CN108642179A - The MiR-210 experimental methods that related target is verified in glioma - Google Patents
The MiR-210 experimental methods that related target is verified in glioma Download PDFInfo
- Publication number
- CN108642179A CN108642179A CN201810481754.1A CN201810481754A CN108642179A CN 108642179 A CN108642179 A CN 108642179A CN 201810481754 A CN201810481754 A CN 201810481754A CN 108642179 A CN108642179 A CN 108642179A
- Authority
- CN
- China
- Prior art keywords
- cell
- mir
- group
- glioma
- renilla
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Toxicology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Hospice & Palliative Care (AREA)
- Genetics & Genomics (AREA)
- Oncology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Experimental method that related target verifies that the invention discloses MiR 210 in glioma, it is characterized in that, include following steps：Utilize glioma cell line, 210 overexpressing cell groups of structure miR, 210 low expression groups of cells of miR and empty plasmid groups of cells respectively, pass through real time qPCR, western blotting experiments detection miR 210 detect the expression of target point protein and its gene to the influence degree of downstream gene.And the related biological that clear miR 210 is tested by MTT, invasion, migration etc. acts on.And it is whether correct by the clear target spot of reporter gene.
Description
Technical field
Experimental method that related target verifies that the present invention relates to MiR-210 in glioma.
Background technology
Human glioma is the most common tumour of central nervous system, accounts for about the 40%-50% of adult's intracranial tumors.At present
Clinical mainly use performs the operation and is aided with the complex treatments such as radiotherapy, chemotherapy.But glioma patient's (especially glioblastoma)
Therapeutic effect is still undesirable, and case fatality rate and recurrence rate are still very high, is worst one of the tumour of prognosis.In recent years, although it is micro-
Neurosurgery technology and related auxiliary treatment means are greatly improved, but therapeutic effect is still bad, this is mainly and people
Glioma quickly grows, infiltrates, invading and drug resistance is related, but mechanism therein is not clear.Inquire into the morbidity of glioma
Mechanism, it is clear its how the mechanism of fast-growth and invasion, be always the more popular topic of neurosurgery research field.
From the point of view of current research, tumor invasiveness relates generally to following three aspects：(1) tumour cell sticks and moves
It moves；(2) degradation (3) tumor neovasculature generation of extracellular matrix.The fast-growth of glioma often makes at tumour cell
In the environment of anoxic, how glioma grows and how to continue to surrounding normal brain tissue invasiveness under anaerobic environment
, current research prompt may be related with tumor neovasculature generation, and current research prompts HIF-1 accesses in anoxic ring
It adjusts under border and plays an important role in tumor neovasculature generation.It lacks
Oxygen inducible factor-l (Hypoxia inducibIe factor-l, HIF-l) is by Semenzal993 in anoxic
A kind of transcription factor found in the nucleus extraction object of induction is widely present in mammal and human body cell under anoxia condition
In, heterodimer is constituted by HIF-l α and HIF-l α, wherein HIF-l α are that main oxygen adjusts subunit [5].HIF-l α can with lack
Oxygen acclimatization has the promoter of correlation gene such as vascular endothelial growth factor (VEGF) or the HIF-l binding site knots on enhancer
It closes, promotes the transcription of these genes.Anaerobic environment is commonly present in human solid cancers, in Several Kinds of Malignancy and precancerous lesion
In detect the overexpression of HIF-l α albumen, then without expression in normal structure and benign lesion.Early stage i.e. blood occurs in tumour
Pipe forms and can occur before invading generation the overexpression of HIF-l α, and has with tumour growth, vascularization, invasion transfer
It closes.HIF-l α are had proven in the apoptosis that anoxic mediates and tumor cell proliferation plays an important role.The HIF-1 in human glioma
α accesses equally play a significant role.MicroRNAs (miRNAs) is a kind of non-volume of endogenous being made of 17~27 nucleotide
Code tiny RNA makes target gene mRNA degrade or mRNA is inhibited to translate by binding purpose gene mRNA 3` noncoding regions.So far
Until, the mankind miRNAs of discovery is more than 2000 (http://www.mirbase.org/).They are related to many relevant lifes
Object process, including cell Proliferation, differentiation, Cycle Regulation etc.；Simultaneously in tumor cells pathology also there are many application,
Including：Tumour generation, differentiation, increment, Cycle Regulation, angiogenesis, chemotactic, migration, invasion and apoptosis.And it wherein studies
MiRNA related with anoxic has miR-210, miR-20a, miR-20b, miR-214, miR-138, miR-145 and miR-503
Deng.In our early-stage studies find miR-210 in human glioma by HIF-l α, and play adjust cell invasion work
With.MiR-210 is the transcription product (http in the regions chromosome numbers 11p15.5://www.microma.org/microma/),
There is substantial connection with growth, angiogenesis, invasion and the apoptosis under many tumor hypoxias.Current result of study prompt, miR-
210 all play an important role in kinds of tumors.Rothe et al. points out the proliferation of miR-210 and breast cancer, transfer, invasion
It is related to poor clinical prognosis, it can be as the potential biology index [14] of prediction breast cancer clinical effectiveness.Camps et al.
The miR-210 for reporting hypoxia inducible is used as the independent diagnostics factor in breast cancer, and expression is with DFS phase and always
Negative correlation is presented in body life cycle, particularly evident in single argument and multi-variables analysis.In addition, miR-210 not only with breast cancer
Prognosis it is related, and also play an important role in terms of Metastasis in Breast Cancer invasion.In cancer of pancreas, HIF-l α inductions generate miR-
210, and the prognosis of miR-210 and patient have substantial connection.MiR-210 mainly connects in clear cell carcinoma of kidney (CCRCC)
It is adjusted by HIF-1, but when HIF-1 is lacked, also receives the adjusting of HIF-2.Target spots of the ISCU as miR-210, expression with
MiR-210 presents negatively correlated.In addition, expression of the miR-210 in CCRCC with preferable clinical prognosis and tumour it is low by stages
Low classification is associated, this is unexpected because many research reports all show expression of the miR-210 in tumour with it is poor
Clinical prognosis it is associated.Tea polyphenols (EGCG) can prevent the ubiquitination of HIF-1 α hydroxylatings dependence and subsequent proteasome
The degradation of mediation, to induce expression of the miR-210 in lung cancer, and the overexpression of miR-210 can influence lung carcinoma cell increasing
It grows and grows, and the high expression of miR-210 indicates poor prognosis.In succinate dehydrogenase (SDH) saltant type incidence pair
In ganglioma (HNPGLs), the functional disturbance of SDH induces the unstable expression of HIF.Merlo etc. has found that hypoxemia can induce HIF-
The expression of l α, then HIF-l α induce tumour cell to be overexpressed miR-210, and then adjust the low expression of downstream targets ISCU, from
And HIF-1 α/miR-210/ISCU signal shaft is formd, it is closely related with the pathogenesis of SDH saltant types HNPGLs.
MiR-210 receives the regulation and control of HIF signal paths, the up-regulated expression of hypoxia inducible miR-210 in oophoroma, and then inhibits downstream
The expression of target spot E2F3 plays an important role in tumour generation as the important regulatory factor of hypoxemia response.It is low in liver cancer
Oxygen/miR-210/ blood vessels Membrane Protein 1 (VMP1) access is proved to play a significant role in liver cancer cells hypoxemia is survived and is invaded,
Important value is played to eliminate the liver cancer cells survived under low-oxygen environment.Greither et al. researchs find miR-210 at soft group
The expression knitted in sarcoma (STS) is related with gender and tumor invasion age.
At present clinically for glioma grading, generally glioma is divided according to 2007 World Health Organization (WHO) standard
For four ranks：Pilocytic astrocytoma (I grade of WHO), diffusivity astrocytoma (II grade of WHO), Alzheimer cells
Tumor (III grade of WHO), glioblastoma (IV grade of GBM, WHO).And wherein glioblastoma accounts for more than half, and the life that is averaged
The phase is deposited less than 15 months.So cell experiment is often chosen representative glioblastoma cell line and is verified.Closely
High-throughput protein chip technology is universal over year, substantially increases the efficiency of detection and the screening of associated downstream target spot.And phase
The technology of pass is also in maturation.The application of Relational database simultaneously is also found target spot for us and is provided conveniently.
The present invention is tested by clinical detection and related biological, studies under hypoxemia miR-210 by adjusting downstream targets,
Find the secret that glioma can grow under hypoxemia, migrate and invade.Present invention has discovered that miR-210 is in glioma
Expression can be used as detection tumour progression, judge tumor prognosis and treat the means of tumour, and only in astroglia
The glioma of origin is just suitble to.The present invention has studied effects of the typical case target spot ROD1 of miR-210 in glioma simultaneously.
But with regard to current research level from the point of view of, it is urgently to be resolved hurrily still to leave problems.Firstly, for miR-210 in glioma
It studies or fewer, secondly, miR-210 target spots is not clear in glioma, cannot provide effect for the treatment of glioma
Target spot.The understanding that a system is formed to miR-210, it is essential to confirm to target spot downstream, confirms downstream targets, completely
Signal path can more clearly from show that the regulation and control of miR-210 influence the mechanism of physiology and pathologic process, to more convenient
Put into clinical application.Especially in recent years, with the continuous development of small nucleic acids pharmaceutical technology, especially nucleic acid drug targeting is passed
The rapid development of feed technique greatly reduces the toxicity of pharmaceutical carrier and improves the stability and targeting of drug conveying, makes small
Nucleic acid patent medicine is simultaneously possibly realized applied to clinical treatment.Finally, the miRNA that miR-210 is closed as a species specificity Anoxic Phase, has
It hopes and provides new target spot for the treatment of glioma, new direction is provided for the research and development of small nucleic acids drug.
Invention content
The technical problem to be solved by the present invention is to：By the control of protein chip technology and database, screen in glioma
The corresponding target spots of miR-210, it was demonstrated that the possibility mechanism of action of miR-210 in glioma.
To solve the above-mentioned problems, the present invention adopts the following technical scheme that：
Using glioma cell line, miR-210 overexpressing cells group, miR-210 low expressions groups of cells and sky are built respectively
Plasmid cell group passes through real-time qPCR, influences of the western blotting experiment detections miR-210 to downstream gene
Degree detects the expression of target point protein and its gene.And the phase of clear miR-210 is tested by MTT, invasion, migration etc.
Close biological action.And it is whether correct by the clear target spot of reporter gene.
More specifically, following experimental procedure may be used：
1. the foundation of plasmid construction, cell model
MiR-210mimics and inhibitors and blank miRNA-FITC is purchased from the sharp rich biology in Guangzhou.U251 and
U87-MG glioma cell lines are cultivated respectively to 24 orifice plates, and transfection changes serum free medium in first 2 hours, with liposome 2000
It is transfected into cell after (Lipofectamine 2000Transfection Reagent, Invitrogen) package plasmid, 6 hours
After change liquid, fluorescence microscopy microscopic observation FITC is expressed after 24 hours, is passed through and is compared the visible light visual field and the fluorescence visual field and calculate transfection
Efficiency, close observation cell growth condition and vigor.1st day, another group of cell is extracted about 1 × 10 respectively5Cell drops to
35mm orifice plates change liquid in 2-4 days daily.By above-mentioned processing, miR-210 overexpressing cells system, miR-210 low expression cells are established
System, empty plasmid cell line and blank control cell line.
2. fluorescence quantitative RT-RCR：
By each group cell line extracted total RNA (Trizol, Invitrogen) to be measured, 50 μ l total serum IgEs, NanoDrop are extracted
2000 ultraviolet specrophotometers (NanoDrop companies) calculate concentration and purity, when OD A260/A280 ratios 1.8-2.0 it
Between when, be just considered extraction RNA purity it is higher.Agarose electrophoresis observes RNA integralities.It is anti-that fluorescence quantitative RT-RCR is carried out again
It answers, it is ensured that the purity and concentration of the RNA of extraction.Reverse transcription is carried out using PrimeScript RT reagent Kit (TaKaRa)
CDNA is obtained, the target point protein PCR using PCR kit (QuantiFast SYBR Green Kit, QIAGEN) and synthesis draws
Object (Invitrogen) carries out expression of the PCR observation target spots in cell line, repeats experiment 3 times.
3. Western blotting detects protein expression：
Experimental cell system extracts total protein after cracking, and supernatant is taken to carry out SDS-PAGE electrophoresis, carbon anode plate after centrifugation
100V, 1h turn pvdf membrane, and after closing plus primary antibody is incubated, antibody closing, rinsing, then secondary antibody is added to be incubated, the colour developing of ECL substrates, X- light
Piece exposure imaging, as a contrast, target point protein expression quantity after observation transfection repeats experiment 3 times to GAPDH.
4. flow cytomery apoptosis of tumor cells and increment situation：
Experimental group cell line is digested with 0.25% pancreatin respectively, through horizontal centrifuge 2000RPM, 15min is centrifuged, collects
Cell abandons culture medium, and cold PBS washings cell is twice.According to cell apoptosis detection kit specification, with 400 μ l1*Binding
Buffer suspension cells, concentration about 5*10cells/ml.5 μ l Annexin V-FITC are added in cell suspending liquid, gently
Mixing is incubated 5min under the conditions of being protected from light in 2-8 DEG C after 15min under the conditions of being protected from light in 2-8 DEG C after mixing, 10 μ l PI of addition.Cold PBS
It is diluted to 2ml.Excitation wavelength 488nm is arranged in flow cytometer, and experiment need to be analyzed for the first time singly contaminates respectively through Annexin V, PI
Cell removes spectra overlapping to adjust fluorescence compensation.Distinguish cell according to untreated cell blank control and through Annexin V, PI
The analysis established standards of single dye control after dyeing.Upper machine repeats experiment 3 times.
5. Matrigel：
MiR-210 overexpressions group, miR-210 low expressions group, empty plasmid cell line and blank control group are put respectively
Enter the holes 6- culture dish to be cultivated, inoculating cell is distinguished in culture dish both sides, carries out cut, continues culture until both sides in culture dish
Cell has the fusion of 80%-90%, the picture of 0 and the situation of each group fusion for 24 hours is taken respectively, using formula [fusion rate=(width0h-
Width24h)/width0h× 100%] fusion rate is calculated.
6. Cell migration assay：
It is first added 1 on the polycarbonate membrane of Transwell upper chambers：2(Matrigel：DMEM after) diluting
Matrigel60 μ l, setting 37 DEG C of 30min makes Matrigel aggregate into gel.It is (thin that chemotactic factor (CF) is added in room under Transwell
Born of the same parents' culture solution) 600 μ l；Single cell suspension is made in cell non-serum culture solution to be checked, and people l × 10 are added in the hole of room5A cell, carefully
100~200 μ l of born of the same parents' suspension volume, every group of cell is parallel to do three holes；It is placed in 5%CO2Incubator cultivates 12h in 37 DEG C.In taking-up
Room discards upper chamber liquid, and the cell that film is not passed through above film is wiped with wet cotton swab.Hematoxylin dyes, after rinsing well
Clearmont bilateral mountings, 80 DEG C are dried.Cell through the membrane, every film center and surrounding are directly observed under inverted microscope
Part is each to take 3 visuals field at random, counts the cell number across 8 μm of micropores in each visual field.
7. reporter gene
Build the reporter plasmid of target point protein.Reporter plasmid is transfected into experiment carefully together with purpose plasmid
Born of the same parents.Reporter gene cell pyrolysis liquid is mixed well, reporter gene cell pyrolysis liquid, abundant lytic cell is added.Fully cracking
Afterwards, 10,000-15,000g is centrifuged 3-5 minutes, takes supernatant for measuring.Melt firefly luciferase detection reagent and
Renilla luciferase assay buffers, and reach room temperature.Renilla luciferase detection substrates are placed on ice bath or ice chest
It is spare.The amount that 100 microlitres are needed according to each sample, takes appropriate Renilla luciferase assay buffers, according to 1:100 are added
Renilla luciferases detection substrate (100X) is configured to Renilla luciferases detection working solution.By instrumentation specification
Fluor tester is opened, measuring interval is set as 2 seconds, minute is set as 10 seconds.Complete said determination firefly luciferin
After enzyme step, 100 microlitres of Renilla luciferases are added and detect working solution, are beaten with rifle or with after other appropriate ways mixings
Measure RLU (relative light unit).Using Renilla luciferases as internal reference, firefly luciferin is used
The RLU values that the RLU values divided by Renilla luciferase assays that enzymatic determination obtains obtain.Check the transcriptional activity of destination protein.
The technical effects of the invention are that：
Using protein chip, existing database and target spot forecasting tool, the clear target that miR-210 is acted in glioma is explored
Point specifies miR-210 and adjusts the mechanism for growing and invading under glioma low-oxygen environment.
Many studies have shown that miRNAs takes part in the regulation and control of tumour, since self-discovery MiR-210, it is studied more and more fiery
Heat.MiR-210 plays a crucial role in terms of adjusting hypoxic response.And protein chip technology high-throughput in recent years is general
And the efficiency of detection and the screening of associated downstream target spot is substantially increased, relevant technology is also gradually ripe.While database
It is provided conveniently using also target spot is found for us.The present invention utilizes novel protein chip technology combination database, is examined by clinic
It surveys and related biological is tested, explore miR-210 under low-oxygen environment influences glioma growth, migration by adjusting downstream targets
With the mechanism of invasion.Western blotting, PCR equimolecular Bioexperiment detects correlation RNA and protein expression level, cell
Culture and cell function experimental analysis its act on and verify.A novel target spot ROD1 of miR-210 is had studied in colloid simultaneously
Effect in tumor.It explores miR-210 and its acts on influence and act on state that related target plays in glioma occurrence and development
It is inside and outside to have not been reported.
Description of the drawings
Attached drawing is used to provide further understanding of the present invention, and a part for constitution instruction, the reality with the present invention
It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings：
Fig. 1 is the Technology Roadmap of the present invention；
Fig. 2 is that PCR detects expression of the miR-210 in normal cerebral tissue and different stage glioma.
Specific implementation mode
Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings, it should be understood that preferred reality described herein
Apply example only for the purpose of illustrating and explaining the present invention and is not intended to limit the present invention.
As shown in Figs. 1-2,1. clinical samples are collected and cell line culture
The initial diagnosis for the attached Wuxi No.2 People's Hospital's operation excision of Nanjing Medical University that past collects is colloid
Blastoma specimens sample washes away the tissue of the necrosis of blood clot and coagulation, is put into liquid nitrogen rapidly, is stored in our hospital
In sample storehouse (- 80 DEG C).30 glioblastoma samples and 10 normal cerebral tissue's samples are taken to carry out protein chip experiment.Glue
Matter oncocyte system (U87 and U251) derives from biomedical laboratory of Nanjing Medical University.Choose the colloids such as U251 and U87-MG
Oncocyte system is cultivated using the D-MEM high glucose mediums that 10% fetal calf serum and 1% penicillin/streptomycin is added, respectively
It is divided into normal group and hypoxia group, normal group is placed in 5%CO2It is normally cultivated with 37 DEG C, hypoxia group is placed in 2%O2, 5%CO2、
93%N2It is cultivated with 37 DEG C.Take hypoxia group cell and each three groups of carry out protein chip of normal group cell in each cell line real
It tests.
2. protein chip is analyzed：
Tissue and cell are cracked to extraction albumen respectively, with sample diluent by 20 times of sample dilutions to be measured, by albumen core
Piece (Tumor Suppressors Array, Full Moon Bio Systems) taking-up is placed in moisture preservation box, to specifications
20 μ l, 37 DEG C of water-bath 30min of sample to be checked are added in corresponding lattice, add 20 μ l fluorescent labeled antibodies per lattice after washing, again
37 DEG C of water-bath 30min.Room temperature is put into microarray scanner (4000B, GenePix) setting excitation wavelength 532nm after drying, every
Chip multiple scanning 5 times, every group is repeated 3 times.Then and group the target spot data obtained in cell and DAVID databases are compared,
The data knitted in sample are compared, and select the consistent target point protein of expression trend, and pre- by target spots such as TargetScan
Surveying software screening method may relevant multiple protein.Then subsequent cytology verification is carried out.
3. the foundation of plasmid construction, cell model
MiR-210mimics and inhibitors and blank miRNA-FITC is purchased from the sharp rich biology in Guangzhou.U251 and
U87-MG glioma cell lines are cultivated respectively to 24 orifice plates, and transfection changes serum free medium in first 2 hours, with liposome 2000
It is transfected into cell after (Lipofectamine 2000 Transfection Reagent, Invitrogen) package plasmid, 6 is small
When after change liquid, fluorescence microscopy microscopic observation FITC is expressed after 24 hours, is passed through to compare the visible light visual field and the fluorescence visual field and calculate and is turned
Contaminate efficiency, close observation cell growth condition and vigor.1st day, another group of cell is extracted about 1 × 10 respectively5Cell drops to
35mm orifice plates change liquid in 2-4 days daily.By above-mentioned processing, miR-210 overexpressing cells system, miR-210 low expression cells are established
System, empty plasmid cell line and blank control cell line.
4. fluorescence quantitative RT-RCR：
By each group cell line extracted total RNA (Trizol, Invitrogen) to be measured, 50 μ l total serum IgEs, NanoDrop are extracted
2000 ultraviolet specrophotometers (NanoDrop companies) calculate concentration and purity, when OD A260/A280 ratios 1.8-2.0 it
Between when, be just considered extraction RNA purity it is higher.Agarose electrophoresis observes RNA integralities.It is anti-that fluorescence quantitative RT-RCR is carried out again
It answers, it is ensured that the purity and concentration of the RNA of extraction.Reverse transcription is carried out using PrimeScript RT reagent Kit (TaKaRa)
CDNA is obtained, the target point protein PCR using PCR kit (QuantiFast SYBR Green Kit, QIAGEN) and synthesis draws
Object (Invitrogen) carries out expression of the PCR observation target spots in cell line, repeats experiment 3 times.
5. Western blotting detects protein expression：
Experimental cell system extracts total protein after cracking, and supernatant is taken to carry out SDS-PAGE electrophoresis, carbon anode plate after centrifugation
100V, 1h turn pvdf membrane, and after closing plus primary antibody is incubated, antibody closing, rinsing, then secondary antibody is added to be incubated, the colour developing of ECL substrates, X- light
Piece exposure imaging, as a contrast, target point protein expression quantity after observation transfection repeats experiment 3 times to GAPDH.
6. flow cytomery apoptosis of tumor cells and increment situation：
Experimental group cell line is digested with 0.25% pancreatin respectively, through horizontal centrifuge 2000RPM, 15min is centrifuged, collects
Cell abandons culture medium, and cold PBS washings cell is twice.According to cell apoptosis detection kit specification, with 400 μ l1*Binding
Buffer suspension cells, concentration about 5*10cells/ml.5 μ l Annexin V-FITC are added in cell suspending liquid, gently
Mixing is incubated 5min under the conditions of being protected from light in 2-8 DEG C after 15min under the conditions of being protected from light in 2-8 DEG C after mixing, 10 μ l PI of addition.Cold PBS
It is diluted to 2ml.Excitation wavelength 488nm is arranged in flow cytometer, and experiment need to be analyzed for the first time singly contaminates respectively through Annexin V, PI
Cell removes spectra overlapping to adjust fluorescence compensation.Distinguish cell according to untreated cell blank control and through Annexin V, PI
The analysis established standards of single dye control after dyeing.Upper machine repeats experiment 3 times.
7. Matrigel：
MiR-210 overexpressions group, miR-210 low expressions group, empty plasmid cell line and blank control group are put respectively
Enter the holes 6- culture dish to be cultivated, inoculating cell is distinguished in culture dish both sides, carries out cut, continues culture until both sides in culture dish
Cell has the fusion of 80%-90%, the picture of 0 and the situation of each group fusion for 24 hours is taken respectively, using formula [fusion rate=(width0h-
Width24h)/width0h× 100%] fusion rate is calculated.
8. Cell migration assay：
It is first added 1 on the polycarbonate membrane of Transwell upper chambers：2(Matrigel：DMEM after) diluting
Matrigel60 μ l, setting 37 DEG C of 30min makes Matrigel aggregate into gel.It is (thin that chemotactic factor (CF) is added in room under Transwell
Born of the same parents' culture solution) 600 μ l；Single cell suspension is made in cell non-serum culture solution to be checked, and people l × 10 are added in the hole of room5A cell, carefully
100~200 μ l of born of the same parents' suspension volume, every group of cell is parallel to do three holes；It is placed in 5%CO2Incubator cultivates 12h in 37 DEG C.In taking-up
Room discards upper chamber liquid, and the cell that film is not passed through above film is wiped with wet cotton swab.Hematoxylin dyes, after rinsing well
Clearmont bilateral mountings, 80 DEG C are dried.Cell through the membrane, every film center and surrounding are directly observed under inverted microscope
Part is each to take 3 visuals field at random, counts the cell number across 8 μm of micropores in each visual field.
9. reporter gene
Build the reporter plasmid of target point protein.Reporter plasmid is transfected into experiment carefully together with purpose plasmid
Born of the same parents.Reporter gene cell pyrolysis liquid is mixed well, reporter gene cell pyrolysis liquid, abundant lytic cell is added.Fully cracking
Afterwards, 10,000-15,000g is centrifuged 3-5 minutes, takes supernatant for measuring.Melt firefly luciferase detection reagent and
Renilla luciferase assay buffers, and reach room temperature.Renilla luciferase detection substrates are placed on ice bath or ice chest
It is spare.The amount that 100 microlitres are needed according to each sample, takes appropriate Renilla luciferase assay buffers, according to 1:100 are added
Renilla luciferases detection substrate (100X) is configured to Renilla luciferases detection working solution.By instrumentation specification
Fluor tester is opened, measuring interval is set as 2 seconds, minute is set as 10 seconds.Complete said determination firefly luciferin
After enzyme step, 100 microlitres of Renilla luciferases are added and detect working solution, are beaten with rifle or with after other appropriate ways mixings
Measure RLU (relative light unit).Using Renilla luciferases as internal reference, firefly luciferin is used
The RLU values that the RLU values divided by Renilla luciferase assays that enzymatic determination obtains obtain.Check the transcriptional activity of destination protein.
Claims (1)
- The 1.MiR-210 experimental methods that related target is verified in glioma, it is characterized in that, include following steps：1) foundation of plasmid construction, cell model：Standard good miR-210mimics, inhibitors and blank miRNA-FITC, U251 and U87-MG glioma cell line It is cultivated respectively to 24 orifice plates, transfection changes serum free medium in preceding 2 hours, with being transfected into cell after the package plasmid of liposome 2000,6 Liquid is changed after hour, fluorescence microscopy microscopic observation FITC is expressed after 24 hours, is calculated by comparing the visible light visual field and the fluorescence visual field Transfection efficiency, close observation cell growth condition and vigor；1st day, another group of cell is extracted about 1 × 10 respectively5Cell drips To 35mm orifice plates, liquid is changed daily within 2-4 days；By above-mentioned processing, it is thin to establish miR-210 overexpressing cells system, miR-210 low expressions Born of the same parents system, empty plasmid cell line and blank control cell line；2) fluorescence quantitative RT-RCR：By each group cell line extracted total RNA Trizol, Invitrogen to be measured, 50 μ l total serum IgEs, ultraviolet specrophotometer meter are extracted Concentration and purity are calculated, when OD A260/A280 ratios are between 1.8-2.0, the RNA purity for being just considered extraction meets the requirements, Agarose electrophoresis observes RNA integralities, then carries out fluorescence quantitative RT-RCR reaction, it is ensured that the purity and concentration of the RNA of extraction is adopted Reverse transcription is carried out with PrimeScript RT reagent Kit and obtains cDNA, utilizes PCR kit and the target point protein of synthesis PCR primer carries out expression of the PCR observation target spots in cell line, repeats experiment 3 times；3) Western blotting detects protein expression：Experimental cell system extracts total protein after cracking, taken after centrifugation supernatant carry out SDS-PAGE electrophoresis, carbon anode plate 100V, 1h turns pvdf membrane, and after closing plus primary antibody is incubated, antibody closing, rinsing, then secondary antibody is added to be incubated, the colour developing of ECL substrates, the exposure of X- mating plates Development, as a contrast, target point protein expression quantity after observation transfection repeats experiment 3 times to GAPDH；4) flow cytomery apoptosis of tumor cells and increment situation：Experimental group cell line is digested with 0.25% pancreatin respectively, through horizontal centrifuge 2000RPM, centrifuges 15min, is collected thin Born of the same parents abandon culture medium, and cold PBS washings cell is twice；With 400 μ l1*Binding Buffer suspension cells, concentration about 5* 10cells/ml；5 μ l Annexin V-FITC are added in cell suspending liquid, gently after mixing under the conditions of 2-8 DEG C is protected from light Mixing is incubated 5min under the conditions of being protected from light in 2-8 DEG C after 15min, 10 μ l PI of addition；Cold PBS is diluted to 2ml, and flow cytometer is set Excitation wavelength 488nm is set, experiment for the first time need to analyze the cell singly contaminated respectively through Annexin V, PI and be removed to adjust fluorescence compensation Spectra overlapping, the analysis of single dye control according to untreated cell blank control and after Annexin V, PI distinguish cell dyeing Established standards, upper machine repeat experiment 3 times；5) Matrigel：MiR-210 overexpressions group, miR-210 low expressions group, empty plasmid cell line and blank control group are respectively put into 6 holes Culture dish is cultivated, and inoculating cell is distinguished in culture dish both sides, carries out cut, continues to cultivate until there is both sides cell in culture dish The fusion of 80%-90% takes the picture of 0h and the situation of each group fusion for 24 hours, using formula [fusion rate=(width respectively0hIt is wide Degree24h)/width0h× 100%] fusion rate is calculated；6) Cell migration assay：Matrigel is first added on the polycarbonate membrane of Transwell upper chambers：DMEM is 1：Matrigel60 μ l after 2 dilutions, Setting 37 DEG C of 30min makes Matrigel aggregate into gel, and 600 μ l of chemotactic factor (CF) cell culture fluid are added in room under Transwell； Single cell suspension is made in cell non-serum culture solution to be checked, and people l × 10 are added in the hole of room5A cell, cell suspension volume 100~ 200 μ l, every group of cell is parallel to do three holes；It is placed in 5%CO2Incubator cultivates 12h in 37 DEG C, takes out upper chamber, discards upper chamber liquid, Wipe the cell that film is not passed through above film with wet cotton swab, rear Clearmont bilaterals mounting is rinsed in hematoxylin dyeing well, 80 DEG C It dries, directly observes cell through the membrane under inverted microscope, every film center and peripheral part is each takes 3 visuals field at random is counted The cell number across 8 μm of micropores in each visual field of number；7) reporter geneThe reporter plasmid for building target point protein, experiment cell is transfected by reporter plasmid together with purpose plasmid, will Reporter gene cell pyrolysis liquid mixes well, addition reporter gene cell pyrolysis liquid, abundant lytic cell, fully after cracking, 10000-15000g is centrifuged 3-5 minutes, is taken supernatant for measuring, is melted firefly luciferase detection reagent and Renilla is glimmering Light element enzyme detects buffer solution, and reaches room temperature, Renilla luciferase detection substrates be placed in it is spare on ice bath or ice chest, according to Each sample needs 100 microlitres of amount, appropriate Renilla luciferase assay buffers is taken, according to 1:100 addition Renilla are glimmering Light element enzyme detection substrate is configured to Renilla luciferases detection working solution, and fluor tester is opened by instrumentation specification, Measuring interval is set as 2 seconds, minute is set as 10 seconds, after completing said determination firefly luciferase step, is added 100 Microlitre Renilla luciferases detect working solution, are beaten with rifle or with measuring RLU after other appropriate ways mixings, with In the case that Renilla luciferases are internal reference, the RLU values divided by Renilla fluorescence that are measured with firefly luciferase The RLU values that plain enzymatic determination obtains, check the transcriptional activity of destination protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810481754.1A CN108642179A (en) | 2018-05-18 | 2018-05-18 | The MiR-210 experimental methods that related target is verified in glioma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810481754.1A CN108642179A (en) | 2018-05-18 | 2018-05-18 | The MiR-210 experimental methods that related target is verified in glioma |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108642179A true CN108642179A (en) | 2018-10-12 |
Family
ID=63756885
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810481754.1A Pending CN108642179A (en) | 2018-05-18 | 2018-05-18 | The MiR-210 experimental methods that related target is verified in glioma |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108642179A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110358827A (en) * | 2019-07-09 | 2019-10-22 | 中国人民解放军第四军医大学 | The preparation of application and its kit of the VMP1 gene in pathological diagnosis glioblastoma |
| CN113355284A (en) * | 2021-05-14 | 2021-09-07 | 重庆医科大学附属第一医院 | Transwell chamber for simulating intracellular infiltration and using method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950658A (en) * | 2016-06-07 | 2016-09-21 | 无锡市第二人民医院 | Experiment method for researching roles of TNFR1/MAP4K4/ARP3 compounds in glioma invasive growth mechanism |
-
2018
- 2018-05-18 CN CN201810481754.1A patent/CN108642179A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950658A (en) * | 2016-06-07 | 2016-09-21 | 无锡市第二人民医院 | Experiment method for researching roles of TNFR1/MAP4K4/ARP3 compounds in glioma invasive growth mechanism |
Non-Patent Citations (4)
| Title |
|---|
| N-S LAI ET AL.: "Serum microRNA-210 as a potential noninvasive biomarker for the diagnosis and prognosis of glioma", 《BR J CANCER》 * |
| ZHANG S ET AL.: "MicroRNA-210 regulates cell proliferation and apoptosis by targeting regulator of differentiation 1 in glioblastma cells", 《FOLIA NEUROPATHOL》 * |
| 任志文等: "下调miR-210的表达对U87细胞增殖、侵袭及凋亡的影响", 《郑州大学学报（医学版）》 * |
| 谢大江: "MiR-27a在胶质瘤的表达及其恶性表型体外研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110358827A (en) * | 2019-07-09 | 2019-10-22 | 中国人民解放军第四军医大学 | The preparation of application and its kit of the VMP1 gene in pathological diagnosis glioblastoma |
| CN113355284A (en) * | 2021-05-14 | 2021-09-07 | 重庆医科大学附属第一医院 | Transwell chamber for simulating intracellular infiltration and using method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xu et al. | CircIL4R facilitates the tumorigenesis and inhibits ferroptosis in hepatocellular carcinoma by regulating the miR‐541‐3p/GPX4 axis | |
| Yang et al. | MiR-1246 promotes metastasis and invasion of A549 cells by targeting GSK-3β‒mediated Wnt/β-catenin pathway | |
| Hu et al. | microRNA-128 plays a critical role in human non-small cell lung cancer tumourigenesis, angiogenesis and lymphangiogenesis by directly targeting vascular endothelial growth factor-C | |
| Chen et al. | miR-338-3p suppresses neuroblastoma proliferation, invasion and migration through targeting PREX2a | |
| Müller-Deile et al. | Podocytes regulate the glomerular basement membrane protein nephronectin by means of miR-378a-3p in glomerular diseases | |
| Su et al. | Long non-coding RNA BANCR regulates growth and metastasis and is associated with poor prognosis in retinoblastoma | |
| Sasayama et al. | MicroRNA‐10b is overexpressed in malignant glioma and associated with tumor invasive factors, uPAR and RhoC | |
| Zhao et al. | MiRNA-125b inhibits proliferation and migration by targeting SphK1 in bladder cancer | |
| CN109852695B (en) | miRNA biogenesis in exosomes for diagnosis and therapy | |
| Zhai et al. | miR‐143 suppresses epithelial–mesenchymal transition and inhibits tumor growth of breast cancer through down‐regulation of ERK5 | |
| Zhao et al. | MiRNA-29c regulates cell growth and invasion by targeting CDK6 in bladder cancer | |
| Wan et al. | The lncRNA NORAD/miR-520a-3p facilitates malignancy in non-small cell lung cancer via PI3k/Akt/mTOR signaling pathway | |
| Liu et al. | CircHIPK3 facilitates the G2/M transition in prostate cancer cells by sponging miR-338-3p | |
| Che et al. | miR‐20a inhibits hypoxia‐induced autophagy by targeting ATG5/FIP200 in colorectal cancer | |
| Wei et al. | miR-503-5p inhibits colon cancer tumorigenesis, angiogenesis, and lymphangiogenesis by directly downregulating VEGF-A | |
| Fan et al. | Late SV40 factor: a key mediator of Notch signaling in human hepatocarcinogenesis | |
| Chen et al. | Long noncoding RNA (lncRNA) FOXD2-AS1 promotes cell proliferation and metastasis in hepatocellular carcinoma by regulating MiR-185/AKT axis | |
| Niu et al. | Overexpressed microRNA-136 works as a cancer suppressor in gallbladder cancer through suppression of JNK signaling pathway via inhibition of MAP2K4 | |
| Yang et al. | The expression and function of miR-424 in infantile skin hemangioma and its mechanism | |
| Wang et al. | Downregulation of circDYNC1H1 exhibits inhibitor effect on cell proliferation and migration in hepatocellular carcinoma through miR‐140‐5p | |
| Gu et al. | CircHIPK3/miR-381-3p axis modulates proliferation, migration, and glycolysis of lung cancer cells by regulating the AKT/mTOR signaling pathway | |
| Peng et al. | LncRNA-MALAT1/miRNA-204-5p/Smad4 Axis Regulates Epithelial–Mesenchymal Transition, Proliferation and Migration of Lens Epithelial Cells | |
| CN108642179A (en) | The MiR-210 experimental methods that related target is verified in glioma | |
| Hu et al. | MiR-21-5p promotes sorafenib resistance and hepatocellular carcinoma progression by regulating SIRT7 ubiquitination through USP24 | |
| CN103920164B (en) | MiR-424-5p is suppressing the application in secondary liver cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20181012 |