MICRORNA REGULATING THE INSULIN SIGNALING PATHWAY, AND METHOD FOR SCREENING MATERIAL FOR CONTROLLING THE ACTION OF A TARGET THEREOF
United States Patent Application
The present invention relates to a miRNA regulating the insulin signaling pathway, and to a method for screening a material for controlling the action of a target gene thereof, and particularly, to a method for screening a material for controlling the action of USH or FOG2, a target gene of miR-8 or miR-200 miRNA for promoting cell growth. The present inventors discovered miR-8, a conserved miRNA for regulating the body of a fruit fly by targeting u-shaped material (USH) in the fat cells of Drosophila. It was also confirmed that a target gene of miR-200, a human homologous gene of Drosophila miR-8 miRNA, is FOG2. It was found that Drosophila miR-8 and USH are also conserved in mammals, and FOG2, a human homologous gene of USH, directly binds to a regulating subunit of PI3K and functions. It was confirmed that when the expression of miR-200 is inhibited or FOG2 is expressed in a human cancer cell line, the activity of PI3K, which promotes cell growth, is decreased. Therefore, miR-200 and FOG2 may be useful in screening regulators of insulin signaling pathways.
Inventors:
Kim, Vic Narry (Seoul, KR)
Lee, Jung Hyun (Seoul, KR)
Hyun, Seogang (Seoul, KR)
Jin, Hua (Seoul, KR)
Application Number:
Publication Date:
01/10/2013
Filing Date:
12/02/2009
Export Citation:
SNU R & DB Foundation (Seoul, KR)
Primary Class:
Other Classes:
International Classes:
G01N33/566; C12Q1/02; C12Q1/68; G01N21/64
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Related US Applications:
January, 2010Takeshita et al.February, 2009TrudeauAugust, 2009Banes et al.November, 2009ChuJuly, 2003KluxenApril, 2008Nobles Jr. et al.December, 2009Chang et al.January, 2002MeyersMarch, 2010Henriques et al.June, 2005Collins et al.December, 2003Pihan
Other References:
Goffrini et al, FOGI and FOG2 genes, required for the transcriptional activation of glucose-repressible genes of Kluyveromyces lactis, are homologous to GAL83 and SNF1 of Saccharomyces cerevisiae, 1996, Curr Genet, 29:316-326
Tevosian et al, FOG-2: A novel GATA-family cofactor related to multitype zinc-finger proteins Friend of GATA-1 and U-shaped, 1999, PNAS, 96: 950-955
Liang et al, Characterization of microRNA expression profiles in normal human tissues, 2007, BMC Genomics, 8: 166, p.1-20
Sorrentino et al, The Friend of GATA protein U-shaped functions as a hematopoietic tumor suppressor in Drosophila, 2007, Developmental Biology, 311: 311-323
Aboobaker et al, Drosophila microRNAs exhibit diverse spatial expression patterns during embryonic development, 2005, PNAS, vol.102, 50:
Primary Examiner:
POLIAKOVA-GEORGAN, EKATERINA
Attorney, Agent or Firm:
CLARK & ELBING LLP (101 FEDERAL STREET
BOSTON MA 02110)
A method of screening an insulin signaling regulator, the method comprising the steps of: 1) treating cell lines expressing miR-200 family miRNAs or miR-8 miRNA wit 2) measuring an expression level or an activity of FOG2 or USH protein in cells treated with the testing compound in the step 1); and 3) identifying the testing compound by which the expression or the activity of the FOG2 or USH protein in the cells in the step 1) has been changed as compared to a control.
The method as claimed in claim 1, wherein the cell lines expressing the miR-200 family miRNAs are human cell lines derived from tissues of heart, brain, testicle, liver, lung and skeletal muscle.
The method as claimed in claim 1, wherein the cell lines expressing the miR-8 miRNA are drosophila cell lines derived from fat body tissues.
The method as claimed in claim 1, wherein the testing compound is any one selected from the group including natural compounds, synthesized compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, antigens, metabolites of bacteria or fungi, and bioactive molecules.
The method as claimed in claim 1, wherein in the step 2), the expression level of the FOG2 or USH protein is measured by any one method selected from the group including western blotting, immunostaining, fluorescent staining, and reporter assay.
The method as claimed in claim 1, wherein in the step 2), the activity of the FOG2 or USH protein is measured by any one method selected from the group including i) a method of measuring an activity of a p85α/p110/IRS-1 ii) a method of measuring an expression level of FOXO mRNA and iii) a method of measuring cell growth and proliferation.
The method as claimed in claim 6, wherein the activity of the p85α/p110/IRS-1 complex is measured by measuring a phosphorylation level of AKT protein.
A method of screening an insulin signaling regulator, the method comprising the steps of: 1) treating cell lines expressing FOG2 or USH wit 2) measuring an expression level or an activity of FOG2 or USH protein in cells in the step 1); and 3) identifying the testing compound by which the expression or the activity of the FOG2 or USH protein in the cells in the step 1) has been changed as compared to a control.
The method as claimed in claim 8, wherein the cell lines expressing the FOG2 are human cell lines derived from tissues of heart, brain, testicle, liver, lung and skeletal muscle.
The method as claimed in claim 8, wherein the cell lines expressing the USH are drosophila cell lines derived from fat body tissues.
The method as claimed in claim 8, wherein the testing compound is any one selected from the group including natural compounds, synthesized compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, antigens, metabolites of bacteria or fungi, and bioactive molecules.
The method as claimed in claim 8, wherein in the step 2), the expression level of the FOG2 or USH protein is measured by any one method selected from the group including western blotting, immunostaining, fluorescent staining, and reporter assay.
The method as claimed in claim 8, wherein in the step 2), the activity of the FOG2 or USH protein is measured by any one method selected from the group including i) a method of measuring an activity of a p85α/p110/IRS-1 ii) a method of measuring an expression level of FOXO mRNA and iii) a method of measuring cell growth and proliferation.
The method as claimed in claim 13, wherein the activity of the p85α/p110/IRS-1 complex is measured by measuring a phosphorylation level of AKT protein.
A method of screening an insulin signaling regulator, the method comprising the steps of: 1) bringing FOG2 protein into contact with p85α protein in the presence o 2) measuring a degree of binding of the FOG2 protein to the p85α and 3) identifying the testing compound by which the degree of binding of the FOG2 protein to the p85α protein in vitro in the step 2) has been changed as compared to a control not treated with the testing compound.
A method of screening an insulin signaling regulator, the method comprising the steps of: 1) bringing USH protein into contact with dp60 protein in the presence o 2) measuring a degree of binding of the USH protein to the dp60 and 3) identifying the testing compound by which the degree of binding of the USH protein to the dp60 (Drosophila p60) protein in vitro in the step 2) has been changed as compared to a control not treated with the testing compound.
The method as claimed in claim 15, wherein the testing compound is any one selected from the group including natural compounds, synthesized compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, antigens, metabolites of bacteria or fungi, and bioactive molecules.
The method as claimed in claim 15, wherein in the step 3), the degree of binding between the proteins is measured by any one method selected from the group including SPR, protein chip, gel shift assay, immunoprecipitation assay, co-immunoassay, fluorescence immuno assay and radioimmuno assay.
19. 19-31. (canceled)
The method as claimed in claim 16, wherein the testing compound is any one selected from the group including natural compounds, synthesized compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, antigens, metabolites of bacteria or fungi, and bioactive molecules.
The method as claimed in claim 16, wherein in the step 3), the degree of binding between the proteins is measured by any one method selected from the group including SPR, protein chip, gel shift assay, immunoprecipitation assay, co-immunoassay, fluorescence immuno assay and radioimmuno assay.
Description:
TECHNICAL FIELDThe present disclosure relates to a method for screening material for controlling the insulin signaling pathway.BACKGROUND ARTAnimal body size is a biological parameter subject to considerable s animals of abnormal size are strongly selected against as less fit for survival. Thus, the way in which body size is determined and regulated is a fundamental biological question. Recent studies using insect model systems have begun to provide some clues by showing that insulin signaling plays an important part in modulating body growth (Ikeya et al., 2002; Rulifson et al., 2002). The binding of insulin (insulin-like peptides in Drosophila) to its receptor (InR) triggers a phosphorylation cascade involving the insulin receptor substrate (IRS; chico in Drosophila), phosphoinositide-3 kinase (PI3K), and Akt/PKB (Edgar, 2006). An active PI3K complex consists of a catalytic subunit (p110; dp110 in Drosophila) and a regulatory subunit (p85α; dp60 in Drosophila). Phosphorylated Akt (p-Akt) phosphorylates many proteins including forkhead box O transcription factor (FOXO) which are involved in cell death, cell proliferation, metabolism, and life span control (Arden, 2008). Once activated, the kinase cascade enhances cell growth and proliferation.Organismal growth is achieved not only by cell-autonomous regulation but also by non-cell-autonomous control through circulating growth hormones (Baker et al., 1993; Edgar, 2006). Recent studies in insects indicate that several endocrine organs, such as the prothoracic gland and fat body, govern organismal growth by coordinating developmental and nutritional conditions (Caldwell et al., 2005; Colombani et al., 2005; Colombani et al., 2003; Mirth et al., 2005). However, detailed mechanisms of how body size is determined and modulated remain largely unknown.microRNAs (miRNAs) are noncoding RNAs of -22 nt that act as posttranscriptional repressors by base-pairing to the 30 untranslated region (UTR) of their cognate mRNAs (Bartel, 2009). The physiological functions of individual miRNAs remain largely unknown. Studies of miRNA function rely heavily on computational algorithms that predict target genes (John et al., 2004; Kim et al., 2006; Kiriakidou et al., 2004; Krek et al., 2005; Lewis et al., 2005; Stark et al., 2003). In spite of their utility, however, these target prediction programs generate many false-positive results, because regulation in vivo depends on target message availability and complementary sequence accessibility. To overcome the difficulties in identifying real targets, various experimental approaches have been developed, including micro-arrays, proteomic analyses, and biochemical purification of the miRNA-mRNA complex (Bartel, 2009). Genetic approaches using model organisms can also be useful tools for studying the biological roles of miRNAs at both the organismal and molecular levels (Smibert and Lai, 2008). Despite these advances, however, it is still a daunting task to understand the biological function of a given miRNA and to identify its physiologically relevant targets.Here, the inventors of the present disclosure found using Drosophila as a model system that conserved miRNA miR-8 positively regulates body size by targeting a fly gene called u-shaped (ush) in fat body cells. The inventors further discover that this function of miR-8 and USH is conserved in mammals and that the human homolog of USH, FOG2, acts by directly binding to the regulatory subunit of PI3K. Moreover, the inventors identify the miR-200, a human homologue gene of Drosophila miR-8 miRNA, is FOG2. The present inventors find that either the inhibition of miR-200 expression or the induction of FOG2 expression in human carcinoma cells contributes to decrease in PI3K activity (capable of promoting cell proliferation) to reduce cell proliferation, indicating that miR-200 and FOG2 are usefully adopted for screening systems to Identify a substance capable of controlling insulin signal pathway.Throughout the specification, a number of publications and patent documents are referred to and cited. The disclosure of the cited publications and patent documents is incorporated herein by reference in its entirety to more clearly describe the state of the related art and the present disclosure.DISCLOSURETechnical ProblemAn object of the present invention is to provide a method of screening an insulin signaling regulator, the method including the steps of:1) treating cell lines expressing miR-200 family miRNAs or miR-8 miRNA wit2) measuring an expression level or an activity of FOG2 or USH protein in cells treated with the testing compound in the step 1); and3) identifying the testing compound by which the expression or the activity of the FOG2 or USH protein in the cells in the step 1) has been changed as compared to a control.Another object of the present invention is to provide a method of screening an insulin signaling regulator, the method including the steps of:1) treating cell lines expressing FOG2 or USH wit2) measuring an expression level or an activity of FOG2 or USH protein in cells in the step 1); and3) identifying the testing compound by which the expression or the activity of the FOG2 or USH protein in the cells in the step 1) has been changed as compared to a control.A further object of the present invention is to provide a method of screening an insulin signaling regulator, the method including the steps of:1) bringing FOG2 protein into contact with p85α protein in the presence o2) measuring a degree of binding of the FOG2 protein to the p85α and3) identifying the testing compound by which the degree of binding of the FOG2 protein to the p85α protein in vitro in the step 2) has been changed as compared to a control not treated with the testing compound.A still further object of the present invention is to provide a method of screening an insulin signaling regulator, the method including the steps of:1) bringing USH protein into contact with dp60 protein in the presence o2) measuring a degree of binding of the USH protein to the dp60 and3) identifying the testing compound by which the degree of binding of the USH protein to the dp60 (Drosophila p60) protein in vitro in the step 2) has been changed as compared to a control not treated with the testing compound.A yet further object of the present invention is to provide a method of screening an insulin signaling regulator, the method including the steps of:1) treating cell lines expressing FOG2 protein wit2) measuring a degree of binding of FOG2 protein to p85α protein in cells treated with the testing compound in the step 1); and3) identifying the testing compound by which the degree of binding of the FOG2 protein to the p85α protein in the cells in the step 1) has been changed as compared to a control not treated with the testing compound.A still yet further object of the present invention is to provide a method of screening an insulin signaling regulator, the method including the steps of:1) treating cell lines expressing USH protein wit2) measuring a degree of binding of USH protein to dp60 protein in cells treated with the testing compound in the step 1); and3) identifying the testing compound by which the degree of binding of the USH protein to the dp60 protein in the cells in the step 1) has been changed as compared to a control not treated with the testing compound.Another still yet further object of the present invention is to provide an insulin signaling regulating composition including a material changing an expression or an activity of FOG2 or USH protein, as an active ingredient.Another still yet further object of the present invention is to provide a use of a material changing an expression or an activity of FOG2 or USH protein, in preparation of an insulin signaling regulating composition.Technical SolutionIn accordance with an aspect of the present invention, there is provided a method of screening an insulin signaling regulator, the method including the steps of:1) treating cell lines expressing miR-200 family miRNAs or miR-8 miRNA wit2) measuring an expression level or an activity of FOG2 or USH protein in cells treated with the testing compound in the step 1); and3) identifying the testing compound by which the expression or the activity of the FOG2 or USH protein in the cells in the step 1) has been changed as compared to a control.In accordance with another aspect of the present invention, there is provided a method of screening an insulin signaling regulator, the method including the steps of:1) treating cell lines expressing FOG2 or USH wit2) measuring an expression level or an activity of FOG2 or USH protein in cells in the step 1); and3) identifying the testing compound by which the expression or the activity of the FOG2 or USH protein in the cells in the step 1) has been changed as compared to a control.In accordance with a further object of the present invention, there is provided a method of screening an insulin signaling regulator, the method including the steps of:1) bringing FOG2 protein into contact with p85α protein in the presence o2) measuring a degree of binding of the FOG2 protein to the p85α and3) identifying the testing compound by which the degree of binding of the FOG2 protein to the p85α protein in vitro in the step 2) has been changed as compared to a control not treated with the testing compound.In accordance with a still further object of the present invention, there is provided a method of screening an insulin signaling regulator, the method including the steps of:1) bringing USH protein into contact with dp60 protein in the presence o2) measuring a degree of binding of the USH protein to the dp60 and3) identifying the testing compound by which the degree of binding of the USH protein to the dp60 (Drosophila p60) protein in vitro in the step 2) has been changed as compared to a control not treated with the testing compound.In accordance with a yet further object of the present invention, there is provided a method of screening an insulin signaling regulator, the method including the steps of:1) treating cell lines expressing FOG2 protein wit2) measuring a degree of binding of FOG2 protein to p85α protein in cells treated with the testing compound in the step 1); and3) identifying the testing compound by which the degree of binding of the FOG2 protein to the p85α protein in the cells in the step 1) has been changed as compared to a control not treated with the testing compound.In accordance with a still yet further object of the present invention, there is provided a method of screening an insulin signaling regulator, the method including the steps of:1) treating cell lines expressing USH protein wit2) measuring a degree of binding of USH protein to dp60 protein in cells treated with the testing compound in the step 1); and3) identifying the testing compound by which the degree of binding of the USH protein to the dp60 protein in the cells in the step 1) has been changed as compared to a control not treated with the testing compound.In accordance with another still yet further object of the present invention, there is provided an insulin signaling regulating composition including a material changing an expression or an activity of FOG2 or USH protein, as an active ingredient.In accordance with another still yet further object of the present invention, there is provided a use of a material changing an expression or an activity of FOG2 or USH protein, in preparation of an insulin signaling regulating composition.Advantageous EffectsThe inventors found using Drosophila as a model system that conserved miRNA, miR-8, regulates body size by targeting u-shaped (ush) in fat body cells. Further, they confirmed that a human homolog (miR-200) of drosophila miR-8 (miRNA) targets a gene of FOG2. Also, they discovered that miR-8 and USH of drosophila are conserved also in mammals, and a human homolog of USH, that is, FOG2, acts by directly binding to the regulatory subunit of PI3K. When in human cancer cell lines, expression of miR-200 was inhibited, or FOG2 was expressed, it was found that PI3K activity facilitating cell growth was reduced. Thus, miR-200 and FOG2 may be usefully used in screening of an insulin signaling regulator such as an anti-cancer agent.DESCRIPTION OF DRAWINGSFIGS. 1 to 4 show the comparison between miR-8 null flies (mir-8Δ2) and wild-type flies in phenotypes of female and male adults (see FIG. 1), body weight (see FIG. 2), pupariation and emergence (see FIG. 3), and wing cell size (see FIG. 4).FIGS. 5 and 6 were obtained as follows: in miR-8 null flies, the activities and gene expression of proteins related to insulin signaling were determined by western blotting (see FIG. 5); and an expression level of 4EBP was determined through Q-PCR in miR-8 null larvae (see FIG. 6).FIGS. 7 and 8 were obtained as follows: in the fat body, cuticle and brain tissues of the third-stage larvae (at 96 hr after egg-laying) of mir-8 enhancer trap GAL4 line drosophila (mir-8 gal4), the spatial expression pattern of miR-8 through immuno-staining was determined (see FIG. 7); and in the total larva, fat body, gut, brain and cuticle, the miR-8 expression level was determined through northern blotting (see FIG. 8).FIGS. 9 and 10 show the comparison of body weight among mir8 CgG4&mir8, mir8 pplG4 and wild-type flies, and the comparison of body weight among mir8 elavG4, and mir8 elavG4&mir8.FIG. 11 shows the homology between human miR-200 family miRNAs and drosophila miR-8.FIGS. 12 and 13 show the confirmation that in human cell lines, MCF7, miR-200 family promotes cell proliferation (see FIG. 12); and the schematic view of an algorithm applied to selection of a target gene and ortholog through a target prediction program (see FIG. 13).FIGS. 13 and 14 were obtained as follows: target gene candidates of drosophila miR-8 and human miR-200 miRNAs were selected using miRanda, miTarget, microT, PicTar and Target Scan (see FIGS. 13); and 7 gene pairs that respond both to miR-8 and to mir-200 family miRNAs were identified by applying 3′UTR regions of the gene candidates to a luciferase expression system (see FIG. 14).FIGS. 16 to 18 were obtained as follows: body weights of transgenic flies of mir8 Cg&ush i, mir8 Cg&Lap11, mir8 Cg&Ced-12 i, mir8 Cg&CG8445 i, mir8 Cg&CG12333 i and mir8 Cg&atro i were measured (see FIG. 16); whether each gene in the flies was knocked down was determined by quantitative RT-PCR (see FIG. 17); and body sizes of flies of w1118, w1118ush1518, mir-8Δ2 and mir-8Δ2ush1518 were measured (see FIG. 18).FIGS. 19 to 21 were obtained as follows: whether the mRNA expression level of USH (selected as a target gene of miR-8) in miR-8 null flies was in actuality increased was determined by quantitative RT-PCR (see FIG. 19); whether the expression of USH protein was in actuality increased was determined by western blotting (see FIG. 20); and the regulation of miR-8 on ush 3′UTR's miR target site was determined by measuring luciferase activity through introduction of a mutation into the miR-8 target site (see FIG. 21).FIG. 22 shows the measurement results of body size and wing cell number of drosophila when in drosophila fat body, insulin signaling was suppressed.FIGS. 23 and 24 were obtained as follows: whether insulin signaling is defective in the fat body of miR-8 null flies was determined by observing PI3K activity and nuclear localization of FOXO in the fat body of miR-8 null flies (see FIG. 23); and in fat body tissues of wild-type larvae or miR-8 null larvae, JNK and Akt phosphorylation levels were determined by western blotting (see FIG. 24).FIG. 25 shows the result when in the mosaic fat cells over-expressing miR-8, PI3K activity and cell size were observed through immunostaining.FIGS. 26 to 29 were obtained as follows: through twin-spot analysis (mitotic clone analysis), it was determined that a twin-spot of miR-8 null clones is more slowly generated than wild-type clones (arrow), and miR-8 null clones shows a lower DNA level than wild-type clones (see FIG. 26); in fat body cells over-expressing USH and wild-type cells, PI3K activity was determined (see FIG. 27); through twin-spot analysis in the wing or eye disc, it was determined whether the growth of the organs was inhibited when miR-8 null clones were generated (see FIG. 28); and in wing precursor cells or the eye disc of drosophila larvae, USH expression was determined (see FIG. 29).FIGS. 30 to 34 were obtained as follows: in USH over-expressing cells, a cell size (see FIG. 30), a PI3K activity (see FIG. 31) and a nuclear FOXO signal (see FIG. 32) were determined through immuno- and in USH knockdown mosaic fat cells, a PI3K activity (see FIG. 33) and a nuclear FOXO signal (see FIG. 34) were determined.FIG. 35 shows the result when it was determined whether insulin signaling in the fat body of miR-8 null larvae was suppressed by an increase in ush expression of fat body cells of CgG4, mir8 CgG4, mir8 CgG4&mir8 and mir8 Cg&ush i flies, through quantitative RT-PCR on FOXO target genes lnr and step mRNA.FIGS. 36 and 37 show the detection result of a predicted binding site for miR-8 in ush mRNA (see FIG. 36), and the detection result of a predicted binding site for miR-200 family miRNAs in FOG2 (see FIG. 37).FIG. 38 shows the result when the regulation of miR-200 miRNAs was determined through measurement of luciferase activity, in a case where one (FOG2 ml, FOG2 m2, FOG2 m3), two ((FOG2 m12, FOG2 m23) or three (FOG2 m123) mutants were introduced into three miR-200 target sites of FOG2 3′UTR.FIG. 39 shows a correlation between the expression of FOG2 protein and miR-200c cluster miRNAs in human cell lines derived from different organs, and an expression of FOG2 protein and miR-200 family miRNAs in the cell lines, which were measured by western blotting and northern blotting, respectively.FIGS. 40 to 42 were obtained as follows: when miR-141/200a or miR-200b/c/429 was transfected into liver hepatocellular carcinoma Huh7 cells, an expression level of FOG and a phosphorylation level of Akt were determined through western blotting (see FIG. 40); when miR-141/200a or miR-200b/c/429 was treated with 2′-O-methyl oligonuclelotides antisense in pancreatic cancer cell lines, AsPC1 cells, an expression level of FOG and a phosphorylation level of Akt were determined through western blotting (see FIG. 41); when FAO cell lines were treated with siFOG2, an expression level of FOG and a phosphorylation level of Akt were determined through western blotting (see FIG. 42).FIG. 43 shows the effect of FOG2 on PI3K activity, which was obtained through immune complex kinase assay after Hep3B cell lines were transfected with pCK-flag or pCK-FOG2.FIGS. 44 to 47 were obtained as follows: after Hep3B cell lines were transfected with pCK-flag or pCK-FOG2, a phosphorylation level of Akt, according to treatment with IGF-1 or FOG2, was determined through western blotting (see FIG. 44); in cells transfected with a luciferase reporter plasmid (pFK1tk-luc) containing eight FOXO binding sites, a change in the activity of luciferase according to transduction of miR-200 miRNAs was measured (see FIG. 45); according to transduction of complementary oligonucleotide, a change in the activity of luciferase was measured (see FIG. 46); and when FOG2 was over-expressed in Hep3B cell lines, a change in the activity of luciferase was measured (see FIG. 47).FIGS. 48 and 49 show the measurement results of cell viability according to introduction of miR-200 family miRNAs (see FIG. 48) and introduction of oligonucleotide complementary to miR-200 family miRNAs (see FIG. 49) in Hep3B cells, which were obtained through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliurn bromide) assay.FIG. 50 shows the result when it was determined whether IRS-1/p85α p110 complex was formed according to expression or non-expression of FOG2 in Hep3B cell lines, through immunoprecipitation assay.FIGS. 51 and 52 were obtained as follows: in the nucleus and cytoplasm fraction in Hela or PANC1 cells, expression of FOG2 was determined through western blotting (see FIG. 51); and in HepG2 cells, localization of FOG2 was determined through immunostaining (see FIG. 52).FIGS. 53 and 54 show the result when it was determined whether FOG2 colocalizes together with p85α in PANC1 cells, through immunoprecipitation assay.FIG. 55 shows FOG2 mutant bound to p85α, which was determined through immunoprecipitation assay when mutants of FOG2 and p85α were co-expressed in human cell lines derived from different tissues.FIGS. 56 to 58 were obtained as follows: when PANC1 cells were treated with mutant FOG2—1-412, mutant FOG2—413-789 or mutant FOG2—802-1151, P13K activity according to an increase of an FOG2 expression level was measured (see FIG. 56); it was determined whether GST-fused recombinant p85α protein is directly bound to mutant FOG2—413-789 or mutant FOG2—802-1151 through in vitro binding assay (see FIG. 57); and when FOG2—413-789 or mutant FOG2—802-1151 was added to the immunoprecipitated PI3K complex, PI3K activity was determined (see FIG. 58).FIGS. 59 and 60 show the result of immunoprecipitation assay, in which drosophila USH physically interacts with drosophila p60 (dp60, drosophila ortholog of p85α) when dp60 and USH were co-expressed in human HEK293T cells (in FIG. 59, IP for USH, and in FIG. 60, IP for dp60).FIG. 61 is a schematic view showing an intracellular signaling process of USH/FOG2.BEST MODEHereinafter, the present invention will be described in detail.In order to achieve the above objects, the present invention provides a method of screening an insulin signaling regulator, the method including the steps of:1) treating cell lines expressing miR-200 family miRNAs or miR-8 nmRNA wit2) measuring an expression level or an activity of FOG2 or USH protein in cells treated with the testing compound in the step 1); and3) identifying the testing compound by which the expression or the activity of the FOG2 or USH protein in the cells in the step 1) has been changed as compared to a control.Also, the present invention provides a method of screening an insulin signaling regulator, the method including the steps of:1) treating cell lines expressing FOG2 or USH wit2) measuring an expression level or an activity of FOG2 or USH protein in cells in the step 1); and3) identifying the testing compound by which the expression or the activity of the FOG2 or USH protein in the cells in the step 1) has been changed as compared to a control.Also, the present invention provides a method of screening an insulin signaling regulator, the method including the steps of:1) bringing FOG2 protein into contact with p85α protein in the presence o2) measuring a degree of binding of the FOG2 protein to the p85α and3) identifying the testing compound by which the degree of binding of the FOG2 protein to the p85α protein in vitro in the step 2) has been changed as compared to a control not treated with the testing compound.Also, the present invention provides a method of screening an insulin signaling regulator, the method including the steps of:1) bringing USH protein into contact with dp60 protein in the presence o2) measuring a degree of binding of the USH protein to the dp60 and3) identifying the testing compound by which the degree of binding of the USH protein to the dp60 (Drosophila p60) protein in vitro in the step 2) has been changed as compared to a control not treated with the testing compound.Also, the present invention provides a method of screening an insulin signaling regulator, the method including the steps of:1) treating cell lines expressing FOG2 protein wit2) measuring a degree of binding of FOG2 protein to p85α protein in cells treated with the testing compound in the step 1); and3) identifying the testing compound by which the degree of binding of the FOG2 protein to the p85α protein in the cells in the step 1) has been changed as compared to a control not treated with the testing compound.Also, the present invention provides a method of screening an insulin signaling regulator, the method including the steps of:1) treating cell lines expressing USH protein wit2) measuring a degree of binding of USH protein to dp60 protein in cells treated with the testing compound in the step 1); and3) identifying the testing compound by which the degree of binding of the USH protein to the dp60 protein in the cells in the step 1) has been changed as compared to a control not treated with the testing compound.As a sole homolog of miR-200 family promoting proliferation of human cancer cells (see FIG. 13), miR-8 is known (Ibanez-Ventoso et al., 2008). Thus, the inventors have tried to discover the biological function of miR-200 by using drosophila. It is generally known that reduced body size in insects is caused by either slow larval growth, precocious early pupariation (pupal formation) that shortens the larval growth period, or both (Colombaniet al., 2003; Edgar, 2006; Mirth and Riddiford, 2007). The inventors found that miR-8 null flies exhibit a significantly small body size and a significantly small body weight, and miR-8 null larvae exhibit a significantly small body volume (see FIGS. 1, 2 and 3). Also, it was found that the small body size of miR-8 null flies is likely to be caused by slower growth during the larval period rather than by precocious pupariation, and also caused by decreased cell number rather than reduced cell size (see FIGS. 3 and 4).Also, in order to understand why miR-8 null flies grow slowly, the inventors examined the activities of the proteins involved in insulin signaling in the miR-8 null flies. As a result, it was found that that insulin signaling was significantly reduced in the miR-8 null flies (see FIGS. 5 and 6).The inventors examined the spatial expression pattern of miR-8 in larvae. As a result, they found a high miR-8 expression level in the fat body of larvae (see FIGS. 7 and 8).Recent studies suggested that Drosophila fat body may be an important organ in the control of energy metabolism and growth (Colombani et al., 2005; Colombani et al., 2003; Edgar, 2006; Leopold, P, and Perrimon, N 2007, Nature 450, 186-188). The inventors confirmed that miR-8 expression in the larval fat body is critical for body size control of drosophila, and the function of miR-8 in the fat body is spatio-temporally specifically distinct from neuronal cells (see FIGS. 10 and 11).To further understand the molecular function of miR-8, the inventors set out to identify miR-8 target genes responsible for body size control. Because miR-8 is a highly conserved miRNA and because the human homologs of miR-8 promote cell proliferation in human cells (see FIG. 6), the inventors assumed that the target genes of miR-8 are related to cell proliferation phenotype. The inventors listed target gene candidates of both fly miR-8 and human miR-200 miRNAs by using a target prediction program (see FIG. 14). The listed candidates were divided into two groups of fly genes and human genes. From the target candidate list, the inventors selected, as target genes, drosophila genes, ush, Lap1, CG8445, dbo, Lar, Ced-12, CG12333, cbt, Scr, pan, pnut, Spri, Shc, Rassf and CG5608, that are known as tumor suppressors or negative regulators of cell proliferation in at least one species, and the respective human orthologs of the drosophila genes, FOG2, SEPT7, RIN2, SHC1, HOXB5, KLF11, TCF7L1, ERBB21P, ELMO2, BAP1, W DR37, KLHL20, PTPRD, RASSF2 and VAC14. The Drosophila gene u-shaped (ush) and its human homolog Friend of GATA 2 (FOG2, also known as ZFPM2) were the most frequently predicted gene pair conserved in both species.From among the listed target genes, the inventors identified, as targets of miR-8 and miR-200 family miRNAs, 7 gene pairs that respond both to miR-8 and to miR-200 family miRNAs in fly and human cells, respectively (see FIG. 15). Then, they confirmed that a target gene of miR-8 miRNA is ush, the function of miR-8 in the prevention of neurodegeneration (Karres et al., 2007) is separate from its function in body growth, not only spatially but also at the molecular level (see FIG. 16), and USH inhibits the body growth (see FIG. 18). Thus, it was found that miR-8 inhibits ush in the fat body (see FIG. 19). Thus, it was found that ush is more strongly suppressed in the fat body than in other body parts. Notably, USH protein levels are more dramatically reduced than the mRNA levels (see FIG. 20), indicating that miR-8 represses USH expression by both mRNA destabilization and translational inhibition (Karres et al., 2007). Also, the inventors confirmed that suppression of USH expression is mediated through direct binding of miR-8 to the predicted target site of USH (see FIG. 21). Further, putative target sites for miR-8 were found in all Drosophilid species examined, including distant species such as D. virilis and D. grimshawi. Thus, they found that ush is an authentic target of miR-8.Several reports have suggested that insulin signaling in the larval fat body controls organismal growth in a non-cell-autonomous manner (Britton, J S et al., 2002, Dev Cell 2, 239-249; Colombani et al., 2005).The inventors observed that suppression of insulin signaling in the fat body of flies yielded smaller flies and fewer cells in the wing (see FIG. 22) in the same manner as those in miR-8 null flies (see FIG. 4). From this result, they assumed that USH suppresses body growth by inhibiting insulin signaling. Then, they investigated whether inhibition of insulin signaling in the larval fat body suppresses the growth of flies. As a result, it was found that insulin signaling was specifically disrupted in the fat bodies of miR-8 null larvae (see FIGS. 18, 23 and 24).Also, the inventors more precisely analyzed miR-8's function in fat cells. As a result, they confirmed that miR-8 activates PI3K in a cell-autonomous manner, thereby promoting cell growth (see FIG. 25), and also the effect of miR-8 on the cell growth depends on the type of expressed tissues (see FIGS. 26, 28, and 29).Also, the inventors investigated whether USH negatively regulates insulin signaling. As a result, they found that USH inhibits insulin signaling upstream of or in parallel with PI3K in a cell-autonomous manner (see FIGS. 30, 31, 32, 33 and 34).Excessive insulin signaling is known to reduce the levels of insulin receptor (Inr) and cytohesin Steppke (step) through negative feedback by FOXO (Fuss, B et al., 2006, Nature 444, 945-948; Puig, 0, and Tjian, R 2005, Genes Dev 19, ). The inventors examined whether reduced insulin signaling caused by the absence of miR-8 could be rescued by knockdown of USH. As a result, it was found that the defect of insulin signaling in the fat body of miR-8 null larvae is at least partially attributable to elevated ush levels (see FIG. 35).According to the result of detection of miRNA target genes, the ush mRNA has one predicted binding site for miR-8, whereas the mammalian ortholog of ush, FOG2, has at least three predicted sites for miR-200 family miRNAs (see FIGS. 36 and 37). The inventors confirmed that human miR-200 family miRNAs regulate the expression by directly binding to 3′UTR of fog2 mRNA in the same manner as drosophila miR-8 miRNA regulates the expression by directly binding to 3′UTR of ush mRNA (see FIG. 38).FOG2 is expressed in heart, brain, testicle, liver, lung and skeletal muscle (Holmes, M et al., 1999, J Biol Chem 274, ; Lu, J R et al., 1999, Mol Cell Biol 19, ; Svensson, E C et al., 1999, Proc Natl Acad Sci USA 96, 956-961; Tevosian, S G et al., 1999, Proc Natl Acad Sci USA 96, 950-955). Despite its relatively broad expression in adult tissues, little is known about the function of FOG2 beyond its role in embryonic heart development (Fossett, N, and Schulz, RA 2001, Trends Cardiovasc Med 11, 185-190). Meanwhile, the miR-200 miRNAs have also been reported to be expressed in various adult organs, including pituitary gland, thyroid, pancreatic islet, testes, prostate, ovary, breast, and liver (Landgraf, P et al., 2007, Cell 129, ). The inventors looked for a correlation between the expression of FOG2 protein and miR-200c cluster miRNAs in human cell lines derived from different organs. As a result, there is generally a negative correlation between miR-200c cluster members and FOG2 in expression level (see FIG. 39).Also, the inventors confirmed that FOG2 is in actuality reduced by miR-200 miRNAs (see FIGS. 40, 41 and 39), and that human miR-200 miRNAs have a conserved role in the modulation of insulin signaling, as in the case of fly miR-8 miRNA (see FIGS. 40, 41 and 42). Also, it was confirmed that FOG2 suppresses PI3K (see FIG. 43), and phosphorylation of Akt is reduced by FOG2 (see FIG. 44).Akt represses FOXO activity. When the effect of miR-200 miRNAs on the downstream transducers of Akt was analyzed, it was found that FOXO activity is reduced by miR-200 miRNAs, and FOXO activity is increased by FOG2 (see FIGS. 45, 46 and 47).The inventors investigated whether miR-200 specifically affects the insulin signaling pathway. As a result, it was found that miR-200 specifically modulates PI3K-Akt-FOXO signaling (see FIGS. 44 and 45).Stimulation of PI3K and AKT is known to facilitate cell proliferation, and antagonize apoptosis (Pollak, 2008). Thus, consistently, the introduction of miR-200 miRNAs increased cell viability (see FIG. 50), whereas the introduction of miRNA inhibitors produced the opposite effect (see FIG. 50). Also, through investigation of the action mechanism of FOG2, it was found that FOG2 acts as a negative regulator of PI3K by interfering with the formation of an IRS-1/p85α/p110 complex (see FIGS. 51 and 52).Although FOG2 is thought to be a nuclear transcriptional co-regulator, several studies have reported that FOG2 also localizes to the cytoplasm (Bielinska, M et al., 2005, Endocrinology 146, 397M 3984; Clugston, R D et al., 2008, Am J Physiol Lung Cell Mol Physiol 294, L665-675). This indicates that FOG2 has a function within the cytoplasm (see FIGS. 53 and 54).It was tested whether FOG2 interacts with PI3K. As a result, it was determined that FOG2 directly binds to p85α, the regulatory subunit of PI3K (see FIGS. 55 to 60).In order to determine FOG2's domain to be bound to p85α, the inventors generated several truncated mutants of FOG. As a result, it was confirmed that the middle region of FOG2 (507-789 aa) mediates the interaction with p85α (see FIG. 57), and has an important role of suppressing PI3K (see FIG. 58). This result suggests that direct binding of FOG2 to p83 leads to the inhibition of PI3K activity. Especially, the inventors found that drosophila USH physically interacted with drosophila p60 (dp60; the fly ortholog of p85α) when dp60 was co-expressed with USH in human HEK293T cells (see FIG. 61). From this result, it can be found that the action mechanism of USH/FOG2 may be conserved across the phyla (see FIG. 61).It is preferable that the cell lines expressing miR-200 family miRNAs are human cell lines derived from the heart, brain, testicle, liver, lung and skeletal muscle tissues, and the cell lines expressing miR-8 miRNA are drosophila cell lines derived from fat body tissues, but the present invention is not limited thereto.The measurement of the expression level of FOG2 or USH protein is preferably carried out by any one method selected from the group including western blotting, immunostaining, fluorescent staining, and reporter assay, and the measurement of bindings between FOG2 and p85α, and USH and dp60, and the measurement of the formation of a p85α/p110/IRS-1 complex are preferably carried out by co-immunoprecipitation assay, but the present invention is not limited thereto.In the step 1), the testing compound is preferably any one selected from the group includingnatural compounds, synthesized compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, antigens, metabolites of bacteria or fungi, and bioactive molecules, whose anti-cancer agent enhancing effect is unknown to the person skilled in the art.The activity of FOG2 or USH protein may be measured by any one method selected from the group including i) a method of measuring activity of p85α/p110 ii) a method of measuring expression level of FOXO mRNA and iii) a method of measuring cell growth and proliferation, and also, the activity of p85α/p110 complex (PI3K) may be measured by measuring kinase activity of PI3K or phosphorylation level of AKT protein, but the present invention is not limited thereto.The binding between proteins may be measured by any one method selected from the group including SPR, protein chip, gel shift assay, immunoprecipitation assay, co-immunoassay, fluorescence immuno assay and radioimmuno assay, but the present invention is not limited thereto. Also, all methods for measuring binding of proteins, conventionally used in the art, may be used.Also, the present invention provides an insulin signaling regulating composition including a material changing expression or activity of FOG2 or USH protein, as an active ingredient.It is preferable that a material changing expression FOG2 or USH protein may be an expression inhibitor of FOG2 or USH protein, and the expression inhibitor of FOG2 or USH protein may be an antisense nucleotide or a small interfering RNA (siRNA) that complementarily binds to mRNA of fog2 or ush gene. Also, the material changing the activity of FOG2 or USH protein may be an activity inhibitor of FOG2 or USH protein, and the activity inhibitor of FOG2 or USH protein may be any one selected from the group including materials to be complementarily bound to FOG2 or USH protein such as cytotoxic compound, peptide, peptide mimetics and antibody, but the present invention it not limited thereto.The siRNA includes a 15- to 30-mer sense sequenceselected from base sequences of mRNA of gene (SEQ NO:42 or SEQ ID: 15) coding FOG2 or USH, and an antisense sequence complementarily bound to the sense sequence. Herein, the sense sequence preferably includes 19 to 27 bases, and FOG2 protein expression inhibitor more preferably has a base sequence represented by SEQ ID: 119, although not particularly limited thereto.The antisense nucleotide is hybridized with a complementary base sequence of DNA, immature-mRNA or mature mRNA as determined by Watson-Crick base pairing, and then, as a protein in DNA, interferes with the stream of genetic information. The antisense nucleotide has specificity for a target sequence. This characteristic allows it to be exceptionally multi-functional. The antisense nucleotide is a long chain of a monomer unit, and thus can be easily synthesized on a target RNA sequence. From many recent studies, it was proved that an antisense nucleotide is useful as a biochemical means for research of target protein (Rothenberg et al., J. Natl. Cancer Inst., 81:, 1999). Since the nucleotide synthesis field related to oligonucleotide chemical and enhanced cell adhesion, target binding affinity and nuclease resistance has been recently considerably advanced, an antisense nucleotide may be considered as a novel inhibitor.The Peptide Mimetics are peptides or non-peptides that inhibit a binding domain of FOG2 or USH protein. They suppress activity of FOG2 or USH protein. Main residues of a non-hydrolyzed peptide analogue may be generated by using (3-turn dipeptide core (Nagai et al. 1985, Tetrahedron Lett 26:647), keto-methylene pseudo peptides (Ewenson et al. 1986, J Med Chem 29:295; and Ewenson et al. 1985, in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill.), azepine (Huffman et al. 1988, in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands), benzodiazepine (Freidinger et al. 1988, in P Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands), β-amino alcohol (Gordon et al. 1985, Biochem Biophys Res Commun 126:419) and substituted gamma lactam ring (Garvey et al., 1988 in Peptides: Chemistry and Biology, G. R. Marshell ed., ESCOM Publisher: Leiden, Netherlands).The inventive composition includes the active ingredient in an amount of 0.0001 to 50 wt % with respect to the total weight of the composition.The inventive composition may include, besides a material changing expression or activity of FOG2 or USH protein, at least one kind of active ingredient showing the same or similar function as that of the material.The inventive composition, for administration, may be prepared by additionally including at least one kind of pharmaceutically acceptable carrier beside the above described active ingredient. As the pharmaceutically acceptable carrier, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, malto dextrine solution, glycerol, ethanol, liposome, and a mixture of one or more thereof may be used, and if necessary, other usual additives such as an anti-oxidant, buffer solution, bacteriostatic, etc. may be added. Also, a diluent, dispersion agent, surfactant, binder, and lubricant may be further added for formulation of injectable forms (such as aqueous solution, suspension, and emulsion), pills, capsules, granules or tablets. Also, the carrier may be used in combination with organ specific antibody or other ligands so that it can specifically act on an organ. Further, the composition may be formulated desirably according to each disease or component by using a proper method in the present field or a method disclosed in Remington's Pharmaceutical Science (updated version), Mack Publishing Company, Easton, Pa.Nucleotide or nucleic acid, used in the present invention, may be prepared for administration of oral, topical, parenteral, intranasal, intravenous, intramuscular, subcutaneous, ocular, transdermal and routes. Preferably, nucleic acid or vector is used in an injectable form. Accordingly, especially for direct injection into a treatment area, it may be used in combination with any pharmaceutically acceptable vehicle for an injectable composition. The inventive composition may include a freeze-dried composition which can have an injectable solution composition according to addition of isotonic sterilized solution, dried sterilized water or proper saline solution. The direct injection of nucleic acid into tumor of a patient is advantageous in that therapeutic efficiency can be focused on infected tissues. The dosage of nucleic acid to be used may be adjusted according to various parameters, especially gene, vector, administration method in use, problematic disease or alternatively required treatment period. Also, the range of the dosage may vary according to body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disorder of a patient. The daily dosage ranges about from 0.01 to 12.5 mg/kg, and preferably from 1.25 to 2.5 mg/kg. Also, it is preferable to administer the composition once to several times a day.The inventive insulin signaling regulating composition may be usefully used in molecular biological research of insulin signaling pathway, and prevention or treatment of diseases that can be caused by abnormal action of insulin signaling pathway, e.g., cancer, diabetes mellitus and obesity.Further, the present invention provides a use of a material changing expression or activity of FOG2 or USH protein in the preparation of an insulin signaling regulating composition.Preferably, the material changing expression of FOG2 or USH protein may be an expression inhibitor of FOG2 or USH protein, and the expression inhibitor of FOG2 or USH protein may be an antisense nucleotide or a small interfering RNA (siRNA) that complementarily binds to mRNA of fog2 or ush gene. Also, preferably, the material changing the activity of FOG2 or USH protein may be an activity inhibitor of FOG2 or USH protein, and the activity inhibitor of FOG2 or USH protein may be any one selected from the group including materials to be complementarily bound to FOG2 or USH protein such as cytotoxic compound, peptide, peptide mimetics and antibody, but the present invention it not limited thereto.The siRNA includes a 15- to 30-mer sense sequence selected from base sequences of mRNA of gene (SEQ ID: 11 or SEQ ID: 38) coding FOG2 or USH, and an antisense sequence complementarily bound to the sense sequence. Herein, the sense sequence preferably includes 19 to 27 bases, and FOG2 protein expression inhibitor more preferably has a base sequence represented by SEQ ID: 119, although not particularly limited thereto.Hereinafter, the present invention will be described in detail with reference to following Examples.However, the following Examples are only for illustrative purposes and are not intended to limit the scope of the invention.Example 1Transgenic Flies and Culture of FliesThe characteristics, suppliers, and literatures of transgenic flies used in the present invention are noted in Table 1. The flies were cultured in a standard drosophila medium, at 25° C. before being used in experiments.TABLE 1drosophilacharacteristicsupplierreference(mir-8Δ2)miR-8 null fliesSteveKarres etCohenal., 2007mir-8 gal4When combined withKyoto StockKarres etUAS-GFP, it expressesCentreal., 2007GFP under control ofmir-8 enhancerush1513It includesPatCubadda,ush1513 hypomorphSimpsonY etexpressing a reduced levelal., 1997of ush as the result of amutation in the promoterregionCg gal4It expresses Gal4 in FatYoung JoonTakata etbody and anterior lymphKimal., 2004glandppl gal4It expresses Gal4M. PankratzZinke etspecifically in fat body,al., 1999which is reduced insalivary gland Example 2Culture of Cell LinesCell lines noted in Table 2, bought from American Type Culture Collection (ATCC), were cultured at 37° C., under the condition of 5% CO2.The cultured cell lines were detached from a 75-cell culture flask by being treated with trypsin-EDTA (Trypsin-ethylenediamine tetraacetic acid, Invitrogen, US), added with serum containing medium so as to inactivate trypsin, and was subjected to precipitation through centrifugation. Supernatant was removed, and then culture medium according to each cell line was added to suspend cells. Live cells were stained with trypan blue dye exclusion test, counted by a hemocytometer, and subcultured in a 100 mm dish (5×105 cells/flask).TABLE 2Cell linemediumHepG2RPMI-1640Huh7AsPC1PANC1SW480MCF7MDA MB 231U2OSHeLaHep3BIMDMHCT116HCT116p53KD* IMDM(Iscove's Modified Dulbecco's Medium, Gibco, US): containing 10% FBS(Gibco)* RPMI-1640(Gibco): containing 10% FBS Example 3Small Body Size Phenotype of mir-8 Mutant FliesIt has been known that when human miRNAs, that is, miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) are transduced into human cell lines, cell growth is promoted (Park, S Y et al., 2009, Nat Struct Mol Biol 16, 23-29). In MCF-7 cell lines not treated with anything, and MCF-7 cell lines transduced with siRNA targeting GFP (siGFP, SEQ ID: 1: 5′-UGAAUUAGAUGGCGAUGUU-3′, Samchonri), miR-200 family [miR-141(has-miR-141, SEQ ID: 2: 5′-UAACACUGUCUGGUAAAGAUGG-3′), miR-200b (has-miR-200b, SEQ ID: 3: 5′-UAAUACUGCCUGGUAAUGAUGA-3′), miR-429(has-miR-429, SEQ ID: 4: 5′-UAAUACUGUCUGGUAAAACCGU-3′)] and miR-29b (known to inhibit cell proliferation), on day 1, 4, 5, 6, 7 and 8, the inventors counted the number of cells and measured cell proliferation. As a result, they observed that cells treated with miR-200 family showed an increased cell proliferation as compared to cells not treated with anything, or cells treated with siGFP, and that in cells treated with miR-29b, cell proliferation almost stopped from the 5th day, thus, it was found again that miR-200 family promotes cell proliferation in human cells (see FIG. 6).The miR-200 family is upregulated in certain cancer cells, including uterine cancer (lorio, MV et al., 2007, Cancer Res 67, ; Nam, EJet al., 2008, Clin Cancer Res 14, ), and is highly conserved in bilaterian animals, with miR-8 being the sole homolog of miR-200 in Drosophila mela-nogaster (Ibanez-Ventoso et al., 2008). Thus, the inventors used drosophila in order to discover the biological function of the miR-200. First, the inventors observed that the phenotype of the miR-8 null flies [mir-8Δ2(Karres, J S et al., 2007, Cell 131, 136-145)] shows a significantly small body size and body weight as compared to that of wild-type flies (see FIGS. 1 and 2). Groups of 5 flies (aged 3 to 5 days old after eclosion) were weighed. All flies were ice-anesthetized before weight measurement. Also, it is known that miR-8 null flies result in increased apoptosis in the brain and frequent occurrence of mal-formed legs and wings (Karres et al., 2007).The determination of the final body size in insects during the larval stage is analogous to that which occurs during the human juvenile period (Edgar, 2006; Mirth and Riddiford, 2007). It is generally known that reduced body size in insects is caused by either slow larval growth, precocious early pupariation (pupal formation) that shortens the larval growth period, or both (Colombani et al., 2003; Edgar, 2006; Mirth and Riddiford, 2007). The inventors observed that at 100 hr after eclosion, miR-8 null larvae exhibit a significantly smaller body volume than do wild-type larvae (see FIG. 3). In each group, at 5 hr after laying eggs, eggs were collected, and the number of new pupae and adults were counted every 12 hr. As a result, it was found that the onset of pupariation in miR-8 null flies was not significantly different from that in wild-type flies (see FIG. 3), and adult emergence (ecdysis of a chrysalis into an adult) was slightly delayed (about 12 hr) (see FIG. 3). Thus, the smaller body size of miR-8 null flies is likely to be caused by slower growth during the larval period rather than by precocious pupariation.Insufficient food intake has been reported to accompany either precocious or delayed pupariation, depending on the onset of reduced feeding (Layalle, S et al., 2008, Dev Cell 15, 568-577; Mirth et al., 2005). However, the levels of Drosophila insulin-like peptides (Dilps), which are known to be reduced in starvation conditions (Colombani et al., 2003; Ikeya et al., 2002), were not downregulated in miR-8 null larvae. Given the unaffected onset time of pupariation and the levels of Dilps in miR-8 null larvae, the small body size of miR-8 null flies is unlikely due to reduced feeding.Accordingly, in order to investigate whether the small body phenotype was caused by a reduction in cell size, cell number or both, the inventors collected 8 to 10 wings of miR-8 null flies and wild-type flies, and photographed them. Then, they counted the number of wing cilia with respect to the total of wing pixels, and calculated the relative size of a cell. Then, they divided the whole wing pixel area by a cell pixel area, and measured the relative size of a cell. As a result, wing cell number was reduced in the wing in miR-8 null flies as compared to that of wild-type flies, whereas wing cell size was not significantly different from that of wild-type flies (see FIG. 4). From this result, it was found that the reduced growth in the peripheral tissues of the miR-8 null flies was ascribed to decreased cell number rather than reduced cell size.Example 4Determination of Reduction of Insulin Signaling in miR-8 Null FliesIn order to understand why miR-8 null flies grow slowly, the inventors investigated the activities and gene expression of the proteins involved in insulin signaling in miR-8 null flies. First, by using an anti-phosphorylation Akt specific antibody, the level of activated Akt (p-Akt) was determined by western blotting (see FIG. 5). Specifically, from wild-type and miR-8 null flies, the same number of male and female flies were homogenized using 300 μl of lysis buffer (1% Triton X-100, 50 mM Tris pH7.4, 500 mM NaCl, 7.5 mM MgCl2, 0.2 mM EDTA, 1 mM NaVO4, 50 mM β-glycerophosphate, 1 mM DTT, and 25% glycerol) according to a known method (Lee, S B et al., 2007, EMBO Rep 8, 360-365), and then each group's protein extracted through RIPA buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) was separated by 6~10% SDS-PAGE. The separated protein was transferred to PVDF membrane through semi-dry transfer, blocked through culture in 2% BSA solution, and cultured in 2% BSA solution containing anti-phosphorylation-Akt (P-Akt) antibody [1:1,000,(#9271S, Ser473) Cell Signaling Technology, US]. Then, the protein was cultured in 2% BSA solution containing anti-mouse secondary antibody (SantaCruz, US) conjugated with Peroxidase, and developed on an X-ray film by using Chemiluminescent substrate (Roche, US) through immunochemistry before determination of a band. As a result, it was found that in miR-8 null flies, the p-Akt level was reduced, suggesting that Akt signaling is impaired in the absence of miR-8 (see FIG. 5).Activated p-Akt is known to deactivate FOXO via phosphorylation. Phosphorylation of FOXO prevents nuclear localization of FOXO, which, in turn, results in the reduction of transcription of FOXO target genes. Thus, the inventors examined the expression level of 4EBP (the FOXO target gene) through Q-PCR of 4EBP gene in miR-8 null larvae. First, from third-stage larvae (at 96 hr after eclosion) of wild-type and miR-8 null flies, total RNA was extracted by using Trizol reagent. 1 μg of total RNA and 1 μl of Oligo-dT (Invitrogen, US) were charged with up to 50 μl of distilled water, and placed into SuperScript First-Strand Synthesis System (Invitrogen, US), followed by RT-PCR reaction. Through the reaction at 65° C. 6 min, at 4° C. 5 min, at 42° C. for 60 min, at 95° C. for 5 min, and at 4° C. for 5 min, cDNA was synthesized. By using the cDNA as template, and using a primer pair of 4ebp sense primer (SEQ ID: 5: 5′-ATGCAGCAACTGCCAAATC-3′) and 4ebp antisense primer (SEQ ID: 6: 5′-CCGAGAGAACAAACAAGGTGG-3′), and ABI SYBR PCR master mix (Applied Biosystems, US), in ABI Prism 7900 Sequence Detection System (Applied Biosystems, US), quantitative PCR on 4ebp mRNA was performed. Herein, by using a primer pair of rp49 sense primer (SEQ ID: 7: 5′-AGGGTATCGACAACAGAGTG-3′) and rp49 antisense primer (SEQ ID: 8: 5′-CACCAGGAACTTCTTGAATC-3′), rp49 gene, as a quantitative control, was subjected to PCR. The expression level of 4ebp mRNA was converted into a relative value with respect to the level of rp49 mRNA, and a Ct (comparative cycle threshold) value was analyzed in accordance with a manual (User Bulletin 2, Applied Biosystems, US). The experiment was repeated three times, and the average value was (see FIG. 6) indicated on a graph. From this result, it was found that in miR-8 null flies, insulin signaling was considerably reduced.Example 5Determination of miR-8 Expression Level in drosophila TissuesThe expression of miR-8 in the brain, wing discs, and leg discs of drosophila was observed by Cohen, et al. (Karres et al., 2007). The inventors examined the spatial expression pattern of miR-8 in larvae, in the fat body, cuticle and brain tissues of the third-stage larvae (at 96 hr after egg-laying) of mir-8 enhancer trap GAL4 line drosophila (mir-8 gal4) (Karres et al., 2007), through immuno-staining according to a known method (Lee, Y et al., 2005, Nat Genet37, 305-310). As a result, the fat body showed the highest expression level of GFP (see FIG. 7). Also, from the total larva, fat body, gut, brain and cuticle, the total RNA was separated, and then miR-8 expression level was determined through Northen blotting. Specifically, from the fat body, gut, brain and cuticle, obtained from the whole of miR-8 Ga14 flies and through dissection of miR-8 Ga14 flies, total RNA was extracted by using Trizol reagent (Invitrogen, US), and 5.4 μg of total RNA of each kind of tissues was developed in 12.5% urea-polyacrylamide gel. The developed RNA was transferred to Zeta probe GT membrane (Biorad, US) via the flow of current. Oligonucleotide corresponding to miR-8 was end-labeled with [γ-32ATP] and used as a probe for Northen blotting. The membrane treated with the probe was exposed to a phosphor imaging plate (fugi film, Japan), and was analyzed by a BAS-2500 system (Fugi). As a result, the larval fat body showed the highest miR-8 expression level (see FIG. 8).Example 6Determination of Increase in drosophila Body Size by Fat Body-Specific Expression of miR-8Recent studies suggested that Drosophila fat body is an important organ in the control of energy metabolism and growth (Colombani et al., 2005; Colombani et al., 2003; Edgar, 2006; Leopold and Perrimon, 2007). Based on the assumption that the miR-8 expression level of larval fat body is important in control of drosophila body size, the inventors carried out the following experiments. They observed body weight of flies when miR-8 was e}