CN104450709B - Inhibit nucleic acid oligomer and its application of HMMR genes - Google Patents
Inhibit nucleic acid oligomer and its application of HMMR genes Download PDFInfo
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- CN104450709B CN104450709B CN201410837262.3A CN201410837262A CN104450709B CN 104450709 B CN104450709 B CN 104450709B CN 201410837262 A CN201410837262 A CN 201410837262A CN 104450709 B CN104450709 B CN 104450709B
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Abstract
The invention discloses the nucleic acid oligomer for inhibiting HMMR genes, the nucleic acid oligomer is double-stranded RNA, and the base composition of the nucleic acid oligomer is：(1) more than the 70% homologous antisense strand containing SEQ.ID NO.9 compositions；(2) positive-sense strand with more than 50% sequence homology of nucleic acid oligomer of antisense strand reverse complemental pairing is contained；And (3) described positive-sense strand and the antisense strand can form duplex structure after annealing.And disclose the DNA containing above-mentioned nucleic acid oligomer, carrier, composition and its application.There is the nucleic acid oligomer for the effect of significantly inhibiting to HMMR the present invention provides one kind, which can realize in zoopery inhibits inflammatory effect, significantly reduces the inflammation factor, is a kind of very promising inflammation treatment drug.
Description
Technical field
The invention belongs to biological technical fields, are specifically related to a kind of nucleic acid oligomer for inhibiting HMMR genes and its application.
Background technology
Osteoarthritis (OA) is the one of the major reasons to disable in the world, and approximately more than 10% the elderly has osteoarthritis
Symptom.Rheumatic arthritis (RA) is a chronic inflammatory diseases, affects in the world about 0.5% to 1% adult, is led to
It often results in joint damage and jeopardizes quality of life.A variety of inflammatory cytokines such as TNF-α .IL-1.IL-6 etc. takes part in RA and OA
Morbidity and progress, generate to promote destruction of joint, synovitis, blood vessel by immune cell activated and inducing metal protease
Hyperplasia etc..A variety of inflammatory cytokines such as TNF-α .IL-1.IL-6 etc. takes part in morbidity and the progress of RA and OA, passes through activation
Immunocyte and inducing metal protease generate and promote destruction of joint, synovitis, blood vessel hyperplasia etc..
HMMR/RHAMM be also known as hyaluronic acid mediated cell migration receptor (receptor for hyaluronan-
Mediated motility), it is distributed in cell membrane surface, cytoplasm and nucleus.Its principal biological behavior includes：①
By with the interactions such as micro-pipe, hyaluronic acid, calmodulin, participate in the adherency of cell and turning for movement and cell signal
It leads；2. adjust the cell cycle；3. stablize centerbody and 4. spindle promotes the formation of new vessels.
In past more than 20 years, the R＆D work of most of novel method for the treatment of is the Antiarthritic on remission
The drug (DMOADs) of the resisting rheumatoid arthritis of drug (DMARDs) and remission.Up to the present, pharmacy industry is being developed
It is on the disease retentivity drug (DMOADs) of effective and safe and unsuccessful, thousands of patient is caused still this to be subject to seriously to disable
Property disease caused by pain.With the application of new way and medication, there will be more development in therapy field expection,
This is can block, reverse disease process and the drug for mitigating disease, as siRNA provides huge development space.
RNA interventions are the discoveries for having in life science revolution meaning, become the important tool of research gene function, it can
Cause organism, the gene inactivation in mammalian cell or even animal.It is related harmful that RNA intervention techniques can be applied to treatment
The disease of Overexpression has the potentiality for creating human disease treatment new method.
In the present invention, we provide a kind of brand-new siRNA as arthritis and the medicine of related inflammation；It is a kind of
Inhibit the method for inflammatory Cytokines Expression by Bones and joints chamber local injection siRNA and its preparation, realize and arthritis are controlled
Treatment acts on.
The content of the invention
The object of the invention is intended to provide a kind of nucleic acid oligomer for inhibiting HMMR gene expressions.
Realize that the technical solution of above-mentioned purpose is as follows.
One kind inhibit HMMR genes nucleic acid oligomer, the nucleic acid oligomer be double-stranded RNA, the base group of the nucleic acid oligomer
Become：
The base composition for including the nucleic acid oligomer is：Include (1) containing more than 70% shown in SEQ.ID NO.9
Homologous antisense strand；(2) justice with more than 50% sequence homology of nucleic acid oligomer of antisense strand reverse complemental pairing is contained
Chain；And (3) described positive-sense strand and the antisense strand can form duplex structure after annealing.
In one of the embodiments, the nucleic acid oligomer includes (1) as anti-shown in SEQ.ID NO.9 base sequences
Adopted chain；(2) positive-sense strand formed containing at least 50% sequence homology shown in SEQ.ID NO.8；And (3) described positive-sense strand and antisense
Chain can form duplex structure after annealing.
In one of the embodiments, the nucleic acid oligomer includes (1) and contains at least 70% sequence shown in SEQ.ID NO.9
Arrange the antisense strand of homologous composition；(2) as the positive-sense strand shown in SEQ.ID NO.8 base sequences；And (3) described positive-sense strand and antisense
Chain can form duplex structure after annealing.
In one of the embodiments, the nucleic acid oligomer includes (1) and contains at least 70% sequence shown in SEQ.ID NO.9
Arrange the antisense strand of homologous composition；(2) positive-sense strand formed containing at least 70% sequence homology shown in SEQ.ID NO.8；And (3) institute
Duplex structure can be formed after annealing by stating positive-sense strand and antisense strand.
In one of the embodiments, the nucleic acid oligomer as antisense strand of the base composition as shown in SEQ.ID NO.9 with
And positive-sense strand composition of the base composition as shown in SEQ.ID NO.8.
In one of the embodiments, the nucleic acid oligomer by base composition such as siRNA-HM-36, siRNA-HM-26,
Shown in siRNA-HM-40, siRNA-HM-39, siRNA-HM-41, siRNA-HM-42.
It is double-stranded RNA that the present invention, which provides nucleic acid oligomer, wherein the feature in the double-stranded RNA is：Antisense strand contains nucleosides
Acid sequence is homologous at least more than 70% for sequence 9 in " 5 '-AGUUUCAUCAACUCCAAGC-3' " such as sequence table or with it
The homologous sequence of property；Positive-sense strand contains the nucleotide sequence having with antisense strand reverse complemental chain more than 50% homology.It is above-mentioned
Nucleic acid oligomer can pass through any one or more modifications in (1)-(4)：
1) phosphodiester bond of the connection nucleotide of the nucleic acid oligomer is modified, preferably by the phosphodiester bond
Oxygen substituted with sulphur；
2) the contained ribose of the nucleic acid oligomer is modified, preferably 2 '-OH of ribose is substituted with methoxyl group or fluorine or
Person is carried out deoxidation modification to the 2 '-OH or is modified with open loop nucleic acid (UNA)；
3) to the modification of the nucleotide base of the nucleic acid oligomer, preferably 5 methyl modifications, 5- acetenyls urine of cytimidine are phonetic
Pyridine, indoles modification；
4) end modified to the nucleic acid oligomer, preferably end connects cholesterol, polypeptide, fluorescence, sugar, antibody molecule, phosphorus
Acidifying modification；
5) nucleic acid oligomer of the present invention can add suspension base in 3 ' ends, and it is dTdT preferably to hang base.
Another object of the present invention, which also resides in, provides a kind of DNA for having and inhibiting HMMR gene expression functions.
Specific technical solution is as follows.
Any of the above-described kind of nucleic acid oligomer can be expressed and there is the DNA for inhibiting HMMR gene expressions.
Another object of the present invention, which also resides in, provides a kind of carrier for having the function of to inhibit HMMR gene expressions.
Specific technical solution is as follows.
Carrier containing any of the above-described kind of nucleic acid oligomer or above-mentioned DNA, the carrier for liposome, polymer material,
Polypeptide, nano material.Wherein liposome vectors material be cationic-liposome commonly used in the art, preferably lipo2000；Polymerization
Object carrier material preferably clear matter acid or polylysine；The preferred RGD of polypeptide material；Nano material is preferably chitosan nano.
Another object of the present invention, which also resides in, provides a kind of composition for having the function of to inhibit HMMR gene expressions.Specific skill
Art scheme is as follows.
A kind of composition for having the function of to inhibit HMMR gene expressions, contains any of the above-described kind of nucleic acid oligomer or above-mentioned
DNA and pharmaceutically acceptable carrier.
Another object of the present invention also resides in the application for providing above-mentioned nucleic acid oligomer, DNA, carrier or composition.
Specific technical solution is as follows.
Above-mentioned nucleic acid oligomer, DNA, carrier or composition the answering in HMMR gene expression products in inhibition cell is prepared
With.
Above-mentioned nucleic acid oligomer, DNA, carrier or composition are preparing the disease HMMR's of diagnosis HMMR gene unconventionality initiations
Application in reagent.
Above-mentioned nucleic acid oligomer, DNA, carrier or composition are prepared in the drug for the disease that treatment HMMR gene unconventionalities trigger
Application.
In one of the embodiments, the disease that the HMMR gene unconventionalities trigger may be selected from inflammation, cancer.
In one of the embodiments, the disease that the HMMR gene unconventionalities trigger is arthritis, rheumatic arthritis or
Synovitis.
The present invention is optimized for the different position in HMMR gene C Ds regions, has selected 9 pairs for people and mouse
The siRNA of homologous gene carries out screening test.Result of study shows：Compared with negative control siRNA, antisense strand contains nucleotide
The siRNA-HM-03 that sequence is 5 '-AGUUUCAUCAACUCCAAGC-3 ' is most strong to HMMR gene mRNA expression inhibitions, right
The mRNA silence efficiencies of HMMR genes are up to 86% (embodiment two).In Western blotting (western blotting) experiment, with
After blank compares addition siRNA-HM-03 with negative control group, HMMR protein expression levels are decreased obviously, and difference has conspicuousness (P
<0.05) (embodiment three).Through adding in siRNA-HM-03 in the post-stimulatory cells of IL 1- α, with adding in negative control nucleic acid oligomer
It compares, cellular inflammation factor TNF, COX-2, IL-1 β content are decreased obviously, and show that siRNA-HM-03 has the effect that diminishes inflammation
(example IV).In embodiment five, sequence is optimized in the present invention, the experimental results showed that:Antisense strand containing " 5 '-
The double-strand oligonucleotide of the homologous sequence of AGUUUCAUCAACUCCAAGC-3 ' has inhibition to HMMR gene mRNAs；Positive-sense strand
Complete complementary reversed with antisense strand or part only partial complementarity also have inhibition to HMMR gene mRNAs, and contain and antisense
The double-stranded RNA of 70% homologous sequence of chain also has inhibition.The expression of the application plasmid vector of embodiment six " 5 '-
AGUUUCAUCAACUCCAAGC-3 ' " sequences also have the mRNA inhibitions of good HMMR genes in cell.Embodiment
Seven have studied influence of the chemical modification to the nucleic acid silence HMMR potencies of gene, the results showed that：Oligomerization core after appropriate modification
Acid has good inhibitory action to HMMR genes.The research of embodiment eight shows after chemical modification, siRNA of the invention
Stability in serum significantly improves.
Embodiment nine is the live body rat test of siRNA, independent in the articular cavity of osteoarthritis rat or mixing
Containing antisense strand, " chemical modification object of 5 '-AGUUUCAUCAACUCCAAGC-3 ' or 5AGUUUCAUCAAUUCCAGGC3 close for injection
Save the detection of liquid Inflammatory Factors Contents experiments have shown that, nucleic acid oligomer has apparent inhibition of inflammation in rat body.
The present invention's has the beneficial effect that：1st, the present invention is with gene silent technology, by largely designing, screening, point
Analysis is found that a kind of nucleic acid oligomer for having the effect of significantly inhibiting to HMMR with verification, in a kind of hFLS cells, inhibits efficiency and reaches
It arrives88%, and the effect of gene silencing can be played with nucleotide sequence that the sequence has more than 70% homology, show this hair
Bright is a kind of nucleotide sequence extremely strong to HMMR silencing efficiencies；2nd, the invention further relates to a kind of nucleic acid chemistry trim, have
High stability, high activity, high specific and low cytotoxicity make it become live body application active drug；3rd, the present invention relates to
And nucleic acid can be realized in zoopery inhibit inflammatory effect.In cellular level to the expression inhibiting rate of inflammatory factor IL-1 β
Up to 83%, in rat osteoarthritic inflammation model, expression of the continuous injection oligonucleotide of the present invention to inflammatory factor and inflammation gene expression
There is apparent inhibitory action, test show that HMMR-siRNA has prevention or therapeutic effect to rat arthritis above.
Description of the drawings
The result schematic diagram of Fig. 1 siRNA-HM-03 immunoblot experiments.
Fig. 2 siRNA-HM-03 lower the result schematic diagram of inflammatory factor.
The result schematic diagram of the expression of target gene amount of Fig. 3 difference siRNA-HM-03 structures.
Fig. 4 chemical modifications enhance the result schematic diagram of nucleic acid oligomer serum stability.
Specific embodiment
With reference to specific embodiment, the invention will be further described, but the present invention is not limited to following embodiments.Under
It states in embodiment, is conventional known method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.HFLS is thin
Born of the same parents are purchased from Cell Applications, 293-T cell purchased from ATCC, the purchase of Human IL-1 β immunoassay detection kits
From Assay Pro, Lipofectamine2000 kits are purchased from Invitrogen, and pancreatin, Trizol, PBS are sigma products,
Male SD rat is Guangdong medical experiment animal center product, and the nucleotide sequences such as primer, siRNA are the sharp rich biological section in Guangzhou
Skill Co., Ltd provides.
" TNF " described in the present invention is " TNF-- α ".
Inhibiting rate=NC (negative contral) compares expression quantity-destination gene expression amount/NC (negative
Contral expression quantity) is compareed.
" pharmaceutically acceptable carrier " of the present invention refers to internal transfection reagent commonly used in the art, such as polyethylene
Imines (PEI), jetPEI (L-PEI), hydroxy fatty acid (PHA), cationic polymer, amino acid, liposome
Deng.
The chemical synthesis of one nucleic acid oligomer of embodiment
(1) RNA applied in embodiment and modification oligonucleotide monomer obtain in accordance with the following methods：
RNA nucleotide in oligonucleotide is prepared with 2 '-O-TBDMS phosphoramidite monomers；DNA nucleotide is sub- with deoxyribonucleoside
It is prepared by phosphinylidyne amine monomers；Nucleotide glycosyl modified nucleoside is with 2 '-OCH3, 2 '-F, lock nucleic acid (LNA), open loop nucleic acid (UNA), 5-
It is prepared by methylcystein, indoles, 5-ethinyluracil phosphoramidite monomer；Skeleton phosphothio nucleic acid is with Beaucage reagents
Or PADS reagents are prepared instead of iodine water；Cholesterol, fluorescent marker, sugar-modified, phosphorylation nucleic acid oligomer are prepared by corresponding to monomer；It is more
Peptide modification oligonucleotide is obtained by sulfydryl modification oligonucleotide with polypeptide by Michael addition reaction, and monomer described above is from Sigma
Aldrich, Chem Genes, Glen research companies buy.
(2) nucleic acid oligomer, glycosyl, base, skeleton thio-modification, cholesterol, fluorescent marker modification oligonucleotide preparation method:
Theoretical yield 1umol synthesis specifications are completed, and weigh general solid support CPG 20mg (carrying capacity 50umol/g), respectively
The monomer of class phosphoramidite is dissolved in anhydrous acetonitrile (0.15M).5- ethylmercapto groups -1H-TETRAZOLE acetonitrile solution is as activation
Agent (0.25M), oxidant iodine water (0.02M), 3% trichloroacetic acid dichloromethane solution.According to standard rna synthesis program, journey is set
Sequence Xun Huan synthesis, often walks Coupling time 2-10 minutes, after 20 Xun Huans, completes oligonucleotides synthesis in solid state.CPG is dried up, is turned
It moves on in 5ml EP pipes, ammonium hydroxide/ethanol solution (3/1) 1-20ml is added in, when 55 DEG C of heating 5~20 are small.In turning for 10000rpm
The lower centrifugation 10min of speed takes supernatant, and white gummy solid is obtained after draining concentrated ammonia liquor/ethyl alcohol.Solid is dissolved in 200 μ l 1M TBAF
THF solution, when room temperature concussion 20 is small.0.5ml 1M Tris-HCl buffer solutions (pH 7.4) are added in, room temperature is shaken 15 minutes, put
Machine is drained in centrifugation and is evacuated to volume as original volume 1/2, removes THF.Solution is extracted 2 times with 0.5ml chloroforms, adds in 1ml 0.1M
Mixed solution is poured into solid-phase extraction column by TEAA sample solutions, according to standard rna desalination flow, removes excess salt in solution, gained
Nucleic acid concentration is measured by micro ultraviolet specrophotometer, confirms nucleic acid structure by mass spectrum.
3) polypeptide, antibody connection oligonucleotide preparation method：
After above method prepares 100nmol HS modification nucleic acid oligomers, 950 μ l 100mM HEPES-KOH bufferings are dissolved in
Liquid (pH1-14).The aqueous solution that the polypeptide or antibody modified with 500nmol containing ethylene linkage are dissolved in 50 μ l mixes.Under nitrogen protection
It 0-100 DEG C, is reacted, reaction efficiency is monitored by HPLC, is used to test after Quantitative yield, after solution is concentrated by ultrafiltration.
Annealing：Oligonucleotide mixed in equal amounts made above is in 1ml water or buffer solution, and 95 DEG C are heated 2-20 minutes, room temperature
Standing is cooled to room temperature spare.
Embodiment two inhibits effective nucleic acid oligomer screening of HMMR gene mRNA expressions
SiRNA designs are carried out to determine to target the siRNA of HMMR, carry out biological information screening, it is ensured that sequence for
HMMR sequences are specificity and are not specific for the sequence from any other gene.Target sequence is provided using NCBI
Blast search engine checked compared with the sequence in GenBank, by substantial amounts of preliminary experiment filter out 9 pairs effectively
SiRNA is further optimized.
HMMR
Si-h-HMMR_001：GAATCTGTTTGAGGAAGAA SEQ 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：GCAGTCTCTTGAGGAGAAT SEQ 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：GCTTGGAGTTGATGAAACT SEQ 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：CCGCTGTCAGCTTGCTAAA SEQ 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：GAGCTCAAATCAAGAATAT SEQ 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：GACCAGGACTAATGAACTA SEQ 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 are as follows.
HFLS cells are digested with 0.25% pancreatin, cell suspension inoculation are made in 12 well culture plates, hFLS is thin
Born of the same parents) grow to exponential phase (grow up to 80% fusion it is in blocks when), after siRNA is mixed with transfection reagent Lip2000, progress
Grouping transfection, experiment are divided into 11 groups：Notarget (NTC) is negative control group, NC is blank control group, siRNA-HM-01
It is the siRNA sequence for the design of HMMR gene orders different position to siRNA-HM-09.HFLS cells are collected in transfection afterwards for 24 hours,
It is centrifuged 5 minutes in 1000rpm, removes supernatant, 1ml Trizol lysates are added in cell pellet, repeatedly piping and druming or violent
Concussion makes cell fully crack, and is then transferred in new EP pipes (1.5ml), and 5 minutes are stood at 15-30 DEG C of room temperature.It adds in
0.2ml chloroforms cover tightly centrifuge tube, and vibration 15s makes its abundant mixing, stands 10 minutes or so at room temperature in 4 DEG C of low-temperature centrifugations
Machine centrifuges, and 12000rpm, 15 minutes, liquid was divided into 3 layers after centrifugation：Upper strata colourless liquid be total serum IgE, about 0.5ml；Middle level white
Protein layer；Lower floor's red material is DNA, and drawing upper strata aqueous phase 0.5ml supernatants with suction pipe is transferred to another spare 1.5mlEP pipes
In, while the isopropanol of 0.5ml precoolings is added in, abundant mixing places 10 minutes after 4 DEG C in 4 DEG C, and 12000rpm centrifuges 10 points
Zhong Hou, careful abandoning supernatant, it is seen that be attached with a small amount of white plates in tube wall bottom.1ml 75% is added in DEPC processing
The ethyl alcohol for the Fresh crossed, washing precipitation, vertical concussion after 4 DEG C, 10000rpm centrifugation 5min, outwell most of supernatant in
4 DEG C, 10000rpm centrifuges 5min again, sucks supernatant and adds in the 20 processed water of μ l DEPC, until sediment is completely dissolved, it is purple
Outer analysis measure 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 IgEs are added in PCR pipe, add DEPC water to 9ul；
It is centrifuged after abundant mixing on centrifuge, in 70 DEG C of warm bath 10 minutes；Then put it into ice in 0 DEG C of mixture of ice and water
Bath makes Oligo dT and template annealing be placed on the taking-up of EP pipes and is mixed on ice with reagent, preparation reaction system, of short duration after mixing
Centrifugation.By above-mentioned reaction system be placed in 42 DEG C reaction 1 it is small when, then water-bath makes reverse transcription in 10 minutes in 70 DEG C of water-baths
Enzyme is inactivated obtained reverse transcription product-cDNA.CDNA, fluorescent dye with upstream and downstream primer are mixed, carry out quantitative PCR
Detection, testing result are shown 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 cellular level HMMR-siRNA target genes
The result shows that compared with negative control group in our 9 couple effectively siRNA sequences by screening acquisition early period,
SiRNA-HM--03 is best to the silencing efficiency of HMMR, it is suppressed that more than 86% gene expression.Wherein siRNA-HM--03's
Positive-sense strand is sequence 8, and antisense strand is sequence 9.
The expression of HMMR albumen in three Western blot methods of embodiment detection hFLS
The cell transfected through siRNA-HM-03 takes out blake bottle, discards cell culture fluid, washed 2 times, outwelled with PBS
PBS adds in 2 × Lysis Buffer of appropriate precooling, and abundant cell lysis on ice under cell scraper, will be placed in cell scraper
30min, in 4 DEG C, 12000g of refrigerated centrifuge, centrifuges 15min, takes supernatant, measures protein concentration with Bradford methods, finally
The final concentration of sample protein is adjusted to 2 μ g/ μ l, is saved backup in -80 DEG C of refrigerators.The sample of 12 μ g total protein concentrations is taken respectively
Product add in isometric 2X loading buffer sample-loading buffers.After the abundant mixing of the two, 10 points of bath is boiled in boiling water
Clock, 4 DEG C of storages are spare.According to destination protein molecular size range prepare respective concentration glue (10% SDS-PAGE separation gels and
5% concentration glue), after glue is waited to prepare, with electrophoresis buffer solution for cleaning loading hole after comb is taken out, the ready sample by before
Product loading adds in protein sample per hole, carries out electrophoresis.After electrophoresis, using electrophoretic blotting device, at 4 DEG C, 400mA constant currents
Under the conditions of electricity turn 2 it is small when, will be on protein delivery to pvdf membrane.It is then developed the color and exposure analysis, analysis result is shown in Fig. 1.
The result shows that siRNA-HM-03 significantly suppresses the protein expression of HMMR, it is follow-up select siRNA-HM-03 do into
The experiment of one step.
Inhibition of the example IV nucleic acid oligomer to inflammatory factor
Cell transfection assays.Original cuiture hFLS and 293T cells to 6 orifice plates, cell density about 50% utilize transfection reagent
LipofectamineTM2000 transfects siRNA-HM-03 and control, concentration 50nM.Transfect 24 it is small when after, change non-serum starved training
Support 24 it is small when.When IL 1- α (10ng/ml) stimulations 24 are small, then extract RNA and utilize fluorescence quantitative PCR detection expression of target gene,
It is identical with two operating procedure of embodiment.Utilize real time PCR detection hFLS and TNF, COX2 and IL-1 β bases in 293T cells
The expression of cause.Collect supernatant.Human IL-1 β immunoassay detection kits detect hFLS and 293T cells IL-
The secretion level (referring to Fig. 2) of 1 β.
The result shows that siRNA-HM--03 can effectively inhibit in 293T and hFLS cells compared with negative control group
The differential expression patterns of COX-2, TNF, IL-1 β the inflammation factor, wherein inhibiting in hFLS cells to the gene of IL-1 β
Rate reaches 83%.
Verification of the five homologous oligonucleotide of embodiment to HMMR gene inhibitions
To verify influence of the homologous ratio to siRNA-HM-03 effects, it is homologous that point three groups of development designs are prepared for its
SiRNA, by these sequences according to two method of embodiment, transfection, fluorescence quantitative PCR detection is to HMMR gene mRNA expressions inhibition effect
Rate.First group of antisense strand be
5 '-AGUUUCAUCAACUCCAAGC-3 ', positive-sense strand are " 5'
GCUUGGAGUUGAUGAAACU 3 ' " homologous sequences, as the following table 2 is noted:S=positive-sense strands, AS=antisense strands.Positive-sense strand
11nt, 15nt, 23nt, 27nt, dT pendency, mispairing are selected respectively.
2 antisense chain group of table
Second group：Positive-sense strand is " 5'GCUUGGAGUUGAUGAAACU 3 ' ", and antisense strand 5 '-
AGUUUCAUCAACUCCAAGC-3 ' homologous sequences, such as the following table 3
The just chain group of table 3
3rd group：Positive-sense strand and antisense strand are above two groups of combinations, such as the following table 3.
4 combination group of table
Referring to Fig. 3, the results showed that the siRNA of three groups of designs plays the role of silencing of target genes, shows have with sequence 2
The siRNA of more than 70% homology can disturb the expression of HMMR genes.Wherein it is with the addition of the siRNA- of the 21nt of suspension base
HM-14 interference effects are best, are 88%；The siRNA-HM-21 interference effects of only 11nt complementations are worst, are 15%, but also function to
The effect of interference expression of target gene.
Six plasmid target gene silencing efficiency of embodiment influences
According to HMMR full length sequences, double-strand widow's core of siRNA Photographing On-line Software for Design sequence containing siRNA-HM-03 is utilized
Nucleotide sequence, such as table 4.Using not to any gene of the mankind generate interference siRNA double-strand as negative control (NC).Note：It draws
Line part is base complementrity region (from 5 ' → 3 '), and bolded section is interference target sequence, and Blocked portion is restriction enzyme position
Point.
The double-strand of 5 sequence containing siRNA-HM-03 of table
To forming double-strand after the oligonucleotides annealing of table 5, it is inserted into SiRNA expression vector, structure is containing siRNA-HM-03
The interference carrier of sequence, and transformed competence colibacillus cell DH5 α.Digestion and sequencing identification are carried out in conversion tablet picking positive colony,
Then interference effect is detected.One day before infection, by the good hFLS cell inoculations of growth conditions in six orifice plates into
Row infection:Serum free medium washes cell and once adds in 1.5mL cell culture mediums afterwards；Respectively plasmid is diluted with 250uL culture mediums
Carrier, gently mixing；By 10uL Lipofectamine2000 reagents with 250uL cell culture mediums after, gently mixing, room
Temperature is incubated 5min, and 48h collects cell, real time quantitative PCR method testing goal gene mRNA expression situation (being shown in Table 6) after infection.
6 plasmid vector interference effect of table detects
Influence of seven chemical modification of embodiment to HMMR inhibitions
Different chemical modifications and combinations thereof modification is carried out to siRNA-HM-03, to improve siRNA stability, is promoted dry
Disturb effect.Halogen modification (2 '-Fs modification) of the chemical modification including ribose, methoxyl group modification (2 '-OMe), thio-modification, courage are solid
Alcohol modification etc..Experimental method such as embodiment two, wherein cholesterol, polypeptide, galactolipin, modification siRNA groups do not add in transfection reagent,
Other groups are mixed with lip2000 or PEI or PHA transfection Materials.
Table 7 modifies species
Influence of 8 chemical modification of table to siRNA silencing efficiencies
As known from Table 8, after all kinds of appropriate chemical modifications, siRNA of the invention plays silence target gene table
The effect reached.Wherein siRNA-HM-36, siRNA-HM-26, siRNA-HM-40, siRNA-HM-39, siRNA-HM-41 are disturbed
Effect is preferable, shows that 2 '-oxygen methyl (2 '-methoxyl group), thio, cholesterol or phosphorylation modification can keep preferably interference effect
Fruit.
Influence of eight chemical modification of embodiment to nucleic acid oligomer serum stability
Serum stability detection is carried out to several chemical modification nucleic acid molecules of embodiment seven.By siRNA molecule through no RNA
Enzyme water, which is diluted to after 5 μM, adds in isometric fresh serum, is then incubated 30 minutes at 37 DEG C, and it is different that sampling carries out electrophoresis observation
SiRNA integralities.Referring to Fig. 4, the results showed that unmodified siRNA-HM-14 obvious degradations 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 are at 30 points
It is decomposed in clock without apparent.
Nine rat articular liquid Inflammatory Factors Contents of embodiment are tested
Further to verify that siRNA drugs to arthritic therapeutic effect, carry out related in RT-PCR detection rat models
The expression of gene.Modeling scheme：Male rat (240 ± 20g, Guangdong Medical Lab Animal Center provide) totally 21,
Promote arthritic formation by the use of II Collagenase Types as derivant.II Collagenase Types (Sigma products) are injected into hindlimb joints
Chamber, disposably gives the II Collagenase Types of 200 μ L (500U), and dosage is 100 μ L/ legs.Healthy group is given PBS as blank pair
According to dosage is identical with inflammatory model group with injection time.Dosage regimen：It is after II Collagenase Types 6d is injected, SD rats is random
It is divided into 7 groups, every group 3.One group is PBS groups, other six groups are administered experimental group, wherein siRNA experimental groups every every time for siRNA
Rat injects the bodies such as the siRNA solution (10nmol/ legs) of 20nmol, each every rat injection of 100 μ L, PBS group of volume injected
Long-pending PBS, every group of equal weekly administration 2 times.The siRNA of wherein administration group siRNA-HM-39 is wrapped up by chitosan nano.
The rat after modeling 3 weeks is taken to carry out real-time PCR detections：Last time puts to death rat every other day after being administered, so
After cut off skin, separating subcutaneous tissue and muscle with tweezers progressively exposes joint, and knee joint is soaked in tissue preserration liquid,
Prevent RNA from degrading.Joint is inserted in the mortar for pouring into liquid nitrogen, is fully ground to bone tissue into powder, according to Qiagen companies
Rneasy Mini kit specifications, extracting RNA, Aglient2200 determine that RNA extracts quality, and ultraviolet specrophotometer measures dense
It can be used for the reverse transcription reaction of next step after degree.Quantitative PCR detect respectively in administration group, control group, model group HMMR genes with
The gene expression amount of inflammatory factor the results are shown in Table 9 after statistical analysis.
The expression quantity of inflammatory factor in 9 rat of table
As known from Table 9, inflammation-related gene HMMR, TNF, COX-2, IL-1 β expression rise, si in arthritis model
RNA-HM-36, siRNA-HM-26, siRNA-HM-40, siRNA-HM-39, siRNA-HM-41, siRNA-HM-42 can significantly under
The expression of the HMMR, TNF, COX-2, IL-1 β in mouse inflammation disease are tuned up, protection cartilage and synovial membrane is played, improves inflammation
Effect, the siRNA molecule for showing the present invention is can potentially to prevent or treat the drug of inflammation.
Embodiment described above only expresses the several embodiments of the present invention, and description is more specific and detailed, but simultaneously
Cannot the limitation to the scope of the claims of the present invention therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (11)
1. inhibiting the nucleic acid oligomer of HMMR genes, the nucleic acid oligomer is double-stranded RNA, it is characterized in that,
Antisense strand and base composition such as SEQ.ID NO.8 of the nucleic acid oligomer as base composition as shown in SEQ.ID NO.9
Shown positive-sense strand composition；The nucleic acid oligomer is made of following base through modification, or the nucleic acid oligomer is passed through by following base
Modification and end addition suspension base composition：
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’；
Wherein s represents S modifications, and m represents the modification of 2- methoxyl groups, and Chol represents cholesterol modification, and-p represents phosphorylation modification.
2. nucleic acid oligomer according to claim 1, it is characterized in that, to 3 ' the ends addition suspension alkali of the nucleic acid oligomer
Base, the suspension base is dTdT.
3. the nucleic acid oligomer of claim 1 or 2 can be expressed and there is the DNA for inhibiting HMMR gene expressions.
4. the carrier containing DNA described in the nucleic acid oligomer of claim 1 or 2 or claim 3, the carrier is lipid
Body, polymer material, polypeptide, nano material.
5. carrier according to claim 4, it is characterized in that, the liposome is cationic-liposome；The polymeric material
Expect for hyaluronic acid or polylysine；Nano material is chitosan nano.
6. a kind of composition for having the function of to inhibit HMMR gene expressions, it is characterized in that, contain the oligomerization of claim 1 or 2
DNA described in nucleic acid or claim 3 and pharmaceutically acceptable carrier.
7. the nucleic acid oligomer of claim 1 or 2 either DNA described in claim 3 or the carrier of claim 4 or 5 or
Application of the composition in inhibition cell is prepared in HMMR gene expression products described in person's claim 6.
8. the nucleic acid oligomer of claim 1 or 2 either DNA described in claim 3 or the carrier of claim 4 or 5 or
Composition described in person's claim 6 is preparing the application for the reagent for diagnosing the disease that HMMR gene unconventionalities trigger.
9. the nucleic acid oligomer of claim 1 or 2 either DNA described in claim 3 or the carrier of claim 4 or 5 or
Application of the composition in the drug for preparing the disease that treatment HMMR gene unconventionalities trigger described in person's claim 6.
10. application according to claim 8 or claim 9, it is characterized in that, the disease that the HMMR gene unconventionalities trigger is inflammation,
Cancer.
11. application according to claim 8 or claim 9, it is characterized in that, the disease that the HMMR gene unconventionalities trigger is Bones and joints
Inflammation, rheumatic arthritis, synovitis.
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