CN104388428A - Double-chain siRNA for interfering hnRNPA2/B1 gene expression and application thereof - Google Patents
Double-chain siRNA for interfering hnRNPA2/B1 gene expression and application thereof Download PDFInfo
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Abstract
The invention discloses double-chain siRNA for interfering hnRNPA2/B1 gene expression and application thereof. Aiming at the nucleotide sequence of the hnRNPA2/B1 genes in non-small-cell lung cancer cells, the siRNA is synthesized. The siRNA is shifted into the non-small-cell lung cancer cells to restrain the proliferation of the cells and the migration of the cells by restraining the expression of AXL genes. The siRNA disclosed by the invention is expected to become the targeted drugs to the gene therapy for cancer patients clinically.
Description
Technical field
The invention belongs to biomedicine technical field, relate to a kind of Double-stranded siRNA molecules and application thereof, more specifically, the present invention relates to a kind of double-strand siRNA and the application thereof of disturbing hnRNPA2/B1 genetic expression.
Background technology
HnRNPs endonuclearly has the ribonucleoprotein of process regulating precursor RNA to shear, splice, transcribe a kind of being mainly present in, and is extensively present in various histocyte.HnRNPA2/B1 is the Major Members of hnRNPs family, hnRNPA2/B1 can with strand Telomerase tumor-necrosis factor glycoproteins specific binding, playing protection Telomerase prevents nuclease from making it degrade, and activate effect [the Ford LP of Telomerase, et al., A model forheterogeneous nuclear ribonucleoproteins in telomere and telomeraseregulation., Oncogene, 2002,21 (4): 580-583; Kamma H, et al., Interaction of hnRNPA2/B1isoforms with telomeric ssDNA and the in vitro function, Biochemical andbiophysical research communications, 2001,280 (3): 625-630; Kamma H, etal., Molecular characterization of the hnRNP A2/B1proteins:tissue-specificexpression and novel isoforms, Experimental cell research, 1999,246 (2): 399-411; He Y, et al., Nuclear functions of heterogeneous nuclear ribonucleoproteins A/B, Cellular andmolecular life sciences:CMLS, 2009,66 (7): 1239-1256; Moran-Jones K, et al., hnRNPA2, a potential ssDNA/RNA molecular adapter at the telomere, Nucleic acidsresearch, 2005,33 (2): 486-496].
AXL is a kind of receptor tyrosine kinase receptor tyrosine kinases (RTKs), AXL and other two kinds of RTKs:Tyro3 and Mer is called TAM tyrosine kinase receptors subfamily jointly, research proves that the activation of TAM and signal transduction work in many biological behaviours of cell, comprises the aspects such as cell survival, propagation, migration, adhesion.There is research to be presented in kinds of tumors the high expression level that there is AXL, comprise acute leukemia, mammary cancer, colorectal carcinoma, lung cancer, ovarian cancer and prostate cancer.Studies have found that AXL also plays an important role in the metastasis process of lung cancer.The research such as Tai confirms that AXL causes the activation of matrix metalloproteinase MMP-9 by activating NF-kappaB path, thus promote the invasive ability [TaiKY of lung cancer cell line, et al., Axl promotes cell invasion by inducing MMP-9activity through activationof NF-kappaB and Brg-1, Oncogene, 2008,27 (29): 4044-4055].Catherine A.Vaughan etc. apply the technique study such as co-immunoprecipitation and RNA interference and confirm that saltant type P53 can play biological effect [the Vaughan CA of its short cell proliferation by raising AXL in lung cancer cell line, Singh S, WindleB, et al.Gain-of-Function Activity of Mutant p53in Lung Cancer throughUp-Regulation of Receptor Protein Tyrosine Kinase Axl.Genes & cancer.2012; 3 (7-8): 491-502].Yi-Shing Shieh is by being significantly higher than the lower CL1-5 clone of grade malignancy to finding after AXL detection of expression in the different lung adenocarcinoma cell system of grade malignancy in the clone CL1-0 that grade malignancy is high that AXL expresses.By the expression of interference AXL in two kinds of clone, and find after utilizing the migration and invasion ability of Transwell systems axiol-ogy cell, reduce AXL and express the transfer ability that significantly can reduce CL1-0 cell, simultaneously, observe this clone migration and invasion ability in artificial process LAN CL1-5 clone after AXL albumen to strengthen, malignant degree strengthens [Shieh YS, et al., Expression of axl inlung adenocarcinoma and correlation with tumor progression, Neoplasia (New York, NY), 2005, 7 (12): 1058-1064].
RNA interference (siRNA) is sequence specific gene silencing phenomenon [Fire A., the et a1.Potent and specific genetic interference bydouble-stranded RNA in Caenorhabditis elegans.Nature 1998 of a kind of double chain RNA mediate extensively existed in body; 391:806-811], because of its have that silence efficiency is high, high specificity and easy and simple to handle etc. be better than the advantage of traditional gene knockout means and developed rapidly, ready-made is research tool [Arziman Z., et al.E-RNAi:a webapplication to design optimized RNAi constructs.Nucleic Acids Res2005 important in life science; 33:W582-W588].SiRNA and protein bound form RNA and induce silencing complex (RNA-induced silencing complex, RISC).The mRNA that this mixture can be attached to homology induce it to degrade.
Y.He in its research in 2009 by high flux gene chip examination technology for detection to the expression artificially being reduced hnRNPA2/B1 in Colo16 squamous cell carcinoma system by RNA perturbation technique, cause the differential expression of 123 target genes in downstream, wherein just comprise AXL [He Y, et al., Downstream targets ofheterogeneous nuclear ribonucleoprotein A2mediate cell proliferation, Molecularcarcinogenesis, 2009, 48 (2): 167-179], there is interactional possibility both us in preliminary prompting.But the research more deep about the two dependency has no report at present.
Summary of the invention
An object of the present invention is openly a kind of double-strand siRNA disturbing hnRNPA2/B1 genetic expression.
Two of object of the present invention is that openly above-mentioned siRNA molecule is preparing the application in inhibition tumor cell hnRNPA2/B1 genetic expression reagent.
Three of object of the present invention is that openly above-mentioned siRNA molecule is preparing the application in inhibition tumor cell AXL genetic expression reagent.
Four of object of the present invention is that openly above-mentioned siRNA molecule is preparing the application in inhibition tumor cell antiproliferative agent.
Five of object of the present invention is that openly above-mentioned siRNA molecule is preparing the application in inhibition tumor cell transfer agent.
To achieve these goals, the present invention adopts following technical scheme:
The invention provides a kind of double-strand siRNA disturbing hnRNPA2/B1 genetic expression.This Double-stranded siRNA molecules is made up of the positive-sense strand of nucleotide sequence and antisense strand, and sense strand sequence is as shown in SEQ ID NO.1, and antisense strand sequence is as shown in SEQ ID NO.2.In particular embodiments, the present invention is detected by mRNA transcriptional level and protein expression level and proves that above-mentioned siRNA molecule jamming effectiveness reaches more than 70%.
The invention provides above-mentioned siRNA molecule and prepare the application in inhibition tumor cell hnRNPA2/B1 genetic expression reagent.In specific embodiment of the present invention, above-mentioned siRNA proceeds to mRNA level in-site and the protein expression level that obviously can reduce hnRNPA2/B1 gene in tumour cell.
The invention provides above-mentioned siRNA molecule and prepare the application in inhibition tumor cell AXL genetic expression reagent.In specific embodiment of the present invention, above-mentioned siRNA proceeds to mRNA level in-site and the protein expression level that obviously can reduce AXL gene in tumour cell.
The invention provides above-mentioned siRNA molecule and prepare the application in inhibition tumor cell antiproliferative agent.In specific embodiment of the present invention, MTT experiment proves that above-mentioned siRNA proceeds in tumour cell and causes tumor cell proliferation to slow down.
The invention provides above-mentioned siRNA molecule and prepare the application in inhibition tumor cell transfer agent.In specific embodiment of the present invention, use Transwell Cell migration assay to prove that above-mentioned siRNA proceeds in tumour cell and cause cell migration slack-off.
The tumour cell that the present invention uses is non-small cell lung cancer cell, and preferably, non-small cell lung cancer cell is H1299 cell.
Advantage of the present invention and beneficial effect as follows:
(1) the present invention devises a kind of siRNA for hnRNPA2/B1 gene, the jamming effectiveness of this siRNA reaches more than 70%, the expression level of hnRNPA2/B1 gene in effective reduction cell, for the function and signal transduction pathway studying hnRNPA2/B1 gene provides the foundation.
(2) present invention finds the dependency of hnRNPA2/B1 gene and AXL gene, thus provide new medicine and drug target for treating with the disease of AXL gene-correlation.
(3) present invention finds the dependency of hnRNPA2/B1 gene and tumor cell proliferation and migration, for treatment tumour provides new medicine and drug target.
Accompanying drawing explanation
Fig. 1 shows the expression of hnRNPA2/B1mRNA in A549, SK-MES-1, H1299 tri-kinds of lung cancer cell lines; Wherein, Figure 1A represents Real-Time PCR detection by quantitative result, and Figure 1B represents western blot detected result;
Fig. 2 show needle is to the jamming effectiveness of the siRNA of hnRNPA2/B1 gene; Wherein, Fig. 2 A represents Real-TimePCR detection by quantitative result, and Fig. 2 B represents western blot detected result;
On the impact that AXL genes protein level is expressed after Fig. 3 display interference hnRNPA2/B1 genetic expression; Wherein, Fig. 3 A represents Real-Time PCR detection by quantitative result, and Fig. 3 B represents western blot detected result.
Fig. 4 display utilizes MTT experiment to detect impact on tumor cell proliferation after interference hnRNPA2/B1 genetic expression.
Fig. 5 display utilizes Transwell to test to detect the impact on tumor cell migration ability after interference hnRNPA2/B1 genetic expression.
Embodiment
Further illustrate the present invention below in conjunction with specific embodiment, embodiments of the invention only for explaining the present invention, and do not mean that and limit the scope of the invention.
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1siRNA is to the test experience of hnRNPA2/B1 Gene interfere effect
1. synthesize siRNA
The mRNA sequence (GenBank-ID:NM_002137.3) of hnRNPA2/B1 gene is found in Genebank, designed by Shanghai Ji Ma genome company and chemosynthesis for the siRNA (siRNA-hnRNPA2/B1) of hnRNPA2/B1 gene, design negative control siRNA (siRNA-NC), concrete sequence is as follows simultaneously:
siRNA-hnRNPA2/B1：
Positive-sense strand: 5 '-GGAGGUGGUUAUGACAACUTT-3 ' (SEQ ID NO.1),
Antisense strand: 5 '-AGUUGUCAUAACCACCUCCTT-3 ' (SEQ ID NO.2);
siRNA-NC：
Positive-sense strand: 5 '-UUCUCCGAACGUGUCACGUTT-3 ' (SEQ ID NO.3),
Antisense strand: 5 '-ACGUGACACGUUCGGAGAATT-3 ' (SEQ ID NO.4).
2.siRNA jamming effectiveness detects
2.1 cell cultures: H1299 is cultured in RPMI-1640 substratum, and A549, SK-MES-1 are cultured in DMEM substratum.All containing 10% foetal calf serum in substratum, not containing microbiotic.5%CO in cell culture incubator
2, cultivate under 37 DEG C of environment, every 2-3 days changes liquid.Several clone is adherent growth, goes down to posterity when growing to 80% fusion.
2.2 plating cells transfection: day before transfection, vegetative period of taking the logarithm be carefully that born of the same parents are with 1 × 10
5individual/hole is inoculated in six orifice plates, carries out transfection when cytogamy degree reaches 60%-70%, respectively by siRNA-hnRNPA2/B1 and
2000 are dissolved in the Opti-MEM substratum of serum-free, after ambient temperature with gentle mixing 15min, are added in six orifice plates by the substratum of mixture and serum-free, make siRNA final concentration be 25nM; After cell normally cultivates 6h, substratum is replaced by perfect medium.By siRNA-NC with simple
2000 as negative control and blank.
2.3Real-time PCR detects
2.3.1 the extraction of total serum IgE
(1) cell centrifugation after transfection 48h gets precipitation, adds the RNAiso Plus liquid (TaKaRa) of 1ml, shakes up rear room temperature and leave standstill 5min;
(2) 12000 turns of 4 DEG C of centrifugal 5min, transfer supernatant liquor is to new EP pipe;
(3) often pipe adds 200 μ l chloroforms, concussion mixing, and room temperature leaves standstill 5min;
(4) 12000 turns of 4 DEG C of centrifugal 15min, draw the superiors' colorless supernatant liquid and are about 600-700 μ l to new EP pipe;
(5) in supernatant liquor, equal-volume Virahol is added, left at room temperature 10min after mixing;
(6) 12000 turns of 4 DEG C of centrifugal 10min, carefully remove supernatant, a small amount of white RNA precipitation in the portion that sees the bottom;
(7) often pipe adds 75% ethanol of 1ml precooling;
(8) 12000 turns of 4 DEG C of centrifugal 5min, abandon supernatant, drying at room temperature 5min;
(9) often pipe adds Rnase-free water 10-20 μ l, and RNA is fully dissolved;
2.3.2RNA purity and concentration determination
Get RNA sample 1.0 μ l, use ultramicron nucleic acid concentration determinator mensuration RNA concentration and wavelength to be OD value during 260nm and 280nm.RNA purity: OD260/OD280 ratio is between 1.8-2.0.The volume of required RNA in 10 μ l reverse transcription systems is calculated according to the concentration of RNA.
2.3.3RNA reverse transcription (10 μ l reaction system)
RNA(0.5μg) xμl
5×PrimeScript RT Master Mix(TaKaRa) 2μl
Add DEPC water and supply 10 μ l reaction systems, centrifugal 5s after mixing, carries out reverse transcription, reaction conditions by Geneamp 9700 type PCR instrument: 37 DEG C of 15min, 85 DEG C of 5s, 4 DEG C.After reverse transcription, cDNA product is in-20 DEG C of preservations.
2.3.4 real-time quantitative PCR reaction
2.3.4.1 primer sequence is as follows:
HnRNPA2/B1 gene:
Upstream 5 '-GCTGTAGCAAGAGAGGAATCTGGA-3 ' (SEQ ID NO.5),
Downstream 5 '-GCTTCTTCACAGTTACATGAGCCC-3 ' (SEQ ID NO.6);
AXL gene:
Upstream 5 '-GCAACCTTCACCTACCGAGTTC-3 ' (SEQ ID NO.7),
Downstream 5 '-GGCCAACATGGTGAAACCCT-3 ' (SEQ ID NO.8);
GAPDH gene:
Upstream 5 '-ACCACAGTCCATGCCATCAC-3 ' (SEQ ID NO.9),
Downstream 5 '-TCCACCACCCTGTTGCTGTA-3 ' (SEQ ID NO.10).
2.3.4.2PCR reaction system:
2.3.4.3 reaction conditions:
2.4 immunoblottings (western blot) detect
2.4.1 cleaning
After cell cultures 48h after transfection, abandon nutrient solution, with PBS cleaning twice.
2.4.2 lysing cell
Every hole adds RIPA lysate 2ml (be the PMSF of 10mg/ml containing final concentration), is blown and beaten by cell, ultrasonication.
2.4.3 cell debris is removed
12000rpm, 4 DEG C of centrifugal 15min, reject precipitates.
2.4.4 determination of protein concentration
BCA method carries out determination of protein concentration (the long microplate reader of Gen5 all-wave), gets the protein lysate (volume × protein concn) of equal in quality, and adds isopyknic electrophoresis sample-loading buffer.
2.4.5 protein denaturation
3-5min is boiled in boiling water bath.
2.4.6 separation gel and concentrated glue is configured
Configuration concentration is the separation gel of 12% and the concentrated glue of 5%.
2.4.7 loading
Every hole adds 50 μ g protein samples.
2.4.8 electrophoresis
Concentrated glue 90v, separation gel 110v, stop electrophoresis when tetrabromophenol sulfonphthalein is down to bottom gel.
2.4.9 transferring film
The pvdf membrane methyl alcohol sheared is soaked into 3min, then uses transferring film liquid saturated sponge, filter paper and pvdf membrane, semidrying transferring film.Electrophoresis chamber is put in ice, 70v electrophoresis 120min, 100v electrophoresis 60min.
2.4.10 close
Film is taken off, closes in 5% skimmed milk after mark, 2h closed by shaking table.
2.4.11 primary antibodie is hatched
1x TBST washes film 3 times, each 10min, adds primary antibodie hnRNPA2/B1 (1:2500); AXL (1:300); β-actin (1:1000), 4 DEG C are spent the night.
2.4.12 two anti-hatch
1x TBST washes film 3 times, each 10min, adds two anti-incubated at room temperature 2h after dilution.
2.4.13 colour developing
1x TBST washes film 3 times, each 10min, carries out luminous reaction, detection, Taking Pictures recording image after the A liquid of ECL luminescence reagent and B liquid being mixed with the ratio of 1:1 with film.
2.5 statistical treatment
Adopt SPSS17.0 statistical software to carry out independent samples t test and one-way analysis of variance to experimental result, P<0.05 is considered as difference significance.
2.6 interpretation of result
As shown in Figure 1, in H1299, A549, SK-MES-1 tri-kinds of lung carcinoma cells, mrna expression level (Figure 1A) and the protein expression level (Figure 1B) of H1299 cell hnRNPA2/B1 gene are the highest, therefore select H1299 cell to carry out follow-up test.
After H1299 cell interference hnRNPA2/B1 genetic expression, blank group is compared with negative control group (siRNA-NC), hnRNPA2/B1mRNA expresses (Fig. 2 A) and protein expression (Fig. 2 B) difference not obvious (p>0.05), negative control group (siRNA-NC) is compared with siRNA interference group (siRNA-hnRNPA2/B1), hnRNPA2/B1mRNA expresses (Fig. 2 A) and protein expression (Fig. 2 B) has notable difference (p<0.05), after transfection siRNA-hnRNPA2/B1, the mrna expression level of hnRNPA2/B1 gene and protein expression level obviously reduce.
After H1299 cell interference hnRNPA2/B1 genetic expression, blank group is compared with siRNA-NC group, AXL gene mRNA expression (Fig. 3 A) and protein expression (Fig. 3 B) difference not obvious (p>0.05), siRNA-NC group is compared with siRNA-hnRNPA2/B1 group, AXL gene mRNA expression (Fig. 3 A) and protein expression (Fig. 3 B) differential expression have notable difference (p<0.05), after transfection siRNA-hnRNPA2/B1, the mrna expression level of AXL gene and protein expression level obviously reduce.
Embodiment 2 disturbs hnRNPA2/B1 genetic expression on the impact of H1299 cell proliferation
1. cell cultures, step is with embodiment 1.
2. cell transfecting, step is with embodiment 1.
3.MTT method detects: after the H1299 cell after transfection is changed perfect medium, carry out MTT cytoactive detection after hatching 24h, 48h, 72h.Under microplate reader 490nm, read light absorption value, repeatedly carry out three experiments, results averaged.
4. interpretation of result:
Result as shown in Figure 4, H1299 ability of cell proliferation after siRNA-hnRNPA2/B1 transfection comparatively negative control group (siRNA-NC) reduces, difference has statistical significance (P<0.05), namely, after siRNA-hnRNPA2/B1 transfection, the H1299 ability of cell proliferation of the low expression of hnRNPA2/B1 significantly reduces.
Embodiment 3 disturbs hnRNPA2/B1 genetic expression on the impact of H1299 cell migration ability
1. cell cultures, step is with embodiment 1.
2. cell transfecting, step is with embodiment 1.
3.Transwell tests: the H1299 cell after transfection is carried out transfer ability detection in Transwell cell, contrasts siRNA interference group (siRNA-hnRNPA2/B1) and negative control group (siRNA-NC) microporous membrane lower floor cell quantity after 36h.
4. interpretation of result:
Result as shown in Figure 5, siRNA-hnRNPA2/B1 group is compared with siRNA-NC group, microporous membrane lower floor cell quantity obviously reduces, difference has statistical significance (P<0.05), namely, after siRNA-hnRNPA2/B1 transfection, the H1299 cell migration ability of the low expression of hnRNPA2/B1 significantly reduces.
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 (7)
1. disturb a double-strand siRNA for hnRNPA2/B1 genetic expression, it is characterized in that, described Double-stranded siRNA molecules is made up of positive-sense strand and antisense strand; The sequence of described positive-sense strand is as shown in SEQ ID NO.1, and the sequence of described antisense strand is as shown in SEQ ID NO.2.
2. Double-stranded siRNA molecules according to claim 1 is preparing the application in inhibition tumor cell hnRNPA2/B1 genetic expression reagent.
3. Double-stranded siRNA molecules according to claim 1 is preparing the application in inhibition tumor cell in AXL genetic expression reagent.
4. Double-stranded siRNA molecules according to claim 1 is preparing the application in inhibition tumor cell antiproliferative agent.
5. Double-stranded siRNA molecules according to claim 1 is preparing the application in inhibition tumor cell transfer agent.
6. the application according to any one of claim 2 to 5, is characterized in that, described tumour cell is non-small cell lung cancer cell.
7. application according to claim 6, is characterized in that, described non-small cell lung cancer cell is H1299 cell.
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| WO2021204109A1 (en) * | 2020-04-06 | 2021-10-14 | The University Of Hong Kong | Utilization of nuclear p70 s6 kinase for diagnosis, prognosis and treatment of cancer |
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| CN107417772A (en) * | 2017-09-07 | 2017-12-01 | 华中科技大学同济医学院附属协和医院 | It is a kind of can antagonism hnRNPU protein rna binding activity polypeptide HIP 20 and its application |
| CN107417772B (en) * | 2017-09-07 | 2020-03-27 | 华中科技大学同济医学院附属协和医院 | Polypeptide HIP-20 capable of antagonizing RNA binding activity of hnRNPU protein and application thereof |
| WO2021007800A1 (en) * | 2019-07-17 | 2021-01-21 | 中国医学科学院基础医学研究所 | Anti-infection effects of hnrnpa2b1 and use thereof |
| WO2021204109A1 (en) * | 2020-04-06 | 2021-10-14 | The University Of Hong Kong | Utilization of nuclear p70 s6 kinase for diagnosis, prognosis and treatment of cancer |
| CN113073137A (en) * | 2021-04-02 | 2021-07-06 | 武汉儿童医院 | Postpartum depression detection reagent, system and application |
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