CN107164378A - Specificity suppresses siRNA and its recombinant vector and the application of SPIB gene expressions - Google Patents
Specificity suppresses siRNA and its recombinant vector and the application of SPIB gene expressions Download PDFInfo
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- CN107164378A CN107164378A CN201710470667.1A CN201710470667A CN107164378A CN 107164378 A CN107164378 A CN 107164378A CN 201710470667 A CN201710470667 A CN 201710470667A CN 107164378 A CN107164378 A CN 107164378A
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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Abstract
Suppress the siRNA and its recombinant vector of SPIB gene expressions and the application in the medicine for reversing oophoroma taxol resistance is prepared the invention discloses a species specificity, belong to molecular biology and biomedicine technical field.The siRNA, including positive-sense strand and antisense strand, positive-sense strand：5’‑AAUCAGGGUAGCUGGAAUGCU‑3’；Antisense strand：5’‑CAUUCCAGCUACCCUGAUUCA‑3’.The siRNA that the present invention is provided can specificity, efficiently suppress the mRNA and protein expression of SPIB genes, reduce cell propagation, increase Apoptosis, reduction cell migration and invasive ability, and can effectively reverse ovarian cancer cell to the resistance of taxol.Present invention also offers the application of the siRNA and its recombinant vector in the medicine for preparing treatment oophoroma, stomach cancer, colorectal cancer, head and neck squamous cell carcinoma or reverse ovarian cancer drug-resistant.
Description
Technical field
The present invention relates to molecular biology and biomedicine technical field, a specially species specificity suppresses SPIB gene tables
The siRNA and its recombinant vector that reach and application.
Background technology
RNA interference (RNA interference, RNAi) is the sequence specific post transcriptional base being widely present in animals and plants
Because of Silencing Mechanisms.American scientist Andrew Fire in 1998 have found first in beautiful new rhabditis axei C.Elegans bodies
The inhibitory action of at least 10 times GEM 132s of gene silencing effect produced by dsRNA, and can be induced in filial generation
Same gene suppression.The study mechanism of RNAi phenomenons is shown, can efficiently, specifically degrade cognate rna to cause sequence
The key molecules of row silencing specific genes are the small double chain oligonucleotide of a length of 21-23 bases, also referred to as siRNA
(siRNA).Effectively start RNAi, Er Qieqi are unable to further study show that being shorter than 21bp or being longer than 25bp double-stranded RNA
As long as in have a base mispairing, just substantially decline even disappears the effect of gene silencing, has fully demonstrated the spy of siRNA effects
The opposite sex.
SiRNA is attracted attention as the great discovery of biotechnology in recent years, exactly because it has specificity and high
Effect property blocks the gene of all homologous geneses to suppress function, is the strong instrument of gene functional research.It is dry using small molecule
Disturb RNA (siRNA) and be widely used in the research of malignant tumour mechanism as the method for gene silencing, and thus promoted with the skill
The research and development process of the efficient targeted therapy strategy of tomour specific based on art.
Oophoroma death rate in genital system ranks first, onset concealment, lacks early symptom and effective early stage
Diagnostic means, malignant progression is fast.The therapeutic scheme of standard is that surgical cytoreduction is aided with postoperative platinum class/PTX medication combinedization
Treat, although 70% patient may occur in which complete incidence graph, but most of recurrences in 18 months.Oophoroma after recurrence, even if adopting again
With the entirely different chemotherapeutics of mechanism of action, resistance can be also produced, so that survival rate cannot be significant after oncotherapy
Improve.The five year survival rate of advanced ovarian cancer is hovered 30% or so all the time.High relapse rate and resistant rate are that ovarian cancer prognosis is poor
Major reason.How to improve the therapeutic effect of oophoroma, find the solution of chemotherapy in ovarian cancer resistance, be that gynecological tumor is ground
The important topic studied carefully.
Molecular targeted agents and chemotherapy combined application are the current raising most promising treatments of malignant tumor patient survival rate
Method.SPIB (Spi-B transcription factor) is Ets transcription factor family members, and gene is located at chromosome 19q,
Only expressed under normal circumstances on ripe B cell, T cell progenitor cells and plasmacytoid dendritic cellss, in mature B cell point
Played an important role in the atomization for turning to thick liquid cell and plasmacytoid dendritic cellss, in the hair tonic in embryo development procedure
Played the role of a nucleus in heart reaction, the mouse development of SPIB gene delections is slow, has substantial amounts of in vivo compared with the mouse of wild type
Apoptotic cell is produced.Research recently finds that SPIB has in entity tumor stomach cancer, colorectal cancer and head and neck squamous cell carcinoma
Height expression.We show that SPIB also has high expression in ovarian cancer cell at research in advance, and it was found that in oophoroma taxol
There is abnormal elevated phenomenon in drug-resistant cell strain A2780/Taxol, but whether the gene is relevant with ovarian tumors to need
Further research.Therefore, SPIB gene expressions in ovarian cancer drug-resistant cell strain are disturbed using RNAi technology, will is to ovum
The important supplement of nest cancer pathogenesis, is the important exploration and application to oophoroma and its resistance treatment.
The content of the invention
It is an object of the invention to provide the siRNA that a species specificity suppresses SPIB gene expressions, for ovarian tumors
Mechanism Study.It is another object of the present invention to provide the siRNA in the medicine for reversing oophoroma taxol resistance is prepared
Application.
To achieve the above object, the present invention is adopted the following technical scheme that：
Present invention design, 3 pairs of specificity of synthesis suppress the siRNA of SPIB gene expressions, are transfected into oophoroma taxol resistance to
In medicine cell line A2780/Taxol, as a result find that the interference effect of S2 suppression SPIB gene expressions is most obvious.
The invention provides the siRNA (S2) that a species specificity suppresses SPIB gene expressions, including positive-sense strand and antisense strand,
The positive-sense strand：5’-AAUCAGGGUAGCUGGAAUGCU-3’(SEQ ID NO.1)；
The antisense strand：5’-CAUUCCAGCUACCCUGAUUCA-3’(SEQ ID NO.2).
Preferably, 3 bases at the positive-sense strand and the 5 ' of antisense strand and 3 ' ends carry out the modification of 2 '-methoxyl group.This hair
Bright research has shown that siRNA (S2) stability increase after the modification of 2 '-methoxyl group can improve its water for resisting ribozyme in vivo
The ability of solution, reduction immunostimulation reaction, extension siRNA disturbs the action time of down regulation of gene expression, its effect is had height
Effect property, specificity.
The invention provides a kind of rnai reagent box for including the DNA sequence dna for encoding the siRNA.The kit
In comprising having cloned the DNA plasmid carrier of the siRNA, using when, the plasmid vector is in eukaryotic needed for transcriptional expression
SiRNA, so as to reach the expression of silence SPIB genes.
The invention provides a kind of recombinant vector containing the DNA sequence dna for encoding the siRNA.Preferably, use
Initial carrier is slow virus carrier pLKO.1puro.
Present invention also offers the construction method of recombinant vector, including：
(1) SPIB-S2 fragments are synthesized, Age I and EcoR two restriction enzyme sites of I is selected, according to S2 sequence, designs it
ShRNA sequences, sequence is as follows：
Positive-sense strand：
5’-CCGGTGAATCAGGGTAGCTGGAATGCTTTCAAGAGAGACATTCCAGCTACCCTGATTCATTTTTTG
GTACC-3’(SEQ ID NO.7)；
Antisense strand：
5’-AATTGGTACCAAAAAATGAATCAGGGTAGCTGGAATGTCTCTCTTGAAAGCATTCCAGCTACCCTG
ATTCA-3’(SEQ ID NO.8)；
(2) annealing obtains SPIB-S2 DNA fragmentation；
(3) pLKO.1-SPIB-sh2 recombinant vectors are built with slow virus carrier pLKO.1puro.
The siRNA that the present invention is provided being capable of the efficiently specific expression for suppressing ovarian cancer cell SPIB genes, reduction cell
Propagation, increases Apoptosis, and reduction cell migration and invasive ability, therefore, the siRNA and recombinant vector are used as SPIB genes
Expression inhibiting agent can apply among the research of tumor disease pathogenesis.
The invention provides the application of the siRNA and recombinant vector in SPIB gene expression inhibitors are prepared.
Treatment oophoroma, stomach cancer, colorectal cancer or incidence are being prepared the invention provides the siRNA and recombinant vector
Application in squamous cell cancer drug.
Present invention research shows that taxol resistance strain A2780/Taxol is transfected after the siRNA, and the cell line is to Japanese yew
The sensitiveness of alcohol is significantly improved, and it is 19.51 to reverse index, illustrates the siRNA of the invention provided to taxol resistance strain A2780/
The reversing effect highly significant of Taxol resistances, therefore, the siRNA are latent to reversing the treatment of oophoroma taxol resistance to have
In application value.
The invention provides described siRNA and recombinant vector in the medicine for reversing oophoroma taxol resistance is prepared
Using.
The beneficial effect that the present invention possesses：
The siRNA that the present invention is provided can specific, efficiently suppress the mRNA and protein expression of SPIB genes, reduce swollen
Tumor cell proliferation, increases Apoptosis, reduction tumor cell migration and invasive ability, and can effectively reverse ovarian cancer cell to purple
The resistance of China fir alcohol, is applied to Tumorigenesis research and prepares oncotherapy and reverse the medicine of ovarian cancer drug-resistant treatment
It is significant in thing.
Brief description of the drawings
Fig. 1 is that qRT-PCR detects SPIB mRNA expressions in A2780 cells and A2780/Taxol cells.
Fig. 2 is that Western Blotting detect SPIB protein expression feelings in A2780 cells and A2780/Taxol cells
Condition.
Fig. 3 is SPIB mRNA expressions in A2780/Taxol cells after qRT-PCR detection S1, S2, S3 transfections 48h.
Fig. 4 is SPIB albumen tables in A2780/Taxol cells after Western Blotting detection S1, S2, S3 transfections 72h
Up to situation.
Fig. 5 is pLKO.1-SPIB-sh2 recombinant plasmids and insertion restriction enzyme site schematic diagram.
Fig. 6 is small hair fastener shRNA schematic diagrames.U6 promoters instruct the small hair fastener shRNA in downstream transcription；Including 23 S2 just
Adopted chain base, 23 S2 antisense strand bases.
Fig. 7 is SPIB eggs in A2780/Taxol cells after Western Blotting detection transfections pLKO.1-SPIB-sh2
White expression.
Fig. 8 is A2780/Taxol cell quantities and form change after phase contrast microscope observation transfection pLKO.1-SPIB-sh2
Change.
Fig. 9 is the propagation that bromine mark method detects A2780/Taxol cells after transfection pLKO.1-SPIB-sh2.
Figure 10 is the apoptosis that Caspase3 Activity determinations transfect A2780/Taxol cells after pLKO.1-SPIB-sh2.
Figure 11 is A2780/Taxol cell migration abilities after cell scratch experiment detection transfection pLKO.1-SPIB-sh2.
Figure 12 be Transwell detection transfection pLKO.1-SPIB-sh2 after A2780/Taxol cell migrations ability (A) and
Invasive ability (B).
Figure 13 is reverse of the A2780/Taxol cells to taxol resistance after transfection pLKO.1-SPIB-sh2.
Embodiment
With reference to embodiment, the invention will be further described.The method purpose used in following embodiment is more preferable
Ground understands the present invention, but is not limited to the present invention.Unless otherwise specified, the experimental method being related in embodiment is conventional side
Method, experiment material used is the purchase of conventional reagent company.
Using the statistical analysis softwares of SPSS 18.0, each sample data is with mean ± standard deviationRepresent, between two groups
Difference examined with T (Independent-Sample T Test), it is multigroup between inspection one-way analysis of variance (One-
Way ANOVA), IC50Using probit regression analyses, P ＜ 0.05 have significant difference.
Ovarian Cancer Cells A2780 and oophoroma taxol resistance cell line A2780/Taxol are by Zhejiang Province's female reproduction
Health research key lab cell bank is preserved；
The anti-human GAPDH primary antibodies (Cat.60004-1-Ig) of rabbit-anti people SPIB primary antibodies (Cat.15768-1-AP), mouse, horseradish mistake
Oxide enzyme mark goat anti-mouse IgG (H+L) secondary antibody (Cat.SA00001-1), horseradish peroxidase-labeled goat antirabbit
IgG (H+L) secondary antibody (Cat.SA00001-2) is purchased from Proteintech companies；
Western Blotting Luminol Reagent detection kits (Cat.sc-2048) are purchased from Santa Cruz
Company；
CDNA Reverse Transcriptase kits PrimeScriptTMRT Master Mix (Cat.RR036A), quantitative fluorescent PCR inspection
Test agent box SYBR Premix Ex Taq (perfect Real time, Cat.DRR041A) are purchased from TaKaRa companies；
Lipofectamine3000 transfection reagents box (Cat.L3000008) is purchased from Invitrogen companies；
The conventional RNAi carrier pLKO.1puro of carrier for expression of eukaryon selection, comes from global scientist's plasmid and shares non-profit group
Knit Addgene；
Restriction enzyme A ge I (Cat.R0552S), EcoR I (Cat.R0101S), Kpn I (Cat.R0142S) purchases
From NEB companies；T4 ligases (Cat.2011A), DNA fragmentation purification kit (Cat.9761), DNA gel QIAquick Gel Extraction Kit
(Cat.9762), DNA small scale purification kit (Cat.9760) is purchased from TaKaRa companies；
SiRNA is synthesized by TaKaRa companies；PCR primer and clone are synthesized with DNA by Shanghai Sheng Gong bio-engineering corporations；
Pre-dyed albumen Marker (Cat.26616) is purchased from Fermentas companies；
Bromine mark method cell proliferation detecting kit Cell Proliferation ELISA, BrdU (colorimetric,
Cat.11647229001 Roche companies) are purchased from；
CaspACE Assay System (colorimetric, Cat.G7351) are purchased from Promega companies；
Cell migration, invasive model Transwell Permeable Supports (Cat.3428) are public purchased from Corning
Department；
Lab-Tek II Chamber Slide System-Lab-Tek chamber slides system is purchased from Nunc companies
(Cat.154526)；
BD MatrigelTMBasement Membrane Matirx matrix membranes (Cat.356234) are purchased from BD companies；
SiRNA negative control AllStars Negative Control SiRNA (Cat.1027281) are public purchased from QIAGEN
Department；
PAGE gel configuration kit (Cat.CW0022M) is ShiJi Co., Ltd purchased from health；
0.45um pvdf membranes (Cat.IPVH00010) are purchased from Millipore companies；
Taxol (Cat.P106868) is purchased from Aladdin companies.
SPIB expression is poor in the Ovarian Cancer Cells A2780 of embodiment 1. and its taxol resistance cell line A2780/Taxol
Different research
First, real-time fluorescence quantitative RT-PCR (qRT-PCR) detection SPIB gene mRNA expressions
The culture medium abandoned in 6 orifice plates is inhaled after culture 48h, Trizol extracted total RNAs, Thermo are used after being washed twice with PBS
Nano Drop2000 spectrophotometric determination RNA concentration, and by SYBR Premix Ex Taq (perfect Real time)
Kit specification is operated.First step RNA is denatured.Reaction system：RNA0.5ug, goes RNase DEPC water to complement to 6.8ul；Instead
Answer condition：It is placed on ice after 70 DEG C of incubation 10min.Second step reverse transcription.Reaction system：According to PrimeScript RT
Master Mix kit specifications carry out reverse transcription；Reaction condition：After 42 DEG C of incubations 60min, 85 DEG C of inactivation 5min, -20 DEG C
Preserve.
1ul reverse transcription products are taken to carry out quantitative fluorescent PCR reaction.PCR primer sequence：
5’-AAGAAGCTGCGCCTGTACC-3’；
5 '-TCTTGGCGTAGTTTCGGAGG-3 ', product length：211bp；
Reaction condition：95 DEG C of 10s, 95 DEG C of 5s, 6 DEG C of 30s, totally 40 circulations.
The expression quantity of SPIB mRNA in each group sample is calculated using 2- △ CT methods.
As a result：As shown in figure 1, A2780/Taxol cells are compared with its parental cell A2780, SPIB mRNA expression increases
High 88.19% (P ＜ 0.05), shows that SPIB mRNA expression is significantly increased in taxol resistance cell line.
2nd, Western Blotting detect SPIB protein expressions
The culture medium abandoned in 6 orifice plates is inhaled after culture 72h, PBS is washed 3 times, adds RIPA protein lysates (100ul/ holes),
Piping and druming for several times, is incubated 5min on ice, is allowed to fully cracking, 4 DEG C, 12000 leave the heart 5 minutes, collects supernatant, dispenses -20 DEG C of storages
Deposit；10ul loadings, 8%SDS-PAGE electrophoresis, 200V, 10min are taken after each 95 DEG C of denaturation 5min of sample；100V, 100min；Turn
To pvdf membrane：110V, 120min；60min is closed with the TBS confining liquids containing 5% skimmed milk power；Primary antibody is incubated：SPIB primary antibodies (1：
2000), GAPDH primary antibodies (1：5000) 2h is incubated at room temperature；TBS washes film 10min × 3 time；Secondary antibody is incubated：Horseradish peroxidase
Mark goat anti-mouse IgG (H+L) secondary antibody (1：10000), horseradish peroxidase-labeled goat anti-rabbit igg (H+L) secondary antibody (1：
10000) it is incubated 1h；TBST washes film 10min × 3 time, and TBS washes film 10min × 1 time；After ECL developments, ImageQuant is used
LAS4000 mini (GE Healthcare) are to scanning of image processing.
As a result：As shown in Fig. 2 A2780/Taxol cells are compared with its parental cell A2780, SPIB protein expressions
Significantly increase (P ＜ 0.05).
Embodiment 2.SPIB siRNA design synthesis
SPIB gene mRNA sequences (NM_003121.4) are discovered and seized in Genebank, with siDirect Ver2.0 softwares
(http://sidirect2.rnai.jp/) 3 couples of siRNA sequence (such as SEQ ID NO.1-SEQ ID of Photographing On-line acquisition
NO.6 shown in).In design process selection simultaneously meet document report three kinds of algorithms (Ui-Tei × Reynolds ×
Amarzguioui sequence), and siRNA action specificity highest 23nt long fragments are selected, the design can avoid body in future
100nt after interferon-like immune response, selection initiation codon occurs during interior experiment, 5 ' and 3 ' end UTR areas, G/C content control are avoided
System is in 30-70%.Select the siRNA of 3 pairs of 23nt length as experiment screening interference fragment altogether, architectural feature shows as positive-sense strand
Respectively have that two bases are plug-in with the end of antisense strand 3 ', design feature is as follows
Then use BLASTN(https://blast.ncbi.nlm.nih.gov/Blast.cgi)It is online to carry out homology
Search, excludes the sequence for having homology, influence of the non-specific fragment to siRNA specific effect effects is avoided as far as possible.
Finally positive-sense strand and the 5 ' of antisense strand and 3 ' continuous 3 purine (pyrimidine) bases in end are carried out in chemical synthesis
2 '-OMe (2 '-methoxyl group) are modified, the chemical stability of increase siRNA molecule in the cell, extension siRNA interference gene expressions
The time of downward and effect.Final sequence and modification such as table 1 and formula (I) are as follows：
Table 1
3. 3 couples of SPIB siRNA of embodiment are disturbed SPIB genes in oophoroma taxol resistance strain A2780/Taxol
The detection and screening of effect
First, experiment packet：
1.A2780/Taxol normal groups (do not transfect siRNA), hereinafter referred to as AR；
2.A2780/Taxol negative control groups (transfection negative control siRNA), hereinafter referred to as AR-N；
3.A2780/Taxol experimental groups (transfection S1), hereinafter referred to as AR-S1；
4.A2780/Taxol experimental groups (transfection S2), hereinafter referred to as AR-S2；
5.A2780/Taxol experimental groups (transfection S3), hereinafter referred to as AR-S3.
2nd, packet transfection
To ensure transfection efficiency, cytotoxicity is reduced, we are carried out using Lipofectamine3000 transfection reagents
SiRNA is transfected.The day before transfection, trypsin digestion cell is simultaneously counted, and plating cells make it in transfection day density in six orifice plates
0.5×106/ ml, cell fusion to 70-90%.Per hole 5ul is diluted with 125 μ l serum-free OPTI-MEM culture mediums
The reagents of Lipofectamine 3000 are simultaneously fully mixed；SiRNA premixed liquids are prepared, with 125 μ l serum-free OPTI-MEM culture mediums
Dilution siRNA is mixed to final concentration of 50nM, and fully；Added in the reagents of Lipofectamine 3000 diluted
SiRNA premixed liquids (1:1), it is incubated at room temperature 5min；Finally siRNA- liposome complexes are added in cell, 37 DEG C, 5%
CO2It is middle to continue to cultivate.SPIB protein expressions are detected after SPIB mRNA expression, 72h are detected after 48h.
3rd, real-time fluorescence quantitative RT-PCR (qRT-PCR) detection SPIB gene mRNA expressions
Detecting step is the same, and the expression quantity of SPIB mRNA in each group sample is calculated using 2- △ CT methods.
As a result：As shown in figure 3, A2780/Taxol cells are transfected after S1, S2, S3 respectively, SPIB mRNA expression has
It is decreased obviously, wherein S2 interference effects are compared with negative control group, SPIB mRNA have lowered 83.07%, with S1 (62.05%)
Compared with S3 (55.75%), there were significant differences (P ＜ 0.05).As a result show, S2 has best interference effect to SPIB.
4th, Western Blotting detect SPIB protein expressions
Detecting step is the same, after ECL developments, with Image Quant LAS4000 mini (GE Healthcare) to figure
As scan process.
As a result：As shown in figure 4, compared with negative control, after S2 transfections, SPIB protein expressions in A2780/Taxol cells
It is remarkably decreased (P ﹤ 0.05), and there were significant differences (P ﹤ 0.05) compared with S1 and S3.As a result show, in taxol resistance strain
In A2780/Taxol, S2 has best interference effect to SPIB protein expression.Therefore pass through screening, after S2 is selected as
The siRNA of continuous research.
The eukaryotic vector pLKO.1-SPIB-sh2 of embodiment 4. structure and the interference effect detection to SPIB gene expressions
First, experiment packet：
1.A2780/Taxol normal groups (do not transfect any carrier), hereinafter referred to as AR；
2.A2780/Taxol negative control groups (transfection pLKO.1puro empty carriers), hereinafter referred to as AR-N；
3.A2780/Taxol experimental groups 1 (transfection pLKO.1-SPIB-sh2), hereinafter referred to as AR-sh2；
2nd, SPIB-S2 fragments are synthesized
Age I and EcoR two restriction enzyme sites of I are selected, according to S2 sequence, its shRNA sequence is designed and is building up to true
In nuclear expression carrier pLKO.1puro.Sequence is as follows：
Positive-sense strand：
5’-CCGGTGAATCAGGGTAGCTGGAATGCTTTCAAGAGAGACATTCCAGCTACCCTGATTCATTTTTTG
GTACC-3’(SEQ ID NO.7)；
Antisense strand：
5’-AATTGGTACCAAAAAATGAATCAGGGTAGCTGGAATGTCTCTCTTGAAAGCATTCCAGCTACCCTG
ATTCA-3’(SEQ ID NO.8)；
3rd, eukaryotic vector pLKO.1-SPIB-sh2 structure
PLKO.1-SPIB-sh2 recombinant expression carriers (Fig. 5 and 6) are built with carrier for expression of eukaryon pLKO.1puro, specifically
Method is referring to U.S.'s Cold Spring Harbor Publications《Molecular Cloning:A Laboratory guide》.
By SPIB-S2 positive-sense strands and the annealed program of antisense strand (95 DEG C of denaturation 2min；Slow cooling is annealed to 25 DEG C), 4
DEG C preserve.Age I and EcoR I complete degestion carriers pLKO.1puro, 37 DEG C overnight；Digestion products DNA gel reclaim reagent
Box reclaims DNA.Annealed product reclaims fragment with carrier digestion and is attached reaction, reaction system (10ul)：T4 ligase 1ul,
T4 ligase buffer solution 1ul, annealed product reclaims fragment mixture (mol ratio 3 with carrier digestion:1)；Deionized water mend to
10ul, 16 DEG C of connections are stayed overnight；5ul connection products are taken to be placed in 100ul JM109 competence bacteriums, ice bath 30min, 42 DEG C of heat are stopped
Gram 90s, ice bath 5min, plus LB culture mediums 1000ul, 37 DEG C of shaking table culture 30min, 5000rpm centrifugation 5min, abandon supernatant, will be thin
Bacterium is spread evenly across on LB flat boards (ampicillin containing 50ug/ml), 37 DEG C of inversion overnight incubations；Some independent clones are selected to connect
Plant in the LB culture mediums containing corresponding resistant, bacterium is expanded in 37 DEG C of concussions overnight；Bacterium is collected, is extracted with plasmid DNA purification kit
DNA is obtained, Kpn I digestions identification obtains pLKO.1-SPIB-sh2 recombinant plasmids.
4th, interference effect is observed after pLKO.1-SPIB-sh2 recombinant plasmid transfected cells
PLKO.1-SPIB-sh2 is transfected into the A2780/Taxol cells of exponential phase.Transfect reference
Lipofectamine3000 operational manuals, 5ul is diluted per hole with 125 μ l serum-free OPTI-MEM culture mediums
Lipofectamine3000 reagents are simultaneously fully mixed；5ug recombinant plasmids are added in 125 μ l serum-free OPTI-MEM culture mediums
DNA, adds P3000 reagent 10ul, fully mixes, Prepare restructuring plasmid premixed liquid；In the Lipofectamine diluted
Recombinant plasmid premixed liquid (1 is added in 3000 reagents:1), it is incubated at room temperature 5mim；Finally by recombinant plasmid-liposome complex
250ul is added in cell, 37 DEG C, 5% CO2It is middle to continue to cultivate.SPIB protein expressions are detected after 72h.
As shown in fig. 7, after transfection pLKO.1-SPIB-sh2 recombinant plasmids, compared with negative control (transfection empty plasmid),
SPIB protein expressions are remarkably decreased (P ﹤ 0.05) in A2780/Taxol cells, as a result show, transfection pLKO.1-SPIB-sh2 weights
Group plasmid can effectively disturb SPIB protein expression.
After the transfection pLKO.1-SPIB-sh2 specific inhibitions SPIB expression of embodiment 5. to tumor cell proliferation, apoptosis, move
The influence for moving and attacking
First, experiment packet：
1.A2780/Taxol normal groups (do not transfect any carrier), hereinafter referred to as AR；
2.A2780/Taxol negative control groups (transfection pLKO.1puro empty carriers), hereinafter referred to as AR-N；
3.A2780/Taxol experimental groups (transfection pLKO.1-SPIB-sh2), hereinafter referred to as AR-sh2.
2nd, packet transfection
Transfection procedure is the same, continues to cultivate cell after transfection, propagation, apoptosis, migration and invasion and attack for detecting cell.
3rd, cell proliferation test
Cell is transfected in 96 orifice plates to be continued to cultivate 72h after pLKO.1-SPIB-sh2, and 10ul BrdU marks are added per hole
Liquid is to the final concentration of 10uM of BrdU, and 37 DEG C are incubated 2h；BrdU marking fluids are absorbed, 200ul FixDenat are added per hole, 20 DEG C incubate
Educate 30min；FixDenat is absorbed, 100ul anti-BrdU-POD, 20 DEG C of incubation 90min are added per hole；Per hole 200ul
Washing Solution are washed 3 times；Add 100ul/ holes substrate solution, 20 DEG C of incubation 20min, Detection wavelength 370nm (references
Wavelength 492nm) survey absorbance (A), the ability A of cell propagationExperimental group/AControl groupRepresent.
As a result as shown in Figure 8 and Figure 9, in A2780/Taxol cells, after transfection pLKO.1-SPIB-sh2, differ micro-
Microscopic observation is visible, and experimental group cell quantity is significantly reduced, and suspension cell quantity increase, cell fragment increases；Bromine mark method is tested
Cell proliferation results are displayed that：Compared with negative control, experimental group ability of cell proliferation have dropped 71.22%, have conspicuousness poor
Different (P ＜ 0.05).After the expression for illustrating specific inhibition SPIB, tumor cell proliferation can be suppressed.
4th, Caspase3 Activity determinations Apoptosis
Cell is collected after transfection 72h, lysate adjustment cell density is 1 × 108/ ml, cracks 15min, 15000g on ice
× 20min, collects supernatant.Prepared simultaneously according to CaspACE Assay System (colorimetric) specifications positive and cloudy
Property control sample, is determined and to adjust each group protein concentration identical.Caspace Assay Buffer are added in 96 orifice plates per hole
32ul, DMSO 2ul, 100nM DTT 10ul, deionized water adjustment volume is 98ul, adds 2ul DEVD-pNA substrates, 37 DEG C
4h is incubated, Detection wavelength 405nM surveys absorbance, every group of sample Caspase3 activity is calculated with Δ A methods.
As a result as shown in Figure 10, after A2780/Taxol cell transfectings pLKO.1-SPIB-sh2, Caspase3 activity increases
4.19 times, compared with negative control, have significant difference (P ＜ 0.05), after the expression for illustrating specific inhibition SPIB, can promote
Enter apoptosis of tumor cells.
5th, cell scratch test detection cell migration ability
Horizontal line is uniformly drawn with ruler behind in six orifice plates, the standardized roads of about 0.5cm cross via, per at least 6, hole horizontal stroke
Line.After cell transfecting pLKO.1-SPIB-sh2, when continuing to cultivate 24h cell fusions into individual layer state, used in selection area
200ul pipette tips vertical cut in six orifice plates, PBS washes 3 cells removed under drawing, adds serum free medium and continues to train
Support.0h, 24h, 48h time point take pictures, and randomly select 6 horizontal lines, calculate iuntercellular apart from average.
As a result as shown in figure 11, after pLKO.1-SPIB-sh2 transfectional cells 24h and 48h, A2780/Taxol iuntercellulars away from
From noticeably greater than negative control group, healing ability is remarkably decreased after cell cut, after the expression for illustrating specific inhibition SPIB, energy
Suppress the migration of tumour cell.
6th, Transwell testing inspections cell migration ability
After cell transfecting 48h, with collected by trypsinisation, it is resuspended with serum free medium, adjustment cell density is 5 × 105/
Ml, upper chamber adds 2ml cell suspensions, and lower room adds 10%FBS complete medium 2ml, continues to cultivate 24h, takes out cell, PBS washes 3
It is secondary, the cell of upper chamber upper surface is carefully removed with cotton swab, inversion is dried, 95% ethanol fixes 25min, haematoxylin dyeing shows
Micro- Microscopic observation, count, take pictures.Each cell counts 10 visuals field, averages and counts and analyze changing for cell migration ability
Become.
Cell migration assay result as illustrated in fig. 12, transfection pLKO.1-SPIB-sh2 after, A2780/Taxol experimental groups with
The cell quantity that negative control group penetrates cell is 46 ± 15 and 279 ± 27 respectively, and both have significant difference (P ＜ 0.05)；
The result shows, after specific inhibition SPIB expression, can suppress the migration of tumour cell.
7th, Transwell testing inspections cell invasion ability
By the matrigel of -20 DEG C of preservations first in 4 DEG C of rewarming liquefaction, take matrigel with OPTI-MEM with 1:6 mix on ice it is dilute
Release, be coated with the upper chamber face of cell bottom film, 37 DEG C of solidification 30min absorb the liquid of small indoor precipitation.Matrigel coating after remaining
Ibid, each cell counts 10 visuals field to step, averages and counts and analyze the change of cell invasion ability.
As shown in Figure 12 B, A2780/Taxol experimental groups penetrate the thin of cell to cell invasion experimental result with negative control group
Born of the same parents' quantity is 36 ± 8 and 177 ± 28 respectively, and both have significant difference (P ＜ 0.05)；The result shows, specific inhibition
After SPIB expression, tumor cell invasion can be suppressed.
To the reverse effect of ovarian cancer drug-resistant after embodiment 6.pLKO.1-SPIB-sh2 specific inhibitions SPIB expression
First, experiment packet：
1.A2780 normal groups (do not transfect any carrier), hereinafter referred to as A；
2.A2780/Taxol normal groups (do not transfect any carrier), hereinafter referred to as AR；
3.A2780/Taxol negative control groups (transfection pLKO.1puro empty carriers), hereinafter referred to as AR-N；
4.A2780/Taxol experimental groups (transfection pLKO.1-SPIB-sh2), hereinafter referred to as AR-sh2.
2nd, packet transfection
Transfection procedure is the same, continues to cultivate cell 24h after transfection.
3rd, detection of the cell to paclitaxel-sensitive after transfection pLKO.1-SPIB-sh2
Each group is taken the logarithm growth period cell, and cell is resuspended after pancreatin digestion, cell count and the density for adjusting cell suspension
For 1 × 105/ ml, is inoculated into 96 orifice plates and continues to cultivate 24h.Taxol is added in next day, each group, concentration gradient is set respectively
200ug/ml, 100ug/ml, 50ug/ml, 25ug/ml, 12.5ug/ml, 6.25ug/ml, 3.125ug/ml, 0ug/ml, effect
After 24h, absorbance (A) is surveyed in wavelength 370nm (reference wavelength 492nm) with bromine mark method, suppression of the taxol to every group of cell is calculated
Rate processed, inhibiting rate=AExperimental group/ANegative control group.Each concentration sets 3 multiple holes, averages.
Drug concentration when inhibiting rate is 50% is half-inhibition concentration (IC50)；
Persister A2780/Taxol IC50With its parental cell strain A2780 IC50Ratio be resistance multiple
(Resistant Folder,RF)；
Persister A2780/Taxol IC50The IC after pLKO.1-SPIB-sh2 (reversal agent) is transfected with it50Ratio be
Drug resistance inversion index (Reversal Index, RI).
As a result as shown in table 2, table 3, Figure 13, ICs of the A2780/Taxol to taxol50(42.65 ± 4.88ug/ml) is notable
Higher than ICs of the parent A2780 to taxol50(1.36 ± 0.29ug/ml), resistance multiple is up to 31.36, points out A2780/Taxol
Sensitiveness to taxol is substantially less than parental cell A2780, height resistance.And when A2780/Taxol transfects pLKO.1-
After SPIB-sh2, the sensitiveness to taxol is significantly improved (2.12 ± 0.47ug/ml), transfects pLKO.1-SPIB-sh2 pairs
Clearly, it is 19.51 to reverse index to the reversing effect of A2780/Taxol taxol resistances.
Drug susceptibilities of the table 2.A2780 and A2780/Taxol to taxol
Reverses of the A2780/Taxol to taxol drug sensitiveness after the transfection of table 3. pLKO.1-SPIB-sh2
SEQUENCE LISTING
<110>Zhejiang University
<120>Specificity suppresses siRNA and its recombinant vector and the application of SPIB gene expressions
<130>
<160> 8
<170> PatentIn version 3.3
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Claims (10)
1. specificity suppresses the siRNA of SPIB gene expressions, including positive-sense strand and antisense strand, it is characterised in that the positive-sense strand
Nucleotide sequence as shown in SEQ ID NO.1, the nucleotide sequence of antisense strand is as shown in SEQ ID NO.2.
2. siRNA as claimed in claim 1, it is characterised in that 3 bases at the positive-sense strand and the 5 ' of antisense strand and 3 ' ends
Carry out the modification of 2 '-methoxyl group.
3. applications of the siRNA as claimed in claim 1 or 2 in SPIB gene expression inhibitors are prepared.
4. siRNA as claimed in claim 1 or 2 is preparing treatment oophoroma, stomach cancer, colorectal cancer or incidence squamous cell
Application in cancer drug.
5. applications of the siRNA as claimed in claim 1 or 2 in the medicine for reversing oophoroma taxol resistance is prepared.
6. a kind of rnai reagent box for including the DNA sequence dna for encoding siRNA as claimed in claim 1 or 2.
7. a kind of recombinant vector containing the DNA sequence dna for encoding siRNA as claimed in claim 1 or 2.
8. application of the recombinant vector as claimed in claim 7 in SPIB gene expression inhibitors are prepared.
9. recombinant vector as claimed in claim 7 is preparing treatment oophoroma, stomach cancer, colorectal cancer or incidence squamous cell
Application in cancer drug.
10. application of the recombinant vector as claimed in claim 7 in the medicine for reversing oophoroma taxol resistance is prepared.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2022005266A1 (en) * | 2020-07-02 | 2022-01-06 | 연세대학교 산학협력단 | Method for suppressing cancer metastasis through alteration of adhesion dependence of cancer cells |
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| CN101701217A (en) * | 2009-11-18 | 2010-05-05 | 王理伟 | SiRNA restraining gene expression of transcription factor Spl and application thereof |
| WO2015097210A1 (en) * | 2013-12-23 | 2015-07-02 | Issazadeh-Navikas Shohreh | Immunosuppressive foxa1-expressing t cells |
| CN106609300A (en) * | 2015-10-23 | 2017-05-03 | 北京乐普基因科技股份有限公司 | Coronary artery disease risk assessment kit and risk assessment method |
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| CN101701217A (en) * | 2009-11-18 | 2010-05-05 | 王理伟 | SiRNA restraining gene expression of transcription factor Spl and application thereof |
| WO2015097210A1 (en) * | 2013-12-23 | 2015-07-02 | Issazadeh-Navikas Shohreh | Immunosuppressive foxa1-expressing t cells |
| CN106609300A (en) * | 2015-10-23 | 2017-05-03 | 北京乐普基因科技股份有限公司 | Coronary artery disease risk assessment kit and risk assessment method |
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| REMKO SCHOTTE等: "The ETST ranscription Factor Spi-B Is Required for Human Plasmacytoid Dendritic Cell Development", 《THE JOURNAL OF EXPERIMENTAL MEDICINE》 * |
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| WO2022005266A1 (en) * | 2020-07-02 | 2022-01-06 | 연세대학교 산학협력단 | Method for suppressing cancer metastasis through alteration of adhesion dependence of cancer cells |
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