CN103667424A

CN103667424A - Usage of human EIF3H gene and related medicaments

Info

Publication number: CN103667424A
Application number: CN201210314226.XA
Authority: CN
Inventors: 韩海雄; 孙琴; 谭畅; 翁仕强; 金杨晟; 瞿红花; 曹跃琼
Original assignee: SHANGHAI GENECHEM CO Ltd
Current assignee: SHANGHAI GENECHEM CO Ltd
Priority date: 2012-08-29
Filing date: 2012-08-29
Publication date: 2014-03-26

Abstract

The invention discloses a usage of a human EIF3H gene and related medicaments. The invention discloses usage of the human EIF3H gene in tumor treatment, tumor diagnosis and medicament preparation. The invention further constructs a human EIF3H gene small interfering RNA (Ribonucleic Acid), a human EIF3H gene interference nucleic acid construction body and a human EIF3H gene inference lentivirus and discloses usage thereof. The siRNA or the nucleic acid construction body containing the siRNA sequence and the lentivirus provided by the invention can be used for specifically inhibiting the expression of the human EIF3H gene, and especially, the lentivirus can be used for efficiently infecting target cells and efficiently inhibiting the expression of the human EIF3H gene in the target cells so as to inhibit the growth of tumor cells and promote the apoptosis of the tumor cells, thereby having important significance in the tumor treatment.

Description

Purposes and the related drugs thereof of people EIF3H gene

Technical field

The present invention relates to biological technical field, relate more specifically to purposes and the related drugs thereof of people EIF3H gene.

Background technology

Eukaryotic translation initiation factor 3(eukaryotic translation initiation factors3, eIF-3) be the most complicated maximum factor in eukaryotic translation initiation factor, in protein translation initiating process, playing a significant role, is the center protein factor connecting each other with other translation initiation factors.EIF-3 is subunit complex body more than, is that separation and purification obtains from rabbit reticulated red blood cells the earliest.EIF-3 molecular weight is 550-700KDa, Mammals eIF-3 comprises at least 12 subunits, there is a(p170, eIF3s10), b(p116, eIF3s9), c(p110), d(p66, eIFs7), e(p48, int6), f(p44, p42, eIF3s4), h(p40, eIF3s3), i(p36), j(p35, eIF3s1), k(p28), (the Fraser CS such as l(p69), Lee JY, Mayeur GL, Bushell M, Doudna JA, Hershey JW.The j-subunit of human translation initiation factor eIF3 is required for the stable binding of eIF3 and its subcomplexes to 40 S ribosomal subunits in vitro.J Biol Chem.2004, 279 (10): 8946-56.).The effect of eIF-3 is to form 43S initiation complex at translation initiation; make 40S small subunit ribosome keep unbound state, stablize ternary complex (the Valasek L that eIF-2 and Met-tRNAi and GTP form; Nielsen KH; Zhang F, et al.Interactions of Eukaryotic Translation Initiation Factor3 (eIF3) Subunit NIP1/c with eIF1 and eIF5 Promote Preinitiation Complex Assembly and Regulate Start Codon Selection.Mol Cell Biol.2004; 24 (21): 9437-9455.).

Quantity research shows greatly, and the expression of a plurality of subunits of eIF-3 has in the different steps of kinds of tumors the unusual phenomenon that increases or reduce, and has ubiquity.EIF3a is the subunit of eIF-3 maximum, studies show that in a large number eIF3a has phenomenon (the Zong ZZ of high expression level in mammary cancer, esophagus cancer, lung cancer, ovarian cancer, colorectal carcinoma and liver cancer, UU Z, Cui P, et a1.Role of eIF3a in regulating cell cycle progression.Exp Cell Res.2009; 315 (11): 1889-1894.Chen GP, Burger MM.p150 overexpression in gastric carcinolna:the association with p53.apoptosis and cell proliferation．Inl J Cancer.2004；112(3)：393-398．）。Int6/eIF3e (p48) is the genomic insertion point of mouse mammary tumour virus, after viral integrase, can make Int6/eIF3e be sheared, the eIF3e that expression is sheared can cause glucagonoma to become, so Int6/eIF3e and tumour there is relation (Mack DL.Boulanger CA, Cauahan R, et a1.Expression of truncated Int6/elF3e in mammary alveolar epithelium lcads to persistent hyperplasia and tumorigenesis.Breast Cancer Res.2007; 9 (4): R42.).The expression of other subunit of eIF-3 in tumour also more to be had extremely, and mostly be rising, for example eIF3b crosses and expresses in mammary cancer, the transcriptional level of eIF3c in carcinoma of testis increases (Dong ZZ, Zhang JT.Initiation factor elF3 and regulation of mRNA translation, cell growth, and cancer.Oncol Hematol.2006; 59 (3): 169-180.), eIF3i expresses and increases (Lei YX in the tumour of hepatocellular carcinoma, squamous cell carcinoma of the head and neck and cadmium induction, Wei L, Wang M, et a1.Malignant trailsformation and abnormal expression of eukaryofic initiation factor in bronchial epithelial cells induced by cadmium chloride.Biomed Environ sci.2008; 21 (4): 332-338.).But eIF3f is different, it is the synthetic negative regulatory factor of a kind of albumen, expression amount in pancreatic tumor cell and melanoma cell reduces, eIf3f is positioned at chromosomal 11p15.4, and phenomenon (the Doldan A that has karyomit(e) 11pl5.4 to lack in kinds of tumors, Chandramouli A, Shanas R, el at.Loss of the eukaryotic initiation factor 3f in pancreatic cancer.Mol Carcinog.2008; 47 (3): 235-244.).

EIF3H is eukaryotic translation initiation factor 3 one of them subunit, is positioned on 8q23 karyomit(e) the chromosomal long-armed amplification phenomenon that occurs in kinds of tumors of 8q.There are some researches show; EIF3H has gene amplification and high expression level phenomenon (Jalava SE in mammary cancer, prostate cancer and liver cancer; Porkka KP; Rauhala HE; Isotalo J; Tammela TL, et al.TCEB1 promotes invasion of prostate cancer cells.Int J Cancer.2009; 124:95 – 102.Zhang L; Smit-McBride Z; Pan X; Rheinhardt J; Hershey JW (2008) An oncogenic role for the phosphorylated h-subunit of human translation initiation factor eIF3.J Biol Chem 283:24047 – 24060.Savinainen KJ; Helenius MA; Lehtonen HJ, Visakorpi T.Overexpression of EIF3S3 promotes cancer cell growth.Prostate.2006; 66:1144 – 1150.).The method that Savinainen etc. disturb by RNA reduces after the expression amount of EIF3H in mammary cancer and prostate cancer; multiplication capacity and clonality (the J Biol Chem 283:24047 – 24060.Savinainen KJ that can obviously suppress mammary cancer and prostate cancer cell; Helenius MA; Lehtonen HJ, Visakorpi T.Overexpression of EIF3S3 promotes cancer cell growth.Prostate.2006; 66:1144 – 1150.).The discoveries such as Alan; in colorectal cancer cells; adopting uses the same method reduces after the expression of EIF3H gene; also can reduce multiplication capacity (the Pittman AM of colorectal cancer cells; Naranjo S; Jalava SE, et al.Allelic variation at the 8q23.3colorectal cancer risk locus functions as a cis-acting regulator of EIF3H.POLS genet.2010; 6 (9): 1-9.).The research of EIF3H high expression level in kinds of tumors tissue shows, EIF3H may be closely related with generation, development, the malignant behaviors of kinds of tumors.Therefore, EIF3H and tumour there is certain relation, be expected to become a novel targets of therapy of tumor.

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 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.).

In order to further investigate the regulatory function of EIF3H in tumour occurs, the present invention chooses mammary cancer, lung cancer, colorectal carcinoma and glioma cell model, take RNAi as means research EIF3H is in mammary cancer, lung cancer, colorectal carcinoma and glioma generation and developing effect.

Summary of the invention

The object of the invention is to open and people EIF3H (eukaryotic translation initiation factor3, subunit H) methods for the treatment of of gene-correlation and medicine, the RNA of take disturbs (RNAi) as means research EIF3H 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 EIF3H 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 EIF3H gene that can specificity suppress, or can specificity suppress the expression of EIF3H 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 EIF3H albumen or activity.Preferably, described tumour cell is selected from the arbitrary of mammary cancer, lung cancer, colorectal carcinoma, 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 EIF3H gene, or enough reduces expression or the active dosage of EIF3H albumen.Further, the expression of described EIF3H 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 EIF3H gene or EIF3H 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 EIF3H 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 EIF3H of specificity gene.

Further, the first chain-ordering of described siRNA and the target sequence in EIF3H gene are basic identical.Preferably, the target sequence in described EIF3H gene contains the arbitrary sequence in SEQ ID NO:1-31.

When the target sequence in described EIF3H gene is the reticent EIF3H genetic expression of described small molecules interference RNA specificity, with the fragment in the corresponding EIF3H gene of mRNA fragment of the described complementary combination of small molecules interference RNA.

Preferably, described EIF3H gene source is in people.

First aspect present invention also discloses a kind of people EIF3H gene of separation at preparation or screening anti-tumor medicine, or the purposes in preparing diagnosing tumor medicine.

Further, described tumour is selected from the arbitrary of mammary cancer, lung cancer, colorectal carcinoma, glioma.

Described by separated EIF3H gene for the preparation of or screening anti-tumor medicine comprise the content of two aspects: one, using EIF3H gene as medicine or preparation for the action target of tumour cell, be applied to prepare anti-tumor medicine or preparation; Its two, using EIF3H gene as medicine or preparation for the action target of tumour cell, be applied to screen anti-tumor medicine or preparation.

Described using EIF3H 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 EIF3H gene as RNA interference effect, develop medicine or preparation for tumour cell, thereby can reduce the expression level of EIF3H gene in tumour cell.

Described using EIF3H 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 EIF3H gene as effective object, medicine or preparation are screened, using to find and can suppress or promote the medicine of people EIF3H genetic expression as oncotherapy drug candidate.EIF3H gene small molecules interference RNA (siRNA) be take people EIF3H 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 EIF3H gene and albumen thereof as effective object.

Described by EIF3H gene for the preparation of diagnosing tumor medicine, refer to the preparation that is applied to diagnosing tumor medicine using EIF3H gene expression product as a diagnosing tumor index.

Described anti-tumor medicine is for can specificity suppressing transcribing or translating of EIF3H gene, or can specificity suppress the expression of EIF3H albumen or the molecule of activity, thereby the expression level of EIF3H 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 EIF3H 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.

The amount of application of described anti-tumor medicine is enough to reduce transcribing or translating of people EIF3H gene, or enough reduces expression or the active dosage of people EIF3H albumen.So that the expression of people EIF3H 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 EIF3H gene, to reach the object for the treatment of.Concrete, during treatment, can effectively reduce the administering substances of people EIF3H gene expression dose in patient.

Second aspect present invention discloses a kind of separated nucleic acid molecule that reduces EIF3H 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 EIF3H gene recombination; Or

B) shRNA, in described shRNA, containing can be under stringent condition and the nucleotide sequence of EIF3H 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 EIF3H gene, 15-27 continuous nucleotide sequence is basic identical.Preferably, in the sequence of described the first chain and EIF3H gene, 19-23 continuous nucleotide sequence is basic identical; Better, in the sequence of described the first chain and EIF3H 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 EIF3H 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 EIF3H gene, 15-27 continuous nucleotide sequence is basic identical.Described shRNA can become siRNA (siRNA) and then play the effect of endogenous EIF3H 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 EIF3H 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 EIF3H gene is basic identical, when the target sequence of described EIF3H gene is siRNA for the reticent EIF3H genetic expression of specificity, by the fragment in the corresponding EIF3H gene of mRNA fragment of described siRNA identification silence.

Preferably, arbitrary sequence that the target sequence in described EIF3H gene contains SEQ IDNO:1-31.

Further, described EIF3H 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:43, is specially 5 '-GCUGUUGCAGAUAAACAUGAA-3 '.

SiRNA shown in SEQ ID NO:43 for take the sequence shown in SEQ ID NO:30 be RNA disturb target sequence design, for a chain of the siRNA of people EIF3H 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 EIF3H 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:44, is specially: 5 '-GCUGUUGCAGAUAAACAUGAAUUCAAGAGAUUCAUGUUUAUCUGCAACAGC-3 '.

ShRNA can become siRNA after enzyme is cut processing, and then plays the effect of endogenous people EIF3H 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:1-31.

Third aspect present invention, discloses a kind of EIF3H 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 EIF3H gene interfere RNA construct can be the gene fragment clone of the aforementioned people EIF3H gene shRNA of coding to be entered to known carrier obtain.Further, described EIF3H gene interfere RNA construct is that EIF3H gene disturbs lentiviral vectors.

It is the DNA fragmentation of the aforementioned EIF3H gene shRNA of coding to be cloned into known carrier obtain that EIF3H gene of the present invention disturbs lentiviral vectors, described known carrier mostly is lentiviral vectors, described EIF3H 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 EIF3H gene of specificity.

Further, described EIF3H 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, 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 EIF3H gene that pGCSIL-GFP is vector construction and disturb lentiviral vectors, called after pGCSIL-GFP-EIF3H-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 mammary cancer, lung cancer, colorectal carcinoma or glioma.

EIF3H 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.EIF3H gene disturbs lentiviral vectors to can be used for preparing described EIF3H 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 EIF3H gene and has disturbed slow virus, by aforementioned EIF3H 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 EIF3H gene by infected tumor's cell, thereby suppresses the propagation of mammary cancer, lung cancer, colorectal carcinoma or glioma tumour cell.This EIF3H 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, EIF3H gene interfere RNA construct, and/or EIF3H gene disturbs slow virus.

Further, described pharmaceutical composition contains double-stranded RNA described in 1～99wt%, shRNA, EIF3H gene interfere RNA construct or EIF3H 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 mammary cancer, lung cancer, colorectal carcinoma or glioma.

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.Further, described tumour is selected from the arbitrary of mammary cancer, lung cancer, colorectal carcinoma, 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 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 EIF3H 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 EIF3H gene interfere RNA construct, and/or described EIF3H gene disturbs slow virus.

In sum, the present invention has designed 31 RNAi target sequences for people EIF3H gene, build corresponding EIF3H RNAi carrier, wherein the RNAi carrier pGCSIL-GFP-EIF3H-siRNA of encoding sequence SEQ ID NO:30 can significantly lower EIF3H 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-EIF3H-siRNA and can the RNAi sequence for EIF3H gene efficiently be imported to MCF-7 Human Breast Cancer Cells, lung cancer H1299 cell, colorectal carcinoma RKO cell and glioma U87 cell target, reduce the expression level of EIF3H gene, significantly suppress the multiplication capacity of above-mentioned tumour cell.Therefore the EIF3H 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 EIF3H gene, especially slow virus, can efficiently infect target cell, suppress expeditiously the expression of EIF3H 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: EIF3H-RNAi slow virus was infected MCF-7 Human Breast Cancer Cells, lung cancer H1299 cell, colorectal carcinoma RKO cell and glioma U87 cell after 5 days, and the expression level of EIF3H mRNA significantly reduces

Fig. 3: EIF3H-RNAi slow virus was infected MCF-7 Human Breast Cancer Cells after 5 days, caused cell inhibitory effect

Fig. 4: EIF3H-RNAi slow virus was infected people's lung cancer H1299 cell after 5 days, caused cell inhibitory effect

Fig. 5: EIF3H-RNAi slow virus was infected human colon carcinoma RKO cell after 5 days, caused cell inhibitory effect

Fig. 6: EIF3H-RNAi slow virus was infected people's glioma U87 cell after 5 days, caused cell inhibitory effect

Embodiment

The present invention relates to one group of small molecules interference RNA for people EIF3H gene (siRNA) sequence, rna interference vector and RNA and disturbed slow virus.Choose people EIF3H 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 EIF3H gene.

Contriver finds, adopts after the expression of the EIF3H gene of transferring person under RNAi method the propagation of inhibition tumor cell effectively, and this achievement in research shows that EIF3H gene is proto-oncogene, can be used as the target spot of oncotherapy.Contriver is further synthetic and tested the multiple siRNA for EIF3H gene, filter out the expression that can effectively suppress EIF3H and then the siRNA that suppresses MCF-7 Human Breast Cancer Cells, lung cancer H1299 cell, colorectal carcinoma 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 EIF3H genes, built can specificity reticent EIF3H genetic expression slow virus.The present invention studies discovery, for siRNA and the RNAi slow virus of people EIF3H gene design, stablizes the expression of also lowering specifically EIF3H gene, and effectively suppresses the propagation of human tumor cells.The present invention shows that EIF3H 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 EIF3H 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 EIF3H gene RNAi slow virus: from Genbank, transfer people EIF3H gene order; Prediction siRNA site; The synthetic effective siRNA sequence for EIF3H 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 EIF3H 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 EIF3H gene.

Based on aforesaid method, the invention provides 31 Effective target sites (specifically as shown in SEQ ID NO 1-31) that disturb EIF3H gene, built the slow virus of special interference people EIF3H gene.

The present invention simultaneously also discloses a kind of people EIF3H gene RNAi slow virus (EIF3H-RNAi) and preparation and application thereof.

This research is found, utilizes the RNAi method of lentivirus mediated, after reducing the expression of EIF3H gene in tumour cell, and the effective propagation of inhibition tumor cell.This research shows, EIF3H gene is a proto-oncogene, can promote tumor cell proliferation, in occurring and develop, tumour there is important biological function, EIF3H gene can be the target of oncotherapy, and the EIF3H 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.

Embodiment 1 is for the preparation of people EIF3H gene RNAi slow virus

1. screening is for the effective siRNA target spot of people EIF3H gene

From Genbank, transfer EIF3H(NM_003756) gene information; Utilize the design software Genechem design of Shanghai JiKai Gene Chemical Technology Co., Ltd for the effective siRNA target spot of EIF3H gene.In encoding sequence (CDS) region of EIF3H gene, every the sequence of 21 bases of an initial acquisition of base, table 1 has been listed wherein 31 effective siRNA target sequences for EIF3H gene.

Table 1 target is in the siRNA target sequence of people EIF3H gene

SEQ ID NO	TargetSeq	Initiation site
				1	AAGTGCAGATAGATGGCCTTG	139
2	GATAGATGGCCTTGTGGTATT	146
			3	AGATGGCCTTGTGGTATTAAA	149
4	AACTGAAGTTGTTCAAGGAGT	203
			5	TGTAGAAGATCGGCTTGAAAT	239
6	TAGAAGATCGGCTTGAAATTA	241
			7	AGCCTTCGCCATGTAAACATT	342
8	CATACTATGGCTCATTCGTTA	394
			9	CACTCCTGGACTCTCAGTTTA	421
10	TACCAGCATGCCATTGAAGAA	444
			11	GAAGAATCTGTCGTTCTCATT	459
12	AAGAATCTGTCGTTCTCATTT	460
			13	GAATCTGTCGTTCTCATTTAT	462
14	AGGCATACAGACTGACTCCTA	520
			15	TAAACTGATGGAAGTTTGTAA	539
16	CACCTTTGAGTACATGTTTGA	602
			17	TGAAGAAGTGCCGATTGTAAT	620
18	AAATGAGCCAAGATATAGTTA	781
			19	TACAACACATACATGAGGAAT	804
20	AACACATACATGAGGAATACT	807
			21	TACATGAGGAATACTAGTAAA	813
22	GAGGAATACTAGTAAACAACA	818
			23	AGCAGCAGAAACATCAGTATC	838
24	AGGAGAATATGCAGCGCCAGA	874
			25	TGCAGGCCAGATAAACACTTA	983
26	TGCCAGAACATCAAGGAGTTC	1005
			27	CCCAAGGATCTCTCTCACTAA	499
28	CGCCATGTAAACATTGATCAT	348
			29	GCAACTCTTGGAAGAAATATA	1140
30	GCTGTTGCAGATAAACATGAA	693
			31	GCTTGAAATTACCAACTGCTT	251

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:30 of take is example) containing AgeI and EcoRI restriction enzyme site cohesive end; With AgeI and EcoRI restriction enzyme, act on pGCSILGFP carrier (Shanghai JiKai Gene Chemical Technology Co., Ltd provides, accompanying drawing Fig. 1), make its linearizing, agarose gel electrophoresis is identified endonuclease bamhi.

Table 2 two ends are containing the double-stranded DNA Oligo of AgeI and EcoRI restriction enzyme site cohesive end

5’

Neck

Ring

Neck

3’

SEQ ID

Positive-sense strand

CCGG

GCTGTTGCAGATAAACATGAA

TTCAAGAGA

TTCATGTTTATCTGCAACAGC

TTTTTG

32

Antisense strand

AATTCAAAAA

GCTGTTGCAGATAAACATGAA

TCTCTTGAA

TTCATGTTTATCTGCAACAGC

33

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, wherein upstream primer sequence: 5 '-CCTATTTCCCATGATTCCTTCATA-3 ' (SEQ ID NO:36); Downstream primer sequence: 5 '-GTAATACGGTTATCCACGCG-3 ' (SEQ ID NO:37), 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 expressed rna i for SEQ ID NO:30 successfully constructing, called after pGCSIL-GFP-EIF3H-siRNA.

Build pGCSIL-GFP-Scr-siRNA negative control plasmid, negative control siRNA target sequence is 5 '-TTCTCCGAACGTGTCACGT-3 ' (SEQ ID NO:38).While building pGCSILGFP-Scr-siRNA negative control plasmid, double-stranded DNA Oligo sequence (table 3) for the synthetic two ends of Scr siRNA target spot containing AgeI and EcoRI restriction enzyme site cohesive end, all the other construction processs, authentication method and condition be same pGCSIL-GFP-EIF3H-siRNA all.

Table 3 two ends are containing the double-stranded DNA Oligo of AgeI and EcoRI restriction enzyme site cohesive end

5’

Neck

Ring

Neck

3’

SEQ ID

Positive-sense strand

CCGG

TTCTCCGAACGTGTCACGT

TTCAAGAGA

ACGTGACACGTTCGGAGAA

TTTTTG

34

Antisense strand

AATTCAAAAA

TTCTCCGAACGTGTCACGT

TCTCTTGAA

ACGTGACACGTTCGGAGAA

35

By T4DNA ligase enzyme 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
AgeI(10U/μl)	1.0

EcoRI(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 EIF3H-siRNA slow virus

The DNA that extracts RNAi plasmid pGCSIL-GFP-EIF3H-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 the first chain of the siRNA containing in virus concentrated solution is as shown in SEQ ID NO:43.The wrapping process of contrast slow virus, with EIF3H-siRNA slow virus, only replaces pGCSIL-GFP-EIF3H-siRNA carrier with pGCSIL-GFP-Scr-siRNA carrier.

Embodiment 2 real-time fluorescence quantitative RT-PCR methods detect the silence efficiency of EIF3H gene

MCF-7 Human Breast Cancer Cells in logarithmic phase, lung cancer H1299 cell, colorectal carcinoma RKO cell and glioma U87 cell carry out trysinization, and (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 infect plural number (MOI, MCF-7:20, H1299:, 10, RKO:20, U87:20) value, add the virus of embodiment 1 preparation of sufficient quantity, change substratum after cultivating 24h, 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 EIF3H gene is as follows: upstream primer 5 '-TCAGCCGTGAAGCAAGTG-3 ' (SEQ ID NO:39) and downstream primer 5 '-GAGGGAAAGGAAAGCAGTTG-3 ' (SEQ ID NO:40).Take house-keeping gene GAPDH as internal reference, and primer sequence is as follows: upstream primer 5 '-TGACTTCAACAGCGACACCCA-3 ' (SEQ ID NO:41) and downstream primer 5 '-CACCCTGTTGCTGTAGCCAAA-3 ' (SEQ ID NO:42).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 is calculated the gene expression abundance that has infected EIF3H mRNA.Infect the cell of contrast virus (Lv-Scr-siRNA) in contrast.Experimental result (Fig. 2) shows, in MCF-7 Human Breast Cancer Cells, lung cancer H1299 cell, colorectal carcinoma RKO cell and glioma U87 cell, the expression level of EIF3H mRNA has lowered 91.7%, 98.1%, 76.3% and 91.2%.

Embodiment 3 detects the multiplication capacity of the tumour cell that infects EIF3H-siRNA slow virus

MCF-7 Human Breast Cancer Cells in logarithmic phase, lung cancer H1299 cell, colorectal carcinoma RKO cell and glioma U87 cell carry out trysinization, and (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 infect plural number (MOI, MCF-7:20, H1299:, 10, RKO:20, U87:20), add the virus of sufficient quantity, change substratum after cultivating 24h, 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 shown in Fig. 3-6).

Experimental result shows, slow virus is infected each tumour of group and cultivates after 5 days at cells in vitro, rate of propagation significantly slows down, rate of propagation far below control group tumour cell, vigor cell number has declined respectively 81.6%, 94.7%, 84.6% and 86.4%, shows that EIF3H 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

1. the separated people EIF3H gene purposes in arbitrary anti-tumor medicine of preparation or screening mammary cancer, lung cancer, colorectal carcinoma, glioma.

2. a separated nucleic acid molecule that reduces EIF3H genetic expression in tumour cell, described nucleic acid molecule comprises:

3. the nucleic acid molecule of separation as claimed in claim 2, 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 EIF3H 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 EIF3H gene is basic identical.

4. the nucleic acid molecule of separation as described in claim 2 or 3, is characterized in that, the target sequence of described EIF3H gene contains arbitrary sequence in SEQ ID NO:1-31.

5. the nucleic acid molecule of separation as described in claim 2 or 3, 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:43.

6. the nucleic acid molecule of separation as described in claim 2 or 3, is characterized in that, the sequence of described shRNA is as shown in SEQ ID NO:44.

7. an EIF3H gene interfere RNA construct, the gene fragment that contains the shRNA in nucleic acid molecule separated described in the arbitrary claim of coding claim 2-6, can express described shRNA.

8. EIF3H gene interfere RNA construct as claimed in claim 7, is characterized in that, described EIF3H gene interfere RNA construct is for disturbing lentiviral vectors.

9. EIF3H gene interfere RNA construct as claimed in claim 8, 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.

10. EIF3H gene disturbs a slow virus, by disturbing lentiviral vectors described in the arbitrary claim of claim 8-9 under slow virus packaging plasmid, clone auxiliary, through virus packing, forms.

11. 1 kinds for preventing or treat the pharmaceutical composition of tumour, its active substance contains nucleic acid molecule separated described in the arbitrary claim of claim 2-6, EIF3H gene interfere RNA construct described in the arbitrary claim of claim 7-9, and/or EIF3H gene claimed in claim 10 disturbs slow virus.

The application of pharmaceutical composition in the anti-tumor medicine of preparation treatment mammary cancer, lung cancer, colorectal carcinoma or glioma described in 12. claims 11.

13. 1 kinds of test kits for reducing EIF3H 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 2-6, EIF3H gene interfere RNA construct described in the arbitrary claim of claim 7-9, and/or EIF3H gene claimed in claim 10 disturbs slow virus.