CN107058318A - MicroRNA molecule miR 6847 5p related to vincristine drug resistance and its application - Google Patents
MicroRNA molecule miR 6847 5p related to vincristine drug resistance and its application Download PDFInfo
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- CN107058318A CN107058318A CN201710123665.5A CN201710123665A CN107058318A CN 107058318 A CN107058318 A CN 107058318A CN 201710123665 A CN201710123665 A CN 201710123665A CN 107058318 A CN107058318 A CN 107058318A
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- vincristine
- colon cancer
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- 229960004528 vincristine Drugs 0.000 title claims abstract description 90
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 title claims abstract description 90
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Abstract
The invention provides a kind of microRNA molecule miR 6847 5p related to vincristine drug resistance and its application, belong to genetic engineering and technical field of cancer biotherapy.The 5p of miR 6847 of the present invention nucleotide sequence is as shown in SEQ ID NO.1, it has high expression, the characteristic in vincristine sensitive cells strain low expression in vincristine mdr cell, vincristine resistance mark can be used as, colon cancer vincristine resistance auxiliary diagnostic box is directed to for preparing, also new target spot is provided for design colon cancer vincristine medicine-resistant medicine.The invention provides application of the miRNA marker in terms of preparation is for treating the vincristine drug resistance auxiliary diagnostic box of colon cancer, screening new treatment of colon cancer medicine, and application of the miRNA inhibitor in antagonism vincristine medicine-resistant medicine is prepared, with preferable clinical value.
Description
Technical field
The present invention relates to genetic engineering and technical field of cancer biotherapy, relate in particular to a kind of resistance to vincristine
The related microRNA molecule miR-6847-5p of the property of medicine and its application.
Background technology
Colorectal cancer is one of most common malignant tumor of digestive tract, and the incidence of disease of colorectal cancer is just raised year by year, and it is sent out
Sick rate is only second to the malignant tumours such as the cancer of the esophagus, stomach cancer and lung cancer.Vincristine (vincristine, VCR) is that have at present extensively
The antineoplastic chemotherapeutics used.However, long-term treatment can make tumour cell to VCR produce drug resistance, this is to cause
The one of the main reasons of chemotherapy failure.Data display, 90% cancer death is relevant with the drug resistance of tumour.
MicroRNA (miRNA) is a class endogenous, non-coding microRNA highly conserved, with adjusting function, length
About 20~25 nucleotides.It has been found that it is relevant with many biological phenomenas, such as develop, break up and apoptosis, tumour occurs also to lead
Relate to the exception of miRNA expressions.In addition, also there are some researches show miRNA and sensitivity phase of the tumour cell to chemicotherapy
Close.MiRNA can wide participation growth cycle, cell propagation and differentiation, Apoptosis, metabolism, neuromodulation, tumour generation
And the various physiology such as interaction and the regulation process of pathology of virus and host.In cell genetic fragment copy number variation,
Insertion, missing and the Genome stability anomaly such as numerical abnormalities of chromosomes can cause some miRNA unconventionality expression or
Regulation and control.MiRNA mutation, abnormal expression or processing modification exception will influence miRNA normal function, cause the table of target gene
Up to changing, the drug susceptibility of tumour cell is influenceed.
Vincristine is played an important role in the therapeutic process of colon cancer, but the resistance phenomenon occurred is to limit it to face
The main cause of bed curative effect.Specify biological functions and mechanism of the new micoRNA in colon cancer cell vincristine resistance
New biological target is provided to effectively improve the clinical efficacy of colon cancer.
The content of the invention
It is an object of the invention to provide a kind of microRNA molecule miR-6847-5p related to vincristine drug resistance
And its application.
The present invention, with reference to bioinformatic analysis method and experimental verification, is passed through using high throughput sequencing technologies of new generation
Colon cancer vincristine drug-resistant cell strain is set up, skill is sequenced with HiSeq 2500 in the research strategy screened using full-length genome
The cell line differential expression of the method for art and bioinformatics, detection and analysis drug-resistant cell strain and non-resistance
MiRNAs, has found miR-663a high expression in vincristine mdr cell.
A kind of microRNA molecule related to vincristine resistance that the present invention is provided, it is miR-6847-5p, its core
Nucleotide sequence contains the sequence shown in SEQ ID NO.1.
The invention provides the biomaterial containing miR-6847-5p, the biomaterial is carrier, host cell, turned
Gene cell system, engineering bacteria.
Biomaterial the invention provides miR-6847-5p molecules or containing it is being used as colon cancer vincristine resistance
Application in mark.
Biomaterial the invention provides miR-6847-5p molecules or containing it is preparing colon cancer vincristine resistance
Application in auxiliary diagnostic box.
In above-mentioned application, the microRNA molecule high expression in vincristine mdr cell.
In above-mentioned application, microRNA molecule low expression in vincristine sensitive cells.
The invention provides the inhibitor of miR-6847-5p molecules answering in the medicine for preparing antagonism vincristine resistance
With.
The present invention provides a kind of medicine for being used to treat colon cancer, contains miR-6847-5p molecule inhibitors.
Detect the specific primer of miR-6847-5p molecules to belonging to protection scope of the present invention for PCR.
In an embodiment of the present invention, for detecting the specific primers of miR-6847-5p molecules to for SEQ ID
Shown in NO.5-7.
The invention provides above-mentioned specific primer in colon cancer vincristine resistance auxiliary diagnostic box is prepared
Application.
The beneficial effects of the present invention are miR-6847-5p of the present invention nucleotide sequence contains such as SEQ ID NO.1 institutes
The sequence shown, it has high expression, the characteristic in vincristine sensitive cells strain low expression in vincristine mdr cell, can
As vincristine resistance mark, colon cancer vincristine resistance auxiliary diagnostic box is directed to for preparing, can be special
MiR-6847-5p expression quantity, is efficiently used for the auxiliary diagnosis of clinical colon cancer vincristine resistance in ground detection tissue；Profit
With the characteristic of the miR-6847-5p of the present invention high expression in vincristine resistance, on this basis, to design and develop
MiR-663a inhibitor is reduced to the application in vincristine drug resistance medicine preparing or screened newly using it as target spot
Application in treatment of colon cancer medicine.It is new in the Changchun that preparation is used to treat colon cancer the invention provides the miRNA marker
Application in terms of the new treatment of colon cancer medicine of alkali drug resistance auxiliary diagnostic box, screening, and the miRNA inhibitor is in system
Application in standby antagonism vincristine medicine-resistant medicine, with preferable clinical value and wide application prospect.
Brief description of the drawings
Fig. 1 is that cell propagation-toxicity test detects VCR sensitive cells HCT-8 and drug-resistant cell strain HCT-8/VCR to VCR
Resistance ability design sketch.
The expression that Fig. 2 is miR-6847-5p in VCR sensitive cells HCT-8 and resistance HCT-8/VCR cells.
Fig. 3 is resistance energy of the cell propagation-toxicity test detection HCT-8 cell transfecting miR-6847-5p analogies to VCR
Power design sketch.
Fig. 4 is that cell propagation-toxicity test is detected after HCT-8/VCR cell transfecting miR-6847-5p inhibitor to VCR's
Resistance ability design sketch.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing substantially from spirit of the invention
In the case of essence, the modifications or substitutions made to the inventive method, step or condition belong to the scope of the present invention.
Unless otherwise specified, chemical reagent used in embodiment is skill used in conventional commercial reagent, embodiment
The conventional meanses that art means are well known to those skilled in the art.Cell line, reagent used etc. are in embodiment and test example
Commercial goods.HCT-8 cells are purchased from Chinese Academy of Sciences's Shanghai cell bank.
Embodiment 1 builds vincristine medicine-resistant cell line HCT-8/VCR
Colon cancer drug-resistant cell strain HCT-8/VCR is set up using the method for being stepped up vincristine (VCR) concentration.Will be quick
Sense cell HCT-8 is incubated in the nutrient solution containing VCR, and initial concentration 5ng/mL gradually increases drug concentration later, is respectively
10th, 100,1 000 and 2 000ng/mL.After each concentration obtains drug resistance, with good thin of limiting dilution method clonal growth
Born of the same parents, subsequently into the culture and screening of next concentration.Last HCT-8 cells persistently cultivated for 20 generations in 2000ng/mL VCR
More than, medicine-resistant cell line HCT-8/VCR is built, tests first 1 week and disables VCR.
The cell of embodiment 2 tests (mtt assay) to VCR drug sensitivity
Take the logarithm the persister HCT-8/VCR and sensitive strain cell HCT-8 in growth period respectively, dilute by serum free medium
It is 1 × 10 that interpretation of the law tally, which counts cell density,5Individual/mL, and with 1 × 104The hole cell training of cell solution inoculation 96 in individual/hole
Support in plate.Blank control group adds the nutrient solution without VCR, and experimental group is separately added into containing 10ng/ml, 20ng/ml, 100ng/
Ml, 200ng/ml, 1000ng/ml, 2000ng/ml, 3000ng/ml, 4000ng/ml vincristine nutrient solution, 100 μ l/ holes.
Each group cell incubates culture 48h in 37 DEG C；Culture plate is taken out, the μ l/ holes of MTT (5mg/mL) solution 20 are added, continued
It is put into 37 degree of cell culture incubators and incubates 4h；Then inhaled with pipettor and abandon culture supernatant, added the μ l/ holes of DMSO 150, fully shake
After swinging, culture plate is put into enzyme-linked immunosorbent assay instrument, Detection wavelength is set as 570nm, absorbance (OD) value in each hole is read.
Background cell mean is calculated, and is returned to zero using this average value as zero point, and calculates each group cell survival rate：Carefully
Born of the same parents' survival rate=(experimental group mean OD value-background group mean OD value)/blank control group mean OD value-background group mean OD value)
× 100%, then calculate drug concentration during 50% cell survival, i.e. half-inhibition concentration IC50.
Colon cancer cell HCT-8 and HCT-8/VCR are handled using the VCR of various concentrations, Fig. 1 is shown in the inhibiting rate of cell.
HCT-8 cells and HCT-8/VCR 503nhibiting concentration IC50 are respectively 140.265 and 2350.469.
Embodiment 3 filters out the miRNA related to vincristine resistance using biology information technology
1st, cell total rna is extracted
Cell is collected, addition 1mL Trizol separation agents in cell culture fluid, each culture hole is discarded, blows and beats cell number
It is secondary to crack cell.5min is incubated at room temperature.0.2mL chloroform reagents are added, lid is covered, 15s are vibrated, 4 DEG C are incubated at room temperature
15min, 15000rpm centrifuge 10min, and solution is separated into three-phase.The upper solution of colourless liquid phase is transferred to a new centrifuge tube
In.0.5mL isopropanols are added, lid is covered, turn upside down makes liquid fully mix for several times, 10min, 4 DEG C is incubated at room temperature
12000rpm centrifuges l5min, abandons supernatant, adds 75% ethanol, and vibration is washed, 4 DEG C, 7500rpm centrifugation 5min, abandons supernatant, does
Dry 5-l0min, removes ethanol.Add the treated ddH of 20 μ L DEPC2O, pipettor is blown and beaten for several times.RNA solution is taken in light splitting
A260, A260/280 are determined on photometer respectively, RNA content and purity is calculated.1 × the TAE handled with 0.2 μ L DEPC,
1% Ago-Gel, 100V constant pressure electrophoresis 30min, observation.
2nd, cDNA library builds and is sequenced
Extract after cell total rna, RNA of the fragment length in 18-30nt scopes is reclaimed, using T4RNA ligases, in fragment
5 ' end add 5 ' sequence measuring joints, 3 ' end add 3 ' sequence measuring joints, then using this with joint fragment as template, using random
Primer synthesizes cDNA, adds 5 ' end connector primers and 3 ' adapter-primers enter performing PCR amplification, set up the complete sequencing libraries of PCR.With
High flux, the high-flux sequence platforms of HiSeq 2500 of highly sensitive Illumina companies exploitation carry out HCT-8 and HCT-8/V
MiRNA sequencing.Sequencing is sequenced by Shanghai Jing Neng bio tech ltd.
3rd, statistical method
MiRNA expression quantity, which is calculated, uses TPM computation measures index (transcript per million), and TPM formula=
(the read numbers that every miRNA is compared)/(the read numbers that sample is always compared) × 106, TPM is meant that with every megabit
MiRNA expression figureofmerits are done with paired sequence, wherein always than being used to normalize expression numerical quantity with paired read numbers.Utilize
DESeq softwares screen the miRNA of differential expression to sample comparison group, calculate sample expression respectively using TPM and measure log2, meet
P≤0.05 and >=2 times of differential expression scopes screen the difference miRNA between two groups.
4th, result is shown, compared with HCT-8 cells, and the miRNA of 48 differential expressions, wherein miR-6847- are filtered out altogether
5p raises 4.32 times in HCT-8/VCR cells, and difference has statistical significance (P<0.01).
MiR-6847-5p expression quantity in the detection HCT-8 and HCT-8/VCR cells of embodiment 4
1st, design of primers
According to acquisition hsa-miR-6847-5p mature sequences (http in miRBaSe databases://
www.mirbase.org,No：MIMAT0027594)：5 '-ACAGAGGACAGUGGAGUGUGAGC-3 ' (SEQ ID NO.1) are set
Count following primer：
Reverse transcriptase primer 1：5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCTCAC-3′(SEQ ID
NO.2)
Reverse transcriptase primer 2：5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCTCACT-3′(SEQ ID
NO.3)
Reverse transcriptase primer 3：5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCTCACTC-3′(SEQ ID
NO.4)
Quantification PCR primer：
Forward primer 1：5′-ACACTCCAGCTGGGACAGAGGACAGTGGAG-3′(SEQ ID NO.5)
Forward primer 2：5′-ACACTCCAGCTGGGACAGAGGACAGTGGAGT-3′(SEQ ID NO.6)
Reverse primer：5′-CTCAACTGGTGTCGTGGAGT-3′(SEQ ID NO.7)
According to ncbi database (https://www.ncbi.nlm.nih.gov/) in U6snRNA house-keeping genes
(Genbank:NR_004394.1 following primer) is designed：
U6snRNA reverse transcriptase primer：5′-CGCTTCACGAATTTGCGTGTCAT-3′；(SEQ ID NO.8)
U6 forward primers：5′-CTCGCTTCGGCAGCACA-3′；(SEQ ID NO.9)
U6 reverse primers：5′-AACGCTTCACGAATTTGCGT-3′；(SEQ ID NO.10)
2nd, cell total rna is extracted
Whole process wears disposable glove.With the non-disposable glassware or plastic ware without RNase.Glassware can
To toast 4h in 150 DEG C of baking oven.Plastic ware can soak 10min in 0.5M NaOH, after being thoroughly rinsed totally with water
Autoclaving is standby.
Whole process is operated on ice, prevents RNA from degrading.Take 5-10 × 106HCT-8 and HCT-8/VCR cells, from
The heart, abandons supernatant, and the Trizol reagents for shifting to an earlier date precooling with pipette plus 1ml are blown and beaten come cell lysis to homogeneous well-illuminated liquid repeatedly
Afterwards, homogenised sample is placed into 5min on ice, it is ensured that cell is sufficiently cracked.200 μ l chloroforms are added, are acutely vortexed 30 seconds, room
Temperature stands 5min.4 DEG C, 12000 × g centrifugations 15min.Careful transfer supernatant is added into a RNase-free1.5ml centrifuge tubes
Isometric isopropanol is mixed.4 DEG C, 12000 × g centrifugation 15min abandon supernatant.Add the ethanol of 750 μ l 75%, 4 degree, 12000 × g
5min is centrifuged, supernatant is abandoned.After ethanol is air-dried, plus 45 μ l DEPC processing water, it is stored at room temperature 2min dissolvings RNA.Denaturing electrophoretic is detected
And concentration mensuration, you can use or -80 DEG C of preservations.
3rd, the following system of DNase I processing configuration：
| RNA | 1μg |
| 10×Reaction Buffer with MgCl2 | 1μl |
| DNase I, RNase-free | 1μl |
| DEPC handles water | To 10 μ l |
| Cumulative volume | 10μl |
37 DEG C of water-bath 30min.65 DEG C of water-bath 10min inactivation DNase I.
4th, reverse transcription
Reverse transcription reaction system (20 μ L)：The μ L of 5 × primer buffer 4, reverse transcriptase 1 μ L, (10 μ of reverse transcriptase primer 1
M) 0.5 μ L, U6snRNA reverse transcriptase primers primer (10 μM) 0.5,1 μ g of μ L, RNA, plus mended without RNase water to 20 μ L systems.Mix
Centrifugation.Reverse transcription reaction condition：42 DEG C of incubations 15min, 85 DEG C of inactivation reverse transcriptase 5s, 4 DEG C of preservations.From reverse transcriptase primer 2
Or 3 respectively according to the method described above carry out reverse transcription reaction can also obtain identical reverse transcription effect.
5、Real-time PCR
The reaction system 20 (μ L) of fluorescence quantitative RT-RCR：The μ L of 2 × SYBR Green PCR Master Mix 10, template
The μ L of DNA 1, upstream and downstream primer (10 μM) each 0.5 μ L, when distilled water is settled to the detection of 20 μ L. real time fluorescent quantitatives, forward primer
1st, 2 expanded respectively with general reverse primer PCR.Template cDNA is respectively that reverse transcriptase primer reverse transcription is produced.
Reaction condition is：95 DEG C of denaturation 10min；95 DEG C of 15s, 60 DEG C of 15s；95 DEG C of 15s, 60 DEG C of 30s, 95 DEG C of 15s, 45
Circulation.Take fluorescent value to draw solubility curve, determine the specificity of amplified production.
6th, has-miR-6847-5p expression quantity statistical analysis
Using U6snRNA house-keeping genes as internal reference, using relative quantification method, the expression quantity of gene is calculated.
The expression quantity F=2 of gene—△△ct
F values are bigger, and expression quantity is higher.Wherein, the △ △ ct=(ct of the target gene of testing sample average values-to be measured
The ct of the house-keeping gene of sample average value)-(house keeper of the ct of the target gene of control sample average value-check sample
The ct of gene average value).
As a result show, compared with HCT-8 cells, has-miR-6847-5p is raised in HCT-8/VCR cells, analysis knot
Fruit is shown in has-miR-6847-5p expression quantity in HCT-8/VCR cells than 3.005 times of HCT-8 cell upregulation, and difference has
Statistical significance (P<0.05), as shown in Figure 2.
It is resistance to after the cell propagation of embodiment 5-toxicity test detection HCT-8 cell transfecting has-miR-6847-5p minics
Medicine characteristic
When HCT-8 cells are in active growth state, the orifice plate of 12 preparation 12 is inoculated into the day before transfection, in 12 orifice plates
Middle inoculation HCT-8 cell suspensions.Second day, transfection miRNA analogies (25nM) were transfected after 8h, and vitellophag simultaneously divides disk to 96
In orifice plate, vincristine (final concentration 10ng/ml, 20ng/ml, 100ng/ml, 200ng/ml, 1000ng/ml, 2000ng/ are added
ml).After 48h, 96 orifice plates are taken out, adding 10 μ L CCK8 solution to every hole (is careful not in hole generate bubble, they can shadow
Ring the reading of OD values).Culture plate is incubated 3h in incubator.The absorbance at 450nm is determined with ELIASA.If temporarily not
OD values are determined, 10 μ L 1%w/v SDS solution can be added into every hole, and cover culture plate and are kept in dark place in room temperature condition
Under.Determined in 24h, absorbance will not change.
As a result show, the resistance ability for transfecting the HCT-8 of has-miR-6847-5p analogies groups cell vincristine shows
Write and be higher than untransfected group (Fig. 3), further illustrate that has-miR-6847-5p can improve the resistance of vincristine of colon cancer cell
Ability.
The cell propagation of embodiment 6-toxicity test detection HCT-8/VCR cell transfectings has-miR-6847-5p
Resistant characterization after inhibitor
By has-miR-6847-5p mortifiers (being purchased from Ji Man biotechnologies (Shanghai) Co., Ltd.) transfection of chemical synthesis
Into HCT-8/VCR cells, has-miR-6847-5p is set to express reduction in HCT-8/VCR cells.As a result show, relative to
Control group non-transfected cells, transfect resistance ability of the HCT-8/VCR cells to vincristine of has-miR-6847-5p mortifiers
(Fig. 4) is significantly reduced, the resistance ability of the vincristine of colon cancer cell can be reversed to a certain extent.Further demonstrate
The resistance ability of the vincristine of has-miR-6847-5p and colon cancer cell is closely related, and miR-6847-5p improves cell pair
The resistance ability of vincristine, miR-6847-5p inhibitor can reduce resistance ability of the cell to vincristine.
The miRNA kits of embodiment 7
MiRNA kits include miR-6847-5p primers (as shown in SEQ ID NO.2-7), pcr reagent
(including thermal starting Taq archaeal dna polymerases (2.5U/ μ L) and its reaction buffer, dNTP (10mM)), U6snRNA internal control primers
(SEQ ID NO.8-10) and fluorescent dye SYBR-Green 1.Using the kit of the present invention, examination is extracted with reference to conventional RNA
Agent and general miRNA reverse transcription reagents, can specifically detect the expression quantity of miR-6847-5p in tissue, be efficiently used for
The auxiliary diagnosis of clinical colon cancer vincristine resistance, precisely to be treated, improved curative effect, reduction side effect.
The effect detection of miRNA kits：
30 colon cancer cases of Henan Prov. Tumour Hospital 2013-2015 are chosen, miRNA has been carried out to its paraffin specimen and carried
Take, sample is analyzed using miRNA kits of the present invention, as a result show that miRNA kits of the present invention can specifically, effectively
Ground amplifies miR-6847-5p sequences, carries out statistical analysis to its expression quantity, as a result shows 14 case sample miR-
MiR-6847-5p expression quantity average out to 2.322 in 12.108 ± 3.032,16 samples of 6847-5p expression quantity average out to ±
2.413, expression quantity differs 5.24 times of (P<0.05).With reference to clinical therapeutic efficacy analysis, the higher sample of miR-6847-5p expression quantity
This is poor using the effect of the treatment colon cancer of vincristine, and the relatively low sample of expression quantity uses the treatment colon of vincristine
The effect of cancer is preferable.It further demonstrate that miR-6847-5p expression is relevant with the drug resistance of colon cancer vincristine.Its reagent
Box can be used for the drug resistance of clinical detection colon cancer vincristine, is further used for instructing clinical application and precisely treats, carries
High curative effect, reduction side effect.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
SEQUENCE LISTING
<110>Henan Prov. Tumour Hospital of Henan Medical college of Zhengzhou University of Zhengzhou Railway Vocational and Technical College
<120>The microRNA molecule miR-6847-5p related to vincristine drug resistance and its application
<130> KHP171110288.9
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> RNA
<213> miR-6847-5p
<400> 1
acagaggaca guggagugug agc 23
<210> 2
<211> 42
<212> DNA
<213>Artificial sequence
<400> 2
ctcaactggt gtcgtggagt cggcaattca gttgaggctc ac 42
<210> 3
<211> 43
<212> DNA
<213>Artificial sequence
<400> 3
ctcaactggt gtcgtggagt cggcaattca gttgaggctc act 43
<210> 4
<211> 44
<212> DNA
<213>Artificial sequence
<400> 4
ctcaactggt gtcgtggagt cggcaattca gttgaggctc actc 44
<210> 5
<211> 30
<212> DNA
<213>Artificial sequence
<400> 5
acactccagc tgggacagag gacagtggag 30
<210> 6
<211> 31
<212> DNA
<213>Artificial sequence
<400> 6
acactccagc tgggacagag gacagtggag t 31
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence
<400> 7
ctcaactggt gtcgtggagt 20
<210> 8
<211> 23
<212> DNA
<213>Artificial sequence
<400> 8
cgcttcacga atttgcgtgt cat 23
<210> 9
<211> 17
<212> DNA
<213>Artificial sequence
<400> 9
ctcgcttcgg cagcaca 17
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<400> 10
aacgcttcac gaatttgcgt 20
Claims (10)
1. the microRNA molecule related to vincristine resistance, it is miR-6847-5p, and its nucleotide sequence contains SEQ ID
Sequence shown in NO.1.
2. the biomaterial containing microRNA molecule described in claim 1, it is characterised in that the biomaterial be carrier,
Host cell, transgenic cell line, engineering bacteria.
3. biomaterial is being used as colon cancer vincristine described in the microRNA molecule or claim 2 described in claim 1
Application in resistance mark.
4. biomaterial described in the microRNA molecule or claim 2 described in claim 1 is preparing colon cancer vincristine
Application in resistance auxiliary diagnostic box.
5. the application as described in claim 4 or 5, it is characterised in that the microRNA molecule is in vincristine mdr cell
Middle high expression.
6. the application as described in claim 4 or 5, it is characterised in that the microRNA molecule is in vincristine sensitive cells
Middle low expression.
7. the inhibitor of the microRNA molecule described in claim 1 answering in the medicine for preparing antagonism vincristine resistance
With.
8. a kind of medicine for being used to treat colon cancer, it is characterised in that the suppression containing the microRNA molecule described in claim 1
Preparation.
9. the specific primer pair for microRNA molecule described in PCR test rights requirement 1.
10. the specific primer described in claim 9 is in colon cancer vincristine resistance auxiliary diagnostic box is prepared
Using.
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| CN112458175A (en) * | 2020-12-01 | 2021-03-09 | 河北仁博科技有限公司 | Application of miR-3689a-5p in preparation of preparation for diagnosing or treating tumor drug resistance |
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| CN104388541A (en) * | 2014-10-21 | 2015-03-04 | 复旦大学附属肿瘤医院 | Application of miR-1914* and miR-1915 |
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| CN104388541A (en) * | 2014-10-21 | 2015-03-04 | 复旦大学附属肿瘤医院 | Application of miR-1914* and miR-1915 |
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| CN112458175A (en) * | 2020-12-01 | 2021-03-09 | 河北仁博科技有限公司 | Application of miR-3689a-5p in preparation of preparation for diagnosing or treating tumor drug resistance |
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