CN103045596A - Down syndrome 21 chromosome-related miRNA, gene, screening method and application - Google Patents

Abstract

The invention relates to a down syndrome 21 chromosome-related miRNA and a precursor sequence thereof, and the precursor sequence includes a nucleotide sequence of SEQ ID NO.1. The miRNA is positioned inside a DS (down syndrome) critical region of a chromosome 21 and is related to occurrence and development of a DS disease. Therefore, the novel miRNA can be widely applied to preparation of a DS diagnosing or detecting gene detection chip or relevant reagents. The invention further relates to a screening method of the miRNA.

Description

No. 21 karyomit(e)s of mongolism be correlated with miRNA, gene, screening method and application

Technical field

The present invention relates to the genetically engineered field, relate in particular to a kind of miRNA, gene, screening method and the application relevant with No. 21 karyomit(e)s of mongolism.

Background technology

Mongolism (Down syndrome, be called for short DS) is againtrisomy 21, and (Trisomy 21, Ts21) syndrome, a kind of inborn defect type disease that causes because having more or No. 21 karyomit(e)s of part.Among the approximately per 700 routine babies 1 example is just arranged.The patient shows as the 80 various clinical symptoms such as the complete and congenital heart defects of congenital mental retardation, hyponea, anthropometic.Biological study has been found to contain more than 500 gene on No. 21 karyomit(e)s, comprises 5 miRNA(microRNA, Microrna) gene: miR-99a, let-7c, miR-125b-2, miR-155 and miR-802.MiRNA is that a class length is the endogenous strand non-coding adjusting RNA of 19 ~ 25 Nucleotide.MiRNA can be combined with the 3 ' terminal specific of target gene mRNA, makes its degraded or transcribes inhibition, has very important effect in the biological procedureses such as differentiation of the increment of cell, differentiation, apoptosis, embryo's growth and tissue.Studies show that crossing of 5 miRNA on No. 21 karyomit(e) express all relevant with the various clinical proterties of DS patient, such as amentia, leukemia of children, immune deficiency, ypotension etc.But the miRNA relevant with No. 21 karyomit(e) also has a lot, therefore, studies these miRNA, and particularly the miRNA relevant with the mongolism specificity has important researching value.

Summary of the invention

Based on this, be necessary to provide a kind of miRNA, gene, screening method and the application relevant with No. 21 karyomit(e)s of mongolism.

A kind of miRNA gene relevant with No. 21 karyomit(e)s of mongolism has the nucleotide sequence such as SEQ ID NO.1.

The precursor of a kind of miRNA relevant with No. 21 karyomit(e)s of mongolism has the nucleotide sequence such as SEQ ID NO.2.

A kind of miRNA relevant with No. 21 karyomit(e)s of mongolism has the nucleotide sequence such as SEQ ID NO.3.

The application in preparation diagnosis or detection mongolism reagent of above-mentioned miRNA, its gene and precursor-gene thereof.

The application in preparation diagnosis or detection mongolism gene detecting chip of above-mentioned miRNA, its gene and precursor-gene thereof.

The screening method of the relevant miRNA of No. 21 karyomit(e)s of a kind of mongolism comprises the steps:

Gather respectively the human umbilical cord blood mononuclear cell sample of mongolism fetus and normal fetus;

Extract the total RNA in the described sample, be not more than the little RNA of 30 Nucleotide with sex change polypropylene amine gel electrophoresis Separation and Recovery length, under the effect of T4RNA ligase enzyme, hold the nucleotide sequence joint that adds such as SEQ ID NO.4 at 5 ' of the little RNA that reclaims, 3 ' end adds the nucleotide sequence joint such as SEQ ID NO.5, behind TBE polypropylene amine gel electrophoresis purifying, behind Solexa little RNA reverse transcription primer and pcr amplification, make the cDNA library of little RNA, re-use the gel electrophoresis of TBE polypropylene amine described cDNA library is carried out purifying and carries out sequencing analysis;

Small RNA molecular to the rear gained that checks order removes joint and the processing that depollutes, and therefrom filter out the nucleotide sequence that length is 18 ~ 30nt, with SOAP software institute's calling sequence and people's whole genome sequence are compared, with the sequence on the coupling according to GenBank Rfam miRBase17.0 repeat exon the priority orders of the intron note of classifying, then with intron and the small RNA molecular sequence of the upper sequence of comparison as the new data source of miRNA, with MIREAP and two kinds of softwares of MiPred the karyomit(e) at described new miRNA place is analyzed, obtained the relevant miRNA of No. 21 karyomit(e)s of described mongolism.

The relevant miRNA of No. 21 karyomit(e)s of above-mentioned mongolism or its precursor are the miRNAs relevant with the Down syndrome in patients specificity, further research discovery, and its growth with the generation of the adjusting of the expression of gene, cytodifferentiation, embryo morphology and heart is relevant.Therefore, the discovery of the miRNA that this is new, for further studying No. 21 chromosomal functions, explore the new treatment target spot of Down syndrome in patients empirical theory basis and a new direction are provided, and can be applicable to preparation diagnosis or detect the field of Down syndrome in patients reagent, for the diagnosis and detection of mongolism disease provides easy means.

Description of drawings

Fig. 1 is the Cord blood chromosome karyotype analysis figure as a result of the mongolism fetus of an embodiment;

Fig. 2 is the Cord blood chromosome karyotype analysis figure as a result of normal fetus;

The seat analysis chart that Fig. 3 is miR-nov21 on No. 21 karyomit(e).

Embodiment

Below main with specific embodiments and the drawings the relevant miRNA of No. 21 karyomit(e)s of mongolism, gene, screening method and application are described in further detail.

The screening method of one embodiment, result and correlation detection comprise the steps:

1, screening

1.1, collection and the preservation of sample collection and umbilical cord blood

Collect in February, 2011 ~ 2011 and be diagnosed as Cord blood sample 5 examples of standard type DS fetus, normal fetus Cord blood 3 examples year June in the antenatal diagnosis of Shenzhen people's hospital row, through the karyotyping of conventional G banding chromosome.In the 5 routine DS umbilical cord bloods, 2 examples are built the storehouse for little RNA and are carried out sequencing analysis, and 3 examples are used for the PCR checking.This research is ratified by Shenzhen people's hospital Ethics Committee, and obtains all pregnant woman's informed consents.

Under ultrasonic guidance, extract 18 ~ 22 pregnant all DS fetuses and normalfetus Cord blood 2 ~ 3mL (BD company) in the EDTA anticoagulant tube, then use lymphocyte separation medium (Invitrogen company) to extract human umbilical cord blood mononuclear cell, incorporate Trizol reagent (Invitrogen company), place cryopreservation tube, frozen for subsequent use in-80 ℃.

1.2, Cord blood RNA extracts and the Solexa order-checking

The foundation in the little RNA of human umbilical cord blood mononuclear cell library and order-checking, bioinformatic analysis are finished under China large gene studies institute assists, and whole experimentation carries out in strict accordance with reagent and instrumentation specification sheets.

Main experiment flow comprises: the total RNA that adopts Invitrogen T rizol test kit extracting 2 routine DS umbilical cord blood samples, again 2 sample balanced mix, get 10 μ g mixing RNA samples are not more than 30 Nucleotide with 15% sex change polypropylene amine gel electrophoresis Separation and Recovery Nucleotide (nucleotide, nt) length little RNA; Again under T4RNA ligase enzyme (Promega company) effect, add respectively top connection at little RNA two ends, wherein, the sequence of 5 ' end connector is that the sequence of 5 '-GUUCAGAGUUCUACAGUCCGACGAUC-3 ' (SEQ ID NO.4) and 3 ' end connector is that (P represents phosphate group (phosphate) to 5 '-pUCGUAUGCCGUCUUCUGCUUGidT-3 ' (SEQ ID NO.5); IdT represents reverse deoxythymidine (inverteddeoxythymidine)), behind the tri-borate-EDTA of 10wt% (TBE) polypropylene amine gel electrophoresis purifying, by the cDNA library of making little RNA behind Solexa little RNA reverse transcription primer and the pcr amplification.Then with the gel electrophoresis of 6wt%TBE polypropylene amine cDNA library is carried out purifying, carry out sequencing analysis with Illumina Hiq-2000 sequenator.

1.3, the prediction of data processing and new miRNA

At first the small RNA molecular sequence of order-checking gained is removed joint, gone inferior quality and the processing such as depollute obtains the clean sequence of 18 ~ 30 length of nucleotides.Then use SOAP(2.0) software compares the clean sequence of gained and people's whole genome sequence, with the sequence on the coupling according to GenBank R fam(10.0) miRBase17.0 repeat exon the priority orders of the intron note of classifying, to the sequence of the upper any annotation information of comparison not with " Unanno " expression.Then the data source of the small RNA molecular sequence of Intron and Unanno being predicted as new miRNA, utilization MIREAP and two kinds of softwares of MiPred carry out the forecast analysis of new miRNA.

1.4, Stem-loop RT-PCR checking

For the expression of new miRNA gene in DS fetus and normal fetus on checking Solexa sequencing analysis result's reliability and No. 21 karyomit(e) changes, use SYBR Green Stem-loop RT-PCR that the ripe body of new miRNA on No. 21 karyomit(e) is verified.Use Invitrogen Trizol test kit to extract respectively total RNA of residue 3 routine DS umbilical cord bloods and 3 routine normal fetus human umbilical cord blood mononuclear cells; On the ABI-7500 real-time PCR, adopt 20 μ L reaction systems, use miRNA molecular specificity stem ring reverse transcriptase primer (Chinese Shanghai Ji Ma company) and M-MLV reverse transcription test kit (Promega company), behind reverse transcription reaction, at 95 ℃ of denaturation 5min, 95 ℃ of sex change 15s, 60 ℃ of annealing 15s, continuously 40 circulations of amplification under the reaction conditions of 72 ℃ of extension 32s, each gene arranges 3 multiple holes, cDNA concentration is 1:20, take rRNAU6 as confidential reference items, adopts 2-Δ Δ^CtThe relative expression quantity of new miRNA gene on No. 21 karyomit(e)s of method calculating.

2, result

2.1, the umbilical cord blood chromosome karyotype analysis

As shown in Figure 1, the dyed body karyotyping of 5 routine DS umbilical cord bloods shows to be standard DS caryogram.Fig. 2 is the Cord blood karyotype of normal fetus.

2.2, small RNA molecular sequence classification note

By the sequencing analysis of Solexa high throughput sequencing technologies to the little RNA of DS fetal cord blood mononuclear cell library, altogether obtain the high quality sequence of 23324424 10 ~ 30 length of nucleotides, through removing joint, going inferior quality and the sequence of depolluting etc. obtains the clean sequence of 21770729 18 ~ 30 length of nucleotides after processing, account for 90.6% of total amount, add up to 280056 kinds of little RNA.Use SOAP (2.0) software that the various little RNA of gained and people's whole genome sequence (hg19) are compared, allow at most the mispairing of 2 bases in the comparison process, obtain 8087250 matching sequences, totally 58523 kinds of small RNA moleculars.Compare with public database, to mate complete genomic little RNA sequence classification note and be miRNA, tRNA, rRNA, cell small nuclear RNA sequence (snRNA), small nucleolar RNA sequence (snoRNA), piRNA, mRNA degradation fragment, tumor-necrosis factor glycoproteins (repeat), the little RNA sequence of cytoplasm (scRNA), the little RNA sequence of signal recognition particle (srpRNA), intron sequences (Intron) and not note sequence (Unanno), totally 12 classes.See Table 1.

The little RNA classification annotation table of table 1 and whole genome sequence coupling

2.3, prediction and the checking of new miRNA

By the analysis of MIREAP and two kinds of softwares of MiPred, find altogether 21 new miRNA, as shown in table 2.

21 new miRNA molecules that table 2 sifts out through MIREAP and MiPred

Annotate: "+" and "-" in the chromosomal localization number the miRNA gene that represents respectively to be correlated with is to be positioned at chromosomal normal chain and minus strand.

21 new miRNA of above-mentioned analysis derive from respectively 12 karyomit(e)s, wherein only there is 1 new miRNA to derive from karyomit(e) No. 21, called after miR-nov21 is positioned at No. 21 chromosomal " DS critical areas " (chr21q22.2-q22.3), as shown in Figure 3.The sequence of this miRNA is SEQ ID NO.3, and its precursor-gene sequence is SEQ ID NO.2, forms hairpin structure (perhaps being called " loop-stem structure ").The Stem-loopRT-PCR the result sees Table 3.

The PCR the result of the ripe body of table 3miR-nov21 in DS and normal fetus human umbilical cord blood mononuclear cell

Annotate: " # " represents DS fetus and normal fetus miRNA relatively, and difference has statistical significance (p＜0.05).

2.4, target gene prediction and biological function analysis

Adopt TargetScan C ustom that miR-nov21 is carried out the target gene prediction, obtain 211 target genes totally 215 binding sites.The analysis of Gene Ont ology biological function is found, miR-nov21 molecular regulation target gene and the expression of gene, the adjusting of cytodifferentiation, the generation of embryo morphology and the growth relevant (p value＜0.001) of heart are as shown in table 4.

Table 4miR-nov21 molecular regulation target gene Gene Ontology biological function analytical results

3 interpretations of result

The precursor hairpin structure sequence length of this new miRNA is 83 Nucleotide, and minimum folding free energy is-32.00kal/mol.MiR-nov21 is ripe, and body length is 23 Nucleotide, is positioned on the stem arm of precursor hairpin structure.With UCSC genetic analysis platform miR-nov21 is further analyzed, find that the miR-nov21 gene is positioned at the beta amyloid precursor protein and shears enzyme 2(beta-siteAPP cleaving enzyme 2, BACE2) on the antisense strand of the 9th intron of gene, also be that the expression of miR-nov21 gene is relevant with the BACE2 gene.BACE2 proteolytic enzyme can be induced the generation of amyloid, causes the accumulation of amyloid to make neurodegeneration, has important in the forming process of alzheimer's disease and DS patient's dementia.

The genetic expression imbalance is the basic reason that causes the various phenotypes of DS on No. 21 karyomit(e)s that research is found to have more, change and be positioned at the intragentic expression of No. 21 karyomit(e)s " DS critical area ", then in the forming process of the various clinical symptom of DS patient, have important effect.Stem-loop RT-PCR result shows: miR-nov21 is high expression level in DS fetal cord blood mononuclear cell, 1.5 times of control group, illustrate that 5 known on miR-nov21 and No. 21 karyomit(e) miRNA are the same, in the forming process of DS disease, have important effect.Find that by the target gene analysis miR-nov21 can regulate the CPG-island and methylate in conjunction with albumen 2(methyl-CpG-bindingprotein 2, expression MeCP2).MeCP2 albumen is a kind of transcription factor, in most tissues and cell expression is arranged, and wherein expression amount is the highest in cerebral tissue.MeCP2 albumen can be combined with the CpG-island dinucleotides that methylates, and induces the enrichment of histone modification and Chromatin Remodeling associated protein complex body, regulates the expression of particular target gene, and neuronic growth is had vital role.It should be noted that MeCP2 also is the target gene of miR-155, miR-802 on No. 21 karyomit(e), let-7c and miR-125b-2, with the negative correlativing relation that is expressed as of miR-155, miR-802, let-7c and miR-125b-2.MECP2 albumen low expression in the DS human fetal brain found in research, thereby DS patient's intelligence growth is had material impact, on No. 21 karyomit(e) miRNA cross express relevant with the congenital amentia of DS patient.

The biological function analysis of miR-nov21 target gene is shown: the generation of the expression of miR-nov21 and gene, the adjusting of cytodifferentiation, embryo morphology is relevant with the growth of heart.In the adjusting of cytodifferentiation, miR-nov21 can regulate the expression of its target gene SMAP1, ETS1, CDK6, FOXO3, PURB and TOB2 by being combined with its target gene 3 ' terminal specific, participates in regulation and control red corpuscle and granulocytic differentiation.Because the expression of miRNA and its target gene is the negative regulation relation, the high expression level of miR-nov21 can make the expression of its target gene SMAP1, ETS1, CDK6, FOXO3, PURB and TOB2 reduce in theory, and then cause red corpuscle and granulocytic differentiation and maturation obstacle, participate in DS ewborn infant " symptomatic leukemia " and the leukemic formation of later stage DS patient.In addition, find also that by bioinformatic analysis miR-nov21 can by the expression of regulation and control BBS4, BMP2, XIRP1, NODAL, RXRA, GATA4, ADIPOR2, PTCH1, SOX6 and GJC1 gene, participate in the adjusting of heart development.Studies show that, the NODAL signal path is relevant with the heterauxesis of heart; The disappearance of GATA4 gene can cause heart bifurcated and embryonic death, then can cause myocardium dysplasia and endocardial cushion defect at the early stage inactivation of heart development, and late period, inactivation can cause the decline of heart function.Therefore, the high expression level of miR-nov21 may be relevant with the formation of DS heart of fetus birth defects.

In sum, the miR-nov21 gene that is positioned at No. 21 karyomit(e)s " DS critical area " is relevant with development with the generation of DS disease.Thereby, this new miRNA(miR-nov21), its gene or its precursor-gene can be widely used in the preparation diagnosis or detect in the preparation process of DS gene detecting chip or related reagent, the gene detecting chip for preparing or reagent can be used for the detection of DS fetus, do not need to carry out chromosome karyotype analysis, convenient and easy.

The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Claims (10)

1. a miRNA gene relevant with No. 21 karyomit(e)s of mongolism has the nucleotide sequence such as SEQ ID NO.1.

2. the application of miRNA gene as claimed in claim 1 in preparation diagnosis or detection mongolism reagent.

3. the application of miRNA gene as claimed in claim 1 in preparation diagnosis or detection mongolism gene detecting chip.

4. the precursor of a miRNA relevant with No. 21 karyomit(e)s of mongolism has the nucleotide sequence such as SEQ IDNO.2.

5. the application of the precursor of miRNA as claimed in claim 1 in preparation diagnosis or detection mongolism reagent.

6. the application of the precursor of miRNA as claimed in claim 1 in preparation diagnosis or detection mongolism reagent.

7. a miRNA relevant with No. 21 karyomit(e)s of mongolism has the nucleotide sequence such as SEQ ID NO.3.

8. the application of miRNA as claimed in claim 1 in preparation diagnosis or detection mongolism reagent.

9. the application of miRNA as claimed in claim 1 in preparation diagnosis or detection mongolism gene detecting chip.

10. the screening method of the relevant miRNA of No. 21 karyomit(e)s of a mongolism is characterized in that, comprises the steps:

Gather respectively the human umbilical cord blood mononuclear cell sample of mongolism fetus and normal fetus;

Extract the total RNA in the described sample, be not more than the little RNA of 30 Nucleotide with sex change polypropylene amine gel electrophoresis Separation and Recovery length, under the effect of T4RNA ligase enzyme, hold the nucleotide sequence joint that adds such as SEQ ID NO.4 at 5 ' of the little RNA that reclaims, 3 ' end adds the nucleotide sequence joint such as SEQ ID NO.5, behind TBE polypropylene amine gel electrophoresis purifying, behind Solexa little RNA reverse transcription primer and pcr amplification, make the cDNA library of little RNA, re-use the gel electrophoresis of TBE polypropylene amine described cDNA library is carried out purifying and carries out sequencing analysis;

Small RNA molecular to the rear gained that checks order removes joint and the processing that depollutes, and therefrom filter out the nucleotide sequence that length is 18 ~ 30nt, with SOAP software institute's calling sequence and people's whole genome sequence are compared, with the sequence on the coupling according to GenBank Rfam miRBase17.0 repeat exon the priority orders of the intron note of classifying, then with intron and the small RNA molecular sequence of the upper sequence of comparison as the new data source of miRNA, with MIREAP and two kinds of softwares of MiPred the karyomit(e) at described new miRNA place is analyzed, obtained the relevant miRNA of No. 21 karyomit(e)s of described mongolism.

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