CN102031255A - Antisense nucleic acid of human miR-223 and applications of antisense nucleic acid - Google Patents

Abstract

The invention discloses antisense nucleic acid of human miR-223 and applications of the antisense nucleic acid, in particular to antisense oligonucleotide for inhibiting expression of microRNA-223 and applications thereof. The antisense oligonucleotide disclosed by the invention comprises the following sequence: 5'-UGGGGUAUUUGACAAACUGACA-3'. The sequence above can specifically bind with human miR-223. The antisense oligonucleotide disclosed by the invention can be ribonucleotide, deoxyribonucleotide or chimaera of the ribonucleotide and the deoxyribonucleotide, and any nucleotide in the chain of the antisense oligonucleotide can be modified. The antisense oligonucleotide of the miR-223 can effectively inhibit the expression of the miR-223 in human brain glioma cells U87, and can also inhibit growth and proliferation of the cells above at the same time, so as to effectively treat brain gliomas and other tumours with highly expressed miR-223.

Description

People miR-223 antisense nucleic acid and application thereof

Technical field

The present invention relates to biomedicine field.Particularly, the present invention relates to the purposes of a kind of small molecules microRNA (miRNA), relate in particular to the application of the antisense oligonucleotide of this miRNA.This antisense oligonucleotide can with people miR-223 complementation, thereby suppress the expression of people miR-223.The invention still further relates to the pharmaceutical composition that contains this miRNA antisense oligonucleotide.

Background technology

MiRNAs is little non-coding RNA, and length is 20-22bp, is normally transcribed by rna plymerase ii (PolII), and general initial product is the big pri-miRNA with cap sequence (7MGpppG) and polyadenylic acid tail (AAAAA).These pri-miRNA are processed into the pre-miRNA precursor product that 70 Nucleotide are formed under the effect of RNase III Drosha and its cofactor Pasha.RAN-GTP and exportin 5 are transported to this precursor molecule in the tenuigenin.Subsequently, another RNase III Dicer shears it and produces the two strands that is about 22 length of nucleotides.This two strands is directed in (miRISC) complex body very soon, wherein contains Argonaute albumen, and sophisticated strand miRNA is retained in this mixture.Sophisticated miRNA is attached to the site of its complementary mRNA and expresses by two kinds of machine-processed negative regulator genes that depend on the sequence complementarity, suppresses its expression with the incomplete complementary miRNA of said target mrna on the protein translation level.Yet, evidence suggests also that recently these miRNA also might influence the stability of mRNA.Use the miRNA binding site of this mechanism to hold non-translational region at 3 ' of mRNA usually.If miRNA and target site complementary fully (perhaps almost completely complementary), the combination of these miRNA often causes the degraded of target molecule mRNA so.MiRNAs is quite conservative in spore, and animal, the miRNAs that finds in plant and the fungi etc. expresses all strict tissue specificity and sequential.

At present, have only the biological function of very little a part of miRNAs to be illustrated.These miRNAs regulate cell growth and tissue differentiation, and are relevant with biological growth and development.A series of studies show that: miRNAs is at cell growth and apoptosis, and hemocyte breaks up, the homeobox generegulation, and neuronic polarity, insulin secretion, the brain form forms, and heart takes place, and plays a significant role in the processes such as late embryogenesis growth.For example, miR-273 participates in the nervous system development process of nematode; MiR-430 participates in the brain development of zebra fish; MiR-181 control Mammals hematopoietic cell is divided into the B cell; MiR-375 regulates mammalian islet cell and grows and insulin secretion; MiR-143 works in the adipocyte differentiation; MiR-196 has participated in the Mammals four limbs and has formed, and miR-1 is relevant with heart development.Other has the researchist to find that many neural miRNAs are subjected to sequential and regulate in pallium is cultivated, and shows its mRNA that may control compartmentation translation.

MiRNA expresses relevant with multiple cancer, and these genes may play tumor suppressor gene or oncogene effect.In B cell chronic lymphatic leukemia (CLL), find to have the change of miRNA expression level at first, in various human tumors, all detect the miRNA changes of expression level subsequently successively.Discover that it is relevant that miRNAs and tumour form, and can bring into play the effect (as mir-15a and mir-16-1) of tumor suppressor gene, also can play the effect (as mir-155 and mir-17-92 bunch) of oncogene.Think at present, in tumour cell, ripe body of some miRNA or precursor expression horizontal abnormality, and the miRNA of abnormal expression plays a role by influencing the said target mrna translation, participates in neoplastic process, and plays an important role.Be subjected to the regulation and control of let-7 family as the Ras proto-oncogene, the BCL2 anti-apoptotic genes expression is subjected to miR-15a-miR-16-1 bunch of regulation and control, and the E2F1 transcription factor is subjected to miR-17-92 bunch of regulation and control, and the BCL6 anti-apoptotic genes expression is subjected to the regulation and control of miR-127 etc.The down-regulated expression of miRNAs also has substantial connection with tumour, and this is indicating that miRNA has the function of oncogene.For example, mir-143 and mir-145 obviously downward modulation in colorectal carcinoma.What is interesting is that the precursor molecule of its hairpin structure is similar with content in the healthy tissues in tumour, this shows, may be because its course of processing is damaged.But the tumor suppressor gene function of mir-143 and mir-145 may not only be confined to colorectal carcinoma, also obviously downward modulation of its expression amount in clones such as mammary cancer, prostate cancer, uterus carcinoma, lymphatic cancer.Another report shows that miR-21 expresses increase in glioblastoma multiforme.Expression amount is than the high 5-100 of healthy tissues doubly in tumor tissues for this gene.

MiRNAs is natural antisense acting factor, can regulate and control the several genes relevant with propagation with eukaryote existence.Aspect oncotherapy, the application prospect light of miRNA.Utilizing miRNA as aspect the treatment target spot, existing experimental data support:, the variation of miRNA express spectra occurs as in the process of gemcitabine (gemcitabine) treatment; The expression level (as making miR-21 cross expression) of regulation and control part miRNA can be promoted the susceptibility of cholangiocarcinoma cell to chemotherapeutics.MiRNAs by in the possible effectively deactivation tumour of introducing and the miRNA complementary synthetic antisense oligonucleotide---anti-miRNA oligonucleotide (AMOs)---with oncogene characteristic delays its growth.Clinically, can methylate or lock nucleic acid modified antisense oligonucleotide administrations such as (LNA) by 2 '-O-frequent or that continue and make the miRNA inactivation.These modifications make oligonucleotide more stable, and are lower than other treatment means toxicity.Use antagomirs (with cholesterol link coupled AMOs), can effectively suppress the miRNA activity in Different Organs behind the injection mouse, thereby may become a kind of medicine likely.Opposite, cross and express the miRNAs that those have the tumor suppressor gene effect, also can be used for the treatment of some specific tumour as let-7 family.

Antisense oligonucleotide (Flanagan WM.Antisense comes of age.Cancer﹠amp; MetastasisReviews 1998; 17 (2): 169-76) be meant one section can with the Nucleotide of the base complementrity of its target gene.Antisense oligonucleotide can be answered expression of gene by inhibitory phase.

Nearly 30 years, although the complex therapy of tumour is very general clinically, but based on operation, chemicotherapy is that the complex therapy of assisting is also not obvious to the survival rate raising of tumour patient, overall survival rate was still lower in 5 years, paced up and down about 30%～55%, did not significantly improve, 5 years survival rates of middle and advanced stage patient are lower, are about 20%.And all there are limitation separately in these methods, particularly to middle and advanced stage and recurrence patient unsatisfactory curative effect, to poorer with distant metastasis person's curative effect.Therefore, seeking safer and more effective treatment approach is the difficult problem that raising tumour patient survival rate and life quality are needed to be resolved hurrily.

Summary of the invention

The subject matter that the present invention will solve just provides the antisense nucleic acid (inhibitor) of a kind of new miR-223, be used for efficient, low toxicity or suppress the expression of miR-223 innocuously, and then treatment and miR-223 overexpression diseases associated, comprise various noumenal tumours, various leukemia etc.

Another problem that the present invention will solve just provides above-mentioned antisense nucleic acid suppresses miR-223 expression and cell growth inhibiting and propagation in cell application.

The inventor has designed and synthesized the antisense nucleic acid of a species specificity at miR-223 by extensive and deep research, and checking has the effect that suppresses miR-223 expression and tumor cell proliferation in culturing cell.Studies show that these antisense nucleic acides can suppress the growth and the malignant proliferation ability of tumour cell.

The present invention has designed the antisense nucleic acid molecule that can be incorporated into the miR-223 different positions in, in culturing cell U87, and the antisense nucleic acid cell growth ability that checking suppresses the miR-223 expression specificity, the influence of multiplication capacity.Antisense nucleic acid molecule length can comprise 13 ~ 22 nucleotide residues, inhibition growth of human tumor cells ability in various degree, the characteristic of multiplication capacity are all arranged, wherein the shortest antisense nucleic acid length is 13 bases, and the antisense nucleic acid of different lengths all has good growth of tumour cell and proliferation inhibition activity.Therefore, above-mentioned antisense nucleic acid all can be used to prepare the preparation that suppresses growth of tumour cell ability, multiplication capacity, the wherein tumour cell of preferred miR-223 high expression level.Finished the present invention on this basis.

A first aspect of the present invention provides the antisense oligonucleotide of a kind of miR-223, and described antisense oligonucleotide suppresses the expression of miR-223 in people's cell.Usually, continuous 13 ~ 22 nucleotide sequence complementations in the described antisense oligonucleotide and 5 ' UGUCAGUUUGUCAAAUACCCCA-3 '.In a preferred embodiment of the invention, the length of described antisense oligonucleotide is 18 ~ 22 Nucleotide.More preferably, the sequence of described antisense oligonucleotide is 5 '-UGGGGUAUUUGACAAACUGACA-3 '.

At present, the hybridization avidity of RNA and miRNA has very high pharmaceutical use than the avidity height of DNA and miRNA hybridization in the nucleic acid hybridization.But the DNA cost of the synthetic cost than synthetic RNA far away is low, also has good market potential.And can adopt the chimeric antisense nucleic acid that forms that links to each other of ribose RNA monomer and ribodesose dna single body to develop as medicine.A series of antisense nucleic acid molecules of the present invention's design had both comprised dna molecular, also comprised the RNA molecule, and two kinds of molecules all have the activity that suppresses the miR-223 expression.

The antisense nucleic acid of the present invention's design, it is active that its sequence has specific biological, its antisense nucleic acid institute complementary length for the site of a certain gene complementation has much relations, long as complementary, then biologic activity can be higher, suppressing effect also can be more better, increasing or reducing one to several bases and antisense nucleic acid complementary and same gene locus, equally also has biologic activity in various degree, also can reach in various degree the inhibition growth of tumour cell and the effect of propagation, of the present invention studies show that the shortlyest reaches 13 bases and still has the effect that miR-223 expresses that suppresses.In the antisense nucleic acid research, various chemical modification methods are a lot.The present invention adopts sulfo-, methoxy modified antisense nucleic acid, mainly be that preclinical studies such as these the two kinds pharmacology of modifying the antisense nucleic acid of modes, pharmacokinetics, toxicology are that research is the most comprehensive in the various chemically modified antisense nucleic acides, modified antisense nucleic acid can prevent effectively in vivo in the human body that a large amount of exonucleases cut the enzyme of antisense nucleic acid and degrade, thus make antisense nucleic acid lose should by biologic activity.The sulfo-antisense nucleic acid also can excite the activity of RNA enzyme in addition, the RNA chain of degraded and its hybridization, and therefore preferred these the two kinds antisense nucleic acides of modifying modes are used in experiment.Will be clear that any modifying method that can increase antisense nucleic acid stability and bioavailability can use, as cholesterol modify, PEG modification etc.

Above-mentioned antisense nucleic acid of the present invention has the effect that suppresses the miR-223 expression.When with after above-mentioned antisense nucleic acid transfection is in the cell strain U87 that expresses miR-223, can effectively suppress the growth and the malignant proliferation ability of tumour cell.

The present invention also provides a kind of pharmaceutical composition, and it contains oligonucleotide of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier includes but not limited to: salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.

In a third aspect of the present invention, the purposes of antisense oligonucleotide of the present invention is provided, be used to prepare the medicine for the treatment of following disease:

(A) treatment human tumor growth;

(B) treatment human body and miR-223 cross the expression diseases associated.

As used herein, " antisense oligonucleotide " refers to the nucleotide oligomer of antisense.Antisense oligonucleotide by base complementrity (G-C) pairing forms three chains (anti-gene) with double-stranded DNA for A-T, A-U, or forms heteroduplex (antisense) with single stranded RNA, thus the processing and the translation of duplicating, transcribing or transcribing back mRNA of blocking gene.Simultaneously, double-stranded RNA can be degraded by intracellular ribonuclease H (RNase H), thereby more effectively blocks target gene expression.Because antisense nucleotide can only combine with the target sequence of reverse complemental, has the specificity height, the characteristics that side effect is little.

The length of antisense oligonucleotide of the present invention is not particularly limited, and in general, in order to reach the specificity of hybridization, antisense oligonucleotide need be known the Nucleotide that 13 monomers are formed.Usually the length of antisense oligonucleotide is 13 ~ 35bp, and for miRNA, that preferable is 18 ~ 22bp.

Description of drawings

Fig. 1 has shown that the miR-223 antisense oligonucleotide suppresses the expression of miR-223 in the tumour cell U87 cell, A is the expression of miR-223 behind the transfection miR-223 antisense oligonucleotide, and B is the expression of miR-223 behind the transfection negative control antisense oligonucleotide; C is the expression of internal control gene U6 behind the transfection miR-223 antisense oligonucleotide, and D is the expression of internal control gene U6 behind the transfection negative control antisense oligonucleotide; E is that miR-223 suppresses the effect histogram behind the transfection antisense oligonucleotide, and wherein sample3 represents the miR-223 expression behind the transfection miR-223 antisense oligonucleotide, and NC represents miR-223 expression behind the transfection negative control.

Embodiment

Antisense oligonucleotide of the present invention, continuous 13 ~ 22 nucleotide sequence complementations in its sequence and 5 ' UGUCAGUUUGUCAAAUACCCCA-3 ', and should be not and the RNA sequence complementation of other genes.In a preferred embodiment of the invention, the sequence of described antisense oligonucleotide is 5 '-UGGGGUAUUUGACAAACUGACA-3 '.Antisense oligonucleotide provided by the invention is a modified outcome, it contains at least two, and at least 4 usually, preferable at least 6, at least 8 better Nucleotide do not have the Nucleotide of the modification of toxic side effects, and described modification mode comprises 2 ' methoxyl group replacement, thio-modification etc.In order to increase the cellular uptake rate of antisense oligonucleotide, can also on the basis of above-mentioned modification, carry out cholesterol and modify or the PEGization modification antisense oligonucleotide.Oligonucleotide after the above-mentioned modification can continue effectively to match with target sequence, and has the longer transformation period in vivo than common not modified Yeast Nucleic Acid or thymus nucleic acid.

The present invention has following advantage:

1, antisense oligonucleotide acts on specific target site, the site of non-specific binding seldom, the specificity height;

2, antisense oligonucleotide provided by the invention is through suitable chemically modified, has that toxicity is low, side effect is little and characteristics such as long half time;

3, antisense oligonucleotide provided by the invention has the good restraining effect, and the inhibiting rate of the expression of miR-223 is reached more than 70%, and the inhibiting rate of growth of tumour cell is surpassed 50%.

Below in conjunction with embodiment and accompanying drawing the present invention is described in further detail.Yet should be appreciated that and enumerate these embodiment, and be not to be used for limiting the scope of the invention just for an illustration.

Embodiment 1, miR-223 antisense nucleic acid suppress the expression of miR223

Real-time quantitative fluorescence detects test and is finished by Shanghai JiMa pharmacy Technology Co., Ltd, and concrete experimental procedure comprises:

Cell cultures: the U87 cell, the 10%FBS-DMEM culture medium culturing, 37 ℃, 5%CO2 cultivates.

Cell transfecting:

1) transfection the day before yesterday, with not containing antibiotic culture medium inoculated culturing cell in right amount, the degree of converging of cell reaches 30～50% when making transfection in 24 orifice plates;

2) the transfection sample is prepared oligomer-Lipofecta mine as follows ^TM2000 mixtures:

A. the Opti-that does not contain serum with 50 μ l

The I substratum dilutes the negative control of miR-223inhibitor, negative control, FAM mark respectively, and final concentration is 50nM, mixing gently, and 3 multiple holes are established in each transfection;

B. mixing Lipofecta mine gently before using ^TM2000, get the Opti-that 2 μ l are diluted to 50 μ l then

In the I substratum, at room temperature hatch 5min gently behind the mixing;

C. after hatching 5min, the Lipofecta mine of dilution ^TM2000 mix with the antisense nucleotide and the contrast of dilution respectively, at room temperature hatch 20min gently behind the mixing, to allow the formation of mixture;

3) mixture is joined in each hole that comprises cell and substratum, the culture plate that rocks back and forth lightly mixes;

4) 37 ℃, 5%CO ₂The incubator overnight incubation is changed the substratum that contains 10% foetal calf serum and is continued to cultivate 24h.Total RNA extracts:

1) centrifugal collecting cell adds 500 μ l Ezol in the centrifuge tube, and with the centrifuge tube mixing that turns upside down, room temperature is placed 10min.

2) trichloromethane of adding 200 μ l RNA special uses, the mixing that acutely turns upside down, the thorough mixing of the liquid in centrifuge tube becomes the oyster white shape.

3) room temperature is placed 5min, the centrifugal 15min of 12000rpm/min.

4) carefully supernatant is transferred in another clean 1.5ml centrifuge tube, avoids inhaling middle level albumen phase and lower floor's organic phase.

5) add the Virahol of the RNA special use of 500 μ l precoolings in the supernatant, room temperature is placed 5min.The centrifugal 10min of 10000rpm/min.

6) carefully abandon most supernatant, add 75% washing with alcohol precipitation of 1ml RNA special use, the centrifugal 10min of 10000rpm/min.

7) carefully abandon most supernatant, place room temperature to dry ethanol, every pipe adds 20 μ l DEPC water dissolution, mixing.

The RNA reverse transcription:

The RNA that above-mentioned extracting is obtained carries out reverse transcription with U6 and two kinds of special reverse transcriptase primers of RNA of hsa-mir-223 respectively, preparation cDNA template.Damping fluid and enzyme used in the reverse transcription are Promega company product.

(i). the reverse transcription system:

Reagent name	Consumption/pipe
		5×Reverse?Transcription?buffer	4μl
RT?Primer?Mix(U6，hsa-mir-223)(1 μM)	1.25μl
		dNTP(10mM)	0.75μl
RNA	2μl
		RTase(200/μl)	0.5μl
DEPC?H 2O	To 20 μ l

(ii). the reverse transcription reaction condition:

Reaction conditions is: 16 ℃ of 30min; 42 ℃ of 30min; 85 ℃ of 10min.

Fluorescence quantitative PCR detection:

(1) dilution of .cDNA template:

The cDNA that obtains behind the above-mentioned reverse transcription is diluted 3 times, in the system of 20 μ l, add

40 μ l RNase/DNase free ddH ₂O, mixing.

(2). the quantitative fluorescent PCR system:

Reagent name	Consumption/pipe
		2×PCR?Master?Mix	10μl
F?Primer(20μM)	0.2μl
		R?Primer(20μM)	0.2μl
Template	2μl
		rTaq?DNA?polymerase(5U/ul)	0.2μl
ddH 2O	Add to 20 μ l

(3). reaction conditions:

(i)95℃3min；

(ii)95℃15s；

(iii)55℃30s；

(iv)72℃30s；

Ii to the iv goes on foot totally 40 circulations.

Embodiment 2, MiRNA antisense oligonucleotide suppress active to human neuroglia glucagonoma clone U87 and detect

Cell cultures:

The U87 cell, the 10%FBS-DMEM culture medium culturing, 37 ℃, 5%CO2 cultivates.Collect the good U87 cell of growth conditions, centrifugal counting is with 2 * 10 ³Every hole is laid in 96 orifice plates, and 37 ℃, 5%CO2 cultivates 24h.

Transfection:

1) transfection the day before yesterday, with not containing antibiotic culture medium inoculated culturing cell in right amount, the degree of converging of cell reaches 30～50% when making transfection in 96 orifice plates;

2) the transfection sample is prepared oligomer-Lipofecta mine as follows ^TM2000 mixtures:

A. the Opti-that does not contain serum with 25 μ l

The I substratum dilutes the negative control of miR-223 antisense oligonucleotide, negative control, FAM mark respectively, and final concentration is 50nM, mixing gently, and 3 multiple holes are established in each transfection;

B. mixing Lipofecta mine gently before using ^TM2000, get the Opti-that 1 μ l is diluted to 25 μ l then

The I substratum is at room temperature hatched 5min gently behind the mixing;

C. after hatching 5min, the Lipofecta mine of dilution ^TM2000 mix with the antisense nucleotide and the contrast of dilution respectively, at room temperature hatch 20min gently behind the mixing, to allow the formation of mixture;

3) mixture is joined in each hole that comprises cell and substratum, the culture plate that rocks back and forth lightly mixes;

4) 37 ℃, 5%CO ₂The incubator overnight incubation is changed the substratum that contains 10% foetal calf serum and is continued to cultivate 72h.

The MTT experiment:

Upwards add MTT (Sigma) 5mg/ml (physiological saline preparation) for preparing in the cell that obtains in the step with 0.9%, every hole adds 20 μ l hatches after 4 hours to inhale and removes substratum and MTT, and every hole adds DMSO100 μ l and reads the absorbance of OD570-OD630 by microplate reader.

The sample negative control

0.35 0.76

0.33 0.70

0.36 0.71

Calculate inhibiting rate:

$\ln \mathrm{hibition\; rate}(\%)=\frac{\mathrm{NC}(\mathrm{OD}570-\mathrm{OD}630)-\mathrm{Micro\; RNA\; inhibitor}(\mathrm{OD}570-\mathrm{OD}630)}{\mathrm{NC}(\mathrm{OD}570-\mathrm{OD}630)}\×100\%$

Calculating inhibiting rate is 52.18 ± 2.9%.

Claims (9)

1. an antisense oligonucleotide is characterized in that described antisense oligonucleotide comprises sequence 5 ' UGGGGUAUUUGACAAACUGACA-3 '.

2. antisense oligonucleotide as claimed in claim 1 is characterized in that Nucleotide wherein is the mosaic of ribonucleotide, deoxyribonucleotide or Nucleotide and deoxyribonucleotide.

3. the modification type of arbitrary antisense oligonucleotide in the claim 1 ~ 2.

4. the oligonucleotide described in claim 3 is characterized in that described chemically modified is selected from the one or more combination in ribose modification, base modification or the phosphoric acid backbone modification.

5. the oligonucleotide described in claim 4 is characterized in that described chemically modified is selected from thio-modification, 2 ' one or more in methoxyl group modification, the cholesterol modification.

6. oligonucleotide as claimed in claim 5, ' methoxyl group is modified, and 5 ' hold two Nucleotide to carry out thio-modification to it is characterized in that carrying out 2 by all nucleic acid, and 3 ' holds four Nucleotide to carry out thio-modification and hold the connection cholesterol 5 ' or 3 '.

7. as each described oligonucleotide of claim 1～6, preparing the application for the treatment of in the mir-223 overexpression relative disease medicine.

8. oligonucleotide as claimed in claim 7 is characterized in that described mir-223 overexpression relative disease is a cerebral glioma.

9. a pharmaceutical composition is characterized in that containing each the described oligonucleotide of claim 1～6 and the pharmaceutically acceptable carrier for the treatment of significant quantity.

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