KR101736025B1 - Reverse-aging induced method using a circulating aging marker - Google Patents
Reverse-aging induced method using a circulating aging marker Download PDFInfo
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Abstract
The present invention relates to a technique for inducing reverse aging of a specific organ by identifying aging markers present in the circulatory system. The microRNA is isolated from whole blood of 2, 6, and 12 months old, and analyzed by NGS. The 27 microRNAs were selected, and 10 microRNAs contained in the networks involved in cancer, organ damage, abnormalities and reproductive system diseases were selected and their control proteins were analyzed. As a result, 6 kinds of microRNAs microRNA was selected and expression of these six microRNAs was significantly reduced with aging. As a result of administering the microRNA to a mouse as a gene regulator, the expression of the microRNA was increased in the liver and the expression of the aging-related marker protein was increased , The microRNA of the present invention can be usefully used as a pharmaceutical composition for reverse aging.
Description
TECHNICAL FIELD The present invention relates to a technique for extracting aging biomarkers existing in a circulatory system to induce reverse aging of a specific organ.
Aging is a process in which the structure and function of the body gradually deteriorates as the age increases and the sensitivity to disease and death increases drastically.
The aging index, which represents the aging phenomenon, has been mainly focused on analyzing the indicators for aging diseases such as diabetes, hypertension and dementia, and studying the mechanism. Therefore, there have been developed anti-aging techniques for delaying aging by treating inhibitors and the like of substances inducing aging, and researches on methods for predicting and diagnosing aging have been widely tried.
Korean Patent Application No. 10-2009-0058459, for example, discloses an aging diagnostic method for evaluating protein expression as an index and a biomarker for aging diagnosis. Korean Patent Application No. 10-2013-0031124 discloses a method for diagnosing muscle aging using protein And the like. However, the conventional diagnostic method as described above is difficult to obtain a consistent result as a diagnostic method using unstable proteins in the surrounding environment such as temperature and pH.
Anti-aging is the prevention, management, and treatment of physical diseases caused by aging, prevention light management and aging. It is widely applied to medicine, food, cosmetics and medical devices. On the other hand, reverse aging technology refers to a technique of restoring the aging state by originally regulating the expression of a genetic element that induces senescence.
For the development of this reverse aging technique, it is possible to obtain more information about the related aging genetic factors and to have an in-depth understanding of its mechanism.
microRNA (miRNA, miRNA) is a 21-25 nt single-stranded RNA molecule that binds to the 3'-UTR of mRNA to control the expression of eukaryotic genes (Bartel DP et al., Cell. 2004.01.23; 116 (2): 281-297). The production of miRNA is made into a stem-loop-structured precursor micorRNA (pre-microRNA) by Drosha (RNase III type enzyme) and is transferred to the cytoplasm and cleaved by Dicer to produce mature microRNA [Kim VN et al ., Nat Rev Mol Cell Biol. 2005. 05; 6 (5): 376-385). MicroRNAs prepared as described above are involved in development, cell proliferation and death, lipid metabolism, tumor formation, etc. by controlling the expression of target proteins (Wienholds E et al., Science, 309 (5732): 310-311 ; Nelson P et al., Trends Biochem Sci., 28: 534-540 (2003); Lee RC et al., Cell, 75: 843-854 (1993); and Esquela-Kerscher A et al., Nat Rev Cancer 6: 259-269 (2006)).
The present invention relates to a technique of identifying an aging mechanism regulated by a circulating gene and inducing reverse aging by administering such a regulatory substance in vivo. These technologies are in the concept phase all over the world and they are the first technology to induce reverse aging by regulating the expression of aging genes of specific organs by administration of specific genetic factors. It will be an important key to induce the reverse aging of the human body by using materials and contribute to healthy life and prolongation of life.
It is an object of the present invention to provide a technique for identifying aging-related gene expression regulating substances and inducing reverse aging of specific organs using the same. Such a method is intended to provide a method for treating aging-related diseases through treatment of diseases caused by aging, and further, by regulating aging genes.
In order to achieve the above object, the present invention provides a microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA- microRNA-21a-5p, and microRNA-30a-5p as an active ingredient.
The present invention also relates to a microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, microRNA-21a-5p, and microRNA-30a-5p as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating an aging-type disease.
The present invention also relates to a microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, a DNA probe or an aptamer which binds complementarily to at least one microRNA selected from the group consisting of microRNA-21a-5p, and microRNA-30a-5p.
The present invention also relates to a microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, Provided is a kit for diagnosing or monitoring an aging or an aging-type disease comprising complementary and amplifiable DNA primers to at least one microRNA selected from the group consisting of microRNA-21a-5p and microRNA-30a-5p.
Further, according to the present invention,
1) In vitro microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, microRNA-21a-5p, and microRNA-30a-5p; And
2) determining that the degree of senescence increases as the level of expression in step 1) is decreased or that the risk of developing an aging-type disease is high;
Further, according to the present invention,
1) microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA- treating the test substance with a cell line expressing at least one microRNA selected from the group consisting of microRNA-21a-5p, and microRNA-30a-5p;
2) measuring the expression level of the microRNA in the cell line; And
3) screening candidate substances for the prevention of aging or aging-type diseases, comprising the step of selecting a test substance whose degree of expression of the microRNA is increased as compared with a control group not treating the test substance.
The present invention also relates to a microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, microRNA-21a-5p, and microRNA-30a-5p.
The present invention also relates to a microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, microRNA-21a-5p, and microRNA-30a-5p to at least one microRNA to induce reverse aging of cells.
The present invention relates to a technique for inducing reverse aging of a specific organ by identifying aging markers present in the circulatory system. In the present invention, an aging mechanism regulated by a circulating gene is identified, and the regulatory substance is administered in vivo We have developed a technique to induce aging. Conventionally, it has been focused on analyzing the index of aging-type diseases such as diabetes, hypertension and dementia, and studying the mechanism thereof. It is also known that anti-aging techniques for delaying aging by treating inhibitors of substances inducing aging Has been developed. Reverse aging, on the other hand, refers to a technique of restoring the aging state by regulating the expression of a genetic element that induces senescence. The present invention was the first invention in the world that was invented as a technique for inducing reverse aging by regulating the expression of an aging gene of a specific organ by administering a specific gene. These technologies will be important key to contribute to healthy life and life extension by inducing the reverse aging of the human body by using various aging gene control materials obtained from research on aging mechanism in the future.
FIG. 1 is a schematic diagram showing a method of next generation sequencing (NGS) analysis of RNA isolated from mouse blood.
FIG. 2 is a diagram showing an analysis of a network involved in miRNA DEGs analyzed with agarose solution. FIG.
Figure 3 is a diagram showing diseases and disorders that are likely to occur based on circulating miRNA DEGs in the blood of aged mice.
4 is a graph showing the amount of miRNA change in the blood of aged mice using the qPCR method.
Fig. 5 shows the histological results of aging mice. Fig.
Fig. 6 is a graph showing the results of confirming the amount of miRNA change between aged mice by the qPCR method. Fig.
FIG. 7 is a graph showing the result of western blot analysis of the amount of protein change between aging mice. FIG.
FIG. 8 is a graph showing the intra-mouse liver and lung miRNA concentrations after injection of gene regulators into 18-month-old mice. miRNA (1) is a single injection of microRNA-21a-5p and miRNA (6) is microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, microRNA- And microRNA-30a-5p were mixed and injected at once.
FIG. 9 is a graph showing changes in protein expression in mice after 18 months old mice were injected with gene regulators. FIG. miRNA (1) is a single injection of microRNA-21a-5p and miRNA (6) is microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, microRNA- And microRNA-30a-5p were mixed and injected at once.
FIG. 10 is a graph showing changes in the expression pattern of aging-related marker proteins in mice after 18-month-old mice were injected with gene regulators. miRNA (1) is a single injection of microRNA-21a-5p and miRNA (6) is microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, microRNA- And microRNA-30a-5p were mixed and injected at once.
FIG. 11 shows the result of confirming telomerase activity of mouse after qPCR method after injecting gene control substance into 18-month-old mouse. miRNA (1) is a single injection of microRNA-21a-5p and miRNA (6) is microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, microRNA- And microRNA-30a-5p were mixed and injected at once.
Hereinafter, the present invention will be described in detail.
The present invention relates to microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, microRNA-21a-5p, and microRNA-30a-5p as an active ingredient.
The microRNA-16-5p comprises the nucleotide sequence of SEQ ID NO: 1, the microRNA-130b-3p comprises the nucleotide sequence of SEQ ID NO: 2, and the microRNA-17-5p comprises the nucleotide sequence of SEQ ID NO: Wherein the microRNA-103-3p comprises the nucleotide sequence of SEQ ID NO: 4, and the microRNA-21a-5p comprises the nucleotide sequence of SEQ ID NO: 5, The microRNA-30a-5p comprises the nucleotide sequence of SEQ ID NO: 6.
The microRNA can be isolated from mouse and human body tissues and tissues, whole blood, serum, blood cells, spleen, urine and the like. According to a specific embodiment of the present invention, it is most preferable to separate the microRNA from whole blood. Whole blood is most suitable as a clinical sample because it is easy to secure, easy to manipulate, small wound, small pain and danger to patients.
According to a specific embodiment of the present invention, the microRNA is a microRNA which consistently changes in 2, 6, and 12 months of a mouse, It is involved in networks involved in organ disease and its expression decreases as the aging progresses.
The reverse aging means the anti-aging of the concept of preventing and treating the aging, and regulating the expression of a genetic factor inducing aging differently, thereby restoring the aging state to the previous state.
The pharmaceutical composition for reverse aging comprises microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, microRNA-21a-5p, and microRNA-30a-5p. It is most preferable that the microRNA is a mixture of six microRNAs.
In a specific example of the present invention, the present inventors isolated microRAN from 2, 6, and 12 months old mouse whole blood and performed NGS (next generation sequencing) analysis to select 27 mircroRNAs that changed consistently according to
The microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, one or more microRNAs selected from the group consisting of microRNA-21a-5p and microRNA-30a-5p are included in complex form with various nucleic acid carriers (viral or nonviral carriers) known in the art to enhance in vivo delivery efficiency . For example, as a recombinant plasmid or viral vector expressing the microRNA. Plasmids usable for this purpose include pSilencer (Ambion), pSiEx (Novagen), siXpress (Takara Bio), pBLOCK-iT (Invitrogen), pcDNA3.1 (Invitrogen), pCEP4 (Invitrogen), SilenCircle Allele, and the like. As the viral carrier, retroviral vectors, adenovirus vectors, adeno-associated viral vectors, vaccinia virus vectors, lentivirus vectors, herpes virus vectors, alphavirus vectors, EB virus vectors, papilloma virus vectors, , But are not limited to. As a non-viral carrier, it is also possible to use Mirus TrasIT-TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, G-fectin, cationic phospholipid nanoparticles, cationic macromolecule, cationic micelle , Cationic emulsions or liposomes, ligand-DNA complexes, geneguns, and the like. The form of the liposome is combined with an amphipathic agent, such as a micelle, an insoluble monolayer, a liquid crystal, or a lipid present as a lamellar layer present in an aqueous solution. Lipids for liposomal formulation include, but are not limited to, monoglyceride, diglyceride, sulfatide, lysolecithin, lecithin phospholipid, saponin, bile acid, lipofectin, and the like.
In order to enhance the stability of the microRNA of the present invention, biocompatible polymers such as polyethylene glycol may be conjugated to increase intracellular absorption, so that it can be formulated so as to utilize general ribonucleic acid intracellular delivery techniques known in the art. The method also includes suspending the cells in a microRNA-containing solution by electroporation and introducing them into cells by passing a pulse of a DC high voltage. In the living body, the solution containing the microRNA can be administered to the desired site, and the DC voltage can be pulsed through the electrode to be introduced into the cell.
The composition of the present invention may contain 0.1 to 99.9% by weight of the pharmaceutical composition of the present invention as an active ingredient based on the total weight of the composition, and may include a pharmaceutically acceptable carrier, excipient or diluent.
The compositions of the present invention may be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of suppositories include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
The composition of the present invention may be administered orally or parenterally, and it is preferable to select the intraperitoneal, rectal, rectal, intravenous, intramuscular, subcutaneous, intrauterine or intracerebral injection methods during parenteral administration.
The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.
The dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, , Preferably 30 to 500 mg / kg, more preferably 50 to 300 mg / kg, and may be administered 1 to 6 times a day.
The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.
The present invention also relates to a microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, microRNA-21a-5p, and microRNA-30a-5p as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating an aging-type disease.
The aging-type disease is preferably hepatic steatosis, diabetes, hypertension and dementia.
The present invention also relates to a microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, a DNA probe or an aptamer which binds complementarily to at least one microRNA selected from the group consisting of microRNA-21a-5p, and microRNA-30a-5p.
The present invention also relates to a microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, Provided is a kit for diagnosing or monitoring an aging or an aging-type disease comprising complementary and amplifiable DNA primers to at least one microRNA selected from the group consisting of microRNA-21a-5p and microRNA-30a-5p.
The kit for diagnosing or monitoring an aging or aging-type disease according to the present invention comprises microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA- microRNA-21a-5p, and microRNA-30a-5p, or by measuring changes in the expression levels of DNA probes, aptamers or primers complementarily binding to the expression of one or more microRNAs selected from the group consisting of microRNA- have.
Further, according to the present invention,
1) In vitro microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, microRNA-21a-5p, and microRNA-30a-5p; And
2) determining that the degree of senescence increases as the level of expression in step 1) is decreased or that the risk of developing an aging-type disease is high;
The measurement of the expression level of the microRNA can be performed by various methods known in the art. For example, RT-PCR, Northern blotting, hybridization using a cDNA microarray, or in situ hybridization reaction can be used. Preferably by real-time PCR or immunoblotting.
Further, according to the present invention,
1) microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA- treating the test substance with a cell line expressing at least one microRNA selected from the group consisting of microRNA-21a-5p, and microRNA-30a-5p;
2) measuring the expression level of the microRNA in the cell line; And
3) screening candidate substances for the prevention of aging or aging-type diseases, comprising the step of selecting a test substance whose degree of expression of the microRNA is increased as compared with a control group not treating the test substance.
The screening was performed using microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA- microRNA-21a-5p, and microRNA-30a-5p, wherein the test substance is selected from the group consisting of a natural compound, a synthetic compound, It is preferably a group consisting of RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, antigens, bacterial or fungal metabolites and bioactive molecules.
The present invention also relates to a microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, microRNA-21a-5p, and microRNA-30a-5p.
The present invention also relates to a microRNA-16-5p, microRNA-130b-3p, microRNA-17-5p, microRNA-103-3p, microRNA-21a-5p, and microRNA-30a-5p to at least one microRNA to induce reverse aging of cells.
Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.
However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.
< Example 1> In aging mouse blood miRNA DEGs analysis
2, 6, and 12 months C57 / bl6 mice were purchased from the Central Laboratory Animals, and RNA was isolated from the whole blood of each individual to perform next generation sequencing (NGS) analysis.
Specifically, as shown in FIG. 1, Illumina HiSeq 2000/2500 was used, TruSeq Small RNA Library Prep Kit was used, and Agilent DNA 1000 kit was used. biotin labeled cRNA synthesis, cRNA fragmentation, expression array hybridization, washing, staining using Genechip Fluidics Station 450, Scanning through Genechip Array scanner 3000 7G, and Data Analysis.
As a result, as shown in Table 1, 27 microRNAs which consistently changed 4 or more were selected when comparing 2 month old and 6 month old and 6 month old and 12 month old (Table 1) . The list of miRNAs listed in Table 1 was uploaded to an IPA program as an input file using IPA (Ingenuity pathway analysis, Quiagen, http://www.engenuity.com/) based on the differentially expressed genes (DEG) , And "Core analysis" provided by the program. 10b-5p, miR-29b-3p, miR-17-5p, miR-16-5p, and miR-16b) of 27 DEGs, which are networks involved in cancer, organ damage and abnormalities, The DEG of the miR-130b-3p, miR-103-3p, miR-21a-5p, miR-148a-3p, miR-30a-5p and miR-181b-5p was analyzed to be contained in this network.
(2 months vs. 6 months)
(6 months vs. 12 months)
< Example 2> Aging Mouse In the solution Circulating miRNA To DEGs Analysis of major functional changes
Based on the miRNA DEG of the mouse blood whose expression was changed according to the aging identified in Example 1, IPA analysis was performed to derive the major changes of the organisms according to their expression.
Specifically, IPA analysis was performed to identify diseases and disorders highly related to the miRNA identified in Example 1 above. For this purpose, the list of miRNAs listed in Table 1 above was uploaded to an IPA program as an input file, and the "Core analysis" provided by the program was performed.
As a result, as shown in FIG. 3, the expression of miRNA DEG (miR-16-5p, miR-130b-3p, miR-17-5p, miR- The major function of related organisms of miR-21a-5p, miR-30a-5p) was analyzed to be highly correlated with hepatic steatosis. It is also known that miR-103-3p and miR-21a-5p are involved in the lyophilization, as shown in Table 2 below, confirming the regulatory proteins (E2F2, PPARA, AKT) have.
< Example 3> Aging miRNA Analysis of expression changes
Real-time quantitative PCR (Real-time Quantitative PCR) was used to determine how the miRNA expression in the blood of the 3, 8, 12 and 24 month old mice was altered in the blood according to the above Example 1 Respectively.
Specifically, RNA (total RNA) contained in mouse blood of 3, 8, 12 and 24 months was isolated and used by Exiqon (USA) miRCURY LNA ™ Universal RT microRNA PCR Kit and Applied Biosystems 7500 Real-Time PCR System Experiments were conducted according to the manual. Table 3 below shows the sequences of miRNAs identified in this experiment.
As a result, as shown in Fig. 4, miRNA expression was significantly decreased with aging (Fig. 4).
< Example 4 > Histological Changes in Mouse Mice Altered by Aging
8, 12 and 24 month old mice were analyzed using immunohistochemical staining to elucidate the functional changes in aging mice.
Specifically, 8, 12 and 24 month-old mice were autopsied to isolate hepatic tissues and then immobilized in 10% formalin. After paraffin embedding, paraffin mouse liver tissue was stained with 3 To 5 [mu] l in thickness, and H & E staining was performed to observe lesions at a ratio of 100-fold and 200-fold.
As a result, as shown in Fig. 5, it was confirmed that lesions of hepatic steatosis were increased as the aging progressed (Fig. 5).
< Example 5> Aging miRNA Analysis of expression changes
The aging-related miRNAs identified in Example 1 were examined for expression in liver of mice at 3, 8 and 12 months.
Specifically, RNA (total RNA) contained in mouse blood of 3, 8, 12 and 24 months was isolated and confirmed by qPCT and Western blotting. QPCR was performed by the same primer and method as in Example 3 Respectively.
Western blotting was performed by Western blotting in the presence of aging-related miRNAs identified in Example 1 as shown in Table 2. The Western blot analysis was performed in accordance with a previous study (Lee KM et al. Acta. Biomater., 9: 5600, 2013). The protein in mouse liver tissue was pulverized with a RIPA buffer (Sigma-Aldrich), and left for 30 minutes. Min for centrifugation to obtain protein. The primary antibodies used were BCL-2, PPARA, E2F1, E2F2, AKT, CCND1, Smad2 / 3 and beta-actin antibodies purchased from the US Cell Signaling Technology. Horseradish peroxidase conjugated secondary antibody was used. To confirm the expression level of the protein, the membrane subjected to western blotting was analyzed according to the manufacturer's protocol using an enhanced chemiluminescence kit (Thermo Scientific, USA).
As a result, as shown in FIG. 6, it was confirmed that miRNA expression was significantly decreased as the aging progressed as in Example 3 (FIG. 6), and the related regulatory proteins were changed according to aging (Fig. 7).
< Experimental Example 1> Analysis of aging index change after administration of gene regulatory substance
MiRNAs whose expression is decreased according to the aging identified in the present invention were administered to mice as gene regulators and then their changes were confirmed.
Specifically, miR-16-5p, miR-130b-3p, miR-17-5p, miR-103-3p, miR-30a-5p and miR-21a-5p mimic were prepared from Bionea Respectively. miR-21a-5p mimic was injected alone at a concentration of 10 nanomoles, and each of the 6 miRNA mimics was mixed at 10 nanomoles each. For artificial injection, the liver in vivo Transfection Reagent of Altogen Biosystems was used to administer to 18-month-old mouse abdominal cavity, and the changes of aging indexes in specific organs after 24 hours were analyzed. In the three groups in which the gene regulatory substance was not administered, the group in which six gene regulatory substances were administered at the time of administration, and the group in which miR-21a-5p was administered alone, the same method as that of Example 3 and Example 5 Lt; / RTI > and qPCR were performed.
As a result, as shown in FIG. 8 and FIG. 9, it was confirmed that the amount of miRNA expression was increased in the group to which the gene regulatory substance was administered compared with the group without gene regulatory substance (FIG. 8) It was confirmed that the expression of the protein expressed in the liver was regulated by the gene regulating substance (FIG. 9).
< Experimental Example 2> Verification of Expression Change of Aging Protein
In
Specifically, in the <Experimental Example 1>, the liver of the mouse to which the gene regulating substance was administered was pretreated and western blotting was carried out in the same manner as in <Example 5>. The agar-related labeled protein antibody used in the Western blot was purchased from Bioworld Technology, Inc. of the United States and purchased from SIRT1 of abcam.
As a result, as shown in Fig. 10, it was confirmed that aging-related labeled proteins mTOR and SIRT1 showed reverse aging results by injection of gene regulating substances (Fig. 10).
< Experimental Example 3> Verification of Expression Change of Aging Protein
In Example 1, telomerase activity in which telomere was elongated in a mouse to which a gene regulating substance was administered was confirmed.
Specifically, in Experimental Example 1, RNA (total RNA) in mouse liver tissues injected with a gene regulatory substance was isolated and then qPCR was performed by synthesizing cDNA. The primers used for confirming the expression level of mRNA were as shown in Table 4 below and were prepared from mMion1, mMrella, mTep1, mTerf2, mRert and mTnks from Bionea Korea, strand cDNA Synthesis Kit and Applied Biosystems 7500 Real-Time PCR System.
(SEQ ID NO: 7)
(SEQ ID NO: 8)
(SEQ ID NO: 9)
(SEQ ID NO: 10)
(SEQ ID NO: 11)
(SEQ ID NO: 12)
(SEQ ID NO: 13)
(SEQ ID NO: 14)
(SEQ ID NO: 15)
(SEQ ID NO: 16)
(SEQ ID NO: 17)
(SEQ ID NO: 18)
As a result, as shown in FIG. 11, it was confirmed that the telomerase activity was decreased with aging, as compared with the report that the telomerase activity was increased with aging (FIG. 11). This result is limited to the liver tissues of rats and it seems to be different from the previous report.
<110> Daegu Gyeongbuk Institute of Science and Technology <120> Reverse-aging induced method using a circulating aging marker <130> 2015P-10-049 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> RNA <213> Mus musculus <400> 1 uagcagcacg uaaauauugg cg 22 <210> 2 <211> 22 <212> RNA <213> Mus musculus <400> 2 cagugcaaug augaaagggc au 22 <210> 3 <211> 23 <212> RNA <213> Mus musculus <400> 3 caaagugcuu acagugcagg uag 23 <210> 4 <211> 23 <212> RNA <213> Mus musculus <400> 4 agcagcauug uacagggcua uga 23 <210> 5 <211> 22 <212> RNA <213> Mus musculus <400> 5 uagcuuauca gacugauguu ga 22 <210> 6 <211> 22 <212> RNA <213> Mus musculus <400> 6 uguaaacauc cucgacugga ag 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mMen1 forward primer <400> 7 gcttcatcac aggcaccaaa 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mMen1 reverse primer <400> 8 gacagggcca gatggacatc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mMre11a forward primer <400> 9 ccaggatggc aatctcaaca 20 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> ≪ 223 > mMre11a reverse primer <400> 10 agggcacaga gggcatctg 19 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mTep1 forward primer <400> 11 cctttggccc cttaaaaatg a 21 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mTep1 reverse primer <400> 12 accgtggtag cctgtttttc tc 22 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mTerf2 forward primer <400> 13 gaacttggcc caccctgtta 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mTerf2 reverse primer <400> 14 gtagggctcc gtgtcatcca 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mTert forward primer <400> 15 acaagcccag ctccacatct 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mTert reverse primer <400> 16 ggccatctcc cctggagtaa 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mTnks forward primer <400> 17 ggctgtccca cacacaaaga 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mTnks reverse primer <400> 18 gccattttca tggtgctgaa 20
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