CN103656674A - Use of human eIF5B gene and related drug thereof - Google Patents
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Abstract
The invention discloses a use of a human eIF5B gene and a related drug thereof. The invention discloses the use of the human eIF5B gene in oncotherapy, tumor diagnosis and drug preparation. The invention further discloses the small molecule interfering RNA (Ribose Nucleic Acid) of the human eIF5B gene, the interfering nucleic acid construct of the human eIF5B gene and the interfering lentivirus of the human eIF5B gene, and discloses uses thereof. The siRNA, or the nucleic acid construct containing the siRNA sequence, or the lentivirus containing the siRNA sequence provided by the invention is capable of specifically inhibiting the expression of the human eIF5B gene; in particular, the lentivirus is capable of infecting the target cells, inhibiting the expression of the eIF5B gene in the target cells at high efficiency, and thus being capable of inhibiting the growth of tumor cells and promoting tumor cell apoptosis; and therefore, the lentivirus plays an important role in tumor therapy.
Description
Technical field
The present invention relates to biological technical field, relate more specifically to purposes and the related drugs thereof of people eIF5B 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 21 to 23 nucleotide intervals.Cell 2000; 101:25-33.).Though tumor patient is through chemotherapy, radiotherapy and Comprehensive Treatment, 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 tumor.In recent years, RNAi has become the available strategy of the gene therapy of tumor.The expression that utilizes RNAi technology can suppress the antioncogene, 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.).
Eukaryotic translation initiation factor (eukaryotic translation initiation factors, eIFs) be that class protein translation in eukaryotic cell is necessary, guarantee the protein that correct mRNA-ribosome complex forms, the protein of the composition eIFs having found at present has more than 20 and plants, great majority are to form different eIFs with a plurality of subunit forms, form with complex plays a role, wherein study more translation initiation factor and have eIF-2, eIF-3, eIF-4 and eIF-5, they bring into play separately different effect (Damoc E in the process of translation initiation, Fraser CS, Zhou M, et a1.Structural eharactefization of the human eukaryotic initiation factor 3 protein complex by mass spectrometry.Mol Ceil Proteom.2007, 10 (7): 1135-1146.).Increasing research in recent years finds, eIFs, except playing an important role in the eukaryotic cell translation initiation stage, also has other effect, and multiple eIFs is all with the generation of tumor with make progress closely related.
Find that the earliest the eIFs of high expressed is eIF-4E in tumor.Compare with normal or benign tumor; eIF-4E is high expressed in some solid malignants; and the height of its expression also to relevant (the LEE JW of progress by stages of tumor; ChoiJJ; Lee KM; Choi CH, et al eIF-4E expression is associated with histopathologic grades in cervical neoplasia.Hum Pathol.2005; 36(11): 1197-203.Hershey J.W.B.Regulation of protein synthesis and the role of eIF3 in cancer.Braz.J.Med.Biol.Res.2010; 43 (10): 920-930.).The expression of eIF-2a in many tumors also usually raises, there is researcher by SABC, to detect the expression of eIF-2a in 88 routine hodgkin's lymphomas, find the expression strong positive of aggressive lymphomas and height aggressive lymphomas eIF-2a, prompting eIF-2a (the Wang S that plays an important role in lymphadenomatous generation and invasive procedure, Rosenwald IB, Hutzler MJ, Pihan GA, Savas L, Chen.JJ, et al.Expression of eukaryotic initiation factor 4E and 2a in non-Hodgkin's lymphomas.Am J Pathol.1999, 155:247-55.).Studies have found that again recently in breast carcinoma, cervical cancer, esophageal carcinoma and the pulmonary carcinoma the mankind; the expression of a subunit p170 of eIF-3 maximum significantly improves (Zong ZZ; Liu Z; Cui P, et a1.Role of eIF3a in regulating cell cycle progression.Exp Cell Res.2009; 315 (11): 1889-1894.).And another subunit p40 of eIF-3 also presents situation (the Tomlinson IP of high expressed in some carcinoma of prostate and primary breast cancer; Webb E; Carvajal-carmona L, el at.A genome-wide association study identifies colorectal cancer susceptibility loci on chromosomes 10p14 and8q23.3.Nat Genet.2008; 40 (5): 623-630.Cappuzzo F, Varella-carcia M.Rossi E.el at.MYC and eIF3H coamplification significantly improve response and survival of non-small cell lung cancer patients (NSCLC) treated with gefitinib.J Thorac Oncol.2009; 4 (4): 472-478.).For eIFs this phenomenon of high expressed in tumor, people have done further research to the dependency of eIF and tumor, find that a lot of eIF also participate in the regulation and control of malignant change of cell, to relevant (the Zhang LL of generation, aggressive and transfer of tumor.Pan XY，Hershey JWB．Individual Overexpression of Five Subunits of Human Translation Initiation Factor elF3 Promotes Malignant Tramformation of Immortal Fibreblast Cells．Biol Chem.2007;282(8)：5790-5800．Zhang L，Smit-Mcbride Z，Pan X，et aL.An oneogenie role for the phosphorylated h-subunit of human translation initiation factor eIF3．J Biol Chem.2008;283(35)：24047-24060．）。These researchs focus on eIF-2, eIF-3 and the several factors of eIF-4.
EIF-5B is a subunit in the eIF-5 factor; it is a kind of GTP enzyme; GTP enzymatic activity (the Lee JH that can specificity activates eIF-2; Pestova TV; Shin BS, et al.Initiation factor eIF5B catalyzes second GTP-dependent step in eukaryotic translation initiation.PNAS.2002; 99 (26): 16689-16694.).About eIF-5B at the report in Tumor-assaciated field seldom.In order to study the dependency of eIF-5B and tumor, the present invention chooses pulmonary carcinoma, gastric cancer, colon cancer and glioma cell model, take RNAi as means research eIF5B is in pulmonary carcinoma, gastric cancer, colon cancer and glioma generation and developing effect.
Summary of the invention
The Therapeutic Method and the medicine that the object of the invention is to open and people eIF5B (eukaryotic translation initiation factor 5B) gene-correlation, the RNA of take disturbs (RNAi) as means research eIF5B gene is in the survival of tumor cell and the effect in apoptotic process.
First aspect present invention, take RNA interference as means, having studied eIF5B gene occurs and developing effect in tumor, a kind of method that suppresses or reduce growth of tumour cell, propagation, differentiation and/or survival is disclosed, the method comprises: to tumor cell, use a kind of transcribing or translating of eIF5B gene that can specificity suppress, or can suppress the expression of eIF5B albumen or the molecule of activity by specificity, with this, come growth, propagation, differentiation and/or the survival of inhibition tumor cell.
Described tumor cell is selected from the tumor cell that its growth is relevant with the expression of eIF5B albumen or activity.Preferably, described tumor cell is selected from the arbitrary of gastric cancer, pulmonary carcinoma, colon cancer, glioma.
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 eIF5B gene, or enough reduces expression or the active dosage of eIF5B albumen.Further, the expression of described eIF5B gene is at least lowered 50%, 80%, 90%, 95% or 99%.
Described molecule can be selected from but be not limited to: nucleic acid molecules, carbohydrate, lipid, micromolecule 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 eIF5B gene or eIF5B 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 eIF5B 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 eIF5B of specificity gene.
Further, the first chain-ordering of described siRNA and the target sequence in eIF5B gene are basic identical.Preferably, the target sequence in described eIF5B gene contains the arbitrary sequence in SEQ ID NO:1-74.
When the target sequence in described eIF5B gene is the reticent eIF5B gene expression of described small molecules interference RNA specificity, with the fragment in the corresponding eIF5B gene of mRNA fragment of the described complementary combination of small molecules interference RNA.
Preferably, described eIF5B gene source is in people.
First aspect present invention also discloses a kind of people eIF5B gene of separation at preparation or screening anti-tumor medicine, or the purposes in preparing diagnosing tumor medicine.
Further, described tumor is selected from the arbitrary of gastric cancer, pulmonary carcinoma, colon cancer, glioma.
Described by separated eIF5B gene for the preparation of or screening anti-tumor medicine comprise the content of two aspects: one, using eIF5B gene as medicine or preparation for the action target of tumor cell, be applied to prepare anti-tumor medicine or preparation; Its two, using eIF5B gene as medicine or preparation for the action target of tumor cell, be applied to screen anti-tumor medicine or preparation.
Described using eIF5B gene as medicine or preparation is applied to prepare anti-tumor medicine for the action target of tumor cell or preparation specifically refers to: the target using eIF5B gene as RNA interference effect, develop medicine or preparation for tumor cell, thereby can reduce the expression of eIF5B gene in tumor cell.
Described using eIF5B gene as medicine or preparation is applied to screen anti-tumor medicine for the action target of tumor cell or preparation specifically refers to: using eIF5B gene as effective object, medicine or preparation are screened, using to find and can suppress or promote the medicine of people eIF5B gene expression as oncotherapy drug candidate.EIF5B gene small molecules interference RNA (siRNA) be take people eIF5B 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 eIF5B gene and albumen thereof as effective object.
Described by eIF5B gene for the preparation of diagnosing tumor medicine, refer to the preparation that is applied to diagnosing tumor medicine using eIF5B gene expression product as a diagnosing tumor index.
Described anti-tumor medicine is for suppressing transcribing or translating of eIF5B gene by specificity, or can suppress the expression of eIF5B albumen or the molecule of activity by specificity, thereby the expression of eIF5B gene in reduction tumor 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 eIF5B gene preparation or screening includes but not limited to: nucleic acid molecules, carbohydrate, lipid, micromolecule 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 eIF5B gene, or enough reduces expression or the active dosage of people eIF5B albumen.So that the expression of people eIF5B gene is at least lowered 50%, 80%, 90%, 95% or 99%.
Adopting the method for aforementioned anti-tumor medicine treatment tumor, is mainly by reducing the propagation of the expression inhibition tumor cell of people eIF5B gene, to reach the object for the treatment of.Concrete, during treatment, can effectively reduce the administering substances of people eIF5B gene expression dose in patient.
Second aspect present invention discloses a kind of separated nucleic acid molecules that reduces eIF5B gene expression in tumor cell, and described nucleic acid molecules comprises:
A) double-stranded RNA, in described double-stranded RNA, containing can be under stringent condition and the nucleotide sequence of eIF5B gene recombination; Or
B) shRNA, in described shRNA, containing can be under stringent condition and the nucleotide sequence of eIF5B 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 eIF5B gene, 15-27 continuous nucleotide sequence is basic identical.Preferably, in the sequence of described the first chain and eIF5B gene, 19-23 continuous nucleotide sequence is basic identical; Better, in the sequence of described the first chain and eIF5B 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 eIF5B 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 eIF5B gene, 15-27 continuous nucleotide sequence is basic identical.Described shRNA can become siRNA (siRNA) and then play the effect of endogenous eIF5B gene expression in the reticent tumor 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 eIF5B 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 eIF5B gene is basic identical, when the target sequence of described eIF5B gene is siRNA for the reticent eIF5B gene expression of specificity, by the fragment in the corresponding eIF5B gene of mRNA fragment of described siRNA identification silence.
Preferably, arbitrary sequence that the target sequence in described eIF5B gene contains SEQ ID NO:1-74.
Further, described eIF5B 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:86, is specially 5 '-CGGCGACUUGAACAUAGUAAA-3 '.
SiRNA shown in SEQ ID NO:86 for take the sequence shown in SEQ ID NO:2 be RNA disturb target sequence design, for a chain of the siRNA of people eIF5B 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 eIF5B gene expression in the reticent tumor 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:87, is specially: 5 '-CGGCGACUUGAACAUAGUAAAUUCAAGAGAUUUACUAUGUUCAAGUCGCCG-3 '.
ShRNA can become siRNA after enzyme action processing, and then plays the effect of endogenous people eIF5B gene expression in the reticent tumor cell of specificity.
The interference slow virus carrier of genetic fragment of shRNA of the present invention of encoding contains arbitrary sequence and the complementary series thereof in SEQ ID NO:1-74.
Third aspect present invention, discloses a kind of eIF5B gene interfere RNA construct, and the genetic fragment of the shRNA in the nucleic acid molecules that contains the separation of the present invention of encoding, can express described shRNA
Described people eIF5B gene interfere RNA construct can be the gene fragment clone of the aforementioned people eIF5B gene shRNA of coding to be entered to known carrier obtain.Further, described eIF5B gene interfere RNA construct is that eIF5B gene disturbs slow virus carrier.
It is the DNA fragmentation of the aforementioned eIF5B gene shRNA of coding to be cloned into known carrier obtain that eIF5B gene of the present invention disturbs slow virus carrier, described known carrier mostly is slow virus carrier, described eIF5B gene disturbs slow virus carrier to become after infectious virion through virus packing, infected tumor's cell, and then transcribe out shRNA of the present invention, by steps such as enzyme action processing, finally obtain described siRNA, for the expression of the reticent eIF5B gene of specificity.
Further, described eIF5B gene disturbs slow virus carrier also to contain the nucleotide sequence of label that can be detected in promoter sequence and/or codes for tumor cell; Preferably, described label that can be detected is as green fluorescent protein (GFP).
Further, described slow virus carrier 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, pcDNA1.2/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 eIF5B gene that pGCSIL-GFP is vector construction and disturb slow virus carrier, called after pGCSIL-GFP-eIF5B-siRNA.
The nucleic acid molecules of separation of the present invention can be used for the medicine of preparation prevention or treatment tumor, and described tumor is gastric cancer, pulmonary carcinoma, colon cancer or glioma.
EIF5B 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 tumor.EIF5B gene disturbs slow virus carrier to can be used for preparing described eIF5B gene siRNA.When the medicine as treatment tumor or preparation, be that the described nucleic acid molecules of safe and effective amount is applied to mammal.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 eIF5B gene and has disturbed slow virus, by aforementioned eIF5B gene, disturbs slow virus carrier under slow virus packaging plasmid, cell line auxiliary, through virus packing, forms.This slow virus can also produce the small molecules interference RNA for eIF5B gene by infected tumor's cell, thereby suppresses the propagation of gastric cancer, pulmonary carcinoma, colon cancer or glioma tumor cell.This eIF5B gene disturbs slow virus to can be used for the medicine of preparation prevention or treatment tumor.
Fifth aspect present invention, disclose a kind of for preventing or treat the pharmaceutical composition of tumor, the nucleic acid molecules that its active substance contains aforesaid separation, eIF5B gene interfere RNA construct, and/or eIF5B gene disturbs slow virus.
Further, described pharmaceutical composition contains double-stranded RNA described in 1～99wt%, shRNA, eIF5B gene interfere RNA construct or eIF5B gene and disturbs slow virus, and pharmaceutically acceptable carrier, diluent or excipient.
In preparation during these compositionss, conventionally by active component and mixed with excipients, or with excipient dilution, wrap in can capsule or the carrier that exists of medicine bag form in.When excipient plays diluent, do the used time, it can be that solid, semisolid or fluent material are as the medium of excipient, carrier or active component.Therefore, compositions can be tablet, pill, powder, solution, syrup, sterilizing injecting solution etc.The example of suitable excipient comprises: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline Cellulose, polyvinylpyrrolidone, cellulose, water, etc.Preparation also can comprise: wetting agent, emulsifying agent, antiseptic (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 gastric cancer, pulmonary carcinoma, colon cancer or glioma.
The treatment that is applied as tumor 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.Further, described tumor is selected from the arbitrary of gastric cancer, pulmonary carcinoma, colon cancer, glioma.
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 tumor, propagation, recurrence and/or shift suppressed.Further, at least 10% of the growth of described tumor, 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 eIF5B gene expression in tumor cell is disclosed, described test kit comprises: be present in the nucleic acid molecules of the described separation in container, and eIF5B gene interfere RNA construct, and/or described eIF5B gene disturbs slow virus.
In sum, the present invention has designed 74 RNAi target sequences for people eIF5B gene, build corresponding eIF5BRNAi carrier, wherein the RNAi carrier pGCSIL-GFP-eIF5B-siRNA of coded sequence SEQ ID NO:2 can significantly lower eIF5B 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-eIF5B-siRNA and can the RNAi sequence for eIF5B gene efficiently be imported to people's pulmonary carcinoma H1299 cell, gastric cancer SGC7901, colon cancer RKO cell and glioma U87 cell targeting, reduce the expression of EIF5B gene, significantly suppress the multiplication capacity of above-mentioned tumor cell.Therefore the eIF5B gene silencing of lentivirus mediated is the potential clinical non-operative treatment mode of malignant tumor.
SiRNA provided by the invention or the nucleic acid construct that comprises this siRNA sequence, slow virus can specificity suppress the expression of people eIF5B gene, especially slow virus, can efficiently infect target cell, suppress expeditiously the expression of eIF5B 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: eIF5B-RNAi slow virus was infected people's pulmonary carcinoma H1299 cell, gastric cancer SGC7901, colon cancer H1299 cell and glioma U87 cell after 5 days, and the expression of eIF5B mRNA significantly reduces
Fig. 3: eIF5B-RNAi slow virus was infected people's pulmonary carcinoma H1299 cell after 5 days, caused cell inhibitory effect
Fig. 4: eIF5B-RNAi slow virus was infected people's gastric cancer SGC7901 cell after 5 days, caused cell inhibitory effect
Fig. 5: eIF5B-RNAi slow virus was infected human colon carcinoma RKO cell after 5 days, caused cell inhibitory effect
Fig. 6: eIF5B-RNAi slow virus was infected people's glioma U87 cell after 5 days, caused cell inhibitory effect
The specific embodiment
The generation development that the present invention is based on multiple eukaryotic translation initiation factor and tumor is closely related, and eIF5B, as a kind of eukaryotic translation initiation factor, infers that it may may participate in generation and the development of malignant tumor.
The present invention relates to one group of small molecules interference RNA for people eIF5B gene (siRNA) sequence, rna interference vector and RNA and disturbed slow virus.Choose people eIF5B 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 eIF5B gene.
Inventor finds, adopts after the expression of the eIF5B gene of transferring person under RNAi method the propagation of inhibition tumor cell effectively, and this achievement in research shows that eIF5B gene is proto-oncogene, can be used as the target spot of oncotherapy.Inventor is further synthetic and tested the multiple siRNA for eIF5B gene, filter out the siRNA that can effectively suppress the expression of eIF5B and then suppress people's pulmonary carcinoma H1299 cell, gastric cancer SGC7901 cell, colon cancer RKO cell and glioma U87 cell proliferation and growth, completed on this basis the present invention.
The invention provides siRNA (siRNA) sequence of a series of interference people eIF5B genes, built can specificity reticent eIF5B gene expression slow virus.The present invention studies discovery, for siRNA and the RNAi slow virus of people eIF5B gene design, stablizes the expression of also lowering specifically eIF5B gene, and effectively suppresses the propagation of human tumor cells.The present invention shows that eIF5B 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 eIF5B 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 eIF5B gene RNAi slow virus: from Genbank, transfer people eIF5B gene order; Prediction siRNA site; The synthetic effective siRNA sequence for eIF5B gene, two ends are containing the double-stranded DNA Oligo of restriction enzyme site cohesive end; After slow virus carrier double digestion, be connected the RNAi plasmid of construction expression eIF5B 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 granule, can make the slow virus of efficient reticent eIF5B gene.
Based on said method, the invention provides 74 Effective target sites (specifically as shown in SEQ ID NO1-74) that disturb eIF5B gene, built the slow virus of special interference people eIF5B gene.
The present invention simultaneously also discloses a kind of people eIF5B gene RNAi slow virus (eIF5B-RNAi) and preparation and application thereof.
This research is found, utilizes the RNAi method of lentivirus mediated, after reducing the expression of eIF5B gene in tumor cell, and the effective propagation of inhibition tumor cell.This research shows, eIF5B gene is a proto-oncogene, can promote tumor cell proliferation, in occurring and develop, tumor there is important biological function, eIF5B gene can be the target of oncotherapy, and the eIF5B 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 manufacturer's suggestion is carried out or configures.
1. screening is for the effective siRNA target spot of people eIF5B gene
From Genbank, transfer EIF5B(NM 015904) gene information; Utilize the design software Genechem design of Shanghai JiKai Gene Chemical Technology Co., Ltd for the effective siRNA target spot of EIF5B gene.In coded sequence (CDS) region of EIF5B gene, every the sequence of 21 bases of an initial acquisition of base, table 1 has been listed wherein 74 effective siRNA target sequences for EIF5B gene.
Table 1 targeting is in the siRNA target sequence of people eIF5B gene
2. the preparation of slow virus carrier
Double-stranded DNA Oligo sequence (table 2) for the synthetic two ends of siRNA target spot (the SEQ ID NO:2 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 linearisation, and agarose gel electrophoresis is identified endonuclease bamhi with Age I and EcoR I restricted 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
| 5’ | Neck | Ring | Neck | 3’ | SEQ | |
| Positive-sense strand | CCGG | CGGCGACTTGAACATAGTAAA | TTCAAGAGA | TTTACTATGTTCAAGTCGCCG | TTTTTG | 75 |
| Antisense strand | AATTCAAAAA | CGGCGACTTGAACATAGTAAA | TCTCTTGAA | TTTACTATGTTCAAGTCGCCG | 76 |
By T4 DNA ligase, by double digestion linearisation, (enzyme action system is as shown in table 4,37 ℃, reaction 1h) the carrier DNA double-stranded DNA Oligo good with purification 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 culture medium, mix and get 1 μ l as template; The upstream and downstream of RNAi sequence in slow virus carrier, design universal PC R primer (forward primer sequence: 5 '-CCTATTTCCCATGATTCCTTCATA-3 ' (SEQ ID NO:79); Downstream primer sequence: 5 '-GTAATACGGTTATCCACGCG-3 ' (SEQ ID NO:80), carries out PCR identification experiment (PCR reaction system is as table 6-1, and reaction condition 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 expression RNAi for SEQ ID NO:2 successfully constructing, called after pGCSIL-GFP-EIF5B-siRNA.
Build pGCSIL-GFP-Scr-siRNA negative control plasmid, negative control siRNA target sequence is 5 '-TTCTCCGAACGTGTCACGT-3 ' (SEQ ID NO:81).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 methods, authentication method and condition be same pGCSIL-GFP-EIF5B-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 | 77 |
| Antisense strand | AATTCAAAAA | TTCTCCGAACGTGTCACGT | TCTCTTGAA | ACGTGACACGTTCGGAGAA | 78 |
By T4 DNA ligase by double digestion linearisation (enzyme action system is as shown in table 4,37 ℃, reaction 1h) carrier
Table 4 pGCSIL-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 coupled reaction 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 buffer | 1.0 | 1.0 | 1.0 |
| T4 phage DNA ligase | 1.0 | 1.0 | 1.0 |
| dd H 2O | 16.0 | 17.0 | 16.0 |
| Total | 20.0 | 20.0 | 20.0 |
Table 6-1 PCR reaction system
| Reagent | Volume (μ l) |
| 10×buffer | 2.0 |
| dNTPs(2.5mM) | 0.8 |
| Forward primer | 0.4 |
| Downstream primer | 0.4 |
| Taq polymerase | 0.2 |
| Template | 1.0 |
| ddH 2O | 15.2 |
| Total | 20.0 |
Table 6-2 PCR reaction system program setting
3. pack eIF5B-siRNA slow virus
The DNA that extracts RNAi plasmid pGCSIL-GFP-eIF5B-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 trypsinization exponential phase, take that containing the DMEM complete medium of 10% hyclone, 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 culture medium of sucking-off, adds the complete 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 culture medium 400 μ l, get the plasmid DNA of the above-mentioned extracting of 20 μ l, add to above-mentioned PVM/PEI/DMEM mixed liquor.
Above-mentioned transfection mixture is at room temperature hatched to 15min, be transferred in the culture medium of HEKC 293T cell, 37 ℃, 5%CO
2in incubator, cultivate 16h.Discard the culture medium that contains transfection mixture, PBS solution washing, adds complete medium 2ml, continues to cultivate 48h.Collecting cell supernatant, Centricon Plus-20 centrifugal ultrafiltration device (Millipore) purification 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, filter cup and filtered solution collection cups are below separated, filter 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 subpackage in-80 degrees Celsius of preservations.The sequence of the first chain of the siRNA containing in virus concentrated solution is as shown in SEQ ID NO:86.The packaging process of contrast slow virus, with eIF5B-siRNA slow virus, only replaces pGCSIL-GFP-eIF5B-siRNA carrier with pGCSIL-GFP-Scr-siRNA carrier.
In people's pulmonary carcinoma H1299 of exponential phase cell, gastric cancer SGC7901 cell, colon cancer RKO cell and glioma U87 cell, carry out trypsinization, (cell number is about 5 * 10 to make cell suspension
4/ ml) be inoculated in 6 orifice plates, be cultured to cell fusion degree and reach approximately 30%.According to infecting plural number (MOI, H1299:20, SGC7901:20, RKO:20, U87:20) value, add the virus of embodiment 1 preparation of Sq, after cultivation 24h, change culture medium, until time of infection, reach after 5 days collecting cell.According to the Trizol operating instruction of Invitrogen company, extracted total RNA.According to the M-MLV operating instruction 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 reverse transcriptase inactivation).
Adopt TP800 type Real time PCR instrument (TAKARA) to carry out real-time quantitative detection.The primer of EIF5B gene is as follows: forward primer 5 '-ACGGAGGATTGAGAAACG-3 ' (SEQ ID NO:82) and downstream primer 5 '-AAGAGGAACATTGGTGGC-3 ' (SEQ ID NO:83).Take house-keeping gene GAPDH as internal reference, and primer sequence is as follows: forward primer 5 '-TGACTTCAACAGCGACACCCA-3 ' (SEQ ID NO:84) and downstream primer 5 '-CACCCTGTTGCTGTAGCCAAA-3 ' (SEQ ID NO:85).Press the proportional arrangement reaction system in table 8.
Table 7 reverse transcription reaction system
| Reagent | Volume (μ l) |
| 5×RT buffer | 4.0 |
| 10mMm dNTPs | 2.0 |
| RNasin | 0.5 |
| M-MLV-RTase | 1.0 |
| DEPC H 2O | 3.5 |
| Total | 11.0 |
Table 8 Real-time PCR reaction system
| Reagent | Volume (μ l) |
| SYBR premix ex taq: | 10.0 |
| Forward 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 method Real-time PCR: 95 ℃ of denaturations, 15s; 95 ℃ of each step degeneration 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 degeneration 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 Ctanalytic process is calculated the gene expression abundance that has infected EIF5B mRNA.Infect the cell of contrast virus (Lv-Scr-siRNA) in contrast.Experimental result (Fig. 2) shows, in people's pulmonary carcinoma H1299 cell, gastric cancer SGC7901 cell, colon cancer RKO cell and glioma U87 cell, the expression of EIF5B mRNA has lowered 95.0%, 91.5%, 74.5% and 91.5%.
In the people H1299 of exponential phase cell, gastric cancer SGC7901 cell, colon cancer RKO cell and glioma U87 cell, carry out trypsinization, (cell number is about 5 * 10 to make cell suspension
4/ ml) be inoculated in 6 orifice plates, be cultured to cell fusion degree and reach approximately 30%.According to infecting plural number (MOI, H1299:20, SGC7901:20, RKO:20, U87:20), add the virus of Sq, after cultivation 24h, change culture medium, until time of infection, reach after 5 days, collect each experimental group cell in exponential phase.The resuspended one-tenth cell suspension (2 * 10 of complete 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 shown in Fig. 3-Fig. 6).Result shows, slow virus is infected each tumor of group and cultivates after 5 days at cells in vitro, growth rate significantly slows down, growth rate far below matched group tumor cell, vigor cell number has declined respectively 95.1%, 56.0%, 97.8% and 82.3%, shows that EIF5B 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 eIF5B gene purposes in preparation or screening anti-tumor medicine.
2. purposes as claimed in claim 1, is characterized in that, described tumor is selected from the arbitrary of gastric cancer, pulmonary carcinoma, colon cancer, glioma.
3. a separated nucleic acid molecules that reduces eIF5B gene expression in tumor cell, described nucleic acid molecules comprises:
A) double-stranded RNA, in described double-stranded RNA, containing can be under stringent condition and the nucleotide sequence of eIF5B gene recombination; Or
B) shRNA, in described shRNA, containing can be under stringent condition and the nucleotide sequence of eIF5B gene recombination.
4. the nucleic acid molecules 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 eIF5B 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 eIF5B gene is basic identical.
5. the nucleic acid molecules of separation as described in claim 3 or 4, is characterized in that, the target sequence of described eIF5B gene contains arbitrary sequence in SEQ ID NO:1-74.
6. the nucleic acid molecules 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:86.
7. the nucleic acid molecules 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:87.
8. an eIF5B gene interfere RNA construct, the genetic fragment that contains the shRNA in nucleic acid molecules separated described in the arbitrary claim of coding claim 3-7, can express described shRNA.
9. eIF5B gene interfere RNA construct as claimed in claim 8, is characterized in that, described eIF5B gene interfere RNA construct is for disturbing slow virus carrier.
10. eIF5B gene interfere RNA construct as claimed in claim 9, is characterized in that, described interference slow virus carrier obtains after the gene fragment clone of the described shRNA of coding is entered to slow virus carrier, and described slow virus carrier 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、
Arbitrary in pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.
11. 1 kinds of eIF5B genes disturb slow viruss, by disturbing slow virus carrier described in the arbitrary claim of claim 9-10 under slow virus packaging plasmid, cell line auxiliary, through virus packing, form.
12. 1 kinds for preventing or treat the pharmaceutical composition of tumor, its active substance contains the separated nucleic acid molecules described in the arbitrary claim of claim 3-7, eIF5B gene interfere RNA construct described in the arbitrary claim of claim 8-10, and/or the eIF5B gene described in claim 11 disturbs slow virus.
The application of pharmaceutical composition in the anti-tumor medicine of preparation treatment gastric cancer, pulmonary carcinoma, colon cancer or glioma described in 13. claim 12.
14. 1 kinds of test kits for reducing eIF5B gene expression in tumor cell, it is characterized in that, described test kit comprises: be present in container, separated nucleic acid molecules described in the arbitrary claim of claim 3-7, eIF5B gene interfere RNA construct described in the arbitrary claim of claim 8-10, and/or the eIF5B gene described in claim 11 disturbs slow virus.
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