CN103215270B - DNA (deoxyribonucleic acid) segment with function of inhibiting cell migration and invasion of glioma, and application of DNA segment - Google Patents
DNA (deoxyribonucleic acid) segment with function of inhibiting cell migration and invasion of glioma, and application of DNA segment Download PDFInfo
- Publication number
- CN103215270B CN103215270B CN201310156550.8A CN201310156550A CN103215270B CN 103215270 B CN103215270 B CN 103215270B CN 201310156550 A CN201310156550 A CN 201310156550A CN 103215270 B CN103215270 B CN 103215270B
- Authority
- CN
- China
- Prior art keywords
- dna fragmentation
- invasion
- dna
- segment
- glioma
- 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.)
- Expired - Fee Related
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a DNA (deoxyribonucleic acid) segment with a function of inhibiting cell migration and invasion of glioma cells, and application of the DNA segment. The DNA segment is the base sequence as shown in SEQ NO. 1. The invention designs interference plasmids of DIAPH1 genes, applies a glioma cell model to research the inhibition effect of the glioma cell model on cell migration and invasion, and provides a new method for treatment application of the glioma.
Description
Technical field
The present invention relates to a kind of DNA fragmentation and the application thereof with the migration of suppression brain glioblastoma cell and invasion and attack function.
Background technology
Cerebral glioma (Gliolma) is a kind of common malignant tumour originating from central nervous system, its feature is wettability growth, with normal cerebral tissue without obvious boundary, majority is not limited to a cerebral lobe, outside cerebral tissue, deeply destroy cerebral tissue in finger-like, its sickness rate accounts for 35% ~ 60% of adult's intracranial tumors.In recent years, because its poor prognosis, case fatality rate high make the survival rate of surgery alone treatment malignant glioma very low.The usual prognosis of patient suffering from malignant glioma is very poor, particularly the elderly, in the U.S., every year nearly 10,000 people can suffer from this disease, but the patient of 50% mean survival time after the treatment, also less than 12 months, faces this present situation, research is a kind of extremely urgent to the effective therapy system of malignant glioma.
DIAPH1 is a kind of homologous genes belonging to the transparent gene of fruit bat.Its albumen of expressing is subordinated to into protein family because having into albumen homology structural domain FH1 with FH2, and at stress fiber, filopodia, focal adhension, plays an important role in the formation of microtubule.This albumen is as Actin muscle nucleating factor simultaneously, it take part in numerous biological procedures based on Actin muscle, comprise form generation, division of cytoplasm, the formation of cell polarity, cell adhesion and motion etc., and often to lack of proper care as expressed in Tumor Differentiation, Infiltration and metastasis in pathological conditions.
RNA interference (RNA interference, RNAi) refers in the cell by double chain RNA mediate, with the mRNA of its sequence homology, selective degradation occurs, thus causes the phenomenon of silenced gene expression, and this is a kind of gene silencing mechanism of post-transcriptional level.
Summary of the invention
An object of the present invention is that providing a kind of has the DNA fragmentation suppressing brain glioblastoma cell migration and invasion and attack function.
Two of object of the present invention is to provide the interference plasmid containing this DNA fragmentation.
Three of object of the present invention is the preparation method providing this interference plasmid.
Four of object of the present invention is to provide the application of this DNA fragmentation in preparation treatment cerebral glioma medicine.
For achieving the above object, the present invention adopts following technical scheme:
Have and suppress brain glioblastoma cell migration and a DNA fragmentation for invasion and attack function, it is characterized in that this DNA fragmentation is for the base sequence shown in SEQ ID NO.1.
A kind of method prepared above-mentioned having and suppress the DNA fragmentation of brain glioblastoma cell migration and invasion and attack function, it is characterized in that the concrete steps of the method are: according to the design requirements in pGPU6/GFP/Neo siRNA Expression Vector Kit, design corresponds to the interference DNA fragmentation of DIAPH1 gene, the i.e. oligonucleotide sequence of the shRNA of GGA ACA AGC ATG AGA TCA TTC is respectively 62bp.
A kind of interference plasmid, is characterized in that this plasmid contains above-mentioned fragment.
A kind of method preparing above-mentioned interference plasmid, it is characterized in that the concrete steps of the method are: the specific requirement building carrier design in test kit according to pGPU6/GFP/Neo siRNA Expression Vector Kit gene plasmid, the oligonucleotide sequence primer of above-mentioned shRNA is connected with pGPU6/GFP/Neo siRNA Expression Vector, obtains the interference plasmid of DIAPH1 gene.
A kind ofly above-mentioned preparing the application for the treatment of in cerebral glioma medicine for suppressing brain glioblastoma cell to move with the DNA fragmentation of invasion and attack.
The present invention designs the interference plasmid of DIAPH1 gene, and applies the restraining effect of its on cell migration of brain glioblastoma cell model research and invasion and attack, and the treatment use for glioma provides a kind of novel method.
Accompanying drawing explanation
Fig. 1 is that pGPU6/GFP/Neo siRNA Expression Vector schemes.
Fig. 2 is the expressing quantity change that Western blot analyzes DIAPH1 in brain glioblastoma cell system U87.Wherein None is: untransfected control group; NC is: negative control group; RNAi is: transfection DIAPH1 Gene interfere plasmid group.
Fig. 3 is the ability that Radius 24-Well Cell Migration Assay analyzes brain glioblastoma cell system U87 migration.Wherein None is: untransfected control group; NC is: negative control group; RNAi is: transfection DIAPH1 Gene interfere plasmid group.
Fig. 4 is the ability that BD BioCoat Matrigel Invasion Chamber analyzes brain glioblastoma cell system U87 invasion and attack.Wherein A is: untransfected control group; B is: negative control group; C is: transfection DIAPH1 Gene interfere plasmid group; D is: statistical study figure, and wherein None is: untransfected control group; NC is: negative control group; RNAi is: transfection DIAPH1 Gene interfere plasmid group.
Embodiment
One, the cultivation of brain glioblastoma cell system U87: brain glioblastoma cell system U87 is incubated at 37 DEG C, 5%CO
2constant incubator.When cell enters logarithmic phase, suck the old nutrient solution in culture dish, with the aseptic Hanks cleaning of 2ml, to remove the serum containing trypsin inhibitor, add 1ml 0.05%Trypsin-EDTA-D-Hanks solution in culture dish, 37 DEG C, 5%CO
2place 2min in incubator, then examine under a microscope cell and whether be separated ware wall (as not being separated completely, can time of proper extension trysinization, until be separated substantially completely); Add 2ml fresh medium, blow and beat gently with rifle, then add appropriate nutrient solution to be easy to mix cell; Get 1ml cell suspension to add and be equipped with in the new culture dish of 2ml fresh culture, put into 37 DEG C, 5%CO
2cultivate in incubator, within about about 3 days, go down to posterity 1 time (mainly seeing that cell is with or without being paved with Tissue Culture Dish).
Two, the design of DIAPH1 Gene interfere plasmid and negative control plasmids and transfection: according to the specific requirement designed in pGPU6/GFP/Neo siRNA Expression Vector Kit, design corresponds to the oligonucleotide sequence of the shRNA of DIAPH1 gene (GGA ACA AGC ATG AGA TCA TTC), be respectively 62bp, and synthesize.
The oligonucleotide sequence of this shRNA is:
Sense： 5’- CAC CGG AAC AAG CAT GAG ATC ATT CTT CAA GAG AGA ATG ATC TCA TGC TTG TTC CTT TTT TG-3’
Anti-sense:：5’- gaT CCA AAA AAG GAA CAA GCA TGA GAT CAT TCT CTC TTG AAG AAT GAT CTC ATG CTT GTT CC-3’；
According to the specific requirement designed in pGPU6/GFP/Neo siRNA Expression Vector Kit, design negative control, namely can not the oligonucleotide sequence primer of the shRNA of any genetic expression (GTT CTC CGA ACG TGT CAC GT) in interference cell, be respectively 59bp, and synthesize.
The sequence that the shRNA of negative control is corresponding is:
Sense： 5’- CAC CGT TCT CCG AAC GTG TCA CGT CAA GAG ATT ACG TGA CAC GTT CGG AGA ATT TTT TG-3’
Anti-sense:：5’-GAT CCA AAA AAT TCT CCG AAC GTG TCA CGT AAT CTC TTG ACG TGA CAC GTT CGG AGA AC-3’
According to the specific requirement designed in pGPU6/GFP/Neo siRNA Expression Vector Kit, the oligonucleotide sequence of design above two kinds of shRNA is connected with pGPU6/GFP/Neo siRNA Expression Vector respectively, then distinguishes transfection brain glioblastoma cell system U87.
Day before transfection, is seeded on 24 porocyte culture plates by cell, with not containing antibiotic DMEM culture medium culturing, when cell confluency degree reaches 60%-70 %, carry out FuGENE
the DNA transfection that HD Transfection Reagent mediates.
100 μ l do not dilute 2 μ g plasmids containing the nonreactive DMEM substratum of serum, mix gently; Dilute 3 μ l FuGENE subsequently
hD Transfection Reagent has added in 100 μ l DMEM nonreactive serum free mediums of plasmid to above-mentioned, gently the rear incubated at room temperature of mixing 15 minutes.Joined by above transfection composite in the hole of each cell to be turned, the culture plate that rocks back and forth gently mixes.Cell at 37 DEG C, 5%CO
2cellar culture under saturated humidity.
Three, experiment grouping and Testing index untransfected control group: the brain glioblastoma cell system U87 normally cultivated; Negative control group: the brain glioblastoma cell system U87 of transfection negative control plasmids.Experimental group: the brain glioblastoma cell system U87 of transfection DIAPH1 Gene interfere plasmid.
The detection of DIAPH1 down regulation of gene expression: by Western blot technology for detection.
The detection of brain glioblastoma cell system U87 transfer ability: detect with Radius 24-Well Cell Migration Assay.
The detection of brain glioblastoma cell system U87 invasive ability: detect with BD BioCoat Matrigel Invasion Chamber.
Four, result
1., in transfection after three days, the total protein of extracting brain glioblastoma cell, carries out Western blot analysis with the specific antibody of DIAPH1.Result shows, in transfection DIAPH1 Gene interfere plasmid group, the expressing quantity of DIAPH1 is starkly lower than the expressing quantity of DIAPH1 in untransfected control group and negative control group, see Fig. 2.
2. after transfection, with Radius 24-Well Cell Migration Assay analysis of cells transfer ability.Result shows, in transfection DIAPH1 Gene interfere plasmid group, the transfer ability of cell is starkly lower than untransfected control group and negative control group, see Fig. 3.
3. in transfection two days later, with BD BioCoat Matrigel Invasion Chamber analysis of cells invasive ability.Result shows, in transfection DIAPH1 Gene interfere plasmid group, the invasive ability of cell is starkly lower than untransfected control group and negative control group, see Fig. 4.
<110> Shanghai University
<120> has the DNA fragmentation and application thereof that suppress brain glioblastoma cell migration and invasion and attack function
<160> 2
<210> 1
<211> 121
<212> DNA
<213> artificial sequence
<400> 1
Sense： 5’- CAC CGG AAC AAG CAT GAG ATC ATT CTT CAA GAG AGA ATG ATC TCA TGC TTG TTC CTT TTT TG-3’
<210> 2
<211> 121
<212> DNA
<213> artificial sequence
<400> 1
Anti-sense:：5’- gaT CCA AAA AAG GAA CAA GCA TGA GAT CAT TCT CTC TTG AAG AAT GAT CTC ATG CTT GTT CC-3’；
Claims (5)
1. have and suppress brain glioblastoma cell migration and a DNA fragmentation for invasion and attack function, it is characterized in that this DNA fragmentation is: Sense:5 '-CAC CGG AAC AAG CAT GAG ATC ATT CTT CAA GAG AGA ATG ATC TCA TGC TTG TTC CTT TTT TG-3 ';
Anti-sense:：5’- GAT CCA AAA AAG GAA CAA GCA TGA GAT CAT TCT CTC TTG AAG AAT GAT CTC ATG CTT GTT CC-3’。
2. prepare the method with the DNA fragmentation suppressing brain glioblastoma cell migration and invasion and attack function according to claim 1 for one kind, it is characterized in that the concrete steps of the method are: according to the design requirements in pGPU6/GFP/Neo siRNA Expression Vector Kit, design corresponds to the interference DNA fragmentation of DIAPH1 gene, the i.e. oligonucleotide sequence of the shRNA of GGA ACA AGC ATG AGA TCA TTC, the oligonucleotide sequence of described shRNA is respectively 62bp.
3. an interference plasmid, is characterized in that this plasmid contains DNA fragmentation according to claim 1.
4. prepare the method for interference plasmid according to claim 3 for one kind, it is characterized in that the concrete steps of the method are: according to the specific requirement of carrier design in pGPU6/GFP/Neo siRNA Expression Vector Kit test kit, DNA fragmentation according to claim 1 is connected with pGPU6/GFP/Neo expression vector, obtains the interference plasmid of DIAPH1 gene.
5. a DNA fragmentation with the migration of suppression brain glioblastoma cell and invasion and attack function according to claim 1 is preparing the application for the treatment of in cerebral glioma medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310156550.8A CN103215270B (en) | 2013-04-28 | 2013-04-28 | DNA (deoxyribonucleic acid) segment with function of inhibiting cell migration and invasion of glioma, and application of DNA segment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310156550.8A CN103215270B (en) | 2013-04-28 | 2013-04-28 | DNA (deoxyribonucleic acid) segment with function of inhibiting cell migration and invasion of glioma, and application of DNA segment |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103215270A CN103215270A (en) | 2013-07-24 |
| CN103215270B true CN103215270B (en) | 2015-02-25 |
Family
ID=48813428
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310156550.8A Expired - Fee Related CN103215270B (en) | 2013-04-28 | 2013-04-28 | DNA (deoxyribonucleic acid) segment with function of inhibiting cell migration and invasion of glioma, and application of DNA segment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103215270B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773767B (en) * | 2014-01-24 | 2016-01-20 | 华东理工大学 | The inhibitory RNA of target ADGB and preparing the application in antitumor drug |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102653762A (en) * | 2011-12-21 | 2012-09-05 | 上海大学 | Gene with function of inhibiting proliferation of gastric cancer cell BGC-823 and preparation method of gene |
-
2013
- 2013-04-28 CN CN201310156550.8A patent/CN103215270B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102653762A (en) * | 2011-12-21 | 2012-09-05 | 上海大学 | Gene with function of inhibiting proliferation of gastric cancer cell BGC-823 and preparation method of gene |
Non-Patent Citations (3)
| Title |
|---|
| DIAPH2和FMNL1在大肠癌组织中的表达及其临床意义;李余发等;《实用医学杂志》;20091231;第25卷(第1期);第47页左栏第1段，第48页左栏第4段 * |
| Ezrin影响胶质瘤在裸鼠脑内的浸润性生长;刘乃杰等;《中国实验诊断学》;20130228;第17卷(第2期);232-236 * |
| NM_001079812;Rai et al.;《GenBank》;20120626;1-11 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103215270A (en) | 2013-07-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111979290B (en) | Application of SPP1 gene in preparation of medicine for enhancing sensitivity of ovarian cancer patient to PARP inhibitor | |
| CN106591306B (en) | Application of the siRNA of targeting interference tumour PTN-PTPRZ1 access in immunotherapy of tumors | |
| WO2012096461A2 (en) | Composition for suspension culturing of stem cells | |
| CN105567686B (en) | Application of miR-126-3p in porcine ovarian granular cells | |
| CN111718995B (en) | Biomarker for nasopharyngeal carcinoma metastasis diagnosis and/or prognosis evaluation | |
| CN111500578A (en) | Circ RNA-FTO for regulating and controlling osteogenic differentiation and tissue regeneration of ADSCs and application thereof | |
| CN104726500A (en) | Application of MicroRNA26b-3p inhibitor in preparation of human umbilical cord derived mesenchymal stem cell | |
| CN109055374B (en) | shRNA for specifically inhibiting OCT1 gene expression and application thereof | |
| CN104388428A (en) | Double-chain siRNA for interfering hnRNPA2/B1 gene expression and application thereof | |
| CN102433336B (en) | Micro-molecular interference ribonucleic acid (RNA) for inhibiting glioma proliferation and promoting glioma apoptosis as well as preparation method and application thereof | |
| CN101353656B (en) | siRNA inhibiting expression of epidermal growth factor receptor genes and use thereof | |
| CN110251529A (en) | MiR-124-3p and its analog are preparing the application in anti-breast cancer disease medicament | |
| CN103215270B (en) | DNA (deoxyribonucleic acid) segment with function of inhibiting cell migration and invasion of glioma, and application of DNA segment | |
| CN101705227B (en) | SiRNA for inhibiting human AP-2alpha gene expression and anti-cervical cancer application thereof | |
| CN109745335A (en) | MiR-218 is preparing the application in mammary cancer chemotherapy drug sensitizer | |
| KR101325186B1 (en) | An Anticancer Agent Comprising Double-stranded miRNAs as an Active Ingredient | |
| CN104995300B (en) | The adjusting of RNA activity and vasopermeability | |
| CN104357451B (en) | For the siRNA of DD3 gene and expression vector establishment thereof and application | |
| CN100352922C (en) | SiRAN and expression carrier for inhibiting human VEGF gene expression and their pharmaceutical use | |
| KR102466691B1 (en) | Composition for treating metastatic solid cancer comprising TSG6 inhibitor | |
| CN103952406B (en) | The siRNA of targeting STAT3 gene of suppression people's malignant glioma propagation and expression vector thereof and application | |
| CN104911183A (en) | Transcription template of shRNA for inhibiting expression of pancreatic cancer TAK1 gene | |
| CN111575384A (en) | Application of human GLT8D1 gene in clinical diagnosis and treatment of tumor | |
| CN104693244A (en) | Application of 4,4'-bipyridine bridged tetranuclear platinum complex in preparation of anti-telomerase negative tumor medicine | |
| KR101613709B1 (en) | Pharmaceutical Compositions for Inhibiting Invasion and Migration of Brain Cancer |
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: 20150225 Termination date: 20180428 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |