CN104450709A - Oligomerization nucleic acid inhibiting HMMR (hyaluronan-mediated motility receptor) gene and application of oligomerization nucleic acid - Google Patents
Oligomerization nucleic acid inhibiting HMMR (hyaluronan-mediated motility receptor) gene and application of oligomerization nucleic acid Download PDFInfo
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Abstract
The invention discloses oligomerization nucleic acid inhibiting an HMMR (hyaluronan-mediated motility receptor) gene. The oligomerization nucleic acid is double-strand RNA (ribonucleic acid), a basic group of the oligomerization nucleic acid comprises an antisense strand and a positive-sense strand, the antisense strand comprises SEQ. ID NO.9 components, more than 70% of the components are homologous, the positive-sense strand comprises oligomerization nucleic acid reversely and complementarily paired with the antisense strand, more than 50% of sequences are homologous, and the positive-sense strand and the antisense strand can form a double-strand structure after annealing. The invention further discloses DNA (deoxyribonucleic acid) containing the oligomerization nucleic acid, a carrier, a composition and an application of the oligomerization nucleic acid. The oligomerization nucleic acid has a remarkable inhibiting function for an HMMR, can achieve an inflammation inhibition function in an animal experiment, remarkably reduces various inflammatory factors and is a quite promising inflammation therapy medicine.
Description
Technical field
The invention belongs to biological technical field, specifically relate to nucleic acid oligomer and application thereof that a class suppresses HMMR gene.
Background technology
Osteoarthritis (OA) is one of major reason disabled in the world, approximately has the symptom of osteoarthritis more than the elderly of 10%.Rheumatic arthritis (RA) is a chronic inflammatory diseases, affects the grownup of in the world about 0.5% to 1%, usually causes joint damage and jeopardize quality of life.Multiple inflammatory cytokine such as TNF-α .IL-1.IL-6 etc. take part in morbidity and the progress of RA and OA, and it promotes destruction of joint, synovitis, blood vessel hyperplasia etc. by immune cell activated and inducing metal protease-producing.Multiple inflammatory cytokine such as TNF-α .IL-1.IL-6 etc. take part in morbidity and the progress of RA and OA, and it promotes destruction of joint, synovitis, blood vessel hyperplasia etc. by immune cell activated and inducing metal protease-producing.
HMMR/RHAMM, also known as the cell migration acceptor (receptor forhyaluronan-mediated motility) of hyaluronic acid mediated, is distributed in surface of cell membrane, cytoplasm and nucleus.Its principal biological behavior comprises: 1. by with the interaction such as microtubule, hyaluronic acid, calmodulin, participate in the adhesion of cell and the transduction of motion and cell signal; 2. the cell cycle is regulated; 3. 4. stable center body and spindle body promote the formation of new vessel.
In in the past more than 20 year, the R&D work of most of novel method for the treatment of is the medicine (DMOADs) about the arthritis medicine (DMARDs) of remission and the resisting rheumatoid arthritis of remission.Up to the present, pharmacy industry is upper and unsuccessful at the disease retentivity medicine (DMOADs) of development effective and safe, the misery causing thousands of patient to be still subject to this serious disabling condition to bring.Along with the application of new way and medication, in this treatment field, expection will have more development, and this is capable of blocking, reverse disease process and relaxes the medicine of disease, as siRNA provides huge development space.
RNA intervention is the discovery of tool revolution meaning in life science, and become the important tool of research gene function, it can cause organism, mammalian cell, the gene inactivation even in animal.RNA intervention techniques can be applicable to treat the disease about deleterious gene overexpression, has the potentiality of creation of mankind disease treatment novel method.
In the present invention, we provide a kind of brand-new siRNA as the medicine of sacroiliitis and related inflammation; Pass through a method for osteoarthrosis chamber local injection siRNA and preparation inflammation-inhibiting factor expression thereof, achieve the therapeutic action to arthritis.
Summary of the invention
The object of the invention aims to provide the nucleic acid oligomer that a class suppresses HMMR genetic expression.
The technical scheme realizing above-mentioned purpose is as follows.
One class suppresses the nucleic acid oligomer of HMMR gene, and described nucleic acid oligomer is double-stranded RNA, and the based composition of described nucleic acid oligomer is:
The based composition including described nucleic acid oligomer for: include (1) antisense strand containing more than 70% homology shown in SEQ.ID NO.9; (2) positive-sense strand of nucleic acid oligomer more than 50% sequence homology matched with described antisense strand reverse complemental is contained; And (3) described positive-sense strand and described antisense strand annealed after can form duplex structure.
Wherein in an embodiment, described nucleic acid oligomer includes (1) by the antisense strand shown in SEQ.ID NO.9 base sequence; (2) containing the positive-sense strand that at least 50% sequence homology shown in SEQ.ID NO.8 is formed; And (3) described positive-sense strand and antisense strand annealed after can form duplex structure.
Wherein in an embodiment, described nucleic acid oligomer includes the antisense strand that (1) is formed containing at least 70% sequence homology shown in SEQ.ID NO.9; (2) by the positive-sense strand shown in SEQ.ID NO.8 base sequence; And (3) described positive-sense strand and antisense strand annealed after can form duplex structure.
Wherein in an embodiment, described nucleic acid oligomer includes the antisense strand that (1) is formed containing at least 70% sequence homology shown in SEQ.ID NO.9; (2) containing the positive-sense strand that at least 70% sequence homology shown in SEQ.ID NO.8 is formed; And (3) described positive-sense strand and antisense strand annealed after can form duplex structure.
Wherein in an embodiment, described nucleic acid oligomer is made up of the antisense strand of based composition as shown in SEQ.ID NO.9 and the positive-sense strand of based composition as shown in SEQ.ID NO.8.
Wherein in an embodiment, described nucleic acid oligomer by based composition as shown in siRNA-HM-36, siRNA-HM-26, siRNA-HM-40, siRNA-HM-39, siRNA-HM-41, siRNA-HM-42.
The invention provides nucleic acid oligomer is double-stranded RNA, feature in wherein said double-stranded RNA is: antisense strand contains nucleotides sequence and is classified as " 5 '-AGUUUCAUCAACUCCAAGC-3' " as sequence in sequence table 9, or has the homologous sequence of more than at least 70% homology with it; Positive-sense strand contains the nucleotide sequence with antisense strand reverse complemental chain with more than 50% homology.Above-mentioned nucleic acid oligomer can be passed through any one or more modification in (1)-(4):
1) phosphodiester bond of the connection Nucleotide of described nucleic acid oligomer is modified, preferably the oxygen sulphur of described phosphodiester bond is replaced;
2) ribose contained by described nucleic acid oligomer is modified, preferably ribose 2 '-OH methoxyl group or fluorine replaced or carry out deoxidation modification to described 2 '-OH, or modifying with open loop nucleic acid (UNA);
3) to the modification of the nucleotide base of described nucleic acid oligomer, 5 the methyl modifications of preferred cytosine(Cyt), 5-ethinyluracil, indoles are modified;
4) end modified to described nucleic acid oligomer, preferred end connects cholesterol, polypeptide, fluorescence, sugar, antibody molecule, phosphorylation modification;
5) nucleic acid oligomer of the present invention can add suspension base at 3 ' end, and preferably hanging base is dTdT.
Another object of the present invention is also that providing a kind of has the DNA suppressing HMMR genetic expression function.
Concrete technical scheme is as follows.
Any one nucleic acid oligomer above-mentioned can be expressed and there is the DNA suppressing HMMR genetic expression function.
Another object of the present invention is also that providing a kind of has the carrier suppressing HMMR genetic expression function.
Concrete technical scheme is as follows.
Carrier containing any one nucleic acid oligomer above-mentioned or above-mentioned DNA, described carrier is liposome, polymer materials, polypeptide, nano material.Wherein liposome vectors material is the cationic-liposome that this area is commonly used, preferred lipo2000; The acid of polymer carrier materials preferably transparent matter or polylysine; The preferred RGD of polypeptide material; Nano material is preferably chitosan nano.
Another object of the present invention is also that providing a kind of has the composition suppressing HMMR genetic expression function.Concrete technical scheme is as follows.
There is the composition suppressing HMMR genetic expression function, containing any one nucleic acid oligomer above-mentioned or above-mentioned DNA, and pharmaceutically acceptable carrier.
Another object of the present invention is also the application providing above-mentioned nucleic acid oligomer, DNA, carrier or composition.
Concrete technical scheme is as follows.
Above-mentioned nucleic acid oligomer, DNA, carrier or composition are preparing the application in T suppression cell in HMMR genetic expression goods.
The application in the reagent of the disease HMMR of preparation diagnosis HMMR gene unconventionality initiation of above-mentioned nucleic acid oligomer, DNA, carrier or composition.
Application in the medicine of the disease that above-mentioned nucleic acid oligomer, DNA, carrier or composition preparation treatment HMMR gene unconventionality causes.
Wherein in an embodiment, the disease that described HMMR gene unconventionality causes can be selected from inflammation, cancer.
Wherein in an embodiment, the disease that described HMMR gene unconventionality causes is sacroiliitis, rheumatic arthritis or synovitis.
The different positions that the present invention is directed in HMMR gene C Ds region is optimized design, have selected 9 and carries out shaker test to for people and the homogenic siRNA of mouse.Result of study shows: compared with negative control siRNA, antisense strand contains nucleotides sequence and is classified as the siRNA-HM-03 of 5 '-AGUUUCAUCAACUCCAAGC-3 ' to HMMR gene mRNA expression inhibition the most by force, reaches 86% (embodiment two) to the mRNA silence efficiency of HMMR gene.In immunoblotting (western blotting) experiment, with blank to compare with negative control group add siRNA-HM-03 after, HMMR protein expression level obviously declines, and difference has significance (P<0.05) (embodiment three).SiRNA-HM-03 is added in the post-stimulatory cell of IL 1-α, with add compared with negative control nucleic acid oligomer, cellular inflammation factor TNF, COX-2, IL-1 β content obviously decline, and show that siRNA-HM-03 has the effect that diminishes inflammation (embodiment four).In embodiment five, the present invention is optimized sequence, and experimental result shows: antisense strand containing " 5 '-AGUUUCAUCAACUCCAAGC-3 ' the few nucleic acid of double-strand of homologous sequence to HMMR gene mRNA, there is inhibition; Positive-sense strand and the reverse complete complementary of antisense strand or part only partial complementarity also have inhibition to HMMR gene mRNA, and containing also there is inhibition with the double-stranded RNA of antisense strand 70% homologous sequence.Embodiment six is applied plasmid vector and is expressed " 5 '-AGUUUCAUCAACUCCAAGC-3 ' " sequence, also has the mRNA inhibition of good HMMR gene in cell.Embodiment seven have studied the impact of chemically modified on the reticent HMMR potency of gene of nucleic acid, and result shows: the nucleic acid oligomer after suitable modification has good restraining effect to HMMR gene.The research of embodiment eight shows, after chemically modified, the stability of siRNA of the present invention in serum significantly improves.
Embodiment nine is the live body rat test of siRNA, in the joint cavity of osteoarthritis rat separately or hybrid injection containing antisense strand " 5 '-AGUUUCAUCAACUCCAAGC-3 ' or the chemical modification object of 5AGUUUCAUCAAUUCCAGGC3; synovial fluid Inflammatory Factors Contents detection experiment shows, nucleic acid oligomer has obvious inhibition of inflammation in rat body.
Of the present invention have beneficial effect to be: 1, the present invention uses gene silent technology, and found that a class has remarkable inhibiting nucleic acid oligomer to HMMR through a large amount of designs, screening, Analysis &Validation, in a class hFLS cell, suppression efficiency reaches
88%, and can play the effect of gene silencing with the nucleotide sequence that this sequence has more than 70% homology, shows that the present invention is a class to the extremely strong nucleotide sequence of HMMR silencing efficiency; 2, the invention still further relates to a class nucleic acid chemistry modifier, its high stability had, high reactivity, high specific and low cytotoxicity make it to become the active drug that live body is used; 3, the nucleic acid that the present invention relates to can realize inflammation-inhibiting effect in experimentation on animals.83% is reached in the expression inhibiting rate of cell levels to inflammatory factor IL-1 β, in rat bone arthritis model, the expression of the few nucleic acid of continuous injection the present invention to inflammatory factor and inflammation gene expression all has obvious restraining effect, and above experiment all shows that HMMR-siRNA has prevention or therapeutic action to rat arthritis.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, but the present invention is not limited to following examples.In following embodiment, if no special instructions, conventional known method is.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.HFLS cell is purchased from Cell Applications, 293-T cell is purchased from ATCC, Human IL-1 β immunoassay detection kit is purchased from Assay Pro, Lipofectamine2000 test kit is purchased from Invitrogen, pancreatin, Trizol, PBS are sigma product, male SD rat is Guangdong medical experiment animal center product, and the nucleotide sequence such as primer, siRNA is Guangzhou Ribo Bio Co., Ltd. and provides.
" TNF " described in the present invention is " TNF--α ".
Inhibiting rate=NC (negative contral) contrasts expression amount-destination gene expression amount/NC (negativecontral) and contrasts expression amount.
" pharmaceutically acceptable carrier " of the present invention refers to transfection reagent in body conventional in this area, as polymine (PEI), and jetPEI (L-PEI), hydroxy fatty acid (PHA), cationic polymers, amino acid, liposome etc.
The chemosynthesis of embodiment one nucleic acid oligomer
(1) RNA applied in embodiment and the few nucleic acid monomer of modification obtain all in accordance with the following methods:
RNA Nucleotide in few nucleic acid is prepared with 2 '-O-TBDMS phosphoramidite monomer; DNA Nucleotide is prepared with deoxynucleoside phosphoramidite monomer; Nucleotide glycosyl modified nucleoside is with 2 '-OCH
3, 2 '-F, lock nucleic acid (LNA), open loop nucleic acid (UNA), 5-methylcytosine, indoles, 5-ethinyluracil phosphoramidite monomer preparation; Skeleton phosphothio nucleic acid replaces the preparation of iodine water with Beaucage reagent or PADS reagent; Cholesterol, fluorescent mark, sugar-modified, phosphorylation nucleic acid oligomer are prepared by corresponding monomer; Peptide modified few nucleic acid is obtained through Michael reaction by the few nucleic acid of sulfydryl modification and polypeptide, and the above monomer is bought from SigmaAldrich, Chem Genes, Glen research company.
(2) nucleic acid oligomer, glycosyl, base, skeleton thio-modification, cholesterol, fluorescent mark modify few nucleic acid preparation method:
Theoretical yield 1umol synthesizes specification to be completed, and take general solid support CPG 20mg (carrying capacity 50umol/g), the monomer of all kinds of phosphoramidite is dissolved in (0.15M) in anhydrous acetonitrile.5-ethylmercapto group-1H-TETRAZOLE acetonitrile solution as activator (0.25M), oxygenant iodine water (0.02M), 3% trichoroacetic acid(TCA) dichloromethane solution.According to standard rna synthesis program setting program circulation synthesis, often walk Coupling time 2-10 minute, after 20 circulations, complete oligonucleotide solid phase synthesis.Dry up CPG, transfer in 5ml EP pipe, add ammoniacal liquor/ethanolic soln (3/1) 1-20ml, 55 DEG C are heated 5 ~ 20 hours.Under the rotating speed of 10000rpm, centrifugal 10min gets supernatant liquor, obtains white gummy solid after draining strong aqua/ethanol.Solid is dissolved in 200 μ l 1M TBAF THF solution, and room temperature shakes 20 hours.Add 0.5ml 1M Tris-HCl damping fluid (pH 7.4), room temperature shakes 15 minutes, is placed in centrifugally to drain machine to be evacuated to volume be original volume 1/2, removing THF.Solution 0.5ml chloroform extraction 2 times, adds 1ml 0.1M TEAA sample solution, pours mixing solutions into solid-phase extraction column, according to standard rna desalination flow process, excess salt in removing solution, gained nucleic acid concentration is measured by micro-ultraviolet spectrophotometer, confirms nucleic acid construct by mass spectrum.
3) polypeptide, antibody connect few nucleic acid preparation method:
Above method is dissolved in 950 μ l 100mM HEPES-KOH damping fluid (pH1-14) after preparing 100nmol HS modification nucleic acid oligomer.The polypeptide modified containing ethylene linkage with 500nmol or antibody are dissolved in the aqueous solution of 50 μ l.0-100 DEG C under nitrogen protection, reacts, and reaction efficiency is monitored by HPLC, after Quantitative yield, will be used for experiment after solution ultrafiltration and concentration.
Annealing: the few nucleic acid balanced mix of above preparation is in 1ml water or damping fluid, and 95 DEG C of heating 2-20 minute, room temperature leaves standstill that to be cooled to room temperature for subsequent use.
Embodiment two suppresses effective nucleic acid oligomer screening of HMMR gene mRNA expression
Carry out siRNA design to determine that target is in the siRNA of HMMR, carries out bioinformation screening, guarantee that sequence is specific for HMMR sequence and is not specific for the sequence from any other gene.The blast search engine that target sequence uses NCBI to provide is checked relative to the sequence in GenBank, filters out 9 to effective siRNA, optimize further through a large amount of preliminary experiments.
HMMR
Si-h-HMMR_001：
GAATCTGTTTGAGGAAGAASEQ ID NO.1
Target sequence: GAATCTGTTTGAGGAAGAA
Positive-sense strand (5'-3'): 5'GAAUCUGUUUGAGGAAGAA dTdT 3'SEQ ID NO.2
Antisense strand (5'-3'): 5 ' UUCUUCCUCAAACAGAUUC dTdT 3'SEQ ID NO.3
Si-h-HMMR_002：
GCAGTCTCTTGAGGAGAATSEQ ID NO.4
Target sequence: GCAGTCTCTTGAGGAGAAT
Positive-sense strand (5'-3'): 5'GCAGUCUCUUGAGGAGAAU dTdT 3'SEQ ID NO.5
Antisense strand (5'-3'): 5 ' AUUCUCCUCAAGAGACUGC dTdT 3'SEQ ID NO.6
Si-h-HMMR_003：
GCTTGGAGTTGATGAAACTSEQ ID NO.7
Target sequence: GCTTGGAGTTGATGAAACT
Positive-sense strand (5'-3'): 5'GCUUGGAGUUGAUGAAACU dTdT 3'SEQ ID NO.8
Antisense strand (5'-3'): 5 '-AGUUUCAUCAACUCCAAGC dTdT-3 ' SEQ ID NO.9
Si-h-HMMR_004：
CCGCTGTCAGCTTGCTAAASEQ ID NO.10
Target sequence: CCGCTGTCAGCTTGCTAAA
Positive-sense strand (5'-3'): 5'CCGCUGUCAGCUUGCUAAA dTdT 3'SEQ ID NO.11
Antisense strand (5'-3'): 5'UUUAGCAAGCUGACAGCGG dTdT 3'SEQ ID NO.12
Si-h-HMMR_005：
GAGCTCAAATCAAGAATATSEQ ID NO.13
Target sequence: GAGCTCAAATCAAGAATAT
Positive-sense strand (5'-3'): 5'GAGCUCAAAUCAAGAAUAU dTdT 3'SEQ ID NO.14
Antisense strand (5'-3')): 5'AUAUUCUUGAUUUGAGCUC dTdT 3'SEQ ID NO.15
Si-h-HMMR_006：
GACCAGGACTAATGAACTASEQ ID NO.16
Target sequence: GACCAGGACTAATGAACTA
Positive-sense strand (5'-3'): 5'GACCAGGACUAAUGAACUA dTdT 3'SEQ ID NO.17
Antisense strand (5'-3')): 5'UAGUUCAUUAGUCCUGGUC dTdT 3'SEQ ID NO.18
Si-h-HMMR_007：GCTAGATATTGCCCAGTTA SEQ ID NO.19
Target sequence: GCTAGATATTGCCCAGTTA
Positive-sense strand (5'-3'): 5'GC UAGAUAUUGCCCAGUUA dTdT 3'SEQ ID NO.20
Antisense strand (5'-3'): 5'UAACUGGGCAAUAUCUAGC dTdT 3'SEQ ID NO.21
Si-h-HMMR_008：GCAAACACTGGATGAGCTT SEQ ID NO.22
Target sequence: GCAAACACTGGATGAGCTT
Positive-sense strand (5'-3'): 5'GCAAACACUGGAUGAGCUU dTdT 3'SEQ ID NO.23
Antisense strand (5'-3'): 5'AAGCUCAUCCAGUGUUUGC dTdT 3'SEQ ID NO.24
Si-h-HMMR_009：GCTGGAGAACTCATCATTA SEQ ID NO.25
Target sequence: GCTGGAGAACTCATCATTA
Positive-sense strand (5'-3'): 5'GCUGGAGAACUCAUCAUUA dTdT 3'SEQ ID NO.26
Antisense strand (5'-3'): 5'UAAUGAUGAGUUCUCCAGC dTdT 3'SEQ ID NO.27
Cell transfection assays is as follows.
The hFLS cell pancreatin of 0.25% digests, make cell suspension inoculation in 12 well culture plates, hFLS cell) grow to logarithmic phase (namely growth reach 80% fusion in blocks time), after siRNA mixes with transfection reagent Lip2000, carry out grouping transfection, experiment is divided into that 11 groups: Notarget (NTC) is negative control group, NC is blank group, siRNA-HM-01 to siRNA-HM-09 is siRNA sequence for the design of HMMR gene order different positions.HFLS cell is collected after transfection 24h, in 1000rpm centrifugal 5 minutes, remove supernatant, 1ml Trizol lysate is added in cell precipitation thing, piping and druming or concuss make the abundant cracking of cell repeatedly, then be transferred in new EP pipe (1.5ml), at room temperature 15-30 DEG C, leave standstill 5 minutes.Add 0.2ml chloroform, cover tightly centrifuge tube, vibration 15s makes it fully mix, and at room temperature standing about 10 minutes centrifugal in 4 DEG C of refrigerated centrifuges, 12000rpm, and 15 minutes, centrifugal rear liquid was divided into 3 layers: upper strata colourless liquid is total serum IgE, about 0.5ml; Middle level white egg lamina albae; Lower floor's red is DNA, drawing upper strata aqueous phase 0.5ml supernatant with suction pipe is transferred in another 1.5mlEP pipe for subsequent use, add the Virahol of 0.5ml precooling simultaneously, 10 minutes are placed in 4 DEG C in 4 DEG C after abundant mixing, 12000rpm is after centrifugal 10 minutes, careful abandoning supernatant, has a small amount of white plates at tube wall Bottomattached as seen.Add the freshly prepared ethanol of 1ml 75% DEPC process, washing precipitation, in 4 DEG C after vertical concussion, the centrifugal 5min of 10000rpm, outwell most of supernatant in 4 DEG C, 10000rpm recentrifuge 5min, suck supernatant and add 20 μ l DEPC treated waters, dissolve completely to throw out, ultra-violet analysis measures the concentration of institute's extracting RNA.
1 μ l Oligo dT (0.5 μ g/ μ l) and 2.0 μ g total serum IgE are joined in PCR pipe, then adds DEPC water to 9ul; Carry out centrifugal after abundant mixing on whizzer, in 70 DEG C of temperature baths 10 minutes; Put it into ice bath in 0 DEG C of mixture of ice and water subsequently, EP pipe is taken out by Oligo dT and template annealing be placed on ice with reagent mix, preparation reaction system, of short duration centrifugal after mixing.Above-mentioned reaction system is placed in 42 DEG C of reactions 1 hour, in 70 DEG C of water-baths, water-bath makes reversed transcriptive enzyme inactivation by obtained reverse transcription product-cDNA for 10 minutes more subsequently.CDNA, fluorescence dye are mixed with upstream and downstream primer, carry out quantitative PCR detection, detected result is in table 1.
Fluorescence quantification PCR primer: F:5 '-ACCAGGACTAATGAACTAC-3 ' SEQ ID NO.41; R:5 '-CCTTCTTGCTTAGCCATC-3 ' SEQ ID NO.42.
The relative expression quantity of table 1hFLS cell levels HMMR-siRNA target gene
Result shows, compare us in the effective siRNA sequence of 9 couple that early stage, screening obtained with negative control group, the silencing efficiency of siRNA-HM--03 to HMMR is best, inhibits the genetic expression of more than 86%.Wherein the positive-sense strand of siRNA-HM--03 is sequence 8, and antisense strand is sequence 9.
Embodiment three Western blot method detects the expression of HMMR albumen in hFLS
Through the cell of siRNA-HM-03 transfection, take out culturing bottle, discard cell culture fluid, wash 2 times with PBS, outwell PBS, add 2 × Lysis Buffer of appropriate precooling, scrape with cell and cell is scraped, be placed on ice fully lysing cell 30min, in refrigerated centrifuge 4 DEG C, 12000g, centrifugal 15min, gets supernatant liquor, measures protein concentration by Bradford method, finally the final concentration of sample protein is all adjusted to 2 μ g/ μ l, for subsequent use in-80 DEG C of Refrigerator stores.Get the sample of 12 μ g total protein concentrations respectively, add isopyknic 2X loading buffer sample-loading buffer.After the two fully mixing, in boiling water, boil bath 10 minutes, deposit for subsequent use for 4 DEG C.According to the glue (the SDS-PAGE separation gel of 10% and the concentrated glue of 5%) of target protein molecular size range preparation respective concentration, after preparing in glue, comb is taken out rear electrophoresis buffer solution for cleaning loading hole, to ready sample loading before, every hole adds protein sample, carries out electrophoresis.After electrophoresis terminates, use electrophoretic blotting device, at 4 DEG C, under 400mA constant current conditions, electricity turns 2 hours, by protein delivery on pvdf membrane.Carry out subsequently developing the color and exposure analysis, analytical results is shown in Fig. 1.
Result shows, siRNA-HM-03 significantly suppress the protein expression of HMMR, and the follow-up siRNA-HM-03 of selecting does further test.
Embodiment four nucleic acid oligomer is to the suppression of inflammatory factor
Cell transfection assays.Original cuiture hFLS and 293T cell to 6 orifice plate, cell density about 50% utilizes transfection reagent lipofectamineTM2000 transfection siRNA-HM-03 and contrast, concentration 50nM.Transfection, after 24 hours, changes non-serum starved cultivation 24 hours.IL 1-α (10ng/ml) stimulates 24 hours, extracts RNA subsequently and utilizes fluorescence quantitative PCR detection expression of target gene, identical with embodiment two operation steps.Real time PCR is utilized to detect the expression level of TNF, COX2 and IL-1 β gene in hFLS and 293T cell.Collect supernatant liquor.Human IL-1 β immunoassay detection kit detects the secretion level (referring to Fig. 2) of hFLS and 293T cell IL-1 β.
Result shows, siRNA-HM--03 all effectively can suppress the differential expression patterns of COX-2, TNF, IL-1 β inflammation factor in 293T and hFLS cell compared with negative control group, wherein in hFLS cell, reaches 83% to the gene inhibition rate of IL-1 β.
The few nucleic acid of embodiment five homology is to the checking of HMMR gene inhibition effect
For checking homology ratio is on the impact of siRNA-HM-03 effect, divide three groups to carry out design and prepared its homology siRNA, by these sequences according to embodiment two method, transfection, fluorescence quantitative PCR detection is to HMMR gene mRNA expression suppression efficiency.First group of antisense strand is
5 '-AGUUUCAUCAACUCCAAGC-3 ', positive-sense strand is " 5'
GCUUGGAGUUGAUGAAACU 3 ' " homologous sequence, as following table 2 note: S=positive-sense strand, AS=antisense strand.Positive-sense strand selects 11nt, 15nt, 23nt, 27nt, dT pendency, mispairing respectively.
Table 2 antisense strand group
Second group: positive-sense strand is " 5'GCUUGGAGUUGAUGAAACU 3 ' ", antisense strand is 5 '-AGUUUCAUCAACUCCAAGC-3 ' homologous sequence, as following table 3
Table 3 positive-sense strand group
3rd group: positive-sense strand and antisense strand are above two groups of combinations, as following table 3.
Table 4 combination group
See Fig. 3, result shows that three groups of siRNA designed all serve the effect of silencing of target genes, shows have the siRNA of more than 70% homology all can disturb the expression of HMMR gene with sequence 2.The siRNA-HM-14 interference effect that wherein with the addition of the 21nt hanging base is best, is 88%; Only the siRNA-HM-21 interference effect of 11nt complementation is the poorest, is 15%, but also serves the effect of interference expression of target gene.
Embodiment six plasmid target gene silencing efficiency affects
According to HMMR full length sequence, utilize siRNA Photographing On-line software design containing the Double stranded oligonucleotide acid sequence of siRNA-HM-03 sequence, as table 4.Adopt and the siRNA double-strand of interference is not produced as negative control (NC) to any gene of the mankind.Note: dashed part is base complementrity region (from 5 ' → 3 '), bolded section is interference target sequence, and Blocked portion is restriction endonuclease sites.
Table 5 is containing the double-strand of siRNA-HM-03 sequence
Form double-strand after the oligonucleotide annealing of his-and-hers watches 5, be inserted in SiRNA expression vector, build the interference carrier containing siRNA-HM-03 sequence, and transformed competence colibacillus cell DH5 α.Carry out enzyme at transformation plate picking positive colony to cut and check order qualification, subsequently interference effect is detected.One day before infection, hFLS cell good for growth conditions is inoculated in six orifice plates and infects: serum free medium is washed cell and once added 1.5mL cell culture medium afterwards; Respectively with 250uL substratum dilution plasmid vector, mix gently; By 10uL Lipofectamine2000 reagent with after 250uL cell culture medium, mix gently, incubated at room 5min, 48h collecting cell after infecting, real time quantitative PCR method testing goal gene mRNA expression situation (see table 6).
Table 6 plasmid vector interference effect detects
Embodiment seven chemically modified is on the impact of HMMR inhibition
Different chemically modifieds and combination modification thereof are carried out to siRNA-HM-03, to improve siRNA stability, promotes interference effect.Chemically modified comprises the halogen of ribose and modifies (2 '-F modifies), methoxyl group modification (2 '-OMe), thio-modification, cholesterol modification etc.Experimental technique is as embodiment two, and wherein cholesterol, polypeptide, semi-lactosi, modification siRNA group do not add transfection reagent, and other groups all mix with lip2000 or PEI or PHA transfection Materials.
Kind modified by table 7
Table 8 chemically modified is on the impact of siRNA silencing efficiency
As known from Table 8, after all kinds of suitable chemically modified, siRNA of the present invention all serves the effect of reticent destination gene expression.Wherein siRNA-HM-36, siRNA-HM-26, siRNA-HM-40, siRNA-HM-39, siRNA-HM-41 interference effect is better, shows that 2 '-oxygen methyl (2 '-methoxyl group), sulfo-, cholesterol or phosphorylation modification can keep good interference effect.
Embodiment eight chemically modified is on the impact of nucleic acid oligomer serum stability
Serum stability detection is carried out to some chemically modified nucleic acid molecule of embodiment seven.SiRNA molecule is added isopyknic fresh serum after being diluted to 5 μMs without RNA enzyme water, then hatches 30 minutes at 37 DEG C, the different siRNA integrity of electrophoresis observation is carried out in sampling.See Fig. 4, result shows unmodified siRNA-HM-14 obvious degradation after 30 minutes, and modification of nucleic acids siRNA-HM-36, siRNA-HM-26, siRNA-HM-40, siRNA-HM-39, siRNA-HM-41, siRNA-HM-42 in 30 minutes without obviously decomposing.
Embodiment nine rat articular liquid Inflammatory Factors Contents is tested
For verifying that siRNA medicine is to arthritic therapeutic action further, carry out the expression level that RT-PCR detects genes involved in rat model.Modeling scheme: male rat (240 ± 20g, Guangdong Medical Lab Animal Center provides) totally 21, promotes arthritic formation with II Collagenase Type as inductor.II Collagenase Type (Sigma product) is expelled to hindlimb joints chamber, the disposable II Collagenase Type giving 200 μ L (500U), dosage is 100 μ L/ legs.Healthy group gives PBS as blank, and consumption is identical with inflammatory model group with inject time.Dosage regimen: after injection II Collagenase Type 6d, SD rat is divided into 7 groups at random, often organizes 3.One group is PBS group, other six groups is siRNA administration experimental group, wherein the siRNA solution (10nmol/ leg) of siRNA experimental group every rat injection 20nmol at every turn, volume injected 100 μ L, PBS group at every turn every rat injects isopyknic PBS, often organizes equal Per-Hop behavior 2 times.The siRNA of wherein administration group siRNA-HM-39 is wrapped up by chitosan nano.
Get the rat of modeling after 3 weeks and carry out real-time PCR detection: put to death rat every other day after last administration, then cut off skin, progressively expose joint with tweezers separation subcutis and muscle, knee joint is soaked in tissue preserration liquid, prevents RNA from degrading.Being inserted in joint pours in the mortar of liquid nitrogen, fully be ground to osseous tissue powdered, according to Qiagen company Rneasy Mini kit specification sheets, extracting RNA, Aglient2200 determines RNA extracting quality, can be used for next step reverse transcription reaction after ultraviolet spectrophotometer mensuration concentration.Quantitative PCR detects the gene expression amount of HMMR gene and inflammatory factor in administration group, control group, model group respectively, the results are shown in Table 9 after statistical study.
The expression amount of inflammatory factor in table 9 rat
As known from Table 9; in arthritis model, inflammation-related gene HMMR, TNF, COX-2, IL-1 β expresses and raises; siRNA-HM-36, siRNA-HM-26, siRNA-HM-40, siRNA-HM-39, siRNA-HM-41, siRNA-HM-42 significantly can lower the expression of HMMR, TNF, COX-2, IL-1 β in rat inflammation disease; serve protection cartilage and synovial membrane; improve the effect of inflammation, show that siRNA molecule of the present invention is the potential medicine preventing or treat inflammation.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It 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.
Accompanying drawing explanation
The result schematic diagram of Fig. 1 siRNA-HM-03 immunoblot experiment.
Fig. 2 siRNA-HM-03 lowers the result schematic diagram of inflammatory factor.
The result schematic diagram of the expression of target gene amount of the different siRNA-HM-03 structure of Fig. 3.
Fig. 4 chemically modified strengthens the result schematic diagram of nucleic acid oligomer serum stability.
Claims (17)
1. suppress the nucleic acid oligomer of HMMR gene, described nucleic acid oligomer is double-stranded RNA, it is characterized in that, the based composition of described nucleic acid oligomer for: include (1) antisense strand containing more than 70% homology shown in SEQ.ID NO.9; (2) positive-sense strand of nucleic acid oligomer more than 50% sequence homology matched with described antisense strand reverse complemental is contained; And (3) described positive-sense strand and described antisense strand annealed after can form duplex structure.
2. nucleic acid oligomer according to claim 1, is characterized in that, described nucleic acid oligomer includes (1) by the antisense strand shown in SEQ.ID NO.9; (2) containing the positive-sense strand that at least 50% sequence homology shown in SEQ.ID NO.8 is formed; And (3) described positive-sense strand and described antisense strand annealed after can form duplex structure.
3. nucleic acid oligomer according to claim 1, is characterized in that, described nucleic acid oligomer includes the antisense strand that (1) is formed containing at least 70% sequence homology shown in SEQ.ID NO.9; (2) by the positive-sense strand shown in SEQ.ID NO.8 base sequence; And (3) described positive-sense strand and antisense strand annealed after can form duplex structure.
4. nucleic acid oligomer according to claim 1, is characterized in that, described nucleic acid oligomer includes the antisense strand that (1) is formed containing at least 70% sequence homology shown in SEQ.ID NO.9; (2) containing the positive-sense strand that at least 70% sequence homology shown in SEQ.IDNO.8 is formed; And (3) described positive-sense strand and antisense strand annealed after can form duplex structure.
5. nucleic acid oligomer according to claim 1, is characterized in that, described nucleic acid oligomer is made up of the antisense strand of based composition as shown in SEQ.ID NO.9 and the positive-sense strand of based composition as shown in SEQ.ID NO.8.
6. the nucleic acid oligomer according to any one of claim 1-5, is characterized in that, described nucleic acid oligomer is modified through following at least one:
(1) the oxygen sulphur of the phosphodiester bond of the connection Nucleotide of described nucleic acid oligomer is replaced;
(2) ribose 2 '-OH contained by described nucleic acid oligomer methoxyl group or fluorine replaced or carry out deoxidation modification to described 2 '-OH, or modifying with open loop nucleic acid;
(3) cytosine(Cyt) 5 methyl modifications of described nucleic acid oligomer, 5-ethinyluracil, indoles are modified;
(4) cholesterol, polypeptide, fluorescence, sugar, antibody molecule, phosphorylation modification are connected to described nucleic acid oligomer end;
(5) suspension base is added to the end of described nucleic acid oligomer.
7. nucleic acid oligomer according to claim 1, is characterized in that, described nucleic acid oligomer forms by following base is modified, or described nucleic acid oligomer by following base, modified and end adds and hangs based composition:
siRNA-HM-25:5 GCCUGGAAUUGAUGAAACUdTdT3
5 AGUUUCAUCAAUUCCAGGCdTdT3; Or
siRNA-HM-26：5’GsCsUsUGGAGUUGAUGAAAsCsUs3’
5 '-AsGsUsUUCAUCAACUCCAAsGsCs-3 '; Or
siRNA-HM-36：5’GmCmUmUGGAGUUGAUGAAAmCmUm 3’
5 ' p-AmGmUmUUCAUCAACUCCAAmGmCm-3 '; Or
siRNA-HM-39：5’GmsCmsUmsUGGAGUUGAUGAAAmsCmsUms 3’
5 '-AmsGmsUmsUUCAUCAACUCCAAmsGmsCms-3 '; Or
siRNA-HM-40：5’Chol-GmCmUmUGGAGUUGAUGAAAmCmUm 3’
5 '-AmGmUmUUCAUCAACUCCAAmGmCm-3 '; Or
siRNA-HM-41：5'Chol-GmCmUmUGGAGUUGAUGAAAmCmUm3’
5 ' p-AmGmUmUUCAUCAACUCCAAmGmCm
-3 '; Or
siRNA-HM-42：5 Chol-GmsCmsCmsUGGAAUUGAUGAAAmsCmsUms3’
5 p-AmsGmsUmsUUCAUCAAUUCCAGmsGmsCms3’；
Wherein s represents that S modifies, and m represents that 2-methoxyl group is modified, and Chol represents that cholesterol is modified, and-p represents phosphorylation modification.
8. the nucleic acid oligomer according to claim 6 or 7, is characterized in that, add 3 ' end of described nucleic acid oligomer and hang base, described suspension base is dTdT.
9. can express claim 1-8 nucleic acid oligomer described in any one and there is the DNA suppressing HMMR genetic expression function.
10. the carrier containing DNA described in claim 1-8 nucleic acid oligomer described in any one or claim 9, described carrier is liposome, polymer materials, polypeptide, nano material.
11. carriers according to claim 10, is characterized in that, described liposome is cationic-liposome; Described polymer materials is hyaluronic acid or polylysine; Nano material is chitosan nano.
12. 1 kinds have the composition suppressing HMMR genetic expression function, it is characterized in that, containing DNA described in claim 1-8 nucleic acid oligomer described in any one or claim 9, and pharmaceutically acceptable carrier.
Described in DNA described in 13. claim 1-8 nucleic acid oligomer described in any one or claim 9 or claim 10 or 11, carrier or composition according to claim 12 are preparing the application in T suppression cell in HMMR genetic expression goods.
The application of the reagent of the disease that carrier described in DNA described in 14. claim 1-8 nucleic acid oligomer described in any one or claim 9 or claim 10 or 11 or composition according to claim 12 cause at preparation diagnosis HMMR gene unconventionality.
Application in the medicine of the disease that carrier described in DNA described in 15. claim 1-8 nucleic acid oligomer described in any one or claim 9 or claim 10 or 11 or composition according to claim 12 cause at preparation treatment HMMR gene unconventionality.
16. application according to claims 14 or 15, is characterized in that, the disease that described HMMR gene unconventionality causes is inflammation, cancer.
17. application according to claims 14 or 15, is characterized in that, the disease that described HMMR gene unconventionality causes is osteoarthritis, rheumatic arthritis, synovitis.
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