CN103757021A - 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
- Publication number
- CN103757021A CN103757021A CN201410021188.8A CN201410021188A CN103757021A CN 103757021 A CN103757021 A CN 103757021A CN 201410021188 A CN201410021188 A CN 201410021188A CN 103757021 A CN103757021 A CN 103757021A
- Authority
- CN
- China
- Prior art keywords
- double
- sirna
- dnmt
- hla
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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 biological medicine technology field, be specifically related to double-stranded siRNA molecule and application thereof, more specifically, the present invention is directed to the synthetic siRNA of nucleotide sequence design of human tumor cells DNMT gene, described siRNA proceeds to the obviously expression of Promote immunity originality associated protein after liver cancer cell.
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 expression of regulating immune molecules and tumour antigen and the immunogenicity of tumour and the immunoreactivity of methylating has clear and definite relation, therefore, reasonably demethylation is the effective means [Teitella that overcomes 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 at present the gene product of 19 CTA families, all there is stronger immunogenicity (as CT10 (cancer/testis10), there is the immune epitope of multiple HLA-I (human leukocyte antigen) I class antigen restriction, experiment in vivo and vitro confirms lymphocytotoxicity reaction that can inducing function, promote disappearing of tumour, in HCC patient's liver cancer tissue, CTA expresses very high [Zhao L, et al.Progress and prospects in hepatocellular carcinoma surgery.Ann Chir, 1988, 52:558-563], therefore for the anti-tumor immunotherapy of CTA, meet liver cancer and other tumour patients carry out immune ideal chose [Chen YT., 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 subject to Regulation by Methylation, by suppressing DNMT, can promote it to express [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 are subject to Regulation by Methylation also not clear in liver cancer cell.
Recently research shows, in Mammals, siRNA RNA (siRNA) can be by the RNA of conserved structure is biologically disturbed, thereby suppress expression of target gene.RNA interference effect (RNAi) technology is considered to the most effective a kind of specificity and lowers one of the method for 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].Disturb little 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 structure, and the sequence of siRNA has homology with the said target mrna sequence acting on; Two strand ends of siRNA are 5 ' end phosphoric acid and 3 ' terminal hydroxy group.SiRNA can be used as a kind of special primer, synthesizes dsRNA under rna dependent rna polysaccharase (RdRp) effect take said target mrna as template, and the latter can be degraded and form new siRNA.Newborn dsRNA is synthetic and degraded repeatedly, constantly produces new siRNA, thereby makes the gradual minimizing of said target mrna, presents gene silencing phenomenon.
In order to probe into CTA and HLA genetic expression in liver cancer cell, whether be subject to methylated regulation and control, we utilize siRNA perturbation technique, reticent DNMT gene, detects CTA and the expression of HLA in liver cancer cell, thereby provides new treatment means for the immunogenicity of recovering liver cancer cell.
Summary of the invention
The object of the invention is to openly a kind of efficient wide spectrum small molecules interference RNA (siRNA) for 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 the double-stranded siRNA molecular composition of open one, 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), the sequence of siRNA3 antisense strand is: 5'-UUUGUUCUCGGCUCUGAUCtt-3 ' (SEQ ID NO:6).
The invention provides double-stranded siRNA or double-stranded siRNA molecular composition and reduce the application in cell DNMT genetic expression reagent in preparation, preferably, DNMT is DNMT1, DNMT3a or DNMT3b.
The invention provides double-stranded siRNA or double-stranded siRNA molecular composition and promote the application in cell CTA protein expression reagent in preparation, preferably, CTA albumen is CT10.
The invention provides double-stranded siRNA or double-stranded siRNA molecular composition and promote cell HLA-I proteinoid to express the application in reagent in preparation, 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, thereby disturb the translation process after transcribing, and then the CTA of promotion DNMT protein regulation and the expression of HLA albumen, strengthen the toxic reaction of immunocyte, disappearing of inducing tumor cell, reaches 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 prepare in the medicine that regulates DNMT gene in cell brings into play the effect that RNA disturbs, and strengthens the toxic reaction of immunocyte, and inducing tumor cell disappears, and reaches the object for the treatment of tumour.
Accompanying drawing explanation
The detected result of the siRNA that Fig. 1 has shown chemosynthesis to DNMT Gene silencing efficacy, wherein
Fig. 1 a shown and utilizes QPCR to detect the reticent effect of siRNA to DNMT gene,
Fig. 1 b shown and utilizes immunoblotting to detect the reticent effect of siRNA to DNMT gene,
Fig. 2 has shown the impact of the siRNA that utilizes immunoblotting to detect chemosynthesis on CTA genetic expression,
The impact of the siRNA that Fig. 3 has shown chemosynthesis on HLA genetic expression, wherein
Fig. 3 a shown and utilizes QPCR to detect the impact of siRNA on HLA genetic expression,
Fig. 3 b shown and utilizes immunoblotting to detect the impact of siRNA on HLA genetic expression,
Embodiment
Describe embodiments of the invention below in detail.Below by the embodiment being described with reference to the drawings, be exemplary, only for explaining the present invention, and can not be interpreted as limitation of the present invention.
Embodiment 1: the test experience of the siRNA of chemosynthesis to 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 extracts purification kit (QIAGEN), liposome 2000 transfection reagents (Invitrogen), DMEM substratum (Gibco), Oligo (dT) (Promega), M-MLV (Promega), Tap enzyme (Takara) etc.
2. synthetic siRNA
In Genebank, find DNMT1, DNMT3a, the gene order (NO:NM-001379, NO:NM-175629, NO:NM-006892) of DNMT3b.By the design of the triumphant biotech firm of upper sea base and chemosynthesis, a pair of siRNA sequence of each gene design, * 2 of 2.0OD (5nmol), and have interference sequence and the negative control of positive control β-actin, 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: liver cancer cell HepG2 is containing in the DMEM substratum of 10%FBS, 37 ℃, 5%CO
2incubator is cultivated.
3.2 cell bed board transfections: cell is pressed to 1 × 10
4/ hole is inoculated in 24 porocyte culture plates, at 37 ℃, 5%CO
2incubator cell cultures 24 hours, without dual anti-containing in the DMEM substratum of 10%FBS, 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 general contrast siRNA, be NC-siRNA, without homology, concentration is 80nM/ hole with the sequence of DNMT gene.Transfection respectively simultaneously.
3.3RT-PCR detects DNMT gene mRNA level: with mRNA, extract purification kit (TurboCapture mRNA kit) and extract purifying cells RNA, operation is undertaken by test kit specification sheets, without RNA enzyme water/hole, dissolve RNA 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 ℃ of sex change 10min.Insert rapidly ice-cold at least 5min; Operation on ice, add following reagent: 6.0 μ L5*buffer, 5.0 μ L2.5 μ M 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 ℃, 70 ℃ of 15min stopped reactions, by DNMT mrna expression level in gene-specific primer detection sample, 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 μ M), 2.5 μ L10* reaction buffers, 2.5 μ L dNTP (0.2mM), use without RNA enzyme water and supply system to 25 μ L.Reaction conditions: 95 ℃ of denaturation 10min, 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 45s, circulate 36 times, fully extend 72 ℃ 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: the HepG2 cell disturbing through RNA, 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 level: by DNMT mrna expression level (primer sequence is as shown in table 1) in gene-specific primer detection sample, 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 μ M), 1.0 μ L20 × Ever Green I, 0.5 μ L Hotstar TapE (5U/ μ L), uses without RNA enzyme water and supplies system to 25 μ L.Reaction conditions: 40 ℃ of reverse transcription 30min, 95 ℃ of denaturation 5min, 94 ℃ of sex change 20s, 60 ℃ of annealing 45s, 72 ℃ are extended 45s, circulate 40 times, fully extend 72 ℃ of 10min.
Interpretation of result: with β-actin internal reference, with real-time quantitative PCR 2
-Δ Δ ctmethod is determined 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 with respect to β-actin; X-coordinate represents that 3 are processed experimental group, wherein " simulation transfection group " represents the negative control of transfection concentration 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 three kinds of DNMT RNA interfering experimental group of cotransfection, and result shows the reticent successful of siRNA of the present invention to DNMT gene.
3.5 immunoblottings (western blot) detected DNMT expression of gene protein level: according to after 3.2 method transfectional cell 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, with BCA protein detection kit, carry out the mensuration of protein concentration, each experimental group is got 50 μ g albumen and is carried out SDS-PAGE, transferring film, 5% skim-milk sealing, add respectively DNMT1 antibody, DNMT3a antibody, DNMT3b antibody detects, take house keeping protein Tubulin as 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, in the group that has DNMT1 to disturb, all there is obvious reduction, the amplitude especially reducing in DNMT1+3a+3b group is more obvious; Simulation transfection group in without reduction.Expression and the DNMT1 of DNMT3a and DNMT3b are similar, in the group that has DNMT to disturb, without corresponding band, illustrate that the DNMT RNA interfering of the present invention's design can significantly reduce the expression level of DNMT albumen, more obviously (Fig. 1 is b) for three kinds of DNMT RNA interfering mixed effects.
The impact of embodiment 2:DNMT gene silencing on CTA genetic expression
1.RT-PCR detects the variation 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, use liposome siNRA to disturb after DNMT gene, two genes of visible CT10 and SSX1 have expressing again in various degree in siRNA1 group with in mixing interference group (siRNA1+2+3), for untreated fish group and simulation transfection group are without respective strap (data do not show).MAGE-1 and MAGE-3 have 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 is (data do not show) obviously.
2.Western blot detects the variation 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 significantly and expresses in siRNA1 group, is mixing more obviously (Fig. 2) of effect of interference group (siRNA1+2+3) expression.
The above results shows, siRNA1 group has promoted CT10 albumen in CTA molecule and the expression of SSX1, mixes interference group (siRNA1+2+3) effect more obvious.
The impact of embodiment 3:DNMT gene silencing on HLA genetic expression
1.RT-PCR detects the variation 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, use liposome siNRA to disturb after DNMT gene, HLA-A, HLA-B in visible HLA-I quasi-molecule, HLA-C gene have expressing again in various degree in siRNA1 group with in 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: take the relative expression that simulates three kinds of HLA-I quasi-molecules of transfection group as 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 variation 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 significantly and expresses in siRNA1 group, and more obviously (Fig. 3 b) for the effect of expressing again in mixing interference group (siRNA1+2+3).
The above results shows, siRNA1 group has promoted HLA-I quasi-molecule to express, and mixes interference group (siRNA1+2+3) effect 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 test kit (QIAGEN), purification column (Promega), Tap enzyme (Takara) etc.
3. experimental procedure
3.1DNA extracts: utilize the step that DNA extraction test kit (QIAGEN) is gone up to specifications to extract the total DNA of cell.
3.2DNA modifies: get 1-10 μ gDNA, be dissolved in 18 μ L sterilized waters 95 ℃ of water-bath sex change 20min, ice bath; Add 3M sodium hydroxide 2 μ L (to mix, pulse is centrifugal), 42 ℃ of water-bath sex change 20min, preparation 5M sodium bisulfite treatment solution (every duplicate samples 0.5ml, take preparation 4ml as example): 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 ℃ of water-baths, are inverted repeatedly until dissolve completely; To above-mentioned 20 μ L DNA sex change liquid, add freshly prepared 5M sodium bisulfite treatment solution 380 μ L (mix, pulse is centrifugal); Add 200 μ L paraffin oils with vaporization prevention, 50 ℃ of water-bath 12-16h;
3.3DNA purification desalination: DNA purification column is installed, mark; The sample of above-mentioned processing is added in purification column (Promega) to vacuum take-off; Add 80% washed with isopropyl alcohol secondary; 12000rpm is centrifugal, removes residual liquid; DNA is eluted to (80 ℃ of water, 80 ℃ of temperature are bathed 5min, centrifugal) in 50 μ L sterilized waters; Add 3M sodium hydroxide 1 μ L, 37 ℃ of water-bath 15min; Add 5M ammonium acetate 166 μ L; Add the dehydrated alcohol of 2.5 times of volumes ,-70 ℃ of precipitation 0.5-1h; The centrifugal 20min of 10000rpm, abandons supernatant; 70% ethanol 200 μ L wash precipitation, and centrifugal 20min, abandons supernatant; DNA is dissolved in 30-50 μ L TE damping fluid to-20 ℃ of preservations.
The PCR reaction 3.4 methylate
3.4.1 design of primers: the primer sequence that methylates 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] primer and the reaction system of document.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 primer designed, designed that methylates of CT10, from the First Exon of www.genecards.org website download CT10 gene, 1000bp sequence and First Exon itself amount to 1190bp; Enter www.methprimer.com,, copy this section of sequence, select MSP primer, export online 5 pairs and methylate and the non-primer that methylates.Select first pair methylate and the non-primer that methylates test (primer sequence is shown in Table 2).
3.4.2 prepare reaction system
In 200 μ L PCR pipes, add following composition, and mix.
3.4.3 set reaction conditions
95 ℃ of sex change 15min, 94 ℃ of sex change 2min subsequently, 60 ℃ of annealing 2min, 72 ℃ are extended 2min, 4 circulations of increasing, then 94 ℃ of sex change 30s, 60 ℃ of annealing 45s, 72 ℃ are extended 20s, 30-35 the circulation of increasing; Finally continue to extend 10min.Methylate primer and the isogenic non-primer that methylates used identical condition.
3.4.4PCR product gel electrophoresis
Prepare 1.5% sepharose, get PCR product 5 μ L, 6* sample-loading buffer 1 μ L, Marker4 μ L.90mv, 30-40min observes electrophoresis situation under UV-light, and Marker distal-most end stops electrophoresis while migrating to gel 3/4 length.
Interpretation of result: experiment finds that CT10 promoter region is in methylated, after DNMT disturbs, the non-band that methylates in various degree can be detected, and meanwhile, band in addition significantly methylates.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, and HLA-B and HLA-C have 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 methylate primer, sequence and length
Although illustrated and described embodiments of the invention, those having ordinary skill in the art will appreciate that: in the situation that not departing from principle of the present invention and aim, can carry out multiple variation, modification, replacement and modification to these embodiment, scope of the present invention is limited by claim and equivalent thereof.
Claims (10)
1. a double-stranded siRNA molecule, is characterized in that, has positive-sense strand and the antisense strand composition of following nucleotide sequence:
Positive-sense strand: 5'-UCAGGAACGCGCACUGAAAtt-3 ' (SEQ ID NO:1),
Antisense strand: 5'-UUUCAGUGCGCGUUCCUGAtt-3 ' (SEQ ID NO:2).
2. a double-stranded siRNA molecular composition, is characterized in that, described composition comprises siRNA claimed in claim 1.
3. siRNA molecular composition claimed in claim 2, is characterized in that, described siRNA molecular composition also comprises siRNA2 and/or siRNA3; Wherein said 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 said siRNA3 comprises positive-sense strand and antisense strand, the sequence of siRNA3 positive-sense strand is: 5'-GAUCAGAGCCGAGAACAAAtt-3 ' (SEQ ID NO:5), the sequence of siRNA3 antisense strand is: 5'-UUUGUUCUCGGCUCUGAUCtt-3 ' (SEQ ID NO:6).
4. in claim 1-3, the double-stranded siRNA described in any one or double-stranded siRNA molecular composition reduce the application in cell DNMT genetic expression reagent in preparation.
5. in claim 1-3, the double-stranded siRNA described in any one or double-stranded siRNA molecular composition promote the application in cell CTA protein expression reagent in preparation.
6. in claim 1-3, the double-stranded siRNA described in any one or double-stranded siRNA molecular composition promote cell HLA-I proteinoid to express the application in reagent in preparation.
7. the application described in claim 4, is characterized in that, described DNMT is DNMT1, DNMT3a or DNMT3b.
8. the application described in claim 5, is characterized in that, described CTA albumen is CT10.
9. the application described in claim 6, is characterized in that, described HLA-I proteinoid is HLA-A, HLA-B or HLA-C.
10. the cell described in any one in claim 2 to 5, is characterized in that, described cell is tumour cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410021188.8A CN103757021B (en) | 2014-01-17 | 2014-01-17 | Double-chain siRNA molecule of silencing DNMT (Desoxvribose Nucleic Acid Methyltransferases) gene and application of double-chain siRNA molecule |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410021188.8A CN103757021B (en) | 2014-01-17 | 2014-01-17 | Double-chain siRNA molecule of silencing DNMT (Desoxvribose Nucleic Acid Methyltransferases) gene and application of double-chain siRNA molecule |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103757021A true CN103757021A (en) | 2014-04-30 |
| CN103757021B CN103757021B (en) | 2015-05-20 |
Family
ID=50524340
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410021188.8A Expired - Fee Related CN103757021B (en) | 2014-01-17 | 2014-01-17 | Double-chain siRNA molecule of silencing DNMT (Desoxvribose Nucleic Acid Methyltransferases) gene and application of double-chain siRNA molecule |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103757021B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881168A (en) * | 2017-11-14 | 2018-04-06 | 中国科学院深圳先进技术研究院 | A kind of Primer composition and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101313065A (en) * | 2005-08-01 | 2008-11-26 | 纽珀滕索有限公司 | Production of reprogrammed cells with restored potential |
| WO2011022413A2 (en) * | 2009-08-18 | 2011-02-24 | Nupotential, Inc. | Reprogramming a cell by activation of the endogenous transcription factor network |
-
2014
- 2014-01-17 CN CN201410021188.8A patent/CN103757021B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101313065A (en) * | 2005-08-01 | 2008-11-26 | 纽珀滕索有限公司 | Production of reprogrammed cells with restored potential |
| WO2011022413A2 (en) * | 2009-08-18 | 2011-02-24 | Nupotential, Inc. | Reprogramming a cell by activation of the endogenous transcription factor network |
Non-Patent Citations (4)
| Title |
|---|
| HIROAKI KAWASAKI & KAZUNARI TAIRA: "Induction of DNA methylation and gene silencing by short interfering RNAs in human cells", 《NATURE》 * |
| Z. SHENG GUO等: "De novo Induction of a Cancer/Testis Antigen by 5-Aza-2′-Deoxycytidine Augments Adoptive Immunotherapy in a Murine Tumor Model", 《CANCER RESEARCH》 * |
| 李宏涛等: "DNA甲基转移酶基因干扰对HepG2 细胞癌-睾丸抗原再表达的影响", 《解放军医学杂志》 * |
| 梁伟等: "DNA 甲基转移酶基因干扰对K562 细胞癌- 睾丸抗原表达的影响", 《现代生物医学进展》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881168A (en) * | 2017-11-14 | 2018-04-06 | 中国科学院深圳先进技术研究院 | A kind of Primer composition and its application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103757021B (en) | 2015-05-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Loss of exosomal miR‐148b from cancer‐associated fibroblasts promotes endometrial cancer cell invasion and cancer metastasis | |
| Yu et al. | Baicalein inhibits cervical cancer progression via downregulating long noncoding RNA BDLNR and its downstream PI3 K/Akt pathway | |
| Li et al. | MicroRNA expression and its implications for diagnosis and therapy of gallbladder cancer | |
| Wei et al. | RETRACTED ARTICLE: MicroRNA-330-3p promotes cell invasion and metastasis in non-small cell lung cancer through GRIA3 by activating MAPK/ERK signaling pathway | |
| Wang et al. | Up-regulation of p21WAF1/CIP1 by miRNAs and its implications in bladder cancer cells | |
| Bach et al. | Targeting nicotinamide N-methyltransferase and miR-449a in EGFR-TKI-resistant non-small-cell lung cancer cells | |
| Lin et al. | Circular RNA circ‐EGLN3 promotes renal cell carcinoma proliferation and aggressiveness via miR‐1299‐mediated IRF7 activation | |
| Yamada et al. | Tumor‐suppressive micro RNA‐135a inhibits cancer cell proliferation by targeting the c‐MYC oncogene in renal cell carcinoma | |
| Kong et al. | RUNX3-mediated up-regulation of miR-29b suppresses the proliferation and migration of gastric cancer cells by targeting KDM2A | |
| Jia et al. | MicroRNA-10a is down-regulated by DNA methylation and functions as a tumor suppressor in gastric cancer cells | |
| Wang et al. | ZEB1 activated-VPS9D1-AS1 promotes the tumorigenesis and progression of prostate cancer by sponging miR-4739 to upregulate MEF2D | |
| Han et al. | MicroRNA-33a-3p suppresses cell migration and invasion by directly targeting PBX3 in human hepatocellular carcinoma | |
| Leite et al. | MicroRNA 100: a context dependent miRNA in prostate cancer | |
| Sun et al. | MiR-19 regulates the proliferation and invasion of glioma by RUNX3 via β-catenin/Tcf-4 signaling | |
| Sun et al. | MicroRNA-4465 suppresses tumor proliferation and metastasis in non-small cell lung cancer by directly targeting the oncogene EZH2 | |
| Unlu et al. | Damage associated molecular pattern molecule-induced microRNAs (DAMPmiRs) in human peripheral blood mononuclear cells | |
| Li et al. | MicroRNA-410 promotes cell proliferation by targeting BRD7 in non-small cell lung cancer | |
| Yi et al. | Epigenetically altered miR-1247 functions as a tumor suppressor in pancreatic cancer | |
| Xiong et al. | Role of miR‑34 in gastric cancer: From bench to bedside | |
| Wang et al. | Downregulation of microRNA‑33a promotes cyclin‑dependent kinase 6, cyclin D1 and PIM1 expression and gastric cancer cell proliferation | |
| Lee et al. | miR-203 downregulates Yes-1 and suppresses oncogenic activity in human oral cancer cells | |
| Zong et al. | PCDH8 inhibits glioma cell proliferation by negatively regulating the AKT/GSK3β/β‑catenin signaling pathway | |
| Olbromski et al. | MicroRNAs modulate the expression of the SOX18 transcript in lung squamous cell carcinoma | |
| Wan et al. | miR-125b promotes cell proliferation by directly targeting Lin28 in glioblastoma stem cells with low expression levels of miR-125b | |
| Zhang et al. | LncRNA SNHG7 participates in osteosarcoma progression by down-regulating p53 via binding to DNMT1. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: NO.1 HOSPITAL ATTACHED TO PLA GEN. HOSPITAL Free format text: FORMER OWNER: XIAO WENHUA Effective date: 20140904 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20140904 Address after: 100048 Beijing city Haidian District Fuchengmen Road No. 51 Applicant after: The First Affiliated Hospital of General Hospital of PLA Address before: 100048 Beijing city Haidian District Fuchengmen Road No. 51 Applicant before: Xiao Wenhua |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150520 Termination date: 20190117 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |