CN101942441A - Structure and application of siRNA for suppressing proliferation and metastasis of human lung cancer cells - Google Patents
Structure and application of siRNA for suppressing proliferation and metastasis of human lung cancer cells Download PDFInfo
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Abstract
The invention provides a structure and application of small interference RNA (siRNA) for suppressing proliferation and metastasis of human lung cancer cells, and relates to the bioengineering pharmaceutical field, in particular to a method for designing, synthesizing and screening the small interference RNA (siRNA) for suppressing (silencing) gene expression of human VEGF so as to suppress the proliferation, invasion and metastasis of malignant cells such as lung cancer cells and the like according to gene sequence information on a vascular endothelial growth factor (VEGF). Through the structure and the application, that the siRNA which is complementary with a specified region of the VEGF mRNA has a suppression effect on the proliferation and the metastasis of the human lung cancer A549 cells cultured in vitro is designed and discovered. Thus, the invention relates to the siRNA sequence, the structure and a novel medicament thereof for preventing and curing the proliferation and the metastasis of the malignant cells such as lung cancer cells and the like aiming at the VEGF gene.
Description
Technical field
The present invention relates to the biotechnology pharmaceutical field, specifically a kind of at vascular endothelial growth factor (vascular endothelial growthfactor, VEGF) siRNA of gene (small interference RNA, sequential structure siRNA) and become the newtype drug of malignant cell propagation such as treatment lung cancer etc. and Invasion and Metastasis.
Background technology
Lung cancer has become and has caused one of human dead common disease, and the incidence and mortality in China big and medium-sized cities all occupies various pernicious midship knurls first place.Although the employing multiple therapy methods, patient 3 years and 5 years survival rates are still very low, cause the major cause of death directly related with transfer with the recurrence of tumour.
The propagation of cancer cells and Invasion and Metastasis are the complex processes of multifactor regulation and control.The propagation of cancer cells is that tumour cell increases cell quantity and the process that increases gross tumor volume by cell fission, and the increase of gross tumor volume can destroy the structure and the function of place histoorgan.The Invasion and Metastasis of cancer cells is meant that tumour cell breaks away from former position, invades and pass matrix (this stage is invasion and attack) on every side, through blood or the lymphokinesis process to diffusion at a distance (this stage is for shifting).
Studies show that, the vascular endothelial growth factor of tumor cell secretion (vascular endothelial growth factor, VEGF) in the propagation of neovascularization and cancer cells and Invasion and Metastasis, bringing into play important effect, no matter VEGF is in the lung carcinoma cell of vitro culture, still in the tumour cell of tumor animal, or high expression level is all arranged in the human body cancerous lung tissue, and vegf expression level and lung cancer for prognosis are closely related.Ohta etc. [OhtaY, et al.Chest, 2002,121:1624-1627] discover have the primary tumor vegf expression level of nodus lymphoideus transferring rate to be higher than no transferrer to lung cancer, the expression of VEGF is higher than primary tumor in metastatic lymph node.VEGF is independent factor of lung cancer for prognosis, patient's prognosis that vegf expression is high relatively poor [Seto T, et al.Lung Cancer, 2006,53 (1): 91-96; SadasiwanC, et al.ASCO MeetingAbstract, 2007,25:7696].
Therefore, utilize the correlation technique means to reduce vegf expression and just might reduce lung cancer propagation and metastasis rate, prolong patient's life, improve patient's survival rate and quality of life.
RNAi is a kind of simple and rapid posttranscriptional gene intervention techniques of setting up in 1998, small molecules double-stranded RNA fragments specific ground by 20～23 base pairs combines with the homologous sequence gene, thereby reticent target gene can replace the gene knockout technology of traditional dna level.Compare with traditional antisense technology, RNAi has the effect high specificity, effective, longer duration, cell inner expression be stable, can heredity etc. advantage, can be widely used in silence [the Liu G of goal gene, et al.Histol Histopathol.2007,22 (2): 211-217; Fuchs U, et al.Adv cancer Res.2007,96:75-102.].Since 2003, RNAi uses increasingly extensive in the research in fields such as metabolic disease, disease of viral infection, multiple malignant tumour, nervous system disorders and drug screening.In view of the immense value and the broad prospect of application of RNAi technology, the U.S. " SCIENCE " magazine in 2002 is chosen as it first of annual ten big sciences progress in whole world.Therefore and the discoverer U.S. scientist Craig Mello of RNAi and Andrew Fire also win Nobel's physiology in 2006 and medical science prize.
The objective of the invention is to seek the novel gene medicine that the energy differential high efficient suppresses proliferation of lung cancer cells, can obviously reduce the small molecules interference RNA (siRNA) of VEGF genetic expression and can improve its bioactive chemically modified derivative.
Summary of the invention
Content of the present invention is to design the siRNA sequence of some target VEGF mRNA according to the sequence information of VEGF mRNA and bioinformatics technique, make up the VEGF-siRNA plasmid vector respectively, transfection A549 cell, set up transfectional cell and stablize strain, with the inhibition effect of fluorescence quantitative PCR detection VEGF-siRNA VEGF mRNA expression.The result shows 1 siRNA sequence to be had comparatively ideal inhibition VEGF mRNA expression and can obviously suppress A549 cell proliferation and transfer in cell in vitro experiment, and prompting can be used for the prevention and the treatment of proliferation of lung cancer cells transfer.
According to the present invention, sequence total length (sequence number: NM001033756 at people VEGF mRNA in the U.S. state-run biotechnology information center ncbi database, CDS:492-1607) siRNA of 1220～1238 sites design can suppress VEGF genetic expression in, might become the novel biological engineering medicine of malignant tumour Invasion and Metastasis such as prevention and treatment lung cancer.
According to the present invention, be the nuclease resistance that strengthens siRNA, bioavailability, tissue target tropism etc., the present invention has comprised the various chemically modifieds of VEGF-siRNA.
According to the present invention, the VEGF-siRNA of invention and modifier thereof can be made into the reagent of parenterai administration by means known in the art.
According to the present invention, the treatment of VEGF-siRNA of the present invention and modifier thereof is formed can use independent effective constituent or composition forms comprises other antisense nucleic acid of associating and derivative thereof.
According to the present invention, treatment of the present invention is formed, comprise pharmacokinetics, pharmacokinetics, mode of administration, route of administration, the receptor's of certain drug age, body weight, hepatic and renal function state, character, degree and the treatment time limit etc. of disease according to different situations, with an amount of dosed administration.
According to the present invention, enforcement of the present invention has important society and economic benefit to the prevention of malignant tumours such as the lung cancer propagation transfer of serious harm human health with treatment.
Embodiment
Experimental design, method and experimental result are as follows:
1, the synthetic and plamid vector construction of siRNA design
In the U.S. state-run biotechnology information center ncbi database, obtain people VEGF mRNA the sequence total length (sequence number: NM 001033756, CDS:492-1607).According to the siRNA principle of design,, 12 VEGF-siRNA target sequences have been designed respectively in conjunction with design software and bibliographical information.Each siRNA target sequence length 19bp, the BLAST comparison of all going checks that to guarantee not having homology with other gene, it is 64bp that synthetic siRNA transcribes template length, for adding forward and reverse target sequence that 9bp stem ring sequence is separated, tail end inserts 5 T as terminator sequence in the middle of sequence.Upstream and downstream has added BamHI and Hind11I restriction enzyme site respectively.Plasmid (through HindIII be connected with the T4DNA ligase enzyme with synthetic expression template dna fragmentation after the BamHI enzyme is cut, reaction conditions: 16 ℃, 8h) transformation can be expressed GFP, transform DH5 α bacterial strain, that mycin agar plate of card-coating, select positive colony, amplification is also extracted plasmid, identifies with BamHI and Hind 11I double digestion.Enzyme is cut and is identified that correct clone send biotech firm's order-checking.PGCsilencerTM U6/Neo/GFP/RNAi carrier is synthetic by the Shanghai triumphant gene of Ji chemical company.
2, screening transfection VEGF-siRNA lung cancer A549 cell is stablized strain
2.1 1. transient transfection gets the A549 cell that the recovery back third generation is in logarithmic phase, trypan blue dyeing, and survival rate＞95%, the microscopically counting is inoculated in 24 orifice plates with 2 * 105 cells/well.Cell degree of converging 60%～80% carries out transfection.2. plasmid DNA 1 μ g adds mixing among the 50 μ lOPTI-MEM; Lipofectamine 20002 μ l add among the 50 μ l OPTI-MEM and mix, and room temperature leaves standstill 5min.The Lipofectamine2000 diluent adds mixing in the plasmid DNA diluent, and room temperature leaves standstill 20min.Nutrient solution in the Tissue Culture Plate is abandoned in suction, and OPTI washes cell twice, adds 500 μ l OPTI-MEM.100 μ l slowly add in the Tissue Culture Plate with mixed solution, rock culture plate simultaneously gently.Cultivate in 37 ℃, 5%CO2 incubator, change the RPMI-1640 that contains 10%FBS behind the 5h.
2.2 strain is stablized in G418 screening transfection
The 24h cell went down to posterity by 1: 3 behind the transient transfection, continue to cultivate 48h, inhaled and abandoned nutrient solution, add contain G418500 μ g/ml contain the 10%FBS1640 nutrient solution.The next day change liquid, a large amount of necrocytosiss behind the 6d, G418 concentration is adjusted to 200 μ g/ml, continues to be cultured to mono-clonal and forms.Pick out the positive colony of expressing GFP under the fluorescent microscope, go to amplification in the culturing bottle.With GFP is report, observation of cell transfection efficiency under the fluorescent microscope.
3, fluorescence quantitative PCR detection transfection VEGF-siRNA expresses influence to A549 cell VEGE mRNA.
4, the Western trace detects transfection VEGF-siRNA influences A549 cell VEGE protein expression.
5, the MTT colorimetry detects and respectively organizes the ability of cell proliferation change.
6, the Transwell model detects and respectively organizes A549 cells in vitro invasive ability
The result:
1 finds that in 12 VEGF-siRNA of design the siRNA in target VEGF mRNA 1220～1238 sites (sequence is " GATCCGCAGACGTGTAAAT ") has the comparatively effect of ideal inhibition VEGF mRNA expression, it suppresses efficient is 73%, still find no the bibliographical information that this site is used to design siRNA by literature search, therefore, the VEGF gene locus found of this patent and have independent intellectual property right based on the designed VEGF-siRNA in this site.
A549 cell endogenous vegf protein expression of results showed before and after 2Western blot detected transfection: compare with control group (0.73 ± 0.01), A549 cell endogenous vegf protein is expressed and is obviously reduced (0.38 ± 0.01, P＜0.01) behind the transfection VEGF-siRNA;
3MTT tests demonstration: simple transfection VEGF-siRNA group 1～4d cell proliferation and control group indifference (P＞0.05), 5～6d is starkly lower than control group level (P＜0.01)
4 cells in vitro invasion and attack experiment shows: 20h behind the inoculating cell in the Transwell cell, compare with control group (108 ± 9), the VEGF-siRNA group is worn the theca cell number and is obviously reduced (56 ± 9, P＜0.01), shows that designed VEGF-siRNA can reduce A549 cell invasion ability.
Relevant nucleotide sequence in the application for a patent for invention " a kind of structure and purposes that suppresses the siRNA of human lung carcinoma cell propagation transfer "
Table. the sequential structure of the action target spot of the siRNA that can effectively suppress transfer of people's lung cancer A549 cell propagation and VEGF mRNA expression of screening
Claims (3)
1. but a specificity is in conjunction with the siRNA (siRNA) of human vascular endothelial growth factor (VEGF) gene, VEGF mRNA (sequence number: the NM 001033756 that it is reticent; CDS:492-1607) site (1220～1238) sequence is: " GATCCGCAGACGTGTAAAT ".
2. contain siRNA sequence in the claim 1 and the pharmaceutical composition of pharmaceutically acceptable carrier.
3. the siRNA in the claim 1 is used to prepare the purposes of anti-lung cancer and other malignant cell propagation diversion medicaments.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1580259A (en) * | 2003-08-07 | 2005-02-16 | 徐根兴 | SiRAN and expression carrier for inhibiting human VEGF gene expression and their pharmaceutical use |
| US20080188437A1 (en) * | 2002-07-24 | 2008-08-07 | The Trustees Of The University Of Pennsylvania | Compositions and Methods for siRNA Inhibition of Angiogenesis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080188437A1 (en) * | 2002-07-24 | 2008-08-07 | The Trustees Of The University Of Pennsylvania | Compositions and Methods for siRNA Inhibition of Angiogenesis |
| CN1580259A (en) * | 2003-08-07 | 2005-02-16 | 徐根兴 | SiRAN and expression carrier for inhibiting human VEGF gene expression and their pharmaceutical use |
Non-Patent Citations (1)
| Title |
|---|
| 《第四军医大学学报》 20091231 李玲 VEGF靶向性siRNA表达载体的构建及其沉寂作用 1-3 2332-2334 第30卷, 第21期 * |
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