CN105803056A - Application of human IARS2 gene and related medicines thereof - Google Patents
Application of human IARS2 gene and related medicines thereof Download PDFInfo
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Abstract
The invention discloses an application of a human IARS2 gene and related medicines thereof. The invention discloses the application of the human IARS2 gene to tumor therapy, tumor diagnosis and medicine preparation. The invention also further constructs human IARS2 gene siRNA (small interfering ribonucleic acid), a human IARS2 gene interfering nucleic acid construct and a human IARS2 gene interfering lentivirus and discloses applications of human IARS2 gene siRNA, the human IARS2 gene interfering nucleic acid construct and the human IARS2 gene interfering lentivirus. The siRNA provided by the invention or the nucleic acid construct and lentivirus containing the sequence of siRNA can specifically inhibit expression of the human IARS2 gene, in particular, the lentivirus can efficiently infect target cells and efficiently inhibit expression of the IARS2 genes in the target cells and then inhibit growth of tumor cells and promote tumor cell apoptosis and has great significance in tumor therapy.
Description
Technical field
The present invention relates to biological technical field, relate more specifically to purposes and the related drugs thereof of people's IARS2 gene.
Background technology
RNA interference (RNAinterference, RNAi) namely carries out PTGS with the short double-stranded RNA (dsRNA) of nucleotide composition.It can block the expression of internal specific gene efficiently, specifically, cause that it is degraded, thus causing the silence of specific gene in organism, making cells show go out the disappearance of certain gene phenotype, being a kind of conventional laboratory technique studying gene function, searching methods for the treatment of diseases emerging in recent years.Research shows, the double-stranded RNA that length is 21-23nt specific with post-transcriptional level can cause RNAi (TuschlT transcribing, ZamorePD, SharpPA, BartelDP.RNAi:double-strandedRNAdirectstheATP-dependentc leavageofmRNAat21to23nucleotideintervals.Cell2000；101:25-33.).Though tumor patient is through chemotherapy, radiotherapy and Comprehensive Treatment, but five year survival rate is still very low, if tumor invasion is intervened with the relevant gene of progress, the treatment that can be tumor is opened up new way.In recent years, RNAi has become the available strategy of the gene therapy of tumor.Utilize RNAi technology that the expression of proto-oncogene, the antioncogene of sudden change, Cell cycle-related genes, anti-apoptotic related gene etc. can be suppressed to suppress tumor progression (Uprichard, SusanL.ThetherapeuticpotentialofRNAinterference.FEBSLett ers2005；579:5996-6007.).
nullIARS2 is a mitochondrial protein (CotterD,GudaP,FahyE,SubramaniamS.2004.MitoProteome:mitochondrialproteinsequencedatabaseandannotationsystem.NucleicAcidsRes32:D463–D467.)，IARS2 gene has significantly high conservative evolutionary sequence，And study and show in several species，Than right IARS2 gene，Collection of illustrative plates showing, 909 sites are conserved positions，Inquiring about p.P909L by SIFT is an effective functional protein，It is likely that destroyed by PolyPhen2HumDiv and PolyPhen2HumVar.Further research shows that the IARS2 albumen of the Skin Cell of patient directly influences p.P909L sudden change [YaoandShoubridge, 1999].The method combined by SABC polyclonal antibody is detected, in the fibrocyte of versus wild type, more in the cell of saltant type exist IARS2 antibody.The transcriptional activity (SasarmanF, ShoubridgeEA.2012.Radioactivelabelingofmitochondrialtran slationproductsinculturedcells.MethodsMolBiol837:207 217) not affecting cyclophorase in IARS2 gene is present in for p.P909L sudden change.
Clinical research shows that the reduction of IARS2 protein level finds at some patient cells, therefore deduces that the change of IARS2 is belonging to the sudden change on pathology, is the initial reason of the Pathology causing some end-stage patients.Some function assessments are tested the minimizing also demonstrating IARS2 albumen and are caused the abnormal translation (KonovalovaS, TyynismaaH.2013.Mitochondrialaminoacyl-tRNAsynthetasesin hu-mandisease.MolGenetMetab108:206 211) of OXPHO respiratory system.
But, at present about the IARS2 gene experimental report in tumor association area or blank out, particularly people's glioma research field.
Summary of the invention
It is an object of the invention to the open Therapeutic Method with people's IARS2 gene-correlation and medicine, with RNA interference (RNAi) for the effect in the survival and apoptotic process of tumor cell of the means research IARS2 gene.
First aspect present invention, with RNA interference for means, have studied IARS2 gene to occur and developing effect in tumor, disclose a kind of method suppressed or reduce growth of tumour cell, propagation, differentiation and/or survival, the method includes: uses to tumor cell and a kind of can suppress the transcribing or translating of IARS2 gene by specificity, or the expression of IARS2 gene or the molecule of activity can be suppressed by specificity, suppress the growth of tumor cell, propagation, differentiation and/or survival with this.
Described tumor cell grows the expression to IARS2 gene or active relevant tumor cell selected from it.Preferably, described tumor cell is selected from glioma.
In the method for described suppression or reduction growth of tumour cell, propagation, differentiation and/or survival, the amount of application of described molecule is enough reduce transcribing or translating of IARS2 gene, or enough reduces the expression of IARS2 gene or the dosage of activity.Further, the expression of described IARS2 gene is at least lowered 50%, 80%, 90%, 95% or 99%.
Described molecule is selected from but is not limited to: nucleic acid molecules, carbohydrate, lipid, small-molecule chemical medicine, antibody medicine, polypeptide, albumen or interference slow virus.
Described nucleic acid includes but not limited to: siRNA (esiRNA) prepared by antisense oligonucleotide, double-stranded RNA (dsRNA), ribozyme, endoribonuclease III or short hairpin RNA (shRNA).
Described double-stranded RNA, ribozyme, esiRNA or shRNA contain the promoter sequence of IARS2 gene or the information sequence of IARS2 gene.
Further, described double-stranded RNA is siRNA (siRNA).Described siRNA comprises the first chain and the second chain, described first chain and described second chain complementation and is collectively forming RNA dimer, and the sequence of described first chain is essentially identical with 15-27 continuous print nucleotide sequence in IARS2 gene.Described small molecules interference RNA energy mRNA fragment coded by specific binding target sequence, and the expression of specificity silence people's IARS2 gene.
Further, the first chain-ordering of described siRNA is essentially identical with the target sequence in IARS2 gene.Preferably, the target sequence in described IARS2 gene contains SEQIDNO:1.
Fragment when target sequence in described IARS2 gene is described small molecules interference RNA specificity silence IARS2 gene expression, in the IARS2 gene corresponding to mRNA fragment being combined with described small molecules interference RNA complementation.
It is also preferred that the left described IARS2 gene source is in people.
First aspect present invention also discloses people's IARS2 gene of a kind of separation at preparation or screening anti-tumor medicine, or the purposes in preparing diagnosing tumor medicine.
Further, described tumor is selected from glioma.
Described the IARS2 gene of separation is used for preparing or screen anti-tumor medicine includes the content of two aspects: one, be applied to prepare anti-tumor medicine or preparation for the action target of tumor cell as medicine or preparation using IARS2 gene；Its two, IARS2 gene is applied to screening anti-tumor medicine or preparation as medicine or preparation for the action target of tumor cell.
Described it is applied to prepare anti-tumor medicine for the action target of tumor cell as medicine or preparation using IARS2 gene or preparation specifically refers to: using the IARS2 gene target as RNA interference effect, develop the medicine for tumor cell or preparation, it is thus possible to reduce the expression of IARS2 gene in tumor cell.
Described IARS2 gene is applied to screening anti-tumor medicine as medicine or preparation for the action target of tumor cell or preparation specifically refers to: using IARS2 gene as effective object, medicine or preparation are screened, to find the medicine that can suppress or promote people's IARS2 gene expression as oncotherapy drug candidate.Namely IARS2 gene small molecules interference RNA (siRNA) as described in the present invention obtains with people's IARS2 gene for effective object screening, can be used as having the medicine suppressing tumor cell proliferation effect.In addition, such as antibody drug, small-molecule drug etc. also can using IARS2 gene and albumen thereof as effective object.
Described it is used for preparing diagnosing tumor medicine by IARS2 gene, refers to the preparation that IARS2 gene expression product is applied to as a diagnosing tumor index diagnosing tumor medicine.
IARS2 gene expression in 4 strain glioma cells is detected by the method for Real-timeQuantitativePCR.Research finds: IARS2 gene expression abundance in 4 strain glioma cells is high.Prompting IARS2, possibly as a kind of oncogene, plays a significant role in tumor develops；The expression of IARS2 gene is likely to become the mark of diagnosing tumor.
Described anti-tumor medicine is can suppress transcribing or translating of IARS2 gene by specificity, or the expression of IARS2 albumen or the molecule of activity can be suppressed by specificity, thus reducing the expression of IARS2 gene in tumor cell, reach to suppress the purpose of the propagation of tumor cell, growth, differentiation and/or survival.
Anti-tumor medicine or diagnosing tumor medicine that the described IARS2 gene preparation by separation or screening obtain include but not limited to: nucleic acid molecules, carbohydrate, lipid, small-molecule chemical medicine, antibody medicine, polypeptide, albumen or interference slow virus.
Described nucleic acid includes but not limited to: siRNA (esiRNA) prepared by antisense oligonucleotide, double-stranded RNA (dsRNA), ribozyme, endoribonuclease III or short hairpin RNA (shRNA).
The amount of application of described anti-tumor medicine is enough reduce transcribing or translating of people's IARS2 gene, or enough reduces the expression of people's IARS2 gene or the dosage of activity.So that the expression of people's IARS2 gene is at least lowered 50%, 80%, 90%, 95% or 99%.
The method adopting forgoing neoplasms medicine treatment tumor, suppresses the propagation of tumor cell to reach the purpose for the treatment of mainly by the expression reducing people's IARS2 gene.Concrete, during treatment, can will effectively reduce the administering substances of people's IARS2 gene expression dose in patient.
Second aspect present invention discloses and a kind of reduces the nucleic acid molecules of the separation of IARS2 gene expression in tumor cell, and described nucleic acid molecules comprises:
A) double-stranded RNA, in described double-stranded RNA containing can under stringent condition with the nucleotide sequence of IARS2 gene recombination；
Or
B) shRNA, in described shRNA containing can under stringent condition with the nucleotide sequence of IARS2 gene recombination.
Further, described double-stranded RNA comprises the first chain and the second chain, described first chain and described second chain complementation and is collectively forming RNA dimer, and the sequence of described first chain is essentially identical with 15-27 continuous print nucleotide sequence in IARS2 gene.It is also preferred that the left the sequence of described first chain is essentially identical with 19-23 continuous print nucleotide sequence in IARS2 gene；More preferably, the sequence of described first chain is essentially identical with 19,20 or 21 continuous print nucleotide sequences in IARS2 gene.
Further, described double-stranded RNA comprises the first chain and the second chain, described first chain and described second chain complementation and is collectively forming RNA dimer, and the sequence of described first chain is essentially identical with the target sequence in IARS2 gene.
The length of described double-stranded RNA the first chain and the second chain is 15-27 nucleotide；It is also preferred that the left length is 19-23 nucleotide；Best, length is 19,20 or 21 nucleotide.
Further, described double-stranded RNA is siRNA (siRNA).Further, the sequence of described siRNA the first chain, such as shown in SEQIDNO:9, is specially 5 '-GUACUUGCAGUCAUCCAUUAA-3 '.
SiRNA shown in SEQIDNO:9 is a chain with the sequence shown in SEQIDNO:1 for RNA interference target sequence design, siRNA for people's IARS2 gene, another chain i.e. sequence of the second chain is complementary with the first chain-ordering, and this siRNA can play the effect of endogenous IARS2 gene expression in specificity silence tumor cell.
Further, described shRNA includes 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 essentially identical with 15-27 continuous print nucleotide sequence in IARS2 gene.SiRNA (siRNA) can be become after described shRNA is processed and then play the effect of endogenous IARS2 gene expression in specificity silence tumor cell.
Further, described shRNA includes 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 essentially identical with the target sequence in IARS2 gene.
It is also preferred that the left described positive-sense strand fragment is essentially identical with 19-23 continuous print nucleotide sequence in IARS2 gene；More preferably, described positive-sense strand fragment is essentially identical with 19,20 or 21 continuous print nucleotide sequences in IARS2 gene.
Further, the sequence of the loop-stem structure of described shRNA is selected from following arbitrary: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU and CCACACC.
Further, the sequence of described shRNA is such as shown in SEQIDNO:14, particularly as follows: 5 '-GUACUUGCAGUCAUCCAUUAAUUCAAGAGAUUAAUGGAUGACUGCAAGUAC-3 '.
ShRNA can become siRNA after enzyme action is processed, and then plays the effect of endogenous people IARS2 gene expression in specificity silence tumor cell.
The interference slow virus carrier of the genetic fragment encoding shRNA of the present invention contains SEQIDNO:1 and complementary series thereof.
First chain of described double-stranded RNA or the positive-sense strand fragment of described shRNA are essentially identical with the target sequence in IARS2 gene, when the target sequence of described IARS2 gene is siRNA for specificity silence IARS2 gene expression, identified the fragment in the IARS2 gene corresponding to also reticent mRNA fragment by described siRNA.
It is also preferred that the left the target sequence in described IARS2 gene contains SEQIDNO:1.
Further, described IARS2 gene source is in people.
Third aspect present invention, discloses a kind of IARS2 gene RNA and builds body, containing the genetic fragment of the shRNA in the nucleic acid molecules encoding separation of the present invention, can express described shRNA.
It can be the gene fragment clone encoding aforementioned people IARS2 gene shRNA is entered known carrier obtain that described people's IARS2 gene RNA builds body.Further, described IARS2 gene RNA builds body is IARS2 gene interference slow virus carrier.
The IARS2 gene interference slow virus carrier of the present invention is the DNA fragmentation encoding aforementioned IARS2 gene shRNA to be cloned into known carrier obtain, described known carrier mostly is slow virus carrier, described IARS2 gene interference slow virus carrier is after virus packaging becomes infectious virion, infected tumor's cell, and then transcribe out shRNA of the present invention, by steps such as enzyme action processing, the described siRNA of final acquisition, for the expression of specificity silence IARS2 gene.
Further, described IARS2 gene interference slow virus carrier is possibly together with the nucleotide sequence of the label can being detected in promoter sequence and/or codes for tumor cell；Preferably, the described label being detected such as green fluorescent protein (GFP).
nullFurther，Described slow virus carrier can be selected from: pLKO.1-puro、pLKO.1-CMV-tGFP、pLKO.1-puro-CMV-tGFP、pLKO.1-CMV-Neo、pLKO.1-Neo、pLKO.1-Neo-CMV-tGFP、pLKO.1-puro-CMV-TagCFP、pLKO.1-puro-CMV-TagYFP、pLKO.1-puro-CMV-TagRFP、pLKO.1-puro-CMV-TagFP635、pLKO.1-puro-UbC-TurboGFP、pLKO.1-puro-UbC-TagFP635、pLKO-puro-IPTG-1xLacO、pLKO-puro-IPTG-3xLacO、pLP1、pLP2、pLP/VSV-G、pENTR/U6、pLenti6/BLOCK-iT-DEST、pLenti6-GW/U6-laminshrna、pcDNA1.2/V5-GW/lacZ、pLenti6.2/N-Lumio/V5-DEST、Arbitrary in pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.
The embodiment of the present invention specifically lists disturbs slow virus carrier, called after pGCSIL-GFP-IARS2-siRNA with the pGCSIL-GFP people's IARS2 gene being vector construction.
The nucleic acid molecules that the present invention separates can be used for preparation prevention or the medicine for the treatment of tumor, and described tumor is glioma.
The IARS2 gene siRNA of the present invention can be used for suppressing the propagation of tumor cell, can serve as the medicine for the treatment of tumor or preparation further.IARS2 gene interference slow virus carrier then can be used for preparing described IARS2 gene siRNA.When being used as medicine or the preparation for the treatment of tumor, it is that the described nucleic acid molecules of safe and effective amount is applied to mammal.Concrete dosage is it is also contemplated that the factor such as route of administration, patient health situation, and these are all within skilled practitioners skill.
Fourth aspect present invention, discloses a kind of IARS2 gene interference slow virus, by aforementioned IARS2 gene disturb slow virus carrier slow virus packaging plasmid, cell line auxiliary under, form through virus packaging.This slow virus can infected tumor's cell produce for the small molecules interference RNA of IARS2 gene, thus suppressing the propagation of glioma tumor cell.This IARS2 gene interference slow virus can be used for preparation prevention or the medicine for the treatment of tumor.
Fifth aspect present invention, discloses a kind of pharmaceutical composition for preventing or treat tumor, and its active substance contains the nucleic acid molecules of aforesaid separation, and IARS2 gene RNA builds one or more the combination in body or IARS2 gene interference slow virus.
Further, described pharmaceutical composition contains double-stranded RNA described in 1～99wt%, shRNA, IARS2 gene RNA builds body or IARS2 gene interference slow virus, and pharmaceutically acceptable carrier, diluent or excipient.
When preparing these compositionss, generally active component is mixed with excipient, or dilute with excipient, 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 includes: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline Cellulose, polyvinylpyrrolidone, cellulose, water, etc..Preparation may also include that wetting agent, emulsifying agent, preservative (such as methyl hydroxybenzoate and propyl ester), sweeting agent etc..
The invention also discloses the application in the anti-tumor medicine of preparation treatment glioma of the described pharmaceutical composition.
The treatment that application is tumor of this pharmaceutical composition provides a method that a kind of method being specially prevention or treatment target in-vivo tumour is applied in object including by the pharmaceutical composition described in effective dose.Further, described tumor is glioma.
When described pharmaceutical composition is for prevention or treatment target in-vivo tumour, it is necessary to the pharmaceutical composition described in effective dose is applied in object.Adopting the method, the growth of described tumor, propagation, recurrence and/or transfer are suppressed.Further, the part of at least the 10% of the growth of described tumor, propagation, recurrence and/or transfer, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% is suppressed.
The object of described method can be people.
Sixth aspect present invention, disclose the test kit of a kind of IARS2 gene expression for reducing in tumor cell, described test kit includes: the nucleic acid molecules of the described separation being present in container, and IARS2 gene RNA builds body and/or described IARS2 gene interference slow virus.
In sum, the present invention devises 1 RNAi target sequence for people's IARS2 gene, building corresponding IARS2 gene RNAi carrier, wherein the RNAi carrier pGCSIL-GFP-IARS2-siRNA of coded sequence SEQIDNO:1 can significantly lower the expression in mRNA level in-site and protein level of the IARS2 gene.Use slow virus (lentivirus, it is abbreviated as Lv) carry RNAi carrier pGCSIL-GFP-IARS2-siRNA as genetic manipulation instrument the RNAi sequence for IARS2 gene can efficiently be imported glioma U251 cell targeting, reduce the expression of IARS2 gene, significantly inhibit the multiplication capacity of above-mentioned tumor cell.Therefore the IARS2 gene silencing of lentivirus mediated is the clinical non-operative treatment mode that malignant tumor is potential.
SiRNA provided by the invention or comprise the nucleic acid construct of this siRNA sequence, slow virus can specificity suppress people's IARS2 gene expression, especially slow virus, can efficiently infect target cell, suppress the expression of IARS2 gene in target cell expeditiously, promote apoptosis, the invasion and attack reducing tumor cell and transfer ability etc., and then the growth of suppression tumor cell, promote apoptosis of tumor cells, significant in oncotherapy.
Accompanying drawing explanation
Fig. 1: pGCSIL-GFP Plasmid diagram
Fig. 2: IARS2-RNAi slow virus infected people's glioma U251 cell after 5 days, and the expression of IARS2mRNA significantly reduces
Fig. 3: IARS2-RNAi slow virus infected people's glioma U251 cell after 5 days, caused cell inhibitory effect
Detailed description of the invention
The present invention relates to one group of small molecules interference RNA for people's IARS2 gene (siRNA) sequence, rna interference vector and RNA and disturb slow virus.Choose people's IARS2mRNA coding region sequence target site as siRNA, design siRNA target sequence according to continuous print 10-30 in target site (preferred 15-27, more preferably 19-23) individual base sequence.By gene clone, the nucleic acid construct of the above-mentioned siRNA of construction expression, the slow virus of the above-mentioned siRNA of packaging expression.Cell experiment proves, above-mentioned siRNA sequence can the expression of endogenous IARS2 gene in specificity silence human tumor cells.
Inventor have found that, adopt the propagation etc. that can effectively suppress tumor cell under RNAi method after the expression of mediator's IARS2 gene, can efficiently controlling the growth course of tumor, this achievement in research shows that IARS2 gene is proto-oncogene, can as the target spot of oncotherapy.Inventor synthesizes further and tests the multiple siRNA for IARS2 gene, has filtered out and can effectively suppress IARS2 to express and then suppress the siRNA of people's glioma U521 cell proliferation and growth, has completed the present invention on this basis.
The invention provides siRNA (siRNA) sequence of a series of interference people's IARS2 gene, constructing can the slow virus of specificity silence IARS2 gene expression.The present invention studies discovery, for siRNA and the RNAi slow virus of people's IARS2 gene design, stable and downward IARS2 gene specifically expression, and effectively suppresses the propagation of human tumor cells.Present invention demonstrates that IARS2 gene can promote growth of tumour cell, be expected to become the target spot of early diagnosis of tumor and treatment.And, by the expression of RNAi mode silence IARS2 gene, can as the effective means suppressing tumor development.
The mentality of designing of the present invention is:
The present invention screens a kind of people IARS2 gene RNAi slow virus of acquisition by the following method: transfer people's IARS2 gene order from Genbank；Prediction siRNA site；Synthesis is for the effective siRNA sequence of IARS2 gene, the two ends double-stranded DNA Oligo containing restriction enzyme site cohesive end；It is connected with double-stranded DNA Oligo after slow virus carrier double digestion, the RNAi plasmid of construction expression IARS2 gene siRNA sequence；RNAi plasmid and slow virus are packed assistant carrier (PackingMix, Sigma-aldrich company) the cotransfection HEKC 293T needed, produces recombinant slow virus granule, can be prepared by the slow virus of efficiently reticent IARS2 gene.
Based on said method, the invention provides the Effective target site (specifically as shown in SEQIDNO:1) of 1 interference IARS2 gene, construct the slow virus of special interference people's IARS2 gene.
Invention additionally discloses a kind of people IARS2 gene RNAi slow virus (IARS2-RNAi) and preparation and application thereof simultaneously.
The study find that, utilize the RNAi method of lentivirus mediated, after reducing the expression in tumor cell of the IARS2 gene, it is possible to effectively suppress the propagation of tumor cell.The study show that, IARS2 gene is a proto-oncogene, tumor cell proliferation can be promoted, occur in tumor and development has important biological function, IARS2 gene can be the target of oncotherapy, and the IARS2 gene specific silence of lentivirus mediated can as a kind of new tool of oncotherapy.
The present invention is expanded on further below in conjunction with embodiment.Should be understood that embodiment is merely to illustrate the present invention, and unrestricted the scope of the present invention.In embodiment, the experimental technique of unreceipted actual conditions and the reagent of undeclared formula are conventionally condition, such as works such as [U.S.] 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 carries out or configures.
Embodiment 1 is for the preparation of people IARS2 gene RNAi slow virus
1. screening is for the effective siRNA target spot of people's IARS2 gene
IARS2 (NM_018060) gene information is transferred from Genbank；Design the effective siRNA target spot for IARS2 gene.Table 1 lists wherein 1 effective siRNA target sequence for IARS2 gene.
Table 1 targets the siRNA target sequence of people's IARS2 gene
| SEQ ID NO | TargetSeq |
| 1 | GTACTTGCAGTCATCCATTAA |
2. the preparation of slow virus carrier
The two ends double-stranded DNA Oligo sequence (table 2) containing AgeI and EcoRI restriction enzyme site cohesive end is synthesized for siRNA target spot (for SEQIDNO:1)；PGCSIL-GFP carrier (Shanghai JiKai Gene Chemical Technology Co., Ltd provides, Fig. 1) is acted on so that it is linearisation, agarose gel electrophoresis identifies endonuclease bamhi with AgeI and EcoRI restricted enzyme.
Table 2 two ends are containing the double-stranded DNA Oligo of AgeI and EcoRI 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) carrier DNA and the good double-stranded DNA Oligo of purification connect, connect overnight in 16 DEG C in suitable buffer system (linked system is as shown in table 5), reclaim and connect product.Fresh competent escherichia coli cell (conversion operation reference: Molecular Cloning: A Laboratory guide second edition 55-56 page) prepared by calcium chloride is converted by connecting product.Growing bacterium clone surface at connection converted product and be stained with, be dissolved in 10 μ lLB culture medium, mixing takes 1 μ l as template；The upstream and downstream of RNAi sequence, designs general PCR primer, forward primer sequence: 5 '-CCTATTTCCCATGATTCCTTCATA-3 ' (SEQIDNO:6) in slow virus carrier；Downstream primer sequence: 5 '-GTAATACGGTTATCCACGCG-3 ' (SEQIDNO:7), carry out PCR identification experiment (PCR reaction system such as table 6-1, reaction condition is table 6-2 such as).PCR being identified, positive clone checks order and comparison analysis, and the clone that comparison is correct is the carrier expressing RNAi for SEQIDNO:1 successfully constructed, called after pGCSIL-GFP-IARS2-siRNA.
Building pGCSIL-GFP-Scr-siRNA negative control plasmids, negative control siRNA target sequence is 5 '-TTCTCCGAACGTGTCACGT-3 ' (SEQIDNO:8).When building pGCSIL-GFP-Scr-siRNA negative control plasmids, the two ends double-stranded DNA Oligo sequence (table 3) containing AgeI and EcoRI restriction enzyme site cohesive end, all same pGCSIL-GFP-IARS2-siRNA of all the other construction methods, authentication method and condition is synthesized for ScrsiRNA target spot.
Table 3 two ends are containing the double-stranded DNA Oligo of AgeI and EcoRI restriction enzyme site cohesive end
By the T4DNA ligase carrier by double digestion linearisation (enzyme action system is as shown in table 4,37 DEG C, reacts 1h)
Table 4pGCSIL-GFP plasmid enzyme restriction reaction system
| Reagent | Volume (μ l) |
| PGCSIL-GFP plasmid (1 μ g/ μ l) | 2.0 |
| 10×buffer | 5.0 |
| 100×BSA | 0.5 |
| Age I(10U/μl) | 1.0 |
| EcoR I(10U/μl) | 1.0 |
| dd H2O | 40.5 |
| Total | 50.0 |
Table 5 carrier DNA and double-stranded DNA Oligo coupled reaction system
| Reagent | Positive control (μ l) | From even comparison (μ l) | Connection group (μ l) |
| Linearizing carrier DNA (100ng/ μ l) | 1.0 | 1.0 | 1.0 |
| The double-stranded DNA Oligo (100ng/ μ l) of annealing | 1.0 | - | 1.0 |
| 10 × T4 phage DNA ligase buffer | 1.0 | 1.0 | 1.0 |
| T4 phage DNA ligase | 1.0 | 1.0 | 1.0 |
| dd H2O | 16.0 | 17.0 | 16.0 |
| Total | 20.0 | 20.0 | 20.0 |
Table 6-1PCR reaction system
| Reagent | Volume (μ l) |
| 10×buffer | 2.0 |
| dNTPs(2.5mM) | 0.8 |
| Forward primer | 0.4 |
| Downstream primer | 0.4 |
| Taq polymerase | 0.2 |
| Template | 1.0 |
| ddH2O | 15.2 |
| Total | 20.0 |
Table 6-2PCR reaction system program setting
3. packaging IARS2-siRNA slow virus
Extract the DNA of RNAi plasmid pGCSIL-GFP-IARS2-siRNA with the plasmid extraction test kit of Qiagen company, be configured to 100ng/ μ l and store liquid.
24h before transfection, with the HEKC 293T cell of trypsinization exponential phase, adjusts cell density for 1.5 × 10 with the DMEM complete medium containing 10% hyclone5Cell/ml, is inoculated in 6 orifice plates, 37 DEG C, 5%CO2Cultivate in incubator.Can be used for transfecting when cell density reaches 70%-80%.2h before transfection, the original culture medium of sucking-off, add the fresh complete medium of 1.5ml.Explanation according to the MISSIONLentiviralPackagingMix test kit of Sigma-aldrich company, PackingMix (PVM) 20 μ l is added in a sterile centrifugation tube, PEI12 μ l, plasma-free DMEM medium 400 μ l, take the plasmid DNA of the 20 above-mentioned extractings of 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%CO216h is cultivated in incubator.Discarding the culture medium containing transfection mixture, PBS solution is washed, and adds complete medium 2ml, continues to cultivate 48h.Collecting cell conditioned medium liquid, CentriconPlus-20 centrifugal ultrafiltration unit (Millipore) purification and concentration slow virus, step is as follows: (1) 4 DEG C, and 4000g is centrifuged 10min, removes cell debris；(2) 0.45 μm of frit supernatant are in 40ml ultracentrifugation pipe；(3) 4000g is centrifuged, 10-15min, to the viral concentration volume needed；(4) after centrifugal end, filter cup and following filtered solution collection cups being separated, tipped upside down on by filter cup on sample collection cup, centrifugal 2min centrifugal force is less than 1000g；(5) Centrifuge Cup is removed from sample collection cup, sample collection cup is viral concentration liquid.By after viral concentration liquid subpackage in-80 degrees Celsius of preservations.The sequence of first chain of the siRNA contained in viral concentration liquid is such as shown in SEQIDNO:9.The packaging process of comparison slow virus, with IARS2-siRNA slow virus, only replaces pGCSIL-GFP-IARS2-siRNA carrier with pGCSIL-GFP-Scr-siRNA carrier.
The silence efficiency of embodiment 2 real-time fluorescence quantitative RT-PCR method detection IARS2 gene
The glioma U251 cell being in exponential phase carries out trypsinization, and (cell number is about 5 × 10 to make cell suspension4/ ml) it is inoculated in 6 orifice plates, it is cultured to cell fusion degree and reaches about 30%.According to infecting plural number (MOI, U251:5) value, add the virus of embodiment 1 preparation of Sq, after cultivating 24h, change culture medium, after time of infection reaches 5 days, collect cell.Trizol operating instruction according to Invitrogen company, extracted total RNA.M-MLV operating instruction according to Promega company, obtains cDNA (reverse transcription reaction system reacts 1h in 7,42 DEG C of Table, and then water-bath 10min makes reverse transcriptase inactivate in 70 DEG C of water-baths) by RNA reverse transcription.
TP800 type RealtimePCR instrument (TAKARA) is adopted to carry out Real_time quantitative detection.The primer of IARS2 gene is as follows: forward primer 5 '-TGGACCTCCTTATGCAAACGG-3 ' (SEQIDNO:10) and downstream primer 5 '-GGCAACCCATGACAATCCCA-3 ' (SEQIDNO:11).With house-keeping gene GAPDH for internal reference, primer sequence is as follows: forward primer 5 '-TGACTTCAACAGCGACACCCA-3 ' (SEQIDNO:12) and downstream primer 5 '-CACCCTGTTGCTGTAGCCAAA-3 ' (SEQIDNO:13).By the proportional arrangement reaction system in table 8.
Table 7 reverse transcription reaction system
| Reagent | Volume (μ l) |
| 5×RT buffer | 4.0 |
| 10mM dNTPs | 2.0 |
| RNasin | 0.5 |
| M-MLV-RTase | 1.0 |
| DEPC H2O | 3.5 |
| Total | 11.0 |
Table 8Real-timePCR reaction system
| Reagent | Volume (μ l) |
| SYBR premix ex taq: | 10.0 |
| Forward primer (2.5 μMs): | 0.5 |
| Downstream primer (2.5 μMs): | 0.5 |
| cDNA | 1.0 |
| ddH2O | 8.0 |
| Total | 20.0 |
Setting program is two-step method Real-timePCR: denaturation 95 DEG C, 15s；Each step degeneration 95 DEG C, 5s afterwards；Annealing extends 60 DEG C, 30s；Carry out 45 circulations altogether.Extending stage reading light absorption value every time.After PCR terminates, 95 DEG C of degeneration 1min, it is subsequently cooled to 55 DEG C, makes DNA double chain fully combine.Starting to 95 DEG C from 55 DEG C, each step increases by 0.5 DEG C, keeps 4s, reads light absorption value simultaneously, makes melting curve.Adopt 2-ΔΔCtAnalytic process calculates the gene expression abundance having infected IARS2mRNA.Infect the cell of comparison virus (Lv-Scr-siRNA) as comparison.Experimental result (Fig. 2) shows, in glioma U251 cell, the expression of IARS2mRNA has lowered 87.3%.
The multiplication capacity of the tumor cell of IARS2-siRNA slow virus has been infected in embodiment 3 detection
The glioma U251 cell being in exponential phase carries out trypsinization, and (cell number is about 5 × 10 to make cell suspension4/ ml) it is inoculated in 6 orifice plates, it is cultured to cell fusion degree and reaches about 30%.According to infecting plural number (MOI, U251:5), add the virus of Sq, after cultivating 24h, change culture medium, after time of infection reaches 5 days, collect the experimental group cell being in exponential phase.The resuspended one-tenth cell suspension (2 × 10 of complete medium4/ ml), it is about 2000/hole with cell density, inoculates 96 orifice plates.The often multiple hole of group 5, every hole 100 μ l.After completing plate, put 37 DEG C, 5%CO2Incubator is cultivated.From after bed board second day, every day read plate once with Cellomics instrument (ThermoFisher) detection, and continuous detecting reads plate 5 days.By adjusting the input parameter of Cellomicsarrayscan, calculate the quantity of the cell with green fluorescence every time scanned in orifice plate exactly, data are carried out statistics and draws, draw cell proliferation curve (result is as shown in Figure 3).It is shown that slow virus infected group tumor cell In vitro culture after 5 days, growth rate significantly slows, and far below the growth rate of matched group tumor cell, vigor cell number have dropped 80.2%, it was shown that IARS2 gene silencing causes that tumor cell proliferation ability is suppressed.
The above; it is only presently preferred embodiments of the present invention; not to any formal and substantial restriction of the present invention; should be understood that; for those skilled in the art; under the premise without departing from the inventive method, also can making some improvement and supplement, these improve and supplement and also should be regarded as protection scope of the present invention.All those skilled in the art, without departing from the spirit and scope of the present invention, the equivalent variations of a little change, modification and the differentiation made when available disclosed above technology contents, it is the Equivalent embodiments of the present invention；Meanwhile, all change of any equivalent variations, modification and differentiation above-described embodiment made according to the substantial technological of the present invention, all still fall within the scope of technical scheme.
Claims (14)
1. the people's IARS2 gene separated is at preparation or a screening anti-tumor medicine, or the purposes in preparing diagnosing tumor medicine.
2. purposes as claimed in claim 1, it is characterised in that described tumor is selected from glioma.
3. reducing a nucleic acid molecules for the separation of IARS2 gene expression in tumor cell, described nucleic acid molecules comprises:
A) double-stranded RNA, in described double-stranded RNA containing can under stringent condition with the nucleotide sequence of IARS2 gene recombination；Or
B) shRNA, in described shRNA containing can under stringent condition with the nucleotide sequence of IARS2 gene recombination.
4. the nucleic acid molecules separated as claimed in claim 3, it is characterized in that, described double-stranded RNA comprises the first chain and the second chain, described first chain and described second chain complementation and is collectively forming RNA dimer, and the sequence of described first chain is essentially identical with the target sequence in IARS2 gene；Described shRNA includes 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 essentially identical with the target sequence in IARS2 gene.
5. the nucleic acid molecules separated as described in claim 3 or 4, it is characterised in that the target sequence of described IARS2 gene contains SEQIDNO:1.
6. the nucleic acid molecules separated as described in claim 3 or 4, it is characterised in that described double-stranded RNA is siRNA, and the sequence of this siRNA the first chain is such as shown in SEQIDNO:9.
7. the nucleic acid molecules separated as described in claim 3 or 4, it is characterised in that the sequence of described shRNA is such as shown in SEQIDNO:14.
8. IARS2 gene RNA builds a body, containing the genetic fragment of the shRNA in the nucleic acid molecules separated described in coding claim 3-7 any claim, can express described shRNA.
9. IARS2 gene RNA builds body as claimed in claim 8, it is characterised in that described IARS2 gene RNA builds body for interference slow virus carrier.
null10. IARS2 gene RNA builds body as claimed in claim 9，It is characterized in that，Described interference slow virus carrier is obtained by after the gene fragment clone encoding described shRNA is entered slow virus carrier，Described slow virus carrier is selected from: pLKO.1-puro、pLKO.1-CMV-tGFP、pLKO.1-puro-CMV-tGFP、pLKO.1-CMV-Neo、pLKO.1-Neo、pLKO.1-Neo-CMV-tGFP、pLKO.1-puro-CMV-TagCFP、pLKO.1-puro-CMV-TagYFP、pLKO.1-puro-CMV-TagRFP、pLKO.1-puro-CMV-TagFP635、pLKO.1-puro-UbC-TurboGFP、pLKO.1-puro-UbC-TagFP635、pLKO-puro-IPTG-1xLacO、pLKO-puro-IPTG-3xLacO、pLP1、pLP2、pLP/VSV-G、pENTR/U6、pLenti6/BLOCK-iT-DEST、pLenti6-GW/U6-laminshrna、pcDNA1.2/V5-GW/lacZ、pLenti6.2/N-Lumio/V5-DEST、Arbitrary in pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.
11. IARS2 gene interference a slow virus, described in claim 9-10 any claim disturb slow virus carrier slow virus packaging plasmid, cell line auxiliary under, through virus packaging form.
12. one kind for preventing or treat the pharmaceutical composition of tumor, its active substance contains the nucleic acid molecules of the separation described in claim 3-7 any claim, IARS2 gene RNA described in claim 8-10 any claim builds body, and/or the IARS2 gene interference slow virus described in claim 11, and pharmaceutically acceptable carrier, diluent or excipient.
13. the application that pharmaceutical composition described in claim 12 is in the anti-tumor medicine of preparation treatment glioma.
14. one kind for reducing the test kit of IARS2 gene expression in tumor cell, it is characterized in that, described test kit includes: be present in container, the nucleic acid molecules of the separation described in claim 3-7 any claim, IARS2 gene RNA described in claim 8-10 any claim builds body and/or the IARS2 gene interference slow virus described in claim 11.
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| CN108424967A (en) * | 2018-05-28 | 2018-08-21 | 陕西中医药大学第二附属医院 | The application for the biomarker that IARS2 genes are detected as leukaemia |
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| CN108424967A (en) * | 2018-05-28 | 2018-08-21 | 陕西中医药大学第二附属医院 | The application for the biomarker that IARS2 genes are detected as leukaemia |
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