CN102690826A - shRNA for specifically reducing human Aurora-A gene expression and application thereof - Google Patents
shRNA for specifically reducing human Aurora-A gene expression and application thereof Download PDFInfo
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Abstract
The invention relates to the field of molecular genetics and biomedicines, in particular to a shRNA for specifically reducing human Aurora-A gene expression and application of the shRNA. The invention provides hairpin interference RNA (ribonucleic acid) for specifically reducing human Aurora-A gene expression, shown by SEQIDNO. 1; an aimed target sequence is the 995th-1015th bit of human Aurora-AmRNA, shown by SEQIDNO. 2; the shRNA can be sheared in vivo or in vitro to form siRNA shown by SEQIDNO. 4 and SEQIDNO. 5; the invention further provides a DNA (deoxyribonucleic acid) oligonucleotide chain for coading the shRNA, shown by SEQIDNO. 3, and a plasmid including the DNA oligonucleotide chain shown by SEQIDNO. 3. According to the invention, after medicines of Aurora-A-shRNA interference carriers transfect human esophageal cancer cells EC9706, an mRNA transcriptional level and a protein expression level of Aurora-A genes in cells are significantly reduced so as to significantly reduce invasiveness of esophageal cancer cells and prompt apoptosis of esophageal cancer cells irradiated and induced by UV (ultra violet), thereby overcoming the defect that the siRNA chemically synthetized in vitro has short acting time, and providing a new way for developing novel antitumor drugs.
Description
Technical field
The present invention relates to molecular genetics and biological medicine technology field, be specially shRNA and application thereof that a specific specificity reduces people Aurora-A genetic expression.
Background technology
RNA perturbation technique (RNAi) is meant that double-stranded RNA brings out with its homologous sequence mRNA molecule specifically and is degraded, causes the phenomenon of corresponding gene expression inhibiting.Transcribe little double-stranded RNA (the small interfering RNA of generation in artificial chemosynthesis or the cell; SiRNA) behind the entering cell; Ability and complementary target gene mRNA combination are with it degraded mRNA thereby mediate specific nickase, reach the purpose that reduces destination gene expression.
At present, reduce though the siRNA of external chemosynthesis can cause expression of target gene, its effect is of short duration relatively.For gene is carried out long-acting inhibition, many Mammals plasmid expression vectors have been used as research tool, in cell, instruct synthetic siRNA.These carriers comprise dependenc RNA polymerase-III U6 or H1 promotor, by a clear and definite transcripting start point and a transcription termination signal (T5) of being made up of continuous 5 thymus pyrimidines.Wherein, to derive from the segment length of target gene mRNA be the unique sequences of 19～21 bases to a pair of specific oligonucleotide.When with forward and the annealing of reverse DNA oligonucleotide, and when being cloned between two restriction endonuclease sites of carrier, forward DNA oligonucleotide will correctly be positioned the downstream of U6 promotor.The transcription product of recombinant vectors is hair clip type RNA (small hairpin RNA; ShRNA) can self fold pairing formation stem length is stem ring (Stem-loop) structure of 19～21 bases, and the precursor of this loop-stem structure is cut the siRNA that is formed with function very soon in cell.The shRNA of this vector expression has through the siRNA that shear to form that expression amount is stable, the characteristics of longer duration, thereby can cause the long-acting inhibition that target gene is expressed.
Aurora-A is the mitotic serine/threonine kinase of a kind of participation; Its biological action mainly is through regulating the function of centrosome and microtubule; Guarantee the correct separation of centrosome and complete division [the Bischoff JR of endochylema; Plowman GD. The Aurora/Ipl1p kinase family:regulators of chromosome segregation and cytokinesis. Trends Cell Biol, 1999,9 (11): 454-459.].In recent years, why Aurora-A receives much attention, and is because it is close with being related of many tumours, is a kind of potential oncogene.For example at mammary cancer [Zhou H, Kuang J, Zhong L; Et al. Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. Nat Genet, 1998; 20 (2): 189-193.], liver cancer [Jeng YM, Peng SY, Lin CY; Et al. Overexpression and amplification of Aurora-A in hepatocellular carcinoma. Clin Cancer Res, 2004,10 (6): 2065-2071.], colorectal carcinoma [Bischoff JR; Anderson L, Zhu Y, et al. A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers. EMBO J; 1998,17 (11): 3052-3065.], bladder cancer [Sen S, Zhou H; Zhang RD, et al. Amplification/overexpression of a mitotic kinase gene in human bladder cancer. J Natl Cancer Inst, 2002; 94 (17): 1320-1329.] and the esophageal carcinoma [Tong T, Zhong Y, Kong J; Et al. Overexpression of Aurora-A contributes to malignant development of human esophageal squamous cell carcinoma. Clin Cancer Res; 2004,10 (21): 7304-7310. Tanaka E, Hashimoto Y; Ito T; Et al. The clinical significance of Aurora-A/STK15/BTAK expression in human esophageal squamous cell carcinoma. Clin Cancer Res, 2005,11 (5): 1827-1834.] etc. all find to have crossing of Aurora-A to express in the tumour.The Aurora-A abnormal expression is not only closely related with the generation of tumour, and with the invasion and attack of tumour with shift and also have dependency significantly.For example, at bladder cancer, liver cancer [Jeng YM, Peng SY; Lin CY, et al. Overexpression and amplification of Aurora-A in hepatocellular carcinoma. Clin Cancer Res, 2004; 10 (6): 2065-2071. Sen S, Zhou H, Zhang RD; Et al. Amplification/overexpression of a mitotic kinase gene in human bladder cancer. J Natl Cancer Inst, 2002,94 (17): 1320-1329.] in; The expression level of Aurora-A with organize classification, invasion and attack and the rate of transform to be proportionate, and relevant with patient's prognosis; In esophageal squamous cell carcinoma, the histological grade and the Invasion Potential of Aurora-A expression level and tumour are proportionate, and relevant with the distant place nodus lymphoideus transferring rate; But also can be used as an individual index [Tong T, Zhong Y, the Kong J that judges patients'prognosis; Et al. Overexpression of Aurora-A contributes to malignant development of human esophageal squamous cell carcinoma. Clin Cancer Res; 2004,10 (21): 7304-7310. Tanaka E, Hashimoto Y; Ito T; Et al. The clinical significance of Aurora-A/STK15/BTAK expression in human esophageal squamous cell carcinoma. Clin Cancer Res, 2005,11 (5): 1827-1834.].Explanation thus, Aurora-A plays crucial effects in the incidence and development of tumour.Therefore, the Aurora-A gene might become one of effective target spot of treatment tumor development.
As everyone knows, the sickness rate of China's esophageal carcinoma ranks first in the world at present, and especially the some areas with Taihang Mountain peripheral Hebei, Henan and Shanxi are the district occurred frequently.Though excision and postoperative chemicotherapy make patient's survival rate obtain bigger raising, overall prognosis is still pessimistic, and the major cause that causes patient death is the recurrence and the transfer of tumour.Therefore, the newtype drug of the development treatment esophageal carcinoma has important economic value and social benefit, and adopts the RNA perturbation technique might become one of effective way of the treatment esophageal carcinoma.
Summary of the invention
The object of the present invention is to provide a specific specificity to reduce the shRNA and the application thereof of people Aurora-A genetic expression.
The present invention adopts following technical scheme to realize: a specific specificity reduces the shRNA of people Aurora-A genetic expression, and its base sequence is shown in SEQ ID NO.1, promptly
5’-GCACCACUUGGAACAGUUUAUUUCAAGAGAAUAAACUGUUCCAAGUGGUGC-3’。
This shRNA can be applicable to treat in the medicine of the esophageal carcinoma.
The invention provides a kind of hair clip type RNA interfering (Aurora-A-shRNA) that can specificity reduces people Aurora-A genetic expression; Shown in SEQ ID NO.1; To the 995th～1015 of target sequence behaviour Aurora-A mRNA; Shown in SEQ ID NO.2, this shRNA in vivo or external shear to form contain the siRNA shown in SEQ ID NO.4 and SEQ ID NO.5; The present invention also provides the DNA oligonucleotide chain of this shRNA that encodes, shown in SEQ ID NO.3, and the plasmid that comprises the DNA oligonucleotide chain shown in SEQ ID NO.3, utilizes this plasmid can directly perhaps external in vivo generation Aurora-A-shRNA.And the present invention also proved utilize contain the medicine transient transfection human esophagus cancer EC9706 cell of Aurora-A-shRNA interference carrier after; The mRNA transcriptional level of Aurora-A gene obviously reduces in the cell; Protein expression level obviously reduces; The invasive ability of esophageal cancer cell is significantly reduced, and can promote the esophageal cancer cell apoptosis of UV radiation-induced.
In a word, the present invention utilizes the RNA perturbation technique, successfully obtains the shRNA to people Aurora-A gene; Carrier for expression of eukaryon so that this sequence is the basis can be stablized inhibition Aurora-A mRNA and the protein expression level that continues in cell, overcome of short duration shortcoming siRNA action time of external chemosynthesis; The carrier for expression of eukaryon that is the basis with this sequence can effectively suppress the invasion and attack of esophageal cancer cell and the esophageal cancer cell apoptosis that promotes the UV radiation-induced in cell, therefore, and for the exploitation of new type antineoplastic medicine provides new way.
Description of drawings
Fig. 1 is the characteristic pattern of rna interference vector of the present invention;
Fig. 2 observes EC9706 cytological map (* 100) down for simple microscope after the transfection;
Fig. 3 observes egfp expression figure (* 100) in the EC9706 cell down for fluoroscope after the transfection;
The RT-PCR that Fig. 4 expresses for Aurora-A mRNA in the EC9706 cell after the transfection is figure as a result;
Fig. 5 is the Western blot of Aurora-A protein expression in the EC9706 cell after transfection figure as a result;
Fig. 6 is control group Transwell invasion and attack experimental result picture (* 100) after the transfection;
Fig. 7 is experimental group Transwell invasion and attack experimental result picture (* 100) after the transfection;
Fig. 8 is Transwell invasion and attack experimental result statistical graph after the transfection;
Fig. 9 is the cellular control unit DAPI colored graph (* 200) of UV radiation-induced after the transfection;
Figure 10 is the experimental group cell DAPI colored graph (* 200) of UV radiation-induced after the transfection.
Embodiment
Following examples only are used to the present invention is described and are not used in restriction scope of the present invention, the experimental technique of unreceipted actual conditions in the following example, the condition of perhaps advising according to production firm according to normal condition usually.
Embodiment 1:
Can reduce the design of the shRNA of people Aurora-A genetic expression
MRNA base sequence according to people Aurora-A gene in the ncbi database (GeneBank numbers NM_003600.2); SiRNA tool software (siDirect version 2.0) by siDirect company provides on Internet is selected target sequence; The 995th～1015 of target sequence behaviour Aurora-A mRNA; Shown in SEQ ID NO.2, i.e. 5 '-GCACCACUUGGAACAGUUUAU-3 '
Corresponding shRNA sequence according to the target sequence design is:
SEQ?ID?NO.1：5’-GCACCACUUGGAACAGUUUAUUUCAAGAGAAUAAACUGUUCCAAGUGGUGC-3’
The dna sequence dna of coding shRNA is:
SEQ?ID?NO.3：5’-GCACCACTTGGAACAGTTTATTTCAAGAGAATAAACTGTTCCAAGTGGTGC-3’
This shRNA sequence in vivo or external shear to form contain siRNA positive-sense strand and the antisense strand shown in SEQ ID NO.4 and SEQ ID NO.5:
SEQ ID NO.4: positive-sense strand is 5 '-GCACCACUUGGAACAGUUUAU-3 ',
SEQ ID NO.5: antisense strand is 5 '-AUAAACUGUUCCAAGUGGUGC-3 '.
Embodiment 2:
Can reduce the structure of the shRNA interference carrier of people Aurora-A genetic expression
According to above sequence, synthesize the DNA shown in the SEQ ID NO:3 by Shanghai Ji Ma ltd through chemical synthesis, and add the restriction enzyme site of BamH I and Bbs I at two ends.The DNA oligonucleotide is dissolved in the water of sterilization, nuclease free, final concentration is 3mg/mL.Annealing reaction is with the annealing buffer of the forward of each 1mL and reverse DNA oligonucleotide and 48ml (10mM Tris, pH 7.5-8.0,50mM NaCl; 1mM EDTA) mixes; At 90 ℃ of incubation 4min, 70 ℃ of incubation 10min slowly cool off annealed oligonucleotide to 10 ℃.Annealing product and empty pGPU6/GFP/Neo plasmid (Ji Ma ltd in Shanghai provides) are carried out double digestion, adopt DNA purification kit purifying enzyme to cut product, respectively get 2 μ L and connect with the T4 dna ligase.The pGPU6/GFP/Neo carrier of reorganization is transformed the agarose plate that contains penbritin, and the picking positive colony confirms whether insert correct sequence in the recombinant plasmid through order-checking.
The plasmid characteristic is as shown in Figure 1, and wherein shDNA represents the dna sequence dna (SEQ ID NO.3) of code book invention sequence shRNA (SEQ ID NO.1).Order-checking confirms that insertion sequence is entirely true.
Embodiment 3:
Contain the preparation of people Aurora-A-shRNA interference carrier medicine
8 μ g plasmids are diluted in 500 μ L not to be had in the substratum, gently mixing.Get Lipofectamine 2 000 (liposome mediated-method) 8 μ L and be diluted in 500 μ L serum free mediums, mixing, incubated at room 5min.The liposome of dilution is mixed incubated at room 20min with the DNA of dilution.
Embodiment 4:
Utilization contains the medicine transient transfection human esophagus cancer EC9706 cell of people Aurora-A-shRNA interference carrier
Get the growth conditions good cell, in transfection previous day with cell inoculation in petridish, cell density reaches about 70% during transfection.Embodiment 3 drug prepared are added in the petridish, add serum free medium to 2mL, gently mixing.Add behind the 6h and contain 20% RPMI 1640 substratum 2mL, behind the 24h, discard former substratum, change and contain 10% RPMI 1640 substratum.Collecting cell is identified behind the 48h.
Embodiment 5:
Microscopically is observed the expression of green fluorescent protein in the EC9706 cell after the transfection
Have the GFP gene on the carrier, expressing green fluorescent protein is beneficial to the transfection efficiency of observing the medicine that contains recombinant vectors.The result shows that along with the prolongation of transfection time, green fluorescence increases enhancing gradually.The expression of green fluorescent protein in (Fig. 3) paired observation cell under the same visual field white light (Fig. 2) and the fluoroscope, when calculating at 48h, the transfection efficiency that contains the medicine of people Aurora-A-shRNA interference carrier is about 50-60%.
Embodiment 6:
Semi-quantitative RT-PCR detects the interference effect that Aurora-A mRNA is expressed after the transfection
Behind the transfection 48h,, get RNA 7.5 μ g with Trizol reagent extracting cell total rna, random hexamers (500 μ g/mL) 2 μ L, 10mM dNTP mix 1 μ l adds DEPC water to 10 μ L, 65 ℃ of effect 5min behind the mixing; Place 3-5min on ice, add 10 * RT buffer, 2 μ l, 25 mmol/L MgCl
24 μ l, 0.1mol/L DTT 2 μ l, 25 ℃ of effect 2min behind the mixing; In every pipe, add SuperscriptII
TMReversed transcriptive enzyme 1 μ l, mixing, 25 ℃ act on 10min successively, 42 ℃ of effect 90min, 70 ℃ of effect 15min, rt becomes cDNA.Be template again with cDNA, PCR amplification Aurora-A gene, upstream primer is: 5 '-AATGATTGAAGGTCGGATGC-3 '; Downstream primer is: 5 '-TTCTCTGAGCATTGGCCTCT-3 '.With house-keeping gene GAPDH is internal reference, and upstream primer is: 5 '-GCTGAGAACGGGAAGCTTGT-3 '; Downstream: 5 '-GCCAGGGGTGCTAAGCAGTT-3 '.Primer is synthetic by the precious biotech firm in Dalian.The PCR reaction system is following:
| Component | Volume (μ L) |
| 10×PCR Buffer | 2.5 |
| MgCl 2 | 1 |
| dNTP (5 mmol/L) | 2 |
| The Taq archaeal dna polymerase | 0.25 |
| The cDNA template | 1 |
| On the goal gene/downstream primer (10 mmol/L) | 2 |
| The GAPDH primer | 1 |
| Deionized water | 15.25 |
| TV | 25 |
Reaction conditions is: 94 ℃ of 30 s, 58 ℃ of 30 s, 72 ℃ of 30 s, totally 30 circulations.The result shows, with negative plasmid transfection group relatively, the mRNA transcriptional level that contains Aurora-A gene in the drug-treated cell of people Aurora-A-shRNA interference carrier obviously reduces (see figure 4).
Embodiment 7:
Western blot method detects the interference effect to the Aurora-A protein expression after the transfection
Collecting cell behind the transfection 48h is got 50 μ g total protein of cell, behind 10% SDS-PAGE; Be transferred to nitrocellulose filter; With the anti-Aurora-A monoclonal antibody of rabbit (1:1000 dilution) is one anti-, and the sheep anti-mouse igg of HRP mark is two anti-, through hatch wash film after ECL develop; Detecting the proteic expression level of Aurora-A in the cell, is internal reference with β-actin.The result shows, with negative plasmid transfection group relatively, contain that the proteic expression level of Aurora-A obviously reduces (see figure 5) in the drug-treated cell of people Aurora-A-shRNA interference carrier.
Embodiment 8:
Transwell invasion and attack experiment detects the influence of pair cell invasive ability after the transfection
Dilution Matrigel to 250 μ g/mL gathers 8 μ m holes that carbon ester film is two-sided to encapsulate each 30min through extracellular matrix Matrigel, takes out air-dry subsequent use.30 μ L RPMI, 1640 substratum are added in chamber under the Boyden cell, and the 8 μ m holes that Matrigel encapsulates are gathered carbon ester film and are laid on down above the chamber, add one deck rubber pad again, and install groove on the Boyden cell.Get the digestion of growth conditions good cell, with 50 μ L 2 * 10
4Individual cell seeding is the chamber under each Boyden cell, in 37 ℃ of 5%CO
2Cultivate in the incubator.Carefully take off film behind the 24h, scrape off the cell that move on the upper strata.Methyl alcohol with 75% is the cell 15min on the film fixedly.Behind 0.5% Viola crystallina (methyl alcohol preparation) dyeing 20min, zero(ppm) water cleans.In the cell count that the microscopically counting gathers carbon ester film lower surface, carry out statistical analysis, take pictures simultaneously.The result shows, the cell count that the drug-treated cell that contains people Aurora-A-shRNA interference carrier passes film is 56 ± 12 (Fig. 7), and the cell count that control group passes film is 520 ± 32 (Fig. 6).The result shows, compares with control cells, and the drug-treated cells in vitro invasive ability that contains people Aurora-A-shRNA interference carrier obviously weakens, and through statistical analysis, difference has the significance (see figure 8).
Embodiment 9:
The DAPI staining detects the influence of UV irradiation pair cell apoptosis capacity after the transfection
UV behind the transfection 48h (30J/m2,254 nm uv lamps) irradiating cell cleans cell with PBS behind the 12h, cold methanol fixed cell 30min.The PBS cells washed, 0.1 μ g/mL DAPI (4', 6'-diamidino-2-phenylindole hydrochloride, 4,6-diamidine-2-phenylindone) transfect cell nuclear, incubated at room 15min.The PBS cells washed is removed excess liq, mounting with the filter paper suction.The result shows; Compare (Fig. 9) with control cells, contain the cell after the drug-treated of people Aurora-A-shRNA interference carrier, after the UV irradiation; The nucleus chromatin condensation appears in a lot of cells; The edge forms crescent, and nuclear membrane collapse and cytolysis form the apoptotic body of cohesion, presents the characteristic (see figure 10) of typical apoptotic.Point out the medicine that contains people Aurora-A-shRNA interference carrier can promote the esophageal cancer cell apoptosis of UV radiation-induced.
Sequence table
< 110>Mountain Western Medicine S University
< 120>one specific specificity reduce the shRNA and the application thereof of people Aurora-A genetic expression
<160>5
<170>?PaUentIn?Version?3.5
<210>?1
<211>?51
<212>?RNA
< 213>artificial sequence
< 223>the shRNA sequence that designs according to the Aurora-A gene siRNA
<400>?1
gcaccacuug?gaacaguuua?uuucaagaga?auaaacuguu?ccaaguggug?c
<210>?2
<211>?21
<212>?RNA
< 213>people (Homo sapiens)
< 223>Aurora-A target-gene sequence
<400>?2
gcaccacttg?gaacagttta?t
<210>?3
<211>?51
<212>?DNA
< 213>artificial sequence
< 223>dna sequence dna of coding Aurora-A gene shRNA
<400>?3
gcaccacttg?gaacagttta?tttcaagaga?ataaactgtt?ccaagtggtg?c
<210>?4
<211>?21
<212>?RNA
< 213>people (Homo sapiens)
< 223>shRNA shears product siRNA positive-sense strand
<400>?4
gcaccacuug?gaacaguuua?u
<210>?5
<211>?21
<212>?RNA
< 213>people (Homo sapiens)
< 223>shRNA shears product siRNA antisense strand
<400>?5
auaaacuguu?ccaaguggug?c
Claims (6)
1. a specific specificity reduces the shRNA of people Aurora-A genetic expression, it is characterized in that its base sequence is shown in SEQ ID NO.1, promptly
5’-?GCACCACUUGGAACAGUUUAUUUCAAGAGAAUAAACUGUUCCAAGUGGUGC-3’。
2. a specific specificity according to claim 1 reduces the shRNA of people Aurora-A genetic expression, it is characterized in that the 995th～1015 of target sequence behaviour Aurora-A mRNA that it is directed against, shown in SEQ ID NO.2, promptly
5’-?GCACCACUUGGAACAGUUUAU-3’。
3. a specific specificity according to claim 1 reduces the shRNA of people Aurora-A genetic expression, it is characterized in that this shRNA transcribes generation through the DNA oligonucleotide chain shown in SEQ ID NO.3, promptly
5’-?GCACCACTTGGAACAGTTTATTTCAAGAGAATAAACTGTTCCAAGTGGTGC-3’。
4. a specific specificity according to claim 1 reduces the shRNA of people Aurora-A genetic expression, it is characterized in that this shRNA transcribes generation through the plasmid that comprises the DNA oligonucleotide chain shown in SEQ ID NO.3.
5. a specific specificity according to claim 1 reduces the shRNA of people Aurora-A genetic expression, it is characterized in that this shRNA can shear formation and contain siRNA positive-sense strand and the antisense strand shown in SEQ ID NO.4 and SEQ ID NO.5, promptly
SEQ ID NO.4: positive-sense strand is 5 '-GCACCACUUGGAACAGUUUAU-3 ',
SEQ ID NO.5: antisense strand is 5 '-AUAAACUGUUCCAAGUGGUGC-3 '.
6. the application of shRNA in treatment esophageal carcinoma medicine of a specificity reduction people Aurora-A as claimed in claim 1 genetic expression.
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