CN1948483A - SiRNA for inhibiting human Rabj gene expression and its application - Google Patents
SiRNA for inhibiting human Rabj gene expression and its application Download PDFInfo
- Publication number
- CN1948483A CN1948483A CNA2005100304281A CN200510030428A CN1948483A CN 1948483 A CN1948483 A CN 1948483A CN A2005100304281 A CNA2005100304281 A CN A2005100304281A CN 200510030428 A CN200510030428 A CN 200510030428A CN 1948483 A CN1948483 A CN 1948483A
- Authority
- CN
- China
- Prior art keywords
- rabj
- small molecules
- interference rna
- molecules interference
- sirna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
This invention relates to an interfering RNA nucleotide sequence aiming directly at oncogene-like molecule RabJ. Exactly to say, this invention relates to a interfering RNA nucleotide sequence which aims directly at the expression and function of oncogene-like molecule RabJ, the interfering RNA nucleotide sequence possesses gene expression and function of target anti- tumour cell RabJ, having the function of restraining tumour cell to grow and oncogenicity of tumour cell, and having the function of promoting apoptosis, thus contributing to cure tumor. This invention also relates to medicine Combination containing this interfering RNA nucleotide sequence, and discloses the method of using this medicine of interfering RNA for disease therapy, especially it is used to cure hyper-expressed malignant tumor.
Description
Technical field
The invention belongs to biotechnology and medical field.Particularly, the present invention relates to a kind of at the expression of oncogene RabJ and the RNA interfering nucleotide sequence of function, this RNA interfering nucleotide sequence has the function of the RabJ genetic expression of target antitumor cell and function, inhibition growth of tumour cell, the tumorigenicity that suppresses tumour cell and promotion apoptosis of tumor cells, thereby plays the effect of treatment tumour.The invention still further relates to the pharmaceutical composition that contains this RNA interfering nucleotide sequence, and disclose, particularly the purposes in crossing the treating malignant tumor of expressing RabJ the purposes of this RNA interfering medicine in oncotherapy.
Background technology
Small G-protein (small G proteins) is meant single subunit G albumen 1-5 of the about 20-30kDa of molecular weight.Present finishing along with the Human Genome Project, such proteinic member has had been found that kind more than 100, its total feature is can be in conjunction with GTP (guanosine triphosphate) (GTP) and guanosine diphosphate (GDP) (GDP), and can hydrolysis GTP γ position phosphoric acid becoming 6DP (is the GTP enzymic activity, GTPase activity) (Wittinghofer and Pai, Trends Biochem Sci.1991,16:382-387; Polakis and McCormick, J Biol Chem.1993,268:9157-9160; Moodie etc., Oncogene.1995,11:447-454; Marshall, Trends Biochem Sci.1993,18:250-254).Small G-protein is in regulating cell growth, propagation, differentiation, migration, and brings into play important effect in the basic vital movement such as intracellular protein transhipment.
The dysfunction of small G-protein can cause the numerous pathological phenomenons and the generation of disease, wherein Ras and tumour take place with and effect in the cell proliferation regulation and control particularly receive publicity.In the tumour cell or tumor tissues of nearly all type, the sudden change of H-Ras, K-Ras or N-Ras gene is all arranged, and in the gene that tumour is undergone mutation, probability the highest (Bos, the Cancer Res.1989 of Ras sudden change; 49:4682-4689).
Rab (rat brain Ras sample albumen, Ras-like protein in rat brain) be the small molecular weight GTPase that a class belongs to the Ras superfamily, its basic biochemical character is can be in conjunction with GTP/GDP and hydrolysis GTP (Simons and Zerial, Neuron.1993,11:789-799; Takai etc., Physiol Rev.2001,81:153-208; Martinez and Goud, Biochim Biophys Acta.1998; 1404:101-112; Stenmark and Olkkonen, Genome Biol.2001,2:REVIEWS3007).The unusual generation that also can cause disease of Rab albumen and modulin thereof and effect protein, disease (Griscelli syndrome), dysthymia disorders, Charcot-Marie-Tooth neurosis, the kidney disease (uriniferous tubules sclerosis) of and dyspigmentation hemorrhage as some, all the dysfunction with the Rab associated protein is relevant for the diseases such as (choroideremia diseases) of losing one's sight; In some pathologic process of blood vessel, lung, thyroid disease, often follow the proteic high expression level of Rab (Seabra etc., Trends Mol Med.2002,8:23-30).
In the research of Griscelli syndrome (a kind of cause unusually follow pigmentation and the unusual disease of T lymphocyte killing ability), find by pigment transhipment, the sudden change of Rab27a is the genetics cause of disease (Menasche etc. that this disease takes place, Blood.2003,101:2736-2742; Bahadoran etc., J BiolChem.2003; 278:11386-11392; Barral etc., J Clin Invest.2002,110:247-257).The proteic metabolic disturbance that can cause lipoprotein unusually of Rab7 causes hyperlipidaemia and vascular conditions; Some intracellular bacterias as tubercule bacillus, can reach infection host and morbific purpose (Runz etc., J Neurosci.2002,22:1679-1689 by the function that suppresses Rab7; Kim etc., Biochem Biophys Res Commun.2000,293:375-382; Choudhury etc., J ClinInvest.2002,109:1541-1550; Via etc., J Biol Chem.1997,272:13326-13331; Clemens etc., Infect Immun.2000,68:5154-5166; Rupper etc., J Cell Sci.2001,114:2449-2460).
Previous research thinks that Rab albumen and tumorigenic relation are not very close, but present research prompting Rab albumen may participate in the generation of tumour.At first, in some tumour cell or tumor type, find to have the proteic abnormal expression of Rab.Rab25 expression ratio in prostate cancer is higher, and differentiation of its expression level and prostate cancer, clinical stages etc. closely related (He etc., Gene Expr.2002,10:231-242); The expression of Rab38 in K-1735 also obviously raise (Jager etc., CancerRes.2000,60:3584-3591; Loftus etc., Proc.Natl.Acad.Sci.USA.2000,99:4471-4476); The expression of Rab3 in pituitary carcinoma also very high (Culine etc., Cancer.1992,70:2552-2556); In hematopoiesis is that the expression of Rab2 is obviously increased in tumour and the solid tumor patient peripheral blood mononuclear cell; In mouse adrenal carcinoma and mice lung cancer, the expression of Rab2 also show as unusually (Culine etc., Cancer Res.1992,52:3083-3088).Secondly, in tumour, find to have the deletion mutantion of Rab gene.Find to have the disappearance in karyomit(e) 1622q11.2 zone in malignant rhabdomyoma, this zone comprises coding region (Mori etc., Biochem Biophys Res Commun.1999, the 254:594-600 of Rab36; Zhou etc., Gene.2000,241:133-141).In addition, the unusual of Rab modulin also arranged in tumour.In neuroblastoma and hematopoiesis is in the tumour, and the expression of a proteic modulin RabGDI of Rab raises obviously.PRC17 (prostate cancer gene 17, prostate cancer gene 17) is a RabGTP enzyme activation albumen (GTPase activating protein, GAP), the proteic GTP hydrolysis of its adjustable Rab5, its expression in prostate cancer is increased, thereby influence the growth of tumor characteristic (Pei etc., CancerRes.2002,62:5420-5424).
People RabJ is the new small G-protein family member of dendritic cell who derives from healthy adult human peripheral blood mononuclear cell source, it is characterized in that in its protein is formed, comprising nuclear localization signal (the nuclear localization signal of three class functional domain: N ends (1-18 amino acid) and middle (210-216 amino acid), NLS), the J functional domain (217-273 amino acid) of intermediary Rab sample functional domain (19-209 amino acid) and C end, and higher homology is arranged with the Ras family molecule.Three class functional domains mediate the position of appraising and deciding of RabJ respectively; With extracellular signals-modulating kinases (extracellular signal-regulated kinase, ERK1/2) kinases (ERK kinase, MEK1/2), protein kinase C (protein kinase C, PKC), phosphatidylinositol 3-kinase (phosphatidylinositol 3-kinase, P85 subunit PI3K), the interaction of P53 subunit; With functions such as HSC70 and Raf (Ras correlation factor, Ras-associated factor) interactions.The nucleotide sequence of RabJ and aminoacid sequence and method for making are disclosed among the Chinese patent application CN01126826.3.
(RNA interference RNAi) is a kind of effective ways that seal genetic expression that newly-developed gets up in the RNA interference.It adopts RNA interfering (the smallinterfering RNA long with goal gene homologous 21-23 Nucleotide, siRNA) transfection is to target cell, induce silencing complex (RISC) with intracellular restriction endonuclease formation, RISC is the special identification of template its homologous gene mRNA with siRNA and it is carried out laddering shearing, form strong effectively water fall effect, the specific mRNA degraded of induced sequence, the defective phenotype of cell performance specific gene.This strategy can be closed the cancer gene, only has a base mutation then to lose the RNA interference effect, and is very little to normal impact cell, high specificity.
In present technical field, the medication of several gene orders of below listing all can be used for the administration of RNA interfering:
1. direct naked DNA injection
(1) new jet injecting systems discharges naked DNA treatment sequence, can adopt a kind of new jet injecting systems, and naked DNA is discharged in the intravital lung tumor.Wal plucked instrument (W.Walter) doctor and colleague have developed a kind of portable " high speed jet syringe " system, provide another selection outside viral vector and liposome gene release system.The investigator uses 3 sections naked DNAs of this system's injection in the Lewis lung tumor of mouse, and first plasmid expression beta galactose kinase gene (LacZ) is expressed green fluorescence (GFP) gene for second, expresses human tumor necrosis factor-alpha (TNF-α) for the 3rd.Each mouse is all accepted 5 injections, and pressure is 3Pa, discharges 3～5 microlitre plasmid DNA.Wal plucked instrument doctor etc. reports that at " gene therapy " (Gene Therpapy) gene on the tumour of injection back is wide expression.LacZ and GFP genetic expression became obviously in injection in back 48 hours, reached peak value at 72～96 hours; LacZ expresses and TNF-α secretion occurred at 24～120 hours.They think that " these discoveries have proved the suitability of jet injection, use the naked DNA of minimum dose to carry out gene therapy for cancer when promptly transmitting gene to tumour in vivo.”；
(2) naked DNA direct injection: naked plasmid dna is injected directly in the muscle, intracutaneous of body, subcutaneous, mucous membrane, intravenously, the knurl body.This method is simple;
2. liposome dna direct injection: the lipid physical efficiency of parcel DNA merges with histocyte generation film, and with the DNA absorption, has reduced the destruction of nuclease to DNA.Injecting pathway is with the naked DNA direct injection;
3. Jin Bao is by DNA particle gun blast technique: plasmid DNA is coated on golden micropartical surface, makes bag be penetrated histocyte at a high speed by the golden micropartical of DNA with particle gun;
4. the breeding unsoundness bacterium is carried the plasmid DNA method: select a kind of bacterium that enters certain histoorgan easily, its breeding gene is removed, transfer bacterium to plasmid DNA then, after these bacteriums enter certain histoorgan, because irreproducible, then self cracking and discharge plasmid DNA.That is: attenuation salmonella that can be oral;
5. replication defective adenoviral carries the target DNA method: select a kind of replication-defective adenoviral that enters certain histoorgan easily, carry purpose RNA interference sequence, approach such as can pass through in muscle, intracutaneous, subcutaneous, mucous membrane, intravenously, the knurl body at host's expression in vivo purpose RNA interference sequence.
In recent years, though in clinical trial and using, RNA disturbs and is obtaining some progress aspect some tumour of treatment and the viral infection, and obtained result is still very not satisfactory.Therefore, this area presses for further exploitation specificity, high efficiency, can effectively suppress the RNA interference medicament of tumour.
Summary of the invention
Technical problem to be solved by this invention is just providing a kind of antineoplastic siRNA, described siRNA is specifically at the horizontal target oncogene of mRNA sample molecule RabJ gene, biological behaviour to the tumour cell of expressing RabJ is intervened, thereby effectively suppress the expression of RabJ, reach antitumous effect.
In a first aspect of the present invention, a kind of small molecules interference RNA at the people Rab GTP enzyme RabJ that has the J functional domain is provided, described small molecules interference RNA and people RabJ gene mRNA reverse complemental, its length is 16-30bp, and described small molecules interference RNA can make the growth of HeLa cell reduce more than 50%.
Better, the length of described small molecules interference RNA is 18-25bp.
In another preference, described small molecules interference RNA is incorporated into the corresponding nucleotide sequences with lower area of RabJ: nuclear localization signal, Rab functional domain, J functional domain or its combination.
In another preference, described small molecules interference RNA contains and is selected from the nucleotide sequence shown in SEQ ID NO:1 or the SEQ IDNO:2.
Preferable, described small molecules interference RNA is selected from down group:
si-RabJ1：5’-GAUUCGUGUCUAAAUACCU-3’
3’-CUAAGCACAGAUUUAUGGA-5’；
si-RabJ2：5’-UAGCAGUGCUAGUUUCACC-3’
3’-AUCGUCACGAUCAAAGUGG-5’。
Better, 3 ' end of described small molecules interference RNA also contains 2-4 dT or contains the extended structure of 2-6 U.
A second aspect of the present invention relates to a kind of composition, and it contains the foregoing small molecules interference RNA of 0.001-99.99wt% and acceptable carrier, thinner or vehicle.
Preferably, described composition is a pharmaceutical composition, and described carrier, thinner or vehicle are pharmaceutically acceptable carrier, thinner or vehicle.
In a third aspect of the present invention, provide the purposes of foregoing small molecules interference RNA, described small molecules interference RNA is used to prepare the pharmaceutical composition that suppresses growth of tumour cell composition or treatment tumour.More preferably, described tumour cell or tumour are expressed RabJ albumen, and are selected from down group: cervical cancer, mammary cancer, colorectal carcinoma.
Description of drawings
Fig. 1: after RabJ siRNA disturbed RabJ to express, RabJ was at the expression analysis of mRNA and protein level, and among the figure, mock is for only adding the negative control of transfection reagent;
Fig. 2: after RabJ siRNA disturbs RabJ to express, to the growth-inhibiting of tumour cell;
Fig. 3: after RabJ siRNA disturbed RabJ to express, to the inhibition of tumour cell cell cycle progression, among the figure, U0126 was a kind of clinical antitumor drug that can suppress the tumour cell cycle process that entered, as positive control (buying from Sigma company);
Fig. 4: after RabJ siRNA disturbs RabJ to express, to the inhibition of the external tumorigenicity of tumour cell;
Fig. 5: after RabJ siRNA disturbs RabJ to express, to the inhibition of tumorigenicity in the tumour cell body.
Embodiment
The inventor finds that through extensive and deep research the expression of RabJ in the tumour cell often raises, and the expression that suppresses RabJ can suppress the propagation of tumour cell and the oncogenic activity of inside and outside.The inventor is also further synthetic and tested the siRNA of multiple RabJ, but has filtered out the expression of strongly inhibited RabJ and then suppressed the propagation of tumour cell and the siRNA sequence of the tumorigenicity of inside and outside.Finished the present invention on this basis.
Particularly, the inventor studies show that, RabJ mainly is distributed in testis tissue in primary and secondary spermatocyte (spermatocyte), the early stage spermatid (spermatids), in mesenchymal cell and sustenticular cell, do not have and express, similar with cyclin cyclin A and the expression pattern of cyclin D1 in mouse testis, prompting RabJ may be relevant with cell cycle regulating.
In NIH3T3 clone, cross expressing of RabJ can promote cyclin to express, promote cell proliferation, promote cell cycle progression, and can promote this clone on soft agar, to form the clone, and in nude mouse, also can be formed into fibrosarcoma, pointing out it may be the small G-protein of an oncogene sample.
Research of the present invention finds that also the expression of RabJ often raises in tumour cell, and the expression that suppresses RabJ can suppress the propagation of tumour cell and the oncogenic activity of inside and outside.Because RabJ can form complex body with multiple protein, therefore suppress each kinase whose activity with kinase inhibitor, find that the MEK/ERK inhibitor can suppress the external tumorigenesis ability of RabJ fully, the signal transduction mechanism that prompting ERK1/2 relies on may be the tumorigenesis mechanism of RabJ.
The test-results of Laser Scanning Confocal Microscope is also pointed out, and RabJ is that the nuclear anchor of a MEK1/2 albumen, by increasing the level of nuclear phosphorylation MEK1/2, in the local continuous activation that forms ERK1/2 of karyon, causes the generation of tumour.
Therefore, the inventor studies show that, RabJ plays an important role in processes such as cell cycle regulating, cell proliferation, tumour generation, and its mechanism may be the effect of the MEK1/2 promotion ERK1/2 mediation of phosphorylation by the nuclear anchor, promotes the generation of cell proliferation and tumour.Therefore, RabJ is the small G-protein of an oncogene sample, can be used as potential candidate target in the diagnosis of clinical tumor and treatment.
Activeconstituents
In the present invention, activeconstituents is the siRNA sequence of RabJ.As used herein, term " activeconstituents " refers to the siRNA sequence of such RabJ: described siRNA and people RabJ gene mRNA reverse complemental, its length is generally 16-30bp, more preferably is 18-25bp, and described siRNA can make the growth of HeLa cell reduce more than 50%.
The cDNA sequence of RabJ shown in SEQ ID NO:9, the coding RabJ protein (shown in SEQ ID NO:10).SEQ ID NO:9 total length is 1787bp, comprises 5 ' the end non-coding region of 24bp and 3 ' the end non-coding region of 316bp, and coding contains 273 amino acid whose polypeptide.
As used herein, " siRNA of RabJ " is meant and the small segment RNA interfering of RabJ dna homolog that its length is generally 16-30bp, more preferably is 18-25bp.But the siRNA transfection is to tumour cell, induce silencing complex (RISC) with intracellular restriction endonuclease formation, RISC is the special identification RabJ gene mRNA of template with this siRNA and it is carried out laddering shearing, form strong effectively water fall effect, the specific mRNA degraded of induced sequence, make the defective phenotype of tumour cell performance RabJ gene, thereby suppress tumour cell and growth of tumor.
One of focus of oncotherapy when carrying out the specific gene silence at oncogene, the RNA perturbation technique is the effective technology of present high specific, no matter is at functional genome research therefore, or gene treatment aspect, tumour ground all has application prospect extensively.The inventor has designed many siRNA according to known siRNA principle of design (as people's such as Cenix principle of design) at the different target sites of RabJ gene.Wherein, 2 expression of disturbing siRNA sequence (si-RabJ1 and si-RabJ2) can suppress oncogene sample molecule RabJ effectively.This confirms that the siRNA of RabJ is expected to become the effective means of treatment tumour.SiRNA of the present invention can use separately or several siRNA associatings, can also unite with other medicines and treatment means, is used for the treatment of malignant tumour.
RNA interference sequence si-RabJ1 that the present invention is selected and si-RabJ2 can obtain by modes such as direct chemosynthesis, in-vitro transcription, plasmid amplification, virus replications, so the present invention should comprise the aforementioned applications form that comprises this sequence.
The siRNA sequence of RabJ of the present invention can effectively be sealed the RabJ gene, thereby suppresses the propagation of tumour cell.Particularly, RabJsiRNA sequence of the present invention, reverses the biological behaviour of the tumour cell of positive expression RabJ at the RabJ gene in the mRNA level.Experimental results show that: the expression of (1) inhibition RabJ can suppress the propagation of tumour cell; (2) the interior expression that suppresses RabJ of body can suppress the external tumorigenicity of tumour cell; (3) expression that suppresses RabJ can suppress the intravital growth of tumour cell; (4) expression that suppresses RabJ can suppress the tumour cell cycle process.
Pharmaceutical composition and medication
The present invention also provides a kind of pharmaceutical composition, and it contains siRNA sequence and pharmaceutically acceptable carrier or the vehicle of the RabJ of the present invention of safe and effective amount (as 0.001-99wt%).This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.
When making pharmaceutical composition, be that siRNA sequence with the RabJ of safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 1 microgram/kg body weight, and in most of the cases be no more than about 5 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
When each pharmaceutical composition of system, usually, these siRNA sequences of the present invention can be formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, preferably pH is about 6-8, although the pH value can change to some extent with being prepared Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): in the knurl, intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
The administering mode of preferred siRNA sequence medicine of the present invention includes but not limited to: (1) is the naked DNA injection directly, as utilizes jet injecting systems to discharge naked DNA treatment sequence, naked DNA direct injection; (2) liposome dna direct injection; (3) Jin Bao is by DNA particle gun blast technique; (4) the breeding unsoundness bacterium is carried the plasmid DNA method; (5) replication defective adenoviral carries the target DNA method.
In addition, siRNA sequence of the present invention can be directly used in disease treatment separately, also can with other treatment agent coupling, as TNF-α, TGF-β, IFN-α, Angiostatin, Endostatin, Glyfosfin, haematoporphyrin, lycobetaine, the kosam seeds breast, etoposide (being etoposide), the dehydration galactitol, Zorubicin, tamoxifen, 5 FU 5 fluorouracil, remove first spot chela element, Tegadifur, cucurbitacin, harringtonine, rubescensine B, Irisquinone A, polysaccharide-peptide, cytosine arabinoside, NSC-241240, taxol, lentinan, flutamide, ifosfamide, ubenimex, leuprorelin acetate, doxifluridine, Glass platinum, Yi Linnuoteken, bend azoles and Vumon etc.
Major advantage of the present invention is:
(a) the present invention is directed to the designed siRNA sequence of the different target sites of RabJ gene, can effectively suppress the expression of oncogene sample molecule RabJ, thereby be used for cancer therapy;
(b) siRNA sequence of the present invention can be used separately or several siRNA sequence association, can also unite with other medicines and treatment means, is used for the treatment of malignant tumour.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Co1d Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Material and method
(a)siRNA
The siRNA sequence of RabJ is as follows:
si-RabJ1：5’-GAUUCGUGUCUAAAUACCU-3’
3 '-CUAAGCACAGAUUUAUGGA-5 ' (+119-+137 position) (SEQ ID NO:1);
Si-RabJ2：5’-UAGCAGUGCUAGUUUCACC-3’
3 '-AUCGUCACGAUCAAAGUGG-5 ' (+588-+606 position) (SEQ ID N0:2).
The sudden change negative control sequence of the siRNA sequence of RabJ is as follows:
Scon?si-RabJl：
(SEQ ID NO:3) (+119-+137 sequence negative control);
Scon?si-RabJ：
3 '-AUCCUCACGAUCAUAGUGG-5 ' (SEQ ID NO:4) (+588-+606 sequence negative control).
All siRNA sequences are synthetic by Shanghai Ji Kai bio-engineering corporation.
(b) clone
HeLa clone: ATCC:CCL-2 (available from ATCC), this is the cervical cancer tumor cell line of a kind of positive expression RabJ.
MCF-7 clone: ATCC:HTB-22 (available from ATCC), this is the breast cancer tumour clone of a kind of positive expression RabJ.
SW480 clone: ATCC:CCL-28 (available from ATCC), this is the colorectal carcinoma tumor cell line of a kind of positive expression RabJ.
The cultivation and the treatment process of cell are as follows: in RPMI1640 (Invitrogen company) nutrient solution that contains 10% calf serum, placing 37 degrees centigrade, volume fraction is 5% CO with cell inoculation
2The conventional cultivation in the incubator, no medicine cultivated for two weeks before the experiment.Optimize transfection conditions, final concentration with 200nmol adds cell culture fluid with si-RabJ1 (SEQ ID NO:1), si-RabJ2 (SEQ ID NO:2), Scon si-RabJ1 (SEQID NO:3) and Scon si-RabJ2 (SEQ ID NO:4) respectively, and harvested cell detects after hatching 24～48h.
Embodiment 1:RabJ siRNA disturbs RabJ to express the expression analysis of back RabJ at mRNA and protein level
Respectively with si-RabJ1 (SEQ ID NO:1), si-RabJ2 (SEQ ID NO:2), sudden change negative control Scon si-RabJ1 (SEQ ID NO:3) and Scon si-RabJ2 (SEQ ID NO:4) with transfection reagent lipofectAMINE (Invitrogen company) transient transfection HeLa, MCF-7, SW480 cell, collect cell behind the 48h and detect.
Present embodiment detects si-RabJ1 and the si-RabJ2 inhibition to oncogene sample molecule RabJ genetic expression from mRNA and two levels of protein level.
(a) RT-PCR and PCR product quantitative analysis method detect mRNA
(1) RT-PCR: carry by the total RNA of TRIzol and to take out test kit and extract total RNA.Electrophoresis is identified the quality of RNA, and the UV-light spectrophotometer is quantitative.Get total RNA2.5 μ g, add 50 μ l reverse transcription reaction systems, put test kit (available from Invitrogen company), by specification operation carrying out reverse transcription with the SuperScriptTMII reverse transcription.
(2) pcr amplification: the PCR reaction system is (reagent is available from Invitrogen company) routinely.
RabJ primer 1:5 '-ATG CCG AAG AGG AAG GAG CCC-3 ' (SEQ ID NO:5);
RabJ primer 2: 5 '-GTT TCA CCA AAG AAC AAG CAG-3 ' (SEQ ID NO:6).
β-actin primer 1:5 '-GCATCGTGATGGACTCCG-3 ' (SEQ ID NO:7);
β-actin primer 2: 5 '-TCGGAAGGTGGACAGCGA-3 ' (SEQ ID NO:8).
Above sequence is given birth to worker bio-engineering corporation by Shanghai and is synthesized.
Cycling condition: 94 ℃ of sex change 45 seconds, 58 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, extended at last 10 minutes.Amplified production with β-actin is the confidential reference items contrasts in addition.
(3) the PCR product is quantitative: the amplified production of getting 10 μ l carries out electrophoresis on the agarose gel electrophoresis of 15g/L, with PCR mark (magnificent company) as the molecular mass standard, voltage 100V, 20 minutes.Ethidium bromide staining, developing, photograph on the ultraviolet reflectance instrument.Carry out the sxemiquantitative (expression level that reflects RabJ mRNA with the ratio of RabJ/ β-actin cDNA) of goal gene with scanning analysis.
(b) expression level of Western blot technology for detection RabJ protein expression
That collects 48h respectively organizes cell, the preparation cell pyrolysis liquid, and how anti-(by the method preparation of embodiment among the CN01126826.3 6) carries out Western blot detection with RabJ.Also can adopt methods such as immunofluorescence label and immunohistochemistry technology to detect the expression of RabJ protein level.
The result
The result shows, cell detects after siRNA handles 48h, compare with negative control group, the RabJ mRNA and the protein expression of si-RabJ1 and si-RabJ2 group obviously descend, show that si-RabJ1 and si-RabJ2 transfection successfully prevented the expression of RabJ, and negative control siRNA oligonucleotide (Scon si-RabJ1 and Scon si-RabJ2) does not influence (see figure 1) to the expression of RabJ.
After embodiment 2:RabJ siRNA disturbs RabJ to express, to the inhibition of growth of tumour cell
Present embodiment adopted [
3H]-thymus pyrimidine mixes method.
With HeLa, the MCF-7 of the transfection of RabJ siRNA described in the embodiment 1, SW480 cell with 5 * 10
4Individual cells/well is inoculated in 24 well culture plates (Falcon company), three holes of every kind of cell inoculation.After cultivating 6h, in serum free medium, continue to cultivate 24-48h, 4h in the end, every hole add 0.5 μ Ci (1Ci=37GBq) [
3H]-thymus pyrimidine (Amersham company).Then, give a baby a bath on the third day after its birth time with PBS, cell is dissolved among the PBS that contains 1%Triton X-100, collects with glass fiber filter, and radioactivity is measured with liquid scintillation instrument.Perhaps behind serum starvation 24h, add the normal substratum that contains 10%FCS, continue to cultivate 24h, last 4h adding [
3H]-thymus pyrimidine, carry out radioassay then.
The result shows that RabJ siRNA can obviously suppress HeLa, MCF-7, the external energy for growth of SW480 tumour cell, and the not influence of negative control siRNA oligonucleotide (Scon si-RabJ1 and Scon si-RabJ2) cell growth.Wherein, HeLa, MCF-7, SW480 cell inhibiting effect are seen Fig. 2.
After embodiment 3:RabJ siRNA disturbs RabJ to express, to the inhibition of tumour cell cell cycle progression
Present embodiment has adopted iodate third ingot (PI) marker DNA content detecting method.
HeLa, MCF-7, SW480 cell (5 * 10 with the transfection of RabJ siRNA described in the embodiment 1
6) with 0.25% pancreatin digest the back wash twice with PBS, slowly add 70% ice ethanol, 4 ℃ are fixedly spent the night.1,000g is centrifugal, and collecting cell is washed one time and re-suspended cell with the PBS that contains 1%BSA, 10%FCS.Add 50 μ g/mlPI and 100mg/ml RNA enzyme A, 37 ℃ of mark 30min upward monitor and analyze with CELLQUEST software at flow cytometer (FACS, BectonDickinson company), and cell is represented with percentage ratio in the distribution in each cycle.
The result shows that RabJ siRNA can obviously suppress the cell cycle progression of HeLa, MCF-7, SW480 cell tumour cells in vitro, and the not influence of negative control siRNA oligonucleotide (Scon si-RabJ) cell cycle process.RabJ siRNA1 to the influence of the cell cycle progression of HeLa, MCF-7 cell as shown in Figure 3.
After embodiment 4:RabJ siRNA disturbs RabJ to express, to the inhibition of the external tumorigenicity of tumour cell
Present embodiment has adopted the soft-agar cloning forming method.
In order to detect the ability of the non-dependence growth of cells contacting, with HeLa, MCF-7, the SW480 cell (5 * 10 of the transfection of RabJ siRNA described in the embodiment 1
4) be suspended in 0.5% soft agar, be laid on bottom and contain in 6 orifice plates of 1% soft agar.After cultivating for 3 weeks, light microscopic is observed down, is designated as the positive more than the clone of 50 cells.
The result shows that RabJ siRNA can obviously suppress HeLa, MCF-7, the external tumorigenicity of SW480 tumour cell, and negative control siRNA oligonucleotide (Scon si-RabJ1 and Scon si-RabJ2) does not influence (see figure 4) to tumour cell tumorigenicity.
After embodiment 5:RabJ siRNA disturbs RabJ to express, to the inhibition of tumorigenicity in the tumour cell body
HeLa, MCF-7, SW480 (1 * 10 with RabJ siRNA transfection described in the embodiment 1 and si-RabJ1 and si-RabJ2 coupling (final concentration respectively is 200nmol) transfection
6) be subcutaneously injected into the Balb/C nude mice (except that the coupling group is 2, all the other each groups are 5/group, available from Shanghai must be triumphant laboratory animal company) the lower right side belly, observed for six weeks then, calculate the incidence (the nude mice number that the mouse number/injection cell of tumour occurs) of tumour.
The result shows, RabJ siRNA can obviously suppress HeLa, MCF-7, SW480 tumour cell tumorigenicity in animal body, and negative control siRNA oligonucleotide (Scon si-RabJ1 and Scon si-RabJ2) is to not influence of tumorigenicity in the tumour cell body.Wherein, RabJ siRNA to the influence of tumorigenicity in the body of HeLa, SW480 cell as shown in Figure 5.
In addition, behind coupling si-RabJ1 and the si-RabJ2, it is single with si-RabJ1 or si-RabJ2 that HeLa, MCF-7, SW480 tumour cell tumorigenicity in animal body are higher than by the inhibition degree, and this shows that the siRNA of coupling RabJ can obtain better to suppress effect.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Second Military Medical University, PLA
＜120〉siRNA and the application thereof of inhibition human Rabj gene expression
<130>057873
<160>10
<170>PatentIn?version?3.1
<210>1
<211>19
<212>RNA
＜213〉oligonucleotide
<400>1
gauucguguc?uaaauaccu 19
<210>2
<211>19
<212>RNA
＜213〉oligonucleotide
<400>2
uagcagugcu?aguuucacc 19
<210>3
<211>19
<212>DNA
＜213〉oligonucleotide
<400>3
gauacguguc?uauataccu 19
<210>4
<211>19
<212>DNA
＜213〉oligonucleotide
<400>4
uaggagugcu?aguaucacc 19
<210>5
<211>21
<212>DNA
＜213〉oligonucleotide
<400>5
atgccgaaga?ggaaggagcc?c 21
<210>6
<211>21
<212>DNA
＜213〉oligonucleotide
<400>6
gtttcaccaa?agaacaagca?g 21
<210>7
<211>18
<212>DNA
＜213〉oligonucleotide
<400>7
gcatcgtgat?ggactccg 18
<210>8
<211>18
<212>DNA
＜213〉oligonucleotide
<400>8
tcggaaggtg?gacagcga 18
<210>9
<211>1787
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
<221>CDS
<222>(25)..(843)
<223>
<400>9
caagcttatg?catgcggccg?ctga?atg?gag?gcc?aac?atg?ccg?aag?cgg?aag 51
Met?Glu?Ala?Asn?Met?Pro?Lys?Arg?Lys
1 5
gag?ccc?ggc?agg?tct?ctc?cgc?atc?aaa?gtc?atc?tcc?atg?ggc?aac?gcc 99
Glu?Pro?Gly?Arg?Ser?Leu?Arg?Ile?Lys?Val?Ile?Ser?Met?Gly?Asn?Ala
10 15 20 25
gaa?gtg?ggg?aaa?agc?tgt?att?ata?aag?cga?tac?tgt?gag?aaa?aga?ttc 147
Glu?Val?Gly?Lys?Ser?Cys?Ile?Ile?Lys?Arg?Tyr?Cys?Glu?Lys?Arg?Phe
30 35 40
gtg?tct?aaa?tac?ctg?gca?aca?att?gga?att?gac?tat?gga?gtc?aca?aag 195
Val?Ser?Lys?Tyr?Leu?Ala?Thr?Ile?Gly?Ile?Asp?Tyr?Gly?yal?Thr?Lys
45 50 55
gta?cac?gtc?aga?gac?aga?gaa?atc?aaa?gtt?aac?atc?ttt?gat?atg?gct 243
Val?His?Val?Arg?Asp?Arg?Glu?Ile?Lys?Val?Asn?Ile?Phe?Asp?Met?Ala
60 65 70
gga?cat?ccc?ttc?ttc?tat?gag?gtt?cga?aat?gag?ttt?tac?aag?gac?aca 291
Gly?His?Pro?Phe?Phe?Tyr?Glu?Val?Arg?Asn?Glu?Phe?Tyr?Lys?Asp?Thr
75 80 85
cag?ggt?gtg?ata?ctg?gtc?tat?gat?gtt?ggg?cag?aaa?gac?tcc?ttt?gac 339
Gln?Gly?Val?Ile?Leu?Val?Tyr?Asp?Val?Gly?Gln?Lys?Asp?Ser?Phe?Asp
90 95 100 105
gcc?ctt?gat?gcg?tgg?ctg?gca?gaa?atg?aag?caa?gag?ctt?gga?cct?cat 387
Ala?Leu?Asp?Ala?Trp?Leu?Ala?Glu?Met?Lys?Gln?Glu?Leu?Gly?Pro?His
110 115 120
gga?aac?atg?gaa?aat?att?ata?ttt?gta?gtt?tgt?gcc?aac?aag?att?gat 435
Gly?Asn?Met?Glu?Asn?Ile?Ile?Phe?Val?Val?Cys?Ala?Asn?Lys?Ile?Asp
125 130 135
tgt?acc?aaa?cat?cgc?tgt?gta?gat?gaa?agt?gaa?gga?cgt?ctt?tgg?gct 483
CysThr?Lys?Hi?s?Arg?Cys?Val?Asp?Glu?Ser?Glu?Gly?Arg?Leu?Trp?Ala
140 145 150
gaa?agc?aaa?ggg?ttc?ctg?tac?ttt?gaa?act?tca?gca?caa?act?gga?gaa 531
Glu?Ser?Lys?Gly?Phe?Leu?Tyr?Phe?Glu?Thr?Ser?Ala?Gln?Thr?Gly?Glu
155 160 165
ggc?att?aat?gag?atg?ttc?cag?acc?ttt?tat?ata?tcc?ata?gtt?gat?tta 579
Gly?Ile?Asn?Glu?Met?Phe?Gln?Thr?Phe?Tyr?Ile?Ser?Ile?Val?Asp?Leu
170 175 180 185
tgt?gaa?aat?ggc?ggg?aaa?cgc?cct?acc?acc?aat?agc?agt?gct?agt?ttc 627
Cys?Glu?Asn?Gly?Gly?Lys?Arg?Pro?Thr?Thr?Asn?Ser?Ser?Ala?Ser?Phe
190 195 200
acc?aaa?gaa?caa?gca?gat?gcc?att?cgc?aga?att?cga?aat?agt?aaa?gac 675
Thr?Lys?Glu?Gln?Ala?Asp?Ala?Ile?Arg?Arg?Ile?Arg?Asn?Ser?Lys?Asp
205 210 215
agt?tgg?gac?atg?ctg?gga?gtc?aaa?cct?ggg?gcc?tca?agg?gat?gaa?gtc 723
Ser?Trp?Asp?Met?Leu?Gly?Val?Lys?Pro?Gly?Ala?Ser?Arg?Asp?Glu?Val
220 225 230
aat?aaa?gcg?tat?cgg?aaa?ctt?gct?gtg?ctt?ctt?cac?cct?gac?aaa?tgt 771
Asn?Lys?Ala?Tyr?Arg?Lys?Leu?Ala?Val?Leu?Leu?His?Pro?Asp?Lys?Cys
235 240 245
gta?gca?cct?ggc?agt?gaa?gat?gcc?ttc?aaa?gca?gtt?gtg?aat?gct?cgg 819
Val?Ala?Pro?Gly?Ser?Glu?Asp?Ala?Phe?Lys?Ala?Val?Val?Asn?Ala?Arg
250 255 260 265
aca?gcc?ctc?ctg?aaa?aac?atc?aag?tagaaagtac?agaaaaaagc?cacatgtggg 873
Thr?Ala?Leu?Leu?Lys?Asn?Ile?Lys
270
actcaaatgc?aaacagactt?tccctagagg?tgaaataacc?aacgtggagt?tttccttccc 933
agaatctcac?tgctcttttc?attcatgtgt?tgtcatttgt?atatcagtaa?ttcaggtacc 993
catttcatag?acattttact?gagaaatgac?ctgcatttgt?atgaagtgaa?ctgagcgtca 1053
caccctgtac?ttcatttcat?atttctagat?aattctgaat?ttttttctca?ttcgtcagct 1113
ctgtaattat?agtatcactt?agacatttca?cttggggaaa?tccacaaggt?tcctggaggg 1173
agggaagaga?ggacaagagg?accctttcac?tttttctttt?ttacggaatt?catcatcaga 1233
gaagaaaata?acaaaaatgg?aagcaaacaa?catcagaacc?cctgtaagtt?tggtgtgacc 1293
ttacagacaa?gttgctgctt?ttacaatgag?ttccttaggt?ggtattttaa?cccatcgatc 1353
tataatgatg?actcttggca?gccctttggg?agtttgtaaa?atgaggtgat?acagttctga 1413
attgagcatt?cctttatgat?attcactctg?ttcctcttct?gcagccacca?gtgggagaga 1473
caagccagtc?ctaagagaaa?aggtggtggc?agccacaaat?tctaggtaca?ctggctgctg 1533
cctatcctgt?ccctggatct?gaggcctttc?ccttgccata?gaaatggttg?ctggtagcag 1593
tagagagcac?tgtgcacctg?ggaatgagga?atcaggcccc?aagacagaag?tacttggagg 1653
agccagctgc?agtagtatcc?gcctgtagtc?ccagctactc?aggaggctga?gacaggagga 1713
ttgcttaagc?ccaggagctc?aagtcccacc?tgggcaacat?agtaagatct?tgtctcttaa 1773
aaaaaaaaaa?aaaa 1787
<210>10
<211>273
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>10
Met?Glu?Ala?Asn?Met?Pro?Lys?Arg?Lys?Glu?Pro?Gly?Arg?Ser?Leu?Arg
1 5 10 15
Ile?Lys?Val?Ile?Ser?Met?Gly?Asn?Ala?Glu?Val?Gly?Lys?Ser?Cys?Ile
20 25 30
Ile?Lys?Arg?Tyr?Cys?Glu?Lys?Arg?Phe?Val?Ser?Lys?Tyr?Leu?Ala?Thr
35 40 45
Ile?Gly?Ile?Asp?Tyr?Gly?Val?Thr?Lys?Val?His?Val?Arg?Asp?Arg?Glu
50 55 60
Ile?Lys?Val?Asn?Ile?Phe?Asp?Met?Ala?Gly?His?Pro?Phe?Phe?Tyr?Glu
65 70 75 80
Val?Arg?Asn?Glu?Phe?Tyr?Lys?Asp?Thr?Gln?Gly?Val?Ile?Leu?Val?Tyr
85 90 95
Asp?Val?Gly?Gln?Lys?Asp?Ser?Phe?Asp?Ala?Leu?Asp?Ala?Trp?Leu?Ala
100 105 110
Glu?Met?Lys?Gln?Glu?Leu?Gly?Pro?His?Gly?Asn?Met?Glu?Asn?Ile?Ile
115 120 125
Phe?Val?Val?Cys?Ala?Asn?Lys?Ile?Asp?Cys?Thr?Lys?His?Arg?Cys?Val
130 135 140
Asp?Glu?Ser?Glu?Gly?Arg?Leu?Trp?Ala?Glu?Ser?Lys?Gly?Phe?Leu?Tyr
145 150 155 160
Phe?Glu?Thr?Ser?Ala?Gln?Thr?Gly?Glu?Gly?Ile?Asn?Glu?Met?Phe?Gln
165 170 175
Thr?Phe?Tyr?Ile?Ser?Ile?Val?Asp?Leu?Cys?Glu?Asn?Gly?Gly?Lys?Arg
180 185 190
Pro?Thr?Thr?Asn?Ser?Ser?Ala?Ser?Phe?Thr?Lys?Glu?Gln?Ala?Asp?Ala
195 200 205
Ile?Arg?Arg?Ile?Arg?Asn?Ser?Lys?Asp?Ser?Trp?Asp?Met?Leu?Gly?Val
210 215 220
Lys?Pro?Gly?Ala?Ser?Arg?Asp?Glu?Val?Asn?Lys?Ala?Tyr?Arg?Lys?Leu
225 230 235 240
Ala?Val?Leu?Leu?His?Pro?Asp?Lys?Cys?Val?Ala?Pro?Gly?Ser?Glu?Asp
245 250 255
Ala?Phe?Lys?Ala?Val?Val?Asn?Ala?Arg?Thr?Ala?Leu?Leu?Lys?Asn?Ile
260 265 270
Lys
Claims (10)
1. small molecules interference RNA at the people Rab GTP enzyme RabJ that has the J functional domain, it is characterized in that, described small molecules interference RNA and people RabJ gene mRNA reverse complemental, its length is 16-30bp, and described small molecules interference RNA can make the growth of HeLa cell reduce more than 50%.
2. small molecules interference RNA as claimed in claim 1 is characterized in that, the length of described small molecules interference RNA is 18-25bp.
3. small molecules interference RNA as claimed in claim 1 is characterized in that, described small molecules interference RNA is incorporated into the corresponding nucleotide sequences with lower area of RabJ: nuclear localization signal, Rab functional domain, J functional domain or its combination.
4. small molecules interference RNA as claimed in claim 1 is characterized in that, described small molecules interference RNA contains and is selected from the nucleotide sequence shown in SEQ ID NO:1 or the SEQ ID NO:2.
5. small molecules interference RNA as claimed in claim 1 is characterized in that, described small molecules interference RNA is selected from down group:
si-RabJ1：5’-GAUUCGUGUCUAAAUACCU-3’
3’-CUAAGCACAGAUUUAUGGA-5’；
si-RabJ2：5’-UAGCAGUGCUAGUUUCACC-3’
3’-AUCGUCACGAUCAAAGUGG-5’。
6. according to arbitrary described small molecules interference RNA among the claim 1-5, it is characterized in that 3 ' end of described small molecules interference RNA also contains 2-4 dT or contains the extended structure of 2-6 U.
7. a composition is characterized in that, it contains each described small molecules interference RNA and acceptable carrier, thinner or vehicle among the 0.001-99.99wt% claim 1-6.
8. composition as claimed in claim 7 is characterized in that described composition is a pharmaceutical composition, and described carrier, thinner or vehicle are pharmaceutically acceptable carrier, thinner or vehicle.
9. the purposes of the described small molecules interference RNA of claim 1 is characterized in that, is used to prepare the pharmaceutical composition that suppresses growth of tumour cell composition or treatment tumour.
10. purposes as claimed in claim 9 is characterized in that, described tumour cell or tumour are expressed RabJ albumen, and are selected from down group: cervical cancer, mammary cancer, colorectal carcinoma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2005100304281A CN1948483B (en) | 2005-10-12 | 2005-10-12 | SiRNA for inhibiting human Rabj gene expression and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2005100304281A CN1948483B (en) | 2005-10-12 | 2005-10-12 | SiRNA for inhibiting human Rabj gene expression and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1948483A true CN1948483A (en) | 2007-04-18 |
| CN1948483B CN1948483B (en) | 2012-01-11 |
Family
ID=38018107
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2005100304281A Expired - Fee Related CN1948483B (en) | 2005-10-12 | 2005-10-12 | SiRNA for inhibiting human Rabj gene expression and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1948483B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009030084A1 (en) * | 2007-09-04 | 2009-03-12 | Second Military Medical University | NEW FUNCTIONS AND USES OF HUMAN ONCOGENE-LIKE SMALL G PROTEIN RabJ |
| CN102703447B (en) * | 2007-06-26 | 2013-10-23 | 长春华普生物技术有限公司 | Oligonucleotide with breast cancer treatment effect |
| CN103484463A (en) * | 2013-09-12 | 2014-01-01 | 四川大学 | shRNA for silencing human c2orf68 gene, and medical use thereof |
| CN109078188A (en) * | 2018-08-21 | 2018-12-25 | 浙江大学 | A kind of action target spot and anti-tumor drug of anti-tumor drug |
| CN111544445A (en) * | 2020-05-31 | 2020-08-18 | 北京瀚梅生物科技有限公司 | Cancer inhibition application of composition of cervical cancer stem cell specific membrane penetrating peptide and interference RabJ gene |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1408724A (en) * | 2001-09-21 | 2003-04-09 | 第二军医大学免疫学研究所 | Novel testicular function relative protein and its use |
-
2005
- 2005-10-12 CN CN2005100304281A patent/CN1948483B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703447B (en) * | 2007-06-26 | 2013-10-23 | 长春华普生物技术有限公司 | Oligonucleotide with breast cancer treatment effect |
| WO2009030084A1 (en) * | 2007-09-04 | 2009-03-12 | Second Military Medical University | NEW FUNCTIONS AND USES OF HUMAN ONCOGENE-LIKE SMALL G PROTEIN RabJ |
| CN103484463A (en) * | 2013-09-12 | 2014-01-01 | 四川大学 | shRNA for silencing human c2orf68 gene, and medical use thereof |
| CN109078188A (en) * | 2018-08-21 | 2018-12-25 | 浙江大学 | A kind of action target spot and anti-tumor drug of anti-tumor drug |
| CN111544445A (en) * | 2020-05-31 | 2020-08-18 | 北京瀚梅生物科技有限公司 | Cancer inhibition application of composition of cervical cancer stem cell specific membrane penetrating peptide and interference RabJ gene |
| CN111544445B (en) * | 2020-05-31 | 2021-03-26 | 富彬生物技术(天津)有限责任公司 | Cancer inhibition application of composition of cervical cancer stem cell specific membrane penetrating peptide and interference RabJ gene |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1948483B (en) | 2012-01-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1267550C (en) | Vascular endothelial growth factor 2 | |
| CN1173745C (en) | Use of methioninase in anti-methionine and anti-homocysteine chemotherapy | |
| CN1231694A (en) | Antitumor antisense sequences directed against R1 and R2 components of ribonucleotide reductase | |
| CN1934256A (en) | Decoy nucleic acid to synoviolin gene promoter | |
| CN1739788A (en) | Modulation of TRIP-BR function and method of treating proliferative disorders | |
| CN1948483A (en) | SiRNA for inhibiting human Rabj gene expression and its application | |
| CN101062952A (en) | Fusion protein comprised of human serum and interferon and its coding gene and application | |
| CN1243018C (en) | Blood vessel generating inhibitor | |
| CN1159440C (en) | Mutants of Rb and p53 genes and uses thereof | |
| CN1071336C (en) | Compositions and methods using unbound mpl receptor for stimulating platelet production | |
| CN1701077A (en) | Use of antisense oligonucleotides to inhibit the expression of Akt-1 | |
| CN1294630A (en) | Oligonucleotide sequences complementary to thioredoxin or thioredoxin reductase genes and methods of using same to modulate cell growth | |
| CN1948482A (en) | Antisensedigonucleotides sequence for inhibiting human Rabj gene expression and its application | |
| CN1894278A (en) | Akt activity specifically inhibiting polypeptide | |
| CN1244595C (en) | Tumor suppressor protein and its application | |
| CN1966684A (en) | Construction of HER2/neu mRNA in vitro transcription vector and use thereof | |
| CN1160370C (en) | A novel human cell cysle control related protein and a sequence encoding the same | |
| CN1948342A (en) | HLA-A2 restriction epi polypeptide originated from post selection cancer gene hRabj and its application | |
| CN1948484A (en) | siRNA for inhibiting human hPEBP4 gene expression and its application | |
| CN1233831C (en) | SiRNA and expression plasmid for inhibiting human bc1-2 gene expression and their use for preparing medicine | |
| CN1704426A (en) | Cancer gene and its medical use | |
| CN1289524C (en) | Human telomerase active inhibitor protein and use thereof | |
| CN1170844C (en) | Human macrobiosis-ensuring protein and its coding sequence and application | |
| CN1152957C (en) | Human 6-phosphoglucosamine isomerase, its code sequence and application | |
| CN1690193A (en) | Use of methioninase in anti-methionine and anti-homocysteine chemotherapy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120111 Termination date: 20161012 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |