CN107164378B - siRNA for specifically inhibiting SPIB gene expression and recombinant vector and application thereof - Google Patents
siRNA for specifically inhibiting SPIB gene expression and recombinant vector and application thereof Download PDFInfo
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Abstract
The invention discloses siRNA for specifically inhibiting SPIB gene expression, a recombinant vector thereof and application of the siRNA in preparation of drugs for reversing drug resistance of ovarian cancer taxol, and belongs to the technical field of molecular biology and biomedicine. The siRNA comprises a sense strand and an antisense strand, wherein the sense strand: 5'-AAUCAGGGUAGCUGGAAUGCU-3', respectively; antisense strand: 5'-CAUUCCAGCUACCCUGAUUCA-3' are provided. The siRNA provided by the invention can specifically and efficiently inhibit the mRNA and protein expression of the SPIB gene, reduce cell proliferation, increase cell apoptosis, reduce cell migration and invasion capacity, and effectively reverse the drug resistance of ovarian cancer cells to paclitaxel. The invention also provides application of the siRNA and the recombinant vector thereof in preparing medicines for treating ovarian cancer, gastric cancer, colorectal cancer, head and neck squamous cell carcinoma or reversing drug resistance of ovarian cancer.
Description
Technical Field
The invention relates to the technical field of molecular biology and biomedicine, in particular to siRNA for specifically inhibiting SPIB 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, found for the first time that the dsRNA produced gene silencing effect is at least 10 times of the inhibition effect of antisense nucleotide in C.elegans of caenorhabditis elegans, and can induce the same gene inhibition phenomenon in offspring. The mechanism research of RNAi phenomenon shows that the key molecule capable of degrading homologous RNA efficiently and specifically to result in sequence specific gene silencing is small double-stranded oligonucleotide with 21-23 base length, 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 has attracted attention as a significant finding in recent biotechnology because it has a gene suppression function of specifically and efficiently blocking all homologous genes and is a powerful tool for gene function studies. The method of using small interfering RNA (siRNA) as gene silencing is widely used for the research of malignant tumor mechanism, and thus the development process of tumor specific high-efficiency targeted therapy strategy based on the technology is promoted.
Ovarian cancer is the leading cause of death in reproductive tumors, has occult disease, lacks early symptoms and effective early diagnosis means, and has rapid malignant progression. The standard treatment regimen is tumor cytoreductive surgery with post-operative platinum/taxol drug combination chemotherapy, and most recurred within 18 months, although complete remission can occur in 70% of patients. Even if the ovarian cancer after relapse is treated by chemotherapeutics with completely different action mechanisms, the ovarian cancer can generate drug resistance, so that the survival rate after tumor treatment cannot be obviously improved. The five-year survival rate of advanced ovarian cancer is consistently around 30%. High recurrence rate and drug resistance are important reasons for poor prognosis of ovarian cancer. How to improve the treatment effect of ovarian cancer and search a solution of chemotherapy resistance of ovarian cancer is an important subject of gynecological tumor research.
The combined application of molecular targeted drugs and chemotherapy is the most promising treatment method for improving the survival rate of malignant tumor patients at present. SPIB (Spi-B transcription factor) is a member of the Ets transcription factor family, and the gene is located on chromosome 19q, is normally expressed only on mature B cells, T cell progenitors, and plasmacytoid dendritic cells, plays an important role in the differentiation process of mature B cells into plasmacytoid cells and plasmacytoid dendritic cells, plays a central role in the germinal center reaction during embryonic development, and is slowly developed in mice with SPIB gene deletion, and a large number of apoptotic cells are produced in vivo as compared with wild-type mice. Recent studies found that SPIB is highly expressed in the solid tumors gastric, colorectal and head and neck squamous cell carcinoma. Our previous studies showed that SPIB is also highly expressed in ovarian cancer cells, and abnormal increase in the paclitaxel-resistant cell line a2780/Taxol in ovarian cancer is found, but whether the gene is related to the onset of ovarian cancer remains to be further studied. Therefore, the interference of the expression of the SPIB gene in the ovarian cancer drug-resistant cell strain by adopting the RNAi technology is an important supplement to the pathogenesis of the ovarian cancer and an important exploration and application to the ovarian cancer and the drug-resistant treatment thereof.
Disclosure of Invention
The invention aims to provide siRNA for specifically inhibiting the expression of an SPIB gene, which is used for researching the pathogenesis of ovarian cancer. The invention also aims to provide the application of the siRNA in preparing a medicine for reversing the drug resistance of the ovarian cancer taxol.
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 the expression of the SPIB gene, and the siRNA is transfected into an ovarian cancer Taxol resistant cell strain A2780/Taxol, and the result shows that the interference effect of S2 for inhibiting the expression of the SPIB gene is most obvious.
The invention provides siRNA (S2) for specifically inhibiting the expression of an SPIB gene, which comprises a sense strand and an antisense strand,
the sense strand: 5'-AAUCAGGGUAGCUGGAAUGCU-3' (SEQ ID NO. 1);
the antisense strand: 5'-CAUUCCAGCUACCCUGAUUCA-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 (S2) 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 invention provides an RNA interference kit comprising a DNA sequence encoding the siRNA. The kit comprises a DNA plasmid vector cloned with the siRNA, and when the kit is applied, the plasmid vector transcribes and expresses the required siRNA in eukaryotic cells, so that the expression of silencing the SPIB gene is achieved.
The invention provides a recombinant vector containing a DNA sequence for encoding the siRNA. Preferably, the original vector used is the lentiviral vector plko.1puro.
The invention also provides a construction method of the recombinant vector, which comprises the following steps:
(1) synthesizing an SPIB-S2 fragment, selecting two enzyme cutting sites of Age I and EcoR I, and designing shRNA sequence according to the sequence of S2, wherein the sequence is as follows:
sense strand:
5’-CCGGTGAATCAGGGTAGCTGGAATGCTTTCAAGAGAGACATTCCAGCTACCCTGATTCATTTTTTGGTACC-3’(SEQ ID NO.7)；
antisense strand:
5’-AATTGGTACCAAAAAATGAATCAGGGTAGCTGGAATGTCTCTCTTGAAAGCATTCCAGCTACCCTGATTCA-3’(SEQ ID NO.8)；
(2) annealing to obtain a DNA fragment of the SPIB-S2;
(3) pLKO.1-SPIB-sh2 recombinant vector was constructed using lentiviral vector pLKO.1puro.
The siRNA provided by the invention can efficiently and specifically inhibit the expression of the SPIB gene of ovarian cancer cells, reduce cell proliferation, increase apoptosis and reduce cell migration and invasion capacity, so that the siRNA and the recombinant vector serving as the SPIB 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 an SPIB gene expression inhibitor.
The invention provides application of the siRNA and the recombinant vector in preparing a medicament for treating ovarian cancer, gastric cancer, colorectal cancer or head and neck squamous cell carcinoma.
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 19.51, which shows that the reversal effect of the siRNA provided by the invention on the paclitaxel resistant strain A2780/Taxol is very obvious, therefore, the siRNA has potential application value in the treatment of reversing the paclitaxel resistance of ovarian cancer.
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 the expression of mRNA and protein of the SPIB gene, reduce the proliferation of tumor cells, increase the apoptosis, reduce the migration and invasion capacity of the tumor cells, and effectively reverse the drug resistance of ovarian cancer cells to paclitaxel, and has important significance in the research of tumor pathogenesis and the preparation of drugs for tumor treatment and drug resistance treatment of the reversed ovarian cancer.
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FIG. 1 shows the expression of SPIB mRNA in A2780 cells and A2780/Taxol cells detected by qRT-PCR.
FIG. 2 shows the detection of the expression of SPIB protein in A2780 cells and A2780/Taxol cells by Western Blotting.
FIG. 3 shows the expression of SPIB mRNA in A2780/Taxol cells 48h after transfection with S1, S2, S3 by qRT-PCR.
FIG. 4 shows the expression of SPIB protein in A2780/Taxol cells after transfection with S1, S2 and S3 for 72h by Western Blotting.
FIG. 5 is a schematic diagram of pLKO.1-SPIB-sh2 recombinant plasmid and inserted restriction 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 2 sense strand bases and 23S 2 antisense strand bases.
FIG. 7 shows the detection of the expression of SPIB protein in A2780/Taxol cells after pLKO.1-SPIB-sh2 transfection by Western Blotting.
FIG. 8 shows the number and morphology of A2780/Taxol cells after pLKO.1-SPIB-sh2 transfection under phase contrast microscope observation.
FIG. 9 shows the proliferation of A2780/Taxol cells detected by bromine labeling method after transfection of pLKO.1-SPIB-sh 2.
FIG. 10 shows the detection of Caspase3 activity in apoptosis of A2780/Taxol cells after pLKO.1-SPIB-sh2 transfection.
FIG. 11 shows the migration ability of A2780/Taxol cells after pLKO.1-SPIB-sh2 transfection in cell scratch assay.
FIG. 12 shows the migration ability (A) and invasion ability (B) of A2780/Taxol cells after transfection of pLKO.1-SPIB-sh2 by Transwell assay.
FIG. 13 shows the reversal of paclitaxel resistance in A2780/Taxol cells after pLKO.1-SPIB-sh2 transfection.
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.
The SPSS 18.0 statistical analysis software is adopted, and the data of each sample are averaged to be +/-standard deviationAs shown, the differences between the two groups were measured by T Test (Independent-Sample T Test), and the differences between the groups were measured by One-Way analysis of variance (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;
rabbit anti-human SPIB primary antibody (Cat.15768-1-AP), mouse anti-human GAPDH primary antibody (Cat.60004-1-Ig), horseradish peroxidase-labeled goat anti-mouse IgG (H + L) secondary antibody (Cat.SA00001-1), and horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) secondary antibody (Cat.SA00001-2) were purchased from Proteintech;
western Blotting Luminol Reagent kit (Cat. sc-2048) was purchased from Santa Cruz;
reverse transcription of cDNAKit PrimeScriptTMRT Master Mix (Cat. RR036A), fluorescent quantitative PCR detection kit SYBR Premix Ex Taq (perfect Real time, Cat. DRR041A) purchased from TaKaRa company; lipofectamine3000 transfection kit (cat. l3000008) was purchased from Invitrogen;
the eukaryotic expression vector selects a conventional RNAi vector pLKO.1puro, and is originated 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;
cell Proliferation ELISA, 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;
Lab-Tek II Chamber Slide System-Lab-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 of the differences in expression of SPIB 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 SPIB 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 Thermo Nano Drop2000 spectrophotometer, according to 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. And a 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’-AAGAAGCTGCGCCTGTACC-3’；
5'-TCTTGGCGTAGTTTCGGAGG-3', product length: 211 bp;
reaction conditions are as follows: 10s at 95 ℃, 5s at 95 ℃ and 30s at 6 ℃ for 40 cycles.
The amount of expression of SPIB mRNA in each group of samples was calculated by the 2-DELTA CT method.
As a result: as shown in FIG. 1, the expression of SPIB mRNA was increased by 88.19% (P < 0.05) in A2780/Taxol cells compared to its parental cell, A2780, indicating a significant increase in SPIB mRNA expression in paclitaxel-resistant cell lines.
Second, Western Blotting detection of SPIB protein expression
After culturing for 72h, absorbing and removing the culture medium in the 6-well plate, washing with PBS for 3 times, adding RIPA protein lysate (100 ul/well), blowing and beating for several times, incubating on ice for 5min to fully crack, centrifuging at 4 ℃, 12000 rpm for 5min, collecting supernatant, and subpackaging at-20 ℃ for storage; denaturing each sample at 95 deg.c for 5min, taking 10ul sample, 8% SDS-PAGE electrophoresis at 200V for 10 min; 100V for 100 min; turning to a PVDF membrane: 110V for 120 min; sealing with TBS sealing solution containing 5% skimmed milk powder for 60 min; primary antibody incubation: incubating SPIB primary antibody (1: 2000) and GAPDH primary antibody (1: 5000) at room temperature for 2 h; washing membrane with TBS for 10min × 3 times; and (3) secondary antibody incubation: incubating a goat anti-mouse IgG (H + L) secondary antibody (1: 10000) marked by horseradish peroxidase and a goat anti-rabbit IgG (H + L) secondary antibody (1: 10000) marked by horseradish peroxidase for 1H; washing membranes with TBST for 10min × 3 times and TBS for 10min × 1 time; after ECL development, the images were scanned with ImageQuantLAS4000 mini (ge healthcare).
As a result: as shown in FIG. 2, the expression of the SPIB protein was also significantly increased in A2780/Taxol cells compared to its parent cell, A2780 (P < 0.05).
Example 2 SPIB siRNA design Synthesis
The mRNA sequence of the SPIB gene (NM-003121.4) was obtained from Genebank, and 3 pairs of siRNA sequences (shown in SEQ ID NO.1-SEQ ID NO.6) were obtained by on-line design using siDirect Ver2.0 software (http:// sidirect2.rnai. jp /). In the design process, a sequence simultaneously meeting three algorithms (Ui-Tei x Reynolds x Amarzguioui) reported in the literature is selected, a 23nt length segment with the highest siRNA action specificity is selected, the design can avoid the generation of an interferon-like immune response in future in-vivo experiments, 100nt after an initiation codon is selected, 5 'and 3' end UTR regions are avoided, and the GC content is controlled to be 30-70%. 3 pairs of siRNA with the length of 23nt are selected as experimental screening interference fragments, the structural characteristics are that the 3' ends of the sense strand and the antisense strand are respectively externally hung with two basic groups, and the structural characteristics are as follows
Followed by BLASTN(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 three pairs of SPIB siRNAs on the interfering Effect of SPIB genes in Taxol-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 Lipofectamine3000 transfection reagent for siRNA transfection. One day before transfection, cells were trypsinized and counted, and cells were plated in six-well plates at a density of 0.5X 10 on the day of transfection6Per ml, cells were fused to 70-90%. Each well was diluted with 125. mu.l of serum-free OPTI-MEM medium to 5ul of Lipofectamine3000 reagent and mixed well; preparing siRNA premix, diluting siRNA with 125 mul of serum-free OPTI-MEM culture medium to a final concentration of 50nM, and fully mixing; adding siRNA premix (1:1) into diluted Lipofectamine3000 reagent, and incubating for 5min at room temperature; finally, the siRNA-liposome complex was added to the cells at 37 ℃ with 5% CO2The culture is continued. After 48h, SPIB mRNA expression was detected, and after 72h, SPIB protein expression was detected.
Third, real-time fluorescent quantitative RT-PCR (qRT-PCR) is used for detecting mRNA expression of the SPIB gene
The detection steps are the same as the previous steps, and the expression quantity of the SPIB mRNA in each group of samples is calculated by adopting a 2-delta CT method.
As a result: as shown in FIG. 3, after the A2780/Taxol cells were transfected with S1, S2 and S3, the expression of the SPIB mRNA was significantly reduced, wherein the S2 interference effect was 83.07% lower than that of the negative control group, and the difference was significant (P < 0.05%) compared with S1 (62.05%) and S3 (55.75%). The results show that S2 has the best interference effect on SPIB.
Fourth, Western Blotting detection of SPIB protein expression
The detection procedure was as before, after ECL development, the Image was scanned with Image Quant LAS4000 mini (GE healthcare).
As a result: as shown in FIG. 4, after transfection with S2, there was a significant decrease in SPIB protein expression in A2780/Taxol cells (P < 0.05) compared to the negative control, and a significant difference (P < 0.05) compared to S1 and S3. The results show that S2 has the best interference effect on protein expression of SPIB in the paclitaxel resistant strain A2780/Taxol. Therefore, S2 was selected as siRNA for subsequent studies.
Example 4 construction of eukaryotic vector pLKO.1-SPIB-sh2 and detection of interference effect on expression of SPIB Gene
Firstly, experimental grouping:
A2780/Taxol normal group (without transfection of any vector), hereinafter referred to as AR;
A2780/Taxol negative control group (pLKO.1puro empty vector transfected), hereinafter referred to as AR-N;
A2780/Taxol experimental group 1 (pLKO.1-SPIB-sh 2 transfection), hereinafter referred to as AR-sh 2;
II, synthesizing the fragment SPIB-S2
Two enzyme cutting sites of Age I and EcoR I are selected, and shRNA sequence is designed according to the sequence of S2 and is constructed into a eukaryotic expression vector pLKO.1puro. The sequence is as follows:
sense strand:
5’-CCGGTGAATCAGGGTAGCTGGAATGCTTTCAAGAGAGACATTCCAGCTACCCTGATTCATTTTTTGGTACC-3’(SEQ ID NO.7)；
antisense strand:
5’-AATTGGTACCAAAAAATGAATCAGGGTAGCTGGAATGTCTCTCTTGAAAGCATTCCAGCTACCCTGATTCA-3’(SEQ ID NO.8)；
thirdly, construction of eukaryotic vector pLKO.1-SPIB-sh2
pLKO.1-SPIB-sh2 recombinant expression vector was constructed using eukaryotic expression vector pLKO.1puro (FIGS. 5 and 6), and the specific method was described in molecular cloning, a laboratory Manual, of Cold spring harbor Press, USA.
The sense and antisense strands of SPIB-S2 were annealed (denaturation at 95 ℃ for 2 min; annealing by slow cooling to 25 ℃) and stored at 4 ℃. Completely digesting the vector pLKO.1puro by Age I and EcoR I, and standing overnight at 37 ℃; and recovering DNA from the enzyme digestion product by using a DNA gel recovery kit. The annealing product and the carrier enzyme digestion recovery fragment are subjected to a connection reaction, and the reaction system (10 ul): 1ul of T4 ligase, 1ul of T4 ligase buffer solution, and carrying out enzyme digestion on the annealing product and a carrier to recover a fragment mixture (the molar ratio is 3: 1); supplementing deionized water to 10ul, and connecting overnight at 16 ℃; placing 5ul of the ligation product in 100ul of JM109 competent bacteria, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 90s, carrying out ice bath for 5min, adding 1000ul of LB culture medium, carrying out shake culture at 37 ℃ for 30min, centrifuging at 5000rpm for 5min, discarding the supernatant, uniformly coating the bacteria on an LB plate (containing 50ug/ml ampicillin), and carrying out inverted culture at 37 ℃ for overnight; selecting a plurality of independent colonies, inoculating the colonies into an LB culture medium containing corresponding resistance, and oscillating the colonies overnight at 37 ℃ for strain expansion; collecting bacteria, extracting by using a plasmid DNA purification kit to obtain plasmid DNA, carrying out enzyme digestion identification by Kpn I to obtain pLKO.1-SPIB-sh2 recombinant plasmid.
Fourth, pLKO.1-SPIB-sh2 recombinant plasmid is used for observing interference effect after transfecting cells
pLKO.1-SPIB-sh2 was transfected into A2780/Taxol cells in logarithmic growth phase. Transfection 5ul Lipofectamine3000 reagent was diluted in 125. mu.l serum-free OPTI-MEM medium per well and mixed well according to Lipofectamine3000 protocol; adding 5ug of recombinant plasmid DNA into 125 mul of serum-free OPTI-MEM culture medium, adding 10ul of P3000 reagent, and mixing to obtain recombinant plasmid premix; the diluted Lipofectamine3000 reagent was added to the recombinant plasmid premix (1:1) and incubated at room temperature for 5 mm; finally, 250ul of the recombinant plasmid-liposome complex was added to the cells at 37 ℃ with 5% CO2The culture is continued. SPIB protein expression was detected after 72 h.
As shown in FIG. 7, after the pLKO.1-SPIB-sh2 recombinant plasmid is transfected, the expression of the SPIB protein in A2780/Taxol cells is remarkably reduced (P < 0.05) compared with that of a negative control (transfection of an empty plasmid), and the result shows that the pLKO.1-SPIB-sh2 recombinant plasmid is transfected to effectively interfere the protein expression of the SPIB.
Example 5 Effect of pLKO.1-SPIB-sh2 transfection on proliferation, apoptosis, migration and invasion of tumor cells after specific blocking of SPIB expression
Firstly, experimental grouping:
A2780/Taxol normal group (without transfection of any vector), hereinafter referred to as AR;
A2780/Taxol negative control group (pLKO.1puro empty vector transfected), hereinafter referred to as AR-N;
A2780/Taxol experimental group (pLKO.1-SPIB-sh 2 transfection), hereinafter referred to as AR-sh 2.
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
Cells are transfected into a 96-well plate pLKO.1-SPIB-sh2 and then cultured for 72h, 10ul BrdU labeling solution is added into each well until the final BrdU concentration is 10uM, and the cells are incubated for 2h at 37 ℃; absorbing BrdU labeling solution, adding 200ul FixDenat into each well, and incubating at 20 deg.C for 30 min; removing FixDenat by suction, adding 100ul anti-BrdU-POD into each well, and incubating at 20 deg.C for 90 min; washing 3 times with 200ul washing Solution per well; adding 100 ul/well substrate solution, incubating at 20 deg.C for 20min, detecting absorbance (A) at wavelength of 370nm (reference wavelength of 492nm), and measuring cell proliferation ability with AExperimental group/AControl groupAnd (4) showing.
As shown in FIGS. 8 and 9, in A2780/Taxol cells, pLKO.1-SPIB-sh2 is transfected, and the observation under a phase contrast microscope shows that the number of cells in an experimental group is obviously reduced, the number of suspended cells is increased, and the cell debris is increased; the results of cell proliferation tested by the bromine standard method also show that: compared with a negative control, the cell proliferation capacity of the experimental group is reduced by 71.22%, and the significant difference is realized (P is less than 0.05). Indicating that the tumor cell proliferation can be inhibited after the expression of the SPIB is specifically blocked.
Fourthly, detecting apoptosis by Caspase3 activity
Collecting cells after transfection for 72h, adjusting cell density to 1 × 10 with lysate8-/ml, split on ice for 15min, 15000g × 20min, collect supernatant. According to CaspACE Assay System (colorimetric)) The instructions prepare positive and negative control samples simultaneously, and determine and adjust the same concentration of each histone. Caspace Assay Buffer32ul, DMSO 2ul, 100nM DTT 10ul, deionized water adjusted to 98ul volume, 2ul DEVD-pNA substrate was added to each well of 96-well plate, incubated at 37 ℃ for 4h, the absorbance was measured at 405nM wavelength, and Caspase3 activity was calculated for each group of samples by the Δ A method.
The result is shown in FIG. 10, after pLKO.1-SPIB-sh2 is transfected by A2780/Taxol cells, Caspase3 activity is increased by 4.19 times, and compared with a negative control, the significant difference (P is less than 0.05) is generated, which indicates that tumor cell apoptosis can be promoted after the expression of the specific blocking SPIB.
Fifth, cell migration ability test
And uniformly marking transverse lines at the back of the six-hole plate by using a straight ruler, wherein one transverse line is marked by about 0.5cm and penetrates through the holes, and each hole is provided with at least 6 transverse lines. After cell transfection pLKO.1-SPIB-sh2, when the cells are continuously cultured for 24h and fused into a single layer state, vertically scratching the selected area in a six-hole plate by using a 200ul pipette tip, washing by PBS for 3 times to remove the scratched cells, and adding a serum-free culture medium for continuous culture. Photographing is carried out at the time points of 0h, 24h and 48h, 6 horizontal lines are randomly selected, and the average value of the distances between cells is calculated.
The result is shown in fig. 11, after cells are transfected by pLKO.1-SPIB-sh2 for 24h and 48h, the distance between A2780 and Taxol cells is obviously greater than that of a negative control group, the healing capacity of the cells after scratching is obviously reduced, and the tumor cell migration can be inhibited after the expression of the specific blocking SPIB.
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 5X 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 pLKO.1-SPIB-sh2 transfection, the cell numbers of the A2780/Taxol experimental group and the negative control group penetrating the chamber are 46 + -15 and 279 + -27 respectively, and the two groups have statistical difference (P < 0.05); the results indicate that the specific blocking of SPIB expression inhibits tumor cell migration.
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 assay are shown in FIG. 12B, the numbers of cells penetrating the chamber of the A2780/Taxol test group and the negative control group are 36 + -8 and 177 + -28, respectively, and the two groups have statistical difference (P < 0.05); the results show that tumor cell infiltration can be inhibited after the expression of the SPIB is specifically blocked.
Example 6 pLKO.1-SPIB-sh2 reversal of drug resistance in ovarian cancer after specific blockade of SPIB expression
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 (pLKO.1puro empty vector transfected), hereinafter referred to as AR-N;
A2780/Taxol experimental group (pLKO.1-SPIB-sh 2 transfection), hereinafter referred to as AR-sh 2.
Two, grouped transfection
The transfection procedure was as before, and cells were cultured for 24h after transfection.
Thirdly, detecting the sensitivity of the cells to the paclitaxel after pLKO.1-SPIB-sh2 transfection
Taking cells in logarithmic growth phase from each group, re-suspending the cells after trypsinization, counting the cells and adjusting the density of the cell suspension to 1 × 105And/ml, inoculating into a 96-well plate and continuing culturing for 24 hours. The next day, paclitaxel was added to each group,setting concentration gradient at 200ug/ml, 100ug/ml, 50ug/ml, 25ug/ml, 12.5ug/ml, 6.25ug/ml, 3.125ug/ml and 0ug/ml, respectively, reacting for 24h, measuring absorbance (A) at wavelength of 370nm (reference wavelength of 492nm) by bromine standard method, and calculating the inhibition rate of paclitaxel on each group of cellsExperimental 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/Taxol50IC with its parent cell line A278050The ratio of (A) to (B) is the Resistance Fold (RF);
IC of drug-resistant strain A2780/Taxol50IC after transfection with pLKO.1-SPIB-sh2 (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(42.65 + -4.88 ug/ml) is significantly higher than the IC of parent A2780 for paclitaxel50(1.36 +/-0.29 ug/ml), the drug resistance times are as high as 31.36, which indicates that the sensitivity of A2780/Taxol to paclitaxel is obviously lower than that of a parent cell A2780, and the drug resistance is high. And after the A2780/Taxol is transfected with pLKO.1-SPIB-sh2, the sensitivity to paclitaxel is obviously improved (2.12 +/-0.47 ug/ml), and the reversal effect of the transfected pLKO.1-SPIB-sh2 to the drug resistance of the A2780/Taxol is very obvious, and the reversal index is 19.51.
TABLE 2 drug sensitivity of A2780 and A2780/Taxol to paclitaxel
TABLE 3 reversion of the sensitivity of A2780/Taxol to paclitaxel drugs after pLKO.1-SPIB-sh2 transfection
SEQUENCE LISTING
<110> Zhejiang university
<120> siRNA for specifically inhibiting SPIB gene expression, recombinant vector and application thereof
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Claims (4)
1. The application of the siRNA for specifically inhibiting the expression of the SPIB 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, wherein the nucleotide sequence of the sense strand is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 2.
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 the expression of SPIB gene in the preparation of drugs for reversing the drug resistance of ovarian cancer taxol 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, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 2.
4. The use of claim 3, wherein 3 bases at the 5 ' and 3 ' ends of the sense and antisense strands are 2 ' -methoxy modified.
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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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| 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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