CN104388428B - A kind of double-strand siRNA disturbing hnRNPA2/B1 gene expression and application thereof - Google Patents
A kind of double-strand siRNA disturbing hnRNPA2/B1 gene expression and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of double-strand siRNA disturbing hnRNPA2/B1 gene expression.The nucleotide sequence design synthesis siRNA, this siRNA that the present invention is directed to the hnRNPA2/B1 gene in non-small cell lung cancer cell proceed to suppress propagation and the cell migration of cell by suppressing the expression of AXL gene in non-small cell lung cancer cell.The siRNA of the present invention will be expected to become the targeted drug of tumor patient gene therapy 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, this
Invention relates to a kind of double-strand siRNA disturbing hnRNPA2/B1 gene expression and application thereof.
Background technology
HnRNPs is that a kind of being primarily present in endonuclear has the regulation precursor RNA process shearing, splice, transcribe
Ribonucleoprotein, is widely present in various histiocyte.HnRNPA2/B1 is the Major Members of hnRNPs family, hnRNPA2/
B1 can be specific binding with strand telomerase repetitive sequence, plays protection telomerase and prevents nuclease from making it degrade, and activates end
Effect [Ford LP, et al., the A model for heterogeneous nuclear of granzyme
ribonucleoproteins in telomere and telomerase regulation.,Oncogene,2002,21
(4):580-583；Kamma H,et al.,Interaction of hnRNP A2/B1isoforms with telomeric
ssDNA and the in vitro function,Biochemical and biophysical research
communications,2001,280(3):625-630；Kamma H,et al.,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 and molecular life
sciences:CMLS,2009,66(7):1239-1256；Moran-Jones K,et al.,hnRNP A2,a potential
ssDNA/RNA molecular adapter at the telomere,Nucleic acids research,2005,33
(2):486-496]。
AXL is a kind of receptor tyrosine kinase receptor tyrosine kinases (RTKs), AXL and other two kinds
RTKs:Tyro3 and Mer is collectively referred to TAM tyrosine kinase receptors subfamily, and the activation of research proof TAM and signal transduction are at cell
Many biological behaviour aspects work, including cell survival, breed, migrate, the aspect such as adhesion.There is research display multiple
Tumor exists the high expressed of AXL, including acute leukemia, breast carcinoma, colon cancer, pulmonary carcinoma, ovarian cancer and carcinoma of prostate.Grind
Study carefully discovery AXL and also function to important function in the invasion and attack and transfer process of pulmonary carcinoma.The research such as Tai confirms that AXL is by activating NF-
KappaB path and cause the activation of matrix metalloproteinase MMP-9, thus promote the invasive ability [Tai of lung cancer cell line
KY,et al.,Axl promotes cell invasion by inducing MMP-9activity through
activation of 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 to confirm in lung cancer cell line, and saltant type P53 can be led to
Cross and raise AXL and play its biological effect [Vaughan CA, Singh S, Windle B, et promoting cell proliferation
al.Gain-of-Function Activity of Mutant p53in Lung Cancer through Up-
Regulation of Receptor Protein Tyrosine Kinase Axl.Genes&cancer.2012；3(7-8):
491-502].Yi-Shing Shieh is by finding evil after AXL detection of expression in the lung adenocarcinoma cell system different to grade malignancy
Property high cell line CL1-0 of degree in AXL express the CL1-5 cell line that grade malignancy to be significantly higher than is relatively low.By interference AXL
Expression in two kinds of cell line, and find after utilizing the detection migration of cell of Transwell system and invasive ability, reduce
AXL expresses the transfer ability that can significantly reduce CL1-0 cell, meanwhile, in artificial process LAN CL1-5 cell line after AXL albumen
Observing that this cell line migrates and invasive ability strengthens, malignant degree strengthens [Shieh YS, et al., Expression
of axl in lung adenocarcinoma and correlation with tumor progression,
Neoplasia(New York,NY),2005,7(12):1058-1064]。
RNA interference (siRNA) is that the sequence specific gene silence of a kind of double chain RNA mediate being widely present in body is existing
As [Fire A., et a1.Potent and specific genetic interference by double-stranded
RNA in Caenorhabditis elegans.Nature 1998；391:806-811], because of it, to have silence efficiency high, special
The opposite sex advantage being better than tradition gene knockout means such as strong and easy and simple to handle and developed rapidly, ready-made lead for life sciences
Research tool [Arziman Z., et al.E-RNAi:a web application to design important in territory
optimized RNAi constructs.Nucleic Acids Res2005；33:W582-W588].SiRNA ties with protein
Close and form RNA induction silencing complex (RNA-induced silencing complex, RISC).This complex can be in conjunction with
On the mRNA of homology and induce it to degrade.
Y.He passes through high flux gene chip examination technology for detection to thin at Colo16 scale cancer in its research in 2009
Born of the same parents system is artificially reduced by RNA perturbation technique the expression of hnRNPA2/B1, causes the difference table of 123 target genes in downstream
Reach, the most just include AXL [He Y, et al., Downstream targets of heterogeneous nuclear
ribonucleoprotein A2mediate cell proliferation,Molecular carcinogenesis,2009,
48 (2): 167-179], there is the probability of interaction both us in preliminary prompting.But about the two dependency deeper into
Research has no report at present.
Summary of the invention
An object of the present invention is to disclose a kind of double-strand siRNA disturbing hnRNPA2/B1 gene expression.
The two of the purpose of the present invention are to disclose above-mentioned siRNA molecule at preparation suppression tumor cell hnRNPA2/B1 gene
Express the application in reagent.
The three of the purpose of the present invention are to disclose above-mentioned siRNA molecule in preparation suppression tumor cell AXL gene expression examination
Application in agent.
The four of the purpose of the present invention are to disclose above-mentioned siRNA molecule answering in preparation suppression tumor cell proliferation reagent
With.
The five of the purpose of the present invention are to disclose above-mentioned siRNA molecule answering in preparation suppression tumor cell migration reagent
With.
To achieve these goals, the present invention adopts the following technical scheme that
The invention provides a kind of double-strand siRNA disturbing hnRNPA2/B1 gene expression.This Double-stranded siRNA molecules is by core
The positive-sense strand of nucleotide sequence and antisense strand composition, sense strand sequence as shown in SEQ ID NO.1, antisense strand sequence such as SEQ ID
Shown in NO.2.In particular embodiments, the present invention proves above-mentioned by mRNA transcriptional level and protein expression level detection
SiRNA molecule jamming effectiveness reaches more than 70%.
The invention provides above-mentioned siRNA molecule in preparation suppression tumor cell hnRNPA2/B1 gene expression reagent
Application.In the specific embodiment of the present invention, above-mentioned siRNA proceeds to substantially reduce in tumor cell hnRNPA2/B1 base
The mRNA level in-site of cause and protein expression level.
The invention provides the application in preparation suppression tumor cell AXL gene expression reagent of the above-mentioned siRNA molecule.?
In the specific embodiment of the present invention, above-mentioned siRNA proceed to tumor cell can substantially reduce AXL gene mRNA level in-site and
Protein expression level.
The invention provides the application in preparation suppression tumor cell proliferation reagent of the above-mentioned siRNA molecule.In the present invention
Specific embodiment in, MTT experiment prove above-mentioned siRNA proceed to tumor cell cause tumor cell proliferation slow down.
The invention provides the application in preparation suppression tumor cell migration reagent of the above-mentioned siRNA molecule.In the present invention
Specific embodiment in, using Transwell Cell migration assay to prove, that above-mentioned siRNA proceeds to cause in tumor cell is thin
Born of the same parents migrate slack-off.
The tumor cell that the present invention uses is non-small cell lung cancer cell, it is preferable that non-small cell lung cancer cell is H1299
Cell.
Advantages of the present invention and having the beneficial effect that:
(1) present invention devises a kind of siRNA for hnRNPA2/B1 gene, and the jamming effectiveness of this siRNA reaches 70%
Above, effectively reduce the expression of hnRNPA2/B1 gene in cell, for function and the signal of research hnRNPA2/B1 gene
Signal Transduction Pathways provides the foundation.
(2) present invention finds the dependency of hnRNPA2/B1 gene and AXL gene, thus for treating and AXL gene phase
The disease closed provides new medicine and drug target.
(3) present invention finds hnRNPA2/B1 gene and tumor cell proliferation and the dependency of migration, for treatment tumor
Provide new medicine and drug target.
Accompanying drawing explanation
Fig. 1 shows hnRNPA2/B1mRNA expression in tri-kinds of lung cancer cell lines of A549, SK-MES-1, H1299；
Wherein, Figure 1A represents Real-Time PCR detection by quantitative result, and Figure 1B represents western blot testing result；
Fig. 2 shows the jamming effectiveness of the siRNA for hnRNPA2/B1 gene；Wherein, Fig. 2 A represents Real-TimePCR
Detection by quantitative result, Fig. 2 B represents western blot testing result；
The impact after Fig. 3 display interference hnRNPA2/B1 gene expression, AXL genes protein level expressed；Wherein, Fig. 3 A
Representing Real-Time PCR detection by quantitative result, Fig. 3 B represents western blot testing result.
Fig. 4 shows and utilizes impact on tumor cell proliferation after MTT experiment detection interference hnRNPA2/B1 gene expression.
Fig. 5 show utilize Transwell experiment detection interference hnRNPA2/B1 gene expression after to tumor cell migration energy
The impact of power.
Detailed description of the invention
Further illustrating the present invention below in conjunction with specific embodiment, embodiments of the invention are only used for explaining the present invention,
It is not intended to limit protection scope of the present invention.
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
The embodiment 1siRNA test experience to hnRNPA2/B1 gene interference effect
1. synthesis siRNA
The mRNA sequence (GenBank-ID:NM_002137.3) of hnRNPA2/B1 gene is found, by upper in Genebank
The design of Hai Jima genome company and chemosynthesis are for the siRNA (siRNA-hnRNPA2/B1) of hnRNPA2/B1 gene, simultaneously
Design negative control siRNA (siRNA-NC), particular sequence is as follows:
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 cells are cultivated: H1299 is cultured in RPMI-1640 culture medium, and A549, SK-MES-1 are cultured in DMEM
In culture medium.Culture medium all contains 10% hyclone, without antibiotic.5%CO in cell culture incubator2, 37 DEG C of environment
Lower cultivation, changes liquid in every 2-3 days.Several cell lines are adherent growth, pass on when length to 80% merges.
2.2 plating cells also transfect: day before transfection, and trophophase of taking the logarithm is carefully that born of the same parents are with 1 × 105Individual/hole is inoculated in
Six orifice plates, cell degrees of fusion transfects when reaching 60%-70%, respectively by siRNA-hnRNPA2/B1 and
2000 are dissolved in the Opti-MEM culture medium of serum-free, after ambient temperature with gentle mixing 15min, by mixture and the culture medium of serum-free
Add in six orifice plates, make the final concentration of 25nM of siRNA；After cell normally cultivates 6h, culture medium is replaced by complete medium.Will
SiRNA-NC is with simple2000 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 takes precipitation, adds RNAiso Plus liquid (TaKaRa) of 1ml, shakes up rear chamber
Gentle and quiet put 5min；
(2) 12000 turns of 4 DEG C of centrifugal 5min, transfer supernatant is managed to new EP；
(3) often pipe adds 200 μ l chloroforms, concussion mixing, and room temperature stands 5min；
(4) 12000 turns of 4 DEG C of centrifugal 15min, draw the superiors' colorless supernatant liquid about 600-700 μ l and manage to new EP；
(5) in supernatant, equal-volume isopropanol, left at room temperature 10min after mixing are added；
(6) 12000 turns of 4 DEG C of centrifugal 10min, carefully remove supernatant, the portion's of seeing the bottom white RNA precipitate on a small quantity；
(7) often pipe adds 75% ethanol of 1ml pre-cooling；
(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, makes RNA fully dissolve；
2.3.2RNA purity and concentration measure
Take RNA sample 1.0 μ l, use ultramicron nucleic acid concentration analyzer to measure RNA concentration and wavelength be 260nm and
OD value during 280nm.RNA purity: OD260/OD280 ratio is between 1.8-2.0.Concentration according to RNA calculates 10 μ l reversions
The volume of required RNA in record system.
2.3.3RNA reverse transcription (10 μ l reaction system)
RNA(0.5μg) xμl
5×PrimeScript RT Master Mix(TaKaRa) 2μl
Adding DEPC water and supply 10 μ l reaction systems, after mixing, centrifugal 5s, inverts by Geneamp 9700 type PCR instrument
Record, reaction condition: 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 condition:
2.4 immunoblottings (western blot) detect
2.4.1 clean
After cell after transfection cultivates 48h, abandon culture fluid, with PBS twice.
2.4.2 cell lysis
Every hole adds RIPA lysate 2ml (containing the PMSF of final concentration of 10mg/ml), is blown and beaten by cell, ultrasonic broken
Broken.
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), take equal in quality protein lysate (volume ×
Protein concentration), and add isopyknic electrophoresis sample-loading buffer.
2.4.5 albuminous degeneration
Boiling water bath boils 3-5min.
2.4.6 configuration separation gel and concentration glue
Configuration concentration is separation gel and the concentration glue of 5% of 12%.
2.4.7 loading
Every hole adds 50 μ g protein samples.
2.4.8 electrophoresis
Concentrate glue 90v, separation gel 110v, when bromophenol blue is down to bottom gel, stop electrophoresis.
2.4.9 transferring film
The pvdf membrane methanol sheared is impregnated with 3min, then by transferring film liquid saturated sponge, filter paper and pvdf membrane, semidry method
Transferring film.Electrophoresis tank is put in ice, 70v electrophoresis 120min, 100v electrophoresis 60min.
2.4.10 close
Film is taken off, closes in 5% skim milk after labelling, shaking table is closed 2h.
2.4.11 one anti-hatches
1x TBST washes film 3 times, each 10min, adds an anti-hnRNPA2/B1 (1:2500)；AXL(1:300)；β-
Actin (1:1000), 4 DEG C overnight.
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, with film after being mixed with the ratio of 1:1 with B liquid by the A liquid of ECL luminescence reagent
Carry out luminescence-producing reaction, detection, Taking Pictures recording image.
2.5 statistical disposition
Use SPSS17.0 statistical software that experimental result carries out independent samples t test and one factor analysis of variance, P <
0.05 is considered as difference significance.
2.6 interpretation of result
As it is shown in figure 1, in tri-kinds of lung carcinoma cells of H1299, A549, SK-MES-1, H1299 cell hnRNPA2/B1 gene
Mrna expression level (Figure 1A) and protein expression level (Figure 1B) the highest, therefore select H1299 cell carry out follow-up test.
After the interference hnRNPA2/B1 gene expression of H1299 cell, blank group and negative control group (siRNA-NC) phase
Ratio, hnRNPA2/B1mRNA expresses (Fig. 2 A) and protein expression (Fig. 2 B) difference inconspicuous (p > 0.05), negative control group
(siRNA-NC) compared with siRNA interference group (siRNA-hnRNPA2/B1), hnRNPA2/B1mRNA expresses (Fig. 2 A) and albumen
Express (Fig. 2 B) and there is notable difference (p < 0.05), after transfection siRNA-hnRNPA2/B1, the mRNA table of hnRNPA2/B1 gene
Reach level and protein expression level substantially reduces.
H1299 cell interference hnRNPA2/B1 gene expression after, blank group compared with siRNA-NC group, AXL gene
Mrna expression (Fig. 3 A) and protein expression (Fig. 3 B) difference inconspicuous (p > 0.05), siRNA-NC group and siRNA-hnRNPA2/B1
Group is compared, and AXL gene mRNA expression (Fig. 3 A) and protein expression (Fig. 3 B) differential expression have notable difference (p < 0.05), transfection
After siRNA-hnRNPA2/B1, the mrna expression level of AXL gene and protein expression level substantially reduce.
Embodiment 2 disturbs the hnRNPA2/B1 gene expression impact on H1299 cell proliferation
1. cell is cultivated, and 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 complete medium, carry out after hatching 24h, 48h, 72h
MTT cytoactive detection.Under microplate reader 490nm, read light absorption value, three experiment, results averaged are repeated.
4. interpretation of result:
Result as shown in Figure 4, siRNA-hnRNPA2/B1 transfection after H1299 ability of cell proliferation relatively negative control group
(siRNA-NC) reducing, difference has statistical significance (P < 0.05), i.e. after siRNA-hnRNPA2/B1 transfection, and hnRNPA2/B1
The H1299 ability of cell proliferation of low expression significantly reduces.
Embodiment 3 disturbs the hnRNPA2/B1 gene expression impact on H1299 cell migration ability
1. cell is cultivated, and step is with embodiment 1.
2. cell transfecting, step is with embodiment 1.
3.Transwell tests: the H1299 cell after transfection is carried out in Transwell cell transfer ability detection,
SiRNA interference group (siRNA-hnRNPA2/B1) and negative control group (siRNA-NC) microporous membrane lower floor cell number is contrasted after 36h
Amount.
4. interpretation of result:
Result is as it is shown in figure 5, siRNA-hnRNPA2/B1 group is compared with siRNA-NC group, and microporous membrane lower floor cell quantity is bright
Aobvious minimizing, difference has statistical significance (P < 0.05), i.e. after siRNA-hnRNPA2/B1 transfection, the low expression of hnRNPA2/B1
H1299 cell migration ability significantly reduces.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
These embodiments can be carried out multiple change in the case of departing from the principle of the present invention and objective, revise, replace and modification, this
The scope of invention is limited by claim and equivalent thereof.
Claims (5)
1. double-strand siRNA disturbing hnRNPA2/B1 gene expression, it is characterised in that described double-strand siRNA is by positive-sense strand
Form with antisense strand；The sequence of described positive-sense strand as shown in SEQ ID NO.1, the sequence of described antisense strand such as SEQ ID NO.2
Shown in.
2. the answering in preparation suppression tumor cell hnRNPA2/B1 gene expression reagent of double-strand siRNA described in claim 1
With, it is characterised in that described tumor cell is non-small cell lung cancer cell, and described non-small cell lung cancer cell is H1299 cell.
3. the application in AXL gene expression reagent in preparation suppression tumor cell of double-strand siRNA described in claim 1, its
Being characterised by, described tumor cell is non-small cell lung cancer cell, and described non-small cell lung cancer cell is H1299 cell.
4. the application in preparation suppression tumor cell proliferation reagent of double-strand siRNA described in claim 1, it is characterised in that
Described tumor cell is non-small cell lung cancer cell, and described non-small cell lung cancer cell is H1299 cell.
5. the application in preparation suppression tumor cell migration reagent of double-strand siRNA described in claim 1, it is characterised in that
Described tumor cell is non-small cell lung cancer cell, and described non-small cell lung cancer cell is H1299 cell.
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