CN103468785A - Uses and relevant drugs of human SEMA4C (semaphorin 4C) gene - Google Patents
Uses and relevant drugs of human SEMA4C (semaphorin 4C) gene Download PDFInfo
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Abstract
The invention discloses uses and relevant drugs of human SEMA4C (semaphorin 4C) gene The invention discloses the uses the human SEMA4C gene in tumor treatment, tumor diagnosis and drug preparation. The invention further constructs small molecule interfering RNA of the human SEMA4C gene, an interfering nucleic acid construction body of the human SEMA4C gene, interfering lentivirus of the human SEMA4C gene, and discloses the uses thereof. The siRNA (small interfering RNA) or the nucleic acid construction body containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibit the expression of the human SEMA4C gene, and particularly the lentivirus can efficiently infect target cells and efficiently inhibit the expression of the SEMA4C gene in the target cells so as to inhibit the growth of tumor cells and promote the apoptosis of the tumor cells, and has vital significance in the tumor treatment.
Description
Technical field
The present invention relates to biological technical field, relate more specifically to purposes and the related drugs thereof of human SEMA 4 C gene.
Background technology
Semaphorin family is a very conservative molecule family; because all thering is the SEMA structural domain of high conservative in family member's molecular structure, gain the name; its family member is distributed in from virus to the organic evolution widely of high Mammals tree; form (Yazdani U, Terman JR.The semaphorins.Genome Biol 2006 by eight subfamilies; 7 (3): 211-233.).Initial research discovery, the Semaphorin molecule can grow by nervous system regulation, promotes the growing tip disintegration; the branch and the cynapse that affect aixs cylinder form, and are targeting signal molecule (Inagaki, the S that regulates axon growth; Ohoka, Y, Sugimoto; H; Fujioka, S, Amazaki; M; Kurinami, H, Miyazalci; N; Tohyama, M.Sema4c, a transmembrane semaphorin; interacts with a post-synaptic density protein, PSD-95.J Biol Chem 2001; 276:9174-9181.).But more and more studies have shown that at present; the Semaphorin molecule not only plays an important role in neural system; also play an important role (Goshima Y at aspects such as the genesis of tumour, cell migration, immune responses; Ito T; Sasaki Y, et al.Semaphorins as signals for cell repulsion and invasion.J Clin Invest 2002; 109 (8): 993-998.Wu, H, Fan; J, Zhu, L; Liu, 5, Wu; Y, Zhao, T; Ding, X, Fan; W, and Fan, M.Sema4C expression in neural stem/Progenitor cells and in adult neurogenesis indueed by cerebral isehemia.J Mol Neurosci 2009; 39:27-39.)
Semaphorin IV family mainly finds in Mammals, belongs to the single transmembrane protein, and many subfamily members of research have SEMA4A and SEMA4D now.SEMA4C (Semaphorin 4C) is No. three member of IV subfamily.Compare the SEMA4D of IV family; relevant research is less; but the field related to is comparatively extensive; guiding (the Tmagnone L that comprises neural axon; Artigiani S; Chen H, He Z, Ming Gl; Song H; ChedotalA, Winberg ML, and Goodman CS; poo M; et al.Plexins are a large family of receptors for transmembrane, seereted, and GPI-anehored semaphorins in vertebrates.Cell 1999; 99 (1): 71-80.), increment and migration (the Deng S of brain granulosa cell precursor cell; HirsehbergA; Worzfeld T; Penaehioni JY; Korostylev A, Swierez JM, et al.Plexin-B2; but not Plexin-B 1, critieally modulates neuronal migration and Patterning of the developing nervous system in vivo.J Neurosei 2007; 27 (23): 6333-6347.), myotube differentiation (Kang JS, Yi MJ, Zhang W, FeinleibJL, Cole F and Kranss RS.Netrins and neogenin promote myotubeformation.Cell Biol 2004; 167:493-504.Kang JS; Feinleib JL; Knox S, Ketteringham MA and Krauss RS.Promyogenic members of the Ig and cadherinfamilies associate to positively regulate differentiation.Proe.Natl.Aead.Sei 2003; 100:3989-3994.2), effect (the Zhang Y of potential adjusting β-catenin signal in pigmentation and innervation; Andl T, Yang SH, Teta; M; Liu F, Seykora JT, Tobias JW; Piccolo S; Schmidt-Ullrich R, Nagy A.Activation of beta-catenin, signaling Programs embryonic epidermis to hair folliele fate.Development 2008; 135:2161-2172.); also have recently bibliographical information SEMA4C to express (Wu H in neural generating process after neural stem cell/precursor cell and the damage of adult rat cerebrum ischemia; Fan J; Zhu S; Wu Y, Zhao T, Ding X; Fan W, Fan M.Sema4C expression in neural stem/Progenitor cells and in adult neurogenesis indueed by cerebral isehemia.J Mol Neurosci 2009; 39:27-39.).
Although the related scope of SEMA4C document is more extensive, study it and tumour generation, development and progress research seldom, also there is no directly related foreign language bibliographical information in NCBI.Therefore, be necessary to further investigate the molecular mechanism that SEMA4C acts on and affects tumor cell proliferation in the tumour cell malignant proliferation.
The short double-stranded RNA (dsRNA) that RNA disturbs (RNA interference, RNAi) to form with Nucleotide carries out PTGS.It can block the expression of specific gene in body efficiently, specifically, cause its degraded, thereby cause the silence of specific gene in organism, making cell show the disappearance of certain gene phenotype, is emerging in recent years research gene function a kind of commonly used, the laboratory technique of searching methods for the treatment of diseases.Research shows; the double-stranded RNA that length is 21-23nt can transcribed and the specific RNAi(Tuschl of the causing T of post-transcriptional level; Zamore PD; Sharp PA, Bartel DP.RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.Cell 2000; 101:25-33.).Though tumour patient is through chemotherapy, radiotherapy and complex therapy, five year survival rate is still very low, if tumor invasion is intervened with the relevant gene of progress, can open up new way for the treatment of tumour.In recent years, RNAi has become the available strategy of the gene therapy of tumour.The expression that utilizes the RNAi technology can suppress the cancer suppressor gene, Cell cycle-related genes, anti-apoptosis-related genes etc. of proto-oncogene, sudden change suppresses tumor progression (Uprichard, Susan L.The therapeutic potential of RNA interference.FEBS Letters 2005; 579:5996-6007.).
In order to further investigate the regulatory function of SEMA4C in tumour occurs, the present invention chooses colorectal carcinoma and stomach cancer cell model, take RNAi as means research SEMA4C in colorectal carcinoma and cancer of the stomach generation and developing effect.
Summary of the invention
The object of the invention is to methods for the treatment of and medicine open and human SEMA 4 C (Semaphorin 4C) gene-correlation, the RNA of take interference (RNAi) as means research SEMA4C gene in the survival of tumour cell and the effect in apoptotic process.
First aspect present invention, take the RNA interference as means, having studied the SEMA4C gene occurs and developing effect in tumour, a kind of method that suppresses or reduce growth of tumour cell, propagation, differentiation and/or survival is disclosed, the method comprises: to tumour cell, use a kind of transcribing or translating of SEMA4C gene that can specificity suppress, or can specificity suppress the expression of SEMA4C albumen or the molecule of activity, come growth, propagation, differentiation and/or the survival of inhibition tumor cell with this.
Described tumour cell is selected from the tumour cell that its growth is relevant with the expression of SEMA4C albumen or activity.Preferably, described tumour cell is selected from the arbitrary of colon cancer cell, stomach cancer cell.
In the method for described inhibition or reduction growth of tumour cell, propagation, differentiation and/or survival, the amount of application of described molecule is enough to reduce transcribing or translating of SEMA4C gene, or enough reduces expression or the active dosage of SEMA4C albumen.Further, the expression of described SEMA4C gene at least is lowered 50%, 80%, 90%, 95% or 99%.
Described molecule can be selected from but be not limited to: nucleic acid molecule, carbohydrate, lipid, small molecules chemical drugs, antibody medicine, polypeptide, albumen or interference slow virus.
Described nucleic acid includes but not limited to: siRNA (esiRNA) prepared by antisense oligonucleotide, double-stranded RNA (dsRNA), ribozyme, endoribonuclease III or short hairpin RNA (shRNA).
The information sequence of the promoter sequence that described double-stranded RNA, ribozyme, esiRNA or shRNA contain the SEMA4C gene or SEMA4C gene.
Further, described double-stranded RNA is siRNA (siRNA).Described siRNA comprises the first chain and the second chain, the complementary common formation RNA dimer of described the first chain and described the second chain, and in the sequence of described the first chain and SEMA4C gene, 15-27 continuous nucleotide sequence is basic identical.The coded mRNA fragment of described small molecules interference RNA energy specific binding target sequence, and the expression of the reticent human SEMA 4 C gene of specificity.
Further, the first chain-ordering of described siRNA and the target sequence in the SEMA4C gene are basic identical.Preferably, the target sequence in described SEMA4C gene contains the arbitrary sequence in SEQ ID NO:1-18.
When the target sequence in described SEMA4C gene is the reticent SEMA4C genetic expression of described small molecules interference RNA specificity, with the fragment in the corresponding SEMA4C gene of mRNA fragment of the complementary combination of described small molecules interference RNA.
Preferably, described SEMA4C gene source is in the people.
First aspect present invention also discloses a kind of human SEMA 4 C gene of separation at preparation or screening anti-tumor medicine, or the purposes in preparing the diagnosing tumor medicine.
Further, described tumour is selected from the arbitrary of colorectal carcinoma, cancer of the stomach.
The described gene of the SEMA4C by separation for the preparation of or the screening anti-tumor medicine comprise the content of two aspects: one, using the SEMA4C gene as medicine or preparation be applied to prepare anti-tumor medicine or preparation for the action target of tumour cell; Its two, using the SEMA4C gene as medicine or preparation be applied to screen anti-tumor medicine or preparation for the action target of tumour cell.
Described using the SEMA4C gene as medicine or preparation is applied to prepare anti-tumor medicine for the action target of tumour cell or preparation specifically refers to: the target using the SEMA4C gene as the RNA interference effect, develop medicine or preparation for tumour cell, thereby can reduce the expression level of SEMA4C gene in tumour cell.
Described using the SEMA4C gene as medicine or preparation is applied to screen anti-tumor medicine for the action target of tumour cell or preparation specifically refers to: using the SEMA4C gene as effective object, medicine or preparation are screened, using and find the medicine that can suppress or promote the human SEMA 4 C gene expression as the oncotherapy drug candidate.SEMA4C gene small molecules interference RNA (siRNA) be take human SEMA 4 C gene as the effective object screening obtains as described in the present invention, can be used as having the medicine of inhibition tumor cell proliferation function.In addition, such as antibody drug, small-molecule drug etc. also can be using SEMA4C gene and albumen thereof as effective object.
Described by the SEMA4C gene for the preparation of the diagnosing tumor medicine, refer to the preparation that is applied to the diagnosing tumor medicine using the SEMA4C gene expression product as a diagnosing tumor index.
Described anti-tumor medicine is for can specificity suppressing transcribing or translating of SEMA4C gene, or can specificity suppress the expression of SEMA4C albumen or the molecule of activity, thereby the expression level of SEMA4C gene in the reduction tumour cell, reach the purpose of propagation, growth, differentiation and/or the survival of inhibition tumor cell.
Anti-tumor medicine or diagnosing tumor medicine that the described SEMA4C gene preparation by separation or screening obtain include but not limited to: nucleic acid molecule, carbohydrate, lipid, small molecules chemical drugs, antibody medicine, polypeptide, albumen or interference slow virus.
Described nucleic acid includes but not limited to: siRNA (esiRNA) prepared by antisense oligonucleotide, double-stranded RNA (dsRNA), ribozyme, endoribonuclease III or short hairpin RNA (shRNA).
The amount of application of described anti-tumor medicine is enough to reduce transcribing or translating of human SEMA 4 C gene, or enough reduces expression or the active dosage of human SEMA 4 C albumen.So that the expression of human SEMA 4 C gene at least is lowered 50%, 80%, 90%, 95% or 99%.
Adopting the method for aforementioned anti-tumor medicine treatment tumour, is mainly the purpose that the propagation of the expression level inhibition tumor cell by reducing human SEMA 4 C gene reaches treatment.Concrete, during treatment, can effectively reduce the administering substances of human SEMA 4 C gene expression level in the patient.
Second aspect present invention discloses a kind of nucleic acid molecule that reduces the separation of SEMA4C genetic expression in tumour cell, and described nucleic acid molecule comprises:
A) double-stranded RNA, in described double-stranded RNA, containing can be under stringent condition and the Nucleotide of SEMA4C gene recombination
Sequence; Perhaps
B) shRNA, in described shRNA, containing can be under stringent condition and the nucleotide sequence of SEMA4C gene recombination.
Further, described double-stranded RNA comprises the first chain and the second chain, the complementary common formation RNA dimer of described the first chain and described the second chain, and in the sequence of described the first chain and SEMA4C gene, 15-27 continuous nucleotide sequence is basic identical.Preferably, in the sequence of described the first chain and SEMA4C gene, 19-23 continuous nucleotide sequence is basic identical; Better, in the sequence of described the first chain and SEMA4C gene, 19,20 or 21 continuous nucleotide sequences are basic identical.
Further, described double-stranded RNA comprises the first chain and the second chain, the complementary common formation RNA dimer of described the first chain and described the second chain, and the target sequence in the sequence of described the first chain and SEMA4C gene is basic identical.
Further, described shRNA comprises positive-sense strand fragment and antisense strand fragment, and the loop-stem structure that connects described positive-sense strand fragment and antisense strand fragment, the sequence complementation of described positive-sense strand fragment and described antisense strand fragment, and in the sequence of described positive-sense strand fragment and SEMA4C gene, 15-27 continuous nucleotide sequence is basic identical.Described shRNA can become siRNA (siRNA) and then play the effect of endogenous SEMA4C genetic expression in the reticent tumour cell of specificity after processing.
Further, described shRNA comprises positive-sense strand fragment and antisense strand fragment, and the loop-stem structure that connects described positive-sense strand fragment and antisense strand fragment, the sequence complementation of described positive-sense strand fragment and described antisense strand fragment, and the target sequence in the sequence of described positive-sense strand fragment and SEMA4C gene is basic identical.
Target sequence in the first chain of described double-stranded RNA or the positive-sense strand fragment of described shRNA and SEMA4C gene is basic identical, when the target sequence of described SEMA4C gene is siRNA for the reticent SEMA4C genetic expression of specificity, by the fragment in the corresponding SEMA4C gene of mRNA fragment of described siRNA identification silence.
Preferably, the target sequence in described SEMA4C gene contains the arbitrary sequence in SEQ IDNO:1-18.
Further, described SEMA4C gene source is in the people.
The length of described double-stranded RNA the first chain and the second chain is 15-27 Nucleotide; Preferably, length is 19-23 Nucleotide; Best, length is 19,20 or 21 Nucleotide.
Further, described double-stranded RNA is siRNA (siRNA).Further, the sequence of described siRNA the first chain, as shown in SEQ ID NO:31, is specially 5 '-GACUUGAUGGAGCCACCUAUA-3 '.
SiRNA shown in SEQ ID NO:31 for take the sequence shown in SEQ ID NO:12 be RNA disturb the target sequence design, for a chain of the siRNA of human SEMA 4 C gene, the sequence of the second chain and the first chain-ordering complementation, this siRNA can play the effect of endogenous SEMA4C genetic expression in the reticent tumour cell of specificity.
Further, the sequence of the loop-stem structure of described shRNA can be selected from following arbitrary: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU and CCACACC.
Further, the sequence of described shRNA, as shown in SEQ ID NO:19, is specially: 5 '-GACUUGAUGGAGCCACCUAUAUUCAAGAGAUAUAGGUGGCUCCAUCAAGUC-3 '.
ShRNA can become siRNA after enzyme is cut processing, and then plays the effect that in the reticent tumour cell of specificity, the endogenous human SEMA 4 C gene is expressed.
The interference lentiviral vectors of gene fragment of shRNA of the present invention of encoding contains arbitrary sequence and the complementary sequence thereof in SEQ ID NO:1-18.
Third aspect present invention, disclose a kind of SEMA4C gene interfere RNA construct, and the gene fragment of the shRNA in the nucleic acid molecule that contains the separation of the present invention of encoding, can express described shRNA
Described human SEMA 4 C gene interfere RNA construct can be the gene fragment clone of the aforementioned human SEMA 4 C gene shRNA of coding to be entered to known carrier obtain.Further, described SEMA4C gene interfere RNA construct is that the SEMA4C gene disturbs lentiviral vectors.
It is the DNA fragmentation of the aforementioned SEMA4C gene shRNA of coding to be cloned into to known carrier obtain that SEMA4C gene of the present invention disturbs lentiviral vectors, described known carrier mostly is lentiviral vectors, described SEMA4C gene disturbs lentiviral vectors after the virus packing becomes infectious virion, infected tumor's cell, and then transcribe out shRNA of the present invention, cut the steps such as processing by enzyme, finally obtain described siRNA, for the expression of the reticent SEMA4C gene of specificity.
Further, described SEMA4C gene disturbs lentiviral vectors also to contain the nucleotide sequence of marker that can be detected in promoter sequence and/or codes for tumor cell; Preferably, described marker that can be detected is as green fluorescent protein (GFP).
Further, described lentiviral vectors can be selected from: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti6-GW/U6-laminshrna, pcDNA 1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, arbitrary in pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.
The embodiment of the present invention has specifically been enumerated and take the human SEMA 4 C gene that pGCSIL-GFP is vector construction and disturb lentiviral vectors, called after pGCSIL-GFP-SEMA4C-siRNA.
The nucleic acid molecule that the present invention separates can be used for the medicine of preparation prevention or treatment tumour, and described tumour is colorectal carcinoma or cancer of the stomach.
SEMA4C gene siRNA of the present invention can be used for the propagation of inhibition tumor cell, further can be as medicine or the preparation for the treatment of tumour.The SEMA4C gene disturbs lentiviral vectors to can be used for preparing described SEMA4C gene siRNA.When the medicine as the treatment tumour or preparation, be that the described nucleic acid molecule of safe and effective amount is applied to Mammals.Concrete dosage also should be considered the factors such as route of administration, patient health situation, and these are all within the skilled practitioners skill.
Fourth aspect present invention, disclose a kind of SEMA4C gene and disturbed slow virus, by aforementioned SEMA4C gene, disturbs lentiviral vectors under slow virus packaging plasmid, clone auxiliary, through the virus packing, forms.But this slow virus infected tumor cell also produces the small molecules interference RNA for the SEMA4C gene, thereby suppresses the propagation of intestinal cancer or cancer of the stomach tumour cell.This SEMA4C gene disturbs slow virus to can be used for the medicine of preparation prevention or treatment tumour.
Fifth aspect present invention, disclose a kind of pharmaceutical composition for prevention or treatment tumour, the nucleic acid molecule that its active substance contains aforesaid separation, and SEMA4C gene interfere RNA construct, and/or the SEMA4C gene disturbs slow virus.
Further, described pharmaceutical composition contains the described double-stranded RNA of 1～99wt%, shRNA, SEMA4C gene interfere RNA construct or SEMA4C gene and disturbs slow virus, and pharmaceutically acceptable carrier, thinner or vehicle.
In preparation during these compositions, usually by activeconstituents and mixed with excipients, or with the vehicle dilution, wrap in can capsule or the carrier that exists of anther sac form in.Do the used time when vehicle plays thinner, 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 arbitrary anti-tumor medicine of preparation treatment colorectal carcinoma or cancer of the stomach.
The treatment that is applied as tumour of this pharmaceutical composition provides a kind of method, is specially a kind of method of prevention or treatment target in-vivo tumour, comprises the described pharmaceutical composition of effective dose is applied in object.Further, described tumour is selected from the arbitrary of colorectal carcinoma, cancer of the stomach.
When described pharmaceutical composition is used for prevention or treatment target in-vivo tumour, the described pharmaceutical composition of effective dose need to be applied in object.Adopt the method, the growth of described tumour, propagation, recurrence and/or shift suppressed.Further, at least 10% of the growth of described tumour, propagation, recurrence and/or transfer, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% part is suppressed.
Further, adopt the object of the method to behave.
Sixth aspect present invention, the RNA that discloses a kind of SEMA4C gene of separation disturbs target sequence, and the fragment that described RNA disturbs target sequence to comprise the SEMA4C gene, except the nucleotide fragments of total length SEMA4C gene.
Preferably, described RNA disturb in the nucleotide sequence of target sequence and total length SEMA4C gene at least 10,15,20,25,30,35,40,45 or 50 continuous Nucleotide substantially identical.
Preferably, described RNA interference target sequence is any sequence in SEQ ID NO:1-18.
The RNA of the SEMA4C gene of described separation disturbs target sequence to can be applicable to screen anti-tumor medicine or preparation.Concrete, can to medicine or preparation, be screened described target sequence as effective object, using and find the medicine that can suppress or promote the human SEMA 4 C gene expression as the oncotherapy drug candidate.As screen the acquisition small molecules interference RNA, it is used as to the medicine with inhibition tumor cell proliferation function.Such as antibody drug, small-molecule drug etc. also can described target oligonucleotide fragment as effective object.
The RNA that the invention also discloses the SEMA4C gene disturbs the application of target sequence in arbitrary anti-tumor medicine of preparation treatment colorectal carcinoma, cancer of the stomach.
Seventh aspect present invention, a kind of test kit for reducing the SEMA4C genetic expression in tumour cell is disclosed, described test kit comprises: be present in the nucleic acid molecule of the described separation in container, and SEMA4C gene interfere RNA construct, and/or described SEMA4C gene disturbs slow virus.
In sum, the present invention has designed 18 RNAi target sequences for human SEMA 4 C gene, build corresponding SEMA4C RNAi carrier, wherein the RNAi carrier pGCSIL-GFP-SEMA4C-siRNA of encoding sequence SEQ ID NO.12 can significantly lower the expression of SEMA4C gene at mRNA level and protein level.Use slow virus (lentivirus, be abbreviated as Lv) carry RNAi carrier pGCSIL-GFP-SEMA4C-siRNA as the genetic manipulation instrument and can will efficiently import for the RNAi sequence of SEMA4C gene human colon carcinoma RKO cell and cancer of the stomach SGC7901 cell in target ground, reduce the expression level of SEMA4C gene, significantly suppress the multiplication capacity of above-mentioned tumour cell.Therefore the SEMA4C gene silencing of lentivirus mediated is the potential clinical non-operative treatment mode of malignant tumour.
The carrier, slow virus that siRNA provided by the invention or bag can be expressed this siRNA sequence can specificity suppresses the expression of human SEMA 4 C gene, especially slow virus, can efficiently infect target cell, suppress expeditiously the expression of SEMA4C gene in target cell, and then the growth of inhibition tumor cell, promote apoptosis of tumor cells, significant in oncotherapy.
The accompanying drawing explanation
Fig. 1: pGCSIL-GFP plasmid DNA collection of illustrative plates
Fig. 2: the SEMA4C-RNAi slow virus was infected human colon carcinoma RKO cell and cancer of the stomach SGC7901 cell after 5 days, and the expression level of SEMA4CmRNA significantly reduces
Fig. 3: the SEMA4C-RNAi slow virus was infected human colon carcinoma RKO cell after 5 days, caused cell inhibitory effect
Fig. 4: the SEMA4C-RNAi slow virus was infected people's cancer of the stomach SGC7901 cell after 5 days, caused cell inhibitory effect
Fig. 5: (a, b, c are colorectal carcinoma to use the immunohistochemical methods detected result of SEMA4C antibody on different tumor tissues tissue samples; D, e, f are cancer of the stomach)
Embodiment
The present invention relates to one group of small molecules interference RNA for human SEMA 4 C gene (siRNA) sequence, rna interference vector and RNA and disturbed slow virus.Choose the target site of human SEMA 4 C mRNA coding region sequence as siRNA, according to the preferred 15-27 of 10-30(continuous in target site, more preferably 19-23) individual base sequence design siRNA target sequence.By gene clone, the nucleic acid construct of the above-mentioned siRNA of construction expression, the slow virus of the above-mentioned siRNA of packaging expression.Cell experiment proves, above-mentioned siRNA sequence can the reticent human tumor cells of specificity in the expression of endogenous SEMA4C gene.
The contriver is synthetic and tested the multiple siRNA for the SEMA4C gene, has filtered out the expression that can effectively suppress SEMA4C and then the siRNA that suppresses human colon carcinoma RKO cell and cancer of the stomach SGC7901 cell proliferation and growth, has completed on this basis the present invention.
The invention provides siRNA (siRNA) sequence of a series of interference human SEMA 4 C genes, but built the slow virus of the reticent SEMA4C genetic expression of specificity.The present invention studies discovery, for siRNA and the RNAi slow virus of human SEMA 4 C gene design, stablizes the expression of also lowering specifically the SEMA4C gene, and effectively suppresses the propagation of human tumor cells.The present invention shows that the SEMA4C gene can promote growth of tumour cell, is expected to become the target spot of early diagnosis of tumor and treatment.And, by the expression of the reticent SEMA4C gene of RNAi mode, can be used as the effective means that suppresses tumor development.
Mentality of designing of the present invention is:
The present invention screens by the following method and obtains a kind of human SEMA 4 C gene RNAi slow virus: transfer the human SEMA 4 C gene sequence from Genbank; Prediction siRNA site; The synthetic effective siRNA sequence for the SEMA4C gene, two ends are containing the double-stranded DNA Oligo of restriction enzyme site cohesive end; Be connected the RNAi plasmid of construction expression SEMA4C gene siRNA sequence after the lentiviral vectors double digestion with double-stranded DNA Oligo; RNAi plasmid and slow virus are packed to assistant carrier (Packing Mix, Sigma-aldrich company) the cotransfection HEKC 293T needed, produce the recombinant slow virus particle, can make the slow virus of efficient reticent SEMA4C gene.
Based on aforesaid method, the invention provides 18 Effective target sites (specifically as shown in SEQ ID NO 1-18) that disturb the SEMA4C gene, built the slow virus of special interference human SEMA 4 C gene.
The present invention simultaneously also discloses a kind of human SEMA 4 C gene RNAi slow virus (SEMA4C-RNAi) and preparation and application thereof.
This research is found, utilizes the RNAi method of lentivirus mediated, after reducing the expression of SEMA4C gene in tumour cell, and the effective propagation of inhibition tumor cell.This research shows, the SEMA4C gene is a proto-oncogene, can promote tumor cell proliferation, in occurring and develop, tumour there is important biological function, the SEMA4C gene can be the target of oncotherapy, and the SEMA4C gene specific silence of lentivirus mediated can be used as a kind of new tool of oncotherapy.
Further set forth the present invention below in conjunction with embodiment.Should be understood that embodiment is only for the present invention is described, but not limit the scope of the invention.In embodiment, the reagent of the experimental technique of unreceipted actual conditions and undeclared formula is according to normal condition, as works such as ［ U.S. ］ Sambrook.J; Huang Peitang etc. translate.The molecular cloning test guide, the third edition.Beijing: the condition of the condition described in Science Press 2002 or manufacturers's suggestion is carried out or configures.
1. screening is for the effective siRNA target spot of human SEMA 4 C gene
Transfer SEMA4C(NM 017789.4 from Genbank) gene information; Utilize the effective siRNA target spot of the design software Genechem design of Shanghai JiKai Gene Chemical Technology Co., Ltd for the SEMA4C gene.In encoding sequence (CDS) zone of SEMA4C gene, every the sequence of 21 bases of an initial acquisition of base, table 1 has been listed wherein 18 effective siRNA target sequences for the SEMA4C gene.
Table 1 target is in the siRNA of human SEMA 4 C gene target sequence
2. the preparation of lentiviral vectors
Double-stranded DNA Oligo sequence (table 2) for the synthetic two ends of siRNA target spot (the SEQ ID NO:12 of take is example) containing Age I and EcoR I restriction enzyme site cohesive end; (Shanghai JiKai Gene Chemical Technology Co., Ltd provides, and Fig. 1), makes its linearizing, and agarose gel electrophoresis is identified endonuclease bamhi to act on the pGCSIL-GFP carrier with Age I and EcoR I restriction enzyme.
Table 2 two ends are containing the double-stranded DNA Oligo of Age I and EcoR I restriction enzyme site cohesive end
By the T4 DNA ligase, by the double digestion linearizing, (it is as shown in table 4 that enzyme is cut system, 37 ℃, reaction 1h) the carrier DNA double-stranded DNA Oligo good with purifying is connected, and in suitable buffer system (linked system is as shown in table 5), in 16 ℃ of connections, spends the night, and reclaims and connects product.
Transform fresh competent escherichia coli cell (conversion operation reference: molecular cloning experiment guide second edition 55-56 page) prepared by calcium chloride by connecting product.Grow bacterium clone surface at the connection converted product and be stained with, be dissolved in 10 μ l LB substratum, mix and get 1 μ l as template; The upstream and downstream of RNAi sequence in lentiviral vectors, design universal PC R primer (upstream primer sequence: 5 '-CCTATTTCCCATGATTCCTTCATA-3 ' (SEQ ID NO:20); Downstream primer sequence: 5 '-GTAATACGGTTATCCACGCG-3 ' (SEQ ID NO:21)), carry out PCR identification experiment (the PCR reaction system is as table 6-1, and reaction conditions is as table 6-2).PCR is identified to positive clone is checked order and compare of analysis, compare correct clone and be the RNAi carrier that contains SEQ ID NO.12 successfully constructed, called after pGCSIL-GFP-SEMA4C-siRNA.
Build pGCSIL-GFP-Scr-siRNA negative control plasmid, negative control siRNA target sequence is 5 '-TTCTCCGAACGTGTCACGT-3 ' (SEQ ID NO:22).While building pGCSIL-GFP-Scr-siRNA negative control plasmid, double-stranded DNA Oligo sequence (table 3) for the synthetic two ends of Scr siRNA target spot containing Age I and EcoR I restriction enzyme site cohesive end, all the other construction processs, authentication method and condition be same pGCSIL-GFP-SEMA4C-siRNA all.
Table 3 two ends are containing the double-stranded DNA Oligo of Age I and EcoR I restriction enzyme site cohesive end
Table 4 pGCSIL-GFP plasmid enzyme restriction reaction system
Table 5 carrier DNA and double-stranded DNA Oligo ligation system
Table 6-1 PCR reaction system
Table 6-2 PCR reaction system program setting
3. slow virus is packed
Extract the DNA of RNAi plasmid pGCSIL-GFP-SEMA4C-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, the DMEM perfect medium adjustment cell density that contains 10% foetal calf serum of take is 1.5 * 10
5cell/ml, be inoculated in 6 orifice plates, and 37 ℃, 5%CO
2in incubator, cultivate.When reaching 70%-80%, cell density can be used for transfection.2h before transfection, the original substratum of sucking-off, add the perfect medium that 1.5ml is fresh.Explanation according to the MISSION Lentiviral Packaging Mix test kit of Sigma-aldrich company, add Packing Mix(PVM in a sterilizing centrifuge tube) 20 μ l, PEI 12 μ l, serum-free DMEM substratum 400 μ l, get the plasmid DNA of the above-mentioned extracting of 20 μ l, add to above-mentioned PVM/PEI/DMEM mixed solution.
Above-mentioned transfection miscellany is at room temperature hatched to 15min, be transferred in the substratum of HEKC 293T cell, 37 ℃, 5%CO
2cultivate 16h in incubator.Discard the developing medium that contains the transfection miscellany, the PBS solution washing, add perfect medium 2ml, continues to cultivate 48h.The collecting cell supernatant liquor, Centricon Plus-20 centrifugal ultrafiltration device (Millipore) purifying and concentrated slow virus, step is as follows: (1) 4 ℃, the centrifugal 10min of 4000g, remove cell debris; (2) 0.45 μ m filter filtering supernatant are in 40ml ultracentrifugation pipe; (3) 4000g is centrifugal, and 10-15min, to the concentrated volume of the virus needed; (4) after centrifugal end, filtering cup and following filtered solution collection cups are separated, filtering cup is tipped upside down on the sample collection cup, centrifugal 2min centrifugal force is no more than 1000g; (5) Centrifuge Cup is removed from the sample collection cup, in the sample collection cup, be viral concentrated solution.By after viral concentrated solution packing in-80 degrees centigrade of preservations.Virus contains the virion that can express shRNA as shown in SEQID NO:19 in concentrated solution, and the shRNA(SEQ ID NO:19 of this expressing viral) in vivo after processing, can obtain siRNA(the first chain-ordering as shown in SEQ ID NO:31).The wrapping process of contrast slow virus, with the SEMA4C-siRNA slow virus, only replaces the pGCSIL-GFP-SEMA4C-siRNA carrier with the pGCSIL-GFP-Scr-siRNA carrier.
Human colon carcinoma RKO cell in logarithmic phase and cancer of the stomach SGC7901 cell carry out trysinization, and (cell count is about 5 * 10 to make cell suspension
4/ ml) be inoculated in 6 orifice plates, be cultured to the cytogamy degree and reach approximately 30%.According to infecting plural number (MOI, RKO:10, SGC7901:20) value, add the virus of embodiment 1 preparation of sufficient quantity, change substratum 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, the RNA reverse transcription is obtained to cDNA(reverse transcription reaction system in Table 7,42 ℃ of reaction 1h, then in 70 ℃ of water-baths, water-bath 10min makes the reversed transcriptive enzyme inactivation).
Adopt TP800 type Real time PCR instrument (TAKARA) to carry out the real-time quantitative detection.The primer of SEMA4C gene is as follows: upstream primer 5 '-ACGGTAGTACGGCGGTTC-3 ' (SEQ ID NO:23) and downstream primer 5 '-CTCGGTCTGGTTGTTCTTCC-3 ' (SEQ ID NO:24).Take house-keeping gene GAPDH as internal reference, and primer sequence is as follows: upstream primer 5 '-TGACTTCAACAGCGACACCCA-3 ' (SEQ ID NO:25) and downstream primer 5 '-CACCCTGTTGCTGTAGCCAAA-3 ' (SEQ ID NO:26).Press the proportional arrangement reaction system in table 8.
Table 7 reverse transcription reaction system
Table 8 Real-time PCR reaction system
Setting program is two-step approach Real-time PCR: 95 ℃ of denaturations, 15s; 95 ℃ of each step sex change afterwards, 5s; Annealing is extended 60 ℃, 30s; Carry out altogether 45 circulations.Read light absorption value in the extension stage at every turn.After PCR finishes, 95 ℃ of sex change 1min, then be cooled to 55 ℃, makes the abundant combination of DNA double chain.Since 55 ℃ to 95 ℃, each step increases by 0.5 ℃, keeps 4s, reads light absorption value simultaneously, makes melting curve.Adopt 2-
Δ Δ Ctanalytical method is calculated the gene expression abundance that has infected SEMA4C mRNA.Infect the cell of contrast virus (Lv-Scr-siRNA) in contrast.Experimental result (Fig. 2) shows, in human colon carcinoma RKO cell and cancer of the stomach SGC7901 cell, the expression level of SEMA4C mRNA has lowered 73.7% and 77.3%.
Human colon carcinoma RKO cell in logarithmic phase and cancer of the stomach SGC7901 cell carry out trysinization, and (cell count is about 5 * 10 to make cell suspension
4/ ml) be inoculated in 6 orifice plates, be cultured to the cytogamy degree and reach approximately 30%.According to infecting plural number (MOI, RKO:10, SGC7901:20), add the virus of sufficient quantity, change substratum after cultivation 24h, after time of infection reaches 5 days, collect each experimental group cell in logarithmic phase.The resuspended one-tenth cell suspension (2 * 10 of perfect medium
4/ ml), with cell density, be about 2000/hole, inoculate 96 orifice plates.Every group 5 multiple hole ，Mei hole 100 μ l.After completing plate, put 37 ℃, 5%CO
2incubator is cultivated.From bed board, second day starts, and detect and read plate once every day with Cellomics instrument (Thermo Fisher), and continuous detecting is read plate 5 days.By adjusting the input parameter of Cellomics arrayscan, calculate exactly the quantity of the cell with green fluorescence in each scanning orifice plate, data are added up to drawing, draw cell proliferation curve (result is as Figure 3-Figure 4).
Result shows, slow virus is infected each tumour of group after cells in vitro is cultivated 5 days, and rate of propagation significantly slows down, far below the rate of propagation of control group tumour cell, the vigor cell number has descended respectively 63.9% and 42.9%, shows that the SEMA4C gene silencing causes the tumor cell proliferation ability suppressed.
The test of SEMA4C gene overexpression in embodiment 4 tumour cells
1. experiment material
1) tissue samples: human colon carcinoma and stomach organization sample
2) SEMA4C antibody: purchased from Sigma company
2. test method:
Take out organization chip, organization chip is toasted 30 minutes in 60 ℃ of thermostat containers.Then to the organization chip dewaxing, dewaxing process is: dimethylbenzene 15 minutes, in the mixed liquid of dimethylbenzene: ethanol=1:1, in dehydrated alcohol, in 95% ethanol, in 85% ethanol, in 75% ethanol, soak in distilled water 10 minutes successively; Then configure fresh 3%H with distilled water or PBS
2o
2, room temperature sealing 10 minutes; Antigen retrieval high fire heating 0.01M sodium citrate buffer (pH6.0) in microwave oven is put into organization chip to boiling, and low fire maintains 20 minutes; After naturally cooling to room temperature, insert in distilled water and soak 10 minutes; 10% serum (TBS preparation) seals 30 minutes; Serum is abandoned in suction, does not wash and adds SEMA4C antibody (1:100 dilution) overnight incubation; TBS washes 2 times, each 5 minutes; Add the goat-anti rabbit two of HRP mark anti-, incubated at room 60 minutes; TBS washes 4 times, each 5 minutes; Add DAB dyeing, until aobvious light yellow, put into the distilled water termination reaction; Soak 30 seconds clear water rinsing 7-8 time with bush; The dehydration mounting, in 75% ethanol, 85% ethanol, 95% ethanol, dehydrated alcohol, dimethylbenzene: ethanol=1:1 mixes liquid, in dimethylbenzene, soaks successively and puts 5 minutes; After taking-up, drip the 30ul neutral gum, use the cover glass mounting, dry, observations, take pictures.
3. experimental result
Experimental result is shown in Fig. 5, and experimental result shows: use SEMA4C antibody to carry out the Immunohistochemical Expression detection to colorectal carcinoma and stomach organization, in the different tissues sample, can find the high expression level of SEMA4C gene coded protein.The representative of Fig. 5 Oxford gray is expressed positive.Based on this experimental result, think and can carry out the auxiliary diagnosis cancer by the expression that detects histocyte SEMA4C gene.
Claims (17)
1. the human SEMA 4 C gene of a separation is at arbitrary anti-tumor medicine of preparation or screening colorectal carcinoma, cancer of the stomach, or the purposes in arbitrary diagnosing tumor medicine of preparation colorectal carcinoma, cancer of the stomach.
2. a nucleic acid molecule that reduces the separation of SEMA4C genetic expression in tumour cell, described nucleic acid molecule comprises:
A) double-stranded RNA, in described double-stranded RNA, containing can be under stringent condition and the nucleotide sequence of SEMA4C gene recombination; Perhaps
B) shRNA, in described shRNA, containing can be under stringent condition and the nucleotide sequence of SEMA4C gene recombination.
3. the nucleic acid molecule of separation as claimed in claim 2, it is characterized in that, described double-stranded RNA comprises the first chain and the second chain, the complementary common formation RNA dimer of described the first chain and described the second chain, and the target sequence in the sequence of described the first chain and SEMA4C gene is basic identical; Described shRNA comprises positive-sense strand fragment and antisense strand fragment, and the loop-stem structure that connects described positive-sense strand fragment and antisense strand fragment, the sequence complementation of described positive-sense strand fragment and described antisense strand fragment, and the target sequence in the sequence of described positive-sense strand fragment and SEMA4C gene is basic identical.
4. the nucleic acid molecule of the described separation of claim as arbitrary as claim 2-3, is characterized in that, described SEMA4C gene source is in the people.
5. the nucleic acid molecule of separation as claimed in claim 4, is characterized in that, the target sequence of described SEMA4C gene contains arbitrary sequence in SEQ ID NO:1-18.
6. the nucleic acid molecule of separation as claimed in claim 4, is characterized in that, described double-stranded RNA is siRNA, and the sequence of this siRNA the first chain is as shown in SEQ ID NO:31.
7. the nucleic acid molecule of separation as claimed in claim 4, is characterized in that, the sequence of described shRNA is as shown in SEQ ID NO:19.
8. a SEMA4C gene interfere RNA construct, the gene fragment of the shRNA in the nucleic acid molecule that contains the described separation of the coding arbitrary claim of claim 2-7, can express described shRNA.
9. SEMA4C gene interfere RNA construct as claimed in claim 8, is characterized in that, described SEMA4C gene interfere RNA construct is for disturbing lentiviral vectors.
10. SEMA4C gene interfere RNA construct as claimed in claim 9, is characterized in that, described interference lentiviral vectors also contains the nucleotide sequence of marker that can be detected in promoter sequence and/or codes for tumor cell.
11. as SEMA4C gene interfere RNA construct as described in claim 9 or 10, it is characterized in that, described interference lentiviral vectors obtains after entering lentiviral vectors by the gene fragment clone of the described shRNA that will encode, 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, pLP 1, 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.
12. a SEMA4C gene disturbs slow virus,, is formed through the virus packing under slow virus packaging plasmid, clone auxiliary by the described interference lentiviral vectors of the arbitrary claim of claim 9-11.
13. the pharmaceutical composition for prevention or treatment tumour, the nucleic acid molecule that its active substance contains arbitrary described separation in claim 2-7, the described SEMA4C gene of the arbitrary claim of claim 8-11 interfere RNA construct, and/or the described SEMA4C gene of claim 12 disturbs slow virus.
14. the application of the described pharmaceutical composition of claim 13 in the anti-tumor medicine of preparation treatment colorectal carcinoma or cancer of the stomach.
15. the RNA of the SEMA4C gene of a separation disturbs target sequence, is arbitrary sequence in SEQ ID NO:1-18.
16. the RNA of the described SEMA4C gene of claim 15 disturbs the application of target sequence in the anti-tumor medicine of preparation treatment colorectal carcinoma or cancer of the stomach.
17. the test kit for reducing SEMA4C genetic expression in tumour cell, it is characterized in that, described test kit comprises: be present in container, the nucleic acid molecule of the described separation of arbitrary claim in claim 2-7, the described SEMA4C gene of the arbitrary claim of claim 8-11 interfere RNA construct, and/or the described SEMA4C gene of claim 12 disturbs slow virus.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105695562A (en) * | 2014-11-27 | 2016-06-22 | 中国科学院上海生命科学研究院 | Application of MST4 gene in diagnosis and cell therapy of infectious diseases and related drugs of MST4 gene |
| CN105695562B (en) * | 2014-11-27 | 2020-01-07 | 中国科学院上海生命科学研究院 | Use of MST4 gene diagnosis and cell treatment for infectious diseases and related medicine thereof |
| CN112843255A (en) * | 2021-03-18 | 2021-05-28 | 华中科技大学同济医学院附属同济医院 | Application of SEMA4C in preparation of antitumor drugs |
| CN114045291A (en) * | 2021-11-01 | 2022-02-15 | 武汉爱博泰克生物科技有限公司 | Recombinant human Sema4C protein, expression vector, host cell and application |
| CN114045291B (en) * | 2021-11-01 | 2022-07-19 | 武汉爱博泰克生物科技有限公司 | Recombinant human Sema4C protein, expression vector, host cell and application |
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