CN104232744A - Application of human CIT gene and EGFR gene in curing tumors and related drugs - Google Patents
Application of human CIT gene and EGFR gene in curing tumors and related drugs Download PDFInfo
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Abstract
The invention relates to the field of biotechnology and specifically discloses a separate human CIT gene and an application of the human CIT gene and a human EGFR gene used as synergistic drug targets in the preparation or screening of drugs for curing tumors. The invention also further constructs a CIT gene small interfering RNA, a CIT gene interfering lentiviral vector and a CIT gene interfering lentivirus and discloses an application of the CIT gene small interfering RNA, the CIT gene interfering lentiviral vector and the CIT gene interfering lentivirus in cooperation with the EGFR gene in curing tumors. The siRNA or the lentiviral vector containing siRNA sequence or the lentivirus containing siRNA sequence provided by the invention can inhibit expression of the human CIT gene and/or the human EGFR gene specifically, especially the lentivirus. The CIT gene interfering lentivirus can inhibit tumor cell growth and promote tumor cell apoptosis separately or in cooperation with the EGFR gene interfering lentivirus, and is of great significance in curing tumors.
Description
Technical field
The present invention relates to biological technical field, relate to independent people CIT gene particularly; And the purposes of people CIT gene and Human epidermal growth factor receptor gene Synergistic treatment tumour and related drugs thereof.
Background technology
The gene therapy of current tumour has achieved and has vigorously developed fast, but up to now, still there is a lot of problem needing to solve.Very little, the gene of energy Tumor suppression growth is few in number, is badly in need of providing more how available gene for the current gene for therapy of tumor.In addition, the process such as generation, development, transfer due to tumour is the complex networks system that a polygene participated in, related to many signal paths, therefore individual gene treatment or single therapy method often offer limited effectiveness.Therefore, the emphasis of following gene therapy development excavates being and identifies the gene having important value clinically, explores multiple gene association with different Antitumor Mechanism and to treat and by polygene targeted drug combination therapy etc.
CIT citron(Rho-interacting, serine/threonine kinase21, serine/threonine kinase 21 interactional with Rho) containing one and ROCK(Rho-associated kinase, Rho connects kinases) similar protein kinase domain, this isomer is called Citron-K, be first known Rho-GTP downstream targets, have the advantages that the dependent cells cycle expresses.Citron-K is gathered in Interphase cells; the mitotic division prometaphase is dispersed in endochylema; later stage is distributed in cell cortex; accumulate in cleavage furrow (Madaule P latter stage; Eda M; Watanabe N, et al.Role of citron kinase as a target of the small GTPase rho in cytokinesis.Nature.1998; 394 (6692): 491-494.Paramasivam M, Chang YJ, Lotrco JJ.ASPM and citron kinase co-localize to midboby ring during cytokinesis.Cell Cycle.2007; 6 (13) 105-1612).Citron kinases adjuster sphaeroprotein in cytokinesis process shrinks near and regulates cell magma motion (Yamashiro S; Totsukawa G; Yamakita Y et al.Citron kinase; a rho-dependent kinase, induces di-phosphorylation of regulatory light chain of myosin II.Mol Biol Cell.2003; 14 (5): 1745-1756.).The varient of process LAN citron gene causes producing syncyte, and the varient losing kinase activity occurs abnormal contraction in cytoplasm moving process.
Studies have reported that; citron gene neurone formed and spermatogenesis part physiological process in play functional effect (Di Cunto F; Imarisio S; Hirsch E, et al.Defective Neurogenesis in Citron Kinase Knockout Mice by Altered Cytokinesis and Massive Apoptosis.Neuron2000; 28 (1): 115-127.Di Cunto F, Imarisio S, Camera P, et al.Essential role of citron kinase in cytokinesis of spermatogenic precursors.J Cell Sci.2002; 115 (Pt24): 4819-4826.); knock out in the birth a few week of the mouse after citron gene and just there will be deformity or death (the Di Cunto F due to ataxia or epilepsy; Imarisio S; Hirsch E, et al.Defective Neurogenesis in Citron Kinase Knockout Mice by Altered Cytokinesis and Massive Apoptosis.Neuron2000; 28 (1): 115 – 127.).Simultaneously; Citron-K is required for not breaking up the expansion of male gamete cell precursors physiology; and guarantee that spermatogonium changes spermatocyte into; Citron gene knockout male mice presents two testicle-shaped infringement; and there is not morphological change (Di Cunto F in the ovary of female mice; Imarisio S, Camera P, et al.Essential role of citron kinase in cytokinesis of spermatogenic precursors.J Cell Sci.2002; 115 (Pt24): 4819-4826.).But also studies have reported that; Citron gene also participates in reproduction process (the Atreya CD of virus; Kulkarni S, Mohan KV.Rubella virus P90associates with the cytokinesis regulatory protein citron-K kinase and the viral infection and constitutive expression of P90protein both induce cell cycle arrest following S phase in cell culture.Arch Virol.2004; 149 (4): 779-789.Atreya CD; Mohan KV, Kulkarni S.Rubella virus and birth defects:molecular insights into the viral teratogenesis at the cellular level.Birth Defects Res A Clin Mol Teratol.2004; 70 (7): 431-437.Loomis RJ, Holmes DA, Elms A et al.Citron kinase, a rho A effector, enhances HIV-1virion production by modulating exocytosis.Traffic.2006; 7 (12): 1643-1653.).Therefore, citron gene plays an important role in a series of important physiological process, the Citron gene dependent cells cycle expresses, important physiological function is played in cell mitogen, and cancerous tissue is cell constantly rises in value formation immortalization, therefore, Citron gene may with tumour there is certain relation, be expected to the novel targets becoming therapy of tumor.
EGF-R ELISA (epidermalgrowth factor receptor; EGFR) EGFR is a kind of transmembrane protein; belong to ErbB receptor family member; its part (EGF, TGF-α etc.) is combined with EGFR extracellular fragment and makes it dimerization; cause the cascade reaction of born of the same parents' inner segment tyrosine kinase activation and a series of signal transduction thus; promote cell proliferation; vasculogenesis; transfer inhibited apoptosis (Liang Houjie, Zou Lan .EGFR targeted drug treatment advanced colorectal cancer latest developments. Chinese prescription drugs 2009; 84:58-61.), thus lead oncogenic generation.Large quantity research finds EGFR gene process LAN or abnormal activation in kinds of tumors tissue, thus tumor cell proliferation, Infiltration and metastasis ability are strengthened.EGFR has become therapy target (Ciardiello F, the Tortora G.EGFR antagonists in cancer treatment.N Engl J Med2008 of the broad variety tumour through clinical verification; 358:1160-1174.).
RNAi is the sequence-specific PTGS phenomenon utilizing double chain RNA mediate; become a kind of emerging effective means of research gene function, and be expected to instrument (the Izquierdo M.Short interfering RNAs a tool for cancer gene therapy.Cancer Gene Ther.2005 becoming the disease gene treatments such as tumour; 12 (3): 217-27.).Slow virus (lentivirus) carrier is efficient gene transfer instrument, is mainly used in infecting the cell lower to ordinary method transfection efficiency, as primary cell etc.Lentiviral particle can the cell of simultaneously infection development and non-proliferative, and infect more stable, lasting.Use lentiviral vectors, the high expression level of specific gene can be realized, also can express hairpin RNA (short hairpin RNA, shRNA) and disturb (RNA interference, RNAi) mode to lower the expression of certain gene with RNA.The present invention intend adopt lentivirus mediated RNAi technology research CIT gene with EGFR gene work in coordination with inhibition tumor cell breed in function, for the No operation clinical treatment of malignant tumour provides theoretical foundation.
Summary of the invention
The object of the invention is to disclose the target of CIT gene as cancer therapy, the purposes for the treatment of tumour and related drugs thereof; And using CIT gene and EGFR gene jointly as the target of cancer therapy, by the expression of reticent CIT gene and EGFR gene simultaneously, realize the purposes of Synergistic treatment tumour, and anti-tumor medicine.
The present invention with RNA interference for means, have studied independent CIT gene to occur and developing effect in tumour, and, CIT gene and EGFR gene occur and developing synergy in tumour, disclose a kind of the suppression or reduction growth of tumour cell, propagation, the method of differentiation and/or survival, the method comprises: use to tumour cell and a kind ofly specificity can suppress transcribing of CIT gene and/or EGFR gene, translation, or specificity can suppress the material of the activity of CIT albumen and/or EGFR protein expression, the growth of inhibition tumor cell is carried out with this, propagation, differentiation or survival.
First aspect present invention, discloses the people CIT gene of separation at preparation or screening anti-tumor medicine, or is preparing the purposes in diagnosing tumor medicine.
Described tumour can be any one tumour that the propagation of its tumour cell is relevant to the expression of people CIT gene; Further, be a kind of malignant tumour, such as, be selected from: colorectal carcinoma.
In the present invention, described people CIT gene is applied to preparation or screening anti-tumor medicine as the action target for tumour cell, or prepares diagnosing tumor medicine.Further, the described action target for tumour cell is RNA interference effect target.
Described by the people CIT gene of separation for the preparation of or screening anti-tumor medicine comprise the content of two aspects: one, to be applied to people CIT gene for the action target of tumour cell as medicine or preparation and to prepare anti-tumor medicine; Its two, people CIT gene is applied to screening anti-tumor medicine as medicine or preparation for the action target of tumour cell.
Described being applied to as medicine or preparation for the action target of tumour cell by CIT gene is prepared anti-tumor medicine and is specifically referred to: using the target of CIT gene as RNA interference effect, develop the medicine for tumour cell or preparation, thus improve or reduce the expression level of CIT gene in tumour cell.
Describedly CIT gene is applied to screening anti-tumor medicine as medicine or preparation for the action target of tumour cell specifically refers to: using CIT gene as effective object, medicine or preparation are screened, can suppress or promote that the medicine of people CIT genetic expression is as oncotherapy drug candidate to find.Namely CIT gene small molecules interference RNA (siRNA) as described in the present invention obtains for effective object screening with people CIT gene, can be used as the medicine with inhibition tumor cell proliferation function.In addition, such as antibody drug, small-molecule drug etc. also can using CIT gene and albumen thereof as effective object.
Described by CIT gene for the preparation of diagnosing tumor medicine, refer to preparation CIT gene expression product being applied to diagnosing tumor medicine as a diagnosing tumor index.
Described anti-tumor medicine is specificity can suppress transcribing or translating of CIT gene, or specificity can suppress the expression of CIT albumen or the molecule of activity, thus the expression level of CIT gene in reduction tumour cell, reach the object of the propagation of inhibition tumor cell, growth, differentiation and/or survival.
First aspect present invention, the people CIT gene and the Human epidermal growth factor receptor gene that also disclose separation are being prepared or screening anti-tumor medicine, or are preparing the purposes in diagnosing tumor medicine.
In the present invention, described people CIT gene and Human epidermal growth factor receptor gene are applied to preparation or screening anti-tumor medicine as the action target for tumour cell, or prepare diagnosing tumor medicine.Further, the described action target for tumour cell is RNA interference effect target.
Described by the people CIT gene of separation and Human epidermal growth factor receptor gene for the preparation of or screening anti-tumor medicine comprise the content of two aspects: one, to be applied to people CIT gene and Human epidermal growth factor receptor gene for the action target of tumour cell as medicine or preparation and to prepare anti-tumor medicine; Its two, people CIT gene and Human epidermal growth factor receptor gene are applied to screening anti-tumor medicine as medicine or preparation for the action target of tumour cell.
Described people CIT gene and Human epidermal growth factor receptor gene being applied to as medicine or preparation for the action target of tumour cell is prepared anti-tumor medicine and is specifically referred to: using CIT gene and Human epidermal growth factor receptor gene jointly as the target of RNA interference effect, development simultaneously for anti-tumor medicine or the preparation of two kinds of genes, thus can improve or reduce the expression level of CIT gene and Human epidermal growth factor receptor gene in tumour cell; Or, using CIT gene and Human epidermal growth factor receptor gene as the target of RNA interference effect, develop respectively for the anti-tumor medicine of two kinds of genes, thus obtain anti-tumor medicine or the preparation that can improve or reduce CIT gene and Human epidermal growth factor receptor gene expression dose in tumour cell.
Describedly people CIT gene and Human epidermal growth factor receptor gene are applied to screening anti-tumor medicine as medicine or preparation for the action target of tumour cell specifically refer to: using people CIT gene and Human epidermal growth factor receptor gene simultaneously as effective object, medicine is screened, the medicine of (suppress or promote) people CIT genetic expression and the medicine of impact (suppress or promote) Human epidermal growth factor receptor genetic expression can be affected respectively, then as oncotherapy alternative compositions medicine to find; Or find the medicine that simultaneously can affect (suppress or promote) people CIT gene and Human epidermal growth factor receptor genetic expression as oncotherapy drug candidate.CIT gene small molecules interference RNA (siRNA) as described in the present invention, Gene interfere nucleic acid construct, slow virus, and EGFR gene small molecules interference RNA (siRNA), Gene interfere nucleic acid construct, slow virus, all with CIT gene and EGFR gene be respectively effective object screening obtain; When they use jointly, there is synergistic effect, better than the result of use of single a kind of material; Can be used as the medicine with inhibition tumor cell proliferation function.In addition, such as antibody drug, small-molecule drug etc. also can using people CIT gene and Human epidermal growth factor receptor gene and albumen thereof as effective object.
Described by people CIT gene and Human epidermal growth factor receptor gene for the preparation of diagnosing tumor medicine, refer to and CIT gene expression product and Human epidermal growth factor receptor gene expression product be applied to the preparation of tumour diagnostic reagent as diagnosing tumor index.
Described anti-tumor medicine of the present invention is specificity can suppress transcribing or translating of people's CIT gene and/or Human epidermal growth factor receptor gene, or specificity can suppress people's CIT gene and/or the expression of Human epidermal growth factor receptor gene or the molecule of activity, thus reduce the expression level of tumour cell people's CIT gene and/or Human epidermal growth factor receptor gene, reach the object of the propagation of inhibition tumor cell, growth, differentiation, survival.
The anti-tumor medicine of the present invention's preparation or screening includes but not limited to: nucleic acid molecule, carbohydrate, lipid, small-molecule chemical medicine (such as inhibitor), antibody medicine, polypeptide, albumen or interference slow virus.
Described nucleic acid molecule 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 reduce transcribing or translating of people CIT gene and Human epidermal growth factor receptor gene, or enough reduces people's CIT albumen and the expression of Human epidermal growth factor receptor albumen or the dosage of activity.50%, 80%, 90%, 95% or 99% is at least lowered to make the expression of people CIT gene and Human epidermal growth factor receptor gene.
In the present invention, when people CIT gene and Human epidermal growth factor receptor gene carry out oncotherapy and drug screening as collaborative dispenser target, be the method disturbed by RNA, using the target gene that people CIT gene and EGFR gene disturb as RAN, reduce the expression level of these two genes.
Described tumour can be any one tumour that the expression of propagation and the people CIT gene of its tumour cell and Human epidermal growth factor receptor gene is relevant; Further, be a kind of malignant tumour, such as, be selected from: colorectal carcinoma.
Adopt the method for forgoing neoplasms medicine treatment tumour to be the object that the propagation of expression level inhibition tumor cell by reducing separately people CIT gene reaches treatment, or reached the object for the treatment of by the propagation of the expression level inhibition tumor cell reducing people CIT gene and Human epidermal growth factor receptor gene simultaneously.Concrete, during treatment, effectively can reduce the material of people CIT gene expression dose; Or, effectively can reduce the administering substances of people CIT gene and Human epidermal growth factor receptor gene expression dose in patient.
Second aspect present invention discloses a kind of nucleic acid molecule medicine for the treatment of tumour, and its effective constituent contains the nucleic acid molecule of the separation reducing CIT genetic expression in tumour cell, and reduces the nucleic acid molecule of the separation that EGFR gene is expressed in tumour cell; In described reduction tumour cell, the nucleic acid molecule of the separation of CIT genetic expression comprises:
A) CIT gene double-stranded RNA, in described CIT gene double-stranded RNA containing can under stringent condition with the nucleotide sequence of CIT gene recombination; Or
B) CIT gene shRNA, in described CIT gene shRNA containing can under stringent condition with the nucleotide sequence of CIT gene recombination;
In described reduction tumour cell, the nucleic acid molecule of the separation that EGFR gene is expressed comprises:
A.EGFR gene double-stranded RNA, containing the nucleotide sequence can hybridized with EGFR gene under stringent condition in described EGFR gene double-stranded RNA; Or
B.EGFR gene shRNA, containing the nucleotide sequence can hybridized with EGFR gene under stringent condition in described EGFR gene shRNA.
Further, described tumour is colorectal carcinoma.
Preferably, described CIT gene double-stranded RNA comprises the first chain and the second chain, described first chain and the complementary common formation RNA dimer of described second chain, and the sequence of described first chain is substantially identical with the target sequence of CIT gene.
Preferably, described EGFR gene double-stranded RNA comprises the first chain and the second chain, described first chain and the complementary common formation RNA dimer of described second chain, and the sequence of described first chain is substantially identical with the target sequence of EGFR gene.
Preferably, described CIT gene shRNA comprises positive-sense strand fragment and antisense strand fragment, and connect the loop-stem structure of described positive-sense strand fragment and antisense strand fragment, the complementary of described positive-sense strand fragment and described antisense strand fragment, and the sequence of described positive-sense strand fragment is substantially identical with the target sequence of CIT gene.
Preferably, described EGFR gene shRNA comprises positive-sense strand fragment and antisense strand fragment, and connect the loop-stem structure of described positive-sense strand fragment and antisense strand fragment, the complementary of described positive-sense strand fragment and described antisense strand fragment, and the sequence of described positive-sense strand fragment is substantially identical with the target sequence of EGFR gene.
More excellent, 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.
More excellent, the target sequence of described CIT gene is sequence shown in SEQ ID NO:1; The target sequence of described EGFR gene is sequence shown in SEQ ID NO:2.
When the target sequence of described CIT gene is siRNA for the CIT genetic expression of specificity silence, the fragment in the CIT gene corresponding to the mRNA fragment combined with described siRNA complementation.In like manner, when the target sequence of described EGFR gene is siRNA for the EGFR gene expression of specificity silence, the fragment in the EGFR gene corresponding to the mRNA fragment combined with described siRNA complementation.
The length of described double-stranded RNA 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).
Optimum, the sequence of CIT gene siRNA first chain, as shown in SEQ ID NO:9, is specially 5 '-GUCCUCAUACCAGGAUAAA-3 '.First chain of the CIT gene siRNA shown in SEQ ID NO:9 for the sequence shown in SEQ ID NO:1 be RNA disturb target sequence design for a chain in the siRNA of people CIT gene, the siRNA that this first chain and the second chain form can play the effect of endogenous CIT genetic expression in specificity silence tumour cell.
Optimum, the sequence of EGFR gene siRNA first chain, as shown in SEQ ID NO:10, is specially 5 '-GGCUGGUUAUGUCCUCAUU-3 '.First chain of the EGFR gene siRNA shown in SEQ ID NO:10 for the sequence shown in SEQ ID NO:2 be RNA disturb target sequence design for a chain in the siRNA of Human epidermal growth factor receptor gene, the siRNA that this first chain and the second chain form can play the effect that in specificity silence tumour cell, endogenous EGFR gene is expressed.
Optimum, the sequence of described CIT gene shRNA, as shown in SEQ ID NO:11, is specially: 5 '-GCGUCCUCAUACCAGGAUAAA CUCGAG UUUAUCCUGGUAUGAGGACGC-3 '.
Optimum, the sequence of described EGFR gene shRNA, as shown in SEQ ID NO:12, is specially: 5 '-GUGGCUGGUUAUGUCCUCAUUCUCGAG AAUGAGGACAUAACCAGCCAC-3 '.
When being used as the medicine for the treatment of tumour, be that the double-stranded RNA of safe and effective amount or shRNA are applied to Mammals.Concrete dosage also should consider the factor such as route of administration, patient health situation, and these are all within skilled practitioners skill.
Third aspect present invention, disclose a kind of lentiviral vectors medicine for the treatment of tumour, its effective constituent contains CIT Gene interfere lentiviral vectors and EGFR gene interference lentiviral vectors, described CIT Gene interfere lentiviral vectors contains the gene fragment of the aforementioned CIT gene shRNA that encodes, and described EGFR gene interference lentiviral vectors contains the gene fragment of the aforementioned EGFR gene shRNA that encodes.
Further, described tumour is colorectal carcinoma.
Described CIT Gene interfere lentiviral vectors the DNA fragmentation of the aforementioned CIT gene shRNA of coding is cloned into known carrier obtain, described known carrier mostly is lentiviral vectors, described CIT Gene interfere lentiviral vectors is after virus packaging becomes infectious virion, infected tumor's cell, and then transcribe out described shRNA, the steps such as processing are cut, the described CIT gene siRNA of final acquisition, for the expression of the reticent CIT gene of specificity by enzyme.EGFR gene interference lentiviral vectors is with CIT gene.
The DNA sequence dna of described CIT gene shRNA gene fragment of encoding contains sequence and complementary sequence thereof shown in SEQ ID NO:1.The DNA sequence dna of described EGFR gene shRNA gene fragment of encoding contains sequence and complementary sequence thereof shown in SEQ ID NO:2.
Further, the nucleotide sequence of Gene interfere lentiviral vectors of the present invention also containing the marker that can be detected in promoter sequence and/or codes for tumor cell; Preferably, the described marker 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.
Fourth aspect present invention, disclose a kind of slow virus medicine for the treatment of tumour, its effective constituent contains CIT Gene interfere slow virus and EGFR gene interference slow virus, described CIT Gene interfere slow virus by aforementioned CIT Gene interfere lentiviral vectors slow virus packaging plasmid, clone auxiliary under, form through virus packaging; Described EGFR gene interference slow virus by aforementioned EGFR gene disturb lentiviral vectors slow virus packaging plasmid, clone auxiliary under, form through virus packaging.
Further, described tumour is colorectal carcinoma.
Slow virus of the present invention can infected tumor's cell producing respectively for the small molecules interference RNA of CIT gene or EGFR gene, thus the propagation of inhibition tumor cell.This CIT Gene interfere slow virus and EGFR gene interference slow virus can be used for the medicine preparing prevention or treatment tumour.
Fifth aspect present invention, disclose a kind of pharmaceutical composition being used for the treatment of colorectal carcinoma, at least one in the nucleic acid molecule of its separation of composition containing CIT genetic expression in aforementioned reduction tumour cell, CIT Gene interfere lentiviral vectors, CIT Gene interfere slow virus; And the nucleic acid molecule of the separation that EGFR gene is expressed in aforementioned reduction tumour cell, EGFR gene interference lentiviral vectors, EGFR gene disturb at least one in slow virus.
Preferably, mentioned component is the effective component of the pharmaceutical composition for the treatment of colorectal carcinoma.
When being used as the medicine for the treatment of tumour, be that the double-stranded RNA of safe and effective amount, shRNA or slow virus are applied to Mammals.Concrete dosage also should consider the factor such as route of administration, patient health situation, and these are all within skilled practitioners skill.
In pharmaceutical composition of the present invention, the reticent CIT genetic expression of specificity, and the effective constituent that the reticent EGFR gene of specificity is expressed has played the effect of Synergistic treatment; The embodiment of the present invention is recorded, dual-gene strike the growth of cancer cells inhibiting rate after subtracting obviously be greater than single-gene strike subtract group add and, show that material that the material of the reticent CIT genetic expression of specificity and specificity silence EGFR gene are expressed works in coordination with the propagation of inhibition tumor cell.
Further, medicine of the present invention or pharmaceutical composition contain siRNA described in 1 ~ 99wt%, shRNA, Gene interfere nucleic acid construct and/or Gene interfere slow virus, and pharmaceutically acceptable carrier, thinner or vehicle.
When preparing medicine, usually by effective constituent and mixed with excipients, or with vehicle dilution, or wrap in can in the carrier that exists of capsule or sachet.When vehicle plays thinner effect, it can be solid, semisolid or the fluent material medium as 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.
Beneficial effect of the present invention is: siRNA provided by the invention or comprise the nucleic acid construct of this sequences of small interfering RNAs, slow virus specificity can suppress the expression of people CIT gene, especially slow virus, can be separately, or work in coordination with the growth of inhibition tumor cell with EGFR target slow virus, promote apoptosis of tumor cells, significant in oncotherapy.CIT gene in the present invention namely can as independent oncotherapy target, also can as EGFR gene Synergistic treatment target, significant in oncotherapy.
Accompanying drawing explanation
Fig. 1: GV115 Plasmid diagram
Fig. 2: GV113 Plasmid diagram
Fig. 3: CIT shRNA plasmid identification figure
1#: negative control (ddH2O); 2#: negative control (unloaded from connecting control group)
3#:Marker is followed successively by 5kb from top to bottom, 3kb, 2kb, 1.5kb, 1Kb, 750bp, 500bp, 250bp, 100bp
4# ~ 8#:CIT shRNA1-5 transformant is identified
Fig. 4: EGFR shRNA plasmid identification figure
1#: negative control (ddH2O)
2#: negative control (unloaded from connecting control group)
3#:Marker is followed successively by 5kb from top to bottom, 3kb, 2kb, 1.5kb, 1Kb, 750bp, 500bp, 250bp, 100bp
4# ~ 8#:EGFR shRNA1-5 transformant is identified
Fig. 5: CIT shRNA slow virus and EGFR shRNA slow virus contaminated human colon carcinoma RKO cell after 5 days simultaneously, remarkable antiproliferative effect
Embodiment
The present invention is set forth further below in conjunction with embodiment.Should be understood that embodiment only for illustration of the present invention, but not limit the scope of the invention.In embodiment, the experimental technique of unreceipted actual conditions and the reagent of undeclared formula are conveniently condition, as works such as ［ beautiful ］ Sambrook.J; Huang Peitang etc. translate.Molecular cloning texts 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 CIT gene and EGFR gene RNAi slow virus
1. lentiviral vectors builds
1) lentiviral vectors for people CIT gene and EGFR gene is built
CIT(NM_007174 is transferred from Genbank) and EGFR gene (NM_201282) information; The design software Genechem of Shanghai JiKai Gene Chemical Technology Co., Ltd is utilized to design effective siRNA target spot for CIT gene and EGFR gene.Preliminary screening obtains having the siRNA target sequence (SEQ ID NO.1) obviously suppressing CIT genetic expression function and the siRNA target sequence (SEQ ID NO.2) obviously suppressing EGFR gene expressive function.Carry out BLAST analysis by Genbank database, determine that it does not produce interference effect to other gene any.
Table 1siRNA target sequence
| SEQ?ID?NO. | TargetSeq | Initiation site | Target gene |
| 1 | GTCCTCATACCAGGATAAA | 5709 | CIT |
| 2 | GGCTGGTTATGTCCTCATT | 499 | EGFR |
Double-stranded DNA Oligo sequence (table 2) of two ends containing Age I and EcoR I restriction enzyme site cohesive end is synthesized for siRNA target spot (SEQ ID NO:1); Act on GV115 carrier (belt carrier green fluorescent label, Shanghai JiKai Gene Chemical Technology Co., Ltd provides, Fig. 1) with Age I and EcoR I restriction enzyme, make its linearizing.
Double-stranded DNA Oligo sequence (table 3) of two ends containing Age I and EcoR I restriction enzyme site cohesive end is synthesized for siRNA target spot (SEQ ID NO:2).Act on GV113 carrier (belt carrier red fluorescence marks, and Shanghai JiKai Gene Chemical Technology Co., Ltd provides, Fig. 2) with Age I and EcoR I restriction enzyme, make its linearizing.
Table 2 two ends contain the double-stranded DNA Oligo of Age I and EcoR I restriction enzyme site cohesive end
Table 3 two ends contain the double-stranded DNA Oligo of Age I and EcoR I restriction enzyme site cohesive end
By T4DNA ligase enzyme, by double digestion linearizing, (it is as shown in table 4 that enzyme cuts system, 37 DEG C, reaction 1h) the carrier DNA double-stranded DNA Oligo good with purifying is connected, spend the night in 16 DEG C of connections in suitable buffer system (linked system is as shown in table 5), reclaim connection product.By fresh competent escherichia coli cell (conversion operation reference: Molecular Cloning: A Laboratory guide second edition 55-56 page) prepared by connection product conversion calcium chloride.
Growing bacterium clone surface at connection converted product is stained with, and be dissolved in 10 μ l LB substratum, mixing gets 1 μ l as template; The upstream and downstream of RNAi sequence, designs general PCR primer upstream primer sequence: 5 '-CCTATTTCCCATGATTCCTTCATA-3 ' (SEQ ID NO:7) in lentiviral vectors; Downstream primer sequence: 5 '-GTAATACGGTTATCCACGCG-3 ' (SEQ ID NO:8), carries out PCR identification experiment (, as table 6, reaction conditions is as table 7 for PCR reaction system).The clone positive to PCR qualification checks order and compare of analysis, and PCR qualification result is shown in Fig. 3 and Fig. 4.The clone that comparison is correct is the RNAi carrier containing SEQ ID NO:1 or SEQ ID NO:2 successfully constructed, respectively called after CIT-shRNA-plasmid and EGFR-shRNA-plasmid.
2) GV115 negative control plasmids is built
Negative control siRNA target sequence is 5 '-TTCTCCGAACGTGTCACGT-3 ' (SEQ ID NO:13).When building negative control plasmids, contain double-stranded DNA Oligo sequence (table 8) of Age I and EcoR I restriction enzyme site cohesive end for Scr siRNA target spot synthesis two ends, all the other construction processs, authentication method and condition are all with CIT-shRNA-plasmid and EGFR-shRNA-plasmid.
Table 4 vector plasmid endonuclease reaction system
| Reagent | Volume (μ l) |
| Vector 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 DNA Oligo ligation system
| Reagent | Positive control (μ l) | From connecting contrast (μ l) | Connecting groups (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 6PCR 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 7PCR reaction system program setting
Table 8 two ends contain the double-stranded DNA Oligo of Age I and EcoR I restriction enzyme site cohesive end
| SEQ?ID | ? | 5’ | Neck | Ring | Neck | 3’ |
| 14 | Positive-sense strand | CCGG | TTCTCCGAACGTGTCACGT | CTCGAG | ACGTGACACGTTCGGAGAA | TTTTTG |
| 15 | Antisense strand | AATTCAAAAA | TTCTCCGAACGTGTCACGT | CTCGAG | ACGTGACACGTTCGGAGAA | ? |
2. packaging obtains CIT Gene interfere slow virus and EGFR gene interference slow virus
1) CIT Gene interfere slow virus
Extract RNAi plasmid CIT-shRNA-plasmid with the plasmid extraction test kit of Qiagen company and be mixed with 100ng/ μ l storage liquid.24h before transfection, with the HEKC 293T cell of tryptic digestion logarithmic phase, with the DMEM perfect medium adjustment cell density containing 10% foetal calf serum for 1.5 × 10
5cell/ml, is inoculated in 6 orifice plates, 37 DEG C, 5%CO
2cultivate in incubator.Transfection is can be used for when cell density reaches 70%-80%.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, Packing Mix(PVM is added in a sterile centrifugation tube) 20 μ l, PEI12 μ l, plasma-free DMEM 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 solution.Above-mentioned transfection miscellany is at room temperature hatched 15min, is transferred in the substratum of HEKC 293T cell, 37 DEG C, 5%CO
216h is cultivated in incubator.Discard the developing medium containing transfection miscellany, PBS solution is washed, and adds perfect medium 2ml, continues to cultivate 48h.
Collecting cell supernatant liquor, Centricon Plus-20 centrifugal ultrafiltration unit (Millipore) purifying and concentrated slow virus, step is as follows: (1) 4 DEG C, the centrifugal 10min of 4000g, removing cell debris; (2) 0.45 μm of frit supernatant liquors are in 40ml ultracentrifugation pipe; (3) 4000g is centrifugal, 10-15min, to the viral concentration volume needed; (4), after centrifugal end, filtering cup and filtered solution collection cups are below separated, tipped upside down on by filtering cup 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 CIT Gene interfere slow virus concentrated solution.By after the packing of viral concentration liquid in-80 degrees Celsius of preservations.
2) wrapping process of EGFR gene interference slow virus is with CIT Gene interfere slow virus.
3) negative control slow virus wrapping process is with CIT Gene interfere slow virus.
The oncotherapy synergy of embodiment 2CIT Gene interfere slow virus and EGFR gene interference slow virus
1. experimental technique
The human colon carcinoma RKO cell being in logarithmic phase carries 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 about 30%.According to infection multiplicity (MOI of RKO is 5), experimental group and control group are set simultaneously, experimental group with the titre of 0.03uL for 5 × 10
8the CIT Gene interfere slow virus of TU/mL and the titre of 0.03uL are 5 × 10
8tU/mL EGFR gene interference slow virus join in cancer cells nutrient solution, control group with the titre of 0.03uL for 5 × 10
8the contrast virus of TU/mL joins in cancer cells nutrient solution.
Replaced medium after cultivation 24h, after time of infection reaches 5 days, collects each experimental group and the cellular control unit that are in logarithmic phase.The resuspended one-tenth cell suspension (2 × 10 of perfect medium
4/ ml), be about 2000/hole with cell density, inoculate 96 orifice plates.Often organize 5 multiple holes, every hole 100 μ l.After completing plate, put 37 DEG C, 5%CO
2incubator is cultivated.From after bed board second day, every day was detected with Cellomics ArrayScan VTI High content screening analyser (Thermo Fisher) and reads plate once, and continuous detecting reads plate 4 days.By the input parameter of adjustment Cellomics arrayscan, calculate in each experimental group and control group the quantity (simultaneously infecting the cell of CIT Gene interfere slow virus and EGFR gene interference slow virus) of the cell of band green fluorescence and red fluorescence while at every turn scanning in orifice plate exactly, the quantity (only infecting the cell of CIT Gene interfere slow virus) of independent band green cells, the quantity (only infecting the cell of EGFR gene interference slow virus) of the independent cell of fluorescence redly, carry out statistics to data to draw, draw cell proliferation curve.
Cell proliferation curve the results are shown in Figure 5, result shows: after the human colon carcinoma RKO cell simultaneously infecing CIT shRNA slow virus and EGFR shRNA slow virus cultivates 4 days in vitro, vigor cell proliferation times have dropped 68.23% compared with control group, after the RKO cell infecing separately EGFR shRNA slow virus cultivates 4 days in vitro, vigor cell proliferation times have dropped 11.28% compared with control group, after the RKO cell infecing separately CIT shRNA slow virus cultivates 4 days in vitro, vigor cell proliferation times have dropped 45.94% compared with control group., this experimental result shows, dual-gene strike the inhibitory rate of cell growth after subtracting obviously be greater than single-gene strike subtract group add and, show that CIT gene shRNA slow virus can work in coordination with the propagation of inhibition tumor cell with EGFR gene shRNA slow virus.
Claims (11)
1. the people CIT gene be separated at preparation or screening anti-tumor medicine, or is preparing the purposes in diagnosing tumor medicine.
2. purposes as claimed in claim 1, is characterized in that, described people CIT gene is applied to preparation or screening anti-tumor medicine as the action target for tumour cell, or prepares diagnosing tumor medicine.
3. purposes as claimed in claim 2, it is characterized in that, the described action target for tumour cell is RNA interference effect target.
4. the people CIT gene be separated and Human epidermal growth factor receptor gene at preparation or screening anti-tumor medicine, or are preparing the purposes in diagnosing tumor medicine.
5. treat a nucleic acid molecule medicine for tumour, its effective constituent contains the nucleic acid molecule of the separation reducing CIT genetic expression in tumour cell, and reduces the nucleic acid molecule of the separation that EGFR gene is expressed in tumour cell;
In described reduction tumour cell, the nucleic acid molecule of the separation of CIT genetic expression comprises:
A) CIT gene double-stranded RNA, in described CIT gene double-stranded RNA containing can under stringent condition with the nucleotide sequence of CIT gene recombination; Or
B) CIT gene shRNA, in described CIT gene shRNA containing can under stringent condition with the nucleotide sequence of CIT gene recombination;
In described reduction tumour cell, the nucleic acid molecule of the separation that EGFR gene is expressed comprises:
A.EGFR gene double-stranded RNA, containing the nucleotide sequence can hybridized with EGFR gene under stringent condition in described EGFR gene double-stranded RNA; Or
B.EGFR gene shRNA, containing the nucleotide sequence can hybridized with EGFR gene under stringent condition in described EGFR gene shRNA.
6. nucleic acid molecule medicine as claimed in claim 5, it is characterized in that, described CIT gene double-stranded RNA comprises the first chain and the second chain, described first chain and the complementary common formation RNA dimer of described second chain, and the sequence of described first chain is substantially identical with the target sequence of CIT gene; Described CIT gene shRNA comprises positive-sense strand fragment and antisense strand fragment, and connect the loop-stem structure of described positive-sense strand fragment and antisense strand fragment, the complementary of described positive-sense strand fragment and described antisense strand fragment, and the sequence of described positive-sense strand fragment is substantially identical with the target sequence of CIT gene; Described EGFR gene double-stranded RNA comprises the first chain and the second chain, described first chain and the complementary common formation RNA dimer of described second chain, and the sequence of described first chain is substantially identical with the target sequence of EGFR gene; Described EGFR gene shRNA comprises positive-sense strand fragment and antisense strand fragment, and connect the loop-stem structure of described positive-sense strand fragment and antisense strand fragment, the complementary of described positive-sense strand fragment and described antisense strand fragment, and the sequence of described positive-sense strand fragment is substantially identical with the target sequence of EGFR gene.
7. nucleic acid molecule medicine as claimed in claim 6, it is characterized in that, the target sequence of described CIT gene, is sequence shown in SEQ ID NO:1; The target sequence of described EGFR gene is sequence shown in SEQ ID NO:2.
8. the nucleic acid molecule medicine as described in claim as arbitrary in claim 5-7, is characterized in that, described double-stranded RNA is siRNA, and the sequence of CIT gene siRNA first chain is as shown in SEQ ID NO:9; The sequence of EGFR gene siRNA first chain is as shown in SEQ ID NO:10; The sequence of described CIT gene shRNA is as shown in SEQ ID NO:11; The sequence of described EGFR gene shRNA is as shown in SEQ ID NO:12.
9. treat the lentiviral vectors medicine of tumour for one kind, its effective constituent contains CIT Gene interfere lentiviral vectors and EGFR gene interference lentiviral vectors, and described CIT Gene interfere lentiviral vectors contains the gene fragment of the CIT gene shRNA in nucleic acid molecule medicine described in the arbitrary claim of coding claim 5-8; Described EGFR gene interference lentiviral vectors contains the gene fragment of the EGFR gene shRNA in nucleic acid molecule medicine described in the arbitrary claim of coding claim 5-8.
10. treat the slow virus medicine of tumour for one kind, its effective constituent contains CIT Gene interfere slow virus and EGFR gene interference slow virus, described CIT Gene interfere slow virus by the CIT Gene interfere lentiviral vectors in lentiviral vectors medicine described in claim 9 slow virus packaging plasmid, clone auxiliary under, form through virus packaging; Described EGFR gene interference slow virus by the EGFR gene in lentiviral vectors medicine described in claim 9 disturb lentiviral vectors slow virus packaging plasmid, clone auxiliary under, form through virus packaging.
11. 1 kinds of pharmaceutical compositions for the treatment of colorectal carcinoma, at least one in the CIT Gene interfere slow virus in slow virus medicine described in the CIT Gene interfere lentiviral vectors in lentiviral vectors medicine described in the nucleic acid molecule of its composition containing the separation of CIT genetic expression in the reduction tumour cell in nucleic acid molecule medicine described in the arbitrary claim of claim 5-8, claim 9, claim 10; And the nucleic acid molecule of the separation that EGFR gene is expressed in reduction tumour cell in nucleic acid molecule medicine described in the arbitrary claim of claim 5-8, EGFR gene interference lentiviral vectors in lentiviral vectors medicine described in claim 9, at least one in EGFR gene interference slow virus in slow virus medicine described in claim 10.
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| WO2003083096A2 (en) * | 2002-03-28 | 2003-10-09 | Qlt Inc. | Cancer associated protein kinases and their uses |
| US20030220224A1 (en) * | 2002-04-12 | 2003-11-27 | Davison Daniel B. | Novel polynucleotides encoding the human citron kinase polypeptide, BMSNKC_0020/0021 |
| WO2006040047A2 (en) * | 2004-10-15 | 2006-04-20 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with rho/rac-interacting citron kinase (cit) |
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|---|---|---|---|---|
| WO2003083096A2 (en) * | 2002-03-28 | 2003-10-09 | Qlt Inc. | Cancer associated protein kinases and their uses |
| US20030220224A1 (en) * | 2002-04-12 | 2003-11-27 | Davison Daniel B. | Novel polynucleotides encoding the human citron kinase polypeptide, BMSNKC_0020/0021 |
| WO2006040047A2 (en) * | 2004-10-15 | 2006-04-20 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with rho/rac-interacting citron kinase (cit) |
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| YINGHUI FU ETAL: "RNA interference targeting CITRON can significantly inhibit the proliferation of hepatocellular carcinoma cells", 《MOLECULAR BIOLOGY REPORT》 * |
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