CN101220359B - SiRNA sequence restraining bak gene expression - Google Patents

Abstract

The invention relates to a siRNA double-strand sequence, which is a siRNA sequence for inhibiting bak gene expression. The invention solves the problem that the prior art has no siRNA sequence which is most appropriate for inhibiting the bak gene expression, the sequence is siRNA1: an antisense strand is 5'-AGUGCACACUUGCUAAAGCtt-3' and a sense strand is 5'-GCUUUAGCAAGUGUGCACUtt-3'; the siRNA double-strand sequence which is provided by the invention can be used for preparing drugs for inhibiting neural cell apoptosis or treating neurodegenerative diseases. The siRNA double-strand sequence enters into a biosome in a certain mode, which is the specific expression for inhibiting disease-related proteins and is more effective than any means; the invention can lead related disease genes to be in silence, carry out effective knockout to the expression of a target gene and achieve treatment purpose, and the specificity of the sequence and the powerful effect of the gene inhibition effect are unbeatable compared with the other drugs.

Description

A kind of siRNA sequence that suppresses bak genetic expression

Technical field

The present invention relates to the double-stranded sequence of siRNA, be specially a kind of siRNA sequence of the bak of inhibition genetic expression.

Background technology

RNA disturbs (RNA interference, RNAi) phenomenon is a kind of defense mechanism of resisting transgenosis or adventitious viruses infringement conservative on evolving, be a kind of sequence-specific PTGS (post-transcriptional gene silencing, PTGS).It is found in the organism of many different generas, and transmits between biological cell, has the opposing poisoning intrusion and keeps the stable effect of genome.The RNAi technology can not only advance the development of human post genome project greatly, can also high-throughput ground screening of medicaments target gene, promote gene therapy, new drug development etc., for diseases such as treatment cancer, inherited disease are opened up new approach.The research of RNAi and application have extremely important theory and practical significance, will produce far-reaching influence to the development of Medical Biology.

Though the research course of RNA perturbation technique is shorter, its tempo is out of people's the imagination.RNAi is widely used in the medical researches such as virus infection, cancer, dominant hereditary disease as a kind of new gene therapy in several years in the past, opened up the new concept of " gene therapy (gene-specific therapeutics) ", siRNA (small interfering RNA, siRNA) even be called as " disease medicine ".At present, people have begun to utilize the RNAi technology to analyze the intragentic function of mammalian body from genomic level, can more promptly identify many and gene disease-related, make our network system of the interior gene regulating of postgraduate's object better, the RNAi technology can successfully have been cured the pathology of mammalian cell, organ and live body now, indicating that in the near future RNAi can successfully be used for the treatment of human diseases, making that the title of " disease medicine " of siRNA is qualified.

Known, the intravital cell of people is doomed to dead, some death is physiological, some death then is pathologic, the research of cells involved death process has become a focus of biology, medical research, up till now for this reason in recent years, people have known that the death of cell has dual mode at least, i.e. necrocytosis and apoptosis.Present research is thought, necrocytosis is that cell is accepted post-stimulatory a kind of passive cell death way, apoptosis then is a kind of procedural cell death way of active, it is a kind of basic biological phenomena of cell, apoptosis is the process of the strict control of polygene, these genes are very conservative between kind, as the Bcl-2 gene family, the caspase gene family, oncogene etc., along with the development of Protocols in Molecular Biology has had suitable understanding to the process of apoptosis of many kinds, but the apoptotic process precise mechanism still imperfectly understands up to now, and the disorder of apoptotic process may with the direct or indirect relation of having of numerous disease, as nerve degenerative diseases, tumour, autoimmune disorder etc.Prevent expression of apoptosis-related genes and protein level thereof specifically if can use the RNA perturbation technique, can effective reticent apoptosis-related genes, help cell to reduce apoptosis, promote cell viability.Therefore, RNA interferential research to apoptosis-related genes has extremely important theory and practical significance with application, the screening of medicaments target gene promotes gene therapy, new drug development etc. effectively, for the diseases relevant with apoptosis such as treatment nerve degenerative diseases are opened up new approach.

The thinking of using RNAi treatment disease is according to disease relevant gene order taking place in the Disease-causing gene sequence of pathogenic agent and the organism, the siRNA (siRNA) that design and preparation and these gene orders have homology, by certain mode siRNA is transferred to then and make diseases related gene generation silence in the animal body, thereby reach the purpose of treatment.It is advantageous that the expression that only suppresses disease related protein, and injuring normal cell function not, the powerful property of its sequence-specific and gene inhibition effect is that other drug is difficult to be equal to.SiRNA can be used for treating any because the disease that gene is undergone mutation or overexpression causes, as long as determined target spot with disease-related, it is target gene, just can make the target gene silence specifically by siRNA, so seek optimum siRNA sequence, most important for the widespread use of RNA interferential.

The bcl-2 gene family is a genoid structural similitude and participates in apoptotic one group of gene, difference according to their pair cell apoptosis results is divided into two classes, one class is to suppress apoptotic gene, as bcl-2, and bcl-xl, mc11, bfel, al etc., another kind of is to promote apoptosis gene, as bax, bak, bok, bcl-xs, bik etc., existing a large amount of research promptings, this protein families mainly by regulating the variation of mitochondrial membrane potential, influences the permeability of mitochondrial membrane, and then the release of change mitochondrial protein, the regulating cell apoptosis.Wherein the overexpression of bak gene can be quickened cell generation apoptosis, and the restraining effect of antagonism bcl-2 pair cell apoptosis, thereby promotes apoptosis, and therefore research and the application to bak RNAi has extremely important theory and practical significance.But act on the bak gene, the action effect of siRNA that is the target gene different loci is widely different, may be relevant with the base distribution of siRNA, two ends free energy size etc., in the experiment of the reticent specific gene of siRNA, for guaranteeing the siRNA target gene of effectively degrading, generally need be at synthetic many not homotactic siRNA of target gene design, therefrom filter out ordered sequence again, so effectively the design of siRNA sequence screening is the key point of this technology, up to the present, the siRNA sequence that does not also have optimum inhibition bak genetic expression.

Summary of the invention

The present invention provides a kind of siRNA sequence of the bak of inhibition genetic expression in order to solve the problem of the siRNA sequence that does not also have optimum inhibition bak genetic expression in the prior art.

The present invention adopts following technical scheme to realize: a kind of siRNA sequence of the bak of inhibition genetic expression, this sequence are any or one or more in the following sequence:

SiRNA1: antisense strand 5 '-AGUGCACACUUGCUAAAGCtt-3 '

Positive-sense strand 5 '-GCUUUAGCAAGUGUGCACUtt-3 ';

SiRNA2: antisense strand 5 '-AUGCCACUCUCAAACAGGCtg-3 '

Positive-sense strand 5 '-GCCUGUUUGAGAGUGGCAUtt-3 ';

SiRNA3: antisense strand 5 '-UAAGGUGACCAUCUCUGGGtc-3 '

Positive-sense strand 5 '-CCCAGAGAUGGUCACCUUAtt-3 '.

In the above-mentioned sequence, the optimum siRNA sequence that is used to suppress bak genetic expression is siRNA1:

Antisense strand 5 '-AGUGCACACUUGCUAAAGCtt-3 ',

Positive-sense strand 5 '-GCUUUAGCAAGUGUGCACUtt-3 '.

The siRNA sequence of inhibition bak provided by the present invention genetic expression is made up of positive-sense strand and antisense strand, the the 1st to the 19th Nucleotide from 5 ' end of its positive-sense strand and antisense strand sequence is ribonucleotide, and the 20th to the 21st Nucleotide is deoxyribonucleotide.Sequence in the sequence table is formed by 21 Nucleotide, and the direction of sequence is 5 ' end from left to right to 3 ' end.

The present invention is directed to the synthetic one group of siRNA of bak gene order design, filter out three couples of bak siRNA (siRNA1, siRNA2, siRNA3), detect through QRT-PCR and immunohistochemical method, can effectively suppress bak genetic expression, thereby significantly reduce the expression of Bak protein level, and on neuroblastoma cell, verified design is synthetic and filter out bak siRNA restraining effect to neuroblastoma cell, wherein with bak siRNA1 best results.Bak siRNA sequence provided by the present invention not only can be used for the RNAi of cultured cell in vitro, also can be used for experiment in the body.

The double-stranded sequence of siRNA provided by the present invention can be used for preparation and bak gene-correlation treatment of diseases medicine, as the medicine of apoptosis relative disease or degenerative disease.When double-stranded sequence of this siRNA or the medicine that obtains thus enter in animal body or the human body by certain mode, can suppress the expression of disease related protein more efficient, special than any means in the past, easily, it is reticent that the diseases related gene is taken place, thereby reach the purpose of treatment, it is advantageous that not injuring normal cell function, the powerful property of its sequence-specific and gene inhibition effect is that other drug is difficult to be equal to.The present invention filters out the siRNA sequence that can efficiently suppress bak genetic expression, can effectively knock out target gene expression.

Description of drawings

Fig. 1 is the light microscopic photo of bak siRNA transfectional cell

Fig. 2 for bak siRNA transfection and after DAPI dyes nuclear the fluorescence photo of cell

Fig. 3 be bak siRNA transfection and behind the Cy3 fluorescent mark fluorescence photo of the cell of transfection success

Fig. 4 is three pairs of bak siRNA sequences in the influence of transfection 24h to the cell viability that dyes the aluminium cell

Fig. 5 is three pairs of bak siRNA sequences in the influence of transfection 48h to the cell viability that dyes the aluminium cell

Fig. 6 is three pairs of bak siRNA sequences in the influence of transfection 72h to the cell viability that dyes the aluminium cell

Fig. 7 for bak siRNA1 behind transfection 48h under the different transfection concentration to dying the influence of aluminium cell viability

Fig. 8 is that three pairs of siRNA sequences suppress bak genetic expression amplification curve

Fig. 9 is that three pairs of siRNA sequences suppress β-actin genetic expression amplification curve

Figure 10 detects the transfection of three pairs of siRNA sequences to bak genetic expression inhibiting rate for QRT-PCR

Figure 11 is the Bak protein expression light microscopic figure (* 200) of bak siRNA1 after 4mM dyes the transfection of aluminium group neuroblastoma cell

Figure 12 is the Bak protein expression light microscopic figure (* 200) of bak siRNA1 after 4mM aluminium+low dosage RNAi group neuroblastoma cell transfection

Figure 13 is the Bak protein expression light microscopic figure (* 200) of bak siRNA1 after 4mM aluminium+middle dosage RNAi group neuroblastoma cell transfection

Figure 14 is the Bak protein expression light microscopic figure (* 200) of bak siRNA1 after 4mM aluminium+high dosage RNAi group neuroblastoma cell transfection

Figure 15 is a Bak expressing quantity synoptic diagram behind the bak siRNA in-vitro transfection

Embodiment

Embodiment 1: the siRNA's of inhibition bak genetic expression is synthetic

The freeware that provides on the net with Ambion company ( Http:// www.ambion.com/), write the predictor of siRNA according to the principle of Reynolds.From Http:// www.ncbi.nlm.nih.gov/The cDNA sequence (GenBANK NM_001188) of last acquisition bak is used predictor prediction siRNA then.On the result of contrast prediction siRNA sequence, select some siRNA.Two chains to these selected siRNA all carry out Blast search (database is selected the nr database of NBCI for use), selected at random three siRNA sequences that suppress bak genetic expression in Search Results, be siRNA1, siRNA2, siRNA3, select for use a stochastic sequence as negative control.A, G, C, U in the bak siRNA sequence represents VITAMIN B4 ribonucleotide, guanosint sugar nucleotide, cytosine(Cyt) ribonucleotide and uracil ribonucleotide, and T represents thymine deoxyribotide.Bak siRNA sequence and negative control are all available from U.S. Ambion company, and three couples of siRNA are as follows for synthetic:

SiRNA1: antisense strand 5 '-AGUGCACACUUGCUAAAGCtt-3 '

Positive-sense strand 5 '-GCUUUAGCAAGUGUGCACUtt-3 ';

SiRNA2: antisense strand 5 '-AUGCCACUCUCAAACAGGCtg-3 '

Positive-sense strand 5 '-GCCUGUUUGAGAGUGGCAUtt-3 ';

SiRNA3: antisense strand 5 '-UAAGGUGACCAUCUCUGGGtc-3 '

Positive-sense strand 5 '-CCCAGAGAUGGUCACCUUAtt-3 '.

Determining of the in-vitro transfection of embodiment 2:bak siRNA and selection of best transfection dosage and the best use of time

Use the transfection reagent of Ambion company to carry out transfection, the working specification that all operations all provides by Ambion company.Concrete steps are: embodiment 1 resulting three pairs of bak siRNA sequences and negative control powder are dissolved in respectively in the RNAase-free sterilized water, are made into the concentrated solution that final concentration is 20 μ mol/L, dilution is the application liquid of 2 μ mol/L during use.Neuroblastoma cell is inoculated in (300,000 cells/well) in 6 well culture plates, after 24 hours, cell culture fluid is changed to the RPMI-1640 that contains 5% foetal calf serum with the RPMI-1640 cultivation that contains 15% foetal calf serum.During transfection, respectively the transfection reagent of 5 μ l siRNA solution (negative controls of the bak-siRNA of 2 μ mol/L or 2 μ mol/L) and 5 μ l is diluted in respectively in the 250 μ l serum-free mediums in (Opti-MEM), and after at room temperature hatching 10 minutes, then above-mentioned siRNA and transfection reagent are mixed, mixture adds cell suspension (the cytogamy degree is 50%～70%) in incubated at room 10 minutes, and adds different concns aluminium porcelain enamelling (0～4.5mM, 37 ℃, 5%CO ₂Cultivate, in cultivating back 24h, 48h, 72h collecting cell carry out cell viability and detect.Detection method is: the SH-SY5Y cell in vegetative period of taking the logarithm, make 1.5 * 10 ⁵Individual/ml initial concentration cell suspension, be added on the 96 porocyte culture plates (Corning company), every hole adds 100 μ l, establishes 2 μ mol/L negative control group, siRNA1 group, siRNA2 group, siRNA3 group.Each siRNA transfection group according to the concentration of transfection reagent (10,20,30nmol/L) be divided into basic, normal, high transfection dosage group, every group 5 hole.After being grouped in transfection, inhale and remove supernatant, add 5 μ l CCK-8 reagent and the fresh cell culture fluid of 100 μ l, to hatch 4 hours for 37 ℃, the 450nm microplate reader reads absorbance.Cell inhibitory rate=(experimental group absorbance/control group absorbance-1) * 100%.

The result is as follows, the light microscopic and the fluorescence photo that have wherein shown bak siRNA transfectional cell among Fig. 1, Fig. 2, Fig. 3, DAPI dye the further clear cells transfected that shown of nuclear, the cell of transfection success behind the Cy3 fluorescent mark, the result shows that the transfectional cell number is more than 90% of total cellular score; Fig. 4 to Fig. 6, with three pairs of bak siRNA transfection neuroblastoma cells, and the different time points after transfection detects cell viability, calculates 4mM respectively and dyes the aluminium group at different sequences and the cell viability inhibiting rate put different detection times, and concrete outcome sees Table 1.Data show, the cell viability inhibiting rate when the cell viability inhibiting rate of neuroblastoma is significantly higher than 24h and 72h detection when 48h detects after three couples of bak siRNA transfection; SiRNA1 is significantly higher than siRNA2 and siRNA3 group at the cell viability inhibiting rate of 48h simultaneously.

The different sequences of table 1 are dyed the inhibiting rate (%) of aluminium cell viability to 4mM in different time points

	siRNA1	siRNA2	siRNA3	The F value	The P value
						24h 48h 72h F value P value	3.947±0.103 9.666±0.756 *# 1.325±0.058 4.032 ＜0.05	3.894±0.634 5.111±0.586 # 1.883±0.083 3.654 ＜0.05	4.631±0.568 7.888±0.846 # 1.558±0.084 3.965 ＜0.05	2.695 3.958 2.456 - -	＞0.05 ＜0.05 ＞0.05 - -

^*: compared significant difference, P＜0.05 with 24h with 72h cell viability inhibiting rate

#: compared significant difference, P＜0.05 with the cell viability inhibiting rate of other two couples of siRNA

(0～4.5mM) adds with in the neuroblastoma cell nutrient solution after the different transfection concentration transfection of siRNA1 with the different concns aluminium porcelain enamelling among Fig. 7, and the 48h after hatching altogether detects cell viability, the result shows, cell viability with transfection reagent 48h after transfection of low dosage, all compare the difference maximum respectively dying aluminium dosage group with control group, the optimal sequence that bak siRNA effect is described is siRNA1, and best transfection dosage is low dose group (10nmol/L).

Bak gene inhibition rate behind the embodiment 3:bak siRNA in-vitro transfection detects and the optimal sequence screening

The SH-SY5Y cell in vegetative period of taking the logarithm makes 1.5 * 10 ⁵Individual/ml initial concentration cell suspension, be added on the 24 porocyte culture plates (Corning company), every hole adds 250 μ l, establishes 2 μ mol/L negative control group, the siRNA1 group, siRNA2 group, siRNA3 group, every group 5 hole, after being grouped in transfection, inhale and remove supernatant, add trysinization and collecting cell.With the mRNA of Trizol method extracting cell, be cDNA with reverse transcription reagent with the mRNA reverse transcription, detect the bak expression of gene with fluorescence quantitative PCR method.Concrete steps are: the collecting cell suspension, the centrifugal 10min of 1500rpm abandons supernatant.Add 600 μ l Trizol mixings, room temperature is placed 20min.Add the 0.12ml chloroform, shake well 15s is to oyster white.Room temperature leaves standstill 3min, 4 ℃ of centrifugal 15min of 12000rpm.Get supernatant, add 300 μ l Virahols, room temperature leaves standstill 10min.4 ℃ of centrifugal 10min of 12000rpm.Abandon supernatant, add 600 μ l, 75% ethanol, mixing, 4 ℃ of centrifugal 5min of 7500rpm abandon supernatant, and room temperature is uncapped and placed 30min, add RNAase-free sterilized water 50 μ l, be diluted in 260nm and 280nm place mensuration its absorbance at 1: 1000, calculate purity and the content of mRNA.Calculation formula is: mRNA purity=A ₂₆₀/ (A ₂₈₀-background), mRNA content (μ g/ml)=A ₂₆₀* 40 * 100.As mRNA purity＞1.8, then further carry out reverse transcription.Add 10 * RT damping fluid, 2 μ l, dNTP mixes (2.5mM) 4 μ l, Oligo-dT primer (10 μ M) 2 μ l, RNase inhibitor (10U/ μ l) 1 μ l, Quant ReverseTranscriptase 1 μ l, RNAase-free sterilized water 4 μ l, template ribonucleic acid 6 μ l, fully mixing is hatched 60min for 37 ℃.Get the cDNA product 3 μ l after the amplification, add each 1 μ l of upstream and downstream primer, 2 * PCR master, 12.5 μ l and RNAase-free sterilized water 7.5 μ l, mixing, of short duration centrifugal.Amplification condition is: 94 ℃ of 5min, and 94 ℃ of 50s, 57 ℃ of 50s, 72 ℃ of 1min, 35 are circulated to 2,72 ℃ of 5min.

QRT-PCR detects three pairs of siRNA sequence transfections inhibition bak genetic expression amplification curves and sees Fig. 8, and Fig. 9 is that three pairs of siRNA sequences suppress β-actin genetic expression amplification curves, and the transfection of three pairs of siRNA sequences is seen Figure 10 to the concrete outcome of bak genetic expression inhibiting rate.The result shows, siRNA1, siRNA2, the inhibiting rate of siRNA3 is respectively 57.76%,, 44.56%, 53.05%, according to difference to siRNA to the inhibiting rate of bak gene as can be known, the inhibiting rate of siRNA1 is higher than all the other two groups, preferred siRNA1.

The proteic expression amount of Bak detects behind the embodiment 4:bak siRNA in-vitro transfection

Each organizes cell with 1.5 * 10 ⁵Individual/ml initial concentration cell suspension inoculation is in 6 well culture plates, a built-in 1.5cm * 1.5cm cell cultures cover glass, press embodiment 1 method transfection after, continue to cultivate 24h, abandon supernatant, 4% Paraformaldehyde 96 is 30min fixedly, 0.01M PBS rinsing.Use 3%H ₂O ₂-methanol solution incubated at room 5～10min is with the sealing endogenous peroxydase; 0.01M the PBST rinsing, 5min * 3; The sealing of 5%～10% normal goats serum, incubated at room 30min; The serum deprivation that inclines is not washed, and adds one of 1%BSA dilution and resists, and 4 ℃ are spent the night; 0.01M the PBST rinsing, 5min * 3; The two anti-incubated at room 1h that add the HRP mark; Add 0.01%H ₂O ₂～0.05%DAB colour developing; After PBS rinsing 3 times, gradient alcohol dehydration, dimethylbenzene is transparent, gelatin glycerine mounting, microscopic examination.

Detecting Bak protein expression light microscopic figure with immunohistochemical method shows, behind the bak siRNA in-vitro transfection Figure 11～14 are seen in the influence of bak protein expression, along with the difference of siRNA consumption, the proteic expression amount of its Bak is also different, the siRNA consumption is more little, and the proteic expression amount of Bak is few more; The column diagram that Figure 15 does for the optical density value of software analysis cell, its result is consistent with morphologic observations, has shown the difference of each histone expression amount, can significantly reduce the proteic expression of Bak after bak siRNA in-vitro transfection is described.

In sum, siRNA sequence of the present invention can effectively suppress bak genetic expression, thereby significantly reduces the proteic expression of Bak.SiRNA sequence provided by the present invention not only can be used for the RNAi of cultured cell in vitro, also can be used for experiment in the body.In addition, siRNA provided by the present invention can provide a kind of medicine that suppresses nerve cell apoptosis, also can be used to prepare the medicine for the treatment of nerve degenerative diseases.

SEQUENCE LISTING

＜110〉Mountain Western Medicine S University

＜120〉a kind of siRNA sequence that suppresses bak genetic expression

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<170>Patentin version 3.2

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＜223〉synthetic according to the design software design of Ambion company is the antisense strand of the siRNA1 sequence of target with the bak gene

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agugcacacu ugcuaaagctt 21

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＜223〉synthetic according to the design software design of Ambion company is the positive-sense strand of the siRNA1 sequence of target with the bak gene

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gcuuuagcaa gugugcacut t 21

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＜223〉synthetic according to the design software design of Ambion company is the antisense strand of the siRNA2 sequence of target with the bak gene

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augccacucu caaacaggct g 21

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＜223〉synthetic according to the design software design of Ambion company is the antisense strand of the siRNA3 sequence of target with the bak gene

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cccagagaug gucaccuuat t 21

Claims (1)

1. siRNA molecule that suppresses bak genetic expression is characterized in that the sequence of this molecule is:

SiRNA1: antisense strand 5 '-AGUGCACACUUGCUAAAGCtt-3 ',

Positive-sense strand 5 '-GCUUUAGCAAGUGUGCACUtt-3 '.

Images