CN107541514B - siRNA for specifically inhibiting CO L12A 1 gene expression, recombinant vector and application thereof - Google Patents
siRNA for specifically inhibiting CO L12A 1 gene expression, recombinant vector and application thereof Download PDFInfo
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Abstract
The invention discloses siRNA for specifically inhibiting CO L A1 gene expression, a recombinant vector thereof and application thereof in reversing ovarian cancer taxol resistance, and belongs to the technical field of molecular biology and biomedicine.
Description
Technical Field
The invention relates to the technical field of molecular biology and biomedicine, in particular to siRNA for specifically inhibiting CO L12A 1 gene expression, a recombinant vector and application thereof.
Background
RNA interference (RNAi) is a widely occurring sequence-specific post-transcriptional gene silencing mechanism in animals and plants. In 1998, Andrew Fire, an American scientist, first found that a mixture of sense and antisense strands (i.e., dsRNA) produced at least 10 times the effect of gene silencing over antisense nucleotides in C.elegans, and induced the same phenomenon in progeny. The mechanism research of RNAi phenomenon shows that trace siRNA can silence a large amount of target RNA through post-transcriptional gene silencing, and the key molecule for efficiently and specifically degrading homologous RNA to cause sequence-specific gene silencing is small double-stranded oligonucleotide with 21-23 bases, also called small interfering RNA (siRNA). Further research shows that the double-stranded RNA shorter than 21bp or longer than 25bp can not effectively start RNAi, and the gene silencing effect is obviously reduced or even eliminated as long as one base is mismatched, so that the specificity of the siRNA effect is fully embodied.
siRNA showing strong gene silencing efficacy has attracted attention as a significant finding in recent biotechnology because it has specificity and high efficiency in blocking gene suppression function of homologous genes and is a powerful tool for gene function studies. siRNA has many incomparable advantages and features over traditional methods. Although there are several methods for inhibiting the expression of specific genes, such as antisense RNA, knock-out (knock-out), etc., siRNA has shown significant advantages over these techniques: it has higher specificity and persistence compared to antisense RNA; compared with complex and time-consuming gene knockout, siRNA is a simpler and more effective means. At present, the method of using siRNA as gene silencing is widely used for the research of malignant tumor mechanisms, the key point of the research is mainly focused on how to improve the gene silencing efficacy, including target gene locus selection, optimization of an introduction system, influence on host cell functions and the like, and the research and development process of tumor specific high-efficiency targeted therapy strategies based on the technology is promoted.
Ovarian cancer is one of the most common gynecological malignant tumors, and the mortality rate of ovarian cancer is the first of all gynecological tumors. The biggest obstacle to ovarian cancer treatment is the development of tumor cell resistance, especially the emergence of multidrug resistance. With the implementation of the platinum/taxol medicament combined chemotherapy, the primary chemotherapy response rate of the ovarian cancer can reach 80%. But most patients relapse within 2-3 years. Even if the ovarian cancer is treated by chemotherapeutics with completely different action mechanisms, the ovarian cancer is resistant, so that the survival rate of the treated tumor cannot be obviously improved. The five-year survival rate of the advanced ovarian cancer always ranges from 20% to 30%. High recurrence rate and high drug resistance rate after recurrence are one of the leading causes of high mortality of ovarian cancer, and the search for a solution to drug resistance in ovarian cancer chemotherapy is an important subject of current tumor research.
The ECM is a macromolecule synthesized and secreted by cells to the extracellular space and distributed on the cell surface or among cells, and mainly comprises a plurality of polysaccharides, proteins or proteoglycans, and the substances form a complex network structure, support and connect tissue structures, and regulate the physiological activities of tissues and cells.
Although the specific action mechanism of CO L A1 in the tumor is not clear so far, the involvement of CO L A1 in the pathogenesis and the development of the tumor is gradually known, previous researches indicate that CO L A1 has an abnormal rise phenomenon in an ovarian cancer Taxol resistant cell strain A2780/Taxol, but whether the gene is related to the pathogenesis of the ovarian cancer and the drug resistance of the ovarian cancer is still to be further researched.
Disclosure of Invention
The invention aims to provide siRNA for specifically inhibiting the expression of CO L12A 1 gene, which is used for researching the pathogenesis of ovarian cancer, and the invention also aims to provide application of the siRNA in preparing medicines for treating ovarian cancer and reversing paclitaxel resistance.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention designs and synthesizes 3 pairs of siRNA for specifically inhibiting CO L12A 1 gene expression, and the siRNA is transfected into an ovarian cancer Taxol resistant cell strain A2780/Taxol, and the result shows that S1 has the most obvious interference effect for inhibiting CO L12A 1 gene expression.
The invention provides siRNA (S1) for specifically inhibiting the expression of a CO L12A 1 gene, which comprises a sense strand and an antisense strand,
the sense strand: 5'-UCAACUUCUGCCUCAAUGGAA-3' (SEQ ID NO. 1);
the antisense strand: 5'-CCAUUGAGGCAGAAGUUGACC-3' (SEQ ID NO. 2).
Preferably, the sense and antisense strands are 2 ' -methoxy modified at the 5 ' and 3 ' ends of the 3 bases. The research of the invention proves that the stability of the siRNA (S1) modified by the 2' -methoxy group is increased, the capability of resisting the hydrolysis of ribozyme in vivo can be improved, the immune stimulation reaction is reduced, the action time of siRNA interfering the expression of gene to be reduced is prolonged, and the action has high efficiency and specificity.
The kit comprises a DNA plasmid vector for cloning the siRNA, and when the kit is applied, the plasmid vector transcribes and expresses the siRNA in a eukaryotic cell so as to silence the expression of a CO L12A 1 gene.
Preferably, the original vector adopted is a lentiviral vector p L KO.1puro.
The invention also provides a construction method of the recombinant vector, which comprises the following steps:
(1) synthesizing a CO L12A 1-S1 fragment, selecting two restriction enzyme cutting sites of Age I and EcoR I, and designing shRNA sequences according to the sequence of S1, wherein the sequences are as follows:
sense strand:
5’-CCGGGGTCAACTTCTGCCTCAATGGAATTCAAGAGATTCCATTGAGGCAGAAGTTGACCTTTTTTGGTACC-3’(SEQ ID NO.7)；
antisense strand:
5’-AATTGGTACCAAAAAAGGTCAACTTCTGCCTCAATGGAATCTCTTGAATTCCATTGAGGCAGAAGTTGACC-3’(SEQ ID NO.8)；
(2) annealing to obtain a DNA fragment of CO L12A 1-S1;
(3) the p L KO.1-CO L12A 1-sh1 recombinant vector was constructed using the lentiviral vector p L KO.1puro.
The siRNA provided by the invention can efficiently and specifically inhibit the expression of the CO L12A 1 gene of ovarian cancer cells, reduce cell proliferation, increase apoptosis and reduce cell migration and invasion capacity, so that the siRNA and a recombinant vector serving as a CO L12A 1 gene expression inhibitor can be applied to the research of pathogenesis of tumor diseases.
The invention provides application of the siRNA and the recombinant vector in preparation of a CO L12A 1 gene expression inhibitor.
The invention provides application of the siRNA and the recombinant vector in preparing a medicament for treating ovarian cancer, colorectal cancer or breast cancer.
The research of the invention shows that after the siRNA is transfected by the paclitaxel resistant strain A2780/Taxol, the sensitivity of the cell strain to paclitaxel is obviously improved, and the reversal index is 4.65, which shows that the siRNA provided by the invention has very obvious reversal effect on the drug resistance of the paclitaxel resistant strain A2780/Taxol, therefore, the siRNA has potential application value in the treatment of reversing the drug resistance of ovarian cancer paclitaxel.
The invention provides application of the siRNA and the recombinant vector in preparation of a medicine for reversing drug resistance of ovarian cancer taxol.
The invention has the following beneficial effects:
the siRNA provided by the invention can specifically and efficiently inhibit mRNA and protein expression of CO L12A 1 gene, reduce tumor cell proliferation, increase apoptosis, reduce tumor cell migration and invasion capacity, effectively reverse drug resistance of ovarian cancer cells to paclitaxel, and has important significance in tumor pathogenesis research and preparation of drugs for tumor treatment and ovarian cancer drug resistance reversal treatment.
Drawings
FIG. 1 shows the qRT-PCR detection of mRNA expression of CO L12A 1 in A2780 cells and A2780/Taxol cells, wherein A is A2780 cells and AR is A2780/Taxol cells.
FIG. 2 shows Western Blotting to detect protein expression of A2780 cells and A2780/Taxol cells CO L12A 1, wherein A is A2780 cells and AR is A2780/Taxol cells.
FIG. 3 shows the expression of mRNA of A2780/Taxol cells CO L12A 1 after 48h transfection of S1, S2 and S3 by qRT-PCR.
FIG. 4 shows Western Blotting to detect the expression of protein in A2780/Taxol cells CO L12A 1 72h after transfection with S1, S2 and S3.
FIG. 5 is a schematic diagram of the p L KO.1-CO L12A 1-sh1 recombinant plasmid and the inserted enzyme cutting sites.
FIG. 6 is a schematic diagram of a small hairpin shRNA. The U6 promoter directs the transcription of downstream small hairpin shRNA; comprises 23S 1 sense strand bases and 23S 1 antisense strand bases.
FIG. 7 shows Western Blotting detection of protein expression of A2780/Taxol cell CO L12A 1 after transfection of p L KO.1-CO L12A 1-sh 1.
FIG. 8 shows the change in the number and morphology of A2780/Taxol cells after transfection with p L KO.1-CO L12A 1-sh1 under phase contrast microscopy.
FIG. 9 shows the proliferation of A2780/Taxol cells after transfection of p L KO.1-CO L12A 1-sh1 by bromine labeling method.
FIG. 10 shows the detection of Caspase3 activity in apoptosis of A2780/Taxol cells after transfection of p L KO.1-CO L12A 1-sh 1.
FIG. 11 is a cell scratch test for detecting the migration ability of A2780/Taxol cells after transfection of p L KO.1-CO L12A 1-sh 1.
FIG. 12 shows the migration and invasion abilities of A2780/Taxol cells after transfection of p L KO.1-CO L12A 1-sh1 by Transwell assay.
FIG. 13 shows the reversal of paclitaxel resistance in A2780/Taxol cells after transfection with p L KO.1-CO L12A 1-sh 1.
Detailed Description
The present invention will be further described with reference to the following examples. The methods used in the following examples are intended to better understand the invention but are not intended to limit it. Unless otherwise specified, the experimental methods mentioned in the examples are all conventional methods, and the experimental materials used are all purchased from conventional reagents companies.
Using SPSS 18.0 statistical analysis software, each Sample data is expressed as mean. + -. standard deviation (x. + -.s), the difference between two groups is tested by T Test (Independent-Sample T Test), and the Test between groups is tested by One-Way ANOVA, IC50Using probit regression analysis, P < 0.05 was statistically different.
Ovarian cancer cell strain A2780 and ovarian cancer Taxol resistant cell strain A2780/Taxol are preserved in a cell bank of a key laboratory of female reproductive health research in Zhejiang province;
mouse anti-human CO L12A 1 primary antibody (Cat.sc-166020) was obtained from Santa Cruz, and mouse anti-human GAPDH primary antibody (Cat.60004-1-Ig) and horseradish peroxidase-labeled goat anti-mouse IgG (H + L) secondary antibody (Cat.SA00001-1) were obtained from Proteitech;
western Blotting L ulinol Reagent kit (Cat. sc-2048) was purchased from Santa Cruz;
cDNA reverse transcription kit PrimeScriptTMRT Master Mix (Cat. RR036A), fluorescent quantitative PCR assay kit SYBR Premix Ex Taq (perfect Real time, Cat. DRR041A) from TaKaRa company, L ipofectamine3000 transfection kit (Cat. L3000008) from Invitrogen company;
the eukaryotic expression vector selects an RNAi vector p L KO.1puro, which is derived from a global scientist plasmid sharing non-profit tissue Addgene;
restriction enzymes Age I (Cat. R0552S), EcoR I (Cat. R0101S), Kpn I (Cat. R0142S) were purchased from NEB; t4 ligase (Cat.2011A), a DNA fragment purification kit (Cat.9761), a DNA gel recovery kit (Cat.9762) and a plasmid DNA small purification kit (Cat.9760) are all purchased from TaKaRa company;
siRNA was synthesized by TaKaRa; PCR primers and DNA for cloning were synthesized by Shanghai Bioengineering Co., Ltd;
prestained protein Marker (cat.26616) available from Fermentas corporation;
bromine-labeling Cell Proliferation assay kit Cell Proliferation E L ISA, BrdU (colorimetric, cat.11647229001) was purchased from Roche;
CaspACE Assay System (colorimetric, Cat. G7351) available from Promega;
cell migration, invasion model Transwell Permeable Supports (Cat.3428) was purchased from Corning;
l ab-Tek II Chamber Slide System-L ab-Tek Chamber Slide System was purchased from Nunc corporation (Cat.154526);
BD MatrigelTMbasic Membrane matrix membranes (cat.356234) available from BD;
siRNA Negative Control AllStars Negative Control SiRNA (Cat.1027281) was purchased from QIAGEN;
SDS-PAGE gel preparation kit (cat. cw0022m) purchased from kazai century corporation;
0.45um PVDF membrane (Cat. IPVH00010) was purchased from Millipore;
paclitaxel (cat. P106868) was purchased from Aladdin.
Example 1 study on the expression difference of CO L12A 1 in ovarian cancer cell line A2780 and paclitaxel resistant cell line A2780/Taxol thereof
First, real-time fluorescent quantitative RT-PCR (qRT-PCR) is used for detecting mRNA expression of CO L12A 1 gene
After 48h incubation, the medium in the 6-well plate was aspirated, washed twice with PBS, and total RNA was extracted with Trizol, and the RNA concentration was determined with a ThermoNano Drop2000 spectrophotometer, according to the SYBR Premix Ex Taq (perfect reaction time) kit instructions. First step RNA denaturation, reaction system: RNA0.5ug, adding DEPC water to 6.8 ul; reaction conditions are as follows: incubate at 70 ℃ for 10min and place on ice. The second step of reverse transcription, reaction system: reverse transcription was performed according to PrimeScript RTMaster Mix kit instructions; reaction conditions are as follows: incubating at 42 deg.C for 60min, inactivating at 85 deg.C for 5min, and storing at-20 deg.C.
Taking 1ul of reverse transcription product to perform fluorescent quantitative PCR reaction. PCR primer sequences:
5’-GAACGAGGTTTGCCAGGAGA-3’；
5'-ATGTGTTAGCCGGAACCTGG-3', product length: 263 bp;
reaction conditions are as follows: 10s at 95 ℃, 5s at 95 ℃ and 30s at 6 ℃ for 40 cycles.
The expression level of CO L12A 1mRNA in each group of samples was calculated by 2- △ CT method.
As shown in FIG. 1, the expression of CO L12A 1mRNA was increased by 95.26% (P < 0.05) in A2780/Taxol cell compared with its parent cell, A2780, indicating that the expression of CO L12A 1mRNA was significantly increased in the paclitaxel-resistant cell strain.
II, detecting the expression of the CO L12A 1 protein by Western Blotting
After 72H of culture, the medium in a 6-well plate is removed, PBS is washed for 3 times, RIPA protein lysate (100 ul/well) is added, the mixture is blown and beaten for several times, incubated on ice for 5min to fully lyse, centrifuged for 5min at 4 ℃ and 12000 times, the supernatant is collected and stored at-20 ℃, 10ul of sample is taken after 5min of denaturation at 95 ℃ and then subjected to electrophoresis by 8% SDS-PAGE, 200V, 10min, 100V, 100min, the mixture is transferred to PVDF membrane, 110V, 120min, the mixture is sealed by 5% skimmed milk powder in TBS sealing solution for 60min, primary antibody incubation is performed by CO L A1 primary antibody (1: 500), GAPDH primary antibody (1: 5000) for 2H at room temperature, TBS membrane washing is performed for 10min × times, secondary antibody is performed by horseradish peroxidase labeled anti-mouse IgG (H + L) secondary antibody (1: 10000) for 1H, TBST 10min × times, membrane washing is performed for 2min, secondary antibody Image development is performed by QUANT 3976, and Image is scanned by QUANTE 64 GE (3875) for 10 min).
As shown in FIG. 2, the expression of the CO L12A 1 protein was also significantly increased in A2780/Taxol cells compared with the A2780 cells of the parent cells (P < 0.05).
Example 2 Synthesis of CO L12A 1siRNA design
The method is characterized in that a CO L12A 1 gene mRNA sequence (NM-004370.5) is searched in Genebank, 3 pairs of siRNA sequences (such as SEQ ID NO.1-SEQ ID NO.6) are obtained by using siDirect Ver2.0 software (http:// sidirect2.rnai. jp /) on-line design, sequences simultaneously meeting three algorithms (Ui-Tei × Reynolds × Amarzguioui) reported by the literature are selected in the design process, 23nt length fragments with the highest siRNA action specificity are selected, the design can avoid the generation of interferon-like immunoreaction in vivo experiments, 100nt after the initial codon is selected, 5 ' and 3 ' end UTR regions are avoided, the GC content is controlled to be 30-70%, 3 to 23nt length siRNAs are selected as experimental screening interference fragments, the structural characteristics are that a sense strand and an antisense strand have two external hanging bases at the 3 ' end respectively, and the structure is as follows
Followed by B L ASTN(https://blast.ncbi.nlm.nih.gov/Blast.cgi)Homology search is carried out on line, sequences with homology are excluded, and the influence of nonspecific fragments on the specific action effect of siRNA is avoided as much as possible.
And finally, carrying out 2 '-OMe (2' -methoxyl) modification on 3 continuous purine (pyrimidine) bases at the 5 'end and the 3' end of the sense strand and the antisense strand during chemical synthesis, so that the chemical stability of the siRNA molecule in cells is increased, and the time and effect of siRNA interfering gene expression down regulation are prolonged. The final sequences and modifications are shown in Table 1 and formula (I) below:
TABLE 1
Example 3 detection and screening of the Effect of three pairs of CO L12A 1 siRNAs on the interference of CO L12A 1 Gene in the Taxol drug-resistant Strain A2780/Taxol for ovarian cancer
Firstly, experimental grouping:
A2780/Taxol normal group (no siRNA transfection), hereinafter referred to as AR;
A2780/Taxol negative control group (transfection negative control siRNA), hereinafter referred to as AR-N;
A2780/Taxol panel (transfection S1), hereinafter referred to as AR-S1;
A2780/Taxol panel (transfection S2), hereinafter referred to as AR-S2;
A2780/Taxol panel (transfection S3), hereinafter referred to as AR-S3.
Two, grouped transfection
To ensure transfection efficiency and reduce cytotoxicity, we used L ipofectamine3000 transfection reagent for siRNA transfection one day before transfection, cells were trypsinized and counted, and cells were plated in six-well plates to give a density of 0.5 × 10 on the day of transfection6The cells were fused to 70-90% by diluting 5ul L ipofectamine3000 reagent per well with 125. mu.l serum-free OPTI-MEM medium and mixing well, preparing siRNA premix, diluting siRNA to a final concentration of 50nM with 125. mu.l serum-free OPTI-MEM medium and mixing well, adding siRNA premix (1:1) to the diluted L ipofectamine3000 reagent, incubating at room temperature for 5min, adding siRNA-liposome complex to the cells, and CO at 37 ℃ and 5%2The culture was continued for 48h before the expression of CO L12A 1mRNA was detected and for 72h after the expression of CO L12A 1 protein was detected.
Third, real-time fluorescent quantitative RT-PCR (qRT-PCR) detection of CO L12A 1 gene mRNA expression
The detection procedure was performed as before, and the expression level of CO L12A 1mRNA in each group of samples was calculated by 2- △ CT method.
As shown in FIG. 3, after the A2780/Taxol cells are transfected with S1, S2 and S3 respectively, the expression of CO L12A 1mRNA is obviously reduced, wherein the S1 interference effect is compared with a negative control group, the CO L12A 1mRNA is down-regulated by 82.54%, and is significantly different (P is less than 0.05) compared with S2 (39.23%) and S3 (64.35%), and the results show that S1 has the best interference effect on CO L12A 1.
Fourthly, detecting the expression of the CO L12A 1 protein by Western Blotting
Detection procedures As before, after EC L development, the images were scanned with Image Quant L AS4000mini (GE Healthcare).
As shown in FIG. 4, after S1 transfection, the expression of CO L A1 protein in A2780/Taxol cells was significantly reduced (P < 0.05) compared to the negative control and significantly different (P < 0.05) compared to S2 and S3. the results show that S1 had the best interference effect on the protein expression of CO L A1 in paclitaxel-resistant strain A2780/Taxol.
Example 4 construction of eukaryotic vector p L KO.1-CO L12A 1-sh1 and detection of interference effect on CO L12A 1 gene expression
Firstly, experimental grouping:
A2780/Taxol normal group (without transfection of any vector), hereinafter referred to as AR;
A2780/Taxol negative control group (transfected p L KO.1puro empty vector), hereinafter referred to as AR-N;
A2780/Taxol panel 1 (transfection p L KO.1-CO L12A 1-sh1), hereinafter referred to as AR-sh 1;
secondly, synthesizing a CO L12A 1-S1 fragment
Selecting two restriction sites of Age I and EcoR I, designing shRNA sequence according to the sequence of S1 and constructing into eukaryotic expression vector p L KO.1puro, wherein the sequence is as follows:
sense strand:
5’-CCGGGGTCAACTTCTGCCTCAATGGAATTCAAGAGATTCCATTGAGGCAGAAGTTGACCTTTTTTGGTACC-3’(SEQ ID NO.7)；
antisense strand:
5’-AATTGGTACCAAAAAAGGTCAACTTCTGCCTCAATGGAATCTCTTGAATTCCATTGAGGCAGAAGTTGACC-3’(SEQ ID NO.8)；
thirdly, construction of eukaryotic vector p L KO.1-CO L12A 1-sh1
Eukaryotic expression vector p L KO.1puro was used to construct p L KO.1-CO L12A 1-sh1 recombinant expression vector (FIGS. 5 and 6), which is described in molecular cloning, A.C. Cold spring harbor Press.
CO L A1-S1 sense strand and antisense strand are subjected to an annealing program (denaturation at 95 ℃ for 2 min; slow cooling annealing to 25 ℃) and stored at 4 ℃, Age I and EcoR I completely digest the vector p L KO.1puro at 37 ℃ overnight, the digested product is recycled by a DNA gel recovery kit, DNA is reacted with the digested recovered fragment of the vector, a reaction system (10ul) comprises 1ul of T4 ligase and 1ul of T4 ligase buffer, a mixture of the annealed product and the digested recovered fragment of the vector (molar ratio is 3:1), deionized water is supplemented to 10ul, ligation is performed at 16 ℃ overnight, 5ul of the ligated product is placed in 100ul of JM109 865 competent bacteria, JM 30min, heat shock at 42 ℃ for 90S, ice bath for 5min, addition of L B medium 1000ul, culture at 37 ℃ for 30min, centrifugation at 5000rpm for 5min, supernatant is discarded, the supernatant is uniformly spread on a JM L B plate (containing 50ug/ml of penicillin), ampicillin is added in an inverted culture medium containing L B, culture medium for 1000ul, culture at 37 ℃ for 30min, a plurality of Escherichia coli strains are obtained, DNA strains are subjected to shaking culture, plasmid strains are obtained, plasmid strains containing 24, plasmid strains are obtained, and the strains are subjected to shake culture at 5000 639 ℃ for overnight, and amplified, the strains are obtained, and subjected to obtain plasmid strains.
Fourthly, observing interference effect after the p L KO.1-CO L12A 1-sh1 recombinant plasmid transfects cells
Transfection of P L KO.1-CO L12A 1-sh1 into A2780/Taxol cells in logarithmic growth phase transfection is described in L ipofectamine3000 operating instructions, each well is diluted with 125. mu.l of serum-free OPTI-MEM medium to 5ul of L ipofectamine3000 reagent and mixed well, 5ug of recombinant plasmid DNA is added to 125. mu.l of serum-free OPTI-MEM medium, 10ul of P3000 reagent is added, mixed well to prepare a recombinant plasmid premix, the diluted L ipofectamine3000 reagent is added with the recombinant plasmid premix (1:1), incubated at room temperature for 5 mm, and finally 250ul of recombinant plasmid-liposome complex is added to the cells at 37 ℃, 5% CO2And culturing for 72h, and detecting the expression of the CO L12A 1 protein.
As shown in FIG. 7, after the P L KO.1-CO L12A 1-sh1 recombinant plasmid is transfected, compared with a negative control (transfected empty plasmid), the expression of the CO L12A 1 protein in A2780/Taxol cells is remarkably reduced (P < 0.05), and the result shows that the P L KO.1-CO L12A 1-sh1 recombinant plasmid can effectively interfere the protein expression of CO L12A 1.
Example 5 Effect of transfection of p L KO.1-CO L12A 1-sh1 on tumor cell proliferation, apoptosis, migration and invasion after specific blocking of CO L12A 1 expression
Firstly, experimental grouping:
A2780/Taxol normal group (without transfection of any vector), hereinafter referred to as AR;
A2780/Taxol negative control group (transfected p L KO.1puro empty vector), hereinafter referred to as AR-N;
A2780/Taxol panel (transfection p L KO.1-CO L12A 1-sh1), hereinafter referred to as AR-sh 1.
Two, grouped transfection
The transfection procedure was as before, and cells were cultured after transfection for detection of proliferation, apoptosis, migration and invasion of cells.
Third, cell proliferation assay
The cells are transfected into a 96-well plate p L KO.1-CO L12A 1-sh1 and then cultured for 72 hours, 10ul BrdU labeling Solution is added into each well until the final BrdU concentration is 10uM, incubation is carried out at 37 ℃ for 2 hours, BrdU labeling Solution is sucked off, 200ul FixDanat is added into each well, incubation is carried out at 20 ℃ for 30 minutes, FixDanat is sucked off, 100ul anti-BrdU-POD is added into each well, incubation is carried out at 20 ℃ for 90 minutes, 200ul washing Solution is washed for 3 times in each well, 100 ul/well bottom Solution is added, incubation is carried out at 20 ℃ for 20 minutes, the absorbance (A) is detected at the wavelength of 370nm (reference wavelength of 492nm), and the cell proliferation capacity is detected by the AExperimental group/AControl groupAnd (4) showing.
As shown in FIG. 8 and FIG. 9, after transfection of P L KO.1-CO L12A 1-sh1 in A2780/Taxol cells, the cell number of the experimental group is reduced under phase contrast microscope observation, and the cell proliferation result tested by a bromine standard method shows that compared with a negative control, the cell proliferation capacity of the experimental group is reduced by 25.53%, and the statistical difference (P < 0.05) shows that the tumor cell proliferation can be inhibited after the specific blocking of the expression of CO L12A 1.
Fourthly, detecting apoptosis by Caspase3 activity
Cells were harvested 72h after transfection and lysate adjusted to 1 × 10 cell density8The positive and negative control samples were prepared simultaneously according to CaspACE Assay System (colorimetric) instructions, and the concentrations of each group of proteins were determined and adjusted to be the same.Caspace Assay buffer32ul, DMSO2ul, 100nM DTT 10ul, deionized water adjusted to a volume of 98ul, 2ul DEVD-pNA substrate was added to each well of a 96 well plate, incubated at 37 ℃ for 4h, absorbance was measured at a detection wavelength of 405nM, and Caspase3 activity was calculated for each group of samples by the Δ A method.
The results are shown in FIG. 10, after the A2780/Taxol cell is transfected with P L KO.1-CO L12A 1-sh1, the activity of Caspase3 is increased by 39.42%, and compared with a negative control, the statistical difference is generated (P is less than 0.05), which indicates that the tumor cell apoptosis is increased after the expression of CO L12A 1 is specifically blocked.
Fifth, cell migration ability test
Uniformly marking transverse lines on the back of a six-hole plate by using a ruler, marking one transverse line by about 0.5cm, crossing through holes, wherein at least 6 transverse lines are arranged in each hole, after cell transfection is carried out on p L KO.1-CO L12A 1-sh1, continuously culturing for 24h, and when the cells are fused into a single-layer state, vertically scratching the selected area in the six-hole plate by using a 200ul gun head, washing by PBS for 3 times to remove the marked cells, adding a serum-free culture medium to continuously culture, taking pictures at time points of 0h, 24h and 48h, randomly selecting 6 horizontal lines, and calculating the average value of the distances between the cells.
The result is shown in figure 11, after p L KO.1-CO L12A 1-sh1 transfects cells for 24 hours and 48 hours, the distance between A2780/Taxol cells is obviously larger than that of a negative control group, the healing capacity after cell scratching is obviously reduced, and the result shows that after the expression of CO L12A 1 is specifically blocked, the migration of tumor cells can be inhibited.
Sixth, Transwell test for detecting cell migration ability
48h after cell transfection, the cells were collected by trypsinization, resuspended in serum-free medium and adjusted to a cell density of 5 × 105Adding 2ml of cell suspension into an upper chamber, adding 2ml of 10% FBS complete culture medium into a lower chamber, continuously culturing for 24h, taking out a small chamber, washing with PBS for 3 times, carefully removing cells on the surface of the upper layer of the upper chamber by using a cotton swab, inverting and airing, fixing with 95% ethanol for 25min, staining with hematoxylin, observing under a microscope, counting and taking a picture. 10 fields were counted per chamber, averaged and analyzed for changes in cell migration capacity.
The results of the cell migration experiments are shown in FIG. 12A, after P L KO.1-CO L12A 1-sh1 is transfected, the cell numbers of the A2780/Taxol experimental group and the negative control group penetrating the chamber are respectively 42 +/-9 and 267 +/-47, and the two groups have statistical difference (P is less than 0.05), and the results show that the tumor cell migration can be remarkably inhibited after the expression of CO L12A 1 is specifically blocked.
Seventhly, cell invasion capacity detection by Transwell test
Liquefying matrigel stored at-20 deg.C at 4 deg.C, mixing matrigel with OPTI-MEM at a ratio of 1:6 ice, diluting, coating the upper surface of the bottom membrane of the chamber, solidifying at 37 deg.C for 30min, and removing the liquid separated out from the chamber. The remaining steps after matrigel coating were as above, 10 fields were counted per chamber, and changes in cell invasiveness were counted and analyzed by averaging.
The results of the cell invasion experiments are shown in FIG. 12B, the numbers of cells penetrating the chamber of the A2780/Taxol experimental group and the negative control group are 33 + -6 and 189 + -43 respectively, and the statistical difference (P < 0.05) is obtained between the A2780/Taxol experimental group and the negative control group, and the results show that the tumor cell infiltration can be inhibited after the specific blocking of the expression of CO L12A 1.
Example 6 reversal of drug resistance to ovarian cancer after specific blockade of CO L12A 1 expression by p L KO.1-CO L12A 1-sh1
Firstly, experimental grouping:
a2780 normal group (without transfection of any vector), hereinafter referred to as a;
A2780/Taxol normal group (without transfection of any vector), hereinafter referred to as AR;
A2780/Taxol negative control group (transfected p L KO.1puro empty vector), hereinafter referred to as AR-N;
A2780/Taxol panel (transfection p L KO.1-CO L12A 1-sh1), hereinafter referred to as AR-sh 1.
Two, grouped transfection
The transfection procedure was as before, and cells were cultured for 24h after transfection.
Thirdly, detecting the sensitivity of cells to paclitaxel after p L KO.1-CO L12A 1-sh1 transfection
Cells in logarithmic growth phase were taken from each group, cells were resuspended after trypsinization, cell counts and cell suspension density was adjusted to 1 × 105And/ml, inoculating into a 96-well plate and continuing culturing for 24 hours. The next day, adding paclitaxel into each group, setting concentration gradient at 200ug/ml, 100ug/ml, 50ug/ml, 25ug/ml, 12.5ug/ml, 6.25ug/ml, 3.125ug/ml, 0ug/ml, after 24h, measuring absorbance (A) at wavelength 370nm (reference wavelength 492nm) by bromine standard method, and calculating the inhibition rate of paclitaxel on each group of cells, wherein the inhibition rate is AExperimental group/ANegative control group.3 replicate wells were set for each concentration and the average was taken.
The drug concentration at 50% inhibition was half the Inhibitory Concentration (IC)50)；
IC of drug-resistant strain A2780/Taxol50Of its parent cell line A2780IC50The ratio of (A) to (B) is the Resistance Fold (RF);
IC of drug-resistant strain A2780/Taxol50IC after transfection with it p L KO.1-CO L12A 1-sh1 (Reversant)50The ratio of (A) to (B) is the resistance Reversal Index (RI).
The results are shown in Table 2, Table 3 and FIG. 13, IC of A2780/Taxol vs. paclitaxel50(36.23 + -4.78 ug/ml) was significantly higher than the IC of parent A2780 for paclitaxel50(1.36 +/-0.37 ug/ml), the drug resistance times are as high as 26.64, the sensitivity of A2780/Taxol to paclitaxel is obviously lower than that of a parent cell A2780, and the drug resistance is high, however, after the A2780/Taxol transfects p L KO.1-CO L12A 1-sh1, the sensitivity to paclitaxel is obviously improved (8.25 +/-2.53 ug/ml), and the reversion effect of the transfected p L KO.1-CO L12A 1-sh1 to the drug resistance of A2780/Taxol is very obvious, and the reversion index is 4.65.
TABLE 2 drug sensitivity of A2780 and A2780/Taxol to paclitaxel
TABLE 3 reversion of the drug sensitivity of A2780/Taxol to paclitaxel after transfection of p L KO.1-CO L12A 1-sh1
SEQUENCE LISTING
<110> Zhejiang university
<120> siRNA for specifically inhibiting CO L12A 1 gene expression, recombinant vector and application thereof
<130>
<160>8
<170>PatentIn version 3.3
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Claims (3)
1. The application of the siRNA for specifically inhibiting the expression of the CO L12A 1 gene in the preparation of the medicine for reversing the drug resistance of the ovarian cancer taxol is characterized in that the siRNA comprises a sense strand and an antisense strand, the nucleotide sequence of the sense strand is shown as SEQ ID NO.1, the nucleotide sequence of the antisense strand is shown as SEQ ID NO.2, and the drug resistance of the ovarian cancer taxol is the abnormal increase of the expression of CO L12A 1.
2. The use of claim 1, wherein 3 bases at the 5 ' and 3 ' ends of the sense and antisense strands are 2 ' -methoxy modified.
3. The application of a recombinant vector containing a DNA sequence of siRNA for specifically inhibiting CO L12A 1 gene expression in the preparation of drugs for reversing drug resistance of ovarian cancer paclitaxel is characterized in that the siRNA comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand is shown as SEQ ID No.1, the nucleotide sequence of the antisense strand is shown as SEQ ID No.2, and the drug resistance of the ovarian cancer paclitaxel is abnormally increased in expression of CO L12A 1.
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| Extracellular Matrix Proteins Expression Profiling in Chemoresistant Variants of the A2780 Ovarian Cancer Cell Line;RadosBaw Januchowski et al;《BioMed Research International》;20140430;摘要，第2页左栏第3、4段，第2、4节，表1 * |
| RadosBaw Januchowski et al.Extracellular Matrix Proteins Expression Profiling in Chemoresistant Variants of the A2780 Ovarian Cancer Cell Line.《BioMed Research International》.2014, * |
| siRNA核糖分子化学修饰对RNAi功能的影响;武文斌;《生物技术通报》;20091231;第3节 * |
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