CN106729750A - Method and medicine and their application of high fat of blood, fatty liver, type-II diabetes and losing weight are treated by miR-183 - Google Patents
Method and medicine and their application of high fat of blood, fatty liver, type-II diabetes and losing weight are treated by miR-183 Download PDFInfo
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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Abstract
The present invention relates to biomedicine field, disclose and suppress miR-183 functions with the application in preventing and/or treating high fat of blood, fatty liver, increased weight, diabetes and the symptom similar to the symptom of these diseases.The method for also disclosing the function of suppressing miR-183, including：MiR-183 inhibitor is contacted with the target cell of expression miR-183, also disclose the inhibitor for suppressing miR-183 functions, the pharmaceutical composition and kit of the inhibitor are included, theirs is preventing and/or treating high fat of blood, fatty liver, diabetes and the symptom similar to the symptom of these diseases and the application in losing weight.By suppressing the function of miR-183, can effectively prevent and/or treat high fat of blood, fatty liver, diabetes and the symptom similar to the symptom of these diseases, additionally it is possible to lose weight.Compared to traditional single hypoglycemic or the medicine of single reducing blood lipid or single fat-reducing, the therapy that the present invention is provided can comprehensively modulation of appetite, digest and assimilate, glycolipid metabolism and Adipocyte Differentiation, thus effect is powerful and Small side effects.
Description
Technical field
The present invention relates to biomedicine field, specifically, it is related to by suppressing high fat of blood, fatty liver, increased weight, the function of the mark miR-183 of type-II diabetes is being prevented and/or is treating high fat of blood, fatty liver, type-II diabetes and the symptom similar to the symptom of these diseases and the application in losing weight, a kind of method of the function of suppression miR-183, a kind of inhibitor based on miR-183, pharmaceutical composition and kit, and their applications in the function of suppressing miR-183, particularly preventing and/or treating high fat of blood, fatty liver, type-II diabetes and the symptom similar to the symptom of these diseases and the application in losing weight.
Background technology
Energy intaking surplus is the most basic reason of high fat of blood, fatty liver and obesity, and obesity is the inducement of generally acknowledged type-II diabetes.Existing research show brain (hypothalamus is even more important)-stomach central shaft to appetite, take food and digest and assimilate and glucose-lipid metabolism plays important adjustment effect [1], thus the change of gene expression is likely to the most basic reason of obesity in hypothalamus-stomach.And the intake surplus of energy can at first cause fatty liver, then change the expression of gene in liver, cause the not normal of lipid metaboli, cause angiocardiopathy.Additionally, there are some researches show brain-stomach-liver central shaft plays important adjustment effect [2] to metabolism of blood glucose, showing that brain-stomach-liver central shaft is probably the new target organ system [3] for treating high fat of blood, fatty liver, obesity and type-II diabetes.
More and more evidences show that microRNA (microRNA, miRNA) plays an important role [4] in energy metabolism regulation.MicroRNA is the non-coding RNA molecule (www.mirbase.org) that a class length is 16-25nt, can recognize the rna expression and/or protein expression of simultaneously silencing of target genes by being matched with target gene partial complementarity.After ripe microRNA is loaded on the silencing complex (RISC) of RNA inductions, it is combined with the complementary series in target gene mRNA 3'-UTR by base pairing, so as to trigger the degraded of mRNA and/or suppress the translation of its protein.The nucleotides of the second at microRNA 5' ends to the 8th is referred to as " core sequence ", and the complementary pairing of this seven nucleotides and target gene is the key for recognizing target gene, and pairing degree is higher, with reference to bigger with the possibility and ability of regulation target gene.Other sequences outside microRNA " core sequence " can also strengthen its ability for combining and regulating and controlling target gene with the complementary pairing of target gene simultaneously.It is the expression for recognizing and adjusting target gene by non-fully matching just because of microRNA, just enables a microRNA intracellular at one while adjusting multiple target genes to some extent.
The content of the invention
The purpose of the present invention is the medicine of control energetic supersession of the exploitation based on microRNA.
To achieve these goals, on the one hand, the function the invention provides suppression high fat of blood, fatty liver, increased weight, the mark miR-183 of type-II diabetes is being prevented and/or is treating high fat of blood, fatty liver, type-II diabetes and the symptom similar to the symptom of these diseases and the application in losing weight.
Second aspect, the invention provides a kind of method for suppressing high fat of blood, fatty liver, increased weight, the function of the mark miR-183 of type-II diabetes, wherein, the method includes：MiR-183 inhibitor is contacted with the target cell of expression miR-183.
The third aspect, the invention provides a kind of miR-183 inhibitor, wherein, the miR-183 inhibitor is ASON, including antisense DNA and antisense RNA, the ASON are complementary with miR-183, and with the 8-23 length of nucleotides；Or the miR-183 inhibitor is the siRNA of miR-183 precursors.
Fourth aspect, present invention also offers a kind of pharmaceutical composition, wherein, the pharmaceutical composition contains miR-183 inhibitor as described above and pharmaceutically acceptable carrier.
5th aspect, present invention also offers a kind of kit, wherein, the kit includes miR-183 inhibitor as described above, and optionally, the kit also includes pharmaceutically acceptable carrier.
6th aspect, present invention also offers the application of method as described above, miR-183 inhibitor as described above, pharmaceutical composition as described above and/or kit as described above in the function of suppressing miR-183；Particularly preventing and/or treating high fat of blood, fatty liver, type-II diabetes and the symptom similar to the symptom of these diseases and the application in losing weight.
7th aspect, present invention also offers the application of miR-183 inhibitor as described above, pharmaceutical composition as described above in the medicine for the function of suppressing miR-183 is prepared；Particularly prepare for prevent and/or treat high fat of blood, the medicine of fatty liver, type-II diabetes and the symptom similar to the symptom of these diseases and the application in preparing for the medicine that loses weight.
The present invention is contacted by by miR-183 inhibitor with the target cell of expression miR-183, the function that miR-183 can fully be suppressed (including suppresses the combination of miR-183 and its target gene or the expression quantity of reduction miR-183, so as to suppress the function of miR-183), when individuality administration, can effectively prevent and/or treat miR-183 expression quantity and raise caused disease, for example, high fat of blood, fatty liver, increased weight, type-II diabetes or the symptom similar to the symptom of these diseases.Compared to the medicine of traditional single reducing blood lipid or single hypoglycemic or single fat-reducing, the therapy that the present invention is provided can comprehensively modulation of appetite, digest and assimilate, sugar, phospholipid metabolism, and be oligonucleotides；Thus effect is powerful and Small side effects.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Brief description of the drawings
Accompanying drawing is, for providing a further understanding of the present invention, and to constitute the part of specification, is used to explain the present invention together with following specific embodiment, but be not construed as limiting the invention.In the accompanying drawings：
Fig. 1 be hypothalamus, stomach and in liver miR-183 expression figure, wherein, the expression of the miR-183 during the expression of miR-183 is significantly higher than the mouse at 2 monthly ages in the 12 monthly age mouse.
Fig. 2A is the linear relationship chart of the functional effect that miR-183ASO dosage suppresses miR-183 with it.
Fig. 2 B are the comparison diagrams of the functional effect that miR-183ASO, random controls nucleotides, miR-183 mispairing ASO suppress miR-183 on a cellular level.
Fig. 3 A are the mouse of injection miR-183ASO and PBS respectively with the comparison diagram of time changes of weight.
Fig. 3 B are the mouse of injection miR-183ASO and PBS respectively with the comparison diagram of time body weight rate of rise.
Fig. 3 C are to inject two comparison diagrams of the blood sugar concentration of the mouse of the miR-183ASO and PBS of first quarter moon respectively.
Fig. 4 A are the comparison diagrams of the weight of kidney, liver, epididymal adipose tissues and the stomach of the mouse for injecting trimestral miR-183ASO and PBS respectively.
Fig. 4 B are the comparison diagrams of the ratio of the weight relative body weight of kidney, liver, epididymal adipose tissues and the stomach of injecting trimestral miR-183ASO and PBS respectively.
Fig. 5 is the comparison diagram of the content of T-CHOL (CHOL), triglyceride (TG), HDL (HDL) and low-density lipoprotein (LDL) in the mice serum for inject respectively trimestral miR-183ASO and PBS.
Fig. 6 is the comparison diagram of siRNA and random controls RNA on miR-183 expression influences of miR-183.
Specific embodiment
Specific embodiment of the invention is described in detail below.It should be appreciated that specific embodiment described herein is merely to illustrate and explain the present invention, it is not intended to limit the invention.
Unless otherwise indicated, scientific and technical terminology used herein has has identical implication with the term of those skilled in the art's routine understanding.
The present inventor has found in the process of research, compared to the mouse at 2 monthly ages, miR-183 in master control appetite, the hypothalamus-stomach axle center for digesting and assimilating with energetic supersession is substantially to raise in 12 monthly age mouse, and expression in liver is also significantly rise.Found based on more than, inventor predicts the target gene of the miR-183 guarded in vertebrate using TargetScanHuman (www.targetscan.org) algorithm again, then contrasted with KEGG paths and found, the target gene of many miR-183 is enriched in gastric acid secretion, salivary secretion, phosphatidylinositols signal path, insulin signaling pathway and type-II diabetes signal path (table 1).It is another to there are 68 target genes of miR-183 to take part in Adipocyte Differentiation, cholesterol transport, type-II diabetes [5,6] positioned at the path in MAPK signal paths；The target base for having 84 miR-183 belongs to regulation glucose metabolism, the PI3K-Akt signal paths [5,7] of Adipocyte Differentiation；The TGF-beta signal path [5,8,9] related to obesity, diabetes, hepatopathy is located to 26 target bases of miR-183；And 34 target bases of miR-183 are located at regulation blood sugar and GnRH signaling pathway [10] (table 1) being metabolized.
Table 1
Studied based on more than, the inventors found that miR-183 can be as the mark of high fat of blood, fatty liver, increased weight and type-II diabetes, and high fat of blood, fatty liver, type-II diabetes and the symptom similar to the symptom of these diseases and losing weight can be prevented and/or be treated to the function of then demonstrating by suppressing miR-183.
Therefore, the invention provides suppressing application of the function of high fat of blood, fatty liver, the mark miR-183 of increased weight and type-II diabetes in preventing and/or treating following disease and/symptom：High fat of blood, fatty liver, increased weight and type-II diabetes and the symptom similar to the symptom of these diseases, a kind of miR-183 inhibitor, by the method for the miR-183 inhibitor to suppress the function of miR-183, and the pharmaceutical composition and kit of miR-183 inhibitor are included, and they are preventing and/or are treating high fat of blood, fatty liver, type-II diabetes and the symptom similar to the symptom of these diseases and the application in losing weight.
Method
The invention provides a kind of method of the function of the mark miR-183 for suppressing high fat of blood, fatty liver, increased weight and type-II diabetes, wherein, the method includes：MiR-183 inhibitor is contacted with the target cell of expression miR-183.
In a preferred case, miR-183 has SEQ ID No：Nucleotide sequence shown in 1 (the miR-183 sequences of people and mouse are completely the same) (UAUGGCACUGGUAGAAUUCACU).
According to the present invention, the function of miR-183 " suppress " refers to the degree that miR-183 is lowered to its expression of target gene in target cell compared to the expression miR-183 of the same race for not using the inventive method to process, at least 0.5 times is reduced to the degree that its expression of target gene is lowered using miR-183 in the target cell of the expression miR-183 of present invention treatment, at least 1 times, such as Fig. 2A and Fig. 2 B can generally be reduced.
The method that the present invention provides suppression miR-183 functions in the target cell of expression miR-183 in vivo or in vitro.Term " suppressing the function of miR-183 " refers to cause that the expression quantity of the target gene adjusted by miR-183 is raised by directly or indirectly acting on miR-183 with reagent.Its method includes but is not limited to following several：
1)
Micromolecular compound
MiR-183 inhibitor includes but is not limited to naturally occurring or artificial synthesized micromolecular compound, this kind of micromolecular compound directly acts on miR-183 and causes that the expression quantity of the target gene adjusted by miR-183 is raised, and typically molecular weight is more than 50 and less than the organic compound of 2500 dalton.This kind of candidate compound possesses and protein, particularly the functional group of interaction of hydrogen bond, and generally comprises at least one amine, carbonyl, hydroxyl or carboxylic group.These small molecules miR-183 inhibitor can be found by suitable screening technique or other methods.
2)
ASON
The ASON can by with target miR-183 directly in conjunction with suppressing the function of target miR-183, including antisense RNA and antisense DNA.Preferably, the ASON is complementary with miR-183, with the 8-23 length of nucleotides, and with the 2-8 sequence of nucleotide complementary with miR-183.
As described in the background section, it is known, microRNA can recognize the expression and/or translation of simultaneously silencing of target genes by being matched with target gene partial complementarity, similarly, miR-183 also can be by the Reverse transcriptase function of its own with reference to the nucleotide sequence with its partial complementarity, so as to raise the expression of the target gene of miR-183.Therefore, in the present invention, term " complementation " not only including complete complementary, also including partial complementarity, as long as can combine with miR-183 and suppress its function.
Therefore, the ASON has following nucleotide sequence：
a)SEQ ID No：Nucleotide sequence (AGTGAATTCTACCAGTGCCATA) shown in 4；
B) in SEQ ID No：Through lacking, replacing or adding one or several nucleotides and the nucleotide sequence complementary with miR-183 in nucleotide sequence shown in 4.
When in the case of non-fully complementation, that is, when the ASON is by SEQ ID No：In the case of nucleotide sequence in nucleotide sequence shown in 4 through lacking, replacing or add one or several nucleotides to obtain, in complementary nucleotide acid region, the ASON preferably should at least have 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% complementation with miR-183.It is more highly preferred to, in the nucleotide region that the 2-8 of miR-183 is, the ASON at most has the nucleotides of 3 and miR-183 mispairing.
As described above, in the case of the ASON and miR-183 non-fully complementation, it is further preferred that with SEQ ID No：4 compare, and will at most have 10,9,8,7,6,5,3,2 or 1 difference of nucleotides in length.
In a preferred case, non-fully complementary ASON has SEQ ID No with miR-183：Nucleotide sequence (AGTGAGCTCTACCAGTGGCATA) described in 5.
In addition, present invention additionally comprises some conventional modifications are carried out to the ASON to improve the stability and activity of the ASON, these belong to the scope of the present invention.
The present invention is it is pointed out that have the complete or non-fully complementary RNA of as above characteristic also within the scope of the present invention.Consider stability in the cell, the present invention preferably ASON is DNA.
Because the ASON can be with miR-183 complementations (complete complementary or partial complementarity), therefore, when target cell of the ASON in vivo or in vitro with expression miR-183 contacts, the ASON can carry out complementary pairing with miR-183, and suppress miR-183 and its target gene combination (namely, suppress the activity of miR-183), so as to break silences of the miR-183 to its target gene.
According to the present invention, methods described includes being incorporated into effective dose and miR-183 complementations ASON in the target cell of expression miR-183.Wherein, " effective dose " is different and different according to the target cell of expression miR-183, and show certain dosage effect, as shown in the lower Fig. 2A of the present invention, those skilled in the art can readily determine the effective dose of the target cell for expression miR-183 according to conventional laboratory facilities and the expected purpose for being reached.
When the contact is for contact in vivo, be administered to ASON of the invention in individuality by the method that can be administered by conventional nucleic acid.It is, for example possible to use following method carries out the administration of the ASON：The ASON can be administered by the method for virus infection, microinjection or Vesicle fusion, or can also be used for the intramuscular delivery of the ASON by the method for jet injection.Alternatively, it is also possible to the ASON is coated on golden particulate, percutaneous dosing is then carried out by the known method such as particle bombardment equipment or " particle gun ".These are this area conventional technology, and this is no longer going to repeat them for the present invention.
Furthermore, the ASON can also be incorporated into the target cell of expression miR-183 in the method for expression vector.This kind of expression vector has the insertion for being located at the convenience restriction site of promotor-proximal sequence in order to the ASON.Wherein, the transcription box in the expression vector can include transcription initiation region, target gene or its fragment and transcription termination region.The carrier for example can be but be not limited to, plasmid that virus etc., those skilled in the art can voluntarily be selected according to actual conditions.
Additionally, the ASON can also be for example administered by way of respiratory tract spray delivery so as to be introduced in the target cell of expression miR-183 by way of being prepared into spray agent.
In addition, the mode that the ASON can also be administered orally is so as to be introduced in the target cell of expression miR-183, for example it is administered by way of being prepared into oral formulations, or is administered orally by way of the ASON is mixed with food.
Individuality as described above can be any mammalian cell, including but not limited to：Ungulate, for example, ox, goat, pig, sheep etc.；Rodent, for example, hamster, mouse, rat, rabbit；Primate, for example, monkey, baboon, mankind etc..
When the contact is for vitro exposure, can by by the ASON or carrier containing the ASON (for example, medicine containing the ASON) be added directly into culture have expression miR-183 target cell matrix in contacted, and the target cell for being imported with the expression miR-183 of the ASON is cultivated under conventional cell culture condition.
3)RNAi
Reagent
In a representative embodiment, precursor molecule (precursor of microRNA, pre-microRNA (sequence of people), such as SEQ ID No of RNAi reagents targeting miR-183：Shown in 2, CCGCAGAGUGUGACUCCUGUUCUGUGUAUGGCACUGGUAGAAUUCACUGUGAACAG UCUCAGUCAGUGAAUUACCGAAGGGCCAUAAACAGAGCAGAGACAGAUCCACGA), the expression of miR-183 is adjusted by the mechanism of RNA interference, that is, suppressing the function of miR-183 indirectly.
It is well known in the art that, RNA interference (RNA interference, RNAi) is phenomenon induced by double-stranded RNA (double-stranded RNA, dsRNA), the efficient selective degradation of homologous mRNA.Because use RNAi technology can be with the expression of specific depletion or closing specific gene, so the technology has been widely used for exploring the therapy field of gene function and communicable disease and malignant tumour.And specific to the application, the application by using the precursor molecule of miR-183 RNA interfering, precursor molecule to miR-183 causes gene silencing, so as to reduce the level of the precursor molecule of miR-183, thus, the level of the ripe miR-183 being changed into by the precursor molecule of miR-183 is reduced, namely, the function of miR-183 is inhibited, so as to raise the expression of miR-183 target genes.
RNAi reagents can be small RNA molecule, the typically one single-stranded deoxy-oligonucleotide (shRNA) that can form bobby pin (small hairpin) structure in theory, its length is general not over 100 nucleotides, typically not over 75 nucleotides；Or a double-strand deoxy-oligonucleotide (siRNA) of 15-30bp, most typically 20-23bp, siRNA (such as SEQ ID No as described by the embodiment 5 in the present invention：Antisense strand and such as SEQ ID No shown in 7：Positive-sense strand shown in 8).
In some applications, RNAi reagents can also be the template DNA for encoding shRNA or siRNA.These template DNAs are likely to be present in the carriers such as carrier, such as plasmid vector or viral vectors；Can also not exist with carrier, simply the template DNA of one section of coding shRNA or siRNA adds a common promoter sequence fragment for controlling it to transcribe.
Wherein, the RNAi reagents can also be to contact in vivo or vitro exposure with the contact of the target cell of expression miR-183.The description that the medication of the RNAi reagents is referred to as above to ASON is carried out, and in order to avoid unnecessary repetition, in this not go into detail for the present invention.
miR-183
Inhibitor
Present invention also offers miR-183 inhibitor, the particular type of the miR-183 inhibitor can be at least one in micromolecular compound as described above, ASON and RNAi reagents, in order to avoid unnecessary repetition, in this not go into detail for the present invention.
Pharmaceutical composition
Present invention also offers a kind of pharmaceutical composition, wherein, the pharmaceutical composition contains miR-183 inhibitor as described above and pharmaceutically acceptable carrier.
In the present compositions, can change in the larger context as the content of the miR-183 inhibitor as described above of active component, for example, can be 0.01-99 weight %, it is preferred that can be 1-70 weight %, it is furthermore preferred that can be 5-30 weight %.
According to the present invention, described pharmaceutical composition can be prepared as the conventional various formulations in this area, the present invention is not particularly limited to this, for example, solid can be configured to, it is semi-solid, liquid or gas form, for example, tablet, capsule, elixir, suspension, syrup, powder, particle, ointment, suppository, injection, inhalant, aerosol etc., of the invention to will not enumerate herein.
Therefore, the difference according to pharmaceutical dosage form can also carry out the administration of diversified forms, such as but not limited to, be administered orally, buccal administration, rectally, parenteral, Intraperitoneal medication, respiratory tract inhalation, intradermal administration, percutaneous dosing.
Wherein, the pharmaceutically acceptable carrier can carry out different selections according to the difference of formulation, and these are known in those skilled in the art.Such as but not limited to, the pharmaceutically acceptable carrier can be starch, colloid, lactose, glucose, sucrose, microcrystalline cellulose, kaolin, mannitol, calcium monohydrogen phosphate, sodium chloride, alginic acid etc..
Furthermore it is also possible to add conventional additive such as solubilizer, isotonic agent, suspending agent, emulsifying agent, stabilizer and preservative.
In addition, the pharmaceutically acceptable carrier can also include improving the targeting agent of the ASON targeting certain organs or tissue or cell, the targeting agent for example can be targeting peptides, can also include that can carry the ASON is easier to wear membrane reagent into the target cell of expression miR-183, such as cell-penetrating peptide, liposome, microcapsule bubble and membrane lipoprotein etc..
According to the present invention, flavor enhancement can also be added with described pharmaceutical composition, for example, peppermint, wintergreen etc..Furthermore it is also possible to add colouring agent in described pharmaceutical composition so that prepared formulation has certain attraction in appearance, or is distinguished with other products.
According to the present invention, the conventional medicine that the ASON can also can play similar effect with other is combined to be prepared into combined medicinal composition.
Kit
The invention provides a kind of kit, wherein, the kit includes ASON as described above, optionally, the kit also includes extra reagent, for example, as described above pharmaceutically acceptable carrier, flavor enhancement and/or colouring agent, solubilizer, isotonic agent, suspending agent, emulsifying agent, stabilizer, preservative, targeting agent or wearing membrane reagent.
According to the present invention, the extra reagent can be combined together with the ASON and be present in the kit, or can also be independent deposit in the kit, mixed again when to be used.
Operation instructions can also be included in kit of the invention, the existence form of the specification is not particularly limited, for example, it can be the paper-based form of printing, can be the form of CD, or be the form of network address, application method is obtained by internet when using.
Using
The invention provides suppressing the application of high fat of blood, fatty liver, the function of the mark miR-183 of increased weight and type-II diabetes below preventing and/or treating at least one disease and/or symptom.
The disease and/or symptom include：High fat of blood, fatty liver, increased weight and type-II diabetes and the symptom similar to the symptom of these diseases.
Particularly, the application includes preparing the medicine and/or food for preventing and/or treating a kind of disease of any of the above and/or symptom.Wherein, the food includes health products.
Present invention also offers the application of miR-183 inhibitor as described above, pharmaceutical composition as described above, kit as above and/or method as described above in the function of suppressing miR-183.
It is further preferred that the application includes preventing and/or treatment a kind of disease of any of the above and/or symptom.
In addition, the application present invention also offers miR-183 inhibitor as described above, pharmaceutical composition as described above in preparing for reducing the medicine that miR-183 is measured.
Preferably, the medicine includes the medicine and/or food for preventing and/or treating a kind of disease of any of the above and/or symptom.Wherein, the food includes health products.
According to the present invention, the treatment refers to the improvement of subject's symptom related to the disease or state caused by miR-183 or is wholly absent, wherein, the improvement on wide significance refers to reduce by least one parameter.Specific to the application, for example, can be mitigation, the reduction of blood fat and/or blood sugar and improvement of fatty liver of body weight etc..
The individual for the treatment of can be by any individuality perplexed by symptom as described above, preferably mammal.
According to the present invention, the function of miR-183 " suppress " refers to the degree that miR-183 is lowered to its expression of target gene in target cell compared to the expression miR-183 of the same race for not using the inventive method to process, at least 0.5 times is reduced to the degree that its expression of target gene is lowered using miR-183 in the target cell of the expression miR-183 of present invention treatment, at least 1 times, such as Fig. 2A and Fig. 2 B can generally be reduced.
The form of administration and composition of the medicine are referred to description as above, and in this not go into detail for the present invention.
Below will the present invention will be described in detail by embodiment.In following examples,
MiR-183 over-express vectors
By SEQ ID No：(people's) miR-183 genes (CCGCAGAGTGTGACTCCTGTTCTGTG shown in 3TATGGCACTGGTAGAATTCACTGTGAACAGTCTCAGTCAGTGAATTACCGAAGGGCCATAAACAGAGCAGAGACAGATCCACGA) it is cloned into pCAG-GFP carriers, obtains the overexpression plasmid pCAG-miR-183-GFP of miR-183 genes.Wherein, SEQ ID No：The synthesis of the miR-183 genes shown in 3 and clone are carried out by Jin Sirui companies.
MiR-183 experiences carrier (miR-183sensor vector)
It is by two miR-183 being combined and being adjusted such as SEQ ID No that miR-183 experiences carrier：Target sequence (TGGAAATGAGATCTTGTGCCATAGCTACGGTAAGGATTTTCAGTGCCATT) shown in 11 is cloned into obtained from the xbaI sites in the fiery luciferase of pGL3-SV40 carriers (Fire luciferase) gene 3' downstreams, so that the expression that miR-183 experiences carrier moderate heat luciferase is just adjusted by miR-183.
Embodiment 1
The present embodiment is used to illustrate the difference with miR-183 expression quantity in 12 monthly age mouse at 2 monthly ages
(1) extraction and reverse transcription of total serum IgE
The monthly age of wild type C57/Bl6 male mices 2 and each three of 12 monthly ages, respectively take the blood of 500 μ l at cervical dislocation after death, dissection rounds a hypothalamus and half stomach, and the liver of 200mg, muscle and epididymal adipose.Plus in Trizol reagents (invitrogen) to the blood of 1ml, and mix.The Trizol reagents of 200 μ l to hypothalamus, stomach, liver, muscle and adipose tissue are separately added into again, then hypothalamus, stomach, liver, muscle and adipose tissue are shredded with scissors, again with electric homogenizer these tissue grinders into powder, afterwards by the specification of Trizol the Total RNAs extraction in whole tissue out.With the water dissolving RNA of nuclease free, then with 260 and the 280 of the Instrument measuring RNA of Nanodrop 2000 ratio, take sample of the ratio more than 1.8 and continue subsequent experimental.After determining the concentration of RNA with Qubit afterwards, the integrality of RNA is detected with biological analyser (bioanalyzer), RNA Perfection Index RIN is greater than 0.9.Wherein, in order to ensure every kind of tissue or organ can carry out subsequent experimental, multiple can be set and repeats Total RNAs extraction.
The total serum IgE of 1 μ g is taken from each sample, the short rna of the 10-40nt in total serum IgE is separated using flashPAGE pulp classifiers (Ambion), then prepare the cDNA library of microRNA with the kit reverse transcription of Illumina, then in determination sample on the second generation sequenator microRNA expression.
Result shows that the expression quantity of miR-183 in hypothalamus, stomach and the liver of 12 monthly age mouse is higher than expression in 2 monthly age mouse, show that the expression quantity of miR-183 is raised with advancing age and in hypothalamus, stomach and liver, growth and obesity with body weight are proportionate.
(2) expression quantity of quantitative PCR detection miR-183
In order to confirm that expression quantity of the miR-183 in hypothalamus, stomach and liver is elevated with advancing age, with the expression quantity of quantitative PCR detection miR-183.The total serum IgE of 1 μ g is taken from each sample, with Catch AllTM miRNA＆mRNA RT-PCR kits (Pengekiphen, Kunshan) reverse transcription microRNAs and the cDNA of mRNA.Primer for detecting has：Such as SEQ ID No：MiR-183 forward primers (5'-TATGGCACTGGTAGAATTCACT-3') shown in 12；Such as SEQ ID No：U6 forward primers (5'-CGCAAGGATGACACGCAAATTCG-3') shown in 13；The universal primer that reverse primer is provided for kit.The instrument of detection is the iQ5 systems of Bio-Rad companies, and reagent is the SYBR Green Mix of TaKaRa companies.Each sample detects three multiple holes simultaneously, using U6 as internal reference, expressions of the miR-183 in each sample is calculated with 2- Δ Δ ct methods.Then the expression of the miR-183 of 2 monthly age each internal organs of mouse is set to 1, and calculates the miR-183 relative expression levels in 12 monthly age mouse, as a result see Fig. 1.
As shown in figure 1, the expression quantity of the miR-183 of the hypothalamus of master control appetite-stomach central shaft is substantially to raise in 12 monthly age mouse, and expression of the miR-183 in liver is also notable rise.Quantitative PCR confirm the hypothalamus of 12 monthly age mouse, liver and in stomach miR-183 expression quantity it is higher by 1.8 than expression difference in 2 monthly age mouse, 1.6 and 2.4 times.It is possible thereby to prove, the upper mediation increased weight and the formation of fatty liver of miR-183 are related.Wherein, in Fig. 1, * * P<0.01, * p<0.05.
Embodiment 2
The present embodiment is used to illustrate external adjustment effect of the ASON to miR-183
By HEKC HEK-293T cultures in the DMEM culture mediums containing 10% hyclone.Cell culture incubator it is constant keep 37 DEG C and 5% CO2.HEK-293T cells are seeded in 24 porocyte culture plates with 100,000, every hole inoculum concentration of cell, volume of culture is 500 μ l.The vigor that the luciferase expressed in carrier is experienced from miR-183 is measured with dual-luciferase assay instrument (Promega) with liposome 2000 (Invitrogen) setting cotransfection KEK-293 cells to specifications by such as table 2 below, after 36 hours within second day.Three repeating holes are set every time, and experiment is in triplicate.
Wherein, in each group, miR-183 experiences the amount of being transferred to of carrier in terms of every hole：MiR-183 experiences carrier 500ng, pCAG-GFP empty vectors 20ng, miR-183 over-express vector 500ng, and oligonucleotides is configured to 50 μM of solution.In addition, when the oligonucleotides being transferred to is miR-183ASO, 50 μM of 0.5 μ l and 1 μ l of oligonucleotide solution is taken respectively, its final concentration is respectively 0.05 μM and 0.1 μM after being added to cell culture fluid.To determine the vigor of luciferase, and with it as ordinate, miR-183ASO concentration is abscissa, draws curve.Result is shown in Fig. 2.
As seen from Figure 2, miR-183ASO can suppress the function of miR-183.
By Fig. 2A as can be seen that cotransfection miR-183 experiences the miR-183ASO of carrier, miR-183 over-express vectors and various concentrations, luciferase vitality testing result shows that miR-183ASO can suppress the function of miR-183, and has dosage effect.
Can be seen that overexpression miR-183 by Fig. 2 B can suppress the expression (left 1 post and left 2 post) that miR-183 experiences carrier；MiR-183ASO and miR-183 mispairing ASO can suppress the function (left 4 post and left 5 post) of miR-183, the inhibition of miR-183ASO is better than miR-183 mispairing ASO's, and random controls nucleotides can not suppress the function (left 3 post) of miR-183, Data=average values ± SEM；N=3；***P<0.001, * * P<0.01.And, in HEK293 cells overexpression miR-183 can suppress the expression of reporter gene luciferase during miR-9 experiences carrier to control group level 46%, and as the miR-183ASO of 0.1 μM of corotation final concentration, reporter gene luciferase expression recovers that to the 76% of control group level, i.e. miR-183ASO the 56% of miR-183 functions can be suppressed during miR-183 can be made to experience carrier.
Table 2
Embodiment 3
The present embodiment is used to illustrate that the ASON of miR-183 adjusts the effect of miR-183 target genes and its subsequent affect energetic supersession in vivo
10 wild type C57/Bl6 male mices (Beijing Vital River Experimental Animals Technology Co., Ltd.) by the weight differences of 12 week old less than 10% are randomly divided into two groups, after raising two weeks (high lipid food of feeding 20%), the miR-183ASO (being dissolved in PBS kinds) of experimental mice tail vein injection 3.5OD/100 μ l, control group mice injects the solvent PBS of 100 μ l.Injected once every three days within first week, injected every two weeks once afterwards.The body weight of mouse is weighed weekly, and changes of weight and body weight increase speed are shown in Fig. 3 A and Fig. 3 B respectively.
After two first quarter moons since injection, the fasting blood-glucose of mouse is measured.In 9 points of morning, the feed of mouse is taken away, after hungry 6 hours, cut tail and take one and bleed, then with Roche brilliance type blood glucose meter (model is ACCU-CHEK Performa) and remarkable golden sharp blood sugar test paper measurement blood sugar concentration, see Fig. 3 C.
Behind three months since injection, mouse anesthesia is put to death, and blood is taken from atrium dextrum, and solution cuts and weigh the weight of stomach, liver, epididymal adipose tissues and kidney, as a result sees Fig. 4 A, and the ratio of relative body weight is shown in Fig. 4 B.Blood 5000rpm takes serum after being centrifuged 15 minutes, the content of T-CHOL (CHOL), triglyceride (TG), HDL (HDL) and low-density lipoprotein (LDL) in serum is measured with automatic clinical chemistry analyzer (Hitachi 7180), Fig. 5 is seen.
As seen from Figure 3, compared with control group, the ASON (miR-183ASO) for being injected intravenously miR-183 can suppress the body weight increase (Fig. 3 A) of mouse and the rate of rise (Fig. 3 B) of body weight.Data=average values ± SD；N=5；*P<0.05.Being injected intravenously the ASON (miR-183ASO) of miR-183 can reduce the hyperglycaemia (Fig. 3 C) of high lipid food induction, Data=average values ± SEM；N=3；*P<0.05.
As seen from Figure 4, compared with control group, intravenous injection miR-183ASO can reduce the ratio (Fig. 4 B) of the weight relative body weight of the weight (Fig. 4 A) and liver and stomach of liver and stomach；MiR-183ASO does not influence (Fig. 4 B) on kidney weight.MiR-183ASO can also reduce the content of T-CHOL and triglyceride in blood, but HDL and low-density lipoprotein are not influenceed (Fig. 5).Data=average values ± SD；N=5；*P<0.05；**P<0.01.
The hypothalamus and the total serum IgE of stomach of contrast experiment's group and miR-183ASO injection group mouse are extracted according to the method in embodiment 1, is sequenced and is analyzed through two generations and learn：Compared with contrast experiment's group, miR-183ASO injection groups have raised many genes (table 3) participated in glycolipid metabolism or type-II diabetes or hepatopathy related PI3K-Akt signal paths, MAPK signal paths, TGF-beta signal paths, GnRH signal paths.The many genes (table 3) in gastric acid secretion, salivary secretion, phosphatidylinositols signal path, insulin signaling pathway and type-II diabetes signal path are additionally raised.
Table 3
In sum, suppressing the function of miR-183 can reduce the growth of body weight, and this provides new thinking for fat treatment.Treatment severe simple obesity the best way is to do stomach bypass surgery at present, and this explanation stomach and intestine is a fat important target organ for the treatment of.And the application demonstrates and suppresses the function of miR-183 and can reduce the weight and its ratio with body weight of stomach, and the secretion of hydrochloric acid in gastric juice and saliva is influenceed to influence digesting and assimilating for food, it could therefore be concluded that it is the fat important target spot of a treatment to go out miR-183.
Known triglyceride and T-CHOL is high can cause angiocardiopathy.The function of these as shown by data suppression miR-183 can reduce the concentration of triglyceride and T-CHOL in blood, so as to reduce the risk of the angiocardiopathy that high fat of blood causes, in addition, as the aforementioned, suppressing the function of miR-183 can reduce the increase of liver weight of high lipid food induction, it is known that miR-183 is the important target spot of a treatment high fat of blood and fatty liver.
And molecule experiments of the invention demonstrate miR-183 have adjusted in vivo control appetite, digest and assimilate, energetic supersession, many genes in Adipocyte Differentiation signal path, this illustrate the reducing blood lipid of miR-183 GEM 132 energy, molecular mechanism that is hypoglycemic and suppressing body weight.
Embodiment 4
The present embodiment is used to illustrate external adjustment effect of the siRNA (siRNA) to miR-183
By HEKC HEK-293T cultures in the DMEM culture mediums containing 10% hyclone.Cell culture incubator it is constant keep 37 DEG C and 5% CO2, volume of culture is 500 μ l.Second day with liposome 2000 (Invitrogen) to specifications by miR-183siRNA (antisense strand SEQ ID No：7：5 ' AGACUGUUCACAGUGAAUUCU ' 3, positive-sense strand SEQ ID No：8：5 ' AGAAUUCACUGUGAACAGUCU ' 3, Shanghai Ji agate synthesis, wherein, in SEQ ID No：7 and SEQ ID No：83 ' ends are respectively provided with the structure of dTdT) and random controls RNA (antisense strand SEQ ID No：9：5’CGUGACACGUUCGGAGAA’3；Positive-sense strand SEQ ID No：10：5 ' UUCUCCGAACGUGUCACGU ' 3, Shanghai Ji agate synthesis, wherein, in SEQ ID No：9 and SEQ ID No：10 3 ' ends are respectively provided with the structure of dTdT) respectively transfection KEK-293 in cell, with Trizol cell lysis and extracted total RNA after 36 hours, be subsequently used for the expression quantity of the identical quantitative PCR kit of embodiment 1 and primer detection miR-183.Three repeating holes are set every time, are tested in triplicate, as a result as shown in fig. 6, * * * P<0.01.
Result shows can lower the 72% of miR-183 expression quantity with miR-183siRNA, it follows that the amount of the miR-183 combined with the target gene of miR-183 has also lowered 72%, so as to improve the expression of target gene.It can be seen that, the function of miR-183 also can be successfully suppressed by way of RNA disturbs miR-183 precursors.
Summarize
The present invention is contacted by by miR-183 inhibitor (including ASON and RNA interfering) with the target cell of expression miR-183 in vivo or in vitro, function (the combination of ASON suppression miR-183 and its target gene of miR-183 in the target cell for express miR-183 can fully be suppressed, RNA interfering can reduce the expression quantity of miR-183, so as to suppress the function of miR-183), when individuality administration, can effectively prevent and/or treat the disease caused by miR-183 amount risings, for example, high fat of blood, fatty liver, increased weight and type-II diabetes.Compared to traditional single hypoglycemic or the medicine of single reducing blood lipid or single fat-reducing, the therapy that the present invention is provided can comprehensively modulation of appetite, digest and assimilate, glucose-lipid metabolism, and be oligonucleotides；Thus effect is powerful and Small side effects.For the treatment of the diseases such as obesity, fatty liver, high fat of blood and type-II diabetes provides new direction, so that with high Social benefit and economic benefit.
The preferred embodiment of the present invention described in detail above; but, the present invention is not limited to the detail in above-mentioned implementation method, in range of the technology design of the invention; various simple variants can be carried out to technical scheme, these simple variants belong to protection scope of the present invention.
It is further to note that each particular technique feature described in above-mentioned specific embodiment, in the case of reconcilable, can be combined by any suitable means.In order to avoid unnecessary repetition, the present invention is no longer separately illustrated to various possible combinations.
Additionally, can also be combined between a variety of implementation methods of the invention, as long as it is without prejudice to thought of the invention, it should equally be considered as content disclosed in this invention.
Bibliography
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2.Wang,P.Y.,et al.,Upper intestinal lipids trigger a gut-brain-liver axis to regulate glucose production.Nature,2008.452(7190):p.1012-6.
3.Beraza,N.and C.Trautwein,The gut-brain-liver axis:a new option to treat obesity and diabetesHepatology,2008.48(3):p.1011-3.
4.Schneeberger,M.,et al.,Hypothalamic miRNAs:emerging roles in energy balance control.Front Neurosci,2015.9:p.41.
5. roc brought up, Zhan Li apricot, the progress .Chinese Journal of Cell Biology 2010,32 (5) of Adipocyte Differentiation and its regulation and control:690-695.
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Claims (10)
1. suppress the mark miR-183's of high fat of blood, fatty liver, increased weight or type-II diabetes
Function is being prevented and/or is treating high fat of blood, fatty liver, type-II diabetes and similar to the symptom of these diseases
Symptom in and the application in losing weight, particularly preparing for preventing and/or treating blood high
Fat, fatty liver, diabetes and the symptom similar to the symptom of these diseases and preparing for reducing body
Application in the medicine and/or food of weight.
2. application according to claim 1, wherein, miR-183 has SEQ ID No：1 institute
The nucleotide sequence for showing.
3. a kind of mark miR-183 for suppressing high fat of blood, fatty liver, increased weight or type-II diabetes
Function method, it is characterised in that the method includes：By miR-183 inhibitor and expression miR-183
Expression miR-183 target cell contact.
4. method according to claim 3, wherein, the miR-183 inhibitor is few antisense
Nucleotides, including antisense DNA and antisense RNA, the ASON are complementary with miR-183,
And with the 8-23 length of nucleotides, and with the 2-8 sequence of nucleotide complementary with miR-183
Row；
Preferably, the ASON has following nucleotide sequence：
a)SEQ ID No：Nucleotide sequence shown in 4；
B) in SEQ ID No：Through missing, substitution or addition one or several in nucleotide sequence shown in 4
Individual nucleotides and the nucleotide sequence complementary with miR-183；Preferably, in SEQ ID No：Shown in 4
Nucleotide sequence in through lacking, replacing or adding one or several nucleotides and at least have with miR-183
The nucleotide sequence of 60% complementation；It is furthermore preferred that at most having 3 with the 2-8 nucleotides of miR-183
The nucleotide sequence of the mispairing of individual nucleotides；Most preferably, SEQ ID No：Nucleotides sequence shown in 5
Row.
5. method according to claim 3, wherein, the miR-183 inhibitor is miR-183
The siRNA of precursor；
Preferably, the miR-183 precursors have such as SEQ ID No：Sequence described in 2；
Preferably, the siRNA length of the miR-183 precursors is 15-30bp；
Preferably, the siRNA of the miR-183 precursors has such as SEQ ID No：Shown in 7
Antisense strand and such as SEQ ID No：Positive-sense strand shown in 8.
6. a kind of miR-183 inhibitor, it is characterised in that the miR-183 inhibitor will for right
Ask the small interference of the miR-183 precursors described in ASON and/or the claim 5 described in 4
RNA。
7. a kind of pharmaceutical composition, it is characterised in that the pharmaceutical composition contains described in claim 6
MiR-183 inhibitor and pharmaceutically acceptable carrier.
8. a kind of kit, it is characterised in that the kit includes the miR-183 described in claim 6
Inhibitor, optionally, the kit also includes pharmaceutically acceptable carrier.
9. the method in claim 3-5 described in any one, the miR-183 described in claim 6
The kit described in pharmaceutical composition and/or claim 8 described in inhibitor, claim 7 is suppressing
Application in the function of miR-183；It is preferred that preventing and/or treating high fat of blood, fatty liver, two type glycosurias
Disease and the symptom similar to the symptom of these diseases in and the application in losing weight.
10. the miR-183 inhibitor described in claim 6, the pharmaceutical composition described in claim 7
Application in the medicine for the function of suppressing miR-183 is prepared；It is preferred that prepare for prevent and/
Or treatment high fat of blood, fatty liver, type-II diabetes and the symptom similar to the symptom of these diseases and
Prepare the application in the medicine for losing weight.
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| CN109394779A (en) * | 2018-10-17 | 2019-03-01 | 河南师范大学 | A kind of miR-183 instrumentality of liver cell and its application adjusted in liver cell drug is treated in preparation |
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