CN104928353A

CN104928353A - Applications of human DGKZ gene and related drugs thereof

Info

Publication number: CN104928353A
Application number: CN201410103009.5A
Authority: CN
Inventors: 朱向莹; 孙琴; 高博; 张晓慧; 金杨晟; 瞿红花; 曹跃琼
Original assignee: SHANGHAI GENECHEM CO Ltd
Current assignee: SHANGHAI GENECHEM CO Ltd
Priority date: 2014-03-19
Filing date: 2014-03-19
Publication date: 2015-09-23
Also published as: CN110643705A

Abstract

The invention discloses applications of human DGKZ gene and related drugs thereof. Applications of human DGKZ gene in tumor treatment, tumor diagnosis, and drug preparation are disclosed. The invention further constructs human DGKZ gene micromolecule interference RNA, human DGKZ gene interference nucleic acid construct, and human DGKZ gene interference slow virus, and discloses the applications thereof. The provided siRNA or nucleic acid construct containing the siRNA and slow virus can inhibit the expression of human DGKZ gene specifically, especially the slow virus can infect target cells efficiently and inhibit the expression of DGKZ gene in target cells efficiently, thus the tumor cell growth is inhibited, apoptosis of tumor cells is promoted, and thus the provided siRNA, nucleic acid construct, and slow virus have an important meaning in tumor treatment.

Description

The purposes of people DGKZ gene and related drugs thereof

Technical field

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

Background technology

The RNA interference (RNA interference, RNAi) phenomenon refer to when with double-stranded RNA (dsRNA) transfered cell of endogenous mRNA coding region section sequence homology after, there is selective degradation in this mRNA, and causes the silence of this genetic expression.Research shows; length is that the double-stranded RNA of 21-23nt specificly with post-transcriptional level can cause RNAi(Tuschl T transcribing; Zamore PD; Sharp PA, Bartel DP.RNAi:double-stranded RNA directs theATP-dependent cleavage of mRNA at21to23nucleotide intervals.Cell2000; 101:25-33.).Tumour is the principal disease threatening human health.Though tumour patient is through chemotherapy, radiotherapy and complex therapy, five year survival rate is still very low, if intervene with the relevant gene of progress tumor invasion, new way is opened up in the treatment that can be tumour.In recent years, RNAi has become the available strategy of the gene therapy of tumour.Utilize RNAi technology can suppress proto-oncogene, generation and development (Uprichard, Susan L.The therapeutic potential of RNA interference.FEBS Letters2005 that the expression of cancer suppressor gene, Cell cycle-related genes, anti-apoptotic genes involved etc. of sudden change carrys out Tumor suppression; 579:5996-6007.).

DGKZ, has another name called DGK-ZETA, belongs to Eukaryotic triglyceride kinase families (Diacylglycerol Kinases, DGKs).DGKs comprises a series of enzyme, and they are DGKalpha, DGKbeta, DGKgamma, DGKdelta, DGKepsilon, DGKzeta, DGKeta, DGKtheta, DGKiota and DGKkappa.DGKs extensively distributes in mammalian body, and DGKs, by the content by dialycerides metabolism being phosphatidic acid regulation and control intracellular glycerol two fat, regulates Cellular Signaling Transduction Mediated, and then responds multiple born of the same parents' external stimulus.[Wade D.Van Horn,Charles R.Sanders.Prokaryoticdiacylglycerol kinase and undecaprenol kinase.Annu Rev Biophys.2012;41:81-101.Li D,LyonsJA,Pye VE,Vogeley L, D,Kenyon CP,Shah ST,Doherty C,Aherne M,Caffrey M.Crystal structure of the integral membrane diacylglycerol kinase.Nature.2013May23;497(7450):521-4.Miller DJ,Jerga A,Rock CO,White SW.Analysis of the Staphylococcusaureus DgkB structure reveals a common catalytic mechanism for the soluble diacylglycerolkinases.Structure.2008Jul;16(7):1036-46.]

Multiple member and the cancer of DGK family occur to develop relevant.DGKgamma and DGKbeta has target site [the Shindo M promoting the phorbol exters that cancer is had an effect; Irie K; Masuda A; Ohigashi H; Shirai Y; Miyasaka K, Saito N.Synthesis and phorbol ester binding of the cysteine-rich domains of diacylglycerol kinase (DGK) isozymes.DGKgamma and DGKbeta are new targets of tumor-promoting phorbol esters.J Biol Chem.2003May16; 278 (20): 18448-54.].Within 2007, there is article to report in the invasion and attack of the thymic carcinoma cell that DGKs induces at HGF and process of growth and play keying action [Filigheddu N, Cutrupi S, Porporato PE, Riboni F, Baldanzi G, Chianale F, Fortina E, Piantanida P, De Bortoli M, Vacca G, Graziani A, Surico N.Diacylglycerol kinase is required for HGF-induced invasiveness and anchorage-independentgrowth of MDA-MB-231breast cancer cells.Anticancer Res.2007May-Jun, 27 (3B): 1489-92.].At the positive regulatory factor that melanomatous research display DGKalpha is NF-kappaB; melanoma cell apoptosis [the Yanagisawa K that TNF-alpha can be suppressed to induce; Yasuda S; Kai M; Imai S; Yamada K; Yamashita T; Jimbow K; Kanoh H, Sakane F.Diacylglycerol kinase alpha suppresses tumor necrosis factor-alpha-inducedapoptosis of human melanoma cells through NF-kappaB activation.Biochim Biophys Acta.2007Apr; 1771 (4): 462-74.].In the research of Endometrial Carcinomas, DGKalpha is to the propagation of cancer cells, it is all essential to move and adhere to, imply that it is potential cancer therapy target gene [Filigheddu N, Sampietro S, Chianale F, PorporatoPE, Gaggianesi M, Gregnanin I, Rainero E, Ferrara M, Perego B, Riboni F, Baldanzi G, GrazianiA, Surico N.Diacylglycerol kinase α mediates17-β-estradiol-induced proliferation, motility, andanchorage-independent growth of Hec-1A endometrial cancer cell line through the Gprotein-coupled estrogen receptor GPR30.Cell Signal.2011Dec, 23 (12): 1988-96.].

Little at the report of tumour association area about DGKZ.In order to study the dependency of DGKZ and tumour, the present invention chooses tumor models, is that means research DGKZ occurs and developing effect in tumour with RNAi.

Summary of the invention

The object of the invention is to open methods for the treatment of with people DGKZ gene-correlation and medicine, disturb (RNAi) for the effect of means research DGKZ gene in the survival and apoptotic process of tumour cell with RNA.

First aspect present invention, with RNA interference for means, have studied DGKZ gene to occur and developing effect in tumour, disclose a kind of method suppressing or reduce growth of tumour cell, propagation, differentiation and/or survival, the method comprises: use to tumour cell and a kind ofly specificity can suppress the transcribing or translating of DGKZ gene, or specificity can suppress the expression of DGKZ albumen or the molecule of activity, come the growth of inhibition tumor cell, propagation, differentiation and/or survival with this.

Described tumour is selected from the propagation of its tumour cell any one tumour relevant to the expression of people DGKZ gene, and preferably, described tumour is a kind of malignant tumour: glioma.

Described suppression or reduce in the method for growth of tumour cell, propagation, differentiation and/or survival, the amount of application of described molecule is enough reduce transcribing or translating of DGKZ gene, or the enough expression of reduction DGKZ albumen or the dosage of activity.Further, the expression of described DGKZ gene is at least lowered 50%, 80%, 90%, 95% or 99%.

Further, described molecule can be selected from but be not limited to: nucleic acid molecule, 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 DGKZ gene or the information sequence of DGKZ gene.

Further, described double-stranded RNA is siRNA (siRNA).Described siRNA 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 15-27 continuous print nucleotide sequence in DGKZ gene.MRNA fragment coded by described small molecules interference RNA energy specific binding target sequence, and the expression of specificity reticent people DGKZ gene.

Further, the first chain-ordering of described siRNA is substantially identical with the target sequence in DGKZ gene.Preferably, the target sequence in described DGKZ gene contains the arbitrary sequence in SEQ ID NO:1-13.

When target sequence in described DGKZ gene is the DGKZ genetic expression of described small molecules interference RNA specificity silence, the fragment in the DGKZ gene corresponding to the mRNA fragment combined with the complementation of described small molecules interference RNA.

Preferably, described DGKZ gene source is in people.

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

Further, described tumour is selected from the propagation of its tumour cell any one tumour relevant to the expression of people DGKZ gene, and preferably, described tumour is a kind of malignant tumour: glioma.

Described by the people DGKZ gene of separation for the preparation of or screening anti-tumor medicine comprise the content of two aspects: one, to be applied to people DGKZ gene for the action target of tumour cell as medicine or preparation and to prepare anti-tumor medicine or preparation; Its two, people DGKZ gene is applied to screening anti-tumor medicine or preparation 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 people DGKZ gene prepares anti-tumor medicine or preparation specifically refers to: using the target of people DGKZ gene as RNA interference effect, develop the medicine for tumour cell or preparation, thus the expression level of people DGKZ gene in tumour cell can be reduced.

Describedly people DGKZ gene is applied to screening anti-tumor medicine as medicine or preparation for the action target of tumour cell or preparation specifically refers to: using people DGKZ gene as effective object, medicine or preparation are screened, can suppress or promote that the medicine of people DGKZ genetic expression is as oncotherapy drug candidate to find.Namely people DGKZ gene small molecules interference RNA as described in the present invention obtains for effective object screening with people DGKZ 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 DGKZ gene and albumen thereof as effective object.

Described by people DGKZ gene for the preparation of diagnosing tumor medicine, refer to preparation people DGKZ gene expression product being applied to diagnosing tumor medicine as a diagnosing tumor index.

The expression level of DGKZ gene in tumor tissues, healthy tissues and tumour normal surrounding tissue is detected by immunohistochemical method.Research finds: the expression amount of DGKZ gene in tumor tissues is significantly higher than healthy tissues and tumour normal surrounding tissue.Prompting DGKZ gene may as a kind of oncogene, plays a significant role in the developing of tumour; The expression level of DGKZ gene may become the mark of diagnosing tumor.

Described anti-tumor medicine is specificity can suppress transcribing or translating of DGKZ gene, or specificity can suppress the expression of DGKZ albumen or the molecule of activity, thus the expression level of DGKZ gene in reduction tumour cell, reach the object of the propagation of inhibition tumor cell, growth, differentiation and/or survival.

The anti-tumor medicine that the described DGKZ gene preparation by separation or screening obtain or diagnosing tumor medicine include but not limited to: nucleic acid molecule, carbohydrate, lipid, small-molecule chemical medicine, antibody medicine, polypeptide, albumen or interference slow virus.

The amount of application of described anti-tumor medicine is enough reduce transcribing or translating of people DGKZ gene, or enough reduces the expression of people DGKZ albumen or the dosage of activity.50%, 80%, 90%, 95% or 99% is at least lowered to make the expression of people DGKZ gene.

Adopt the method for forgoing neoplasms medicine treatment tumour, mainly reached the object for the treatment of by the propagation of the expression level inhibition tumor cell reducing people DGKZ gene.Concrete, during treatment, effectively can reduce the administering substances of people DGKZ gene expression dose in patient.

Second aspect present invention discloses a kind of nucleic acid molecule reducing the separation of DGKZ genetic expression in tumour cell, and described nucleic acid molecule comprises:

A) double-stranded RNA, in described double-stranded RNA containing can under stringent condition with the nucleotide sequence of DGKZ gene recombination; Or

B) shRNA, in described shRNA containing can under stringent condition with the nucleotide sequence of DGKZ gene recombination.

Further, described 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 15-27 continuous print nucleotide sequence in DGKZ gene.Preferably, the sequence of described first chain is substantially identical with 19-23 continuous print nucleotide sequence in DGKZ gene; Better, the sequence of described first chain is substantially identical with 19,20 or 21 continuous print nucleotide sequences in DGKZ gene.

Further, described 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 in DGKZ gene.

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).Further, the sequence of described siRNA first chain, as shown in SEQ ID NO:25, is specially 5 '-CUCUGAAAGCAAGCAAGAA-3 '.

SiRNA shown in SEQ ID NO:25 for the sequence shown in SEQ ID NO:1 be RNA disturb target sequence design, for a chain of the siRNA of people DGKZ gene, another chain i.e. sequence of the second chain and the complementation of the first chain-ordering, this siRNA can play the effect of endogenous DGKZ genetic expression in the reticent tumour cell of specificity.

Further, described 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 15-27 continuous print nucleotide sequence in DGKZ gene.Described shRNA can become siRNA (siRNA) and then play the effect of endogenous DGKZ genetic expression in the reticent tumour cell of specificity after processing.

Further, described 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 in DGKZ gene.

Preferably, described positive-sense strand fragment is substantially identical with 19-23 continuous print nucleotide sequence in DGKZ gene; Better, described positive-sense strand fragment is substantially identical with 19,20 or 21 continuous print nucleotide sequences in DGKZ gene.

Further, the sequence of the loop-stem structure of described shRNA is selected from following arbitrary: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC.It is stem ring that the present invention specifically lists with UUCAAGAGA.

Further, the sequence of described shRNA, as shown in SEQ ID NO:26, is specially: 5 '-CUCUGAAAGCAAGCAAGAA UUCAAGAGAUUCUUGCUUGCUUUCAGAG-3 '.

ShRNA cuts after processing can become siRNA through enzyme, and then plays the effect of endogenous people DGKZ genetic expression in the reticent tumour cell of specificity.

The interference lentiviral vectors of gene fragment of shRNA of the present invention of encoding contains arbitrary sequence in SEQ ID NO:1-13 and complementary sequence thereof.

First chain of described double-stranded RNA or the positive-sense strand fragment of described shRNA substantially identical with the target sequence in DGKZ gene, when the target sequence of described DGKZ gene is siRNA for the DGKZ genetic expression of specificity silence, identified the fragment in the DGKZ gene corresponding to mRNA fragment of also silence by described siRNA.

Preferably, the target sequence in described DGKZ gene contains arbitrary sequence of SEQ ID NO:1-13.

Further, described DGKZ gene source is in people.

Third aspect present invention, discloses a kind of people DGKZ Gene interfere nucleic acid construct, comprises the gene fragment of the people DGKZ gene small molecules interference RNA of separation of the present invention of encoding, can express described shRNA.

Described people DGKZ Gene interfere nucleic acid construct can be the gene fragment clone of the aforementioned people DGKZ gene small molecules interference RNA of coding is entered known carrier obtain.Further, described people DGKZ Gene interfere nucleic acid construct behaviour DGKZ Gene interfere lentiviral vectors.

DGKZ Gene interfere lentiviral vectors of the present invention the gene fragment clone of the described people DGKZ gene small molecules interference RNA of coding is entered known carrier obtain, described known carrier mostly is lentiviral vectors, described DGKZ Gene interfere lentiviral vectors is after virus packaging becomes infectious virion, infected tumor's cell, and then transcribe out shRNA of the present invention, the steps such as processing are cut, the described siRNA of final acquisition, for the expression of the reticent DGKZ gene of specificity by enzyme.

Further, the nucleotide sequence of described DGKZ Gene interfere lentiviral vectors 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).

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.

It is the people DGKZ Gene interfere lentiviral vectors of vector construction that the embodiment of the present invention specifically lists with pGCSIL-GFP, called after pGCSIL-GFP-DGKZ-siRNA.

The nucleic acid molecule that the present invention is separated can be used for the medicine preparing prevention or treatment tumour, and described tumour is selected from the propagation of its tumour cell any one tumour relevant to the expression of people DGKZ gene, and preferably, described tumour is a kind of malignant tumour: glioma.

DGKZ gene siRNA of the present invention can be used for the propagation of inhibition tumor cell, can be used as medicine or the preparation for the treatment of tumour further.DGKZ Gene interfere lentiviral vectors then can be used for preparing described DGKZ gene siRNA.When being used as medicine or the preparation for the treatment of tumour, be that the described nucleic acid molecule of safe and effective amount is 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.

Fourth aspect present invention, discloses a kind of people DGKZ Gene interfere slow virus, by aforementioned people DGKZ Gene interfere lentiviral vectors slow virus packaging plasmid, clone auxiliary under, form through virus packaging.This slow virus can also produce for people DGKZ gene small molecules interference RNA by infected tumor's cell, thus the propagation of inhibition tumor cell.This DGKZ Gene interfere slow virus can be used for the medicine preparing prevention or treatment tumour.Described tumour is selected from the propagation of its tumour cell any one tumour relevant to the expression of people DGKZ gene, and preferably, described tumour is a kind of malignant tumour: glioma.

Fifth aspect present invention, also disclose a kind of pharmaceutical composition for preventing or treat tumour, its active substance contains the nucleic acid molecule of aforesaid separation, one or more the combination in DGKZ Gene interfere nucleic acid construct or DGKZ Gene interfere slow virus.

Further, described pharmaceutical composition contains the foregoing double-stranded RNA of 1 ~ 99wt%, shRNA, DGKZ Gene interfere nucleic acid construct or DGKZ Gene interfere slow virus, and pharmaceutically acceptable carrier, thinner or vehicle.

When preparing these compositions, usually by activeconstituents 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.

The invention also discloses the application of described pharmaceutical composition in any one anti-tumor medicine that the propagation of its tumour cell of preparation treatment is relevant to the expression of people DGKZ gene.

The treatment being applied as tumour of this pharmaceutical composition provides a kind of method, is specially a kind of method of prevention or treatment target in-vivo tumour, comprises and being applied in object by the pharmaceutical composition described in effective dose.Further, described tumour is selected from the propagation of its tumour cell any one tumour relevant to the expression of people DGKZ gene.Preferably, described tumour is a kind of malignant tumour: glioma.

When described pharmaceutical composition is used for prevention or treatment target in-vivo tumour, need the pharmaceutical composition described in effective dose to be applied in object.Adopt the method, growth, propagation, the recurrence of described tumour and/or shift suppressed.Further, the growth of described tumour, propagation, recurrence and/or transfer at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% part suppressed.

The object of described method can be people.

Sixth aspect present invention, disclose a kind of test kit for reducing the DGKZ genetic expression in tumour cell, described test kit comprises: the nucleic acid molecule being present in the described separation in container, DGKZ Gene interfere nucleic acid construct, and/or described DGKZ Gene interfere slow virus.

In sum, the present invention devises 13 RNAi target sequences for people DGKZ gene, build corresponding DGKZRNAi carrier, wherein the RNAi carrier pGCSIL-GFP-DGKZ-siRNA of encoding sequence SEQ ID NO:1 significantly can lower the expression of DGKZ gene at mRNA level in-site and protein level.Use slow virus (lentivirus, be abbreviated as Lv) carry RNAi carrier pGCSIL-GFP-DGKZ-siRNA as genetic manipulation instrument the RNAi sequence for DGKZ gene can efficiently be imported people glioma U87 cell target, reduce the expression level of DGKZ gene, significantly suppress the multiplication capacity of glioma U87 cell.Therefore the DGKZ gene silencing of lentivirus mediated is the potential clinical non-operative treatment mode of malignant tumour.

SiRNA provided by the invention or comprise the nucleic acid construct of this siRNA sequence, slow virus specificity can suppress the expression of people DGKZ gene, especially slow virus, efficiently can infect target cell, suppress the expression of DGKZ gene in target cell expeditiously, promote apoptosis, reduce the Infiltration and metastasis ability etc. of tumour cell, and then the growth of inhibition tumor cell, promote apoptosis of tumor cells, significant in oncotherapy.

Accompanying drawing explanation

Fig. 1 represents pGCSIL-GFP Plasmid diagram

Fig. 2 represents that DGKZ-RNAi slow virus infected people glioma U87 cell after 5 days, and the expression level of DGKZ mRNA significantly reduces

Fig. 3 represents that DGKZ-RNAi slow virus infected people glioma U87 cell after 5 days, causes cell inhibitory effect

Fig. 4 is that colony formation detects tumour cell infection DGKZ-RNAi slow virus rear clone Forming ability result schematic diagram

Fig. 5 is apoptosis situation schematic diagram after TTP detection tumour cell infection DGKZ-RNAi slow virus

Embodiment

The present invention is based on the research that zinc finger protein may be relevant to the propagation of tumour cell, resistance and vascularization etc., DGKZ, as a newfound zinc finger protein, infers its generation that may take part in malignant tumour and development.

The present invention relates to one group of small molecules interference RNA for people DGKZ gene (siRNA) sequence, rna interference vector and RNA and disturb slow virus.Choose the target site of people DGKZ mRNA coding region sequence as siRNA, according to the preferred 15-27 of continuous print 10-30(, more preferably 19-23 in target site) individual base sequence design siRNA target sequence.By gene clone, the nucleic acid construct of the above-mentioned siRNA of construction expression, the slow virus of the above-mentioned siRNA of packaging expression.Cell experiment proves, above-mentioned siRNA sequence can the expression of endogenous DGKZ gene in the reticent human tumor cells of specificity.

Contriver finds, can the propagation of inhibition tumor cell effectively after the expression of DGKZ gene of transferring person under adopting RNAi method, promotes apoptosis, reduces the Infiltration and metastasis ability etc. of tumour cell, effectively can control the growth course of tumour.This achievement in research shows that DGKZ gene is proto-oncogene, can be used as the target spot of oncotherapy.Contriver synthesizes further and tests the multiple siRNA for DGKZ gene, has filtered out the expression that effectively can suppress DGKZ and then the siRNA suppressing people's glioma U87 cell proliferation and growth, has completed the present invention on this basis.

The invention provides siRNA (siRNA) sequence of a series of interference people DGKZ gene, constructing can the slow virus of the reticent DGKZ genetic expression of specificity.The present invention studies discovery, for siRNA and the RNAi slow virus of people DGKZ gene design, and stable expression of also lowering DGKZ gene specifically, and effectively suppress the propagation of human tumor cells.The present invention shows that DGKZ gene can promote growth of tumour cell, is expected to the target spot becoming early diagnosis of tumor and treatment.And, by the expression of the reticent DGKZ gene of RNAi mode, can be used as the effective means of Tumor suppression development.

Mentality of designing of the present invention is:

The present invention screens by the following method and obtains a kind of people DGKZ gene RNAi slow virus: from Genbank, transfer people DGKZ gene order; Prediction siRNA site; Synthesize the effective siRNA sequence for DGKZ gene, two ends are containing the double-stranded DNA Oligo of restriction enzyme site cohesive end; Be connected with double-stranded DNA Oligo after lentiviral vectors double digestion, the RNAi plasmid of construction expression DGKZ gene siRNA sequence; By assistant carrier (Packing Mix, Sigma-aldrich company) the cotransfection HEKC 293T that RNAi plasmid and slow virus packaging need, produce recombinant slow virus particle, the slow virus of efficient reticent DGKZ gene can be obtained.

Based on aforesaid method, the invention provides the Effective target site (specifically as shown in SEQ ID NO1-13) of 13 interference DGKZ genes, construct the slow virus of special interference people DGKZ gene.

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

This research finds, utilizes the RNAi method of lentivirus mediated, after reducing the expression in tumour cell of DGKZ gene, and can the propagation of effective inhibition tumor cell.This research shows, DGKZ gene is a proto-oncogene, can promote tumor cell proliferation, occurs and have important biological function in development in tumour, DGKZ gene can be the target of oncotherapy, and the DGKZ gene specific silence of lentivirus mediated can be used as a kind of new tool of oncotherapy.

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: for the preparation of people DGKZ gene RNAi slow virus

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

DGKZ(NM_003646.3 is transferred from Genbank) gene information; Design the effective siRNA target spot for DGKZ gene.Table 1 lists wherein 13 effective siRNA target sequences for DGKZ gene.

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

2. the preparation of lentiviral vectors

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 (for SEQ ID NO:1); Act on pGCSIL-GFP carrier (Shanghai JiKai Gene Chemical Technology Co., Ltd provides, Fig. 1) with Age I and EcoR I restriction enzyme, make its linearizing, 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

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:18) in lentiviral vectors; Downstream primer sequence: 5 '-GTAATACGGTTATCCACGCG-3 ' (SEQ ID NO:19), carries out PCR identification experiment (, as table 6-1, reaction conditions is as table 6-2 for PCR reaction system).The clone positive to PCR qualification checks order and compare of analysis, and the clone that comparison is correct is the RNAi carrier containing SEQ ID NO.1 successfully constructed, called after pGCSIL-GFP-DGKZ-siRNA.

Build pGCSIL-GFP-Scr-siRNA negative control plasmids, negative control siRNA target sequence is 5 '-TTCTCCGAACGTGTCACGT-3 ' (SEQ ID NO:20).When building pGCSIL-GFP-Scr-siRNA negative control plasmids, double-stranded DNA Oligo sequence (table 3) of Age I and EcoR I restriction enzyme site cohesive end is contained, all same pGCSIL-GFP-DGKZ-siRNA of all the other construction processs, authentication method and condition for Scr siRNA target spot synthesis two ends.

Table 3 two ends contain the double-stranded DNA Oligo of Age I and EcoR I restriction enzyme site cohesive end

By the carrier of T4DNA ligase enzyme by double digestion linearizing (it is as shown in table 4 that enzyme cuts system, 37 DEG C, reaction 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 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 6-1PCR reaction system

Reagent	Volume (μ l)
		10×buffer	2.0
dNTPs(2.5mM)	0.8
		Upstream primer	0.4
Downstream primer	0.4
		Taq polysaccharase	0.2
Template	1.0
		ddH ₂O	15.2
Total	20.0

Table 6-2PCR reaction system program setting

3. pack DGKZ-siRNA slow virus

Extract the DNA of RNAi plasmid pGCSIL-GFP-DGKZ-siRNA with the plasmid extraction test kit of Qiagen company, 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 viral concentration liquid.By after the packing of viral concentration liquid in-80 degrees Celsius of preservations.The sequence of first chain of the siRNA contained in viral concentration liquid is as shown in SEQ ID NO:25.The wrapping process of contrast slow virus, with DGKZ-siRNA slow virus, only replaces pGCSIL-GFP-DGKZ-siRNA carrier with pGCSIL-GFP-Scr-siRNA carrier.

Embodiment 2: real-time fluorescence quantitative RT-PCR method detects the silence efficiency of DGKZ gene

The people glioma U87 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 infecting plural number (MOI, U87:10) value, virus prepared by the embodiment 1 adding sufficient quantity, replaced medium after cultivation 24h, after time of infection reaches 5 days, collecting cell.According to the Trizol process specifications of Invitrogen company, extracted total RNA.According to the M-MLV process specifications of Promega company, RNA reverse transcription is obtained cDNA(reverse transcription reaction system in table 7,42 DEG C of reaction 1h, then in 70 DEG C of water-baths, water-bath 10min makes reversed transcriptive enzyme inactivation).

TP800 type Real time PCR instrument (TAKARA) is adopted to carry out Real_time quantitative detection.The primer of DGKZ gene is as follows: upstream primer 5 '-AGCAAGCAAGAAGAAGAAGAGG-3 ' (SEQ ID NO:21) and downstream primer 5 '-GGATTGAGATACCAGAGGAAAGAC-3 ' (SEQ ID NO:22).With house-keeping gene GAPDH for internal reference, primer sequence is as follows: upstream primer 5 '-TGACTTCAACAGCGACACCCA-3 ') (SEQ ID NO:23) and downstream primer 5 '-CACCCTGTTGCTGTAGCCAAA-3 ' (SEQ ID NO:24).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 H ₂O	3.5
		Total	11.0

Table 8Real-time PCR reaction system

Reagent	Volume (μ l)
		SYBR premix ex taq:	10.0
Upstream primer (2.5 μMs):	0.5
		Downstream primer (2.5 μMs):	0.5
cDNA	1.0
		ddH ₂O	8.0
Total	20.0

Setting program is two-step approach Real-time PCR: denaturation 95 DEG C, 15s; Each step sex change 95 DEG C afterwards, 5s; Annealing extension 60 DEG C, 30s; Carry out 45 circulations altogether.Read light absorption value in the extension stage at every turn.After PCR terminates, 95 DEG C of sex change 1min, are then cooled to 55 DEG C, and DNA double chain is fully combined.To 95 DEG C from 55 DEG C, each walks increase by 0.5 DEG C, keeps 4s, reads light absorption value simultaneously, makes melting curve.Adopt 2- ^{Δ Δ Ct}analytical method calculates the gene expression abundance having infected DGKZ mRNA.Infect the cell of contrast virus (Lv-Scr-siRNA) in contrast.Experimental result (Fig. 2) shows, in people glioma U87 cell, the expression level of DGKZ mRNA has lowered 76.5%.

Embodiment 3: detect the multiplication capacity infecting the tumour cell of DGKZ-siRNA slow virus

The people glioma U87 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 infecting plural number (MOI, U87:10), adding the virus of sufficient quantity, replaced medium after cultivation 24h, after time of infection reaches 5 days, collecting each experimental group cell being 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 instrument (Thermo Fisher) and reads plate once, and continuous detecting reads plate 5 days.By the input parameter of adjustment Cellomics arrayscan, calculate the quantity of the cell of the band green fluorescence in each scanning orifice plate exactly, statistics is carried out to data and draws, draw cell proliferation curve (result as shown in Figure 3).Result shows, slow virus infects group tumour at cell injuring model after 5 days, and rate of propagation significantly slows down, far below the rate of propagation of control group tumour cell, vigor cell number have dropped 60.1% respectively, shows that DGKZ gene silencing causes tumor cell proliferation ability suppressed.

Embodiment 4 infects the detection of the tumor cell clone Forming ability of DGKZ-siRNA slow virus

To be inoculated in 12 orifice plates after the enzymic digestion of people glioma U87 cell tryptase, cell density be 10-15%.Within second day, be changed to fresh substratum, include 5ug/ml polybrene.By DGKZ-siRNA slow virus according to MOI(MOI, U87:2) join in culture plate, renew fresh substratum after infecting 12-24h.After infecting 72h, fluorescence microscopy Microscopic observation fluorescence, efficiency of infection reaches 90%.

By be in logarithmic phase infection virus after cell tryptase enzymic digestion after, the resuspended one-tenth cell suspension of perfect medium; Be inoculated in after cell counting (200 cells/well) in 6 orifice plates, the cell inoculated is continued in incubator cultivation and be greater than 50 to cell count in 14 days or most single clones, midway carries out changing liquid and observation of cell state every 3day; Under fluorescent microscope, cell clone is taken pictures before experiment stops; Use paraformaldehyde fixed cell when experiment stops, after PBS washed cell, Giemsa dyes, and takes pictures.

As shown in Fig. 4 result, colony formation detects tumour cell infection DGKZ-siRNA rear clone Forming ability result schematic diagram, for U87 cell, compared with disturbing (control) with contrast, after RNA interference reduces the expression (KD group) of DGKZ gene, the volume that clone's spot number that tumour cell is formed significantly reduces, clones spot obviously reduces; Show that the ability that DGKZ silence causes tumour cell to form clone declines.

After plate clone forms the expression of experiment detection reduction DGKZ, the clonality of U87 cell tumor cells declines.The tumor cell level of apoptosis that embodiment 5 infects DGKZ-siRNA slow virus detects

By be in logarithmic phase cell tryptase enzymic digestion after, the resuspended one-tenth cell suspension of perfect medium; Be inoculated in 96 orifice plates, every hole 100ul; Put 37 DEG C of 5%CO2 incubators to cultivate; After cultivating 36h, abandon nutrient solution, 85% ethanol fixed cell of 4 DEG C of precoolings; PBS washes plate twice; After RNase process cell, PI dyes, lucifuge 15min.By 96 orifice plates on TTP instrument, use the template of default analysis subG1 phase to carry out scanning analysis, obtain result.

As shown in apoptosis situation schematic diagram result after TTP detection tumour cell infection DGKZ-siRNA in Fig. 5, for U87 cell.After PI dyeing-TTP method detects the expression reducing DGKZ, the change of apoptosis corpusculum (subG1 phase) ratio of U87 cell tumor cells.After finding to lower DGKZ genetic expression, the apoptosis ratio of tumour cell increases.Disturb (control) with contrasting, after RNA interference reduces the expression (KD group) of DGKZ gene, the number of the apoptotic body that apoptotic tumor cell produces significantly increases; Show that DGKZ silence causes apoptosis of tumor cells.After PI dyeing-TTP method detects the expression reducing DGKZ, the change of apoptosis corpusculum (subG1 phase) ratio of U87 cell tumor cells.

The above; be only preferred embodiment of the present invention; not to any formal and substantial restriction of the present invention; should be understood that; for those skilled in the art; under the prerequisite not departing from the inventive method, also can make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.All those skilled in the art, without departing from the spirit and scope of the present invention, a little change made when utilizing disclosed above technology contents, the equivalent variations of modifying and developing, be Equivalent embodiments of the present invention; Meanwhile, all according to substantial technological of the present invention to the change of any equivalent variations that above-described embodiment is done, modify and differentiation, all still belong in the scope of technical scheme of the present invention.

Claims

1. the people DGKZ gene be separated at preparation or a 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 tumour is selected from the propagation of its tumour cell any one tumour relevant to the expression of people DGKZ gene.

3. purposes as claimed in claim 1, it is characterized in that, described tumour is selected from: glioma.

4. reduce a nucleic acid molecule for the separation of DGKZ genetic expression in tumour, described nucleic acid molecule comprises:

5. the nucleic acid molecule be separated as claimed in claim 4, it is characterized in that, described 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 in DGKZ gene; Described 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 in DGKZ gene.

6. the nucleic acid molecule be separated as described in claim 4 or 5, it is characterized in that, the target sequence of described DGKZ gene contains arbitrary sequence in SEQ ID NO:1-13.

7. the nucleic acid molecule be separated as described in claim 4 or 5, it is characterized in that, described double-stranded RNA is siRNA, and the sequence of this siRNA first chain is as shown in SEQ ID NO:25.

8. the nucleic acid molecule be separated as described in claim 4 or 5, it is characterized in that, the sequence of described shRNA is as shown in SEQ ID NO:26.

9. a people DGKZ Gene interfere nucleic acid construct, the gene fragment containing the shRNA in the nucleic acid molecule be separated described in the arbitrary claim of coding claim 4-8, can express described shRNA.

10. people DGKZ Gene interfere nucleic acid construct as claimed in claim 9, is characterized in that, described people DGKZ Gene interfere nucleic acid construct behaviour DGKZ Gene interfere lentiviral vectors.

11. people DGKZ Gene interfere nucleic acid constructs as claimed in claim 10, is characterized in that, described interference lentiviral vectors obtains after the gene fragment clone of the described shRNA of coding is entered lentiviral vectors, and described lentiviral vectors is selected from:

pLKO.1-puro、pLKO.1-CMV-tGFP、pLKO.1-puro-CMV-tGFP、pLKO.1-CMV-Neo、

pLKO.1-Neo、pLKO.1-Neo-CMV-tGFP、pLKO.1-puro-CMV-TagCFP、

pLKO.1-puro-CMV-TagYFP、pLKO.1-puro-CMV-TagRFP、pLKO.1-puro-CMV-TagFP635、

pLKO.1-puro-UbC-TurboGFP、pLKO.1-puro-UbC-TagFP635、pLKO-puro-IPTG-1xLacO、

pLKO-puro-IPTG-3xLacO、pLP1、pLP2、pLP/VSV-G、pENTR/U6、

pLenti6/BLOCK-iT-DEST、pLenti6-GW/U6-laminshrna、pcDNA1.2/V5-GW/lacZ、

Arbitrary in pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.

12. 1 kinds of people DGKZ Gene interfere slow viruss, by people DGKZ Gene interfere lentiviral vectors described in the arbitrary claim of claim 10-11 slow virus packaging plasmid, clone auxiliary under, form through virus packaging.

13. 1 kinds for preventing or treat the pharmaceutical composition of tumour, the nucleic acid molecule of the separation described in the arbitrary claim of claim 4-8 containing treatment significant quantity, people DGKZ Gene interfere slow virus described in people DGKZ Gene interfere nucleic acid construct described in the arbitrary claim of claim 9-11 and/or claim 12, and pharmaceutically acceptable carrier, thinner or vehicle.

14. application of pharmaceutical composition in preparation tumor as claimed in claim 13, described tumour is any one tumour that the propagation of its tumour cell is relevant to the expression of people DGKZ gene.

15. application of pharmaceutical composition in preparation treatment glioma medicine as claimed in claim 14.

16. 1 kinds of test kits for reducing people DGKZ genetic expression in tumour cell, it is characterized in that, described test kit comprises: be present in container, people DGKZ gene small molecules interference RNA described in the arbitrary claim of claim 4-8, people DGKZ Gene interfere slow virus described in people DGKZ Gene interfere nucleic acid construct described in the arbitrary claim of claim 9-11 and/or claim 12.