CN103757022A - Interfering RNA (ribonucleic acid) molecule and application thereof - Google Patents
Interfering RNA (ribonucleic acid) molecule and application thereof Download PDFInfo
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Abstract
The invention relates to an interfering RNA (ribonucleic acid) molecule and application thereof. The interfering RNA molecule is siRNA which is designed and synthesized against the nucleotide sequence of a human tumor cell DNMT gene. The siRNA molecule comprises a nucleotide sequence sense strand and a nucleotide sequence antisense strand, wherein the sequence of the sense strand is as shown in a sequence table SEQ ID NO:1, and the sequence of the antisense strand is as shown in a sequence table SEQ ID NO:2. When the siRNA molecule is transferred into a liver cancer cell, expression of immunogenicity related proteins can be obviously promoted.
Description
Technical field
The present invention relates to biological medicine technology field, be specifically related to a kind of 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
It is sequence-specific gene silencing phenomenon [Fire A., the et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature1998 of the extensive a kind of double chain RNA mediate existing in body that RNA disturbs (siRNA); 391:806-811], because it has that silence efficiency is high, high specificity and the advantage that is better than traditional gene knockout means such as easy and simple to handle developed rapidly, ready-made is research tool [Arziman Z., et al.E-RNAi:a web application to design optimized RNAi constructs.Nucleic Acids Res2005 important in life science; 33:W582-W588].SiRNA and protein bound form RNA and induce reticent mixture (RNA-induced silencing complex, RISC).The mRNA that this mixture can be attached to homology goes up and induces its degraded.
DNA methylation refers to by dnmt rna (DNA methyltransferases, DNMTs) catalysis methyl of (C5) on CpG nucleoside diphosphate cytosine(Cyt) ring and modifies, is an important content in epigenetics.In differentiation and maturation cell, be relatively-stationary, have inheritability, by gene expression regulation, participate in various kinds of cell biological procedures, abnormal DNA methylation is extensively to exist in tumour.Abnormal and the tumorigenesis close relation of DNA methylation, participate in the inactivation of cancer suppressor gene and/or the activation of oncogene together with genetics event, another important mechanisms [Laird of tumorigenesis, PW., et al.Cancer epigenetics.Human Molecular Genetics, 2005; 14:5-76Plass, C.Cancer epigenomics.Human Molecular Genetics, 2002; 11:2479-2488Esteller M.Epigenetic lesions causing genetic lesions in human cancer:promoter hypermethylation of DNA repair genes.Eur J Cancer2000; 36:2294-2300].Methylate is also 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].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].DNMT1 finds the earliest, study the most deep, participate in DNA methylation maintain and again methylate aspect compared with other two kinds, play prior effect [Laird, PW., et al.Cancer epigenetics.Human Molecular Genetics, 2005; 14:5-76Plass, C.Cancer epigenomics.Human Molecular Genetics, 2002; 11:2479-2488Bestor, TH.The DNA methyltransferase of mammals.Human molecular genetics, 2000; 9:2395-2402].DNMT3a and DNMT3b are considered to again methylated enzyme.
Cancer/testis antigen (cancer/testis antigen, CTA) be a kind of tumor associated antigen, except testis, in healthy tissues, do not express [Zhao L., et al.Progress and prospects in hepatocellular carcinoma surgery.Ann Chir, 1988; 52:558-563Chen YT., et al.A testicular antigen aberrantlye expressed in human cancers detected by autologous antibody screening.Proc Natl Acad Sci USA, 1997; 94:1914-1918Korangy F., et al.Spontaneous rumor-specific humoral and cellular immune responses to NY-ESO-1in Hepatocellular carcinoma.Clin Cancer Res, 2004; 10:4332-4341Peng j., et al.Expression of cancer/testis (CT) antigens in Chinese hepatocellular carcinoma and its correlation with clinical parameters.Cancer Letters, 2005; 219:223-232Matoba K., et al.Tumor HLA-DR expression linked to early intrahepatic recurrence of hepatocellular carcinoma.Int.J.Cancer., 2005; 115:231-240 Xu Tao etc., liver cell cancerous swelling NY-SAR-35NY-TLU-57 and NY-ESO-1 genetic expression and meaning. Chinese clinical tumor, 2006; 33:545-548Luo GM., et al.Expression of cancer-testis genes in human hepatocellular carcinomas.Cancer Immunity2002; 2:11-20].Determine at present the gene product of 19Ge CTA family, all there is stronger immunogenicity (as CT10 (cancer/testis10), the immune epitope that has the restriction of a plurality of HLA-I (human leukocyte antigen) I class antigen, 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 [ZhaoL, 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.[Nagai M., et al.Expressionof DNA (5-cytosin)-methyltransferases (DNMTs) in hepatocellular carcinomas.Hepatology Research, 2003 such as Leu; 26:186-191] in people's Ovarian Carcinoma Cells strain CP70, by siRNA technology, the mRNA level of DNMT1 and DNMT3b is disturbed, find that the effect that methylates in cell disappears.Utilize siRNA to suppress DNMT active, just likely recover or strengthen the expression of liver cancer cell CTA and HLA albumen, thereby provide new treatment means for recovering the immunogenicity of liver cancer cell.
Summary of the invention
The present invention is intended to solve the problem of immunological tolerance and immunologic escape in liver cancer cell.For this reason, one object of the present invention is to disclose a kind of efficient wide spectrum small molecules interference RNA (siRNA) for DNMT gene.Thereby, according to an aspect of the present invention, the invention provides a kind of double-stranded siRNA molecule, according to embodiments of the invention, this two strands siRNA molecule is comprised of positive-sense strand and the antisense strand with following nucleotide sequence:
siRNA1：
Positive-sense strand: 5 '-CGAGUCUGGCUUUGAGAGUtt-3 ' (SEQ ID NO:1),
Antisense strand: 5 '-ACUCUCAAAGCCAGACUCGtt-3 ' (SEQ ID NO:2);
According to another aspect of the present invention, the invention provides a kind of double-stranded siRNA molecular composition, said composition comprises siRNA1, also comprises
siRNA2：
Positive-sense strand: 5 '-GCCUCAAGAGCAGUGGAAAtt-3 ' (SEQ ID NO:3),
Antisense strand: 5 '-UUUCCACUGCUCCUGAGGCtt-3 ' (SEQ ID NO:4); And/or
siRNA3：
Positive-sense strand:: 5 '-ACCAGGACUCGUUCAGAAAtt-3 ' (SEQ ID NO:5),
Antisense strand: 5 '-UUUCUGAACGAGUCCUGGUtt-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, preferentially, 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.
According to embodiments of the invention, siRNA1 can obviously reduce the transcript and expression level of DNMT1, and the double-stranded siRNA composition of siRNA1+2+3 can obviously reduce DNMT1, the transcript and expression level of DNMT3a and DNMT3b.
According to embodiments of the invention, siRNA1 can obviously increase the transcript and expression level of CT10 gene.
According to embodiments of the invention, the double-stranded siRNA composition of siRNA1+2+3 can obviously increase the transcript and expression level of HLA-A, B, C.
Additional aspect of the present invention and advantage in the following description part provide, and part will become obviously from the following description, or recognize by practice of the present invention.
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 siRNA on CTA genetic expression that utilizes immunoblotting to detect chemosynthesis,
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) is (Promega), M-MLV (Promega), Tap enzyme (Takara) etc.
2. synthesize siRNA
In Genebank, find DNMT1, DNMT3a, the gene order of DNMT3b (NO:NM-001379, NO:NM-175629, NO:NM-006892).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 2.00D (5nmol), and have interference sequence and the negative control of positive control β-actin, as follows:
siRNA1：
Positive-sense strand: 5 '-CGAGUCUGGCUUUGAGAGUtt-3 ' (SEQ ID NO:1),
Antisense strand: 5 '-ACUCUCAAAGCCAGACUCGtt-3 ' (SEQ ID NO:2);
siRNA2：
Positive-sense strand: 5 '-GCCUCAAGAGCAGUGGAAAtt-3 ' (SEQ ID NO:3),
Antisense strand: 5 '-UUUCCACUGCUCCUGAGGCtt-3 ' (SEQ ID NO:4);
siRNA3：
Positive-sense strand:: 5 '-ACCAGGACUCGUUCAGAAAtt-3 ' (SEQ ID NO:5),
Antisense strand: 5 '-UUUCUGAACGAGUCCUGGUtt-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%C02 incubator are cultivated.
3.2 cell bed board transfections: cell is pressed to 1 * 10
4/ hole is inoculated in 24 porocyte culture plates, 37 ℃, 5%C02 incubator 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), and wherein negative control group siRNA is general contrast siRNA, i.e. 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, with 80 μ L, without RNA enzyme water/hole, dissolve RNA, get 2 μ g RNA and add 0.6 μ L Oligo (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, 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 LTap E, each forward and reverse primer of 1 μ L5 ' (0.5 μ M), 2.5 μ L10* reaction buffers, 2.5 μ LdNTP (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%).DNMT gene wherein
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: with gene-specific primer, detect DNMT mrna expression level (primer sequence is as shown in table 1) in 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 cDNA, each forward and reverse primer of 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 DNMTmRNA expression amount, and makes histogram, the results are shown in Figure 1a.As shown in the figure, ordinate zou represents that DNMT is with respect to the mrna expression level of 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 siRNA experimental group for DNMT of independent transfection, " siRNA1+2+3 " represents three kinds of siRNA experimental group for DNMT 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)+1mM NaVanadate (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: the method for use western blot detects 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 siRNA1+2+3 group is more obvious; Simulation transfection group in without reduction.Expression and the DNMT1 of DNMT3a and DNMT3b are similar, illustrate that the siRNA for DNMT of the present invention's design can obviously reduce the expression level of DNMT albumen, use siRNA composition interference effect more obviously (Fig. 1 b).
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 siNRA to disturb after DNMT gene, two genes of CT10 and SSX1 have expressing again in various degree in siRNA1 group and siRNA1+2+3 group, in untreated fish group with simulate transfection group 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 Western blot, 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, and the effect of expressing at siRNA1+2+3 is (Fig. 2) more obviously.
The above results shows, siRNA1 group has promoted CT10 albumen in CTA molecule and the expression of SSX1, and siRNA1+2+3 effect is 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 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 and siRNA1+2+3, and 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: the relative expression who simulates three kinds of HLA-I quasi-molecules of transfection group of take is 1.In siRNA1 group and siRNA1+2+3, relative expression's degree of HLA-A is respectively 8 and 12; Relative expression's degree of HLA-B is respectively 4 and 10; Relative expression's degree of HLA-C is respectively 35 and 50, 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 the effect of expressing again at siRNA1+2+3 is (Fig. 3 b) more obviously.
The above results shows, siRNA1 group has promoted HLA-I quasi-molecule to express, and siRNA1+2+3 group 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 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 g DNA, 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, the preparation 4ml of take is 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; The dehydrated alcohol that adds 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 LTE 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 ofMAGE promotors during gastric cancer progression.British Journal ofcancer, 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 LPCR 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 increase, then 94 ℃ of sex change 30s, 60 ℃ of annealing 45s, 72 ℃ of extension 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, positive-sense strand and the antisense strand with following nucleotide sequence form:
Positive-sense strand: 5 '-CGAGUCUGGCUUUGAGAGUtt-3 ' (SEQ ID NO:1),
Antisense strand: 5 '-ACUCUCAAAGCCAGACUCGtt-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 '-GCCUCAAGAGCAGUGGAAAtt-3 ' (SEQ ID NO:3), and the sequence of siRNA2 antisense strand is: 5 '-UUUCCACUGCUCCUGAGGCtt-3 ' (SEQ ID NO:4); Wherein said siRNA3 comprises positive-sense strand and antisense strand, the sequence of siRNA3 positive-sense strand is: 5 '-ACCAGGACUCGUUCAGAAAtt-3 ' (SEQ ID NO:5), the sequence of siRNA3 antisense strand is: 5 '-UUUCUGAACGAGUCCUGGUtt-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.
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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
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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》, 9 September 2004 (2004-09-09) * |
| 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》, 31 December 2006 (2006-12-31) * |
| 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》, 1 December 2009 (2009-12-01) * |
| 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》, 31 October 2011 (2011-10-31) * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684800A (en) * | 2018-11-02 | 2020-01-14 | 深圳益世康宁生物科技有限公司 | Recombinant adeno-associated virus vector carrying tumor-testis antigen 10 gene and application value thereof |
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