CN103667422A - Use and related medicament of human CUL4B gene - Google Patents
Use and related medicament of human CUL4B gene Download PDFInfo
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Abstract
The invention discloses a use and a related medicament of a human CUL4B gene. The invention discloses the use of the human CUL4B gene in tumor treatment, tumor diagnosis and medicament preparation. The invention further constructs small interfering RNA (ribonucleic acid) of the human CUL4B gene, an interfering nucleic acid construct of the human CUL4B gene and interfering slow virus of the human CUL4B gene, and further discloses the uses thereof. The siRNA (small interfering RNA) or the nucleic acid construct containing the siRNA sequence, and the slow virus, provided by the invention can specifically inhibit the expression of the human CUL4B gene; particularly, the slow virus can efficiently infect target cells, efficiently inhibit the expression of the CUL4B gene in the target cells, further inhibit the growth of tumor cells, promote the apoptosis of the tumor cells and realize important significance in tumor treatment.
Description
Technical field
The present invention relates to biological technical field, relate more specifically to purposes and the related drugs thereof of people CUL4B gene.
Background technology
RNA disturbs (RNA interference, RNAi) with the short double-stranded RNA (dsRNA) that Nucleotide forms, to carry out PTGS.It can block the expression of specific gene in body efficiently, specifically, cause its degraded, thereby cause the silence of specific gene in organism, making cell show the disappearance of certain gene phenotype, is emerging in recent years a kind of conventional research gene function, the laboratory technique of searching methods for the treatment of diseases.Research shows; length is that the double-stranded RNA of 21-23nt can transcribed and the specific RNAi(Tuschl of the causing T of post-transcriptional level; Zamore PD; Sharp PA, Bartel DP.RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21to 23 nucleotide intervals.Cell 2000; 101:25-33.).Though tumour patient is through chemotherapy, radiotherapy and complex therapy, five year survival rate is still very low, if tumor invasion is intervened with the relevant gene of progress, can open up new way for the treatment of tumour.In recent years, RNAi has become the available strategy of the gene therapy of tumour.The expression that utilizes RNAi technology can suppress the cancer suppressor gene, Cell cycle-related genes, anti-apoptosis-related genes etc. of proto-oncogene, sudden change suppresses tumor progression (Uprichard, Susan L.The therapeutic potential of RNA interference.FEBS Letters 2005; 579:5996-6007.).
CUL4B(cullin 4b) belong to cullin family, the division growth of this family member and cell has substantial connection, its variation or unconventionality expression will cause the unsuitable propagation of cell, and cullin gene family member has abnormal expression (Salon C in kinds of tumors tissue, Brambilla E, Brambilla C, et al.Altered pattern of cul-1protein expression and neddylation in human lung tumors:relationships with CAND 1 and cyclin E protein levels.J Pathol.2007, 213 (3): 303-310.Laura PS, Lorenzo M, Francisco J, et al.Abstract 91:The CUL4A ubiquitin ligase, a putative target gene at the 13q34 amplicon and its role in breast cancer pathogenesis & progression.Cancer Research.2012, 72 (8): 91.).
In people cullin family, CUL4B and CUL4A protein sequence homology be up to 83%(Sarikas A, Hartmann T, Pan ZQ.The cullin protein family.Genome Biol.2011; 12:220.), often using both as one, (CUL4) studies in the research in past, to the functional study of CUL4 CRLs is many, by CUL4A, obtained, CUL4A is up-regulated (Laura PS in mammary cancer and hepatocellular carcinoma, Lorenzo M, Francisco J, et al.Abstract 91:The CUL4A ubiquitin ligase, a putative target gene at the 13q34 amplicon and its role in breast cancer pathogenesis & progression.Cancer Research.2012; 72 (8): 91.), show that CUL4A may play a significant role aspect regulation of cell proliferation.But recent research points out both on partial function and concrete mechanism of action, to have larger difference.The research of 2007 is found, CUL4B transgenation can cause the chain mental retardation syndromes of a kind of X, pointing out this gene may be a common chain mental retardation syndromes Disease-causing gene (Patrick ST of X, F.Lucy R, Sarah O, Mutations in CUL4B, Which Encodes a Ubiquitin E3 Ligase Subunit, Cause an X-linked Mental Retardation Syndrome Associated with Aggressive Outbursts, Seizures, Relative Macrocephaly, Central Obesity, Hypogonadism, Pes Cavus, and Tremor.Am.J.Hum.Genet.2007, 80:345 – 352.), so far the importance of CUL4B is just familiar with gradually, and therefore the function of CUL4B has caused the great interest of people.Studies show that the low expression of CUL4B may cause cell proliferation obstacle, occur that the S phase blocks and cause cyclin cyclin E Increased Plasma Half-life.Research also finds that CUL4B is mainly expressed in nucleus, its N-terminal
37kKRK
40nuclear localization signal for CUL4B albumen; and the performance of CUL4B function and its correctly enter closely related (the Zou YX of core; Mi J; Cui JP, et al.Characterization of Nuclear Localization Signal in the N Terminus of CUL4B and Its Essential Role in Cyclin E Degradation and Cell Cycle Progression.J Biol Chem.2009 November 27; 284 (48): 33320 – 33332.).Above result of study shows, CUL4B plays an important role in regulating cell propagation.But up to the present, for CUL4B, the effects anb Mechanism in tumor cell proliferation regulation and control is known little about it.
In order to further investigate the regulatory function of CUL4B in tumor cell proliferation, the present invention chooses osteosarcoma and colon cancer cell model, take RNAi as means research CUL4B is in osteosarcoma and colorectal carcinoma generation and developing effect.
Summary of the invention
The object of the invention is to open and people CUL4B(cullin 4B) methods for the treatment of and the medicine of gene-correlation, the RNA of take interference (RNAi) is as means research CUL4B gene is in the survival of tumour cell and the effect in apoptotic process.
First aspect present invention, take RNA interference as means, having studied CUL4B gene occurs and developing effect in tumour, a kind of method that suppresses or reduce growth of tumour cell, propagation, differentiation and/or survival is disclosed, the method comprises: to tumour cell, use a kind of transcribing or translating of CUL4B gene that can specificity suppress, or can specificity suppress the expression of CUL4B albumen or the molecule of activity, with this, come growth, propagation, differentiation and/or the survival of inhibition tumor cell.
Described tumour cell is selected from the tumour cell that its growth is relevant with the expression of CUL4B albumen or activity.Preferably, described tumour cell is selected from the arbitrary of osteosarcoma, colorectal carcinoma.
In the method for described inhibition or reduction growth of tumour cell, propagation, differentiation and/or survival, the amount of application of described molecule is enough to reduce transcribing or translating of CUL4B gene, or enough reduces expression or the active dosage of CUL4B albumen.Further, the expression of described CUL4B gene is at least lowered 50%, 80%, 90%, 95% or 99%.
Described molecule can be selected from but be not limited to: nucleic acid molecule, carbohydrate, lipid, small molecules chemical drugs, antibody medicine, polypeptide, albumen or interference slow virus.
Described nucleic acid includes but not limited to: siRNA (esiRNA) prepared by antisense oligonucleotide, double-stranded RNA (dsRNA), ribozyme, endoribonuclease III or short hairpin RNA (shRNA).
The information sequence of the promoter sequence that described double-stranded RNA, ribozyme, esiRNA or shRNA contain CUL4B gene or CUL4B gene.
Further, described double-stranded RNA is siRNA (siRNA).Described siRNA comprises the first chain and the second chain, the complementary common formation RNA dimer of described the first chain and described the second chain, and in the sequence of described the first chain and CUL4B gene, 15-27 continuous nucleotide sequence is basic identical.The coded mRNA fragment of described small molecules interference RNA energy specific binding target sequence, and the expression of the reticent people CUL4B of specificity gene.
Further, the first chain-ordering of described siRNA and the target sequence in CUL4B gene are basic identical.Preferably, the target sequence in described CUL4B gene contains the arbitrary sequence in SEQ ID NO:1-18.
When the target sequence in described CUL4B gene is the reticent CUL4B genetic expression of described small molecules interference RNA specificity, with the fragment in the corresponding CUL4B gene of mRNA fragment of the described complementary combination of small molecules interference RNA.
Preferably, described CUL4B gene source is in people.
First aspect present invention also discloses a kind of people CUL4B gene of separation at preparation or screening anti-tumor medicine, or the purposes in preparing diagnosing tumor medicine.
Further, described tumour is selected from osteosarcoma or colorectal carcinoma.
Described by separated CUL4B gene for the preparation of or screening anti-tumor medicine comprise the content of two aspects: one, using CUL4B gene as medicine or preparation for the action target of tumour cell, be applied to prepare anti-tumor medicine or preparation; Its two, using CUL4B gene as medicine or preparation for the action target of tumour cell, be applied to screen anti-tumor medicine or preparation.
Described using CUL4B gene as medicine or preparation is applied to prepare anti-tumor medicine for the action target of tumour cell or preparation specifically refers to: the target using CUL4B gene as RNA interference effect, develop medicine or preparation for tumour cell, thereby can reduce the expression level of CUL4B gene in tumour cell.
Described using CUL4B gene as medicine or preparation is applied to screen anti-tumor medicine for the action target of tumour cell or preparation specifically refers to: using CUL4B gene as effective object, medicine or preparation are screened, using to find and can suppress or promote the medicine of people CUL4B genetic expression as oncotherapy drug candidate.CUL4B gene small molecules interference RNA (siRNA) be take people CUL4B gene as effective object screening obtains as described in the present invention, can be used as having the medicine of inhibition tumor cell proliferation function.In addition, such as antibody drug, small-molecule drug etc. also can be using CUL4B gene and albumen thereof as effective object.
Described by CUL4B gene for the preparation of diagnosing tumor medicine, refer to the preparation that is applied to diagnosing tumor medicine using CUL4B gene expression product as a diagnosing tumor index.
Described anti-tumor medicine is for can specificity suppressing transcribing or translating of CUL4B gene, or can specificity suppress the expression of CUL4B albumen or the molecule of activity, thereby the expression level of CUL4B gene in reduction tumour cell, reaches the object of propagation, growth, differentiation and/or the survival of inhibition tumor cell.
Described anti-tumor medicine or the diagnosing tumor medicine obtaining by separated CUL4B gene preparation or screening includes but not limited to: nucleic acid molecule, carbohydrate, lipid, small molecules chemical drugs, antibody medicine, polypeptide, albumen or interference slow virus.
Described nucleic acid includes but not limited to: siRNA (esiRNA) prepared by antisense oligonucleotide, double-stranded RNA (dsRNA), ribozyme, endoribonuclease III or short hairpin RNA (shRNA).
The amount of application of described anti-tumor medicine is enough to reduce transcribing or translating of people CUL4B gene, or enough reduces expression or the active dosage of people CUL4B albumen.So that the expression of people CUL4B gene is at least lowered 50%, 80%, 90%, 95% or 99%.
Adopting the method for aforementioned anti-tumor medicine treatment tumour, is mainly by reducing the propagation of the expression level inhibition tumor cell of people CUL4B gene, to reach the object for the treatment of.Concrete, during treatment, can effectively reduce the administering substances of people CUL4B gene expression dose in patient.
Second aspect present invention discloses a kind of separated nucleic acid molecule that reduces CUL4B genetic expression in tumour cell, and described nucleic acid molecule comprises:
A) double-stranded RNA, in described double-stranded RNA, containing can be under stringent condition and the nucleotide sequence of CUL4B gene recombination; Or
B) shRNA, in described shRNA, containing can be under stringent condition and the nucleotide sequence of CUL4B gene recombination.
Further, described double-stranded RNA comprises the first chain and the second chain, the complementary common formation RNA dimer of described the first chain and described the second chain, and in the sequence of described the first chain and CUL4B gene, 15-27 continuous nucleotide sequence is basic identical.Preferably, in the sequence of described the first chain and CUL4B gene, 19-23 continuous nucleotide sequence is basic identical; Better, in the sequence of described the first chain and CUL4B gene, 19,20 or 21 continuous nucleotide sequences are basic identical.
Further, described double-stranded RNA comprises the first chain and the second chain, the complementary common formation RNA dimer of described the first chain and described the second chain, and the target sequence in the sequence of described the first chain and CUL4B gene is basic identical.
Further, described shRNA comprises positive-sense strand fragment and antisense strand fragment, and the loop-stem structure that connects described positive-sense strand fragment and antisense strand fragment, the sequence of described positive-sense strand fragment and described antisense strand fragment is complementary, and in the sequence of described positive-sense strand fragment and CUL4B gene, 15-27 continuous nucleotide sequence is basic identical.Described shRNA can become siRNA (siRNA) and then play the effect of endogenous CUL4B genetic expression in the reticent tumour cell of specificity after processing.
Further, described shRNA comprises positive-sense strand fragment and antisense strand fragment, and the loop-stem structure that connects described positive-sense strand fragment and antisense strand fragment, the sequence of described positive-sense strand fragment and described antisense strand fragment is complementary, and the target sequence in the sequence of described positive-sense strand fragment and CUL4B gene is basic identical.
Target sequence in the first chain of described double-stranded RNA or the positive-sense strand fragment of described shRNA and CUL4B gene is basic identical, when the target sequence of described CUL4B gene is siRNA for the reticent CUL4B genetic expression of specificity, by the fragment in the corresponding CUL4B gene of mRNA fragment of described siRNA identification silence.
Preferably, arbitrary sequence that the target sequence in described CUL4B gene contains SEQ IDNO:1-18.
Further, described CUL4B gene source is in people.
The length of described double-stranded RNA the first chain and the second chain is 15-27 Nucleotide; Preferably, length is 19-23 Nucleotide; Best, length is 19,20 or 21 Nucleotide.
Further, described double-stranded RNA is siRNA (siRNA).Further, the sequence of described siRNA the first chain, as shown in SEQ ID NO:30, is specially 5 '-AGCAGUGGAAGCUAUUCAGAA-3 '.
SiRNA shown in SEQ ID NO:30 for take the sequence shown in SEQ ID NO:4 be RNA disturb target sequence design, for a chain of the siRNA of people CUL4B gene, another chain i.e. sequence and the complementation of the first chain-ordering of the second chain, and this siRNA can play the effect of endogenous CUL4B genetic expression in the reticent tumour cell of specificity.
Further, the sequence of the loop-stem structure of described shRNA can be selected from following arbitrary: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU and CCACACC.
Further, the sequence of described shRNA, as shown in SEQ ID NO:31, is specially: 5 '-AGCAGUGGAAGCUAUUCAGAAUUCAAGAGAUUCUGAAUAGCUUCCACUGCU-3 '.
ShRNA can become siRNA after enzyme is cut processing, and then plays the effect of endogenous people CUL4B genetic expression in the reticent tumour cell of specificity.
The interference lentiviral vectors of gene fragment of shRNA of the present invention of encoding contains arbitrary sequence and the complementary sequence thereof in SEQ ID NO:1-18.
Third aspect present invention, discloses a kind of CUL4B gene interfere RNA construct, and the gene fragment of the shRNA in the nucleic acid molecule that contains the separation of the present invention of encoding, can express described shRNA.
Described people CUL4B gene interfere RNA construct can be the gene fragment clone of the aforementioned people CUL4B gene shRNA of coding to be entered to known carrier obtain.Further, described CUL4B gene interfere RNA construct is that CUL4B gene disturbs lentiviral vectors.
It is the DNA fragmentation of the aforementioned CUL4B gene shRNA of coding to be cloned into known carrier obtain that CUL4B gene of the present invention disturbs lentiviral vectors, described known carrier mostly is lentiviral vectors, described CUL4B gene disturbs lentiviral vectors to become after infectious virion through virus packing, infected tumor's cell, and then transcribe out shRNA of the present invention, by enzyme, cut the steps such as processing, finally obtain described siRNA, for the expression of the reticent CUL4B gene of specificity.
Further, described CUL4B gene disturbs lentiviral vectors also to contain the nucleotide sequence of marker that can be detected in promoter sequence and/or codes for tumor cell; Preferably, described marker that can be detected is as green fluorescent protein (GFP).
Further, described lentiviral vectors can be selected from: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti6-GW/U6-laminshrna, pcDNA 1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, arbitrary in pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.
The embodiment of the present invention has specifically been enumerated and take the people CUL4B gene that pGCSIL-GFP is vector construction and disturb lentiviral vectors, called after pGCSIL-GFP-CUL4B-siRNA.
The nucleic acid molecule of separation of the present invention can be used for the medicine of preparation prevention or treatment tumour, and described tumour is osteosarcoma or colorectal carcinoma.
CUL4B gene siRNA of the present invention can be used for the propagation of inhibition tumor cell, further can be as medicine or the preparation for the treatment of tumour.CUL4B gene disturbs lentiviral vectors to can be used for preparing described CUL4B gene siRNA.When the medicine as treatment tumour or preparation, be that the described nucleic acid molecule of safe and effective amount is applied to Mammals.Concrete dosage also should be considered the factors such as route of administration, patient health situation, and these are all within skilled practitioners skill.
Fourth aspect present invention, discloses a kind of CUL4B gene and has disturbed slow virus, by aforementioned CUL4B gene, disturbs lentiviral vectors under slow virus packaging plasmid, clone auxiliary, through virus packing, forms.This slow virus can also produce the small molecules interference RNA for CUL4B gene by infected tumor's cell, thereby suppresses the propagation of osteosarcoma or colorectal carcinoma tumour cell.This CUL4B gene disturbs slow virus to can be used for the medicine of preparation prevention or treatment tumour.
Fifth aspect present invention, disclose a kind of for preventing or treat the pharmaceutical composition of tumour, the nucleic acid molecule that its active substance contains aforesaid separation, CUL4B gene interfere RNA construct, and/or CUL4B gene disturbs slow virus.
Further, described pharmaceutical composition contains double-stranded RNA described in 1~99wt%, shRNA, CUL4B gene interfere RNA construct or CUL4B gene and disturbs slow virus, and pharmaceutically acceptable carrier, thinner or vehicle.
In preparation during these compositions, conventionally by activeconstituents and mixed with excipients, or with vehicle dilution, wrap in can capsule or the carrier that exists of anther sac form in.When vehicle plays thinner, do the used time, it can be that solid, semisolid or fluent material are as the medium of vehicle, carrier or activeconstituents.Therefore, composition can be tablet, pill, pulvis, solution, syrup, sterilizing injecting solution etc.The example of suitable vehicle comprises: lactose, glucose, sucrose, sorbyl alcohol, N.F,USP MANNITOL, starch, Microcrystalline Cellulose, polyvinylpyrrolidone, Mierocrystalline cellulose, water, etc.Preparation also can comprise: wetting agent, emulsifying agent, sanitas (as methyl hydroxybenzoate and propyl ester), sweeting agent etc.
The invention also discloses the application of described pharmaceutical composition in arbitrary anti-tumor medicine of preparation treatment osteosarcoma, colorectal carcinoma.
The treatment that is applied as tumour of this pharmaceutical composition provides a kind of method, is specially a kind of method of prevention or treatment target in-vivo tumour, comprises the described pharmaceutical composition of effective dose is applied in object.
When described pharmaceutical composition is used for prevention or treatment target in-vivo tumour, the described pharmaceutical composition of effective dose need to be applied in object.Adopt the method, the growth of described tumour, propagation, recurrence and/or shift suppressed.Further, at least 10% of the growth of described tumour, propagation, recurrence and/or transfer, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% part is suppressed.
Sixth aspect present invention, a kind of test kit for reducing the CUL4B genetic expression in tumour cell is disclosed, described test kit comprises: be present in the nucleic acid molecule of the described separation in container, and CUL4B gene interfere RNA construct, and/or described CUL4B gene disturbs slow virus.
In sum, the present invention has designed 18 RNAi target sequences for people CUL4B gene, build corresponding CUL4BRNAi carrier, wherein the RNAi carrier pGCSIL-GFP-CUL4B-siRNA of encoding sequence SEQ ID NO:4 can significantly lower CUL4B gene in the expression of mRNA level and protein level.Use slow virus (lentivirus, be abbreviated as Lv) as genetic manipulation instrument, carry RNAi carrier pGCSIL-GFP-CUL4B-siRNA and can the RNAi sequence for CUL4B gene efficiently be imported to human osteosarcoma SaoS2 cell and colorectal carcinoma RKO cell target, reduce the expression level of CUL4B gene, significantly suppress the multiplication capacity of above-mentioned tumour cell.Therefore the CUL4B gene silencing of lentivirus mediated is the potential clinical non-operative treatment mode of malignant tumour.
SiRNA provided by the invention or the nucleic acid construct that comprises this siRNA sequence, slow virus can specificity suppress the expression of people CUL4B gene, especially slow virus, can efficiently infect target cell, suppress expeditiously the expression of CUL4B gene in target cell, and then the growth of inhibition tumor cell, promote apoptosis of tumor cells, significant in oncotherapy.
Accompanying drawing explanation
Fig. 1: pGCSIL-GFP plasmid DNA collection of illustrative plates
Fig. 2: CUL4B-RNAi slow virus was infected human osteosarcoma SaoS2 cell and colorectal carcinoma RKO cell after 5 days, and the expression level of CUL4BmRNA significantly reduces
Fig. 3: CUL4B-RNAi slow virus was infected human osteosarcoma SaoS2 cell after 5 days, caused cell inhibitory effect
Fig. 4: CUL4B-RNAi slow virus was infected human colon carcinoma RKO cell after 5 days, caused cell inhibitory effect
Embodiment
The present invention relates to one group of small molecules interference RNA for people CUL4B gene (siRNA) sequence, rna interference vector and RNA and disturbed slow virus.Choose people CUL4B mRNA coding region sequence as the target site of siRNA, according to the preferred 15-27 of 10-30(continuous in target site, more preferably 19-23) individual base sequence design siRNA target sequence.By gene clone, the nucleic acid construct of the above-mentioned siRNA of construction expression, the slow virus of the above-mentioned siRNA of packaging expression.Cell experiment proves, above-mentioned siRNA sequence can the reticent human tumor cells of specificity in the expression of endogenous CUL4B gene.
Contriver finds, adopts after the expression of the CUL4B gene of transferring person under RNAi method the propagation of inhibition tumor cell effectively, and this achievement in research shows that CUL4B gene is proto-oncogene, can be used as the target spot of oncotherapy.Contriver is further synthetic and tested the multiple siRNA for CUL4B gene, filter out the expression that can effectively suppress CUL4B and then the siRNA that suppresses human osteosarcoma SaoS2 cell and colorectal carcinoma RKO cell proliferation and growth, completed on this basis the present invention.
The invention provides siRNA (siRNA) sequence of a series of interference people CUL4B genes, built can specificity reticent CUL4B genetic expression slow virus.The present invention studies discovery, for siRNA and the RNAi slow virus of people CUL4B gene design, stablizes the expression of also lowering specifically CUL4B gene, and effectively suppresses the propagation of human tumor cells.The present invention shows that CUL4B gene can promote growth of tumour cell, is expected to become the target spot of early diagnosis of tumor and treatment.And, by the expression of the reticent CUL4B gene of RNAi mode, can be used as the effective means that suppresses tumor development.
Mentality of designing of the present invention is:
The present invention screens by the following method and obtains a kind of people CUL4B gene RNAi slow virus: from Genbank, transfer people CUL4B gene order; Prediction siRNA site; The synthetic effective siRNA sequence for CUL4B gene, two ends are containing the double-stranded DNA Oligo of restriction enzyme site cohesive end; After lentiviral vectors double digestion, be connected the RNAi plasmid of construction expression CUL4B gene siRNA sequence with double-stranded DNA Oligo; RNAi plasmid and slow virus are packed to assistant carrier (Packing Mix, Sigma-aldrich company) the cotransfection HEKC 293T needing, produce recombinant slow virus particle, can make the slow virus of efficient reticent CUL4B gene.
Based on aforesaid method, the invention provides 18 Effective target sites (specifically as shown in SEQ ID NO:1-18) that disturb CUL4B gene, built the slow virus of special interference people CUL4B gene.
The present invention simultaneously also discloses a kind of people CUL4B gene RNAi slow virus (CUL4B-RNAi) and preparation and application thereof.
This research is found, utilizes the RNAi method of lentivirus mediated, after reducing the expression of CUL4B gene in tumour cell, and the effective propagation of inhibition tumor cell.This research shows, CUL4B gene may be a proto-oncogene, can promote tumor cell proliferation, in occurring and develop, tumour there is important biological function, CUL4B gene can be the target of oncotherapy, and the CUL4B gene specific silence of lentivirus mediated can be used as a kind of new tool of oncotherapy.
Below in conjunction with embodiment, further set forth the present invention.Should be understood that embodiment is only for the present invention is described, but not limit the scope of the invention.In embodiment, the experimental technique of unreceipted actual conditions and the reagent of undeclared formula are according to normal condition, as works such as [ U.S. ] Sambrook.J; Huang Peitang etc. translate.Molecular cloning test guide, the third edition.Beijing: the condition of the condition described in Science Press 2002 or manufacturers's suggestion is carried out or configures.
1. screening is for the effective siRNA target spot of people CUL4B gene
From Genbank, transfer CUL4B(NM_003588) gene information; Utilize the design software Genechem design of Shanghai JiKai Gene Chemical Technology Co., Ltd for the effective siRNA target spot of CUL4B gene.In encoding sequence (CDS) region of CUL4B gene, every the sequence of 21 bases of an initial acquisition of base, table 1 has been listed wherein 18 effective siRNA target sequences for CUL4B gene.
Table 1 target is in the siRNA target sequence of people CUL4B gene
2. the preparation of lentiviral vectors
Double-stranded DNA Oligo sequence (table 2) for the synthetic two ends of siRNA target spot (the SEQ ID NO:4 of take is example) containing Age I and EcoR I restriction enzyme site cohesive end; (Shanghai JiKai Gene Chemical Technology Co., Ltd provides, and Fig. 1), makes its linearizing, and agarose gel electrophoresis is identified endonuclease bamhi with Age I and EcoR I restriction enzyme, to act on pGCSIL-GFP carrier.
Table 2 two ends are containing the double-stranded DNA Oligo of Age I and EcoR I restriction enzyme site cohesive end
By T4DNA ligase enzyme, by double digestion linearizing, (it is as shown in table 4 that enzyme is cut system, 37 ℃, reaction 1h) the carrier DNA double-stranded DNA Oligo good with purifying is connected, and in suitable buffer system (linked system is as shown in table 5), in 16 ℃ of connections, spends the night, and reclaims and connects product.By connecting product, transform fresh competent escherichia coli cell (conversion operation reference: molecular cloning experiment guide second edition 55-56 page) prepared by calcium chloride.
At connection converted product, grow bacterium clone surface and be stained with, be dissolved in 10 μ l LB substratum, mix and get 1 μ l as template; The upstream and downstream of RNAi sequence in lentiviral vectors, design universal PC R primer (upstream primer sequence: 5 '-CCTATTTCCCATGATTCCTTCATA-3 ' (SEQ ID NO:23); Downstream primer sequence: 5 '-GTAATACGGTTATCCACGCG-3 ' (SEQ ID NO:24), carries out PCR identification experiment (PCR reaction system is as table 6-1, and reaction conditions is as table 6-2).PCR is identified to positive clone checks order and compare of analysis, compare the carrier that correct clone is the corresponding RNAi of the expression for SEQ ID NO:4 target sequence successfully constructing, called after pGCSIL-GFP-CUL4B-siRNA.
Build pGCSIL-GFP-Scr-siRNA negative control plasmid, negative control siRNA target sequence is 5 '-TTCTCCGAACGTGTCACGT-3 ' (SEQ ID NO:25).While building pGCSIL-GFP-Scr-siRNA negative control plasmid, double-stranded DNA Oligo sequence (table 3) for the synthetic two ends of Scr siRNA target spot containing Age I and EcoR I restriction enzyme site cohesive end, all the other construction processs, authentication method and condition be same pGCSIL-GFP-CUL4B-siRNA all.
Table 3 two ends are containing the double-stranded DNA Oligo of Age I and EcoR I restriction enzyme site cohesive end
5’ | Neck | Ring | Neck | 3’ | SEQ | |
Positive-sense strand | CCGG | TTCTCCGAACGTGTCACGT | TTCAAGAGA | ACGTGACACGTTCGGAGAA | TTTTTG | 21 |
Antisense strand | AATTCAAAAA | TTCTCCGAACGTGTCACGT | TCTCTTGAA | ACGTGACACGTTCGGAGAA | 22 |
By T4 DNA ligase by double digestion linearizing (it is as shown in table 4 that enzyme is cut system, 37 ℃, reaction 1h) carrier
Table 4pGCSIL-GFP plasmid enzyme restriction reaction system
Reagent | Volume (μ l) |
PGCSIL-GFP plasmid (1 μ g/ μ l) | 2.0 |
10×buffer | 5.0 |
100×BSA | 0.5 |
Age I(10U/μl) | 1.0 |
EcoR I(10U/μl) | 1.0 |
dd H 2O | 40.5 |
Total | 50.0 |
Table 5 carrier DNA and double-stranded double-stranded DNA Oligo ligation system
Reagent | Positive control (μ l) | From connecting contrast (μ l) | Connection group (μ l) |
Linearizing carrier DNA (100ng/ μ l) | 1.0 | 1.0 | 1.0 |
The double-stranded DNA Oligo (100ng/ μ l) of annealing | 1.0 | - | 1.0 |
10 * T4 phage DNA ligase enzyme damping fluid | 1.0 | 1.0 | 1.0 |
T4 phage DNA ligase enzyme | 1.0 | 1.0 | 1.0 |
dd H 2O | 16.0 | 17.0 | 16.0 |
Total | 20.0 | 20.0 | 20.0 |
Table 6-1PCR reaction system
Reagent | Volume (μ l) |
10×buffer | 2.0 |
dNTPs(2.5mM) | 0.8 |
Upstream primer | 0.4 |
Downstream primer | 0.4 |
Taq polysaccharase | 0.2 |
Template | 1.0 |
ddH 2O | 15.2 |
Total | 20.0 |
Table 6-2PCR reaction system program setting
3. pack CUL4B-siRNA slow virus
The DNA that extracts RNAi plasmid pGCSIL-GFP-CUL4B-siRNA with the plasmid extraction test kit of Qiagen company, is mixed with 100ng/ μ l storage liquid.24h before transfection, with the HEKC 293T cell of tryptic digestion logarithmic phase, take that containing the DMEM perfect medium of 10% foetal calf serum, to adjust cell density be 1.5 * 10
5cell/ml, is inoculated in 6 orifice plates, and 37 ℃, 5%CO
2in incubator, cultivate.When reaching 70%-80%, cell density can be used for transfection.2h before transfection, the original substratum of sucking-off, adds the perfect medium that 1.5ml is fresh.According to the explanation of the MISSION Lentiviral Packaging Mix test kit of Sigma-aldrich company, in a sterilizing centrifuge tube, add Packing Mix(PVM) 20 μ l, PEI 12 μ l, serum-free DMEM substratum 400 μ l, get the plasmid DNA of the above-mentioned extracting of 20 μ l, add to above-mentioned PVM/PEI/DMEM mixed solution.Above-mentioned transfection miscellany is at room temperature hatched to 15min, be transferred in the substratum of HEKC 293T cell, 37 ℃, 5%CO
2in incubator, cultivate 16h.Discard the developing medium that contains transfection miscellany, PBS solution washing, adds perfect medium 2ml, continues to cultivate 48h.
Collecting cell supernatant liquor, Centricon Plus-20 centrifugal ultrafiltration device (Millipore) purifying and concentrated slow virus, step is as follows: (1) 4 ℃, the centrifugal 10min of 4000g, removes cell debris; (2) 0.45 μ m filter filtering supernatant are in 40ml ultracentrifugation pipe; (3) 4000g is centrifugal, and 10-15min, to the concentrated volume of the virus needing; (4) after centrifugal end, filtering cup and filtered solution collection cups are below separated, filtering cup is tipped upside down on sample collection cup, centrifugal 2min centrifugal force is no more than 1000g; (5) Centrifuge Cup is removed from sample collection cup, in sample collection cup, be viral concentrated solution.By after viral concentrated solution packing in-80 degrees Celsius of preservations.The sequence of siRNA the first chain containing in virus concentrated solution is as shown in SEQID NO:30.The wrapping process of contrast slow virus, with CUL4B-siRNA slow virus, only replaces pGCSIL-GFP-CUL4B-siRNA carrier with pGCSIL-GFP-Scr-siRNA carrier.
In the human osteosarcoma SaoS2 of logarithmic phase cell and colorectal carcinoma RKO cell, carry out trysinization, (cell count is about 5 * 10 to make cell suspension
4/ ml) be inoculated in 6 orifice plates, be cultured to cytogamy degree and reach approximately 30%.According to infecting plural number (MOI, SaoS2:10, RKO:10) value, add the virus of embodiment 1 preparation of sufficient quantity, after cultivation 24h, change substratum, until time of infection, reach after 5 days collecting cell.According to the Trizol process specifications of Invitrogen company, extracted total RNA.According to the M-MLV process specifications of Promega company, RNA reverse transcription is obtained to cDNA(reverse transcription reaction system in Table 7,42 ℃ of reaction 1h, then in 70 ℃ of water-baths, water-bath 10min makes reversed transcriptive enzyme inactivation).
Adopt TP800 type Real time PCR instrument (TAKARA) to carry out real-time quantitative detection.The primer of CUL4B gene is as follows: upstream primer 5 '-GGGAAAGGAATGGTGAA-3 ' (SEQ ID NO:26) and downstream primer 5 '-TGCATAGAGCCGGTTAG-3 ' (SEQ ID NO:27).Take house-keeping gene GAPDH as internal reference, and primer sequence is as follows: upstream primer 5 '-TGACTTCAACAGCGACACCCA-3 ' (SEQ ID NO:28) and downstream primer 5 '-CACCCTGTTGCTGTAGCCAAA-3 ' (SEQ IDNO:29).Press the proportional arrangement reaction system in table 8.
Table 7 reverse transcription reaction system
Reagent | Volume (μ l) |
5×RT buffer | 4.0 |
10mM dNTPs | 2.0 |
RNasin | 0.5 |
M-MLV-RTase | 1.0 |
DEPC H 2O | 3.5 |
Total | 11.0 |
Table 8Real-time PCR reaction system
Reagent | Volume (μ l) |
SYBR premix ex taq: | 10.0 |
Upstream primer (2.5 μ M): | 0.5 |
Downstream primer (2.5 μ M): | 0.5 |
cDNA | 1.0 |
ddH 2O | 8.0 |
Total | 20.0 |
Setting program is two-step approach Real-time PCR: 95 ℃ of denaturations, 15s; 95 ℃ of each step sex change afterwards, 5s; Annealing is extended 60 ℃, 30s; Carry out altogether 45 circulations.In the extension stage, read light absorption value at every turn.After PCR finishes, 95 ℃ of sex change 1min, are then cooled to 55 ℃, make the abundant combination of DNA double chain.Since 55 ℃ to 95 ℃, each step increases by 0.5 ℃, keeps 4s, reads light absorption value simultaneously, makes melting curve.Adopt 2-Δ Δ Ct analytical method to calculate the gene expression abundance that has infected CUL4B mRNA.Infect the cell of contrast virus (Lv-Scr-siRNA) in contrast.Experimental result (Fig. 2) shows, in human osteosarcoma SaoS2 cell and colorectal carcinoma RKO cell, the expression level of CUL4B mRNA has lowered 90.1% and 48.3%.
In the human osteosarcoma SaoS2 of logarithmic phase cell and colorectal carcinoma RKO cell, carry out trysinization, (cell count is about 5 * 10 to make cell suspension
4/ ml) be inoculated in 6 orifice plates, be cultured to cytogamy degree and reach approximately 30%.According to infecting plural number (MOI, SaoS2:10, RKO:10), add the virus of sufficient quantity, after cultivation 24h, change substratum, until time of infection, reach after 5 days, collect each experimental group cell in logarithmic phase.The resuspended one-tenth cell suspension (2 * 10 of perfect medium
4/ ml), with cell density, be about 2000/hole, inoculation 96 orifice plates.Every group 5 multiple holes, every hole 100 μ l.
Complete after plate, put 37 ℃, 5%CO
2incubator is cultivated.From bed board, second day starts, and detect and read plate once every day with Cellomics instrument (Thermo Fisher), and continuous detecting is read plate 5 days.By adjusting the input parameter of Cellomics arrayscan, calculate exactly the quantity of the cell with green fluorescence in each scanning orifice plate, data are added up to drawing, draw cell proliferation curve (result is as Figure 3-Figure 4).Result shows, slow virus is infected each tumour of group after cells in vitro is cultivated 5 days, and rate of propagation significantly slows down, far below the rate of propagation of control group tumour cell, vigor cell number has declined respectively 55.4% and 86%, shows that CUL4B gene silencing causes tumor cell proliferation ability suppressed.
The above; it is only preferred embodiment of the present invention; not to any formal and substantial restriction of the present invention; should be understood that; for those skilled in the art; do not departing under the prerequisite of the inventive method, also can make some improvement and supplement, these improvement and supplement and also should be considered as protection scope of the present invention.All those skilled in the art, without departing from the spirit and scope of the present invention, a little change of making when utilizing disclosed above technology contents, the equivalent variations of modifying and developing, be equivalent embodiment of the present invention; Meanwhile, the change of any equivalent variations that all foundations essence technology of the present invention is done above-described embodiment, modification and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (14)
1. the separated people CUL4B gene purposes in preparation or screening anti-tumor medicine.
2. purposes as claimed in claim 1, is characterized in that, described tumour is selected from osteosarcoma or colorectal carcinoma.
3. a separated nucleic acid molecule that reduces CUL4B genetic expression in tumour cell, described nucleic acid molecule comprises:
A) double-stranded RNA, in described double-stranded RNA, containing can be under stringent condition and the nucleotide sequence of CUL4B gene recombination; Or
B) shRNA, in described shRNA, containing can be under stringent condition and the nucleotide sequence of CUL4B gene recombination.
4. the nucleic acid molecule of separation as claimed in claim 3, it is characterized in that, described double-stranded RNA comprises the first chain and the second chain, the complementary common formation RNA dimer of described the first chain and described the second chain, and the target sequence in the sequence of described the first chain and CUL4B gene is basic identical; Described shRNA comprises positive-sense strand fragment and antisense strand fragment, and the loop-stem structure that connects described positive-sense strand fragment and antisense strand fragment, the sequence of described positive-sense strand fragment and described antisense strand fragment is complementary, and the target sequence in the sequence of described positive-sense strand fragment and CUL4B gene is basic identical.
5. the nucleic acid molecule of separation as described in claim 3 or 4, is characterized in that, the target sequence of described CUL4B gene contains arbitrary sequence in SEQ ID NO:1-18.
6. the nucleic acid molecule of separation as described in claim 3 or 4, is characterized in that, described double-stranded RNA is siRNA, and the sequence of this siRNA the first chain is as shown in SEQ ID NO:30.
7. the nucleic acid molecule of separation as described in claim 3 or 4, is characterized in that, the sequence of described shRNA is as shown in SEQ ID NO:31.
8. a CUL4B gene interfere RNA construct, the gene fragment that contains the shRNA in nucleic acid molecule separated described in the arbitrary claim of coding claim 3-7, can express described shRNA.
9. CUL4B gene interfere RNA construct as claimed in claim 8, is characterized in that, described CUL4B gene interfere RNA construct is for disturbing lentiviral vectors.
10. CUL4B gene interfere RNA construct as claimed in claim 9, is characterized in that, described interference lentiviral vectors obtains after the gene fragment clone of the described shRNA of coding is entered to lentiviral vectors, and described lentiviral vectors is selected from: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti6-GW/U6-laminshrna, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, arbitrary in pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.
11. 1 kinds of CUL4B genes disturb slow viruss, by disturbing lentiviral vectors described in the arbitrary claim of claim 9-10 under slow virus packaging plasmid, clone auxiliary, through virus packing, form.
12. 1 kinds for preventing or treat the pharmaceutical composition of tumour, its active substance contains the separated nucleic acid molecule described in the arbitrary claim of claim 3-7, CUL4B gene interfere RNA construct described in the arbitrary claim of claim 8-10, and/or the CUL4B gene described in claim 11 disturbs slow virus.
The application of pharmaceutical composition in the anti-tumor medicine of preparation treatment osteosarcoma or colorectal carcinoma described in 13. claims 12.
14. 1 kinds of test kits for reducing CUL4B genetic expression in tumour cell, it is characterized in that, described test kit comprises: be present in container, separated nucleic acid molecule described in the arbitrary claim of claim 3-7, CUL4B gene interfere RNA construct described in the arbitrary claim of claim 8-10, and/or the CUL4B gene described in claim 11 disturbs slow virus.
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