CN105154445A - Novel serum miRNA biomarker of myodystrophy - Google Patents

Abstract

The invention provides a novel serum miRNA biomarker of myodystrophy, and specifically provides a separated miRNA or a set of miRNA, wherein the miRNA is selected from the miRNA of which sequence is shown as SEQ ID NO.:1, the miRNA of which sequence is shown as SEQ ID NO.:2, and/or the miRNA of which sequence is complemented the sequences shown as SEQ ID NO.:1 and SEQ ID NO.:2. The invention also provides a biochip and a kit containing the sequences. The micro RNA biomarker in the invention can distinguish tissue samples of patients with myodystrophy and the normal samples quite effectively proved by tests, and DMD myodystrophy and BMD myodystrophy can be distinguished effectively by using the micro RNA in the invention.

Description

The serum miRNA marker of new muscular dystrophy myopathy

Technical field

The invention belongs to biomedicine field, specifically, the present invention relates to the serum miRNA marker of two new muscular dystrophy myopathies.

Background technology

Microrna (microRNA is called for short miRNA) is in recent years nematode, and fruit bat and plant, a kind of endogenic length found in the eukaryotes such as Mammals is the non-coding strand tiny RNA about 22 Nucleotide.It has the specificity of tissue and time on expressing, on post-transcriptional level, negative regulation is carried out to the expression of gene by the base pair complementarity with said target mrna, causing degraded or the Translational repression of mRNA, is the important regulating and controlling molecule regulating other functional genes to express.Although increasing evidence shows that miRNA is small, it is by being formed complete or not exclusively unpaired mutually with said target mrna thus having vital effect to the various vital processes of organism.Some miRNA have been proved to be specific expressed in muscle, and play very important effect in muscle development process.In muscle differentiation process, specificity miRNA is by relying on into flesh factor of determination MyoD, and the function of Mef2 and Myogenin regulates the balance between muscle reproduction restraint.MiR-208b and miR-499 is arranged in the intron of slow muscle Myosin heavy chain gene (Myh7 and Myh7b), and the type of they and myofibrillar differentiation has close relationship.

But nearest research shows, no matter be in the recycle system of animal or people, the stable existence of miRNA also can be detected, and the change of miRNA kind in these recycle systems and quantity, relevant to the physiological and pathological situation of organism.The miRNAs disclosed in the recycle system possesses the Potential feasibility of the non-invasive diagnosis mark of disease thus.By the discovery of serum miRNAs and the relation of disease, and the further research to miRNAs target, and then novel treatment plan is designed for some diseases.

The method of research miRNA is a lot, the most frequently used is uses high-throughout chip to screen the miRNA interested to special, the present inventor, then the method for small throughput is used, such as real-timePCR, no rthernblot, marker detection (fluorescent mark or nanometer particle to mark etc.) are expressed specific miRNA and are verified, also can be detected locus and the timing of specific miRNA expression in addition by situ hybridization.Functionally, the relation of miRNA and target gene can be studied by the method for the no ckdown of k or process LAN, thus illustrate the concrete function of miRNA in organism.

Muscular dystrophy myopathy is a kind of lethal X-sex-linked recessive inheritance neuromuscular disease.His major pathologic features is that the distortion of skeletal muscle Progressive symmetric erythrokeratodermia is downright bad, fibrosis, fatty infiltration, progressively loses locomotor activity subsequently and finally breathes and cardiac failure death.Sickness rate is about 1/3500 life birth boy baby.

This disease causes because muscular dystrophy sudden change causes this protein expression to lack.Dystrophin is a kind of cytoskeletal protein, and myofiber is connected to the extracellular matrix of surrounding by it by cytolemma.When this hypoproteinosis, the weak necrosis of myofiber of initial damage, and along with the activity of series of complex, comprise phagolysis, inflammatory cell infiltration and fibrosis subsequently and fat substituted muscle and then cause follow-up myofibrillar atrophy cause breathing or cardiac failure thus make patient's premature death.

The sudden change of dystrophin gene, if cause the dystrophin albumen that can give expression to functional defect or quantity not sufficient, and the clinical manifestation of patient is gentleer, is referred to as becker type muscular dystrophy (BMD); If transgenation causes dystrophin hypoproteinosis and clinical manifestation is serious, is duchenne muscular dystrophy (DMD).

Owing to not having the effective methods for the treatment of for DMD patient at present, therefore early find, early contact co-treatment, there is more importantly meaning the time extending its ambulation for patient.Therefore, those skilled in the art be devoted to develop a kind of can accurately, rapid detection DMD judge the technology in its clinical development stage.

Summary of the invention

Object of the present invention be just to provide a kind of newly, can be used for microRNA mark distinguishing muscular dystrophy patients with myopathy sample and normal sample and uses thereof.

Another object of the present invention is to provide the chip and test kit that detect described disease.

In a first aspect of the present invention, provide a kind of miRNA or miRNA set of separation, described miRNA is selected from:

The miRNA of (i) sequence as shown in SEQIDNO.:1;

(ii) miRNA of sequence as shown in SEQIDNO.:2, and/or

(iii) with the miRNA of miRNA sequence complementation shown in (i) or (ii).

In another preference, described miRNA is separated from people.

A second aspect of the present invention, that provide a kind of separation or artificial constructed precursor miRNA, described precursor miRNA can be sheared and be expressed as miRNA or miRNA set as described in the first aspect of the invention in people's cell.

A third aspect of the present invention, provides a kind of polynucleotide of separation, and described polynucleotide can be become precursor miRNA by people's cell transcription, and described precursor miRNA can be sheared and be expressed as miRNA or miRNA as claimed in claim 1 in people's cell to be gathered.

In another preference, described polynucleotide have the structure shown in formula I:

Seq _forward-X-Seq _oppositelyformula I

In formula I,

Seq _forwardfor the nucleotide sequence of miRNA described in becoming at people's cells;

Seq _oppositelyfor with Seq _forwardsubstantially the nucleotide sequence of complementation or complete complementary;

X is for being positioned at Seq _forwardand Seq _oppositelybetween intervening sequence, and described intervening sequence and Seq _forwardand Seq _{instead to}not complementary;

And the structure shown in formula I is after proceeding to people's cell, form the secondary structure shown in formula II:

Formula II,

In formula II, Seq _forward, Seq _oppositelywith the definition of X as above-mentioned,

|| represent at Seq _forwardand Seq _oppositelybetween formed base pair complementarity relation.

A fourth aspect of the present invention, provides a kind of carrier, and described carrier contains miRNA as described in the first aspect of the invention, or the polynucleotide as described in third aspect present invention.

A fifth aspect of the present invention, provides the purposes of miRNA or the miRNA set described in first aspect present invention, and described miRNA or miRNA set is for the preparation of chip or the test kit of distinguishing DMD sample, BMD sample and normal sample.

A sixth aspect of the present invention, provides a kind of miRNA chip, and described miRNA chip comprises:

Solid phase carrier; And

Be fixed on the oligonucleotide probe on described solid phase carrier in order, described oligonucleotide probe corresponds to the sequence shown in SEQIDNO.:1 and/or SEQIDNO.:2 specifically.

In another preference, described oligonucleotide probe contains:

Complementary land; And/or

The joining region be connected with solid phase carrier.

A seventh aspect of the present invention, provides the purposes of the miRNA chip described in a kind of sixth aspect present invention, and described miRNA chip is for the preparation of the test kit distinguishing DMD sample, BMD sample and normal sample.

A eighth aspect of the present invention, provides a kind of test kit, containing the miRNA chip described in sixth aspect present invention in described test kit; Or

MiRNA or miRNA set described in first aspect present invention.

A ninth aspect of the present invention, provides a kind of method that screening is used for the treatment of the drug candidate of muscular dystrophy myopathy, comprises step:

A () provides a test group and a control group, wherein candidate substances is applied to cell or the animal of test group in described test group, and measure the expression level using miR-499 and/or miR-208b in rear described test group, and described control group adopts the condition identical with test group, but candidate substances is not applied to cell or the animal of control group;

B the expression level of miR-499 and/or miR-208b of test group and miR-499 and/or miR-208b of control group compares by ();

Wherein, when expression level significantly lower than control group of the expression level of miR-499 and/or miR-208b of test group, then show that this candidate substances is the drug candidate being used for the treatment of DMD.

A tenth aspect of the present invention, provides a kind of method diagnosing muscular dystrophy myopathy, comprises step:

A () detects the expression level of miR-499 and/or miR-208b in sample;

B (), according to the detected result of step (a), judge that whether the sample detected is from muscular dystrophy patients with myopathy, determination methods is:

If miR-499 expression level is higher than the expression level in normal sample, and/or

If the expression level of miR-208b is higher than the expression level in normal sample,

Then judge that this detection sample comes from DMD patient.

In another preference, if described miR-499 expression level is higher than 3 times of normal sample, preferably higher than 4 times of normal sample, more preferably higher than 5 times of normal sample, best higher than 6.5 times (specificity reaches 100%, and susceptibility reaches 100%) of normal sample; And/or

If the expression level of miR-208b is higher than 2 times (specificity reaches 90%, and susceptibility reaches 95%) of normal sample, more preferably higher than 3 times of normal sample,

Then judge that this detection sample comes from muscular dystrophy patients with myopathy.

The expression level of miR-499 and/or miR-208b in normal sample mentioned in the present invention is the mean value of miR-499 and/or miR-208 expression level in normal sample.

A eleventh aspect of the present invention, provides a kind of method diagnosing differentiation DMD and BMD muscular dystrophy myopathy, comprises step:

A () detects the expression level of miR-499 and/or miR-208b in sample;

B (), according to the detected result of step (a), judge that whether the sample detected is from DMD patient or BMD patient, determination methods is:

If miR-499 expression level is higher than the expression level in BMD sample, and/or

If the expression level of miR-208b is higher than the expression level in BMD sample,

Then judge that this detection sample comes from DMD patient.

In another preference, described miR-499 expression level is higher than normal average value 27 times (specificity reaches 92%, and susceptibility reaches 43%); And/or

If the expression level of miR-208b is higher than normal average value 4.5 times (specificity reaches 83%, and susceptibility reaches 62%),

Then judge that this detection sample comes from DMD patient instead of BMD patient.

Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.

Accompanying drawing explanation

Fig. 1 shows the change of miRNA in muscular dystrophy patients serum, and figure A shows the change of miR-499 in muscular dystrophy patients serum; Figure B shows the change of miR-208b in muscular dystrophy patients serum.

Fig. 2 shows miRNA (figure A:miR-499; Figure B:miR-208b) for the ROC curve of normal people, BMD and DMD patient.

Fig. 3 shows the dependency at miRNA and age.

Fig. 4 shows the dependency at myofiber and age.

Fig. 5 shows the expression change of miRNA in change lever.

Fig. 6 shows the dependency of serum miRNA and IIc type muscular fiber type in DMD infant.

Fig. 7 shows the dependency of serum miRNA and I type muscular fiber type in DMD infant.

Embodiment

The present inventor, through studying for a long time and widely, by detecting the microRNA express spectra level of muscular dystrophy clinical samples serum and normal sample serum, therefrom filters out specific microRNA:miR-499 and miR-208b first.Through testing identity, miR-499 and miR-208b effectively can distinguish muscular dystrophy patient tissue sample and normal sample as microRNA mark.And use microRNA of the present invention effectively can distinguish DMD and BMD muscular dystrophy myopathy.Complete the present invention on this basis.

The measurable sample of this microRNA is that its prediction accuracy reaches more than 80% from DMD sample or normal sample.Based on microRNA of the present invention, test kit can be developed to, for distinguishing DMD sample and normal sample.

MiRNA and precursor thereof

The invention provides the miRNA (microRNA) found from people that a class is new.As used herein, described " miRNA " refers to a kind of RNA molecule, from the transcript processing that can form miRNA precursor.Ripe miRNA has 18-26 Nucleotide (nt) (more particularly about 19-22nt) usually, does not also get rid of the miRNA molecule with other number Nucleotide.MiRNA can be detected by no rthern trace usually.

MiRNA is the tiny RNA of a kind of Inner source property, at post-transcriptional level regulate gene expression.Increasing evidence shows that miRNAs plays important regulating and controlling effect in muscle tissue, comprises the generation development etc. of the propagation of muscle, differentiation, growth and some relevant muscle diseases.Research in recent years finds, except the miRNA in tissue and cell, the expression of peripheral blood miRNA also has significant dependency, specificity and stability with numerous disease, meets the requirement of disease diagnosis marker.The present inventor is changed by the expression of comparing miR-208b and miR-499 in normal mouse and Duchenne muscular dystrophy mouse model mdx mice serum; two kinds of muscular dystrophy myopathy DuchenneShi muscular dystrophy (DMD) and Becker'sShi muscular dystrophy (BMD) patient change with the expression of miRNA in the serum of normal infant respectively; also analyze the dependency of these two miRNA serum expression levels Muscle fiber density different from patient, and then miR-208b and miR-499 can well identify the patient of muscular dystrophy myopathy certainly simultaneously.Further, the Muscular pathology situation of patient can be reflected at these two miRNA of certain age level.

In the two kinds of muscle mentioned in the present invention, the miRNA (miR-208b and miR-499) of enrichment also can as the serum biomarkers of DMD diagnosis.And the expression of miR-208b and miR-499 in the patients serum of 2 to 6 years old and their age and myofiber somatotype have close dependency.These two miRNA of proposing enrichment in muscle thus not only can may be used for inferring the pathological change of this disease and effective Biological indicators of Development Process as the diagnosis marker of DMD disease.

In a preference, miR-208b nucleotide sequence of the present invention is:

UUAAGACUUGCAGUGAUGUUU(SEQIDNO.:1)

Making in a preference, miR-499 nucleotide sequence of the present invention is:

AUAAGACGAACAAAAGGUUUGU(SEQIDNO.:2)

The miRNA in people source can be separated by from people's cell.As used herein, " separation " refers to that material is separated from its primal environment (if natural substance, namely primal environment is natural surroundings).As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials existed separately, then for separation and purification.

MiRNA can process from precursor miRNA (PrecursormiRNA, Pre-miRNA), and described precursor miRNA can be folded into a kind of stable stem ring (hair clip) structure, and described loop-stem structure length is generally between 50-100bp.Described precursor miRNA can be folded into stable loop-stem structure, and the stem both sides of loop-stem structure comprise substantially complementary two sequences.Described precursor miRNA can be natural or synthetic.

Precursor miRNA can be sheared generation miRNA, described miRNA can be substantially complementary with the sequence at least partially of the mRNA of encoding gene.As used herein, " substantially complementary " refers to that the sequence of Nucleotide is enough complementary, can interact, as formed secondary structure (as loop-stem structure) in the foreseeable mode of one.Usually, the nucleotide sequence of two " substantially complementary " mutually between have at least the Nucleotide of 70% to be complementary; Preferably, the Nucleotide of 80% is had at least to be complementary; Preferred, have at least the Nucleotide of 90% to be complementary; Preferred further, have at least the Nucleotide of 95% to be complementary; As 98%, 99% or 100%.Usually, maximum 40 unmatched Nucleotide can be had between two enough complementary molecules; Preferably, there are maximum 30 unmatched Nucleotide; Preferred, there are maximum 20 unmatched Nucleotide; Preferred further, there are maximum 10 unmatched Nucleotide, as having 1,2,3,4,5,8,11 unmatched Nucleotide.

As used herein, " stem ring " structure is also referred to as " hair clip " structure, refer to a kind of nucleic acid molecule, it can form the secondary structure that one comprises double-stranded region (stem), described double-stranded region is formed by two regions (being positioned on same a part) of this nucleic acid molecule, the both sides of two region apportion double stranded section; It also comprises at least one " ring " structure, comprises non-complementary nucleic acid molecule, i.e. single-stranded regions.Even if two of this nucleic acid molecule regions are not complete complementaries, the double stranded section of Nucleotide also can keep double-stranded state.Such as, insertion, disappearance, replacement etc. can cause the not complementary of a zonule or this zonule self to form the secondary structure of loop-stem structure or other form, but these two regions still can be substantially complementary, and interact in foreseeable mode, form the double-stranded region of loop-stem structure.Loop-stem structure is well-known to those skilled in the art, usually obtain one there is the nucleic acid of the nucleotide sequence of primary structure after, those skilled in the art can determine whether this nucleic acid can form loop-stem structure.

MiRNA of the present invention has the sequence as shown in SEQIDNO.:1.In order to improve stability or other character of miRNA, also can add at least one protectiveness base at least one end of described miRNA, as " TT " etc.

Antisense oligonucleotide

According to miRNA sequence provided by the present invention, can have devised its antisense oligonucleotide, described antisense oligonucleotide can lower the expression of corresponding miRNA in vivo.As used herein, " antisense oligonucleotide (antisense-oligonucleotides; AS-Ons or ASO) " is also called " antisense nucleotide ", refers to that length is about the DNA molecular of 18-26nt (more particularly about 19-22nt) or RNA molecule or its analogue.

In the present invention, described " antisense oligonucleotide " also comprises the modified antisense nucleotide adopted as obtained based on means such as nucleic acid lock or nucleic acid chains backbone modification technology, described modification does not change the activity of antisense oligonucleotide substantially, more preferably, described modification can improve the stability of antisense oligonucleotide, activity or result for the treatment of.Nucleic acid lock (lockednucleicacid, LNA) typically refers to the modification technique 2 ' of ribose Sauerstoffatom and 4 ' carbon atom coupled together by a methylene bridge.LNA can extend the serum half-life of miRNA, improves target affinity, reduces scope and the degree of the effect of missing the target.The antisense drug developed based on the modification technique of nucleic acid chains skeleton is in solubility, and the aspects such as nuclease-resistant degraded are improved greatly, and are easy to a large amount of synthesis.The backbone modification method of oligonucleotide has multiple, comprises sulfo-method, such as, be sulfo-deoxynucleotide chain by deoxynucleotide chain thio-modification.The method is substituted by the Sauerstoffatom sulphur atom of the phosphate bond on DNA skeleton, can resist nuclease degradation.Should be understood that and anyly the major part of described antisense oligonucleotide or all active modification can be kept to be included in the present invention.

As optimal way of the present invention, nucleic acid lock is carried out to antisense oligonucleotide and modifies; More preferably also carry out thio-modification.

Transferred to after in human body by antisense oligonucleotide of the present invention, they obviously can lower the expression of relevant miRNA.

Polynucleotide construction

According to miRNA sequence provided by the present invention, can design the polynucleotide construction that can be processed to the miRNA that can affect corresponding mrna expression after being imported into, also namely described polynucleotide construction can raise the amount of corresponding miRNA in vivo.Therefore, the invention provides a kind of polynucleotide (construction) of separation, described polynucleotide (construction) can become precursor miRNA by people's cell transcription, and described precursor miRNA can be expressed as described miRNA by people's cell shearing.

As a kind of optimal way of the present invention, described polynucleotide construction contains the structure shown in formula I:

Seq _forward-X-Seq _oppositely

Formula I

In formula I,

Seq _forwardfor the nucleotide sequence of miRNA described in can becoming at cells, Seq _oppositelyfor with Seq _forwardsubstantially complementary nucleotide sequence; Or, Seq _oppositelyfor the nucleotide sequence of miRNA described in can becoming at cells, Seq _forwardfor with Seq _forwardsubstantially complementary nucleotide sequence;

X is for being positioned at Seq _forwardand Seq _oppositelybetween intervening sequence, and described intervening sequence and Seq _forwardand Seq _oppositelynot complementary;

Structure shown in formula I, after proceeding to cell, forms the secondary structure shown in formula II:

Formula II

In formula II, Seq _forward, Seq _oppositelywith the definition of X as above-mentioned;

|| represent at Seq _forwardand Seq _oppositelybetween formed base pair complementarity relation.

Usually, described polynucleotide construction is positioned on expression vector.Therefore, the present invention also comprises a kind of carrier, and it contains described miRNA, or described polynucleotide construction.Described expression vector is usually also containing promotor, replication orgin and/or marker gene etc.Method well-known to those having ordinary skill in the art can be used for building expression vector required for the present invention.These methods comprise recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.Described expression vector preferably comprises one or more selected marker, to be provided for the phenotypic character selecting the host cell transformed, as kalamycin, gentamicin, Totomycin, amicillin resistance.

Chip

MicroRNA chip of expression spectrum containing reaching a hundreds of probe, contains multiple microRNA usually, utilizes the principle of DNA double chain homologous complementary in full-length genome level, detect the content of contained various microRNA in sample.Therefore, can detect the transcriptional level of the microRNA within the scope of full-length genome in sample to be tested at one time.

Utilize miRNA sequence of the present invention, corresponding miRNA chip can also be prepared, and then study the regulative mode of its express spectra and miRNAs.

On the other hand, the present invention also provides a kind of chip for analyzing miRNA express spectra, and described chip can be used for distinguishing DMD sample and normal sample.

The oligonucleotide probe that described miRNA chip of the present invention comprises solid phase carrier and is fixed in order on described solid phase carrier, described oligonucleotide probe comprises the sequence shown in SEQIDNO.:1.

Particularly, according to miRNA of the present invention, applicable probe can be designed, be fixed on solid phase carrier, be formed " oligonucleotide arrays ".Described " oligonucleotide arrays " refers to the array with addressable point (namely with distinctive, addressable address is the position of feature), and each addressable point is all containing a coupled characteristic oligonucleotide.As required, oligonucleotide arrays can be divided into multiple sub-battle array.

Described solid phase carrier can adopt the various common used materials in gene chip field, such as but not limited to nylon membrane, and the slide, plastic sheet etc. of the slide modified through active group (as aldehyde radical, amino etc.) or silicon chip, unmodified.

The preparation of described miRNA chip can adopt the common manufacturing method of biochip known in the art.Such as, if what solid phase carrier adopted is modify slide or silicon chip, 5 ' end of probe is containing amido modified poly-dT string, oligonucleotide probe can be mixed with solution, then point sample instrument is adopted to modify on slide or silicon chip by its point, be arranged in predetermined sequence or array, then being spent the night by placement is fixed, and just can obtain miRNA chip of the present invention.If nucleic acid is not containing amido modified, then its preparation method also can refer to: " the gene diagnosis technology-on-radiation operational manual " of Wang Shenwu chief editor; The no micscale.Science of J.L.erisi, V.R.Iyer, P.O.BROWN.Exploringthemetabolicandgeneticcontrolofgeneex pressiononage, 1997; 278:680 and Ma Li people, Jiang Zhonghua edits. biochip. and Beijing: Chemical Industry Press, 2000,1-130.

On the other hand, present invention also offers a kind of method by miRNA express spectra in miRNA chip detection people tissue, comprise step:

(1) RNA sample be separated from people's tissue is provided, described RNA arranges marker;

(2) RNA of (1) is contacted with described chip, make the oligonucleotide probe generation hybridization on described RNA and solid phase carrier, thus form " oligonucleotide probe-RNA " binary complex on solid phase carrier;

(3) detect the marker of binary complex that (2) are formed, thus determine people organize in the express spectra of corresponding miRNA.

The method extracting RNA from people's tissue is method well known to those skilled in the art, comprises Trizol method.

Preferred, in step (1), after isolate RNA sample from people's tissue tissue, suitably process RNA sample, have the RNA of certain length with enrichment, described length generally between 10-100 (small fragment RNA).After above-mentioned process, utilize these small fragment RNAs to carry out follow-up hybridization, the accuracy that chip catches miRNA can be improved like this.Those skilled in the art can isolate the RNA with certain fragment length easily, such as can adopt gel electrophoresis to be separated.

Marking RNA is also method well known to those skilled in the art, and it realizes with the method for the marker of RNA specific binding by adding when hybridizing, and described marker is such as labelling groups.Described labelling groups includes but not limited to: digoxin molecule (DIG), biotin molecule (Bio), fluorescein and derivative biomolecules (FITC etc.) thereof, other fluorescence molecule (as Cy3, Cy5 etc.), alkaline phosphatase (AP), horseradish peroxidase (HRP) etc.These marks and marking method thereof have been routine techniques well-known in the art all.

When being hybridized by above-mentioned RNA and miRNA chip, first miRNA chip and pre-hybridization buffer can be carried out prehybridization.

Solid-phase hybridization between RNA and miRNA chip of the present invention carries out according to the classical way of this area, and the general personnel in this area empirically easily determine the optimum condition of related buffers, probe and concentration of specimens, prehybridization temperature, hybridization temperature and time etc.Or also can with reference to described in " Molecular Cloning: A Laboratory guide ".

Then measurement information is treated according to acquisition of informations such as the position of marking signal on miRNA chip, intensity.If amplified production fluorophor marks, also directly can obtain with fluorescence detection device (as laser confocal scanner Scanarray3000 etc.) and treat measurement information.

Detection kit

Present invention also offers a kind of test kit, containing chip of the present invention in described test kit.Described test kit can be used for the express spectra detecting miRNA; Or for distinguishing DMD sample and normal sample, preferably, also containing the marker for labeled rna sample in described test kit, and the substrate corresponding with described marker.

In addition, also can comprising in described test kit for extracting the required all ingredients such as RNA, PCR, hybridization, colour developing, including but not limited to: extract, amplification liquid, hybridization solution, enzyme, contrast liquid, nitrite ion, washing lotion, antibody etc.

In addition, working instructions and/or chip image analysis software can also be comprised in described test kit.

Diagnostic method

On the one hand, the method for diagnosis muscular dystrophy myopathy provided by the invention, comprises step:

A () detects the expression level of miR-499 and/or miR-208b in sample;

B (), according to the detected result of step (a), judge that whether the sample detected is from muscular dystrophy patients with myopathy, determination methods is:

If miR-499 expression level is higher than the expression level in normal sample, and/or

If the expression level of miR-208b is higher than the expression level in normal sample,

Then judge that this detection sample comes from muscular dystrophy patients with myopathy.

In another preference, if described miR-499 expression level is higher than 3 times of normal sample, preferably higher than 4 times of normal sample, more preferably higher than 5 times of normal sample, best higher than 6.5 times (specificity reaches 100%, and susceptibility reaches 100%) of normal sample; And/or

If the expression level of miR-208b is higher than 2 times (specificity reaches 90%, and susceptibility reaches 95%) of normal sample, more preferably higher than 3 times of normal sample,

Then judge that this detection sample comes from muscular dystrophy patients with myopathy.

The expression level of miR-499 and/or miR-208b in normal sample mentioned in the present invention is the mean value of miR-499 and/or miR-208 expression level in normal sample.

On the other hand, the invention provides a kind of method diagnosing differentiation DMD and BMD muscular dystrophy myopathy, comprise step:

A () detects the expression level of miR-499 and/or miR-208b in sample;

B (), according to the detected result of step (a), judge that whether the sample detected is from DMD patient or BMD patient, determination methods is:

If miR-499 expression level is higher than the expression level in BMD sample, and/or

If the expression level of miR-208b is higher than the expression level in BMD sample,

Then judge that this detection sample comes from DMD patient.

In another preference, described miR-499 expression level is higher than normal average value 27 times (specificity reaches 92%, and susceptibility reaches 43%); And/or

If the expression level of miR-208b is higher than normal average value 4.5 times (specificity reaches 83%, and susceptibility reaches 62%),

Then judge that this detection sample comes from DMD patient instead of BMD patient.

Major advantage of the present invention

A () the invention provides a kind of microRNA mark that can be used for distinguishing muscular dystrophy myopathy sample and normal sample.

B () effectively can distinguish muscular dystrophy myopathy sample and normal sample by microRNA mark of the present invention.

C () microRNA of the present invention mark can also judge that sample to be detected comes from DMD patient or BMD patient.

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.

Embodiment 1 experiment material and method

(1) human serum sample collects the detection with clinical indices

15 routine BMD used in experiment and 52 routine patients DMD and the routine serum sample of control group 23 all gather from Children's Hospital, Fudan University, and this research through the accreditation of paediatrics clinical pharmacology Ethics Committee of Fudan University, and possesses informed consent book.Serum keeping is in-80 DEG C of refrigerators.Simultaneously ATP enzyme dyeing added up the dissimilar Muscle fiber density of patient, the level of creatine kinase CK in clinical biochemical systems axiol-ogy infant serum.

(2) extracting of RNA and real-time quantitative PCR detect

Serum miRNA is by the serum miRNA extraction agent box extracting of Tian Gen company.RNA reverse transcription is transcribed by the inverting method of neck ring structure, detects the expression change of miRNA in serum finally by real-time quantitative PCR.

(3) statistical method

Relatively the variance analysis of two groups of experimental group is analyzed by t-test, p value be less than 0.05 be considered to have noticeable change.The assessment as diagnosis effect of ROC curve.Region area below ROC curve is used as the accuracy that diagnosis marker distinguishes basic, and dependency is by perarson check analysis.

Embodiment 2 blood serum designated object screens

(1) selection of miRNA

From the miRNA organizing miRNA chip of expression spectrum to screen muscle tissue high expression level, analyze the document reported, determine that the miRNA studied is miR-208b and miR-499.

(2) extracting of serum miRNA

The blood plasma 4 DEG C collected is left standstill 15 minutes, then 4 DEG C, and the centrifugal 30min of 3000 × rpm gets serum and retains for subsequent use.

A. sample preparation: often add isopyknic lysate MZ in pipe serum, vibrator concussion mixing 30s.

B. room temperature places 5min, and nucleic acid-protein mixture is separated completely.

C. room temperature 12,000rpm (~ 13,400 × g) centrifugal 10min, gets supernatant, proceed to one new in the EP pipe of RNase.

D. add isopyknic chloroform with serum, build pipe lid, thermal agitation 15s, room temperature places 5min.

E. room temperature 12,000rpm (~ 13,400 × g) centrifugal 15min, sample divides three layers, is transferred in new pipe by the aqueous phase on upper strata.

F. measure the volume of transfer liquid, slowly add the dehydrated alcohol that transfer liquid amasss 1/3 volume, mixing (now may occur precipitation).The solution obtained is proceeded to adsorption column miRspin together with precipitation, room temperature places 2min, room temperature 12,000rpm (~ 13,400 × g) centrifugal 30s, centrifugal rear reservation effluent liquid and adsorption column miRspin (adsorption column to finally all transfer liquid is centrifugal complete after discard).

G. measure effluent volume, slowly add the dehydrated alcohol of effluent volume 2/3 volume, mixing (now may occur precipitation).The solution obtained is proceeded to adsorption column miRelute together with precipitation, and room temperature places 2min, and room temperature 12,000rpm (~ 13,400 × g) centrifugal 30s, discards effluent liquid after centrifugal, retains adsorption column miRelute.

H. in adsorption column miRelute, add the protein liquid removal MRD (check whether and add ethanol) of 500 μ l, room temperature leaves standstill 2min, and room temperature 12,000rpm (~ 13,400 × g) centrifugal 30s, abandons waste liquid.

I. in adsorption column miRelute, add the rinsing liquid RW (check whether and add ethanol) of 600 μ l, room temperature leaves standstill 2min, and room temperature 12,000rpm (~ 13,400 × g) centrifugal 30s, abandons waste liquid.

J. repeating step (9).

K. adsorption column miRelute is put into collection tube, room temperature 12,000rpm (~ 13,400g) centrifugal 1min, remove residual liquid.(note: the object of this step is removed by rinsing liquid remaining in adsorption column, remains, may affect the experimental implementation such as follow-up RT if any rinsing liquid) is simultaneously by the ddH of RNase-free ₂o is preheating to 50 ~ 60 DEG C.

L. adsorption column miRelute is proceeded in the EP pipe of a new 1.5ml, add the ddH of the RNase-free of 20 μ l ₂o, room temperature places two minutes, room temperature 12,000rpm (~ 13,400g) centrifugal 2min.

M. repeating step l), improve the yield of RNA.-80 DEG C save backup.

(3) qRT-PCR detects

A. reverse

In this experiment, micrRNA reversion uses stem-loop reversion system

From miRNADatabase:http: find out sequence //microrna.sanger.ac.uk/ as follows:

miR-208b：AUAAGACGAACAAAAGGUUUGU

miR-499:UUAAGACUUGCAGUGAUGUUU

Then forward primer and the universal primer of these two miRNA is designed

Reverse transcriptase primer (RT-Primer):

miR-208bRT-primer：

CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACAAACCT

miR-499RT-primer：

CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAAACATCA

Real-timePCR primer:

miR-208bprimer：ACACTCCAGCTGGGATAAGACGAACAAAAG

miR-499primer：ACACTCCAGCTGGGTTAAGACTTGCAGTGA

Universal primer Universalprimer:CTCAAGTGTCGTGGAGTCGGCAA

Preparation PCR reaction system

(2) RT-PCR response procedures

1	42℃	15min
			2	95℃	2min
3	16℃	0min

After having reversed, by cDNA dilute, mixing, be kept at-20 DEG C for subsequent use.

b.Real-timePCR

The internal reference that in relative quantification, serum miRNA uses is miR-223.

Reaction system and program

(4) myofiber somatotype ATP enzyme dyeing

A. the beaker filling iso-pentane is not put into liquid nitrogen, not submergence, below iso-pentane, be frozen into the muscle tissue that can to start freezing Tragacanth when being in a liquid state above solid and fix.

B. careful freezing organization embedding block to be taken out, put into rapidly the container sealed type storage of precooling or cook frozen section in time.Or long-term preservation below-80 DEG C.

C. frozen section 8-10um, puts into the preincubate liquid of three kinds of different PH respectively, room temperature lower 15 minutes, 5 minutes, 5 minutes.PH value is respectively 10.5,4.6 and 4.2.

D. add Veronal sodium liquid (PH9.4-9.7) to clean gently by 1 minute for 30 seconds.

E. Incubating Solution is added 45 minutes, 37 DEG C.

F.1% calcium chloride wash 3 times each 3 minutes.

G.2% cobalt chloride reacts 3 minutes.

H. washing 3 times 3 minutes/time is distilled.

I.1% ammonium sulfide (used time preparation) 1 minute.

J. running water.

K. dehydration of alcohol at different levels, transparent, mounting.

L. basis of microscopic observation PH be 10.5 dye black for II type myofiber, white be I type myofiber; PH be 4.6 dye black for I type myofiber, white is IIa type myofiber, in view of therebetween be IIb and IIc myofiber; PH be 4.2 white colourings for IIa or IIb myofiber, in view of therebetween be IIc myofiber.

M. preincubate liquid:

Veronal sodium PH is the calcium chloride of 10.5:20mM Veronal sodium, 18mM;

Acetate buffer solution PH is the Veronal sodium of 4.6:50mM sodium-acetate, 30mM, is adjusted to pH value is adjusted to 4.6 with hydrochloric acid.

Acetate buffer solution PH is 4.2: the same only pH value is adjusted to 4.2.

Experimental result

The change of 1miRNA in muscular dystrophy patients serum

The present inventor have collected the two class infants i.e. serum sample of 15 routine BMDs and 52 routine DMD patients of age from the routine healthy children in 23 between 1 to 14 years old and muscular dystrophy, and the information such as relevant infant gene diagnosis are summarised in table 1.Fig. 1 shows the change of miRNA in muscular dystrophy patients serum.As can be seen from the figure, miR-208b and miR-499 expresses and all significantly raises in BMD and DMD patients serum.Further these serum of prompting the present inventor miRNA can as the possibility of the potential new serum biological marker of this kind of disease.

Table 1 patient information

Patient code

Disease type

Age (moon)

Whether can walk about

Hormonotherapy

Muscle biopsy

Annotation

BMD-01

BMD

11

Be

No

Be

MFT:N/A

BMD-02

BMD

24

Be

No

Be

MFT:N/A

BMD-03

BMD

36

Be

No

Be

MFT:N/A

BMD-04

BMD

36

Be

No

Be

BMD-05

BMD

36

Be

No

Be

BMD-06

BMD

40

Be

No

Be

MFT:N/A

BMD-07

BMD

48

Be

No

Be

BMD-08

BMD

55

Be

No

Be

MFT:N/A

BMD-09

BMD

56

Be

No

Be

MFT:N/A

BMD-10

BMD

75

Be

No

Be

MFT:N/A

BMD-11

BMD

98

Be

No

Be

MFT:N/A

BMD-12

BMD

101

Be

No

Be

BMD-13

BMD

110

Be

No

Be

BMD-14

BMD

120

Be

No

Be

BMD-15

BMD

165

Be

No

Be

MFT:N/A

DMD-01

DMD

7

Be

No

Be

DMD-02

DMD

8

Be

No

Be

DMD-03

DMD

15

Be

No

Be

DMD-04

DMD

17

Be

No

Be

DMD-05

DMD

18

Be

No

Be

MFT:N/A

DMD-06

DMD

22

Be

No

Be

DMD-07

DMD

24

Be

No

Be

DMD-08

DMD

24

Be

No

Be

MFT:N/A

DMD-09

DMD

31

Be

No

Be

MFT:N/A

DMD-10

DMD

36

Be

No

Be

DMD-11

DMD

36

Be

No

Be

DMD-12

DMD

36

Be

No

Be

DMD-13

DMD

40

Be

No

Be

MFT:N/A

DMD-14

DMD

43

Be

No

Be

DMD-15

DMD

43

Be

No

Be

DMD-16

DMD

46

Be

No

Be

DMD-17

DMD

46

Be

No

Be

DMD-18

DMD

48

Be

No

Be

DMD-19

DMD

51

Be

No

Be

DMD-20

DMD

52

Be

No

Be

MFT:N/A

DMD-21

DMD

55

Be

No

Be

DMD-22

DMD

55

Be

No

Be

MFT:N/A

DMD-23

DMD

59

Be

No

Be

DMD-24

DMD

62

Be

No

Be

MFT:N/A

DMD-25

DMD

65

Be

No

Be

DMD-26

DMD

66

Be

No

Be

DMD-27

DMD

67

Be

No

Be

MFT:N/A

DMD-28

DMD

68

Be

No

Be

DMD-29

DMD

73

Be

No

Be

DMD-30

DMD

77

Be

No

Be

DMD-31

DMD

77

Be

No

Be

DMD-32

DMD

79

Be

No

Be

DMD-33

DMD

82

Be

No

Be

MFT:N/A

DMD-34

DMD

83

Be

No

Be

MFT:N/A

DMD-35

DMD

84

Be

No

Be

DMD-36

DMD

90

Be

No

Be

DMD-37

DMD

90

Be

No

Be

DMD-38

DMD

90

Be

No

Be

MFT:N/A

DMD-39

DMD

92

Be

No

Be

MFT:N/A

DMD-40

DMD

92

Be

No

Be

MFT:N/A

DMD-41

DMD

93

Be

No

Be

MFT:N/A

DMD-42

DMD

94

Be

No

Be

MFT:N/A

DMD-43

DMD

96

Be

No

Be

MFT:N/A

DMD-44

DMD

96

Be

No

Be

MFT:N/A

DMD-45

DMD

96

Be

No

Be

MFT:N/A

DMD-46

DMD

98

Be

No

Be

MFT:N/A

DMD-47

DMD

104

Be

No

Be

DMD-48

DMD

104

Be

No

Be

MFT:N/A

DMD-49

DMD

125

Be

No

Be

DMD-50

DMD

128

Be

No

Be

DMD-51

DMD

138

No

No

Be

MFT:N/A

DMD-52

DMD

144

Be

No

Be

DMD-53

DMD

41

Be

No

Be

miRNAs:N/A

DMD-54

DMD

56

Be

No

Be

miRNAs:N/A

DMD-55

DMD

60

Be

No

Be

miRNAs:N/A

DMD-56

DMD

92

Be

No

Be

miRNAs:N/A

DMD-57

DMD

151

Be

No

Be

miRNAs:N/A

Note: N/A: invalid; MFT: muscular fiber type

2 serum miRNA are as the assessment of the diagnostic marker of muscular dystrophy

Analyzed by Receiver Operating Characteristics (ROC) curve (Fig. 2), miR-499 and miR-208b can be good at distinguishing normal child and muscular dystrophy patient, especially DMD infant (AUC:0.9913 and 0.9130, the equal <0.0001 of P), they also can well distinguish BMD and DMD patient (AUC:0.6987 and 0.7115, P=0.0198 and P=0.0131) simultaneously.

Fig. 2 shows the ROC curve of miRNA for normal people, BMD and DMD patient, as shown in the figure, the ROC curve of the 52 routine BMD patients of routine DMD patient 15 and 23 routine normal children, the numeric representation in figure be area under a curve AUC.Wherein blue, red and green curve represents DMD and normal child, the ROC curve of BMD and normal child and BMD and DMD respectively.Can find out that from Fig. 2 A serum miR-499 level height can well distinguish DMD and normal child (AUC=0.99, p<0.0001), also BMD and normal child (AUC=0.9913 can well be distinguished, p<0.001), also certain value (AUC=0.6987, p=0.0198) is had for differentiation BMD and DMD; Can find out that from Fig. 2 B serum miR-208b level height can well distinguish DMD and normal child (AUC=0.9323, p<0.001), also BMD and normal child (AUC=0.913 can well be distinguished, p<0.001), for differentiation BMD and DMD relative to miR-499 value slightly high (AUC=0.7115, p=0.0131).These results show, the level of serum miR-499 and miR-208b not only can distinguish normal child and DMD/BMD, and also have clinical reference value for differentiation DMD and BMD.

3 serum miRNA, myofiber and the dependency at age

DMD patient's cardinal symptom is gradual degeneration of skeletal muscle necrosis, and cardiac muscle is gradually by fibrous tissue replacement, and fatty infiltration, eventually through breathing or cardiac failure death.In order to confirm that the level of miRNA in serum can be associated with the process of disease further, first the present inventor analyzes the dependency between age and serum miRNA.Just in time be proportionate with the age in period (2-6 year) time of infant early diagnosis as the expression level of miR-499 and miR-208b in the serum of Fig. 3 display, and during this period of time in the fast muscle fiber per-cent (IIa type and IIb type) of patient reduce, first this necrose consistent at the early stage fast muscle fiber of patient with observing clinically.The present inventor finds that the IIc type Muscle fiber density with regenerated fibre characteristic increases (Fig. 4) gradually in this age bracket simultaneously, illustrates that these two the miRNA levels in serum may be relevant with anathreptic situation to muscle injury severity thus.

Fig. 3 shows the dependency at miRNA and age, as can be seen from the figure along with increase miR-499 and the rising of miR-208b expression level at age.

Fig. 4 shows the dependency at myofiber and age, and the IIc type Muscle fiber density as can be seen from the figure along with increase fast muscle fiber per-cent (IIa type and the IIb type) minimizing at age with regenerated fibre characteristic increases.

Table 2 and table 3 respectively illustrate the level of these two serum miRNA and the dependency between muscle fiber types and Different age group is summed up.As can be seen from the table, CK does not all have dependency with the age in different age brackets, miR-499 and miR-208b has positive correlation with the age during DMD patient 2-6 year, and IIa+IIb type Muscle fiber density and age are negative correlation during this period, and IIc type myofiber and age are proportionate.

Table 2miRNA and the dependency at age

The dependency at table 3 muscular fiber type and age

4 serum miRNA and myofibrillar dependency

According to the pathological change of DMD, patient's initial stage muscular death mainly occurs in fast muscle fiber, and then the present inventor infers that whether these miRNA are relevant to muscle fiber types.As shown in Figure 5, Fig. 5 shows the expression change of miRNA in change lever, as can be seen from the figure miR-1 and miR-133 expresses higher in the tibialis anterior muscle (TA) based on fast muscle fiber, and miR-499, miR-208b and miR-206 then express very high in the soleus muscle (SOL) based on slow switch fibers.The special miR-208a of cardiac muscle and be not that the miR-21 of specifically expressing in muscle expresses no significant difference in two kinds of myofibers.These data show that the expression of miR-499 and miR-208b detected in serum is exactly mainly come from slow muscle.Following the present inventor analyzes the dependency of miR-499 and miR-208b and different muscular fiber type in the Different age group of DMD patient, found that in the infant of DMD early stage (2 to 6 years old), the level of serum miR-499 and miR-208b and IIc type myofiber positive correlation (Fig. 6).And miR-499 and miR-208b is proportionate (Fig. 7) with the myofiber (I type) of slow muscle in older infant (being greater than 6 years old) serum.

Fig. 6 shows the dependency of serum miRNA and IIc type muscular fiber type in DMD infant, as can be seen from the figure along with the expression amount of increase miR-499 and miR-208b of IIc Muscle fiber density raises.

Fig. 7 shows the dependency of serum miRNA and I type muscular fiber type in DMD infant.As can be seen from the figure along with the expression amount of increase miR-499 and miR-208b of I type Muscle fiber density also obviously raises.Illustrate that having a high proportion of slow muscle could secrete miRNAs specific expressed in more slow muscle thus.

The dependency of DMD patient serum miRNA and muscular fiber type in Different age group is summed up in table 4.Comprehensive these information the present inventor finds that serum miRNA expression level is not quite identical in the change of different age brackets.As can be seen from form, in slow muscle, the expression level of these two miRNA (miR-499 and miR-208b) in serum of high expression level is more relevant to the pathological condition of patient.Be proportionate at these two miRNA of patient's commitment (2 to 6 years old) and the IIc type Muscle fiber density with regenerated muscle fibers characteristic, can illustrate that the expression level of these two miRNA in serum is higher, the anathrepsis ability of patient is stronger, and the expression level of this two miRNA that (are greater than 6 years old) in older patient and IIa, IIb type Muscle fiber density is negative correlation, and be proportionate with I type myofiber, illustrate that the expression level of these two miRNA in serum in this age bracket is got over Gao Ze and represented patient muscle mainly based on I type myofiber.

Serum miRNA and CK and myofibrillar dependency in table 4DMD infant

Discuss

This patent describes the expression level of two miRNA in serum can as the biomarker of muscular dystrophy.In this research, the present inventor finds that these two miRNA all significantly raise in the mouse models and muscular dystrophy patients serum of muscular dystrophy.

Although the level of creatine kinase CK is by the conventional major bio-markers doing muscle disease in serum, thus evaluate damage and the degree of necrosis of muscle, but it is always not reliable, because the impact that it is often subject to extraneous factor is such as moved, and at this time its change is just not necessarily relevant with disease.And the level of serum miRNA is not too be subject to extraneous factor on the impact of the pressure of health compared with CK level.

The present inventor studies further and finds while the per-cent of fast muscle fiber from the DMD patient of 2 to 6 years old reduces, the serum expression level of the miRNA (miR-208b and miR-499) of enrichment in slow switch fibers and age with and the IIc type myofiber that regenerates be proportionate.In addition, research report display in the past, TNF-α, IL-6 and the GPX up-regulated in DMD patient muscle.In sum, the present inventor infers why the miRNA of these enrichments in slow switch fibers in the early stage infant serum of DMD raises, and may be the secretion of the muscle satellite cell of propagation coming from the lighter slow switch fibers of degree of injury that inflammatory cytokine stimulates and be activated.Therefore, in the early stage slow switch fibers of patient, the expression of the miRNA of enrichment in serum can reflect the corresponding inflammatory reaction level of muscle and anathrepsis level.In addition, the present inventor also demonstrates the expression level of miR-499 and miR-208b in the infant serum more than 6 years old and slow switch fibers ratio is proportionate.Infer that these miRNA expression levels in now serum are then the indexs representing remaining muscle quality thus.

These results make the present inventor draw, and some miRNA in serum can as valuable prognostic marker thus monitoring DMD progression of disease.The abnormal expression of the serum miRNA expression level that the present inventor has been found that an early stage patient (age was at about 4 years old) in the process of analytical data comparatively with age bracket other people and age less patient is low, infers stage (being greater than 6 years old) that its change of illness state has reached more late period to find this patient by follow-up investigation (about 8 years old) change of illness state is more serious compared with other patients with age bracket at present.The stronger explanation serum miRNA of this case may be used for the pathological change of monitoring DMD patient.Owing to not having the effective methods for the treatment of for DMD patient at present, therefore early find, early contact co-treatment, there is more importantly meaning the time extending its ambulation for patient.

MiR-499 and miR-208b is the special miRNA of skeletal muscle slow muscle.The level representation of serum miR-499 and the miR-208b total amount of skeletal muscle slow muscle.First impaired at the early stage skeletal muscle fast muscle of morbidity, the content of the special miRNA of fast muscle in serum is caused to raise rapidly, to remain unchanged after reaching peak value very soon, and even its serum level can be caused to decline, so the special miRNA of fast muscle is not suitable for judgement disease because fast muscle total amount reduces.On the contrary, skeletal muscle slow muscle is slowly impaired, can react disease preferably, serum miR-499 and miR-208b level higher, illustrate be in a bad way or develop fast.In the morbidity later stage, skeletal muscle total amount reduces, and the content of the special miRNA of all skeletal muscle in serum declines, and the skeletal muscle now remained mostly is slow muscle, and therefore slow muscle means that disease is slow more.If serum miR-499 is relative with miR-208b level high, illustrate that PD is slow.Clinically, can judge that the relative all infants of its disease are fast or slow according to specific period different infant serum miR-499 with miR-208b level, also can judge that its course of disease stabilizes or is degrading according to same infant in the level of specific period serum miR-499 and miR-208b, and whether pharmacological agent is effective.

Therefore, the biomarker of serum miRNA that the present inventor proposes is that muscular dystrophy patient early diagnosis not only can propose clinical diagnosis suggestion, and for their treatment process, also can propose corresponding suggestion or assessment.

Embodiment 3 prepares miRNA chip

Convert miRNA sequence provided by the invention (SEQIDNO.:1) to complementary sequence, add the catenation sequence of 10-20nt according to the feature such as GC ratio producing sequence at sequence two ends; Core sequence is different, and catenation sequence is also different.Catenation sequence can be produced at random by program, and the probe that catenation sequence and core sequence are formed meets the following conditions:

1), in probe sequence, the quantity of same Nucleotide (A, C, G, T) can not exceed 50% of sequence sum;

2) quantity of any continuous print A, T or C, G can not exceed 25% of sequence sum;

3) G, C content account for the 40%-60% of sequence sum;

4) probe sequence can not from hybridization, and namely in probe sequence, the length of complementary fragment can not exceed 30% of probe length.

Be combined on slide for what make the probe steady of synthesis, adopt 5 ' end of conventional method probe in post synthesis to carry out glycosyl modified.

The point system of chip: first alkylation modification is carried out on the surface of slide, to improve binding ability.Adopt conventional chip spotting methods to carry out point sample, in order to detect the repeatability of cross experiment, 3-6 hybridization point put by each probe on slide.

Embodiment 4

Prepared by test kit

By good for the Chip Packaging of preparation in embodiment 3, be placed in a box together with working instructions, form test kit.

Embodiment 5

The detection validation of chip

(30 routine muscular dystrophy myopathy samples and the normal sample of 30 examples are comprised to the multiple samples from infection from hospital, wherein in 30 routine muscular dystrophy myopathy samples, 15 examples are DMD sample, 15 examples are BMD sample), by method preparation and the mark microRNA of embodiment 1-2, by chip prepared by the method for embodiment 3, detect by double-blind method.According to the existence of illustrated microRNA mark whether and upper mediation downward situation carry out judgement sample.Wherein, positive control and negative control are respectively known DMD sample and normal sample.

Result shows, comprises the chip of species specificity microRNA of the present invention, and its exactness is 90%, effectively can distinguish DMD sample and normal sample.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (10)

1. miRNA or the miRNA set be separated, it is characterized in that, described miRNA is selected from:

The miRNA of (i) sequence as shown in SEQIDNO.:1;

(ii) miRNA of sequence as shown in SEQIDNO.:2, and/or

(iii) with the miRNA of miRNA sequence complementation shown in (i) or (ii).

2. that be separated an or artificial constructed precursor miRNA, it is characterized in that, described precursor miRNA can be sheared and be expressed as miRNA or miRNA as claimed in claim 1 and gather in people's cell.

3. the polynucleotide be separated, it is characterized in that, described polynucleotide can be become precursor miRNA by people's cell transcription, and described precursor miRNA can be sheared and be expressed as miRNA or miRNA as claimed in claim 1 in people's cell to be gathered.

4. method as claimed in claim 3, wherein, described polynucleotide have the structure shown in formula I:

Seq _forward-X-Seq _oppositelyformula I

In formula I,

Seq _forwardfor the nucleotide sequence of miRNA described in becoming at people's cells;

Seq _oppositelyfor with Seq _forwardsubstantially the nucleotide sequence of complementation or complete complementary;

X is for being positioned at Seq _forwardand Seq _oppositelybetween intervening sequence, and described intervening sequence and Seq _forwardand Seq _{instead to}not complementary;

And the structure shown in formula I is after proceeding to people's cell, form the secondary structure shown in formula II:

Formula II,

In formula II, Seq _forward, Seq _oppositelywith the definition of X as above-mentioned,

|| represent at Seq _forwardand Seq _oppositelybetween formed base pair complementarity relation.

5. a carrier, is characterized in that, described carrier contains the present invention miRNA as claimed in claim 1, or polynucleotide as claimed in claim 3.

6. the purposes of miRNA or miRNA set described in claim 1, is characterized in that, described miRNA or miRNA set is for the preparation of chip or the test kit of distinguishing DMD sample, BMD sample and normal sample.

7. a miRNA chip, is characterized in that, described miRNA chip comprises:

Solid phase carrier; And

Be fixed on the oligonucleotide probe on described solid phase carrier in order, described oligonucleotide probe corresponds to the sequence shown in SEQIDNO.:1 and/or SEQIDNO.:2 specifically.

8. the purposes of miRNA chip according to claim 7, is characterized in that, described miRNA chip is for the preparation of the test kit distinguishing DMD sample, BMD sample and normal sample.

9. a test kit, is characterized in that, containing miRNA chip according to claim 7 in described test kit; Or

MiRNA or miRNA set according to claim 1.

10. screening is used for the treatment of a method for the drug candidate of muscular dystrophy myopathy, it is characterized in that, comprises step:

A () provides a test group and a control group, wherein candidate substances is applied to cell or the animal of test group in described test group, and measure the expression level using miR-499 and/or miR-208b in rear described test group, and described control group adopts the condition identical with test group, but candidate substances is not applied to cell or the animal of control group;

B the expression level of miR-499 and/or miR-208b of test group and miR-499 and/or miR-208b of control group compares by ();

Wherein, when expression level significantly lower than control group of the expression level of miR-499 and/or miR-208b of test group, then show that this candidate substances is the drug candidate being used for the treatment of DMD.