CN103757021B - Double-chain siRNA molecule of silencing DNMT (Desoxvribose Nucleic Acid Methyltransferases) gene and application of double-chain siRNA molecule - Google Patents
Double-chain siRNA molecule of silencing DNMT (Desoxvribose Nucleic Acid Methyltransferases) gene and application of double-chain siRNA molecule Download PDFInfo
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Abstract
The invention relates to the technical field of biological medicines, and in particular relates to double-chain siRNA molecule of silencing DNMT (Desoxvribose Nucleic Acid Methyltransferases) gene and an application of the molecule, and more particularly siRNA is synthesized through nucleotide sequence design aiming at human tumor cell DNMT gene. After siRNA is transferred into liver cancer cells, the expression of immunogenicity related protein is prompted.
Description
Technical field
The present invention relates to biomedicine technical field, be specifically related to Double-stranded siRNA molecules and application thereof, more specifically, the present invention is directed to the nucleotide sequence design and synthesis siRNA of human tumor cells DNMT gene, described siRNA proceeds to after liver cancer cell can the expression of obvious Promote immunity originality associated protein.
Background technology
Dnmt rna (DNA methyltransferases, DNMTs) be catalytic dna methylate modify enzyme.The DNMTs with catalytic methylation function has 3 kinds, is respectively DNMT1, DNMT3a and DNMT3b [Plass, C.Cancer epigenomics.Human Molecular Genetics, 2002; 11:2479-2488].DNA methylation is differentiation and the functional expression of regulation and control immunocyte.The methylate expression of regulating immune molecules and tumour antigen and the immunogenicity of tumour and immunoreactivity has clear and definite relation, therefore, rational demethylation is the effective means [Teitella overcoming immunological tolerance and immunologic escape, M., et al.DNA methylation in immune system.Clinical immunology, 2003; 109:2-5].
Cancer/testis antigen (cancer/testis antigen, CTA) be a kind of tumor associated antigen, determine the gene product of 19 CTA families at present, all there is stronger immunogenicity (as CT10 (cancer/testis10), there is the immune epitope that multiple HLA-I (human leukocyte antigen) I class antigen limits, experiment in vivo and vitro confirm can inducing function lymphocytotoxicity reaction, promote disappearing of tumour, in the liver cancer tissue of HCC patient, CTA expresses very high [Zhao L, et al.Progress and prospects in hepatocellular carcinoma surgery.Ann Chir, 1988, 52:558-563], therefore meet for the anti-tumor immunotherapy of CTA ideal chose [the Chen YT. that liver cancer and other tumour patients thereof carry out immunity, et al.A testicular antigen aberrantlye expressed in human cancers detected by autologous antibody screening.Proc Natl Acard Sci USA, 1997, 94:1914-1918Korangy F., et al.Spontaneous tumor-specific humoral and cellular immune responses to NY-ESO-l in Hepatocellular carcinoma.Clin Cancer Res, 2004, 10:4332-4341Oh BK., et al.DNA methyltransferase expression and DNA methylation in human hepatocellular carcinoma and their clinicopathological correlation].Although have been reported title, the expression of CTA and HLA gene is all by Regulation by Methylation, by suppressing DNMT, can promote that it expresses [Guo ZS., et al.De novo induction of a Cancer/Testis Antigen by5-Aza-2'-Deoxycytidine Augments Adoptive Immunotherapy in a Murine Tumor Mode.Cancer Res., 2006; 66:1105-1113].But whether CTA, HLA be also not clear by Regulation by Methylation in liver cancer cell.
Recent research shows, in Mammals, siRNA RNA (siRNA) is by disturbing the RNA of biologically conserved structure, thus suppresses expression of target gene.RNA interference effect (RNAi) technology is considered to one of a kind of method of the most effective specific downregulation expression of target gene [Noori-Daloii MR., et al.Use of siRNA in knocking down of dopamine receptors, a possible therapeutic option in neuropsychiatric disorders.Molecular biology reports2012; 39:2003-2010].Interference tiny RNA (siRNA) be RNA interference effect (RNAi) rely occur important intermediate effect molecule.SiRNA is special double-stranded RNA (dsRNA) molecule that a class is about 21 ~ 25 Nucleotide (nt), has characteristic structural, and namely the sequence of siRNA has homology with the said target mrna sequence acted on; SiRNA two single stranded end are 5 ' end phosphoric acid and 3 ' terminal hydroxy group.SiRNA can be used as a kind of special primer, is templated synthesis dsRNA with said target mrna under rna dependent rna polysaccharase (RdRp) effect, and the latter can be degraded and form new siRNA.Newborn dsRNA repeatedly synthesizes and degrades, and constantly produces new siRNA, thus makes the gradual minimizing of said target mrna, present gene silencing phenomenon.
Whether methylated regulation and control are subject in order to probe into CTA and HLA genetic expression in liver cancer cell, we utilize siRNA perturbation technique, reticent DNMT gene, detects the expression of CTA and HLA in liver cancer cell, thus provides new treatment means for the immunogenicity recovering liver cancer cell.
Summary of the invention
The object of the invention is to openly a kind of efficiently for the wide spectrum small molecules interference RNA (siRNA) of DNMT gene, sequence is as follows:
siRNA1：
Positive-sense strand: 5'-UCAGGAACGCGCACUGAAAtt-3 ' (SEQ ID NO:1),
Antisense strand: 5'-UUUCAGUGCGCGUUCCMGAtt-3 ' (SEQ ID NO:2).
Another object of the present invention is to open a kind of Double-stranded siRNA molecules composition, said composition comprises above-mentioned siRNA1, also comprises siRNA2 and/or siRNA3; Wherein siRNA2 comprises positive-sense strand and antisense strand, the sequence of siRNA2 positive-sense strand is: 5'-CAGAUGUUCUUCGCUAAUAtt-3 ' (SEQ ID NO:3), and the sequence of siRNA2 antisense strand is: 5'-UAUUAGCGAAGAACAUCUGtt-3 ' (SEQ ID NO:4); Wherein siRNA3 comprises positive-sense strand and antisense strand, the sequence of siRNA3 positive-sense strand is: 5 '-GAUCAGAGCCGAGAACAAAtt-3 ' (SEQ ID NO:5), and the sequence of siRNA3 antisense strand is: 5'-UUUGUUCUCGGCUCUGAUCtt-3 ' (SEQ ID NO:6).
The invention provides double-strand siRNA or the application of Double-stranded siRNA molecules composition in preparation reduction cell DNMT genetic expression reagent, preferably, DNMT is DNMT1, DNMT3a or DNMT3b.
The invention provides double-strand siRNA or the application of Double-stranded siRNA molecules composition in preparation promotion cell CTA protein expression reagent, preferably, CTA albumen is CT10.
The invention provides double-strand siRNA or the application of Double-stranded siRNA molecules composition in preparation promotion cell HLA-I proteinoid expression reagent, preferably, HLA-I proteinoid is HLA-A, HLA-B or HLA-C.
Preferably, the cell that the present invention uses is tumour cell.
Experiment in vitro proves, the antisense strand of siRNA molecule of the present invention can be combined with the mRNA of DNMT gene specifically, degraded mRNA, thus disturb the translation process after transcribing, and then the expression of CTA and the HLA albumen of promotion DNMT protein regulation, strengthen the toxic reaction of immunocyte, disappearing of inducing tumor cell, reach the object for the treatment of tumour.
The invention has the beneficial effects as follows: siRNA molecule of the present invention can be applied to preparation and regulate the effect playing RNA interference in the medicine of DNMT gene in cell, strengthen the toxic reaction of immunocyte, inducing tumor cell disappears, and reaches the object for the treatment of tumour.
Accompanying drawing explanation
Fig. 1 shows the siRNA of chemosynthesis to the detected result of DNMT Gene silencing efficacy, wherein
Fig. 1 a shows and utilizes QPCR to detect siRNA to the silencing efficiency of DNMT gene,
Fig. 1 b shows the silencing efficiency utilizing immune-blotting method siRNA to DNMT gene,
Fig. 2 shows the impact utilizing the siRNA of immune-blotting method chemosynthesis on CTA genetic expression,
Fig. 3 shows the siRNA of chemosynthesis to the impact of HLA genetic expression, wherein
Fig. 3 a shows and utilizes QPCR to detect siRNA to the impact of HLA genetic expression,
Fig. 3 b shows the impact utilizing immune-blotting method siRNA on HLA genetic expression,
Embodiment
Embodiments of the invention are described below in detail.Being exemplary below by the embodiment be described with reference to the drawings, only for explaining the present invention, and can not limitation of the present invention being interpreted as.
Embodiment 1: the siRNA of chemosynthesis is to the test experience of DNMT Gene silencing efficacy
1. key instrument, reagent and material.
1.1 instruments: PCR instrument (ABI company); Real-time PCR (Bio-Rad); Cell culture incubator (Thermo), gel imaging instrument (Alpha Innothech) etc.
1.2 materials and reagent: mRNA extraction purification test kit (QIAGEN), liposome 2000 transfection reagent (Invitrogen), DMEM substratum (Gibco), Oligo (dT) (Promega), M-MLV (Promega), Tap enzyme (Takara) etc.
2. synthesize siRNA
DNMT1 is found, the gene order (NO:NM-001379, NO:NM-175629, NO:NM-006892) of DNMT3a, DNMT3b in Genebank.By upper sea base, triumphant biotech firm designs and chemosynthesis, each gene design a pair siRNA sequence, and 2.0OD (5nmol) * 2, and the interference sequence and the negative control that have positive control β-actin are as follows:
siRNA1：
Positive-sense strand: 5'-UCAGGAACGCGCACUGAAAtt-3 ' (SEQ ID NO:1),
Antisense strand: 5'-UUUCAGUGCGCGUUCCUGAtt-3 ' (SEQ ID NO:2).
siRNA2：
Positive-sense strand: 5 '-CAGAUGUUCUUCGCUAAUAtt-3 ' (SEQ ID NO:3),
Antisense strand: 5'-UAUUAGCGAAGAACAUCUGtt-3 ' (SEQ ID NO:4).
siRNA3：
Positive-sense strand: 5 '-GAUCAGAGCCGAGAACAAAtt-3 ' (SEQ ID NO:5),
Antisense strand: 5'-UUUGUUCUCGGCUCUGAUCtt-3 ' (SEQ ID NO:6).
β-actin siRNA：
Positive-sense strand: 5'-ACCAGGUGCUACGCUCAGAAAtt-3 ',
Antisense strand: 5'-UCUGACUACUGUGAGGUCUtt-3 '.
Negative control (NC-siRNA)
Positive-sense strand: 5'-UUAAGUAGCUUGGCCUUGAtt-3 ',
Antisense strand: 5'-UCAAGGCCAAGCUACUUAAtt-3 '.
3. silence efficiency checking
3.1 cell cultures: hepatocellular carcinoma H22 containing 10%FBS DMEM substratum in, 37 DEG C, 5%CO
2incubator is cultivated.
3.2 plating cells transfection: by cell by 1 × 10
4/ hole is inoculated in 24 porocyte culture plates, at 37 DEG C, 5%CO
2incubator cell cultures 24 hours, containing in the DMEM substratum of 10%FBS without dual anti-, transfection is according to the specification sheets transfection of lipofectamine 2000 (purchased from Invitrogen company), experiment is divided into untransfected group, negative control group and experimental group (80nM), wherein negative control group siRNA is universal control siRNA, i.e. NC-siRNA, with the sequence of DNMT gene without homology, concentration is 80nM/ hole.Transfection respectively simultaneously.
3.3RT-PCR detects DNMT gene mRNA levels: with mRNA extraction purification test kit (TurboCapture mRNA kit) extraction purification cell RNA, operation is undertaken by test kit specification sheets, RNA is dissolved without RNA enzyme water/hole with 80 μ L, get 2 μ gRNA and add 0.6 μ LOligo (dT), aseptic DEPC water supplements volume to 20 μ L, 70 DEG C of sex change 10min.The ice-cold at least 5min of rapid insertion; Operate on ice, add following reagent: 6.0 μ L5*buffer, 5.0 μ L2.5 μM dNTPs, 1.0 μ L RNasin (40U/ μ L) and 1.0 μ L M-MLV are after mixing, of short duration centrifugal, hatch 1h for 37 DEG C, 70 DEG C of 15min stopped reactions, detect DNMT mrna expression level in sample with gene-specific primer, and the house-keeping gene β-actin that simultaneously increases is as internal reference contrast (primer sequence is as shown in table 1).Each reaction do 3 parallel.Set up following 25 μ L reaction systems: 1 μ L template cDNA, 0.5 μ L Tap E, the forward and reverse primer of each 1 μ L5 ' (0.5 μM), 2.5 μ L10* reaction buffers, 2.5 μ L dNTP (0.2mM), supply system to 25 μ L with without RNA enzyme water.Reaction conditions: 95 DEG C of denaturation 10min, 94 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C extend 45s, circulate 36 times, fully extend 72 DEG C of 10min.Each PCR sample of equal volume carries out agarose gel electrophoresis (gum concentration: 1%).Wherein DNMT gene
Interpretation of result: from RT-PCR electrophoresis result: through the HepG2 cell of RNA interference, in corresponding group, all decrease, the DNMT of untreated fish group and simulation control group changes no significant difference, and data do not show.
3.4 fluorescence real-time quantitative PCRs (QPCR) detect DNMT gene mRNA levels: detect DNMT mrna expression level (primer sequence is as shown in table 1) in sample with gene-specific primer, and the house-keeping gene β-actin that simultaneously increases contrasts as internal reference.Each reaction do 3 parallel.Set up following 25 μ L reaction systems: 1.0 μ L template eDNA, the forward and reverse primer of each 1 μ L5 ' (0.5 μM), 1.0 μ L20 × Ever Green I, 0.5 μ L Hotstar TapE (5U/ μ L), supplies system to 25 μ L with without RNA enzyme water.Reaction conditions: 40 DEG C of reverse transcription 30min, 95 DEG C of denaturation 5min, 94 DEG C of sex change 20s, 60 DEG C of annealing 45s, 72 DEG C extend 45s, circulate 40 times, fully extend 72 DEG C of 10min.
Interpretation of result: with β-actin internal reference, with real-time quantitative PCR 2
-Δ Δ ctmethod determines DNMT mrna expression amount, and makes histogram, the results are shown in Figure 1a.As shown in the figure, ordinate zou represents the mrna expression level of DNMT relative to β-actin; X-coordinate represents 3 process experimental group, wherein " simulation transfection group " represents the negative control of transfection concentrations 80nM NC-siRNA, other two groups is siRNA1 transfection experiment group, " siRNA1 " represents a kind of DNMT RNA interfering of independent transfection experimental group, " siRNA1+2+3 " represents cotransfection three kinds of DNMT RNA interfering experimental group, and it is obvious that result shows the silencing efficiency of siRNA of the present invention to DNMT gene.
3.5 immunoblottings (western blot) detected DNMT expression of gene protein level: according to after the method transfectional cell of 3.2 72 hours, with cell pyrolysis liquid (150mM sodium-chlor+1%NP-40 (washing agent)+0.1%SDS (washing agent)+2 μ g/ml Aprotinin (proteinase inhibitor) (adding before use)+2 μ g/ml Leupeptin (proteinase inhibitor) (adding before use) or 1mM PMSF (proteinase inhibitor)+1.5mM EDTA (proteinase inhibitor)+1mMNaVanadate (phospholipase inhibitor)) collecting cell, the mensuration of protein concentration is carried out with BCA protein detection kit, each experimental group is got 50 μ g albumen and is carried out SDS-PAGE, transferring film, 5% skim-milk is closed, add DNMT1 antibody respectively, DNMT3a antibody, DNMT3b antibody detects, with house keeping protein Tubulin for internal reference, observe the expression of DNMT albumen.
Interpretation of result: use the method for western blot to detect the expression level of each experimental group DNMT, DNMT1 is the biobelt of a 190kDa, consistent with the description of antibody specification sheets, all have obvious reduction in the group disturbed there being DNMT1, the amplitude especially reduced in DNMT1+3a+3b group is more obvious; Then without reduction in simulation transfection group.Expression and the DNMT1 of DNMT3a and DNMT3b are similar, in the group disturbed there being DNMT, without corresponding band, illustrate that DNMT RNA interfering that the present invention designs significantly can reduce the expression level of DNMT albumen, three kinds of DNMT RNA interfering mixed effects more obviously (Fig. 1 b).
Embodiment 2:DNMT gene silencing is on the impact of CTA genetic expression
1.RT-PCR detects the change of CTA gene transcription level
1.1 key instruments, reagent and consumptive material are with embodiment 1
1.2RT-PCR experimental procedure is with embodiment 1 (corresponding primer sequence is in table 1)
Interpretation of result: in HepG2 cell, after using liposome siNRA to disturb DNMT gene, visible CT10 and SSX1 two genes have expressing again in various degree in siRNA1 group and mixing interference group (siRNA1+2+3), for untreated fish group with simulate transfection group then without respective strap (data do not show).MAGE-1 and MAGE-3 has no significant change in each group.NY-ESO-1 is also without considerable change, and its excess-three kind CTA, CTp11, SCP1 and SSX4 do not detect expression (data do not show).
1.3 fluorescence real-time quantitative PCRs (QPCR) experimental procedure is with embodiment 1 (corresponding primer sequence is in table 1)
Interpretation of result: QPCR detection display, MAGE1 is without considerable change.The trend of expressing again of CT10 and SSX1 obviously (data do not show).
2.Western blot detects the change of CTA expression of gene protein level
The step of Westernblot, with embodiment 1, utilizes antibody for the CT10 protein antibodies in CTA albumen.
Interpretation of result: the expression of CT10 albumen is consistent with the result of RT-PCR, has in siRNA1 group and significantly expresses again, in the effect more obvious (Fig. 2) that mixing interference group (siRNA1+2+3) is expressed.
The above results shows, siRNA1 group facilitates the expression of CT10 albumen in CTA molecule and SSX1, and mixing interference group (siRNA1+2+3) effect is more obvious.
Embodiment 3:DNMT gene silencing is on the impact of HLA genetic expression
1.RT-PCR detects the change of HLA gene transcription level
1.1 key instruments, reagent and consumptive material are with embodiment 1
1.2RT-PCR experimental procedure is with embodiment 1 (corresponding primer sequence is in table 1)
Interpretation of result: in HepG2 cell, after using liposome siNRA to disturb DNMT gene, HLA-A, HLA-B, HLA-C gene in visible HLA-I quasi-molecule has expressing again in various degree in siRNA1 group and mixing interference group (siRNA1+2+3), HLA-II quasi-molecule and CIITA gene are all without band, and data do not show.
1.3 fluorescence real-time quantitative PCRs (QPCR) experimental procedure is with embodiment 1 (corresponding primer sequence is in table 1)
Interpretation of result: to simulate the relative expression of transfection group three kinds of HLA-I quasi-molecules for 1.In siRNA1 group and mixing interference group (siRNA1+2+3), relative expression's degree of HLA-A is respectively 5 and 8; Relative expression's degree of HLA-B is respectively 2.5 and 6; Relative expression's degree of HLA-C is respectively 30 and 45, and (Fig. 3 a).
2.Western blot detects the change of HLA expression of gene protein level
The step of Western blot, with embodiment 1, utilizes antibody for HLA-I proteinoid antibody.
Interpretation of result: the expression of HLA-I proteinoid is consistent with the result of RT-PCR, has in siRNA1 group and significantly expresses again, in the effect more obvious (Fig. 3 b) that mixing interference group (siRNA1+2+3) is expressed again.
The above results shows, siRNA1 group facilitates HLA-I quasi-molecule and expresses, and mixing interference group (siRNA1+2+3) effect is more obvious.
The detection of the promoter methylation state of embodiment 4:CTA and HLA
1. instrument: PCR instrument (ABI company), gel imaging instrument (Alpha Innothech)
2. material and reagent: DNA extraction kit (QIAGEN), purification column (Promega), Tap enzyme (Takara) etc.
3. experimental procedure
3.1DNA extracts: the step utilizing DNA extraction kit (QIAGEN) to go up to specifications extracts cell STb gene.
3.2DNA modifies: get 1-10 μ gDNA, be dissolved in 18 μ L sterilized waters, 95 DEG C of water-bath sex change 20min, ice bath; Add 3M sodium hydroxide 2 μ L (mixing, pulse is centrifugal), 42 DEG C of water-bath sex change 20min, preparation 5M sodium bisulfite treatment solution (every increment product 0.5ml, to prepare 4ml): get 1.9g sodium bisulfite and add in 2.5ml sterilized water, add 2M sodium hydroxide 0.7ml, add 1M Resorcinol (110mg/ml) 0.5ml, 50 DEG C of water-baths, are inverted repeatedly until dissolve completely; Freshly prepared 5M sodium bisulfite treatment solution 380 μ L (mixing, pulse is centrifugal) is added to above-mentioned 20 μ L DNA sex change liquid; Add 200 μ L paraffin oils with vaporization prevention, 50 DEG C of water-bath 12-16h;
3.3DNA purification desalination: install DNA purification column, mark; The sample of above-mentioned process is added in purification column (Promega), vacuum take-off; Add 80% washed with isopropyl alcohol secondary; 12000rpm is centrifugal, removes residual liquid; DNA is eluted to (80 DEG C of water, 80 DEG C of temperature bathe 5min, centrifugal) in 50 μ L sterilized waters; Add 3M sodium hydroxide 1 μ L, 37 DEG C of water-bath 15min; Add 5M ammonium acetate 166 μ L; Add the dehydrated alcohol of 2.5 times of volumes ,-70 DEG C of precipitation 0.5-1h; The centrifugal 20min of 10000rpm, abandons supernatant; 70% ethanol 200 μ L washes precipitation, and centrifugal 20min, abandons supernatant; DNA is dissolved in 30-50 μ L TE damping fluid ,-20 DEG C of preservations.
The PCR reaction 3.4 methylate
3.4.1 design of primers: the methylated primers sequence of HLA-I quasi-molecule is with reference to [Nie Y. such as Nie, et al.DNA hypermethylation is a mechanism for loss of expression of HLA CTAss I genes in human esophageal squamous cell carcinomas.Carcinogenesis, 2001; 22:1615-1623] the primer of document and reaction system.MAGE primer sequence is with reference to [HondaT., et al.Demethylation of MAGE promotors during gastric cancer progression.British Journal of cancer, 2004 such as Honda; 90:838-843] document.The methylated primers designed, designed of CT10, download the First Exon of CT10 gene from www.genecards.org website before, 1000bp sequence and First Exon itself amount to 1190bp; Enter www.methprimer.com, copying this section of sequence, select MSP primer, namely exporting 5 online to methylating and non-methylated primers.Select first to methylate and non-methylated primers test (primer sequence is shown in Table 2).
3.4.2 reaction system is prepared
In 200 μ L PCR pipe, add following composition, and mix.
3.4.3 reaction conditions is set
95 DEG C of sex change 15min, subsequently 94 DEG C of sex change 2min, 60 DEG C of annealing 2min, 72 DEG C extend 2min, 4 circulations of increasing, then 94 DEG C of sex change 30s, 60 DEG C of annealing 45s, and 72 DEG C extend 20s, 30-35 circulation of increasing; Finally continue to extend 10min.Methylated primers and isogenic non-methylated primers use identical condition.
3.4.4PCR product gel electrophoresis
Prepare 1.5% sepharose, get PCR primer 5 μ L, 6* sample-loading buffer 1 μ L, Marker4 μ L.90mv, 30-40min, observe electrophoresis situation under ultraviolet light, stops electrophoresis when Marker distalmost end migrates to gel 3/4 length.
Interpretation of result: experiment find CT10 promoter region be in methylated, through DNMT interference after, the non-band that methylates in various degree can be detected, meanwhile, significantly methylate band in addition.The promoter region of MAGE-1 is the non-band that methylates, and does not see the band that methylates (data do not show).
HLA-A can detect the non-band that methylates and methylate, HLA-B and HLA-C has the non-band that methylates, for the band that methylates (data do not show) being detected.
Table 1RT-PCR primer sequence, condition and length
Table 2 methylated primers, sequence and length
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (8)
1. a Double-stranded siRNA molecules, is characterized in that, is made up of the positive-sense strand of following nucleotide sequence and antisense strand:
Positive-sense strand: 5 '-UCAGGAACGCGCACUGAAAtt-3 ',
Antisense strand: 5 '-UUUCAGUGCGCGUUCCUGAtt-3 '.
2. a Double-stranded siRNA molecules composition, is characterized in that, described composition is made up of siRNA, siRNA2 and siRNA3 according to claim 1; Wherein said siRNA2 comprises positive-sense strand and antisense strand, and the sequence of siRNA2 positive-sense strand is: the sequence of 5 '-CAGAUGUUCUUCGCUAAUAtt-3 ', siRNA2 antisense strand is: 5 '-UAUUAGCGAAGAACAUCUGtt-3 '; Wherein said siRNA3 comprises positive-sense strand and antisense strand, and the sequence of siRNA3 positive-sense strand is: the sequence of 5 '-GAUCAGAGCCGAGAACAAAtt-3 ', siRNA3 antisense strand is: 5 '-UUUGUUCUCGGCUCUGAUCtt-3 '.
3. the application of double-strand siRNA according to claim 1 in preparation reduction hepatocellular carcinoma H22 in DNMT genetic expression reagent.
4. the application of double-strand siRNA according to claim 1 in preparation promotion hepatocellular carcinoma H22 in CTA protein expression reagent.
5. in preparation, double-strand siRNA according to claim 1 promotes that in hepatocellular carcinoma H22, HLA-I proteinoid expresses the application in reagent.
6. the application described in claim 3, is characterized in that, described DNMT is DNMT1, DNMT3a or DNMT3b.
7. the application described in claim 4, is characterized in that, described CTA albumen is CT10.
8. the application described in claim 5, is characterized in that, described HLA-I proteinoid is HLA-A, HLA-B or HLA-C.
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