KR101330448B1 - Method for regulation of metabolic homeostasis, stress resistance, and lifespan by the endogenous siRNA(endo-siRNA) pathway - Google Patents

Abstract

The present invention relates to a method for regulating metabolic homeostasis, external stress resistance or lifespan, comprising regulating dicer gene expression involved in endo-siRNA production. The present invention described above can be usefully used for the development of therapeutic agents for metabolic disorders and the study of physiological function of endo-siRNA.

Description

Method for regulation of metabolic homeostasis, stress resistance, and lifespan by the endogenous siRNA (endo-siRNA) pathway}

The present invention relates to a method in which metabolic homeostasis, external stress resistance or lifespan is regulated through gene expression regulation by endo-siRNA produced by the activity of Dicer protein. More specifically, the present invention provides Dicer-2 (hereinafter, “Dcr-2”) which inhibits generation of endogenous small interfering RNA (hereinafter referred to as “endo-siRNA” or “endo-siRNA”) produced in vivo. Metabolic homeostasis, external stress resistance, or life control methods using mutations.

In many animals, small RNAs with a length of about 21 nucleotides (hereinafter referred to as "nt") are expressed, and these small RNAs act on sequential complementary target mRNAs to inhibit degradation or translation of the transcript. Inducement (Development 135: 1201-1214). These small RNAs are known to play important roles in a variety of biological processes, viral resistance and genome stability (Development 135: 1201-1214).

Small RNAs can be largely divided into small interfering RNAs (hereinafter referred to as "siRNAs") and micro RNAs (microRNAs referred to as "miRNAs") (Development 135: 1201-1214) (FIG. 12). Reference). Drosophila expresses two RNase III enzymes, Dicer-1 (hereinafter referred to as “Dcr-1”) and Dicer-2 (hereinafter referred to as “Dcr-2”), which are miRNA and siRNA, respectively. It has been reported to play an important role in production (Cell 117: 69-81).

Initially, siRNAs were thought to be produced by two strands of RNA (double stranded RNA) or viral infections, which have been experimentally delivered intracellularly, but recent further research has been done in Drosophila somatic cells and germ cells themselves. L. has identified endo-siRNA (Mol. Cell 31: 309-312, Curr. Biol. 18: 795-802, Nature 453: 798-802, Science 320: 1077-1081, Nature 453: 793 -797, Nat. Struct. Mol. Biol. 15: 581-590, Nature 453: 803-806). In Drosophila, these endo-siRNAs are responsible for the activity of Dcr-2 from binding sites between transposons, transcripts with long hairpin structures, and sequential complementary transcripts. Is generated. This finding suggests the potential of various endo-siRNAs to function as defense mechanisms of transposons.

Endo-siRNA has also been identified in C. elegans and mammals (Nat. Rev. Mol. Cell Biol. 9: 673-678). Endo-siRNA (Endo-siRNA) has been reported to be involved in maintaining resistance to temperature changes and in the formation of heterochromatin in Drosophila, and in nematodes, it plays an important role in spermatogenesis. Has been reported (Proc. Natl. Acad. Sci. USA 106: 21258-21263, PLoS One 4: e7576, Genetics 183: 1283-1295). However, the overall function of end-siRNA (endo-siRNA) in vivo is not known yet.

As a background technology of the present invention, there are techniques described in the following prior art document, but there is no specific description about the physiological function of endo-siRNA (endo-siRNA) in vivo.

Yoshinori Kawamura et al., Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells, Nature 453: 793-797 (2008.6) Doh Hwan Lim, Functional Analysis of Mistake Mutations in Dicer-2 in the siRNA-induced Silencing Pathway in Drosophila, Master's Thesis, Graduate School of Korea University (2009.2)

The present invention is to solve the problems and demands of the prior art as described above, the present inventors in Dcr-2 using two lines of mutant Drosophila deficient in Dcr-2 that does not produce endo-siRNA (endo-siRNA) The present invention was identified by identifying 21 proteins regulated by the gene, and confirming that gene expression regulation by endo-siRNA is involved in the regulation of external stress and lifespan and plays an important role in maintaining energy metabolism. Completed.

Accordingly, an object of the present invention is to provide a method of controlling metabolic homeostasis, external stress resistance or lifespan by regulating the activity and expression of Dicer genes involved in endo-siRNA production.

Another object of the present invention is to provide a method for producing a model animal in which metabolic homeostasis, external stress, or lifespan are controlled by controlling the expression of the Dicer gene.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the invention provides for metabolic homeostasis, external stress resistance, or in a non-human animal comprising the step of regulating the expression of the dicer gene involved in endo-siRNA production. Provides a life control method.

According to another aspect of the present invention, the present invention provides for the production of a model animal with controlled metabolic homeostasis, external stress resistance or lifespan, comprising the step of regulating the expression of Dicer genes involved in endo-siRNA production. Provide a method.

According to another aspect of the invention, the invention provides a metabolic related disorder model animal due to a deficiency in endo-siRNA produced by the activity of Dicer protein.

In the present invention, the dicer gene collectively refers to the RNase III gene involved in endo-siRNA production, for example, the fruit fly dicer- 2 gene. The animal in the method of the present invention is not particularly limited as long as it has a Dicer gene, but may be, for example, a fruit fly.

In the present invention, the dicer gene expression control includes both dicer gene expression inhibition and overexpression. Inhibiting Dicer gene expression inhibits the production of endo-siRNA, reducing its resistance to external stress, shortening its lifespan, reducing triglycerides and glucose, and reducing glycogen Is increased.

In the present invention, the external stress is not particularly limited, and examples thereof include oxidative stress, vesicle stress, hunger stress, and low temperature stress.

The dicer gene expression suppression is a conventional gene deletion method known in the art, for example, by using a light such as chemicals or ultraviolet light, or by using a genetic recombination technology through homologous recombination technology, the dicer gene region is mutated Deleted mutants can be obtained and gene pop-out techniques can be used. Mutations of the present invention may be prepared by a point mutagenesis method, but are not limited thereto, and may be performed by selecting a suitable method from among known mutation methods in the art. In an embodiment of the present invention, the EMS mutation line dcr- 2 mutation ( dcr- 2 ^R416X and dcr-2 ^L811fsX ) Drosophila was used.

According to another aspect of the present invention, there is provided a metabolic related disorder modulator comprising a dicer inhibitor or an activity enhancer involved in endo-siRNA production.

The dicer inhibitor is, but is not limited to, a dicer mutant protein or polynucleotide encoding the dicer gene.

The dicer activity enhancer is, but is not limited to, a dicer mutant protein or polynucleotide encoding the dicer gene.

In the present invention, metabolic related disorders include, but are not limited to, type I diabetes, type II diabetes, inadequate glucose tolerance, insulin resistance, hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, dyslipidemia Hyperemia or syndrome X.

Modulators of the invention may be mixed with a pharmaceutically acceptable carrier. The method of administering the modulator of the present invention is not particularly limited, but may be parenterally administered (for example, intravenously, subcutaneously, intraperitoneally, or topically) or orally according to a desired method. Preferably, administration by intravenous injection is more preferred. Dosage varies depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of disease. The daily dosage is about 0.1 to 100 mg / kg for the compound, preferably 0.5 to 10 mg / kg, and more preferably administered once to several times a day.

Hereinafter, the present invention will be described in detail.

We found two different dcr- 2 mutations ( dcr- 2 ^R416X and one) that ^lacked the production of endo-siRNA (endo-siRNA). dcr -2 ^L811fsX) using proteins extracted from the head and body of the fruit flies were subjected to two-dimensional gel analysis (hereinafter, "2D-gel"quot;) and MALDI-TOF mass spectrometry analysis, as compared to normal flies dcr- 2 mutations ( dcr-2 with ^R416X dcr- 2 ^L811fsX ) We identified 21 proteins that showed a 1.5-fold difference in expression (increase or decrease) in Drosophila. As a result of analyzing the identified proteins by their function, most of them were proteins related to metabolism, external stress, and lifespan (FIGS. 2, 3, and Table 1).

Therefore, in order to confirm the in vivo physiological function of endo-siRNA (endo-siRNA) based on the function of proteins exhibiting this expression change, first dcr- 2 mutation ( dcr- 2 ^L811fsX ) Drosophila was used to measure resistance to various external stresses (oxidative stress, endoplasmic reticulum stress, hunger stress, cold stress). It was confirmed that the resistance to external stresses (oxidative stress, ER stress, starvation stress, cold stress) decreases from a Drosophila cancer, dcr -2 mutation compared to normal flies in both male (dcr -2 ^L811fsX) fruit flies, such dcr In the fruit flies re-introduced in the dcr- 2 gene into the -2 mutant fruit flies, resistance to external stress was restored similarly to the normal fruit flies (FIGS. 6-8).

In addition, the sensitivity of Drosophila to external stress is closely related to Drosophila lifespan, so the dcr- 2 mutation ( dcr- 2 ^R416X and dcr -2 ^L811fsX ) Drosophila lifespan was compared with normal Drosophila. dcr -2 mutation (with dcr -2 ^R416X dcr -2 ^L811fsX) and trans-2-dcr hetero Xi Goat ^{(transheterozygote, dcr-2 R416X /} dcr-2 L811fsX) mutant Drosophila decreased life span as compared to normal fruit fly Drosophila, which reintroduce the dcr-2 gene is an external stress It was confirmed that the resistance was restored to a level similar to the lifespan of normal fruit flies, such as resistance (FIG. 10).

In addition, 2D-gel and MALDI-TOF mass spectrometry analysis showed that most of the proteins with different expressions in endo-siRNA-deficient Drosophila compared to normal Drosophila were proteins involved in metabolism, and also resistant to external stress. The difference in resistance to starvation during the measurement is very closely related to lipid accumulation in the body. Therefore, measuring the amount of storage in the body lipid (lipid) into triglyceride (triglyceride) and was dcr -2 mutation ^(R416X dcr-2 compared to the normal adult and larvae of the fruit fly dcr-2 ^L811fsX ) and low levels of triglycerides in the body and adults of larva was confirmed. The levels of glycogen, an energy storage form of another carbohydrate, were measured, indicating that dcr-2 mutations ( dcr-2 ^R416X and dcr-2 ^L811fsX ) larvae throughout the body was confirmed to increase compared to the normal larvae. The levels of glucose in body fluids, which are closely linked to the levels of triglyceride and glycogen in the body, are regulated by dcr-2 mutations ( dcr-2 ^R416X and dcr-2 ^L811fsX ) was confirmed to be reduced in the body fluid of the larvae (FIG. 11).

As described above, the endo-siRNA of the present invention (endo-siRNA) Metabolic homeostasis, external stress and lifespan can be regulated by regulating the expression and activity of Dicer genes involved in the production, and dicer mutations can be used to provide a model animal with controlled metabolic homeostasis, external stress and lifespan. In addition, the present invention can be usefully used for the development of therapeutic agents for metabolic related disorders and the study of physiological function of endo-siRNA.

1 shows a dcr- 2 mutant ( dcr- 2 ^R416X with two different lines) from a normal Drosophila. dcr-2 ^L811fsX ) Western blot of RNAi related proteins in Drosophila. β-Tubulin was used as loading control. * Marks are nonspecific bands.
2 is an image of a proteomic analysis of a protein regulated by Dcr-2. Normal Drosophila and two different lines of the dcr- 2 mutation ( dcr- 2 with ^R416X dcr -2 ^L811fsX ) Representative images of 2D-gel analysis in the head (A) and body (B) of Drosophila. MALDI-TOF was performed to identify proteins for spots indicated by arrows and circles. The protein corresponding to each spot number is shown in Table 1.
Figure 3 shows the normal Drosophila and dcr- 2 mutant ( dcr- 2 ^R416X with 2D-gel analysis). dcr -2 ^L811fsX ) is an enlarged image of spots showing differences in expression in the head (H) and body (B) of Drosophila. Each arrow and circle marks the point of difference. The protein corresponding to each spot number is shown in Table 1.
Figure 4 shows the dcr- 2 mutant ( dcr- 2 ^R416X with two different lines from normal Drosophila). dcr-2 ^L811fsX ) Data comparing the expression levels of mRNA transcripts corresponding to proteins with different expressions in Drosophila. Head (A) and body (B) were analyzed using quantitative RT-PCR method and normalized to the expression level of rp49 . Independently repeated 3 data were expressed as mean ± standard error of transcript expression levels in dcr-2 mutants based on 1 transcript expression level in normal Drosophila.
5 shows the dcr- 2 mutant ( dcr- 2 ^R416X with two different lines from normal Drosophila). dcr-2 ^L811fsX ) Western blot image of dG9a protein in Drosophila. β-Tubulin was used as loading control.
6 shows the effect of the deletion of the dcr- 2 gene on resistance to various external stresses. Survival was analyzed when exposed to 10 mM paraquat (A), 100 mM DTT (B), starvation (C), cold stress (D) in 10-day-old male fruit flies. Three sets of 20 animals per genotype were run to create survival curves with mean ± standard error. Statistical analysis showed that the log-rank test showed p <0.001.
7 shows the effect of the deletion of the dcr- 2 gene on resistance to various external stresses. Survival was analyzed when exposed to 10 mM paraquat (A), 100 mM DTT (B), starvation (C) and cold stress (D) in 10-day-old female Drosophila. Three sets of 20 animals per genotype were run to create survival curves with mean ± standard error. Statistical analysis showed that the log-rank test showed p <0.001.
FIG. 8 measured resistance to oxidative stress of a dcr- 2 transheterozygote mutant. Survival was analyzed when exposed to 10 mM paraquat in 10 day old male (A) and female (B) fruit flies. Three sets of 20 animals per genotype were run to create survival curves with mean ± standard error. Statistical analysis showed that the log-rank test showed p <0.001.
Figure 9 shows the results of the dcr -2 reintroduce the genetic background of the mutant Drosophila dcr -2 (dcr -2 ^L811fsX) verifying the restoration of the RNAi phenomenon. (A) Drosophila (B) dcr- 2 mutant ( dcr- 2 ^L811fsX ) Drosophila (C) dcr- 2 reintroduction Drosophila
10 shows the effect of deletion of the dcr- 2 gene on longevity. 15 sets of 20 animals for each genotype were made to create survival curves with mean ± standard error. Statistical analysis showed that the log-rank test showed p <0.001.
11 shows the results of analyzing the effect of Dcr-2 activity on energy metabolism regulation. Triglyceride levels were analyzed in the entire body of three larvae (A, left) and adult females and males (A, right). In addition, trehalose and glucose levels were compared and analyzed in hemolymphs of three larvae (B). Glycogen levels were analyzed in the whole body of male fruit flies (C). The data were expressed as mean ± standard error, and statistical analysis was performed by Dunnett's test after ANOVA *, p <0.05; **, p <0.01; ***, p <0.0001.
12 is a view showing briefly the RNAi pathway in Drosophila.
Figure 13 is a schematic diagram of a method for controlling metabolism, stress resistance and lifespan by inhibiting the production of endo-siRNA by the present invention.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

< Example  1> dcr -2  In mutant fruit flies RNAi  Identification of changes in expression of proteins involved in the phenomenon

In the dcr- 2 mutant Drosophila, Western blot was used to identify changes in RNAi (including miRNA and siRNA) related proteins other than R2D2 protein, which is affected by protein stability through interaction with Dcr-2. The expression of proteins was compared and analyzed.

<1-1> fruit fly line

Drosophila was maintained at 25 ° C. in normal Drosophila medium. Genotype of normal Drosophila is yw eyFLP ; FRT42D (hereinafter referred to as "normal or wildtype"). Genotypes of dcr- 2 mutant Drosophila are yw eyFLP ; FRT42D , dcr- 2 ^R416X (hereinafter referred to as “ dcr- 2 ^R416X ”) and yw eyFLP ; FRT42D , dcr- 2 ^L811fsX (hereinafter referred to as “ dcr- 2 ^L811fsX ”), is a Drosophila line induced mutations using EMS, and a normal Dcr-2 protein is formed by abnormal termination codon of the dcr- 2 gene due to mutation. These are non-forming mutants (Cell 117 (1): 69-81).

And after producing the DNA to the original genomic promoters Dcr-2 attached hemagglutinin (hereinafter, "HA" hereinafter) at the amino terminus by (genomic promoter) for expression experiments recovery through the introduction of genetic material, dcr -2 ^Inserted into the ^L811fsX Drosophila to create a dcr- 2 reintroduced Drosophila, genotypes of which are as follows: yw eyFLP ; FRT42D , dcr -2 ^L811fsX ; HA - dcr -2 (hereinafter “ dcr -2 ^L811fsX ; HA - dcr -2 ” Called)

<1-2> Western Blot  ( western blot )

We used normal Drosophila, two different dcr- 2 mutations ( dcr- 2 ^R416X and dcr- 2 ^L811fsX ) Drosophila were used. Proteins were obtained by homogenizing each genotype Drosophila in lysis buffer (100 mM potassium acetate, 30 mM HPES-KOH (pH 7.2), 2 mM magnesium acetate, 5 mM DTT, 1 mg / ml Pefabloc SC). As follows. anti-Dcr-2, anti-R2D2 (from Q. Liu), anti-Dcr-1 (Abcam), anti-Ago1 (Abcam), anti-Ago2 (from M. Siomi), anti-dFMR, anti- TSN (obtained from G. Hannon) and anti-β-tubulin (Developmental Studies Hybridoma Bank).

As a result, the dcr -2 mutation ( dcr -2 ^R416X and dcr -2 ^L811fsX ) Drosophila did not express R2D2, which affects protein stability by interacting with Dcr-2 and Dcr-2, but the expression levels of other RNAi proteins related to miRNA and siRNA pathway were not changed (Fig. One).

< Example  2> Dcr -2 Protein Identification and Regulation of Proteins Transcript  Expression level investigation

We used the dcr- 2 mutation ( dcr- 2 ^R416X and dcr- 2 ^L811fsX ) Proteins were identified with 2D-gel and MALDI-TOF to investigate proteins with varying expression levels in Drosophila.

<2-1> Two - dimensional gel  Analysis (hereinafter, “2D- gel And protein identification)

2D-gel analysis showed that 10 days of the same number of cancers, male Drosophila (normal Drosophila, two different lines of dcr- 2 mutation ( dcr- 2 ^R416X and dcr- 2 ^L811fsX ) About 200 heads and bodies were separated and repeated three times independently. Protein concentration was determined by Bradford assay. Briefly describing the 2D-gel analysis, the primary separation was separated by isoelectric focusing on a 24 cm strip (Genomine Inc.) with a pH 4-10 nonlinear gradient. Secondary separation was performed by electrophoresis on 20- × 24-cm, 10-16% linear gradient sodium dodecyl sulfate-polyacrylamide gel. These isolated proteins were identified by silver staining. Image analysis and quantitative analysis of protein spots were performed using PDQuest software (version 7.0, Bio-Rad).

Protein identification involves in-gel trypsin digestion of spots with differences in expression and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry analysis (hereinafter referred to as "MALDI-TOF", Ettan MALDI-TOF mass spectrometer). GE Healthcare Bioscience). Peptide mass spectra were also compared to the Drosophila NCBI non-redundant database using the ProFound program (http://prowl.rockefeller.edu/prowl-cgi/profound.exe).

As a result, 2D-gel analysis of 3 iterations of the dcr-2 mutant ( dcr-2 ^R416X and dcr-2 ^L811fsX ) In the fruit flies, 15 spots showing more than 1.5-fold expression change were confirmed in 15 heads and 16 body parts, respectively (FIGS. 2 and 3). Proteins corresponding to each spot were identified using MALDI-TOF. When the proteins analyzed were classified by their function, most of them were proteins involved in metabolism, response to external stress, development and development (Table 1).

<2-2> quantitative RT - PCR

Normal Drosophila and two different lines of the dcr- 2 mutation ( dcr- 2 with ^R416X dcr- 2 ^L811fsX ) Drosophila head and body were separated from each tissue using TRIzol reagent (Invitrogen). 1 μg of the isolated RNA was treated with DNaseI (Invitrogen), and first-stranded cDNA was synthesized using random hexamer (Promega) and MMLV-reverse transcriptase (Invitrogen). Quantitative RT-PCR was performed using SYBR green supermix (Bio-rad) as a template of this cDNA. Primers for each gene used in PCR are as follows (Table 2).

As a result, three genes (CG4408, CG11841, CG6776) in the head, and three genes (CG6084, CG11661, CG4466) in the body were present with the dcr- 2 mutation ( dcr- 2 ^R416X and dcr -2 ^L811fsX ) Drosophila both lines showed a 1.5-fold difference in the same direction as the protein expression pattern. The other genes did not show a statistically significant difference (FIG. 4). All data were obtained through independent 3 replicates.

<2-3> Western Blot  ( western blot )

Western blots were performed to reconfirm the proteins with different expressions in dcr- 2 mutants using the method of <2-1>. The antibody was used in the same manner as in <1-2> and the antibodies used were as follows. anti-dG9a (obtained from A. Lambertsson), anti-β-tubulin (Developmental Studies Hybridoma Bank).

As a result, the dG9a protein was expressed only in normal Drosophila, and the dcr- 2 mutation ( dcr- 2 ^R416X and dcr -2 ^L811fsX ) was not found in both Drosophila lines (Figure 5).

<2-4> Statistical Analysis

Prism 5 software (GraphPad) was used to statistically verify differences in expression levels. Statistical processing was performed by Dunnett's test with analysis of variance (hereinafter referred to as “ANOVA”) and multiple tests.

< Example  3> dcr -2  Investigation of resistance to various external stresses in mutant fruit flies

The inventors used a dcr- 2 mutant ( dcr- 2 ^L811fsX ) Drosophila to analyze the ^{biophysiological} function of endo-siRNA (endo-siRNA) using various external stresses (oxidative stress, vesicle stress, hunger stress, cold stress). Resistance to was compared with normal fruit flies.

<3-1> oxidative stress ( Oxidative stress Resistance measurement

Sixty-day-old Drosophila cancers and 60 males each were measured, and 20 animals per vial were progressed in three sets. All fruit flies were fasted for three hours in an empty vial containing no food prior to measuring their resistance to stress, followed by a vial containing 5% sucrose with 10 mM paraquat. ), The survival rate of the fruit flies was measured every hour.

As a result, it was found that in both cancer and male compared with normal fruit flies Drosophila mutant dcr -2 (dcr -2 ^L811fsX) decreased resistance to oxidative stress, dcr -2 reintroduced in Drosophila fruit flies with normal oxidative stress similarly It was confirmed that the resistance to increase (FIGS. 6 and 7). And further dcr -2 trans hetero Xi Goth ^{(transheterozygote, dcr -2 R416X / dcr} -2 L811fsX) of cancer mutations, it was confirmed that both males decreased in resistance to oxidative stress than normal fruit flies (Fig. 8).

<3-2> endoplasmic reticulum stress ( ER stress) Resistance to

Drosophila used for the measurement is the same as <3-1>. All Drosophila were fasted for 3 hours in an empty vial containing no food before measuring resistance to stress, then a vial containing 5% sucrose containing 100 mM dithiothreitol (DTT) vial) to measure the survival rate of fruit flies every hour.

As a result, and in both cancers were male fruit flies have mutations dcr -2 (dcr -2 ^L811fsX) compared to normal fruit flies confirm that the resistance to the endoplasmic reticulum stress (ER stress) decreased, dcr -2 reintroduction of a normal fruit fly Drosophila It was confirmed that the resistance to ER stress was restored to the level (FIGS. 6 and 7).

<3-3> starvation Starvation Resistance measurement

Drosophila used for the measurement is the same as <3-1>. To measure the resistance to stress, the fruit flies were transferred to empty vials containing no food to measure the survival rate of fruit flies every hour.

As a result, cancer, dcr -2 mutations in both males (dcr -2 ^L811fsX) it was found that fruit flies are resistant to starvation decreased than normal fruit flies, dcr -2 reintroduced in Drosophila is resistant to starvation similar to the normal fruit flies It was confirmed that the improvement (Fig. 6, 7).

<3-4> low temperature stress ( Cold stress Resistance measurement

Drosophila used for the measurement is the same as <3-1>. To measure the resistance to stress, the fruit flies were transferred to a vial containing normal fruit fly foods, and 8 hours of the day were subjected to cold stress at 4 ° C., and then 16 hours were recovered at 25 ° C. The daily survival of the fruit flies was measured by applying cold stress every day.

As a result, cancer, and it was confirmed that the mutant fruit flies dcr -2 (dcr -2 ^L811fsX) in both male resistance to cold stress decreased more than normal fruit fly, Drosophila dcr -2 reintroduction is resistant to cold stress similar to the normal fruit flies It was confirmed that the (Fig. 6, 7).

<3-5> dcr -2  Reintroduction Fruit fly RNAi  Recovery verification of the phenomenon

The present inventors attempted to determine whether the RNAi phenomenon was normally restored in the dcr- 2 reintroduced Drosophila. Normal Drosophila, dcr- 2 mutation ( dcr) in the background of the introduction of the GMR-wIR gene, which can induce the long hairpin dsRNA, which can inhibit the expression of the white gene responsible for the color of fruit flies in vivo -2 ^L811fsX ) Drosophila, dcr-2 re-introduced Drosophila eye color. Under normal conditions, when the expression of the Drosophila white gene due to the inhibitory dsRNA in Drosophila eye color is changed to yellow (inhibition) in the red (top), when the RNAi phenomenon defect in the mutant dcr -2 expression of the white gene suppression Drosophila's eye color stays red (normal). When the RNAi phenomenon was restored by re-introducing dcr- 2 into the fruit flies, the white gene expression by dsRNA was suppressed again, and the eye color was changed from red (normal) to yellow (inhibited). 9).

<3-6> Statistical Analysis

Statistically, Prism 5 software (GraphPad) was used to verify the difference in survival rate for each external stress. Statistical processing was performed log-rank test.

< Example  4> dcr -2  Mutation In the fruit fly  Life survey

<4-1> Drosophila Life Span Comparison Analysis

We used the dcr- 2 mutation ( dcr- 2 ^R416X and dcr- 2 ^L811fsX ) Drosophila life was compared with normal Drosophila. Drosophila used in the measurement was a female and male for one day to breed after the adult fly (adult fly) was mated, and then the life span was measured by dividing the male and female. The life span of fruit flies was measured in 300 females and 30 males and 25 ℃, 65% humidity, and 12 hours of varying contrast.

As a result, dcr- 2 mutations ( dcr- 2 ^R416X and dcr- 2 ^L811fsX ) Drosophila was confirmed to have a shorter lifespan than normal Drosophila, and dcr- 2 reintroduced Drosophila was found to have a lifespan similar to that of normal Drosophila (FIG. 8). In addition, it was confirmed that in cancer, both male further dcr-2 mutant trans hetero Xi Goat ^{(transheterozygote, dcr-2 R416X /} dcr-2 L811fsX) of the life is decreased than normal fruit flies (Fig. 10).

<4-2> Statistical Analysis

Prism 5 software (GraphPad) was used to statistically verify the difference in lifespan. Statistical processing was performed log-rank test.

< Example  5> dcr -2 Mutation In the fruit fly  Energy metabolism investigation

We used the dcr- 2 mutation ( dcr- 2 ^R416X and dcr -2 ^L811fsX ) Triglyceride, glycogen, and glucose levels were measured to investigate changes in energy metabolism in fruit flies.

<5-1> Triglyceride ( Triglyceride Level measurement

dcr -2 mutation (with dcr -2 ^R416X dcr -2 ^L811fsX) a third instar larva 30 rabbits and six sets of 10 days-old male rats 30 15 occurs at the same time to measure the triglyceride (triglyceride) in the whole body of a normal fruit fly, dcr-2 reintroduced in Drosophila, including Drosophila Set, and 10 females of 30 females were used. Each sample was homogenized in 600 μl lysis buffer (lysis buffer: 150 mM NaCl, 10 mM Tris-HCl, pH 8.0, and 0.1% Triton X-100) and then filtered with 0.45 μm-filter (Sartorius). Triglyceride was measured using Triglyceride Assay Kit (ThermoElectron). In brief, about 7 μl of the filtered sample was mixed with 150 μl triglyceride assay solution and maintained at 37 ° C. for 10 minutes, and then absorbance was measured at 200 nm (SpectraMax M5, Molecular Devices). The levels of triglycerides were normalized to the amount of protein in each sample.

As a result, the dcr -2 mutation ( dcr -2 ^R416X and The larvae of dcr -2 ^L811fsX ) showed 28-50% triglyceride reduction in the whole body compared to the normal larvae, and 21-49% triglyceride in both the female and the female body in the adult body. Was found to decrease. And The dcr- 2 reintroduced larvae and adults recovered to levels similar to those of normal larvae and adults (FIG. 11A).

<5-2> glycogen ( Glycogen Level measurement

dcr -2 mutation (with dcr -2 ^R416X 330 10-day-old females and males were used to measure glycogen levels in normal Drosophila, including dcr- 2 ^L811fsX ) Drosophila, and dcr-2 Re-introduced Drosophila. Each sample was homogenized on ice with 300 ml of phosphate buffered saline (PBS) containing 0.5% Tween 20 and immediately maintained at 70 ° C. for 5 minutes. It was then centrifuged at 4 ° C. and 5 μl of the supernatant was added to 50 μl Colorimetric Reaction Mixture (Abcam). The mixture was maintained at 37 ° C. for 30 minutes and then its absorbance was measured at 570 nm (SpectraMax M5, Molecular Devices). Glycogen levels were normalized to the amount of protein in each sample.

As a result, the dcr -2 mutation ( dcr -2 ^R416X and dcr- 2 ^L811fsX ) Drosophila males were found to have increased levels of 25-28% glycogen (Glycogen) compared to normal Drosophila males. In males of the dcr- 2 reintroduced Drosophila, the levels were restored to the levels similar to those of the normal Drosophila (FIG. 11C).

<5-3> glucose ( Glucose Level measurement

dcr -2 mutation (with dcr -2 ^R416X dcr -2 ^L811fsX ) 3-4 larvae per set to measure glucose levels in the hemolymph of three larvae at the same time in normal and dcr- 2 reintroduced fruit flies, including fruit flies. Was used. Fasted at 2% agar prior to measurement and about 0.4 μl of hemolymph was converted to porcine kidney trehalase (Sigma) to convert trehalose (a disaccharide of glucose) to glucose (pH 6.8, 0.16). 200 μl of Infinity Glucose Reagent (ThermoElectron) with a concentration of U / ml was incubated at 37 ° C. for one day. Glucose concentration in total body fluid (hemolymph) was obtained by measuring the absorbance at 340 nm (SpectraMax M5, Molecular Devices). Standard curves were obtained at glucose concentrations of 0-100 mg / ml.

As a result, the dcr -2 mutation ( dcr -2 ^R416X and dcr -2 ^L811fsX ) Larvae It was confirmed that the level of circulating glucose (glucose) is reduced in 10-14% body fluid compared to the normal larvae in both lines. And in dcr- 2 reintroduced larvae recovered to levels similar to normal larvae (FIG. 11B).

<5-4> Statistical Analysis

Statistically, Prism 5 software (GraphPad) was used to verify the difference between the levels of triglyceride, glycogen, and glucose. Statistical analysis was performed by Dunnett's test with ANOVA and multiple tests.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is obvious that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. It is therefore intended that the scope of the present invention be defined by the appended claims and their equivalents.

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Claims (5)

Inducing mutations that inhibit the expression of the Drosophila dicer-2 gene involved in endo-siRNA production and reduce the metabolic homeostasis of Drosophila, oxidative stress, endoplasmic reticulum Metabolic homeostasis, external stress resistance, or life control method in Drosophila characterized by reducing the resistance of Drosophila to external stress selected from the group consisting of stress, starvation stress and cold stress, or reducing Drosophila life. . The method of claim 1, wherein the Dicer-2 gene mutation is dcr-2 ^R416X Or dcr-2 ^L811fsX . The method of controlling metabolic homeostasis, external stress resistance, or lifespan in Drosophila. Inducing mutations that inhibit the expression of the Drosophila dicer-2 gene involved in endo-siRNA production and reduce metabolic homeostasis, oxidative stress, vesicle stress, hunger A method for producing fruit flies having reduced resistance to external stress selected from the group consisting of stress and cold stress, or reduced lifespan. delete delete

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