CN107326067A - A kind of miRNA labels of NASH - Google Patents
A kind of miRNA labels of NASH Download PDFInfo
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- CN107326067A CN107326067A CN201710231790.8A CN201710231790A CN107326067A CN 107326067 A CN107326067 A CN 107326067A CN 201710231790 A CN201710231790 A CN 201710231790A CN 107326067 A CN107326067 A CN 107326067A
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Abstract
The invention discloses a kind of miRNA labels, the miRNA labels are miRNA 653.MiRNA 653 can be used for the process and the order of severity for judging NASH.Inspection proves that, miRNA 653 can effectively distinguish NASH sample and normal sample.On this basis, miRNA 653 can be also used for preparing the medicine for suppressing NASH.The present invention provides new diagnostic method for the clinical NASH of diagnosis on a molecular scale, while providing new drug target for the treatment of NASH.
Description
Technical field
The invention belongs to biomedicine field, it is related to a kind of NASH miRNA labels and its application, specifically
It is related to a kind of miRNA-653 label related to NASH and its application.
Background technology
MiRNA is naturally present in internal 21-22nt non-coding RNA molecule, is that a class is heavy by posttranscriptional gene
The silent RNA that expression of target gene is adjusted.It is estimated that the gene that 1/3 is there are about in organism is regulated and controled by miRNA.miRNA
It can be combined, pressed down with the complementary series in target gene mRNA5 '-UTR or 3 '-UTR by base pairing with RISC complex
Protein translation processed, or trigger mRNA degradeds, so that the expression of negative regulation target gene.
Alcohol and fatty mistake in the liver cell caused by other clearly damage liver factors except non-alcoholic fatty liver refers to
Degree is deposited as the clinical pathology syndrome of principal character, the closely related acquired metabolism with insulin resistance and genetic predisposition
Irritability hepatic injury.Including simple fatty liver (SFL), nonalcoholic fatty liver disease (NASH) and its related liver cirrhosis.With
The fashion trend of fat and its associated metabolic syndrome globalization, non-alcohol fatty liver turns into the prosperities such as America and Europe
Country and the Important cause of disease of China's areas of well-being chronic liver disease, average adult NAFLD illness rates 10%~30%, wherein 10%~
20% is NASH, and hepatic sclerosis incidence is up to 25% in the latter 10 years.
Non-alcohol fatty liver except can directly result in decompensated liver cirrhosis, hepatocellular carcinoma and liver transplantation recurrence in addition to,
The progress of other chronic liver diseases can be also influenceed, and participates in the morbidity of diabetes B and atherosclerosis.Metabolic syndrome is related
Malignant tumour, arteriosclerotic cardiovascular and cerebrovascular disease and hepatic sclerosis are influence patients with nonalcoholic fatty liver disease quality of life
With the key factor of life expectancy.Therefore, non-alcohol fatty liver is the new challenge in contemporary medical science field, non-wine at no distant date
Harm of the essence fatty liver disease to human health will be continuously increased.
MiRNA is the endogenous non-coding microRNA of regulatory gene expression, and gene expression is carried out in post-transcriptional level
Regulation and control, participate in the physiology courses such as cell cycle, apoptosis, development, differentiation and metabolism.MiRNA expresses imbalance meeting in cell
Cause the generation of a variety of diseases including NASH, recent studies have shown that, some miRNA are in NASH
Unconventionality expression in the blood of patient, but which kind of miRNA is relevant with generation, the development of NASH does not reach common understanding yet.
It is therefore desirable to find with NASH to occur, develop relevant miRNA, so that clinically to judge and treat non-wine
Essence fatty liver provides a kind of effective means.
The content of the invention
It is an object of the invention to provide a kind of miRNA labels that can be used for judging NASH.
To achieve these goals, present invention employs following technical scheme：
The invention provides a kind of for anticipation NASH risk, the miRNA marks of diagnosis NASH
Thing, the miRNA labels are miRNA-653.The miRNA-653 is selected from at least one of the following group：：miRNA-653
Initial miRNA, miRNA-653 precursor miRNA, maturation miRNA-653；The initial miRNA of miRNA-653 can be in people's cell
It is sheared and is expressed as ripe miRNA-653；The miRNA-653 precursor miRNAs can be sheared and be expressed as in people's cell
Ripe miRNA-653.
It should be known that the miRNA-653 of the present invention includes the functional equivalent of composing type nucleic acid molecules, i.e. variant, it shows
Show complete miRNA-653 nucleic acid molecules identical function, although they are by the missing of nucleotide residue, displacement or insertion
Mutation.
Those skilled in the art know, in order to ensure miRNA stability, can be protected in miRNA one end or two ends increase
Shield property base, such as TT, can also be modified miRNA bases, but do not influence miRNA function.Therefore, people in the art
Member knows, under conditions of miRNA-653 functions are not influenceed, and base modification is carried out to miRNA-653 or increases alkali at two ends
The sequence that base is obtained is also contained within protection scope of the present invention.
In some specific embodiments of the present invention, the miRNA-653 is ripe miRNA-653.The maturation
MiRNA-653 includes miRNA-653-5p, miRNA-653-3p, and they enjoy common Seed Sequences jointly.
Although that used in some embodiments is ripe miRNA-653, those skilled in the art can
With expection, initial miRNA (pi-miRNA-653), precursor miRNA (pre-miRNA-653) can obtain and maturation
Technique effect same miRNA-653, because cell is had the ability further by initial miRNA (pi-miRNA-653), precursor
MiRNA (pre-miRNA-653) is processed as ripe miRNA-653.
The miRNA-653 nucleic acid molecules of the present invention can exist in single-stranded or double-stranded form.Ripe miRNA-653 master
To be in single stranded form, and miRNA-653 precursors are that part is certainly complementary, to form duplex structure.The nucleic acid molecules of the present invention can
In the form of being RNA, DNA, PNA, LNA.
The invention provides applications of the miRNA-653 in the instrument for preparing anticipation NASH risk.
Present invention also offers applications of the miRNA-653 in the instrument for preparing diagnosis NASH.
The experiment proves that the level for having occurred miRNA-653 in the urine of NASH is substantially less than and is
The level of miRNA-653 in the blood, serum or blood plasma of NASH does not occur.Therefore, with non-alcoholic does not occur
MiRNA-653 level is compared in the blood of fatty liver, serum or blood plasma, if in subject's urine miRNA-653 level
Significantly reduce, then then may determine that NASH has occurred for the subject, so as to take prevention non-alcoholic fatty
The scheme of liver provides diagnostic base for the formulation of clinical treatment.
Present invention also offers applications of the miRNA-653 in the instrument of NASH is judged.With do not occur it is non-
MiRNA-653 level is compared in the blood of alcoholic fatty liver, serum or blood plasma, if subject's blood, serum or blood plasma
The middle miRNA-653 notable condition of level, then show that NASH occurs for subject.
Further, above-mentioned anticipation NASH risk, judge the kit whether NASH occurs
Include but be not limited to, chip, kit.The instrument includes being used for the reagent of miRNA-653 expressions in sample to be tested.It is described
Reagent can be the primer or probe for miRNA-653.
Present invention also offers applications of the above-mentioned miRNA-653 in high-flux sequence platform.Pass through high-flux sequence energy
Know the expression of miRNA-653 in blood to be detected, serum or plasma sample, the result of sample to be tested is non-with not occurring
Blood, serum or the plasma sample of alcoholic fatty liver are compared, and easily determine that sample to be tested whether there is NASH
Risk or easily determine whether sample to be tested has occurred that NASH.Therefore, obtained by high-flux sequence
Obtain miRNA-653 and the application of NASH correlation is also contained within protection scope of the present invention.
It is used for whether anticipation NASH risk, diagnosis NASH to be sent out present invention also offers one kind
Raw chip, the chip includes solid phase carrier；And it is fixed on the oligonucleotide probe on the solid phase carrier, the few core
Thuja acid probe includes specifically corresponding to miRNA-653 part or all of sequence.The oligonucleotide probe may also include
For in the prior art it has been reported that can be used for judge the miRNA whether NASH occurs oligonucleotides spy
Pin.A variety of miRNA detection probe is placed on the same chip by detecting that a variety of miRNA indexs joints judge non-alcoholic
The situation of fatty liver is also contained within protection scope of the present invention.
Further, the solid phase carrier, which includes the solid phase carrier, can use the various common used materials in genetic chip field,
Such as, but not limited to nylon membrane, the slide or silicon chip modified through active group (such as aldehyde radical, amino), unmodified slide, modeling
Tablet etc..
Preparing for described miRNA chips can use the common manufacturing method of biochip known in the art, for example, such as
Fruit solid phase carrier uses modification slide or silicon chip, and 5 ' ends of probe are gone here and there containing amido modified poly- dT, can be by oligonucleotides
Probe is configured to solution, then using point sample instrument by its point on modification slide or silicon chip, be arranged in predetermined sequence or array,
Then fixed by standing overnight, so that it may obtain the miRNA chips of the present invention.
It is used for whether anticipation NASH risk, diagnosis NASH to be sent out present invention also offers one kind
Raw kit, the kit includes being used to detect miRNA-653 expression in subject's blood, serum or blood plasma
Reagent.Compared with the expression of the miRNA-653 in the blood of NASH, serum or blood plasma does not occur, if logical
Crossing the expression of miRNA-653 in kit detection blood, serum or blood plasma significantly reduces, then judges the non-wine of the subject
Essence fatty liver risk is very high or has occurred NASH.
Further, the reagent includes the primer and/or probe for miRNA-653.The reagent is also included for existing
Have in technology it has been reported that can be used for judge NASH risk, or judge whether NASH occurs
MiRNA primer and/or probe.A variety of miRNA detection primer and/or probe are placed in same reagent box and passed through
Detect that a variety of miRNA indexs joints judge that the situation of NASH is also contained within protection scope of the present invention.
The miRNA-653 of the present invention can be natural or artificial synthesized, or use can express miRNA-653
DNA fragmentation carrier transfectional cell obtain.The carrier includes viral vector, eukaryotic vector.
Viral vector can be any appropriate carrier, including but not limited to retroviral vector, adenovirus vector, gland
Viral related viral vectors, herpesviral (such as herpes simplex virus, vaccinia virus and Epstein-Barr virus) carrier, alphavirus vectors.
Carrier for expression of eukaryon can be any appropriate expression vector, including but not limited to pCMV-Myc expression vectors,
PcDNA3.0 expression vectors, pcDNA3.1 expression vectors, pEGFP expression vectors, pEFBos expression vectors, pTet expression vectors,
PTRE expression vectors or engineered carrier on the basis of known expression vector, such as pBin438, pCAMBIA1301
Deng.
MiRNA-653 DNA fragmentation, which can be expressed, to be obtained in the following way：From miRNA databases
(http://microrna.sanger.ac.uk/sequences/) find positions of the miRNA-653 on genome and specific
Sequence information, the initial miRNA of miRNA-653 position is determined according to genome sequence, in the initial miRNA positions of miRNA-653
Upstream and downstream 500-800bp it is interval in design specific primer, the sequence in the middle of amplimer can obtain expression miRNA-653
DNA fragmentation.
The medicine of suppression or treatment NASH is being prepared present invention also offers foregoing miRNA-653
In application.
The experiment proves that miRNA-653 is related to NASH, on this basis, by promoting miRNA-
653 expression can be used for the risk for suppressing generation or the development of NASH.
Further, the medicine includes miRNA-653 activators.The miRNA-653 activators can promote miRNA-
653 expression or the function that miRNA-653 can be activated.The suppression target of the miRNA-653 activators is not limited to
MiRNA-653 in itself, includes miRNA-653 upstream and downstream, for example：The genome sequence of coding miRNA -653, miRNA-
653 target genes, the albumen or gene for regulating and controlling miRNA-653.
Further, miRNA-653 inhibitor includes albumen, oligonucleotides, micromolecular compound.
Preferably, the miRNA-653 inhibitor is miRNA-653 ASON or miRNA-653 simulation
Thing.
Its ASON is easily designed according to miRNA-653 sequences, ASON is transferred to human body
After interior, they can substantially lower miRNA-653 expression." ASON (antisense-
Oligonucleotides, AS-Ons or ASO) " be also known as " GEM 132 ", refer to length be about 18-26nt (particularly
About 19-22nt) DNA molecular or RNA molecule or its analog.
In the present invention, described " ASON " is also included using as based on nucleic acid lock or nucleic acid chains backbone modification
The GEM 132 through modification that the means such as technology are obtained, described modification does not change the activity of ASON substantially, more
Goodly, the modification can improve the stability, activity or therapeutic effect of ASON.Nucleic acid locks (locked nucleic
Acid, LNA) typically refer to the modification skill that the 2 ' oxygen atoms and 4 ' carbon atoms of ribose is connected by a methylene bridge
Art.The antisense drug that modification technique based on nucleic acid chain backbone is developed has greatly in terms of solubility, nuclease-resistant degraded to be changed
It is kind, and it is easy to a large amount of synthesis.The backbone modification method of oligonucleotides has a variety of, including thio method, such as by deoxynucleotide chain
Thio-modification is thio deoxynucleotide chain.This method is to substitute the oxygen atom of the phosphate bond on DNA skeletons with sulphur atom, can
Resist nuclease degraded.It should be understood that any largely or entirely active modification that can keep the ASON is all
Comprising in the present invention.
The medicine of the suppression NASH of the present invention is also comprising pharmaceutically acceptable carrier, the carrier
Including but not limited to：Diluent, buffer, supensoid agent, emulsion, granule, encapsulation agents, excipient, filler, adhesive, spray
Mist agent, cutaneous permeable agent, wetting agent, disintegrant, sorbefacient, surfactant, colouring agent, flavouring or absorption carrier.
Including but not limited to microinjection agent, the formulation suitable for transfection, parenteral solution, tablet, powder can be made in the medicine
Agent, granula, capsule.The medicine of above-mentioned various formulations can be prepared according to the conventional method of pharmaceutical field.
The medicine can be administered alone；Or the medicine that with other can suppress NASH is combined and applied
With.
The medicine can be applied in vitro：MiRNA-653 or miRNA-653 expression vector is imported or turned in vitro
Contaminate human body itself or variant cell, after vitro cell expansion, defeated the Huis' body.
The medicine can be applied in vivo：MiRNA-653 or miRNA-653 expression vector are introduced directly into vivo.
This carrier can be virus type or non-viral, even naked DNA or RNA.
Described subject can be the mankind or other mammals.More specifically, subject be organ, it is tissue, thin
Born of the same parents.
Brief description of the drawings
Fig. 1 is expressions of the miRNA-653 in the serum of NASH crowd and normal population；
Fig. 2 is expressions of the miRNA-653 in the serum of rats with nonalcoholic fatty liver disease model；
Fig. 3 is the colored graph that hepatic cell line HepG2 cells are added after oleic acid；
Fig. 4 is expressions of the miRNA-653 in the hepatic cell line HepG2 for adding oleic acid culture.
Sequence table
Beijing * * * * institutes
A kind of microRNA labels of NASH
miR-653
guguugaaacaaucucuacug
Claims (10)
- Applications of the 1.miRNA-653 in anticipation NASH risk, diagnosis NASH instrument is prepared, its It is characterised by, the miRNA-653 is selected from at least one of the following group：Initial miRNA, miRNA-653 precursors of miRNA-653 MiRNA, maturation miRNA-653；The initial miRNA of miRNA-653 can be sheared in people's cell and be expressed as maturation miRNA-653；The miRNA-653 precursor miRNAs can be sheared in people's cell and be expressed as ripe miRNA-2166.
- 2. the application according to claim, it is characterised in that the miRNA-653 is ripe miRNA-653.
- 3. applications of the miRNA-653 in high-flux sequence platform described in claim 1, it is characterised in that pass through high flux Blood to be measured, the expression of miRNA-653 described in serum or plasma sample are known in sequencing, and the miRNA- is known in analysis 653 with the correlation of NASH.
- 4. a kind of anticipation NASH risk, the chip for diagnosing NASH, it is characterised in that the chip Including solid phase carrier；And the oligonucleotide probe on the solid phase carrier is fixed on, the oligonucleotide probe includes special Property correspond to claim 1 described in miRNA-653 part or all of sequence.
- 5. a kind of anticipation NASH risk, the kit for diagnosing NASH, it is characterised in that the examination Agent box includes being used to detect the examination of the expression of the miRNA-653 in subject's blood, serum or blood plasma described in claim 1 Agent；With the blood of NASH does not occur, the expression of miRNA-653 described in serum or blood plasma compared, if blood MiRNA-653 expression is significantly reduced described in liquid, serum or blood plasma, then judges the NASH of the subject Risk it is high or occurred NASH.
- 6. kit according to claim 5, it is characterised in that the reagent includes drawing for the miRNA-653 Thing and/or probe.
- 7. applications of the miRNA-653 in the medicine for suppressing NASH is prepared described in claim 1.
- 8. application according to claim 7, it is characterised in that the medicine includes the miRNA-653 activators.
- 9. application according to claim 8, it is characterised in that the miRNA-653 activators can promote miRNA-653 Expression or miRNA-653 function can be activated.
- 10. application according to claim 9, it is characterised in that the miRNA-653 activators are the miRNA-653 Oligonucleotides or the miRNA-653 analogies.
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| CN107858355A (en) * | 2017-11-24 | 2018-03-30 | 北京大学首钢医院 | A kind of miRNA marker of NASH and its application |
| CN110042157A (en) * | 2019-04-24 | 2019-07-23 | 河北仁博科技有限公司 | Application of the miR-380-3p in the product of preparation diagnosis, prevention and/or treatment nonalcoholic fatty liver |
| CN110734971A (en) * | 2019-11-27 | 2020-01-31 | 广州中医药大学(广州中医药研究院) | group biomarker for diagnosing metabolic fatty liver mediated vascular diseases and application thereof |
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| CN107858355A (en) * | 2017-11-24 | 2018-03-30 | 北京大学首钢医院 | A kind of miRNA marker of NASH and its application |
| CN107858355B (en) * | 2017-11-24 | 2021-05-25 | 北京大学首钢医院 | miRNA marker of non-alcoholic fatty liver disease and application thereof |
| CN110042157A (en) * | 2019-04-24 | 2019-07-23 | 河北仁博科技有限公司 | Application of the miR-380-3p in the product of preparation diagnosis, prevention and/or treatment nonalcoholic fatty liver |
| CN110734971A (en) * | 2019-11-27 | 2020-01-31 | 广州中医药大学(广州中医药研究院) | group biomarker for diagnosing metabolic fatty liver mediated vascular diseases and application thereof |
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