CN101054580A - Imitation miRNA sequence and preparation method thereof - Google Patents
Imitation miRNA sequence and preparation method thereof Download PDFInfo
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- CN101054580A CN101054580A CNA2007100720036A CN200710072003A CN101054580A CN 101054580 A CN101054580 A CN 101054580A CN A2007100720036 A CNA2007100720036 A CN A2007100720036A CN 200710072003 A CN200710072003 A CN 200710072003A CN 101054580 A CN101054580 A CN 101054580A
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Abstract
The present invention relates to quasi-miRNA sequence and its preparation method which solve the defect of miRNA lacking gene speciality. quasi-miRNA has 22nt,the 5' end of which has 1-8 nucleotide, 3' end of which has 7 nucletides, both of which are supplementary to target mRNA 3' UTR extended sequence. Thecis prepared according to the following steps: 1) determining target mRNA 3'UTR extended sequence, 2) designing quasi-miRNA sequence containing 22nt according to extension sequence speciality,in which 5' end of quasi-miRNA has 1-8 nucleotide, 3' end has 7 nucletides, both of which are supplementary to target mRNA 3' UTR extended sequence.The present invention provides quasi-miRNA sequence with gene speciality and novel method for researching miRNA function and can be widely used in gene therapy and treatment and biological field.
Description
Technical field
The present invention relates to a kind of RNA sequence and preparation method thereof.
Background technology
MiRNAs is the noncoding single stranded RNA molecule that a class of generation is about 21～25nt after being processed through enzyme by the single stranded RNA precursor of about 70～90 the base sizes with hairpin structure, extensively is present in plant and the cell from the nematode to the mankind.The molecule that Microrna (miRNA) is a kind of extensive existence, finely tune genetic expression, the miRNA gene accounts for 1% in human genome, the target gene majority of miRNA be participate in transcribing, signal transduction, tumorigenic gene, be that a class is evolved and gone up molecule conservative, play the important regulating and controlling effect in life.MiRNA has following advantage to the fine setting of target gene: 1. more save energy than the adjusting from protein level; 2. with respect to transcriptional regulatory, the effect of miRNA is faster and be reversible; 3. for some cell functions that only needs trace of albumin to regulate, just can reach the regulation and control purpose by miRNA; 4. the miRNA that encodes in the intron is a resource in a kind of cell that can efficiently utilize.
Although the effect of miRNA also is special, particularly 5 ' 2～8 bases of end, selectively targeted in its target gene, but the specificity of different with siRNA (small inference RNA) is miRNA is not strong, often a miRNA acts on a plurality of target genes or target gene of a plurality of miRNA regulation and control in vivo, therefore lacks gene specific.
Summary of the invention
The objective of the invention is in order to solve the defective that miRNA lacks gene specific, and a kind of plan miRNA sequence that provides and preparation method thereof.
Intending the miRNA sequence is 22nt, and sequence 5 ' end has 1～8 Nucleotide, 3 ' end that the extension sequence specificity complementation of the 3 ' UTR of 7 Nucleotide and target mRNA is arranged.Above-mentioned plan miRNA sequence prepares according to the following steps: (one) measures the extension sequence of the 3 ' UTR of target mRNA; (2) contain the plan miRNA sequence of 22 nt according to extension sequence specificity design, wherein intending miRNA sequence 5 ' end has 1～8 Nucleotide, 3 ' end that 7 Nucleotide and the complementation of extension sequence specificity are arranged, and promptly obtains intending the miRNA sequence.
The present invention measures the extension sequence of the 3 ' UTR of target mRNA by gene sequencing software such as MirScan, miRseeker, BLAST and mfold and online gene order comparison instrument, and finding the specificity segment of target mRNA, the miRNA sequence is intended in design according to the extension sequence specificity again.
Plan miRNA sequence among the present invention is compared with traditional exogenous small molecules interference RNA (siRNA) has following obvious difference:
Though 1. intending miRNA and siRNA all is exogenous rna, siRNA must match the effect that just can play gene silencing fully with the target gene sequence, and intends that miRNA only needs and 6 Nucleotide of target gene sequence pairing just can play the effect of gene silencing.It is easier than designing siRNA that miRNA is intended in design, because it is obviously much higher than seeking one section probability than long gene specific sequence to seek one section short gene specific sequence on target gene.
2. active position: intend consistent 3 ' the UTR district that mainly acts on target gene of miRNA, and siRNA can act on any position of mRNA with endogenous miRNA.
3. the mode of action: intend the translation that miRNA and miRNA mainly suppress target gene, also can cause target gene mRNA degraded under certain conditions, promptly intend miRNA and transcribe the back inhibition at target gene and work; And siRNA plays the effect of gene silencing by degraded target gene mRNA, does not directly influence the translation process of target gene.Therefore in case eliminated intend miRNA after complete target gene mRNA can recover translation process and synthetic proteins, and must treat that new elimination gene mRNA is finished and just can restart after transcribing eliminating after the siRNA protein translation process.
Synthetic is intended miRNA and had following difference with endogenous miRNA: the plan miRNA of synthetic is that the sequences Design according to goal gene forms, and gene action has specificity, and plan miRNA downstream target for modulation is single and special; And the effect of endogenous miRNA lacks selectivity, and a miRNA can act on a plurality of genes, produces multiple follow-up effect.Plan miRNA sequence provided by the invention for the function of studying miRNAs provides new research means, can be widely used in the gene diagnosis and the treatment of disease, and expanded application is in field of biology because of having gene specific.
The present invention intend miRNA have the transduction stablize, efficiently express, safe advantage.
Description of drawings
Fig. 1 is miR-HCN2 gene order figure; Fig. 2 is miR-HCN4 gene order figure; Fig. 3 is the mRNA level detection figure that transcribes HCN2 in the H9c2 cell, the solid black bar shaped post level of mRNA in the H9c2 cell of representing to transduce behind the miR-HCN2 among the figure, and white hollow bar shaped post is represented the level of mRNA in the blank group H9c2 cell among the figure; Fig. 4 is the mRNA level detection figure that transcribes HCN4 in the H9c2 cell, the solid black bar shaped post level of mRNA in the H9c2 cell of representing to transduce behind the miR-HCN4 among the figure, and white hollow bar shaped post is represented the level of mRNA in the blank group H9c2 cell among the figure.
Embodiment
Embodiment one: it is 22nt that present embodiment is intended the miRNA sequence, and sequence 5 ' end has 1～8 Nucleotide, 3 ' end that the extension sequence specificity complementation of the 3 ' UTR of 7 Nucleotide and target mRNA is arranged.
Present embodiment is intended the miRNA sequence and can be imported in the following manner:
1, directly will intend miRNA and import intravital method:
(a) dystopy imports: will intend the cell that miRNA imports non-pathology, and as subcutaneous, muscle etc.;
(b) original position imports: will intend the position that miRNA directly imports pathology, as tumour cell, medullary cell etc.;
2, intend the external importing (feedback) of miRNA treatment: the portion of tissue or the cell that are about to patient take out in vitro culture, are fed back in the body again after miRNA is intended in importing;
3, somatocyte is intended the miRNA importing: will intend miRNA and transfer to somatocyte (making it to bring into play endogenous miRNA function, to reach the purpose of treatment);
4, the plan miRNA of sexual cell imports: will intend in sexual cell (spermoblast or ovum) that miRNA transfers to the patient or the middle body early embryo (make it can develop into normal individual, generally adopt microinjection).
Present embodiment is intended the miRNA sequence and be can be used for multiple disease, as the assistance diagnosis and the treatment of tumour, hypertension, asthma, diabetes, obesity, heart trouble, senile dementia etc.
Through many group contrast experiments prove intend 5 ' end among the miRNA sequence 22nt have 1～8 Nucleotide, 3 ' end have 7 Nucleotide must with extension sequence specificity complementation among the 3 ' UTR of target mRNA, otherwise the plan miRNA that synthesizes does not have influence to the expression of target mRNA.
Embodiment two: present embodiment is intended the miRNA sequence and prepared according to the following steps: (one) measures the extension sequence of the 3 ' UTR of target mRNA; (2) contain the plan miRNA sequence of 22nt according to extension sequence specificity design, wherein intending miRNA sequence 5 ' end has 1～8 Nucleotide, 3 ' end that 7 Nucleotide and the complementation of extension sequence specificity are arranged, and promptly obtains intending the miRNA sequence.
On the basis of cardiovascular disordeies such as the known hypertension that causes because of the miRNA disorder, heart trouble, can adopt and intend miRNA diagnosis and treatment.Change as miRNA such as the intravital miR-29c of patient, miR-93, miR-150, miR-195 in the myocardial hypertrophy pathogenic process, the intravital miR-24 of patient, miR-125b, miR-195, miRNA such as miR-199a, miR-214 change in the heart failure process, and therefore the plan miRNA sequence that adopts the present embodiment method to prepare above-mentioned miRNA is treated these diseases at gene level.
The plan miRNA sequence of present embodiment preparation is used for the Clinics and Practices of tumour, for example miR-15 and miR-16 are positioned at karyomit(e) 13q14 section, discover that 65% chronic lymphatic leukemia (CLL) patient carries the cancer cells that miR-15 and miR-16 lose or suddenly change, and also often has losing of this section in chronic lymphatic leukemia, multiple myeloma and prostate cancer.These two kinds of miRNA regulate apoptosis process by suppressing the proteic expression of Bcl-2, Bcl-2 albumen mainly realizes that by acting on plastosome it suppresses the biologic activity of apoptosis, therefore lose or suddenly change as miR-15 and miR-16, the proteic expression amount of Bcl-2 rises, apoptosis of tumor cells is obstructed, so cause tumour to take place.Therefore adopt the present embodiment preparation to intend the effect that miRNA can reach the above-mentioned tumour of treatment.
The plan miRNA sequence of present embodiment preparation is used for the Clinics and Practices of nervous system disorders.As locating the pathogenic miRNA of Alzheimer ' s disease (degenerative brain disorder, senile dementia), Parkinson ' s disease nervous system disorderss such as (Parkinson's disease, Parkinsonisies), just can adopt plan miRNA to treat.
By synthetic and import corresponding plan miRNA and regulate out of control genetic transcription and translation, the disease that can Clinics and Practices be caused by inherited genetic factors is as diseases such as diabetes, obesity, asthma.
Therefore some miRNA is stem cell distinctive (as mouse stem cells specifically expressing miR-290～295, human stem cell specifically expressing miR-371～373), but synthetic is intended miRNA accordingly and carried out stem cell induction-differential therapy relative disease.The same miRNA that intends can be applied to other field of biology, very important in multicellular organism grows as the Notch signal pathway, Notch genes encoding helix-loop-helix suppresses son and feather protein in fruit bat, 3 ' UTR K-box of these genes of recent findings, GY-box, the Brd-box block is also regulated by miRNA, K-box is by miR-2, miR-11 regulates, GY-box is regulated by miR-7, Brd-box is by miR-4, miR-79 regulates, and then influence wing blood vessel spatial distribution, the thin and thick isophenous, but so plan miR-2 of synthetic, miR-11, miR-7, miR-4 and miR-79 etc. can regulate the organ dysfunction of other organism.
Embodiment three: the difference of present embodiment and embodiment two is: intending miRNA sequence 5 ' end in the step (two) has 2～7 Nucleotide, 3 ' end that 7 Nucleotide and the complementation of extension sequence specificity are arranged.Other step and selected parameter are identical with embodiment two.
Embodiment four: the difference of present embodiment and embodiment two is: intending miRNA sequence 5 ' end in the step (two) has 3～6 Nucleotide, 3 ' end that 7 Nucleotide and the complementation of extension sequence specificity are arranged.Other step and selected parameter are identical with embodiment two.
Embodiment five: the difference of present embodiment and embodiment two is: intending miRNA sequence 5 ' end in the step (two) has 4～5 Nucleotide, 3 ' end that 7 Nucleotide and the complementation of extension sequence specificity are arranged.Other step and selected parameter are identical with embodiment two.
Embodiment six: present embodiment confirms after testing that miR-1 and miR-133 can suppress the transcribing of HCN2, miR-1 can suppress HCN4 transcribes, miR-1 and miR-133 also can be used to reduce ventricular muscle cell pacemaker activity.At first measure the extension sequence of the 3 ' UTR of target mRNA by gene sequencing software, and find the specificity segment of target mRNA, according to the plan miRNA sequence miR-HCN2 (the miR-HCN2 gene order as shown in Figure 1) of extension sequence specificity design 22nt, the 5 ' end of intending miRNA sequence miR-HCN2 has 8 Nucleotide, 3 ' end that the extension sequence specificity complementation of the 3 ' UTR of 7 Nucleotide and target mRNA is arranged again.And then make up miRNA target spot-luciferase indicator carrier (PGL3-target DNA): with the 3 ' UTR of target mRNA be template by round pcr synthetic can with miRNA bonded oligonucleotide sequence, and with reference to Promega company luciferase detection system product description (former English technical manual number: construction of carrier is inserted into synthetic target oligonucleotide sequence on the multiple clone site of downstream luciferase genes (adopting HindIII and SpeI double digestion) TB281).10nM being intended miRNA sequence miR-HCN2 transduces into American Type Culture Collection (ATCC) (ATCC again, Manassas, VA) H9c2 cell (rat ventricular myocytes strain), again 1 μ g PGL3-target DNA and 0.1 μ g PRL-TK (the Renilla luciferase expression carrier that thymidine kinase drives) are gone into the H9c2 cell with lipofectamine 2000 (Invitrogen) cotransfection, in 48 hours of transfection, measure the activity (two luciferase reporter gene system normalizing react) of test kit (Promega) (Lumat LB9507) measurement luciferase on luminometer with dual luciferase indicator.
Cell all is to carry out transfection after hungry 24 hours in the nutrient solution of serum-free in the present embodiment.Every group of data of present embodiment are represented by mean ± standard error, adopt non-matching student t-check to add up, and two tail p＜0.05 is thought obvious statistical significance, carries out curve fitting with nonlinear least square method in GraphPad Prism.
Its HCN2 expression level of the rat cell of handling with 10nM miR-HCN2 in the present embodiment changes, and endogenous HCN2 albumen reduces about 75%.Adopt the method identical to observe the protein expression that plan miRNA sequence miR-HCN4 (the miR-HCN4 gene order as shown in Figure 2) at HCN4 can suppress HCN4 equally, suppress 70% approximately with present embodiment.The mRNA level of HCN2 and HCN4 of transcribing in the H9c2 cell is after testing distinguished as shown in Figure 3 and Figure 4, has confirmed the real action rule that meets natural miRNAs of effect of simulation miRNA.
The function association of miR-HCN2/miR-HCN4: estimate the influence that these factor pair pacemaker currents (If), myocardial cell's contraction frequency produce for clear and definite miR-HCN2/miR-HCN4 can disturb the expression of HCN2 and HCN4, present embodiment.At first these factor transfections are entered the suckling mouse cell, with full cell patch pincers record If electric current, all detect under the current potential, with respect to negative control group, transfection its If electric current of cell of miR-HCN2/miR-HCN4 obviously reduce, miR-HCN2 (10nM) reduces If electric current about 66% and 41% respectively with miR-HCN4 (10nM) when-120mV.Neonatal rat myocardial cell is commissioned to train in culture dish Central Plains and is kept primary characteristic when supporting, and in the time of in changing the culture dish that is coated with ln over to, contraction synchronous frequency in basis is obviously stable, is 59 ± 7 times/minute.Use TFO-HCN4 before measuring shrinking percentage, not 48 hours (not adding liposome) of miR-HCN2/miR-HCN4 incubated cell, the monolayer cell contraction frequency is suppressed in various degree by miR-HCN2 and miR-HCN4, using miR-HCN2 and miR-HCN4 simultaneously can increase the inhibition degree, and experimental data conforms to the result of protein expression and fluorescence intensity.
Claims (4)
1, intend the miRNA sequence, it is characterized in that intending the miRNA sequence is 22nt, and sequence 5 ' end has 1～8 Nucleotide, 3 ' end that the extension sequence specificity complementation of the 3 ' UTR of 7 Nucleotide and target mRNA is arranged.
2, intend the preparation method of miRNA sequence according to claim 1, it is characterized in that intending the miRNA sequence prepares according to the following steps: (one) measures the extension sequence of the 3 ' UTR of target mRNA; (2) contain the plan miRNA sequence of 22nt according to extension sequence specificity design, wherein intending miRNA sequence 5 ' end has 1～8 Nucleotide, 3 ' end that 7 Nucleotide and the complementation of extension sequence specificity are arranged, and promptly obtains intending the miRNA sequence.
3, the preparation method of plan miRNA sequence according to claim 2, it is characterized in that intending in the step (two) miRNA sequence 5 ' end has 2～7 Nucleotide, 3 ' end that 7 Nucleotide and the complementation of extension sequence specificity are arranged.
4, the preparation method of plan miRNA sequence according to claim 2, it is characterized in that intending in the step (two) miRNA sequence 5 ' end has 3～6 Nucleotide, 3 ' end that 7 Nucleotide and the complementation of extension sequence specificity are arranged.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008074328A2 (en) * | 2006-12-21 | 2008-06-26 | Exiqon A/S | Microrna target site blocking oligos and uses thereof |
| EP2113567A1 (en) * | 2006-12-21 | 2009-11-04 | Exiqon A/S | MicroRNA target site blocking oligos and uses thereof |
| CN104940954A (en) * | 2015-06-23 | 2015-09-30 | 同济大学 | Application of MicroRNA-7 to preparation of anti-gliosis drug |
| US9464106B2 (en) | 2002-10-21 | 2016-10-11 | Exiqon A/S | Oligonucleotides useful for detecting and analyzing nucleic acids of interest |
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2007
- 2007-04-06 CN CNA2007100720036A patent/CN101054580A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9464106B2 (en) | 2002-10-21 | 2016-10-11 | Exiqon A/S | Oligonucleotides useful for detecting and analyzing nucleic acids of interest |
| WO2008074328A2 (en) * | 2006-12-21 | 2008-06-26 | Exiqon A/S | Microrna target site blocking oligos and uses thereof |
| WO2008074328A3 (en) * | 2006-12-21 | 2008-08-07 | Exiqon As | Microrna target site blocking oligos and uses thereof |
| EP2113567A1 (en) * | 2006-12-21 | 2009-11-04 | Exiqon A/S | MicroRNA target site blocking oligos and uses thereof |
| EP3536788A1 (en) * | 2006-12-21 | 2019-09-11 | QIAGEN GmbH | Microrna target site blocking oligos and uses thereof |
| CN104940954A (en) * | 2015-06-23 | 2015-09-30 | 同济大学 | Application of MicroRNA-7 to preparation of anti-gliosis drug |
| WO2016206172A1 (en) * | 2015-06-23 | 2016-12-29 | 同济大学 | Use of microrna-7 in preparation of drug for resisting gliosis |
| CN104940954B (en) * | 2015-06-23 | 2017-12-26 | 同济大学 | Applications of the MicroRNA 7 in anticol matter chemical drug thing is prepared |
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