KR101133559B1 - NOVEL miRNA FROM HUMAN MESENCHYMAL STEM CELL - Google Patents

Abstract

The present invention relates to novel miRNAs or precursor miRNAs derived from human mesoderm stem cells, by regulating the activity of miRNAs or precursor miRNAs that have been specifically expressed in specific developmental stages or in specific tissues to control expression of target genes. The mechanism of cell differentiation and development can be identified as well as the process can be controlled. In addition, the present invention relates to a composition for regulating the activity of the miRNA or precursor-type miRNA derived from human mesoderm stem cells, and a method for regulating cell differentiation by regulating its activity.

Description

Novel MiRNA FROM HUMAN MESENCHYMAL STEM CELL from Human Mesoderm Stem Cells

The present invention relates to novel miRNAs derived from human mesodermal stem cells and precursor-type miRNAs thereof that can effectively identify cell differentiation and developmental mechanisms as well as regulate cell differentiation and developmental processes.

microRNAs (miRNAs) are small endogenous noncoding RNAs involved in many biological processes, including development, differentiation, proliferation, etc. (Laurent LC et al. 2008. Stem Cells. 26 (6): 1506 -1516). Recent studies suggest the importance of miRNAs. Mature miRNAs are about 19-25 nucleotides in length and were cleaved by Dicer Rnase III from 60- to 80-nucleotide double stranded RNA fold backs (dsRNA hairpin structures) (Grishok A et al. 2001 Cell. 106). (1): 23-34).

miRNA was predicted to bind up to 3 'untranslated regions (3'UTR) of target mRNAs to upregulate more than 30% of genes in the human genome (Stark A et al. 2007. Genome Res. 17 ( 12): 1865-79). In addition, miRNA has been shown to play an important role in stem cells as well as cancer, diabetes, viral infections, heart disease (Huang KM et al. 2008. Mamm Genome. 19 (7-8): 510-516). miRNA is also associated with the development of psoriasis and atopic eczema, the most common skin acute inflammatory diseases.

In lower organisms, after the first eggplant, they are assigned to the germ line. In mammals, however, germ lineages are isolated near the time of late cystogenesis during development (Gu P et al. 2008. PLoS ONE. 3 (7): e2548). In mammals, in the case of Dicer-/-(Dicer endonuclease gene defect), the embryo is killed at 7.5 on embryonic day, and ES (Dicer-/-) cells (Dicer-/-) are proliferated and differentiated And no mRNA maturation occurs (Hatfield SD et al. 2005. Nature. 435 (7044): 974-978). Recently, pyro sequencing of small RNAs isolated from ES and ES (Dicer-/-) has revealed 46 new miRNAs out of about 110,000 miRNA transcripts in ES cells, most of which have identified six distinct miRNA loci. Occupy. These miRNA loci or human homologues have been shown to play a major role in cell cycle regulation or tumorigenesis, suggesting that the main function of the miRNA pathway in ES cells is to define the cell cycle (Calabrese JM et al. 2007. Proc Natl Acad Sci US A. 104 (46): 18097-18102).

miRNAs have been known to bind to antisense complementarity sites in the 3 ′ untranslated region of target mRNAs to sequence-specifically regulate transcription of target mRNAs (Lagos-Quintana M et al. 2002. Curr Biol. 12: 735-739. ). miRNAs can bind to specific recognition sequences at 3'-UTR of 200 or more transcripts, and each mRNA can have recognition sequences for many miRNAs (Wienholds E et al. 2005. FEBS Lett. 579 (26): 5911-5922).

Embryonic stem cells (ESCs) are becoming a valuable resource for regenerative medicine because they have unlimited proliferation and pluripotency (Cho SW et al. 2007. Circulation. 116 (21): 2409-2419). ES cells have immortal totipotent, but the specific patterns of gene expression that contribute to this specific physiological state are unknown (Chen C et al. 2007. Mamm Genome. 18 (5): 316-327). Analyzing these cells reveals interesting facts that mammalian cell differentiation and genetic reprogramming are mediated by RNAi (Kanellopoulou C et al. 2005. Genes Dev. 19 (4): 489-501 ).

Functional studies of miRNAs have academic significance in that they can be a new breakthrough in understanding eukaryotic gene expression regulation. Functional studies of miRNAs are important for the study of diseases caused by functional defects of miRNAs. In addition, many miRNAs are known to regulate development and differentiation in cells. In this aspect, identifying and identifying the function of miRNA by separating and identifying the miRNA from stem cells is important in medical aspects as it can be used not only for the study of differentiation mechanism but also for cell therapy.

The present invention is to provide a miRNA that can effectively regulate the process of cell differentiation and development. In the present invention, new miRNAs were identified from human mesoderm stem cells, and the difference in expression of miRNAs in differentiation cells was analyzed to determine whether they influence the differentiation and development processes.

The present invention relates to miRNAs or precursor miRNAs derived from human mesoderm stem cells.

In the present invention, "pre-form miRNA" refers to a miRNA precursor having a stem-loop structural form before being cleaved by Dicer to become a miRNA. By “miRNA” is meant short non-coding RNA derived from an endogenous gene, which acts as a post-transcriptional regulator of gene expression. miRNA acts as a post-transcriptional regulator of gene expression by pairing bases with mRNA of the target gene. The precursor miRNA of the present invention is converted into miRNA through a series of processing in the cell. That is, the miRNA is processed from a longer (70-80 nt) hairpin form precursor called precursor-miRNA by Dicer, an RNAse III enzyme, which recognizes the target miRNA of the target gene by antisense complementarity Mediate downregulation.

In the present invention, "target (target) gene" or "target (target) mRNA" means an mRNA target site recognized by miRNA. Depending on the degree of complementarity between the “target gene” or “target mRNA” and miRNA, miRNAs inhibit the activity of target genes by a mechanism that cleaves target mRNAs or inhibits translation of target mRNAs into proteins.

The present invention relates to miRNAs that act on various target genes expressed during differentiation and / or development, specifically, human mesoderm stem cells selected from the group consisting of nucleic acids having nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 22 Provided are miRNAs derived or human precursors derived from human mesoderm stem cells selected from the group consisting of nucleic acids having nucleotide sequences of SEQ ID NOs: 23 to 44.

The expression of miRNA isolated in the present invention is observed to be different in human embryonic stem cells (ESCs), differentiated NPCs (Neuronal Precursor Cells) and endothelial cells (ECs). In quantitative analysis, most miRNAs were upregulated as they differentiated from ES to EC, while some miRNA expressions were upregulated and some miRNA expressions were downregulated as they differentiated into NPCs. From this, it can be seen that miRNA of the present invention affects gene expression and activity during cell differentiation and development.

In addition, the present invention can be used as a disease marker miRNA whose expression is increased or decreased in certain diseases. That is, the present invention provides a disease marker selected from the group consisting of miRNA having the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 22 and its precursor miRNA. One example of a disease marker is a miRNA or a precursor miRNA of the invention wherein the cancer marker is increased or decreased in cancer cells can be used as a cancer marker. In addition, miRNAs or their precursor miRNAs of the present invention whose expression is reduced or increased in certain differentiation processes and / or in certain tissues can be used as cell differentiation markers.

The present invention also provides a miRNA having increased or decreased expression in somatic cells, which is selected from the group consisting of miRNA having a nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 22 and its precursor miRNA. MiRNA of the present invention or its precursor-type miRNA may increase or decrease expression in somatic cells as differentiation progresses. By investigating the differentiation relationship between expression patterns of differentiation miRNAs and target mRNAs of the present invention, dynamic expression information analysis of genes, large scale network construction, and integrated analysis of miRNA and mRNA expressions are possible.

The present invention also provides a miRNA for regulating aging of a cell selected from the group consisting of a nucleic acid having a nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 22 and its precursor type miRNA. The miRNA of the present invention or its precursor type miRNA acts on aging of cells, for example, senescence of mesodermal stem cells and the like.

In addition, the expression and distribution of miRNAs or their precursor miRNAs of the present invention not only regulate cell differentiation and development, but are also associated with the development of cancer, viral infections, inflammatory diseases, metabolic diseases, and the like. Therefore, the miRNA of the present invention or its precursor miRNA may be a target of disease treatment, and manipulation to miRNA has therapeutic potential. More specifically, miRNAs of the present invention and their precursor miRNAs are involved in the development of diseases such as cancer or stroke and have therapeutic potential for these diseases. The present invention also regulates miRNAs and their precursor miRNA cell death.

miRNAs function by specifically selecting, binding, or reacting to target residues in cells. Thus, the expression and activity of the miRNA-targeted genes can be enhanced in a manner that increases or decreases the intracellular expression of the miRNA, or in a manner that increases or decreases the intracellular activity of the miRNA. Can be controlled. In view of the above, the present invention provides a composition for regulating cell differentiation as follows.

(i) The present invention relates to a miRNA derived from a human mesoderm stem cell selected from the group consisting of nucleic acids having a nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 22 or a nucleic acid having a nucleotide sequence of SEQ ID NO: 23 to SEQ ID NO: 44 It relates to a composition for regulating cell differentiation comprising a precursor-type miRNA derived from selected human mesoderm stem cells.

(ii) The present invention provides a vector expressing a miRNA derived from a human mesoderm stem cell selected from the group consisting of nucleic acids having a nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 22 or a nucleic acid having a nucleotide sequence of SEQ ID NO: 23 to SEQ ID NO: 44 It relates to a composition comprising a vector expressing a precursor miRNA derived from human mesoderm stem cells selected from the group consisting of.

(iii) The present invention relates to a composition for regulating cell differentiation comprising an agent for regulating the activity of miRNAs derived from human mesoderm stem cells selected from the group consisting of nucleic acids having the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 22.

Specifically, the compositions of (i) and (ii) directly increase the concentration of miRNA or precursor type miRNA that will be processed in the cell to act as miRNA, resulting in inhibition of the activity of the target gene. .

The miRNA or precursor miRNA introduced into the cell is a miRNA of SEQ ID NO: 1 to SEQ ID NO: 44, or a nucleotide sequence in which some nucleotide residues have a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof Can have In addition, for the miRNA selected from SEQ ID NO: 1 to SEQ ID NO: 44, it may include additional nucleotides adjacent to the 5 'or 3' side.

The nucleotides that form the miRNAs or precursor miRNAs may also be nucleotide analogues that function similarly to nucleotides but have portions having non-naturally occurring moieties. Nucleotide analogs can have modified sugar moieties or sugar-to-sugar bonds. For example, there are phosphorothioates and other sulfur-containing species whose use is known in the art. Nucleotide analogs may also include species comprising at least slightly modified base forms. Thus, purines and pyrimidines other than those found in nature may be used. Such analogs may be functionally replaced with natural nucleotides (or synthesized nucleotides according to the natural class). Nucleotide analogues generally have a structure similar to natural nucleotides that can bind to the target, but have modifications, nuclease resistance that enhance the ability of miRNAs or precursor miRNAs to penetrate the intracellular space of the cells in which they reside. It can be modified to. Also the 5 'end cap, 3' end can be structurally or chemically modified.

In addition to the method of synthesizing extracellular and miRNA miRNAs or precursor miRNAs, miRNAs derived from human mesoderm stem cells of SEQ ID NOs: 1 to 22 or precursor miRNAs of SEQ ID NOs: 23 to 44 are expressed temporarily or permanently in cells. There is a method of transforming or infecting a miRNA expression vector into a cell.

In the present invention, "vector" means a means for introducing a nucleic acid sequence (eg, DNA, RNA, etc.) encoding miRNA or precursor miRNA into a host cell, and may have the form of a virus (viral particle). . The vector of the present invention includes a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, an enhancer, and can be variously produced according to the purpose. . The expression vector also includes a selectable marker and, in the case of a replicable expression vector, a replication origin. The vector can either replicate itself or integrate into the host DNA. Vectors of the present invention can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art.

The vector of the present invention includes both viral vectors and nonviral vectors such as plasmid vectors and cozmid vectors. Viral vectors include, but are not limited to, adenovirus vectors, adenovirus (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors. The composition of the present invention is more preferably provided as a virus (viral particle).

In the present invention, the agent that modulates the activity of the miRNA is used to inhibit, block, destroy, or reduce the biological activity of the miRNA or precursor miRNA by acting on the expression, processing, or binding process of the miRNA or precursor miRNA. Inhibitors and promoters that increase the biological activity of the miRNA or precursor miRNA. The mechanism that acts as an inhibitor or promoter in the cell is not particularly limited, and examples thereof include a substance that affects transcription and processing processes, a substance that degrades, and a substance that changes or converts an active form into an inactive form. . Such modulators include single compounds, such as organic or inorganic compounds; Biopolymer compounds such as proteins, nucleic acids, carbohydrates and lipids; And complexes of plural compounds.

One embodiment of the miRNA inhibitor is a miRNA specific antisense nucleic acid, wherein inhibition of miRNA activity results in increased activity of the target gene.

As used herein, the term “antisense nucleic acid” refers to an oligonucleotide that is a DNA or RNA or a derivative thereof comprising a nucleic acid sequence complementary to a sequence of a particular miRNA, and binds to a complementary sequence in the miRNA to translate the miRNA to a protein. It has the characteristic to inhibit. The sequence and length of the antisense nucleic acid are not particularly limited. In order for the antisense nucleic acids of the present invention to efficiently inhibit target miRNAs, the antisense nucleic acids of the present invention can modulate the degree of complementarity between target miRNAs. For example, one or more sequences that do not correspond to miRNAs may be included in one or more forms, and the sequences corresponding to some sequences that do not correspond to one another may be produced.

It is based on the finding that using oligonucleotides designed to bind with high affinity to miRNA targets of the present invention very efficiently mitigates the inhibition of mRNA by miRNA in vivo. Targeting miRNAs with high efficiency in vivo can be accomplished by designing antisense nucleic acids that form duplexes that are highly stable with miRNA targets in vivo.

The composition of the present invention comprising an antisense nucleic acid of the present invention provides a method of de-repression of a target mRNA of miRNA in a cell or organism.

Antisense nucleic acids of the invention can be designed such that the miRNA target is not recruited by an RNA-induced silencing complex (RISC) or mediates RISC-directed cleavage. Antisense nucleic acids can compete with target mRNAs in terms of RISC complex binding, thereby mitigating miRNA inhibition of miRNA target mRNAs by the introduction of antisense nucleic acids that compete as substrates for miRNAs.

In addition, the present invention provides a nucleic acid sequence capable of almost irreversibly binding to miRNA, thereby preventing such undesirable target mRNA cleavage and translation inhibition into proteins. In addition, forms can be constructed that protect against degradation or cleavage (eg, RISC or RNAseH or other endo or exo nucleases).

In addition, antisense nucleic acids can be modified at any position for the purpose of increasing specificity, stability and the like. In particular, one or more bases, sugars or backbones may be optionally modified. For example, the nucleic acid backbone can be modified with phosphorothioate, phosphoroester, methyl phosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic intersaccharide linkages, and the like. In addition, antisense nucleic acids may comprise one or more substituted sugar moieties. Antisense nucleic acids can include modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-me pyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosil HMC, 2-aminoadenine, 2 Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine, etc. There is this. In addition, the antisense nucleic acids of the present invention may be chemically bound to one or more moieties or conjugates that enhance the activity and cellular adsorption of the antisense nucleic acids.

In the case of antisense RNA, it may be synthesized in vitro in a conventional manner to be administered in vivo or to allow antisense RNA to be synthesized in vivo. One example of synthesizing antisense RNA in vitro is using RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose origin of the recognition site (MCS) is in the opposite direction. Such antisense RNA is desirable to ensure that there is a translation stop codon in the sequence so that it is not translated into the peptide sequence. Vectors that can be used are as described above.

In addition, the present invention comprises (i) a miRNA derived from a human mesoderm stem cell selected from the group consisting of a nucleic acid having a nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 22 or a nucleic acid having a nucleotide sequence of SEQ ID NO: 23 to SEQ ID NO: 44 A vector or sequence number expressing a miRNA derived from a human mesoderm stem cell selected from the group consisting of a precursor miRNA derived from a human mesoderm stem cell selected from the group, and (ii) a nucleic acid having a nucleotide sequence from SEQ ID NO: 1 to SEQ ID NO: 22 A vector expressing a precursor miRNA derived from a human mesoderm stem cell selected from the group consisting of nucleic acids having a nucleotide sequence of 23 to SEQ ID NO: 44, or (iii) a nucleic acid having a nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 22 Phosphorus selected from group The present invention relates to a method for controlling cell differentiation by treating an agent or a composition comprising the same, which regulates the activity of miRNAs derived from hepatic mesodermal stem cells.

Such cell differentiation control can control the activity of specific miRNAs and target genes regulated by miRNAs at each stage of differentiation, thereby effectively controlling cell differentiation. Accordingly, it is possible to understand the mechanism of differentiation and development according to the expression of target genes regulated by miRNA, and based on this understanding, it is possible to study diseases caused by a defect in metabolism or function of the target gene. And based on these disease research results, it is possible to treat and prevent disease.

Due to the present invention, it was confirmed that the mesodermal mesenchymal stem cell-derived miRNA acts as a regulatory factor in the process of cell differentiation. In addition, by regulating the activity of the miRNA of the present invention or its precursor-type miRNA that is specifically expressed at a specific developmental stage and controls the expression of a target gene, not only cell differentiation and developmental mechanisms can be identified, but also the process can be effectively controlled. have.

Hereinafter, the present invention will be described in detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1-Materials and Methods

1-1: Culture of Mesenchymal Stem Cells

Human bone marrow-derived mesenchymal stem cells were purchased from Cambrex Bio Science (Warlkersville, MD, USA). Cells were α-MEM supplemented with 10% FBS (Fetal bovine serum; Hyclone, Logan, UT, USA) and 20 μg / ml gentamicin (Invitrogen, Carlsbad, CA, USA) (Invitrogen, Carlsbad, CA, USA). US).

1-2: human ES  Cell culture

Two human ES cell lines (CHA-hES3, Cha Medical University, Korea) were used (for specific details see Ahn, SE et al. 2006. Biochem Biophys Res Commun 340: 403-408.). Human ES cells are 100 mM MEM non-essential amino acid, 100 U / ml penicillin, 0.1 mg / ml streptomycin, 55 mM β-mercaptoethanol, 20% Knockout Serum Replacement and 4 ng / ml recombinant human Cultured on mitotically inactivated STO cells (ATCC CRL-1503) in ES cell medium consisting of DMEM / F12 (1: 1) supplemented with bFGF (recombinant human basic fibroblast growth factor) (R & D systems, USA). . Feeder cells are supplemented with 100 mM MEM non-essential amino acids, 100 U / ml penicillin, 100 μg / ml streptomycin and 55 mM β-mercaptoethanol, and 10% FBS (FBS, Hyclone, Logan, UT) Incubated in DMEM. Medium was changed daily. Human ES cells were passaged at 7 day intervals over freshly prepared STO cells using flame sterilized Pasteur pipettes. All cultures were maintained in 37 ° C., 5% CO ₂ air. Unless otherwise stated, all formulations were purchased from Gibco BRL.

1-3: Differentiation of human ES cells into neural precursor cells

Differentiation of human ES cells into neuroprogenitor cells was performed according to the previously described report (Kim S et al. 2004. Reprod Dev Biol 28 (4): 247-252). In brief, EB (embryo body) formation can be achieved by replacing ES cell clumps with N2 adjuvant (Gibco BRL) in a bacterial plate coated with F-127 (Sigma, St. Louis, MO, USA). , BFGF consisting of DMEM / F12 (1: 1) supplemented with B27 adjuvant (Gibco BRL) and 2 mM L-glutamine (Gibco BRL), and neurobasal medium (Geurcosal medium; Gibco BRL, Gaithersburg, MD, USA) 10 ng / ml) in suspension in N2B27 medium (Ying QL et al., 2003. Nat Biotechnol 21: 183-186). The medium was changed once every two days. If EB (Embryoid body) was cultured for 14 days, the medium was changed every other day. The 14 day old EBs were exchanged into culture plates coated with 0.2% gelatin and incubated for 14 days in neurosphere (NS) medium. NS medium is 2 mM-glutamine, 25 μg / ml human insulin (Sigma), 100 μg / ml human transferrin (Sigma), 20 nM progesterone (Sigma), 60 μM putrescine (Sigma), 30 nM selenite ( Sigma), 2 μg / ml heparin (Sigma), recombinant human EGF (epidermal growth factor; R & D Systems) and DMEM / F12 (1: 1) supplemented with 20 ng / ml FGF-2. The medium was changed once every two days. After 7 days of culture, neuroepithelium cells with rosette structures were removed from the medium and transferred to F-127 coated plates in NS medium (Zhang SC et al. 2001. Nat Biothechnol 19: 1129- 1133). Neurospheres were formed in suspension medium within 24 hours, and neurospheres were incubated for 7 days before use. The medium was changed once every two days.

1-4: Differentiation of Human ES Cells into Endothelial Cells

Isolation and characterization of endothelial cells, CHA3-EC (Cha Medical University, Korea), was performed using the method described in the previous paper (Cho, SW, et al. 2007. I Circulation 116: 2409-2419.) Undifferentiated hESC (CHA-3hESC line, Cha Medical University, Korea), MEM / F12 (50:50; Gibco BRL) supplemented with 20% (v / v) serum replacement; And basic ES medium comprising 1 mM L-glutamine (Gibco), 1% non-essential amino acid (Gibco), 100 mM β-mercaptoethanol (Gibco) and 4 ng / ml bFGF (Sigma, St. Louis, MO) Were cultured on mitotically inactivated STO cells (ATCC CRL-1503). The medium was changed every 24 hours. hESCs were transferred to new feeder cells every 5-7 days using a desecting pipette. For EB (embryo body) formation, hESCs were isolated from feeder cells and suspended for 9 days in bFGF-free hESC culture medium. To isolate the central site, EB at day 9 was attached to a 0.2% gelatin coated dish for 7-9 days and the outgrowth region and its resulting using a desecting pipette. The central site was isolated and the isolated cells were cultured in defined medium (EGM-2; LONZA). Mouse anti-human monoclonal vWF antibody, using a FACS Vantage flow cytometer (BD Bioscience, San Jose, Calif., USA) to purify von Willebrand factor-positive cells from centrally isolated cells Cell sorting was performed with (Chemicon). Sorted cells were cultured in EGM-2, and cell passages were performed using trypsin-EDTA at 2-3 day intervals.

1-5: RNA isolation from obtained cells

Total RNA was extracted from human mesenchymal stem cells, obtained from three different donors. 1 × 10 ⁶ cells were mixed by pipetting with 500 μl TRI reagent (Molecular Research Center Inc., USA) in a 1.5 ml cap tube. 100 μl chloroform was added to the tube and vibrated for 10 seconds. After 5 minutes of incubation at room temperature, the mixture was centrifuged at 4 ° C and 13000 rpm for 10 minutes. After centrifugation, the separated supernatants were transferred to fresh RNAase-free-tubes. Samples were mixed by adding the same volume of cold isopropanol as the supernatant. The pellet was incubated for 20 minutes at -20 ° C and centrifuged at 4 ° C for 10 minutes at the highest speed. After centrifugation, the supernatant was discarded and the remaining pellet was washed with 70% ethanol and centrifuged twice with the previous conditions. RNA pellets were dissolved in DEPC-treated water and analyzed on a 1.2% agarose gel containing 0.1% ethidium bromide (EtBr) and the density was determined via nano drop using a spectrophotometer. For novel miRNA cloning, for RNA enrichment of less than 200 nt, mirVana RNA Isolation Kit (Ambion, Texas, USA) was used according to the manufacturer's instructions.

1-5: Cloning of New miRNAs

As mentioned above, total RNA was prepared from human mesenchymal stem cells to enhance new miRNA acquisition. Small RNA fragments of about 200 nucleotides in size were isolated from total RNA. The isolated small RNA was cloned into DynaExpressmiRNA cloning kit (BioDynamics Laboratory Inc. Japan) according to the manufacturer's instructions. Total RNA isolated from mesenchymal stem cells (MSC), used for cloning, was concentrated twice by acrylamide gel electrophoresis. Figure 1 schematically shows the cloning process of miRNA according to the present invention.

1-6: miRNA Folding  Structure discovery

Cloned miRNAs were analyzed for homology with the genome. Sequences with about 80-100 nucleotides around the miRNA were analyzed and matched at the folding structure prediction website ( http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi .).

1-7: cDNA synthesis by reverse transcription

The total RNA obtained in the previous step was prepared at a concentration of 1.0 mg, treated with DNase I (Roche, Basel, Switzerland) and incubated at 37 ° C. for 20 minutes and at 65 ° C. for 10 minutes to remove genomic DNA. Total RNA was reverse transcribed into cDNA using the Superscript ^™ First-strand Synthesis System as RT-PCR kit according to the manufacturer's instructions. The reaction mixture was added 0.25 ml of SuperScript II reverse transcriptase (200 units), 1 ml of oligo dT (0.5 μg / ml), 2 ml of 10x RT buffer, 2 ml of 0.1 M DTT and 1 ml of 10 mM dNTP. Incubate at 42 ° C. for 50 minutes and then incubate at 70 ° C. for 15 minutes. To prevent contamination by RNA, 1 μl of RNase H was treated with cDNA samples at 37 ° C. for 10 minutes after cDNA synthesis was terminated.

1-8: Analysis of miRNA Expression Using Taqman miRNA Assay

MiRNA gene expression of SEQ ID NOS: 1 to 13 was quantified by real time PCR analysis using TaqMan miRNA assay (Applied Biosystems, Foster City, CA) (FIG. 2A). Custom RT-primers were synthesized complementary to mature miRNAs and provided by a Custom TaqMan Small RNA assay (Applied Biosystems) (Chen, C. et al. 2005. Nucleic Acids Res 33: e179). Complementary DNA templates were normalized using RNU6B (Applied Biosystems, USA) according to the manufacturer's manual and subjected to 40 cycles of PCR analysis. Data was generated using the Rotor-Gene 6 program (Corbett Research, Sydney, Australia).

1-9: Analysis of miRNA expression using RT-PCR

Small RNAs isolated from hES cells, CHA-EC and CHA3-NPC were isolated using mir Vana miRNA separation kit. Expression of the miRNAs of SEQ ID NOs: 14-22 was performed by poly A tailing RT-PCR described in Ro et al. (Ro, S., C. et. Al. 2006. Biochem Biophys Res Commun 351: 756-763.) Quantification (Figure 2b). Small RNA molecules were polyadenylated using polyA polymerase according to the manufacturer's manual, and polyA tailed RNA was purified using Nucway spin column. Purified polyA tailed RNA was reverse transcribed into single-stranded cDNA using Superscript III transcriptase (Invitrogen) with RTQ adapter. The reverse primer of each miRNA is the same sample tailing sequence (RTQ-UNIr sequence of SEQ ID NO: 70 in Table 3), the forward primer is mature miRNA specific, and the RNU6B sequence used as a positive control is ctgcgcaagg atgacacgca aattcgtgaa gcgttccata ttttt (SEQ ID NO: 45).

Example 2-Results

2-1: miRNA isolation from human MSC

Total RNA was isolated from human MSC and concentrated twice using RNA precipitation. RNA was dissolved in DEPC solution to prevent from RNAse contamination. 447.3 μg and 404.2 μg of RNA, respectively, were obtained and isolated using urea-acrylamide gels. The region corresponding to the average length (18-24 nucleotides) of mature miRNA was purified and cloned. Artificially synthesized adapters for cloning new miRNAs (MI-5 '' linker: 5 'AUCGUCUCGGGAUGAAA (RNA-17mer; SEQ ID NO: 46) and MI-3' 'linker: 5' 'pCTGTAACTCGGGTCAATddC (DNA-18mer,' p '' Refers to phosphorylation residues, 'dd' means 2 ', 3'-dideoxy: SEQ ID NO: 47) is linked to isolated RNAs, and adapter-RNA is isolated by urea-acrylamide electrophoresis. Since the sum of the and 3 adapters is 35 nt (17 nt + 18 nt) and the mature miRNA is 18-25 nt, about 50-60 nt of miRNA was isolated, which was linked to the cloning vector. Proliferation was achieved in the cells, and the proliferated product was isolated to determine the sequence (Macrogen, South Korea) Using the protocol mentioned above, approximately 300 small RNA sequences were obtained from whole human MSCs (FIG. 1). .

2-2: Identifying new miRNAs using a web program

The novel miRNAs of the present invention met three conditions: size, stem-loop structure, and cell expression (Chen, C., et al. 2005. Nucleic Acids Res 33: e179; Lagos-Quintana, et al. 2001 Science 294: 853-858; Lai, EC, et al. 2003. C Genome Biol 4: R42). First, the newly cloned small RNA fragments were isolated 18 to 24 nt in length. Then, using a web program (NCBI blast, Genome browser) to confirm the location on the chromosome of these small RNA fragments. Precursor miRNAs were identified via modified stem-loop structures.

In the present invention, 22 novel miRNAs were identified, and the secondary structure of the precursor miRNA was predicted using the RNA fold program ( http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi ). (Fig. 3a), miRNA containing 18 ~ 24 nt was arranged by size (Fig. 3b). Sequences with stem-loop folding structures were classified as miRNAs and others as small non-coding RNAs. These sequences were compared with human gene sequences using computers and classified into two groups, miRNA or small RNA. Redundant sequences were removed and new miRNA sequences were identified through a web program (NCBI blast, Genome browser). The results are shown in Table 1 below. Precursor miRNA sequences corresponding to each mature miRNA are set forth in SEQ ID NOs: 23-44.

TABLE 1 Novel miRNA sequences, sizes, positions on chromosomes

As can be seen in Table 1 and FIG. 4A above, mi93 is on chromosome 1, mi234 is on chromosome 2, mi107 is on chromosome 3, mi174, mi87, mi144 and mi263 are on chromosome 11 , mi206, mi216 are on chromosome 12, mi81, mi168, mi2 and mi84 are on chromosome 14, mi242 is on chromosome 18, mi6, miS28 and mi90 are on chromosome 19, mi11 and mi77 are 22 It can be seen that it is on chromosome and mi9, mi89_2 and mi28 are located on X chromosome. It can be seen that the novel miRNA according to the present invention is particularly present on chromosomes 11 and 14. And it can be seen that the novel miRNA according to the present invention does not exist on 4, 5, 6, 7, 8, 9, 13, 15, 16, 17, 20, 21 and Y chromosome. In addition, mi81 and mi144 are found on two (mi81) or three (mi144) chromosomes with mature miRNA, respectively. Its multiple hits are shown in Table 2 below.

[Table 2] Multiple hit information of miRNA

2-3: miRNA specific PCR primers and probe construction

Twenty-two new miRNAs were quantified using Taqman miRNA assays (SEQ ID NOS: 1-13) and poly A tailed RT-PCR (SEQ ID NOS: 14-22) (Figures 5 and 6). Custom semi qRT-primers (Table 3) and PCR probes were made on request from Applied Biosystems (USA). In other words, the stem is a method for detecting a mature miRNA, a stem that adds a specific nucleotide sequence to a mature nucleotide sequence, rather than a linear primer form, which is a conventional method for specific priming effects. Loop-type primer probes were synthesized by Applied Biosystems. And miRNA expression was measured in each cell using this. First, 13 Taqman probes for mature miRNAs of SEQ ID NOS: 1 to 13 were purchased according to the manufacturer's protocol (Chen, C., et al. 2005. Nucleic Acids Res 33: e179.). Primer sets of nine miRNAs of SEQ ID NOs: 14-22 were then designed (Table 3, Bioneer, South Korea), and the amount of 9 miRNA expression in each cell was measured using SYBR Green (Applied Biosystems, USA).

TABLE 3 Primers

2-4: hES , CHA3 - EC  And CHA3 - NPC in miRNA  Expression

As described in Examples 1-3, the differentiation of hES cells into neuronal cells is called CHA3-NPCs, and the differentiation of hES cells into endothelial cells as described in Examples 1-4 is called CHA-ECs. The degree of intracellular expression of new miRNAs was expressed as a relative ratio between undifferentiated ES cells and their differentiated cells. The novel miRNAs of the invention showed expression differences in ES, EC and NPC. The results are shown in Table 3.

Table 4 Expression Differences in ES / EC / NPC of New miRNAs

As can be seen from the above table, when using the Tagman assay, most miRNAs except mi28 and mi107 were overexpressed in EC and NPC. In particular, mi111 and mi107 were not expressed in EC, mi111 was overexpressed in NPC, and mi107 was underexpressed in NPC (FIG. 5). In Poly A tailed PCR, new miRNAs were overexpressed in EC and NPCs, similar to the results of Taqman assays (FIG. 6). mi206, miS28, mi242 and mi89-2 showed different expressions depending on the cell type, which was overexpressed in EC and underexpressed in NPC. mi263 was underexpressed in both EC and NPC. These results indicate that novel miRNAs are differentially regulated in many stages of cell differentiation.

2-5: miRNA expression in various cell lines

How the novel miRNA of the present invention is expressed in various cell lines was confirmed by the aforementioned Taqman assay (Fig. 7-9) and poly A tailed RT-PCR (Fig. 10) method, the results are shown in Figs. 7 to 10, it was confirmed that the novel miRNA according to the present invention is expressed in several cell lines. From the results of Figure 7, it can be seen that the miRNA of the present invention can be used as lung cancer, a kind of cancer marker. Referring to FIG. 7, except for the error, the expression of mi87 is increased in adenocarcinoma of lung cancer cells, and the expression of mi87 and mi168 is increased in squamous cell carcinoma.

In addition, the present inventors performed experiments using blood cells of stroke patients in order to utilize the miRNA of the present invention as a stroke-specific marker in connection with a clinical professor research team of Bundang Cha Hospital for the diagnosis and treatment of diseases. 8, a number of new miRNAs were expressed in normal and patient groups in peripheral blood monocytes isolated from stroke patients, and expression of mi2, 9, 84, 87, 93, and 111 was increased in the patient group, and mi6, 77, In the case of 107 and 168, there is a pattern of decreased expression in the patient group. Therefore, it can be seen that the miRNA of the present invention is involved in the development of brain diseases such as stroke or cerebral infarction. 9, it can be seen that the miRNA expression of the present invention is different in the cell line expression, which suggests the possibility of using the miRNA of the present invention for cell differentiation regulation.

2-6: in senescent cells miRNA Expression of

      Taqman assay shows that the novel miRNA of the present invention is expressed in senescent cells (3 types-human mesenchymal stem cells (hMSC), lung-derived fibroblasts (WI-38), and skin-derived fibroblasts (CCD39-SK)). It was confirmed.

Continuous passage was carried out to establish human mesenchymal stem cells (hMSC), lung-derived fibroblasts (WI-38), and dermal-derived fibroblasts (CCD39-SK) as senescent cells. The experimental method is as follows. After 1 × 10 ⁵ cells were placed in a 6-well culture dish, all the culture medium was removed the next day, washed with PBS buffer, and then confirmed to be senescent cells using β-Galactocidase Staining Kit (Biovision, USA). 0.5 ml of cell fixative was added to the washed culture dish, treated at room temperature for 10 to 15 minutes, and washed twice with PBS, followed by 0.47 ml of dye solution, 0.005 ml of dye solution, and 0.025 ml of X-gal. After the mixture was put into a culture dish and treated at 37 ° C. for about a day, the dish was washed twice with PBS and photographed with an optical microscope. Referring to FIG. 11, it can be seen that the senescent cells are shown to be positive using the β-gal assay. In other words, when the senescent cells become β-gal assay, the nuclei of the cells around the nuclear staining can be confirmed.

After preparing the senescent cells, the expression pattern of miRNA was confirmed in each cell. Specifically, the novel miRNA of the present invention is expressed in senescent cells (three types-human mesenchymal stem cells (hMSC), lung-derived fibroblasts (WI-38), skin-derived fibroblasts (CCD39-SK) prepared above) It was confirmed by Taqman assay. Referring to FIG. 12A, expression of nine miRNAs (mi2, mi9, mi28, mi77, mi87, mi90, mi107, mi111) in human mesenchymal stem cells increased with aging, and expression decreased in the case of mi168. Referring to FIG. 12B, in the fibroblasts derived from lung, seven miRNAs (mi2, mi28, mi77, mi87, mi107, mi111, mi168) increased as aging progressed and expression decreased in the case of mi9. Referring to Figure 12c, 7 miRNAs (mi28, mi77, mi87, mi90, mi107, mi111, mi168) in fibroblast-derived fibroblasts increased expression as aging progressed and the expression of three miRNAs (mi2, mi84, mi93) Decreased. Accordingly, it is determined that five miRNAs, i.e., mi28, mi77, mi87, mi107, and mi111, which are commonly increased in FIGS. 12A, 12B, and 12C, are associated with genes with reduced expression in senescent cells. This means that the miRNAs of the present invention are used to regulate the aging of cells.

23 new miRNAs or candidates were found from human mesenchymal stem cells. Small RNAs that do not have a hairpin folding structure are not considered miRNAs. All 22 novel miRNAs of the present invention have a unique hairpin structure (FIG. 3A). The 22 miRNAs of the present invention are located on various chromosomes, especially on the 11 and 14 chromosomes.

MiRNA of the present invention shows a specific expression pattern when differentiation of human embryonic stem cells (ESC) (Table 3). When comparing undifferentiated ES cells with differentiated endothelial cells (ECs), 18 miRNAs were overexpressed in EC and two miRNAs were underexpressed. Similarly, 13 miRNAs were overexpressed in differentiated neuroprogenitor cells (NPCs) in human EC and 7 miRNAs were underexpressed.

Twelve miRNA-mi9, mi168, mi2, mi93, mi84, mi87, mi234, mi90, mi6, mi174, mi81 and mi144- of the novel miRNAs of the present invention were overexpressed simultaneously in NP cells and EC. In contrast, two miRNAs, mi28 and mi263, were underexpressed in both NP cells and EC. These results indicate that miRNAs play various roles in stem cell differentiation. If not, when ES differentiates into EC and NPC, it will share a common key gene regulated by a particular miRNA.

MiRNAs of the present invention shows that the expression pattern is also different according to the differentiation line. For example, mi77, mi206. mi28, mi242 and mi89-2 increase when they differentiate into endothelial cells (EC) and decrease when they differentiate into neurons (NPC). These miRNAs are thought to be necessary for the regulation of genes involved in specific differentiation processes. That is, miRNAs may be characterized as having expression specificity depending on the cell type or lineage. Thus, it can be concluded that new miRNAs have cell specific regulatory patterns. This means that the cloned miRNAs of the invention are involved in cell differentiation and cell lineage regulation.

The novel miRNA from the human mesoderm stem cells of the present invention is very useful not only in the academic aspect for understanding eukaryotic gene expression regulation, but also in medical aspects such as disease research and gene therapy caused by miRNA metabolism or functional defect.

1 shows the overall process for cloning new miRNAs.

Figure 2 shows schematically a method for analyzing miRNA expression using (a) Taqman miRNA assay and (b) RT-PCR, two methods used to measure expression of miRNA of the present invention: srcDNA in (b) is small RNA cDNA, Q-PCR means real time quantitative PCR, srSP means small RNA soecific RNA, and RTQ-UNIr means universal reverse primer.

Figure 3a shows the precursor miRNA of the present invention is shown in yellow is a mature miRNA.

Figure 3b is a graph showing the size distribution of the 22 miRNA clones obtained from hMSCs.

4A shows the position on the chromosome of novel miRNAs.

4B is a graph showing the chromosomal distribution of novel miRNAs.

5 is a graph showing the expression levels of hES cells, CHA-3C cells and CHA3-NPC cells of novel miRNAs. Taqman assays were used and the expression rate in each cell was determined based on ES cells.

6 is a graph showing the expression levels of hES cells, CHA-3C cells and CHA3-NPC cells of novel miRNAs. Poly A tailed RT-PCR was performed to measure the expression rate of each cell based on ES cells.

Figure 7 shows the miRNA expression of the present invention in lung-derived normal cells and cancer cell lines.

Figure 8 shows the expression pattern in peripheral blood mononuclear cells (PBMC) isolated from cerebral infarction patients.

9 shows quantitatively the expression patterns of four miRNAs in various cell lines based on WI-38 40p cells, which are normal cells derived from lungs.

10 shows quantitative expression of miRNAs in various cell lines.

Figure 11 shows the results of confirming the senescent cells using the β-gal assay method.

Figure 12 shows the quantitative expression of miRNAs in three types of senescent cells, human mesenchymal stem cells (a), lung-derived fibroblasts (b) and skin-derived fibroblasts (c).

<110> CHA University Industry-Academic Cooperation Foundation <120> NOVEL miRNA FROM HUMAN MESENCHYMAL STEM CELL <130> P09-7125 <160> 71 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (20) <223> mi9 miRNA <400> 1 ggagguuggg aagggcagag 20 <210> 2 <211> 19 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (19) <223> mi168 miRNA <400> 2 uugaucucgg aagcuaagc 19 <210> 3 <211> 19 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (19) <223> mi2 miRNA <400> 3 ucuccuuccc cacucggcc 19 <210> 4 <211> 20 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (19) <223> mi93 miRNA <400> 4 ucaccgcagc ggcccuccua 20 <210> 5 <211> 24 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (24) <223> mi84 miRNA <400> 5 gggcggggaa gcuugggaga gcuc 24 <210> 6 <211> 21 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (21) <223> mi77 miRNA <400> 6 gcggcccuga cggggcgcgg g 21 <210> 7 <211> 20 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (20) <223> mi111 miRNA <400> 7 gcggagagag aauggggagc 20 <210> 8 <211> 19 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (19) <223> mi87 miRNA <400> 8 ggaggaagag ggaaagggc 19 <210> 9 <211> 18 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (18) <223> mi234 miRNA <400> 9 gucuuccucc uucucuuu 18 <210> 10 <211> 18 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (18) <223> mi90 miRNA <400> 10 uggccucgga uggagccc 18 <210> 11 <211> 20 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (20) <223> mi6 miRNA <400> 11 cccgccggac gaccguccuc 20 <210> 12 <211> 18 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (18) <223> mi28 miRNA <400> 12 ugaggcgggg gggcgagc 18 <210> 13 <211> 20 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (20) <223> mi107 miRNA <400> 13 gggcuggggc gcggggaggu 20 <210> 14 <211> 20 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (10) <223> mi206 miRNA <400> 14 gccgccgccg cuuccucucc 20 <210> 15 <211> 19 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (19) <223> mi174 miRNA <400> 15 gaguguggac gugaacggg 19 <210> 16 <211> 24 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (24) <223> mi81 miRNA <400> 16 ggcggcggcg ggcggcgggc cggc 24 <210> 17 <211> 19 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (19) <223> mi144 miRNA <400> 17 ggcggggcgc ggggcaggg 19 <210> 18 <211> 19 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (19) <223> mi216 miRNA <400> 18 gacaguggug gcggcggcg 19 <210> 19 <211> 20 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (20) <223> miS28 miRNA <400> 19 agagaugaag cgggggggcg 20 <210> 20 <211> 22 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (22) <223> mi242 miRNA <400> 20 cgggcggcgg cgggcggcgg gc 22 <210> 21 <211> 24 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (24) <223> mi89_2 miRNA <400> 21 ggaggccggg guggggcggg gcgg 24 <210> 22 <211> 19 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (19) <223> mi263 miRNA <400> 22 ggggagcgag gggcggggc 19 <210> 23 <211> 55 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (55) <223> mi9 preform miRNA <400> 23 aggagguugg gaagggcaga gaugagcaua aaguuuuugc cuuguuuuuc uuuuu 55 <210> 24 <211> 53 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (53) <223> mi168 preform miRNA <400> 24 guuugaucuc ggaagcuaag cagggucggg ccugguuagu acuuggaugg gag 53 <210> 25 <211> 84 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (84) <223> mi2 preform miRNA <400> 25 ucugagggua ggaggcggga gcaaaggcga uuagcaugcu gggugcuguc ugaugaaugg 60 cucucuccuu ccccacucgg ccca 84 <210> 26 <211> 62 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (62) <223> mi93 preform miRNA <400> 26 acucaccgca gcggcccucc uacuagacgc auagcgucca cggggcuggc gggcgggggg 60 gu 62 <210> 27 <211> 61 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (61) <223> mi84 preform miRNA <400> 27 gggcggggaa gcuugggaga gcucauagga ugcagcugcg aguuacaaga guuagcaugc 60 u 61 <210> 28 <211> 75 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (75) <223> mi77 preform miRNA <400> 28 cgcggcccga cccgcuuccc ucagggcgcg gagcucggcg cggcggcccu gacggggcgc 60 gggcccggcc gcgga 75 <210> 29 <211> 80 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (80) <223> mi111 preform miRNA <400> 29 gguuggcuau aacuaucauu uccaagguug ugcuuuuagg aaauguuggc uguccugcgg 60 agagagaaug gggagccagg 80 <210> 30 <211> 53 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (53) <223> mi87 preform miRNA <400> 30 uacggaggaa gagggaaagg gcucuggccc ccucggcguc augucuucgg ugc 53 <210> 31 <211> 67 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (67) <223> mi234 preform miRNA <400> 31 ugucuuccuc cuucucuuug ucagcaacac uuuuccaggg uuuacaaaag accaggcagg 60 cugggca 67 <210> 32 <211> 67 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (67) <223> mi90 preform miRNA <400> 32 ccuggagcca gcggccacuc acgaagcagg cucugacgcc ugguggccuc ggauggagcc 60 cugacuc 67 <210> 33 <211> 48 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (48) <223> mi6 preform miRNA <400> 33 ucccgccgga cgaccguccu ccgccucagg ggacgcgccc aggacgca 48 <210> 34 <211> 49 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (49) <223> mi28 preform miRNA <400> 34 ggugaggcgg gggggcgagc ccugaggggc ucucgcuucu ggcgccaag 49 <210> 35 <211> 55 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (55) <223> mi107 preform miRNA <400> 35 gggggcuggg gcgcggggag gugcuagguc ggccucggcu cccgcgccgc acccc 55 <210> 36 <211> 53 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (53) <223> mi206 preform miRNA <400> 36 cgccgccgcc gcuuccucuc ccacacuugu uccugagucg cucuccugug gcu 53 <210> 37 <211> 61 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (61) <223> mi174 preform miRNA <400> 37 ccagcacggu gacagaccug gaggacacca agcgggacag gaguguggac gugaacgggg 60 g 61 <210> 38 <211> 56 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (56) <223> mi81 preform miRNA <400> 38 ugagccgccc cccgcgcccg gcaugcccgg cccggcccgc cgcccgccgc cgccca 56 <210> 39 <211> 58 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (58) <223> mi144 preform miRNA <400> 39 cccggguccu ccgcgacucg caggccggcu ggggcggggc gcggggcagg ggccgcgg 58 <210> 40 <211> 50 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (50) <223> mi216 preform miRNA <400> 40 ggcaguggug gcggcggcgg cggugaguga ccgcgguggu ggcgccggcg 50 <210> 41 <211> 51 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (51) <223> miS28 preform miRNA <400> 41 agagaugaag cgggggggcg gggucuugcu cuauugccua cgcugaucuc a 51 <210> 42 <211> 57 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (57) <223> mi242 preform miRNA <400> 42 gggcgccggg gcccggggcg cgcagccggg gggcgggcgg cggcgggcgg cgggccg 57 <210> 43 <211> 64 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (64) <223> mi89_2 miRNA <400> 43 ccccgggccc ggcguucccu ccccuuccgu gcgccagugg aggccggggu ggggcggggc 60 gggg 64 <210> 44 <211> 60 <212> RNA <213> Homo sapiens <220> <221> mRNA (222) (1) .. (60) <223> mi263 preform miRNA <400> 44 cgcugggucc gcgcgcccug ggccgggcga uguccgcuug ggggagcgag gggcggggcg 60                                                                           60 <210> 45 <211> 45 <212> DNA <213> Homo sapiens <400> 45 ctgcgcaagg atgacacgca aattcgtgaa gcgttccata ttttt 45 <210> 46 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> MI-5 'Linker <400> 46 aucgucucgg gaugaaa 17 <210> 47 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> MI-3 'Linker <400> 47 ctgtaactcg ggtcaatc 18 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mi9 primer <400> 48 ggaggttggg aagggcagag 20 <210> 49 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> mi168 primer <400> 49 ttgatctcgg aagctaagc 19 <210> 50 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> mi2 primer <400> 50 tctccttccc cactcggcc 19 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mi93 primer <400> 51 tcaccgcagc ggccctccta 20 <210> 52 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> mi84 primer <400> 52 gggcggggaa gcttgggaga gctc 24 <210> 53 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mi77 primer <400> 53 gcggccctga cggggcgcgg g 21 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mi111 primer <400> 54 gcggagagag aatggggagc 20 <210> 55 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> mi87 primer <400> 55 ggaggaagag ggaaagggc 19 <210> 56 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> mi234 primer <400> 56 gtcttcctcc ttctcttt 18 <210> 57 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> mi90 primer <400> 57 tggcctcgga tggagccc 18 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mi6 primer <400> 58 cccgccggac gaccgtcctc 20 <210> 59 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> mi28 primer <400> 59 tgaggcgggg gggcgagc 18 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mi107 primer <400> 60 gggctggggc gcggggaggt 20 <210> 61 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mi206 primer <400> 61 gccgccgccg cttcctctcc 20 <210> 62 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> mi174 primer <400> 62 gagtgtggac gtgaacggg 19 <210> 63 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> mi81 primer <400> 63 ggcggcggcg ggcggcgggc cggc 24 <210> 64 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> mi144 primer <400> 64 ggcggggcgc ggggcaggg 19 <210> 65 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> mi216 primer <400> 65 gacagtggtg gcggcggcg 19 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> miS28 primer <400> 66 agagatgaag cgggggggcg 20 <210> 67 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mi242 primer <400> 67 cgggcggcgg cgggcggcgg gc 22 <210> 68 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> mi89_2 primer <400> 68 ggaggccggg gtggggcggg gcgg 24 <210> 69 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> mi263 primer <400> 69 ggggagcgag gggcggggc 19 <210> 70 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> RTQ UNIr primer <400> 70 cgaattctag agctcgaggc agg 23 <210> 71 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> RTQ-adapter primer <400> 71 cgaattctag agctcgaggc aggcgacatg gctggctagt taagcttggt accgagctcg 60 gatccactag tccttttttt tttttttttt ttttttttvn 100  

Claims (14)

A miRNA derived from a human mesoderm stem cell for regulating cellular senescence selected from the group consisting of SEQ ID NOs: 6 (mi77), 8 (mi87) and 13 (mi107). Cell aging composition comprising a miRNA derived from human mesoderm stem cells of claim 1. Cell aging control composition comprising a miRNA-expressing vector derived from human mesoderm stem cells of claim 1. delete delete delete delete delete delete delete delete delete delete delete

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