analysis of clones overexpressing miR-8 confirmed the functional relationship between miR-8 and the target gene by confirming that the acidic portion of the miR8 overexpressing clone was markedly decreased similarly to the case of decreasing v-ATPase genes . In addition, concanamycin A (CMA), known as a v-ATPase inhibitor, was similarly treated with 150 nM for 10 min at room temperature, indicating a decrease in lysotracker staining.

Therefore, it was confirmed that miR-8 plays a role in reducing intracellular acidification, and miR-8 can affect intracellular acidification by controlling v-ATPase, which is a target gene.

Example  6: In the larva stage miR -8 expression level of each tissue

In order to investigate the expression level of miR-8 in the larval stage, we compared the expression level of mcherry by using a fluorescent image system in which expression of mcherry was regulated by miR-8GAL4 as a reporter system, -8 were compared. The results are shown in Fig.

As shown in FIG. 10, miR-8 was expressed in most larval tissues but it was found to be highly expressed in fat bodies, salivary glands and ring glands.

Example 7: Confirmation of generation of giant larvae by miR-8 or v-ATPase

In order to confirm whether or not the growth of Drosophila is regulated by miR-8 or its target gene v-ATPase, as shown in Example 6 above, among tissues in which miR-8 appears to be highly expressed, 8 was performed in the following manner to confirm the function of miR-8 in the annular gland, which is known to be an important tissue for synthesizing and secreting ecdysone.

(Mir-8) and miR-8 target v-ATPase (Vha26, VhaAC45) in phantom-GAL4, which is capable of expressing the gene in a tissue-specific manner, , Vha55, and Vha100-2) was observed in the 5th day and 15th day of the RNAi group knocked down, and the observation results are shown in FIG. The control group (+) used wild-type flies.

As shown in FIG. 11, when miR-8 overexpression or v-ATPase knockdown was performed in the phyllotaxis of the fruit flies, it was confirmed that the fruit flies could not be converted into the pupa stage and continued to grow in the larval stage to become a giant larva. On the other hand, in the case of control (+), it was confirmed that after 5th day, it entered the pupa stage.

As a result of analyzing the mouth hooks of the larvae, it was confirmed that the 3 ^rd instar larva stage, which is the last stage of the larval development stage, develops to the same level as the control group as shown in FIG.

Example  8: miR -8 or v- To ATPase  Of BR-C expression

The expression level of BR-C was measured by miR-8 or v-ATPase in order to confirm whether the phenotype identified in Example 7 was related to the synthesis and secretion of exfoliation hormone. BR-C is an ecdysone-induced factor that increases expression by epithelial hormone. FIG. 13 shows the results of immunostaining of expression levels of BR-C in the annular spleen upon miR-8 overexpression or v-ATPase decrease. The annular spots are shown in dotted lines, BR-C in red, and DAPI in blue.

As shown in Fig. 13, it was confirmed that the expression level of BR-C in the annular spleen remarkably decreased upon miR-8 overexpression or v-ATPase decrease.

Example 9: Confirmation of change of exfoliation hormone by miR-8 overexpression and decrease of v-ATPase

In the miR-8 overexpression group and v-ATPase knockout group, enzyme immunoassay (EIA) was performed to determine the level of substantial elimination hormone by miR-8 or v-ATPase. The synthesis of hormones was measured. Fig. 14 shows the measurement results of the peel-off hormone synthesis. Dunnett's test was performed with ANOVA and multiple tests.

As shown in Fig. 14, the results of the 5th day of measurement showed that the miR-8 overexpression group (mir-8) or v-ATPase knockdown group (Vha26-RNAi, VhaAC45-RNAi, Vha55- RNAi, Vha100-2 - RNAi) was significantly reduced in the cyclic gland of the brain. It is also confirmed that this result is the same on day 7.

Therefore, it was confirmed that miR-8 and v-ATPase play an important role in the synthesis of exfoliation hormone in the annular gland.

Example 10: Confirmation of the change of gene related to epithelial hormone synthesis by miR-8 overexpression and decrease of v-ATPase

Immunostaining was performed in the annular spleen to confirm the level of expression of nerverland and shroud genes, which are known to be important for the synthesis of exfoliation hormone according to the changes of miR-8 or v-ATPase, and the results are shown in Fig.

As shown in FIG. 15, when miR-8 was over-expressed or knocked down by v-ATPase, the level of expression of the virulence-related gene (neverland, shroud) was decreased at the protein level.

Example 11 Confirmation of Phenotype Recovery by Overexpression of miR-8 and Reduction of v-ATPase through Ectopic Hormone Intake

To determine whether a series of phenotypes caused by miR-8 overexpression and v-ATPase knockdown as described above was due to a decrease in exfoliation hormone, 1 mg / ml 20E (active form of exfoliation hormone) Yeast were added together. The phenotype of Drosophila was then observed and the results are shown in FIG.

As shown in FIG. 16, about 50% of individuals of miR-8 overexpression group (mir-8) or v-ATPase knockdown group (Vha26, VhaAC45, Vha55, Vha100-2) The larval stage was changed from the larva to the pupa stage.

Thus, miR-8 regulates its target gene, v-ATPase, and miR-8 or v-ATPase gene can regulate the steroid hormone, the excretory hormone. Therefore, miR-8 or v- The preparation of an animal model of Drosophila in which the exfoliation hormone is regulated, or a screening method of a therapeutic agent for growth diseases.

Although the present invention has been described in terms of the preferred embodiments mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also to be understood that the appended claims are intended to cover such modifications and changes as fall within the scope of the invention.

<110> Korea University Research and Business Foundation <120> Composition for regulating ecdysis and method for regulating          ecdysis <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> microRNA-8 <400> 1 uaauacuguc agguaaagau guc 23

Claims (8)

v-ATPase (Vacuolar-type H + -ATPase) inhibitor or microRNA-8 (miR-8).
A microRNA-8 (miR-8) inhibitor or a v-ATPase (Vacuolar-type H + -ATPase).
2. The composition of claim 1, wherein the microRNA-8 comprises the nucleotide sequence of SEQ ID NO: 1.
The composition of claim 1, wherein the microRNA-8 inhibits the expression of the v-ATPase gene.
The method of claim 1, wherein the v-ATPase is selected from the group consisting of VhaM8.9 (Vacuolar H + ATPase M8.9), Vha26 (Vacuolar H + ATPase 26 kD subunit), VhaAC45 (Vacuolar H + ATPase AC45 accessory subunit), Vha55 (Vacuolar H + ATPase 55 kD subunit) And Vha100-2 (Vacuolar H + ATPase 100 kD subunit 2).
Promoting the expression of microRNA-8 (miR-8); Or v-ATPase in the presence of the compound.
Inhibiting the expression of microRNA-8 (miR-8); Or promoting the expression of v-ATPase.
A large larval model with increased expression of microRNA-8 (miR-8) or suppressed v-ATPase expression.

Images