CN101705227A - SiRNA for inhibiting human AP-2alpha gene expression and anti-cervical cancer application thereof - Google Patents

Abstract

The invention discloses siRNA for inhibiting human AP-2alpha gene expression and anti-cervical cancer application thereof. In the invention, a biological database is utilized to obtain a human AP-2alpha gene sequence, a group of siRNA capable of inducing RNA interference of human AP-2alpha is designed, and a certain amount of siRNA is synthesized by a chemical method, so that the mRNA level and protein expression level of the human AP-2alpha gene are specifically reduced; meanwhile, the siRNA greatly reduces the activation of transcription level and protein level of a transcription factor AP-2alpha in cervical cancer cells HeLa to a cancer gene ErbB2 to inhibit the propagation of the cancer cells. Therefore, AP-2alpha siRNA is expected to be developed into a novel high-efficiency medicament for treating cervical cancer.

Description

The siRNA and the anti-cervical cancer application thereof that suppress human AP-2 alpha gene expression

Technical field:

The present invention relates to a kind of siRNA, particularly relate to a kind of siRNA and application in the medicament for resisting cervical cancer preparation thereof that suppresses human AP-2 alpha gene expression.

Background technology:

Along with Economic development, the raising of living standards of the people raises year by year in whole global cervical cancer patient's mortality ratio.Cervical cancer is as the malignant tumour of a ubiquity, and its height lethality extensively betides among the whole world women.Therefore, the research that cervical cancer is carried out gene therapy has very important using value.Cervical cancer is a disease that is subjected to the polygene network regulation.AP-2 studies show that as an important transcription factor family AP-2 plays important regulation in cervical cancer.Wherein AP-2alpha in cell growth and tumour generation, play an important role (Cancer Res, 2004,64:631-638).The function part of AP-2alpha is by combining with AP-2alpha downstream gene promoter region, promoting it to express and realize.Oncogene ErbB2 extensive mistake in cervical cancer expressed, and is the target spot of research treatment all the time.ErbB2 function in tumour cell is lost and will be caused the cell growth-inhibiting, and cause apoptosis (Cancer Research, 1997,57:3804-3811).The downward modulation of current research report ErbB2 expression of gene has promoted the apoptosis of cervical cancer cell, thereby reaches the purpose (Med Oncol, 2009) of treatment cervical cancer.There are some researches show that AP-2alpha combines with oncogene ErbB2 gene promoter, promote ErbB2 gene transcription and protein expression, thus shown AP-2alpha cervical cancer take place developing regulating and controlling effect (Oncogene, 1996,13:1701-1707).Nearly 2O, RNAi (RNA interference) has shown strong functions in the tumour Mechanism Study, also shown bright prospect as the novel tumor therapeutic strategy.RNAi is the gene silencing mechanism on the mRNA level of high special, bring out by endogenous or exogenous dsRNA (double-stranded RNA), in cell, be cut into the little RNA of 21-25nt interference (small interfering RNA), siRNA mediation identification and target cutting homology said target mrna molecule, thus cause this gene not expressed.SiRNA is because strong specific RNA interference effect has now become a kind of ideal cell levels gene knockout means.The special genetic expression of generation in the mammalian cell that appears at of siRNA suppresses, and makes that siRNA is that human treatment of diseases has been started the new world.

Therefore how to obtain that a kind of to suppress the siRNA and the carrier thereof of human AP-2 alpha gene expression and be applied to them in the medicament for resisting cervical cancer preparation be present problem demanding prompt solution.

Summary of the invention

Purpose one of the present invention provides a kind of siRNA that suppresses human AP-2 alpha gene expression; The 2nd, can produce the expression vector of above-mentioned siRNA; The 3rd, the purposes of the expression vector of described siRNA or siRNA in the medicament for resisting cervical cancer preparation.

The siRNA of inhibition human AP-2 alpha gene expression provided by the invention, its base sequence and site of action thereof are as follows:

Positive-sense strand 5 ' CGAAGU CUU CUG UUC AGU U 3 '

Antisense strand 5 ' AAC UGAACA GAA GAC UUC G 3 '

Act on the 622-642 position of human AP-2 alpha mRNA

Described siRNA can pass through synthetic, and this siRNA has 2-6 dT to modify or 2-6 U modification 3 '.

The expression vector of described siRNA.

The application of described siRNA in the medicament for resisting cervical cancer preparation.

The application of the expression vector of described siRNA in the medicament for resisting cervical cancer preparation.

The present invention is the target sequence with siRNA, based on the RNA perturbation technique, obtain the cDNA sequence of human AP-2 alpha gene from GenBank, fundamental principle according to the selection of siRNA target sequence, at the AP-2alpha gene design 21 Nucleotide, siRNA 3 ' end adds two deoxyribonucleotides (dTdT) and is the single catenary suspension structure, with strengthen this siRNA in vivo with external stability, prevent degraded.Employed siRNA entrusts Shanghai Ji Ma company synthetic by chemical method by the inventor oneself design among the present invention.This siRNA that experimental results show that by the contriver has greatly reduced transcription factor AP-1-2alpha activation to transcriptional level and the protein level of oncogene ErbB2 in cervical cancer cell HeLa, MTT analysis revealed AP-2alpha crosses and expresses the growth that has promoted the HeLa cervical cancer cell simultaneously, and AP-2alpha siRNA has then suppressed the propagation of cervical cancer cell.Soft-agar cloning forms experimental result and shows as one man that also AP-2alphasiRNA adding back cervical cancer cell forms the number of cloning and sharply descends.Therefore, AP-2alpha siRNA provided by the invention can specificity suppress the AP-2alpha expression of gene, thereby reaches the generation of inhibition cervical cancer and the purpose of growth.This siRNA can be used for preparing the medicament for resisting cervical cancer of high specificity.

Description of drawings

Fig. 1 is that RT-PCR detects the inhibition of AP-2alpha siRNA to the mRNA level of AP-2alpha1 gene.

Fig. 2 is that Western Blotting detects the inhibition of AP-2alpha siRNA to the AP-2alpha protein level.

Fig. 3 is that Luciferase and Western Blotting analyze AP-2alpha siRNA to the transcriptional level of AP-2alpha downstream oncogene ErbB2 and the influence of protein level.

Fig. 4 is that MTT analyzes the influence of AP-2alpha siRNA to cervical cancer cell propagation.

Fig. 5 is that soft-agar cloning forms the influence of experimental analysis AP-2alpha siRNA to the cervical cancer cell growth.

Embodiment

The selection of embodiment 1:siRNA target sequence

AP-2alpha siRNA provided by the present invention obtains according to the human AP-2 alpha sequence that the contriver finds in GenBank, these RNA sequences meet following principle substantially: from the AUG initiation codon of transcript (mRNA), seek " AA " two and connect sequence, and write down its 3 ' end 19 base sequences, as potential siRNA target site.Design during siRNA not at 5 ' and the non-coding region of 3 ' end, reason is that these places have abundant modulin calmodulin binding domain CaM, thereby and these UTR are conjugated protein or translation initiation complex may influence siRNP endonuclease enzyme complex influences siRNA in conjunction with mRNA effect.GC content in general 21 Nucleotide finds effective fragment easilier in the lower fragment of GC content in the scope of 30%-70%.And to avoid the siRNA target sequence to form the continuous repetition of secondary structure and identical base, these structures can influence annealing pairing and the target spot specificity of siRNA.In addition, selected sequence and corresponding genome database (people, perhaps mouse, rat or the like) are compared, get rid of those and other encoding sequences/EST homologous sequence.For example use BLAST (www.ncbi.nlm.nih.gov/BLAST/).Also need RNA space structure picture at last with reference to the Sfold recommendation.We generally add the tail of two dTdT at 3 ' end of positive-sense strand, instruct siRNA to form the reticent mixture of RNAi, also avoid being subjected to the degraded of nuclease.

The contriver is according to above principle, and pair of sequences has been synthesized in design, and its concrete sequence is as follows:

Positive-sense strand 5 ' CGAAGU CUU CUG UUC AGU UdTdT 3 '

Antisense strand 5 ' AAC UGAACA GAA GAC UUC GdTdT 3 '

Act on the 622-642 position of human AP-2 alpha mRNA

It is synthetic to serve Hai Jima company after sequences Design is good, and the purity of the siRNA two strands that the present invention uses is greater than 99%, and the distilled water dissolving with DEPC handled just can be directly used in cell transfecting.

Embodiment 2:RT-PCR detects the gene silencing effect of AP-2alpha siRNA

The HEK293FT cell is that preserve in this laboratory, with 6 orifice plates at 37 ℃, 5%CO ₂, cultivate with DMEM complete culture solution (adding 10% new-born calf serum, 2mM L-L-glutamic acid, penicillin and Streptomycin sulphate) in the 90% relative humidity incubator, treat cell density to 70%.With liposome Lipofectamine 2000 transfection reagent boxes AP-2alpha siRNA is changed in the HEK293FT cell.The liposome transfection method is the nutrient solution that cell is changed into 1.5mL serum-free antibiotic-free before transfection.Every hole siRNA consumption is 100pM, is diluted to 250 μ L respectively with the nutrient solution of serum-free antibiotic-free, mixing gently, and room temperature leaves standstill 5min.Simultaneously liposome is diluted in the nutrient solution of 250 μ L serum-free antibiotic-frees and contains 5 μ L liposomes, mixing gently, room temperature leaves standstill 5min.The liposome of dilution is added equal-volume siRNA mixing, and room temperature leaves standstill 20min.With 500 μ L siRNA/ liposome complexes mixing gently, be added dropwise in the cell, mixing gently, normal condition is cultivated.Allow behind DNA/ liposome complex and the cells contacting 6h, changed serum and antibiotic nutrient solution into.Continue to cultivate 36h, collecting cell.

Cell is directly added 500 μ L TRIzol lysing cell in culture plate, inhale with pipettor and beat several times.Place 10min in room temperature, the nucleic acid-protein mixture is separated fully.Add 100 μ L chloroforms then, thermal agitation 15sec, room temperature is placed 3min.4 ℃ of centrifugal 15min of 10000 * g.Sample is divided into three layers: bottom is yellow organic phase, and the upper strata is colourless water and a middle layer.RNA is mainly at aqueous phase, and the water volume is about 60% of used TRIzol reagent.Water is transferred in the new pipe, with the RNA of isopropanol precipitating aqueous phase.Add 250 μ L Virahols, room temperature is placed 10min.4 ℃ of centrifugal 10min of 10000 * g do not see the RNA precipitation before centrifugal, and centrifugal back gelatinous precipitate occurs in the pipe side and the pipe end.Remove supernatant.Precipitate with 75% washing with alcohol RNA.Every use 1mL TRIzol adds 1mL 75% ethanol at least.4 ℃ are no more than the centrifugal 5min of 7500 * g, abandon supernatant.Room temperature places drying or vacuum is drained the RNA precipitation, and the 5-10min that approximately dries in the air gets final product.Do not want the traditional vacuum drying, too drying can cause the solvability of RNA to reduce greatly.Adding 25-200 μ L does not have the water of RNase, inhales with the rifle head and beats several times, places 10min for 55-60 ℃ and makes the RNA dissolving.Running glue detects the quality of mRNA and measures concentration with ultraviolet spectrophotometer.

With total RNA is template, carries out reverse transcription with AMV ThermoScript II and random primer, first chain of synthetic cDNA.The reverse transcription system is 20 μ L, wherein contains the total RNA of 1 μ g, reverse transcription damping fluid, 5mMMgCl ₂, 1mM dNTP, 3 ' the end random primer of 200pM, AMV ThermoScript II (Promega), RNA enzyme inhibitors.Reverse transcription program: 42 ℃ of 5min, 37 ℃ of incubation 50min reverse transcription, 75 ℃ of 5min deactivation ThermoScript II.Gained cDNA is in-20 ℃ of PCR reactions that store for future use or carry out immediately.Its reaction cumulative volume is under the condition of 20 μ L, wherein all contains in the reaction system: 2 μ L reaction buffers, 1U exTaqDNA polysaccharase, 1 μ L template cDNA, AP-2alpha and-the actin primer respectively adds 1 μ L.Response procedures: 94 ℃ of pre-sex change 3min at first, after carry out 30 circulations: 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 1min; Last 72 ℃ are extended 5min, 4 ℃ of terminations.After reaction is finished product being carried out agarose electrophoresis, takes pictures and analytical results with gel imaging system in EB dyeing back.The result shows that AP-2alphasiRNA1 can reduce the mRNA level of AP-2alpha gene effectively, and the siRNA of negative control is to the not influence (Fig. 1) of mRNA level of AP-2alpha gene.

Embodiment 3:Western Blotting detects the influence of AP-2alpha siRNA to the AP-2alpha protein level.

The pCMV-Myc-AP-2alpha plasmid is made up by the laboratory and checks order proves the exactness of its sequence.The cultivation and the transfection of HEK293FT cell are described with the front, and every hole adds 2 μ g DNA and 50pM siRNA.Inhale after transfection is finished and remove nutrient solution, PBS rinsing cell, exhaustion PBS, 1 * the lysis buffer (50mM Tris-HCl (pH 7.2), 150mM NaCl, 1% (v/v) Triton X-100 that add 100 μ L again, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS) shake up.Place 15min in-80 ℃ of refrigerators, incubation 5min in 37 ℃ of incubators repeat to freeze once molten, make the abundant cracking of cell.Scrape with cell cell is scraped from aperture, change cell pyrolysis liquid and cell the centrifuge tube of 1.5mL over to liquid-transfering gun, place on ice.Vortex concussion 10-15sec, 12000 * g is in 4 ℃ of centrifugal 2min.Getting supernatant liquor is transferred in the clean centrifuge tube.Protein sample at first used 15% SDS-polyacrylamide gel (SDS-PAGE) electrophoresis.To pvdf membrane, the protein with the film surface carries out specific detection with antigen antibody reaction to method (constant voltage 100v, 4 ℃ of electrotransfer 1.5h) by electrotransfer more then with the protein transfer printing.(10mM Tris-HCl (pH 7.4) is 0.9%NaCl) as encapsulant sealing 60min with the TBS damping fluid that contains 10% skim-milk for the nylon membrane that transfer printing is good.The film submergence adds the mouse monoclonal antibody Myc by usefulness encapsulant dilution in 1: 1000, shakes 1h and allow the antibody of dilution fully combine with pvdf membrane in shaking table.Wash film 4 times with the TBS damping fluid, each 10min.The sheep anti-mouse igg (diluting 2000 times) of horseradish peroxidase-labeled is added encapsulant as second antibody, make the film submergence wherein in shaking table, continue to shake 1h.Wash film 4 times with the TBS damping fluid.At last with the colour developing of DAB solution.The result shows that AP-2alpha siRNA can suppress the expression of AP-2alpha albumen in cell, and the siRNA of negative control points out AP-2alpha1 siRNA1 specificity to suppress the expression of AP-2alpha to the not influence of AP-2alpha protein level; GAPDH contrasts as last sample, is consistent (Fig. 2) with the applied sample amount that guarantees the total protein that each is organized.

Embodiment 4:Luciferase and Western Blotting analyze AP-2alpha siRNA to the transcriptional level of AP-2alpha downstream oncogene ErbB2 and the influence of protein level.

The HeLa cell is preserved for this laboratory.With the DMEM culture medium culturing that contains 5 μ g/ml Regular Insulin, the cell of 24 orifice plates is cultivated and transfection, and 1 * lysis buffer (the Promega test kit provides) that the results back adds 100 μ L shakes up.Place 15min in-80 ℃ of refrigerators, incubation 5min in 37 ℃ of incubators repeat to freeze once molten, make the abundant cracking of cell.Pipette 20 μ L cell extracts with liquid-transfering gun and carry out the beta-galactosidase enzymes check and analysis, add 180 μ L analysis buffer,, have an amount of yellow substance to occur at 37 ℃ of incubation 10min.Because transfection has added the expression vector of LacZ simultaneously, and LacZ is a reporter gene that uses in the cell transfecting experiment usually, its coding beta-galactosidase albumen, this proteic expression level is very stable and detects easily, can by with the substrate ONPG rapid reaction yellowly product of sensitivity, writing time is with 100 μ L 1M yellow soda ash termination reactions.Survey the OD value at the 420nm place with ultraviolet spectrophotometer, carry out quantitatively determining the concentration of cell extract according to the OD value.Use luciferase detection kit (Promega), every pipe sample is got 20 μ L and is added 100 μ L luciferase substrates measurement fluorescence intensity under TD-20/20Luminometer (Turner Designs), record experimental data.Found that, found that transcription factor AP-1-2 has activated the transcriptional activity of downstream gene ErbB2, but its activation is removed basically after adding AP-2 siRNA1, and the siRNA of negative control is to the not influence of promoter activity of AP-2; And Western Blotting result has also as one man shown the variation (Fig. 3) of ErbB2 protein level.

Embodiment 5:MTT analyzes the influence of AP-2alpha siRNA to the cervical cancer cell growth.

The HeLa cell is inoculated in 24 orifice plates equally, carry out cell transfecting, put 37 ℃ afterwards, cultivate 24～72h under the 5%CO2 condition, 4h adds MTT10 μ L/ hole (concentration is 5mg/ml) before finishing, and abandons supernatant liquor after continuing to cultivate 4h, add dimethyl sulfoxide (DMSO) (DMSO) 100 μ L/ holes, carry out light absorption value on the microplate reader in vibrating about 10min on the vibrator, putting and detect, wavelength is 570nm.As shown in Figure 4, when crossing, AP-2alpha expresses the growth that has promoted cervical cancer cell; Basically do not breed and add AP-2alpha siRNA1 cervical cancer cell, prompting AP-2alpha siRNA can suppress the growth of cervical cancer cell.

Embodiment 6: soft-agar cloning forms the influence of experimental analysis AP-2alpha siRNA to the cervical cancer cell growth.

The HeLa cell in vegetative period of taking the logarithm, with 0.25% tryptic digestion and piping and druming gently, make it to become unicellular, make viable count, adjust cell density to requirement of experiment with the RPMI-1640 that contains 20% foetal calf serum. prepare the LMP agar liquid glucose of 1.2% and 0.7% two concentration respectively with distilled water, behind the autoclaving, maintain in 40 ℃ and can not solidify. after in 1: 1 ratio 1.2% agarose and 2 * 1640 substratum (calf serum that contains 2 * microbiotic and 20%) being mixed, get the 3mL mixed solution and inject diameter 6cm plate, cooled and solidified can be put CO as bottom-layer agar ₂Standby in the incubator.Allow 0.7% agarose with after 2 * 1640 substratum mix in sterile test tube mutually in 1: 1 ratio, add the cell suspension of 0.2mL again in pipe, abundant mixing injects and is covered with 1.2% agarose bottom plate, by forming two agar layer.After treating that top-layer agar solidifies, insert in 37 ℃ of 5%CO2 incubators and cultivated 10～14 days.Plate is placed under the inverted microscope, and observation of cell clone number calculates rate of formation.As shown in Figure 5, AP-2alpha crosses the formation that expression has promoted the cervical cancer tumer line cell clone effectively, and disturbs back cell clone number sharply to descend AP-2alpha.

The application of embodiment 7:AP-2alpha siRNA in the preparation medicament for resisting cervical cancer.

All experiments show that all the AP-2alpha siRNA of contriver's design can suppress the expression of mRNA and the protein level of AP-2alpha effectively, experimental results show that further this siRNA can suppress the growth of cervical cancer cell.Illustrating that this little double-stranded RNA can be used for preparing with transcription factor AP-1-2alpha expresses relevant cervical cancer medicine.Be used for human vivo medicine-feeding, can use separately or unite use with other medicines.

And this siRNA has also reduced the transcriptional activation of transcription factor AP-1-2 pair downstream gene ErbB2 promotor specifically, MTT analysis revealed AP-2alpha crosses and expresses the growth that has promoted the HeLa cervical cancer cell simultaneously, and AP-2alpha siRNA has then suppressed the propagation of cervical cancer cell.Soft-agar cloning forms experimental result and shows as one man that also AP-2alpha siRNA adding back cervical cancer cell forms the number of cloning and sharply descends.

SEQUENCE?LISTING

＜110〉Hunan Normal University

＜120〉siRNA and the anti-cervical cancer application thereof of inhibition human AP-2 alpha gene expression

＜130〉biological field

<160>2

<170>PatentIn?version?3.3

<210>1

<211>19

<212>RNA

<213>Homo?sapiens

<220>

＜223〉description of artificial sequence: the little double-stranded RNA of synthetic

<400>1

cgaagucuuc?uguucaguu?19

<210>2

<211>19

<212>RNA

<213>Homo?sapiens

<220>

＜223〉description of artificial sequence: the little double-stranded RNA of synthetic

<400>2

aacugaacag?aagacuucg?19

Claims (5)

1. siRNA who suppresses human AP-2 alpha gene expression, its base sequence and site of action thereof are as follows:

Positive-sense strand 5 ' CGA AGU CUU CUG UUC AGU U 3 ';

Antisense strand 5 ' AAC UGA ACA GAA GAC UUC G 3 ';

Act on the 622-642 position of human AP-2 alpha mRNA.

2. according to the siRNA described in the claim 1, can pass through synthetic, it is characterized in that this siRNA has 2-6 dT to modify or 2-6 U modification 3 '.

3. the expression vector of a siRNA as claimed in claim 1 or 2.

4. the application of the siRNA described in claim 1 or 2 in the medicament for resisting cervical cancer preparation.

5. the application of expression vector in the medicament for resisting cervical cancer preparation as siRNA as described in the claim 3.

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