CN107541515B - siRNA for specifically inhibiting PRV1 gene expression and recombinant vector and application thereof - Google Patents

Abstract

The invention discloses siRNA for specifically inhibiting PRV1 gene expression, a recombinant vector thereof and application thereof in reversing ovarian cancer taxol resistance, belonging to the technical field of molecular biology and biomedicine. The siRNA comprises a sense strand and an antisense strand, wherein the sense strand: 5'-UUCUUAGGGGUCCAUUGCCGG-3', respectively; antisense strand: 5'-GGCAAUGGACCCCUAAGAACA-3' are provided. The siRNA provided by the invention can specifically and efficiently inhibit the mRNA and protein expression of PRV1 gene in tumor cells, reduce the proliferation of the 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. The invention also provides application of the siRNA and the recombinant vector thereof in preparing medicines for treating ovarian cancer, gastric cancer, colorectal cancer, neuroblastoma or reversing ovarian cancer drug resistance.

Description

siRNA for specifically inhibiting PRV1 gene expression and recombinant vector and application thereof

Technical Field

The invention relates to the technical field of molecular biology and biomedicine, in particular to siRNA for specifically inhibiting PRV1 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 combined chemotherapy of the platinum/taxol medicaments, 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 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. Research shows that a subgroup of tumor cells, tumor stem-like cells (CSCs), can cause tumor formation and drug resistance, and the drug resistance of the CSCs and the expression of some key genes have obvious correlation. Some CSCs marker molecules and drug resistance related genes are also sequentially found in ovarian cancer cells, and one of the genes of the neutrophil surface molecule PRV1 is. The PRV1 gene belongs to a member of the leukocyte antigen 6 gene superfamily, is usually expressed on the cell membrane of neutrophilic granulocytes, and is originally found to be abnormally increased on the neutrophilic granulocytes of patients with primary thrombocythemia and polycythemia vera, so that the PRV1 gene is used for differential diagnosis of secondary thrombocythemia and secondary polycythemia. However, in later researches, PRV1 in progressive gastric cancer cells is obviously highly expressed, the expression level and clinical prognosis indexes of gastric cancer are obviously related, and the higher the expression level, the worse the prognosis. Recent literature reports that the PRV1 gene is also highly expressed in colorectal cancer, neuroblastoma and ovarian cancer. Meanwhile, the structure of PRV1 is very similar to that of urokinase-type plasminogen activator receptor (uPAR) family members, and the uPAR-type genes are closely related to the adhesion and migration characteristics of cells, and the abnormal adhesion and migration capabilities are the characteristics of tumor cells different from normal cells.

Our previous studies showed that PRV1 was abnormally elevated in the paclitaxel-resistant cell line A2780/Taxol for ovarian cancer, but whether the gene is related to the onset and resistance of ovarian cancer remains to be further studied. Therefore, interference of PRV1 gene expression in the drug-resistant cell strain of ovarian cancer by adopting RNAi technology is an important supplement to the pathogenesis of ovarian cancer and an important exploration and application to the treatment of ovarian cancer and drug resistance thereof.

Disclosure of Invention

The invention aims to provide siRNA for specifically inhibiting PRV1 gene expression, which is used for researching the pathogenesis of ovarian cancer. The invention also aims to provide application of the siRNA in preparing medicaments for treating ovarian cancer and reversing paclitaxel drug 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 PRV1 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 PRV1 gene expression.

The invention provides siRNA (S1) for specifically inhibiting PRV1 gene expression, which comprises a sense strand and an antisense strand,

the sense strand: 5'-UUCUUAGGGGUCCAUUGCCGG-3' (SEQ ID NO. 1);

the antisense strand: 5'-GGCAAUGGACCCCUAAGAACA-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 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 siRNA in eukaryotic cells, so that the expression of PRV1 gene is silenced.

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 PRV1-S1 fragment, selecting two restriction enzyme sites of Age I and EcoR I, and designing the shRNA sequence according to the sequence of S1, wherein the sequence is as follows:

sense strand:

5’-CCGGTGTTCTTAGGGGTCCATTGCCGGTTCAAGAGACCGGCAATGGACCCCTAAGAACATTTTTTGGTACC-3’(SEQ ID NO.7)；

antisense strand:

5’-AATTGGTACCAAAAAATGTTCTTAGGGGTCCATTGCCGGTCTCTTGAACCGGCAATGGACCCCTAAGAACA-3’(SEQ ID NO.8)；

(2) annealing to obtain a DNA fragment of PRV 1-S1;

(3) the p L KO.1-PRV1-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 PRV1 gene in 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 PRV1 gene expression inhibitors 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 PRV1 gene expression inhibitor.

The invention provides application of the siRNA and the recombinant vector in preparing medicines for treating ovarian cancer, gastric cancer, colorectal cancer or neuroblastoma.

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 12.97, 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 on 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 the expression of mRNA and protein of PRV1 gene, reduce the proliferation of tumor cells, increase the apoptosis of tumor cells, reduce the migration and invasion capacity of tumor cells, and effectively reverse the drug resistance of ovarian cancer cells to paclitaxel, and has important significance in the study of tumor pathogenesis and the preparation of drugs for tumor treatment and ovarian cancer drug resistance treatment.

Drawings

FIG. 1 shows the detection of PRV1mRNA expression in A2780 cells and A2780/Taxol cells by qRT-PCR, wherein A is A2780 cells and AR is A2780/Taxol cells.

FIG. 2 shows Western Blotting to detect PRV1 protein expression in A2780 cells and A2780/Taxol cells, wherein A is A2780 cells, and AR is A2780/Taxol cells.

FIG. 3 shows the expression of PRV1mRNA in A2780/Taxol cells 48h after transfection with S1, S2, S3 by qRT-PCR.

FIG. 4 shows the expression of PRV1 protein in A2780/Taxol cells after transfection with S1, S2 and S3 for 72h by Western Blotting.

FIG. 5 is a schematic diagram of the p L KO.1-PRV1-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 the Western Blotting detection of the expression of PRV1 protein in A2780/Taxol cells after p L KO.1-PRV1-sh1 transfection.

FIG. 8 shows the change in the number and morphology of A2780/Taxol cells after transfection of p L KO.1-PRV1-sh1 by phase contrast microscopy.

FIG. 9 shows the proliferation of A2780/Taxol cells after transfection of p L KO.1-PRV1-sh1 by bromine labeling.

FIG. 10 shows the detection of Caspase3 activity on apoptosis of A2780/Taxol cells after transfection of p L KO.1-PRV1-sh 1.

FIG. 11 shows the migration ability of A2780/Taxol cells after transfection of p L KO.1-PRV1-sh1 in a cell scratch test.

FIG. 12 shows the migratory capacity (A) and the invasive capacity (B) of A2780/Taxol cells after transfection of p L KO.1-PRV1-sh1 by Transwell assay.

FIG. 13 shows the reversal of paclitaxel resistance in A2780/Taxol cells after transfection of p L KO.1-PRV1-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.

The SPSS 18.0 statistical analysis software is adopted, and the data of each sample are averaged to be +/-standard deviation

As 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;

mouse anti-human PRV1 primary antibody (Cat. sc-374291) was purchased from Santa Cruz, mouse anti-human GAPDH primary antibody (Cat.60004-1-Ig), horseradish peroxidase-labeled goat anti-mouse IgG (H + L) secondary antibody (Cat. SA00001-1) was purchased from Proteitech;

western Blotting L ulinol Reagent kit (Cat. sc-2048) was purchased from Santa Cruz;

cDNA reverse transcription kit PrimeScript^TMRT 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 Matrigel^TMbasic 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 PRV1 expression differences 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 PRV1 gene mRNA expression

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’-GCCTGGAGTCTCTCACTTG-3’；

5'-CCCCAGTGTTGCTGTTAGGT-3', product length: 248 bp;

reaction conditions are as follows: 10s at 95 ℃, 5s at 95 ℃ and 30s at 6 ℃ for 40 cycles.

The expression level of PRV1mRNA in each group of samples was calculated by 2- △ CT method.

As a result: as shown in FIG. 1, PRV1mRNA expression was 89.58% higher in A2780/Taxol cells compared to its parent cell, A2780 (P < 0.05), indicating that PRV1mRNA expression was significantly higher in the paclitaxel-resistant cell line.

Second, Western Blotting detection of PRV1 protein expression

After 72H of culture, the culture medium in a 6-well plate is sucked away, PBS is washed for 3 times, RIPA protein lysate (100 ul/well) is added, the mixture is blown and beaten for several times, the mixture is incubated for 5min on ice to be fully cracked, the mixture is centrifuged for 5min at 4 ℃ and 12000 times, supernatant is collected and stored at 20 ℃ below zero, 10ul of sample is taken after 5min of denaturation at 95 ℃ of each sample, 8 percent SDS-PAGE electrophoresis is performed at 200V and 10min, 100V and 100min are performed, the mixture is transferred to PVDF membrane at 110V and 120min, 5 percent skim milk powder-containing TBS blocking solution is used for blocking for 60min, primary antibody incubation is performed at PRV1 primary antibody (1: 200), GAPDH primary antibody (1: 395000) for 2H at room temperature, TBS membrane washing is performed for 10min for 3 times, secondary antibody incubation is performed at 10min, goat anti-mouse IgG (H + L) IgG (1: 10000), horseradish peroxidase-goat anti-rabbit IgG (H + L) (1H), TBST 3 min, 10min, goat anti-mouse anti-rabbit IgG (H + 395925) is performed with Image washing by using a goat anti-TBGE (1: 10-TBS) and 10 min), and then Image scanning is performed on a goat anti-TBS.

As a result: as shown in FIG. 2, PRV1 protein expression was also significantly increased (P < 0.05) in A2780/Taxol cells compared to its parent cell, A2780.

Example 2 PRV1siRNA design Synthesis

Searching PRV1 gene mRNA sequence (NM-020406.3) in Genebank, using siDirect Ver2.0 software (http:// siDirect2.rnai.jp /) to design on line and obtain 3 pairs of siRNA sequences (as shown in SEQ ID NO.1-SEQ ID NO. 6). The sequence satisfying three algorithms reported in literature (Ui-Tei × Reynolds × Amarzguio) at the same time is selected in the design process, and 23nt length segment with highest siRNA action specificity is selected, the design can avoid the generation of interferon-like immunoreaction in vivo experiment, 100nt after the initial codon is selected, 5 ' and 3 ' end UTR region are avoided, GC content is controlled at 30-70%, the externally hung siRNA with 3 to 23nt length is selected as experiment screening interference segment, the structural feature is that the sense strand and the antisense strand have two bases at the 3 ' end, the structure is characterized 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 interference Effect of three pairs of PRV1siRNA on PRV1 Gene in 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 is continued. PRV1mRNA expression was detected after 48h, and PRV1 protein expression was detected after 72 h.

Third, real-time fluorescent quantitative RT-PCR (qRT-PCR) is used for detecting PRV1 gene mRNA expression

The detection procedure was performed as before, and the expression level of PRV1mRNA in each group of samples was calculated by 2- △ CT method.

As a result: as shown in FIG. 3, after the A2780/Taxol cells are transfected with S1, S2 and S3 respectively, the expression of PRV1mRNA is obviously reduced, wherein the interference effect of S1 is compared with that of a negative control group, PRV1mRNA is down-regulated by 84.33%, and is significantly different (P is less than 0.05) compared with S2 (46.64%) and S3 (41.04%). The results show that S1 has the best interference effect on PRV 1.

Fourth, Western Blotting detection of PRV1 protein expression

Detection procedures As before, after EC L development, the images were scanned with Image Quant L AS4000mini (GE Healthcare).

As a result: as shown in FIG. 4, PRV1 protein expression was significantly reduced in A2780/Taxol cells after transfection with S1 (P < 0.05) compared to the negative control, and significantly different from S2 and S3 (P < 0.05). The results show that S1 has the best interference effect on protein expression of PRV1 in paclitaxel-resistant strain A2780/Taxol. Therefore, S1 was selected as siRNA for subsequent studies.

Example 4 construction of eukaryotic vector p L KO.1-PRV1-sh1 and detection of interference effect on PRV1 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-PRV1-sh1), hereinafter referred to as AR-sh 1;

II, synthesis of PRV1-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’-CCGGTGTTCTTAGGGGTCCATTGCCGGTTCAAGAGACCGGCAATGGACCCCTAAGAACATTTTTTGGTACC-3’(SEQ ID NO.7)；

antisense strand:

5’-AATTGGTACCAAAAAATGTTCTTAGGGGTCCATTGCCGGTCTCTTGAACCGGCAATGGACCCCTAAGAACA-3’(SEQ ID NO.8)；

thirdly, construction of eukaryotic vector p L KO.1-PRV1-sh1

Eukaryotic expression vector p L KO.1puro was used to construct p L KO.1-PRV1-sh1 recombinant expression vector (FIGS. 5 and 6), which is described in molecular cloning, A.C. Cold spring harbor Press.

PRV1-S1 sense strand and antisense strand are annealed (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 recovered by a DNA gel recovery kit, DNA is recovered by the annealed product and the digested recovered fragment of the vector are subjected to ligation reaction, the reaction system (10ul) comprises 1ul of T4 ligase and 1ul of T4 ligase buffer, the 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 overnight at 16 ℃, 5ul of the ligated product is placed in 100ul of JM109 competent bacteria, ice bath is performed for 30min, heat shock is performed at 42 ℃ for 90S, ice bath is performed for 5min, 1000ul of L B medium is added, incubation is performed for 30min at 37 ℃, centrifugation is performed for 5min at 5000rpm, supernatant is removed, the bacteria are evenly spread on L B plate (containing 50 ug/ml), penicillin is cultured at 37 ℃ for 1000ul, ampicillin is performed for 30min, a plurality of inverted culture medium containing penicillin, plasmid is performed overnight, plasmid containing bacterium is obtained, plasmid is inoculated in a colony amplification for overnight, plasmid is obtained, plasmid is subjected to obtain plasmid DNA, plasmid 8 ℃, plasmid is subjected to obtain plasmid purification, plasmid is subjected to shake culture, plasmid purification at 37.

Fourthly, observing interference effect after the p L KO.1-PRV1-sh1 recombinant plasmid transfects cells

P L KO.1-PRV1-sh1 was transfected into A2780/Taxol cells in logarithmic growth phase according to the L ipofectamine3000 instruction, each well was diluted with 125. mu.l of serum-free OPTI-MEM medium to 5. mu.l of L ipofectamine3000 reagent and mixed well, 5ug of recombinant plasmid DNA was added to 125. mu.l of serum-free OPTI-MEM medium, 10ul of P3000 reagent was added to prepare a recombinant plasmid premix, the diluted L ipofectamine3000 reagent was added to the recombinant plasmid premix (1:1), incubated at room temperature for 5 mm, and finally 250. mu.l of recombinant plasmid-liposome complex was added to the cells at 37 ℃ and 5% CO₂The culture is continued. PRV1 protein expression was detected after 72 h.

As shown in figure 7, after the P L KO.1-PRV1-sh1 recombinant plasmid is transfected, compared with a negative control (transfection empty plasmid), the PRV1 protein expression in A2780/Taxol cells is remarkably reduced (P < 0.05), and the result shows that the P L KO.1-PRV1-sh1 recombinant plasmid can effectively interfere with the protein expression of PRV 1.

Example 5 Effect of transfection of p L KO.1-PRV1-sh1 on tumor cell proliferation, apoptosis, migration and invasion after specific blocking of PRV1 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-PRV1-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 p L KO.1-PRV1-sh1 in a 96-well plate and then cultured for 72 hours, 10ul BrdU labeling Solution is added into each well until the final BrdU concentration is 10uM, the cells are incubated for 2 hours at 37 ℃, the BrdU labeling Solution is sucked off, 200ul FixDenat is added into each well, the cells are incubated for 30 minutes at 20 ℃, the FixDenat is sucked off, 100ul anti-BrdU-POD is added into each well, the cells are incubated for 90 minutes at 20 ℃, 200ul washing Solution is washed for 3 times in each well, 100 ul/well bottom Solution is added, the cells are incubated for 20 minutes at 20 ℃, the absorbance (A) is measured at the wavelength of 370nm (reference wavelength of 492nm), and the cell proliferation capacity is measured by the A_{Experimental group}/A_{Control group}And (4) showing.

As shown in FIG. 8 and FIG. 9, after the cells A2780/Taxol are transfected with P L KO.1-PRV1-sh1, the number of the cells in the experimental group is reduced by observation under a phase-contrast microscope, and the cell proliferation results tested by a bromine standard method show that compared with a negative control, the cell proliferation capacity of the experimental group is reduced by 50.47%, and the significant difference (P < 0.05) shows that the tumor cell proliferation can be inhibited after the expression of the PRV1 is specifically blocked.

Fourthly, detecting apoptosis by Caspase3 activity

Cells were harvested 72h after transfection and lysate adjusted to 1 × 10 cell density⁸(ii)/ml, ice lysis for 15min, 15000g × 20min, and collection of supernatant Positive and negative control samples were prepared simultaneously according to CaspACE Assay System (colorimetric) instructions, and the concentrations of each histone determined and adjusted to be the same, Caspace Assay buffer32ul, DMSO2ul, 100nM DTT 10ul was added to each well of 96-well plate, and the volume of deionized water adjusted to be equal98ul, 2ul of DEVD-pNA substrate is added, incubation is carried out for 4h at 37 ℃, the absorbance is measured at the detection wavelength of 405nM, and the Caspase3 activity of each group of samples is calculated by the delta A method.

The results are shown in FIG. 10, after the A2780/Taxol cell is transfected with P L KO.1-PRV1-sh1, the activity of Caspase3 is increased by 2.92 times, and compared with a negative control, the significant difference (P is less than 0.05) is generated, which indicates that after the expression of PRV1 is specifically blocked, the tumor cell apoptosis can be promoted.

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 each hole is provided with at least 6 transverse lines, after cell transfection is carried out on p L KO.1-PRV1-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 for 3 times by using PBS (phosphate buffer solution) 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-PRV1-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 of the cells after scratching is obviously reduced, and the result shows that the cells can inhibit the migration of tumor cells after the expression of PRV1 is specifically blocked.

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 cell migration experiments are shown in FIG. 12A, the cell numbers of the A2780/Taxol experimental group and the negative control group penetrating the chamber after P L KO.1-PRV1-sh1 transfection are 76 + -21 and 301 + -42 respectively, and the two are statistically different (P < 0.05), and the results show that the tumor cell migration can be inhibited after the expression of PRV1 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 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 42 + -17 and 203 + -39 respectively, and the two groups have statistical difference (P < 0.05); the results show that the tumor cell infiltration can be inhibited after the expression of PRV1 is specifically blocked.

Example 6 reversal of drug resistance to ovarian cancer after specific blockade of PRV1 expression by p L KO.1-PRV1-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-PRV1-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 the cells to the paclitaxel after the p L KO.1-PRV1-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, paclitaxel is added into each group, the concentration gradient is respectively set at 200ug/ml, 100ug/ml, 50ug/ml, 25ug/ml, 12.5ug/ml, 6.25ug/ml, 3.125ug/ml and 0ug/ml, after 24h of action, absorbance (A) is measured at wavelength 370nm (reference wavelength 492nm) by bromine standard method, and Taxus cuspidata is calculatedInhibition rate of alcohol on each group of cells, inhibition rate being A_{Experimental group}/A_{Negative 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 with it after transfection of p L KO.1-PRV1-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(35.89 + -5.27 ug/ml) was significantly higher than the IC of parent A2780 for paclitaxel₅₀(1.26 +/-0.48 ug/ml) and the drug resistance times are as high as 28.48, 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, while the sensitivity of A2780/Taxol to paclitaxel is obviously improved (2.68 +/-0.71 ug/ml) after the A2780/Taxol transfects p L KO.1-PRV1-sh1, and the reversion effect of the transfected p L KO.1-PRV1-sh1 to the drug resistance of A2780/Taxol is very obvious, and the reversion index is 12.97.

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-PRV1-sh1

SEQUENCE LISTING

<110> Zhejiang university

<120> siRNA for specifically inhibiting PRV1 gene expression, recombinant vector and application thereof

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<160>8

<170>PatentIn version 3.3

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Claims (4)

1. The application of the siRNA for specifically inhibiting PRV1 gene expression in the preparation of the medicine 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.

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 PRV1 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, 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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