CN104225619A - Application of human ILK gene in curing tumors and related drugs - Google Patents
Application of human ILK gene in curing tumors and related drugs Download PDFInfo
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- CN104225619A CN104225619A CN201310232216.6A CN201310232216A CN104225619A CN 104225619 A CN104225619 A CN 104225619A CN 201310232216 A CN201310232216 A CN 201310232216A CN 104225619 A CN104225619 A CN 104225619A
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Abstract
The invention relates to the field of biotechnology and specifically discloses a separate human ILK gene and an application of the human ILK 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 an ILK gene small interfering RNA, an ILK gene interfering lentiviral vector and an ILK gene interfering lentivirus and discloses an application of the ILK gene small interfering RNA, the ILK gene interfering lentiviral vector and the ILK 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 ILK gene and/or the human EGFR gene specifically, especially the lentivirus. The ILK 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 ILK gene particularly; And the purposes of people ILK gene and Human epidermal growth factor receptor gene Synergistic treatment tumor and related drugs thereof.
Background technology
The gene therapy of current tumor 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 tumor is the complex networks system that a polygenes 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 polygenes targeted drug therapeutic alliance etc.
Role of integrin-linking kinase (integrin-linked kinase, ILK) is a kind of relative molecular mass is 59 × 10
3, non-receptor Ser/Thr protein kinase; with relevant (the Duxbury MS of multi-signal transduction in cell; Ito H; Benoit E, et al.RNA interference demonstrates a novel role for integrin-linked kinase as a determinant of pancreatic adenocarcinoma cell gemcitabine chemoresistance.Clin Cancer Res.2005May1; 11 (9): 3433-8.).There are some researches show: ILK has the function of the intracellular signaling in mediated integration element born of the same parents, its energy coupling integrin and growth factor receptors, the transmembrane signal participating in mediated cell and basement membrane conducts, intracellular signaling, Wnt signal path and integral protein that regulation and control somatomedin mediates, regulate the adhesion of cell.Growing multiplication, the fibrosis etc. of breaking up, stick together and attacking and organizing.Experiment in vitro shows; formation, the apoptosis of ILK and many tumors, stick, breed, closely related (Persad S, Dedhar S.The role of integrin-linked kinase (ILK) the in cancer progression.Cancer Metastasis Rev.2003Dec of Infiltration and metastasis; 22 (4): 375-84.).
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; angiogenesis; 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 polytype tumor 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 tumor; 12 (3): 217-27.).Slow virus (lentivirus) carrier is efficient gene transfer instrument, is mainly used in infecting the cell lower to conventional 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 slow virus carrier, the high expressed 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 ILK gene with EGFR gene work in coordination with inhibition tumor cell breed in function, for the No operation clinical treatment of malignant tumor provides theoretical foundation.
Summary of the invention
The object of the invention is to disclose the target of ILK gene as treatment of cancer, the purposes for the treatment of tumor and related drugs thereof; And using ILK gene and EGFR gene jointly as the target for the treatment of of cancer, by the expression of reticent ILK gene and EGFR gene simultaneously, realize the purposes of Synergistic treatment tumor, and anti-tumor medicine.
The present invention with RNA interference for hands section, have studied independent ILK gene to occur and developing effect in tumor, and, ILK gene and EGFR gene occur and developing synergism in tumor, disclose a kind of the suppression or reduction growth of tumour cell, propagation, the method of differentiation and/or survival, the method comprises: to tumor cell use a kind of can the transcribing of specific inhibition of IL-6 K gene and/or EGFR gene, translation, or can the material of activity of specific inhibition of IL-6 K 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 ILK gene of separation at preparation or screening anti-tumor medicine, or is preparing the purposes in diagnosing tumor medicine.
In the present invention, described people ILK gene is applied to preparation or screening anti-tumor medicine as the action target for tumor cell, or prepares diagnosing tumor medicine.Further, the described action target for tumor cell is RNA interference effect target.
Described by the people ILK gene of separation for the preparation of or screening anti-tumor medicine comprise the content of two aspects: one, to be applied to people ILK gene for the action target of tumor cell as medicine or preparation and to prepare anti-tumor medicine; Its two, people ILK gene is applied to screening anti-tumor medicine as medicine or preparation for the action target of tumor cell.
Described being applied to as medicine or preparation for the action target of tumor cell by ILK gene is prepared anti-tumor medicine and is specifically referred to: using the target of ILK gene as RNA interference effect, develop the medicine for tumor cell or preparation, thus improve or reduce the expression of ILK gene in tumor cell.
Describedly ILK gene is applied to screening anti-tumor medicine as medicine or preparation for the action target of tumor cell specifically refers to: using ILK gene as effective object, medicine or preparation are screened, can suppress or promote that the medicine of people ILK gene expression is as oncotherapy drug candidate to find.Namely ILK gene small molecules interference RNA (siRNA) as described in the present invention obtains for effective object screening with people ILK 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 ILK gene and albumen thereof as effective object.
Described by ILK gene for the preparation of diagnosing tumor medicine, refer to preparation ILK gene expression product being applied to diagnosing tumor medicine as a diagnosing tumor index.
Described anti-tumor medicine is can the transcribing or translating of specific inhibition of IL-6 K gene, or can the expression of specific inhibition of IL-6 K albumen or the molecule of activity, thus the expression of ILK gene in reduction tumor cell, reach the object of the propagation of inhibition tumor cell, growth, differentiation and/or survival.
Described tumor can be any one tumor that the propagation of its tumor cell is relevant to the expression of people ILK gene; Further, be a kind of malignant tumor, such as, be selected from: pulmonary carcinoma or colon cancer.
First aspect present invention, the people ILK 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 ILK gene and Human epidermal growth factor receptor gene are applied to preparation or screening anti-tumor medicine as the action target for tumor cell, or prepare diagnosing tumor medicine.Further, the described action target for tumor cell is RNA interference effect target.
Described by the people ILK 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 ILK gene and Human epidermal growth factor receptor gene for the action target of tumor cell as medicine or preparation and to prepare anti-tumor medicine; Its two, people ILK gene and Human epidermal growth factor receptor gene are applied to screening anti-tumor medicine as medicine or preparation for the action target of tumor cell.
Described people ILK gene and Human epidermal growth factor receptor gene being applied to as medicine or preparation for the action target of tumor cell is prepared anti-tumor medicine and is specifically referred to: using ILK 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 of ILK gene and Human epidermal growth factor receptor gene in tumor cell; Or, using ILK 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 ILK gene and Human epidermal growth factor receptor gene expression dose in tumor cell.
Describedly people ILK gene and Human epidermal growth factor receptor gene are applied to screening anti-tumor medicine as medicine or preparation for the action target of tumor cell specifically refer to: using people ILK gene and Human epidermal growth factor receptor gene simultaneously as effective object, medicine is screened, the medicine of (suppress or promote) people ILK gene expression and the medicine of impact (suppress or promote) Human epidermal growth factor receptor gene 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 ILK gene and Human epidermal growth factor receptor gene expression as oncotherapy drug candidate.ILK 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 ILK 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 ILK gene and Human epidermal growth factor receptor gene and albumen thereof as effective object.
Described by people ILK gene and Human epidermal growth factor receptor gene for the preparation of diagnosing tumor medicine, refer to and ILK 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 can suppress transcribing or translating of people's ILK gene and/or Human epidermal growth factor receptor gene by specificity, or people's ILK gene and/or the expression of Human epidermal growth factor receptor gene or the molecule of activity can be suppressed by specificity, thus reduce the expression of tumor cell people's ILK 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 molecules, carbohydrate, lipid, small-molecule chemical medicine (such as inhibitor), antibody medicine, polypeptide, albumen or interference slow virus.
Described nucleic acid molecules 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 ILK gene and Human epidermal growth factor receptor gene, or enough reduces people's ILK 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 ILK gene and Human epidermal growth factor receptor gene.
In the present invention, when people ILK 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 ILK gene and EGFR gene disturb as RAN, reduce the expression of these two genes.
Described tumor can be any one tumor that the expression of propagation and the people ILK gene of its tumor cell and Human epidermal growth factor receptor gene is relevant; Further, be a kind of malignant tumor, such as, be selected from: pulmonary carcinoma or colon cancer.
Adopt the method for forgoing neoplasms medicine treatment tumor to be the object that the propagation of expression inhibition tumor cell by reducing separately people ILK gene reaches treatment, or reached the object for the treatment of by the propagation of the expression inhibition tumor cell reducing people ILK gene and Human epidermal growth factor receptor gene simultaneously.Concrete, during treatment, effectively can reduce the material of people ILK gene expression dose; Or, effectively can reduce the administering substances of people ILK gene and Human epidermal growth factor receptor gene expression dose in patient.
Second aspect present invention discloses a kind of nucleic acid molecules reducing the separation of ILK gene expression in tumor cell, and described nucleic acid molecules comprises:
B) ILK gene double-stranded RNA, in described ILK gene double-stranded RNA containing can under stringent condition with the nucleotide sequence of ILK gene recombination; Or
C) ILK gene shRNA, in described ILK gene shRNA containing can under stringent condition with the nucleotide sequence of ILK gene recombination.
Preferably, described ILK 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 ILK gene.
Preferably, described ILK 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 ILK 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 ILK gene is sequence shown in SEQ ID NO:1.
When the target sequence of described ILK gene is siRNA for the ILK gene expression of specificity silence, the fragment in the ILK 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 ILK gene siRNA first chain, as shown in SEQ ID NO:9, is specially 5 '-CGAAGCUCAACGAGAAUCA-3 '.First chain of the ILK 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 ILK gene, the siRNA that this first chain and the second chain form can play the effect of endogenous ILK gene expression in specificity silence tumor cell.
Optimum, the sequence of described ILK gene shRNA, as shown in SEQ ID NO:11, is specially: 5 '-gaCGAAGCUCAACGAGAAUCA CUCGAG UGAUUCUCGUUGAGCUUCGUC-3 '.
Third aspect present invention, discloses a kind of ILK Gene interfere nucleic acid construct, and the genetic fragment of the ILK gene shRNA in the nucleic acid molecules containing the aforementioned separation of coding, can express described ILK gene shRNA.
More excellent, described ILK Gene interfere nucleic acid construct is ILK Gene interfere slow virus carrier.
Described ILK Gene interfere slow virus carrier the DNA fragmentation of the aforementioned ILK gene shRNA of coding is cloned into known carrier obtain, described known carrier mostly is slow virus carrier, described ILK Gene interfere slow virus carrier is after virus packaging becomes infectious virion, infected tumor's cell, and then transcribe out described shRNA, by steps such as enzyme action processing, the described ILK gene siRNA of final acquisition, for the expression of the reticent ILK gene of specificity.
The DNA sequence of described ILK gene shRNA genetic fragment of encoding contains sequence and complementary series thereof shown in SEQ ID NO:1.
Further, the nucleotide sequence of described ILK Gene interfere slow virus carrier also containing the label that can be detected in promoter sequence and/or codes for tumor cell; Preferably, the described label be detected is as green fluorescent protein (GFP).
Further, described slow virus carrier can be selected from: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti6-GW/U6-laminshrna, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, arbitrary in pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.
Fourth aspect present invention, discloses a kind of ILK Gene interfere slow virus, by the ILK Gene interfere slow virus carrier in aforementioned ILK Gene interfere nucleic acid construct slow virus packaging plasmid, cell line auxiliary under, form through virus packaging.
Slow virus medicine of the present invention can infected tumor's cell producing for the small molecules interference RNA of ILK gene, thus the propagation of inhibition tumor cell.This ILK Gene interfere slow virus can be used for the medicine preparing prevention or treatment tumor.
Fifth aspect present invention, disclose a kind of pharmaceutical composition being used for the treatment of pulmonary carcinoma or colon cancer, its active substance contains at least one in the nucleic acid molecules of the separation of ILK gene expression in aforementioned reduction tumor cell, ILK Gene interfere nucleic acid construct, ILK Gene interfere slow virus.
Preferably, the pharmaceutical composition of described treatment pulmonary carcinoma or colon cancer, its effective ingredient also comprises at least one in nucleic acid molecules, EGFR gene interfere RNA construct and the EGFR gene interference slow virus reducing the separation that EGFR gene is expressed in tumor cell.
More excellent, described ILK Gene interfere nucleic acid construct is ILK Gene interfere slow virus carrier.
Further, in described reduction tumor cell, the nucleic acid molecules of the separation that EGFR gene is expressed is:
1) 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
2) EGFR gene shRNA, containing the nucleotide sequence can hybridized with EGFR gene under stringent condition in described EGFR gene shRNA.
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 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.
Preferred, 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.
Preferred, the target sequence of described EGFR gene is sequence shown in SEQ ID NO:2.
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).
Most preferred, 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 tumor cell, endogenous EGFR gene is expressed.
Most preferred, 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 tumor, be that the double-stranded RNA of safe and effective amount, shRNA or slow virus are applied to mammal.Concrete dosage also should consider the factor such as route of administration, patient health situation, and these are all within skilled practitioners skill.
Preferably, described EGFR gene interfere RNA construct is the genetic fragment containing the aforementioned EGFR gene shRNA of coding, can express described EGFR gene shRNA.
More excellent, described EGFR gene interfere RNA construct is EGFR gene interference slow virus carrier.
Preferably, described EGFR gene interference slow virus by EGFR gene disturb slow virus carrier slow virus packaging plasmid, cell line auxiliary under, form through virus packaging.
More excellent, described EGFR gene interference slow virus carrier contains the genetic fragment of coding aforementioned point of EGFR gene shRNA, can express described EGFR gene shRNA.
The DNA sequence of described EGFR gene shRNA genetic fragment of encoding contains sequence and complementary series thereof shown in SEQ ID NO:2.
In pharmaceutical composition of the present invention, the reticent ILK gene expression of specificity, and the effective ingredient 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 suppression ratio after subtracting obviously be greater than single-gene strike subtract group add and, show that material that the material of the reticent ILK gene 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, diluent or excipient.
When preparing medicine, usually by effective ingredient and mixed with excipients, or with excipient dilution, or wrap in can in the carrier that exists of capsule or sachet.When excipient plays diluent effect, it can be solid, semisolid or the fluent material medium as excipient, carrier or active component.Therefore, compositions can be tablet, pill, powder, solution, syrup, sterilizing injecting solution etc.The example of suitable excipient comprises: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline Cellulose, polyvinylpyrrolidone, cellulose, water etc.Preparation also can comprise: wetting agent, emulsifying agent, antiseptic (as methyl hydroxybenzoate and propyl ester), sweeting agent etc.
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 can suppress the expression of people ILK gene by specificity, especially slow virus, can be separately, or work in coordination with the growth of inhibition tumor cell with EGFR targeting slow virus, promote apoptosis of tumor cells, significant in oncotherapy.ILK 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: ILK shRNA plasmid identification figure
1#: negative control (ddH2O); 2#: negative control (unloaded from connecting matched group)
3#:Marker is followed successively by 5kb from top to bottom, 3kb, 2kb, 1.5kb, 1Kb, 750bp, 500bp, 250bp, 100bp
4# ~ 8#:ILK shRNA1-5 transformant is identified
Fig. 4: EGFR shRNA plasmid identification figure
1#: negative control (ddH2O)
2#: negative control (unloaded from connecting matched 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: represent that ILK shRNA slow virus and EGFR shRNA slow virus contaminate people pulmonary carcinoma H1299 cell after 5 days, remarkable antiproliferative effect simultaneously
Fig. 6: represent that ILK shRNA slow virus and EGFR shRNA slow virus contaminate human colon carcinoma RKO cell after 5 days, remarkable antiproliferative effect simultaneously
Detailed description of the invention
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 manufacturer's suggestion is carried out or configures.
Embodiment 1 is for the preparation of people ILK gene and EGFR gene RNAi slow virus
1. build slow virus carrier
1) slow virus carrier for people ILK gene and EGFR gene is built
ILK(NM_004517 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 ILK gene and EGFR gene.Preliminary screening obtains having the siRNA target sequence (SEQ ID NO.1) obviously suppressing ILK gene expression function and the siRNA target sequence (SEQ ID NO.2) obviously suppressing EGFR gene expressive function.Carry out BLAST analysis by Genbank data base, 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 | CGAAGCTCAACGAGAATCA | 770 | ILK |
| 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 restricted enzyme, make its linearisation, agarose gel electrophoresis qualification endonuclease bamhi.
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 labelling, Shanghai JiKai Gene Chemical Technology Co., Ltd provides, Fig. 2) with Age I and EcoR I restricted enzyme, make its linearisation, agarose gel electrophoresis qualification endonuclease bamhi.
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, by double digestion linearisation, (enzyme action system is as shown in table 4,37 DEG C, reaction 1h) the carrier DNA double-stranded DNA Oligo good with purification 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 culture medium, mixing gets 1 μ l as template; The upstream and downstream of RNAi sequence, designs general PCR primer forward primer sequence: 5 '-CCTATTTCCCATGATTCCTTCATA-3 ' (SEQ ID NO:7) in slow virus carrier; Downstream primer sequence: 5 '-GTAATACGGTTATCCACGCG-3 ' (SEQ ID NO:8), carries out PCR identification experiment (, as table 6, reaction condition 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 ILK-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 methods, authentication method and condition are all with ILK-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 coupled reaction 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 buffer | 1.0 | 1.0 | 1.0 |
| T4 phage DNA ligase | 1.0 | 1.0 | 1.0 |
| dd?H 2O | 16.0 | 17.0 | 16.0 |
| Total | 20.0 | 20.0 | 20.0 |
Table 6PCR reaction system
| Reagent | Volume (μ l) |
| 10×buffer | 2.0 |
| dNTPs(2.5mM) | 0.8 |
| Forward primer | 0.4 |
| Downstream primer | 0.4 |
| Taq polymerase | 0.2 |
| Template | 1.0 |
| ddH 2O | 15.2 |
| Total | 20.0 |
Table 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 ILK Gene interfere slow virus and EGFR gene interference slow virus
1) ILK Gene interfere slow virus
Extract RNAi plasmid ILK-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 trypsinization exponential phase, with the DMEM complete medium adjustment cell density containing 10% hyclone 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 culture medium of sucking-off, adds the complete medium that 1.5ml is fresh.
According to the explanation of the MISSION Lentiviral Packaging Mix test kit of Sigma-aldrich company, 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 liquor.Above-mentioned transfection mixture is at room temperature hatched 15min, is transferred in the culture medium of HEKC 293T cell, 37 DEG C, 5%CO
216h is cultivated in incubator.Discard the culture medium containing transfection mixture, PBS solution is washed, and adds complete medium 2ml, continues to cultivate 48h.
Collecting cell supernatant, Centricon Plus-20 centrifugal ultrafiltration unit (Millipore) purification 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 are in 40ml ultracentrifugation pipe; (3) 4000g is centrifugal, 10-15min, to the viral concentration volume needed; (4), after centrifugal end, filter cup and filtered solution collection cups are below separated, tipped upside down on by filter 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 ILK Gene interfere slow virus concentrated solution.By after viral concentration liquid subpackage in-80 degrees Celsius of preservations.
2) packaging process of EGFR gene interference slow virus is with ILK Gene interfere slow virus.
3) negative control slow virus packaging process is with ILK Gene interfere slow virus.
The oncotherapy synergism of embodiment 2ILK Gene interfere slow virus and EGFR gene interference slow virus
1. experimental technique
Carry out trypsinization to the people pulmonary carcinoma H1299 cell and colon cancer RKO cell that are in exponential phase respectively, (cell number is about 5 × 10 to make cell suspension
4/ ml) be inoculated in 6 orifice plates, be cultured to cell fusion degree and reach about 30%.According to infection multiplicity (H1299MOI is the MOI of 5, RKO is 5), experimental group and matched group are set simultaneously, experimental group with the titre of 0.03uL for 5 × 10
8the ILK 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 cancerous cell culture fluid, matched group with the titre of 0.03uL for 5 × 10
8the contrast virus of TU/mL joins in cancerous cell culture fluid.
Replaced medium after cultivation 24h, after time of infection reaches 5 days, collects each experimental group and the cellular control unit that are in exponential phase.The resuspended one-tenth cell suspension (2 × 10 of complete 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 5 days.By the input parameter of adjustment Cellomics arrayscan, calculate in each experimental group and matched group the quantity (simultaneously infecting the cell of ILK 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 ILK 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 5-6, result shows: after the people pulmonary carcinoma H1299 cell simultaneously infecing ILK shRNA slow virus and EGFRshRNA slow virus cultivates 5 days in vitro, vigor cell proliferation times have dropped 95.6% compared with matched group, after the H1299 cell infecing separately EGFR shRNA slow virus cultivates 5 days in vitro, vigor cell proliferation times have dropped 14.5% compared with matched group, after the H1299 cell infecing separately ILK shRNA slow virus cultivates 5 days in vitro, vigor cell proliferation times have dropped 16.2% compared with matched group.
After the human colon carcinoma RKO cell simultaneously infecing ILK shRNA slow virus and EGFR shRNA slow virus cultivates 5 days in vitro, vigor cell proliferation times have dropped 82.84% compared with matched group, after the RKO cell infecing separately EGFR shRNA slow virus cultivates 5 days in vitro, vigor cell proliferation times have dropped 52.23% compared with matched group, after the RKO cell infecing separately ILK shRNA slow virus cultivates 5 days in vitro, vigor cell proliferation times have dropped 18.87% compared with matched group.
Above-mentioned 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 ILK gene shRNA slow virus can work in coordination with the propagation of inhibition tumor cell with EGFR gene shRNA slow virus.
Sequence table
Claims (14)
1. the people ILK 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 ILK gene is applied to preparation or screening anti-tumor medicine as the action target for tumor cell, or prepares diagnosing tumor medicine.
3. purposes as claimed in claim 2, it is characterized in that, the described action target for tumor cell is RNA interference effect target.
4. the people ILK 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. reduce a nucleic acid molecules for the separation of ILK gene expression in tumor cell, described nucleic acid molecules comprises:
A) ILK gene double-stranded RNA, in described ILK gene double-stranded RNA containing can under stringent condition with the nucleotide sequence of ILK gene recombination; Or
B) ILK gene shRNA, in described ILK gene shRNA containing can under stringent condition with the nucleotide sequence of ILK gene recombination.
6. the nucleic acid molecules be separated as claimed in claim 5, it is characterized in that, described ILK 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 ILK gene; Described ILK 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 ILK gene.
7. the nucleic acid molecules be separated as claimed in claim 6, it is characterized in that, the target sequence of described ILK gene is sequence shown in SEQ ID NO:1.
8. the nucleic acid molecules of the separation as described in claim as arbitrary in claim 5-7, is characterized in that, described double-stranded RNA is siRNA, and the sequence of ILK gene siRNA first chain is as shown in SEQ ID NO:9; The sequence of described ILK gene shRNA is as shown in SEQ ID NO:11.
9. an ILK Gene interfere nucleic acid construct, the genetic fragment containing the ILK gene shRNA in the nucleic acid molecules be separated described in the arbitrary claim of coding claim 5-8, can express described ILK gene shRNA.
10. nucleic acid construct as claimed in claim 9, it is characterized in that, described ILK Gene interfere nucleic acid construct is ILK Gene interfere slow virus carrier.
11. 1 kinds of ILK Gene interfere slow viruss, by the ILK Gene interfere slow virus carrier in ILK Gene interfere nucleic acid construct described in claim 10 slow virus packaging plasmid, cell line auxiliary under, form through virus packaging.
12. 1 kinds of pharmaceutical compositions for the treatment of pulmonary carcinoma or colon cancer, its effective ingredient contain reduce the separation of ILK gene expression in tumor cell described in the arbitrary claim of claim 5-8 nucleic acid molecules, ILK Gene interfere nucleic acid construct described in the arbitrary claim of claim 9-10 and claim 11 described at least one in ILK Gene interfere slow virus.
13. pharmaceutical compositions as claimed in claim 12, it is characterized in that, the effective ingredient of described pharmaceutical composition also comprises at least one in nucleic acid molecules, EGFR gene interfere RNA construct and the EGFR gene interference slow virus reducing the separation that EGFR gene is expressed in tumor cell.
14. pharmaceutical compositions as claimed in claim 13, is characterized in that, in described reduction tumor cell, the nucleic acid molecules of the separation that EGFR gene is expressed is:
1) 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
2) EGFR gene shRNA, containing the nucleotide sequence can hybridized with EGFR gene under stringent condition in described EGFR gene shRNA;
Described EGFR gene interfere RNA construct is the genetic fragment containing the aforementioned EGFR gene shRNA of coding, can express described EGFR gene shRNA;
Described EGFR gene interference slow virus by EGFR gene disturb slow virus carrier slow virus packaging plasmid, cell line auxiliary under, form through virus packaging; Described EGFR gene interference slow virus carrier contains the genetic fragment of coding aforementioned point of EGFR gene shRNA, can express described EGFR gene shRNA.
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