CN114292844B - shRNA interfering with U2AF2 gene and application thereof in preparation of anti-triple negative breast cancer drugs - Google Patents
shRNA interfering with U2AF2 gene and application thereof in preparation of anti-triple negative breast cancer drugs Download PDFInfo
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Abstract
The invention discloses shRNA interfering with a U2AF2 gene and application thereof in preparation of anti-triple negative breast cancer drugs, and belongs to the technical field of biological medicines. The invention determines the high expression level of U2AF2 in triple negative breast cancer tissues through experiments, provides an action target for targeted treatment of breast cancer, efficiently inhibits the expression of U2AF2 in drug-resistant cells of human triple negative breast cancer cells and obviously enhances the sensitivity of the cells to chemotherapeutic drugs taxol by designing siRNA sequences for targeted inhibition of U2AF2 genes and corresponding siRNA expression plasmids and shRNA lentiviruses. The invention has great significance for preparing the drug for inhibiting the drug resistance of the triple negative breast cancer cells.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to shRNA interfering with a U2AF2 gene and application thereof in preparation of anti-triple negative breast cancer medicines.
Background
Recent cancer data in 2020 shows that breast cancer has become the first cancer worldwide and is the first leading cause of cancer death in women. Although advances have been made in the recent years of integrated breast cancer treatment, metastasis and recurrence remain major obstacles to increasing patient survival, particularly in Triple-negative breast cancer (TNBC) with a poor prognosis. TNBC accounts for about 15% -20% of breast cancers, and due to lack of ER, PR and HER-2 receptors, endocrine therapy and targeted therapy cannot be performed, and chemotherapy is still the most important treatment means. However, it is troublesome that only 20% of TNBC patients are sensitive to standard chemotherapy regimens, and a significant proportion of patients are very susceptible to chemotherapy resistance. Therefore, targeting key genes for TNBC chemotherapy resistance is an important measure for improving patient survival.
Variable shear regulatory factor U2AF2 (also known as U2AF 65), having a molecular weight of 65KD, regulates U2 snRNP binding to pre-mRNA during selective shear. The mis-regulation of alternative splicing is closely related to the occurrence and development of tumors, such as high expression of U2AF2, and is positively related to the poor prognosis of lung cancer and liver cancer. However, the expression and function of U2AF2 in breast cancer, especially in triple negative breast cancer, has not been reported.
The method for inhibiting the U2AF2 gene reported in the current research only has gene knockout, but the method has long research period, high requirements on technical conditions and difficult application to clinical treatment, so the method has obvious limitations. Ribonucleic acid interference (RNAi) is a gene silencing technology widely used in recent years, and through a specific small interfering ribonucleic acid (short interfering RNA, siRNA) composed of 21 bases, the expression of specific genes in vivo can be blocked simply, efficiently and specifically, so that cells show the deletion of corresponding gene phenotypes, and the RNA interference technology has been widely used in aspects of gene function research, gene therapy of tumors, research and development of new drugs and the like. However, since the half-life of the siRNA fragment is too short, the gene inhibition exerted by the in vitro synthesized siRNA fragment is generally short after it is transferred into the cell. Therefore, in clinical studies, a strategy of constructing a vector capable of expressing an siRNA fragment in vitro in advance and transferring the vector into cells to express the siRNA fragment is generally adopted. Vectors commonly used today include retroviruses, adenoviruses and lentiviruses. Compared with other expression vectors, the lentiviral expression vector has the advantages of low immunogenicity, high infection efficiency, long infection time, stable infection effect, capability of infecting dividing phase cells and non-dividing phase cells, and the like. At present, lentiviral vectors have been developed to clinical experimental stages and become a powerful means for application technology development in the field of tumor treatment.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide shRNA for interfering U2AF2 gene, which can effectively interfere the expression of U2AF2 gene in cells. The invention aims to provide an application of shRNA interfering with the U2AF2 gene in preparing a medicine for resisting triple negative breast cancer.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
an shRNA interfering with a U2AF2 gene, comprising a target sequence as follows:
further, the shRNA interfering with the U2AF2 gene has the following nucleic acid sequence:
sense strand:
antisense strand:
a vector containing the shRNA interfering with the U2AF2 gene.
Further, the vector is GV298.
A recombinant lentivirus comprising a nucleic acid sequence encoding a shRNA of the interfering U2AF2 gene.
The shRNA interfering the U2AF2 gene or the vector containing the shRNA interfering the U2AF2 gene or the recombinant lentivirus is applied to the preparation of anti-triple negative breast cancer drugs.
Further, the anti-triple negative breast cancer drug is a drug for improving the sensitivity of triple negative breast cancer cells to chemotherapeutic drugs.
Further, the anti-triple negative breast cancer drug is a drug for inhibiting invasion capacity of triple negative breast cancer cells.
Further, the anti-triple negative breast cancer drug is a drug for inhibiting the migration capacity of triple negative breast cancer cells.
Compared with the prior art, the invention has the beneficial effects that:
the invention detects the expression of U2AF2 in human breast cancer tissues through immunohistochemical staining, and proves that the U2AF2 is highly expressed in the breast cancer tissues and is closely related to chemotherapy drugs. The invention further designs the siRNA sequence of the human U2AF2 gene, constructs corresponding siRNA plasmid and shRNA slow virus, and after the slow virus of the shRNA interference of the U2AF2 is used for transfecting human triple negative breast cancer drug-resistant cells, experiments prove that the sensitivity of the cells to taxol is obviously improved, which proves that the U2AF2 is a key gene of triple negative breast cancer drug resistance, and the U2AF2 shRNA slow virus can obviously inhibit the chemotherapy drug resistance, thereby having great potential as a drug for treating triple negative breast cancer.
Drawings
FIG. 1 is a graph showing the results of immunohistochemical detection of U2AF2 expression in human triple negative breast cancer tissue;
FIG. 2 is a graph showing the results of Wcsternblot detection of U2AF2 expression in human triple negative breast cancer cells;
FIG. 3 is a graph showing the expression of U2AF2 in normal breast epithelial cells, triple negative breast cancer cells, and triple negative breast cancer drug resistant cells;
FIG. 4 is a graph of GV289 plasmid DNA;
FIG. 5 is a block diagram of helper1.0 lentiviral packaging helper plasmids;
FIG. 6 is a block diagram of helper2.0 lentiviral packaging helper plasmids;
FIG. 7 is a diagram of the bright field and fluorescent field of a null control lentivirus (sh-U2 AF2 NC) and U2AF2 interfering lentivirus (sh-U2 AF 2) infected human triple negative breast cancer resistant cell line (MDA-MB-231/PTX) following lentivirus infection of cells;
FIG. 8 is a graph showing the efficiency of Westembot detection of sh-U2AF2 interference by infection of MDA-MB-231/PTX cells with sh-U2AF2 lentivirus;
FIG. 9 is a graph showing the results of changes in IC50 of cells against paclitaxel after infection of MDA-MB-231/PTX cells with sh-U2AF2 lentivirus;
FIG. 10 is a graph showing the results of a significant decrease in the invasive capacity of MDA-MB-231/PTX cells following U2AF2 interference;
FIG. 11 is a graph showing the significant decrease in MDA-MB-231/PTX cell migration ability following U2AF2 interference.
Detailed Description
The invention is further described below in connection with specific embodiments.
Example 1:
1. immunohistochemical detection of expression of U2AF2 in triple negative breast cancer and paracancerous tissues
1) Paraffin specimens pathologically diagnosed as triple negative breast cancer and corresponding paracancerous tissue specimens were routinely sectioned.
2) Tissue paraffin sections dewaxed to water: paraffin sections were placed in an oven at 60 ℃ for 1 hour, xylene I, II, 10 minutes each, gradient alcohol: 100% alcohol 2 minutes, 95% alcohol 2 minutes, 80% alcohol 2 minutes, 70% alcohol 2 minutes, distilled water washing: 5 min x 2 times.
3) Hydrogen peroxide blocks endogenous peroxidases: 3%H 2 O 2 Room temperature for 10 minutes in the dark.
4) Washing with distilled water: 5 min x 2 times.
5) Antigen retrieval: sodium citrate buffer (10 mM, PH6.0) submerged the sections, covered with a lid, boiled in an autoclave, steamed for 3 min, cooled slowly and then placed in PBS 5 min X3 times.
6) Serum blocking: the sections were removed, the back side of the sections and the surrounding tissue on the front side of the sections were wiped off, but the tissue was kept moist, 100. Mu.L of goat serum was added dropwise to each section, and incubated at 37℃for 15 minutes.
7) Dropwise adding the U2AF2 antibody: serum was removed by blotting with filter paper, without washing, rabbit anti-human antibody U2AF2 (1:1000) was added directly dropwise, and incubated overnight at 4 ℃.
8) PBS:5 min X3 times (on shaker).
9) HRP-labeled secondary antibody was added dropwise at 37 ℃ for 40 min.
10 PBS): 5 min X3 times (on shaker).
11 DAB color development, observation under a mirror, and timely termination (running water flushing termination).
12 Hematoxylin counterstain, room temperature, 30 seconds, tap water rinse.
13 Tap water rinse back to blue for 15 minutes.
14 Gradient alcohol dehydration): 80%, 95% for 2 minutes, 100% for 2 minutes, 2 times, 5 minutes.
15 Xylene clear: xylene I, II for 5 minutes each.
16 Sealing plate): and (5) sealing the neutral resin.
FIG. 1 shows the results of immunohistochemical detection of U2AF2 expression in human triple negative breast cancer tissue, and the results show that U2AF2 is expressed in both cytoplasm and nucleus and the expression in human triple negative breast cancer tissue is obviously higher than that in normal breast tissue.
2. Western blot detects the expression of the U2AF2 protein in triple negative breast cancer tissues and corresponding paracancerous tissues.
1) Protein extraction: 1mL of RIPA lysate is added to each 100mg of tissue specimen, the mixture is placed on a homogenizer for homogenization, the mixture is placed on ice for 10 minutes, and the mixture is centrifuged at 12000rpm and 4 ℃ for 10 minutes in a centrifuge, and the supernatant is sucked and placed in a precooled 1.5mL centrifuge tube.
2) Concentration measurement: mu.L of the protein was aspirated, and the protein concentration was measured by BCA method.
3) Boiling protein: the protein was mixed with 5×loading in proportion and boiled on a protein heater at 95 ℃ for 5 minutes.
4) 10% SDS-Page gel was prepared, 50. Mu.g of sample was applied per well, electrophoresis was performed at 80V for 120 min, and PVDF membrane was transferred at 260mA for 90 min.
5) After the transfer, the PVDF membrane was subjected to 5% skim milk blocking for 2 hours.
6) The TBST solution was washed 5 min X2 times.
7) Incubation resistance: rabbit anti-human U2AF2 antibody was diluted 1:1000 and incubated overnight at 4 ℃.
8) The TBST solution was washed 5 min. Times.2 times, 10 min. Times.2 times.
9) Secondary antibody incubation: goat anti-rabbit secondary antibody was diluted 1:10000 and incubated for 2 hours at room temperature.
10 TBST solution was washed 5 min×2 times, 10 min×2 times.
11 ECL development and strip analysis.
FIG. 2 is a graph showing the Westembot assay of U2AF2 expression in 2 pairs of human triple negative breast cancer tissues and corresponding paracancerous tissues, suggesting that U2AF2 expression in cancerous tissues is significantly higher than paracancerous tissues. N represents a paracancerous tissue, T represents a cancerous tissue, GD represents GAPDH, and serves as an internal reference.
3. Westernblot detection of U2AF2 expression in human triple negative breast cancer drug resistant cells and IC50 determination of sensitivity of cells to Paclitaxel (PTX)
The result of the Westernblot detection method is shown in the left graph of FIG. 3, which shows that U2AF2 is expressed in normal breast cells MCF10A, triple negative breast cancer cells MDA-MB-231 and triple negative breast cancer drug resistant cells MDA-MB-231/PTX, but the expression level is highest in the drug resistant cells.
Diluting cells in logarithmic growth phase to give a concentration of 4×10 4 cell suspensions of cell/mL were inoculated in 96-well plates, 100. Mu.L of each well was placed in an incubator for 24h, and then the corresponding drug was added thereto, and 3 multiplex wells were set for each concentration. After 72 hours, 20. Mu.LCCK 8 assay reagent was added to each well and the culture was continued in the medium. After 3 hours the microplate reader detects the cell proliferation data and analyses it. As a result, the IC50 of the drug-resistant cell MDA-MB-231/PTX was 5 times that of the parent cell MDA-MB-231 as shown in the right panel of FIG. 3.
4. Designing the siRNA sequence of U2AF2 gene and constructing U2AF2-siRNA and negative control plasmid
Firstly, the sequence (NM_ 007279) of the U2AF2 gene is called from Genebank, then 3 siRNA sequences aiming at the U2AF2 gene are designed by using Genechem which is design software of Shanghai Ji Kai gene chemical technology company, and the siRNA sequence with the best interference effect is selected from the siRNA sequences: 5'-cgCCTTCTGTGAGTACGTGGA-3'. Subsequently, a double-stranded DNA oligo comprising the siRNA sequence of the U2AF2 gene was designed, and the sequence and structure thereof are shown in Table 1.
TABLE 1 double stranded DNA oligo comprising the siRNA sequence of the U2AF2 gene
And (3) synthesizing the double-stranded DNA oligo, dissolving the DNA dry powder obtained by synthesis in an annealing buffer solution, carrying out water bath at 90 ℃ for 15 minutes, and naturally cooling to room temperature to form the DNA double strand containing the interference target sequence. The GV298 vector (vector plasmid DNA map, see FIG. 4) was linearized with Age I and BamH I restriction enzymes, and the cleavage reaction system, see Table 2, was identified by agarose gel electrophoresis.
TABLE 2 cleavage reaction System
| Reagent(s) | Volume (mu L) |
| ddH 2 O | 41 |
| 10×CutSmart Buffer | 5 |
| Purified plasmid DNA (1. Mu.g/. Mu.L) | 2 |
| Age I(10U/μL) | 1 |
| BamH I(10U/μL) | 1 |
| Total | 50 |
The linearized GV298 plasmid and the purified double-stranded DNAoligo were ligated by T4 DNA ligase, the ligation reaction system was ligated overnight at 16℃as shown in Table 3, and the ligation product was recovered. Fresh competent E.coli cells prepared by transforming the ligation product with calcium chloride. Dipping the surface of the growing strain of the ligation transformation product, dissolving in 10 mu L of LB culture medium, uniformly mixing, taking 1 mu L of the mixture as a template, and identifying by adopting a PCR method, wherein the primers for PCR identification are as follows: upstream, 5'-CCATGATTCCTTCATATTTGC-3'; downstream, 5'-GTAATACGGTTATCCACGCG-3';
TABLE 3 ligation reaction System
And then sequencing and comparing clones positive to the PCR identification, wherein the correctly-compared clones are successfully constructed vectors containing the U2AF2 interference fragments and are named as sh-U2AF2.
When the siRNA negative control target sequence is 5'-TTCTCCGAACGTGTCACGT-3' and the siRNA control plasmid is constructed, a double-chain DNAoligo sequence (table 4) is synthesized aiming at the siRNA negative control target sequence, and the rest construction methods, the identification methods and the conditions are the same as those of sh-U2AF2, so that the successful negative control is named as sh-U2AF2 NC.
TABLE 4 siRNA negative control double-stranded DNAoligo sequence
5. Construction of sh-U2AF2 lentiviruses and sh-U2AF2 NC control lentiviruses
The bacterial liquid with correct sequence is transferred into 10mL LB liquid medium containing antibiotics, cultured overnight at 37 ℃, and plasmid extraction is carried out by using a small-extraction medium-amount kit without endotoxin. Plasmid stock solutions of 100 ng/. Mu.L were prepared, respectively. The pHelper 1.0 (FIG. 5) and pHelper2.0 (FIG. 6) lentiviral packaging helper plasmids were extracted in the same way. 24 hours prior to transfection, human embryonic kidney cells 293T cells in logarithmic growth phase were digested with trypsin and conditioned with DMEM medium containing 10% FBSCell density of 5X 10 6 Cell/15 mL, re-seeded in 10cm cell culture dish at 37deg.C, 5% CO 2 Culturing in an incubator, and using the cell to transfect after 24 hours and the cell density reaches 70% -80%. Cell culture medium was replaced with PBS-free medium 2 hours prior to transfection. The prepared sh-U2AF2 plasmid or negative control plasmid and each packaged plasmid solution (20. Mu.g of sh-U2AF2 plasmid or negative control plasmid, 15. Mu.g of Helper1.0 plasmid, 10. Mu.g of Helper2.0 plasmid) were added to a sterile centrifuge tube, mixed uniformly with the corresponding volumes of transfection reagent, adjusted to a total volume of 1mL, and incubated at room temperature for 15 minutes. Slowly dripping the mixture into 293T cell culture solution (the addition process is uniform and the cells are prevented from blowing up), mixing, and adding 5% CO at 37deg.C 2 After 6 hours of incubation in the cell incubator, the medium containing the transfection mixture was discarded, and 10mL of PBS was added for washing once, and the dish was gently rocked to wash the residual transfection mixture and discarded. Cell culture medium containing 10% FBS 20mL was slowly added at 37deg.C with 5% CO 2 Culturing in the incubator is continued for 48-72 hours, and then 293T cell supernatant is collected. Cell debris was removed by centrifugation at 4000g for 10 min at 4 ℃. The supernatant was filtered through a 0.45 μm filter in a 40mL ultracentrifuge tube and centrifuged at 25000rpm at 4℃for 2 hours. After centrifugation, the supernatant is discarded, the liquid remained on the tube wall is removed as much as possible, and virus preservation liquid is added, and the resuspension is gently and repeatedly performed (the virus is prevented from being exposed to room temperature for a long time as much as possible, and the virus loss is reduced). After full dissolution, the supernatant is taken and split-packed as required after high-speed centrifugation at 10000rpm for 5 minutes. Lentiviruses comprising sh-U2AF2 plasmid and sh-U2AF2 NC negative control plasmid were designated sh-U2AF2 lentivirus and sh-U2AF2 NC control virus, respectively.
6. Infection efficiency and inhibition efficiency of sh-U2AF2 lentivirus infected human triple negative breast cancer drug-resistant cell line
The MDA-MB-231/PTX human triple negative breast cancer drug-resistant cell line in the logarithmic growth phase is digested by pancreatin, the cell suspension is prepared by a culture medium, and then the cell suspension is inoculated into a 12-hole plate, and the cell fusion degree reaches about 30 percent. According to MOI (complex infection) =50, add 1×10 8 1.25 mu L of TU/mL sh-U2AF2 lentivirus, and after 12 hours of culture, the medium was changed to give a senseInfection efficiency was assessed by fluorescent photographing after 72h of staining, and interference efficiency of U2AF2 was detected by Westernblot after 3 passages of cells collected. The experimental results are shown in figure 7, and the efficiency of the sh-U2AF2 and sh-U2AF2 NC infected cells reaches over 60% under the condition of cell red fluorescent tracer virus infection. As shown in FIG. 8, the protein level suggested that the expression level of U2AF2 in the sh-U2AF2 group was about 50% of that in the control group, and that the inhibition effect of sh-U2AF2 in MDA-MB-231/PTX cells was ideal.
7. Effect of sh-U2AF2 on chemotherapy sensitivity of triple negative breast cancer resistant cells
The results of FIG. 9, which shows that the sensitivity of MDA-MB-231/PTX cells to paclitaxel is significantly improved and the cell viability is reduced by about 30% after U2AF2 interference, by the detection of the IC50 of MDA-MB-231/PTX cells and the parental cells, as described above.
8. Effect of sh-U2AF2 on the ability of triple negative breast cancer drug resistant cells to invade
Paving matrix glue: matrigel gel was diluted with serum-free L15 cell culture medium at 4℃in a ratio of 1:8, 100. Mu.L was uniformly smeared on the surface of the polycarbonate membrane in the upper chamber, and allowed to stand at 37℃for 0.5 hours to polymerize into a gel. Taking the cells to be tested in logarithmic growth phase, washing with PBS, suspending the cells with serum-free L15 medium, and adjusting the cell density to 1×10 5 /mL. 650. Mu.L of 5% FBS-containing L15 medium was added to the 24-well plate lower chamber, then the Transwell chamber was placed in the 24-well plate with forceps, 200. Mu.L of the cell suspension was added to the upper chamber, and finally the mixture was placed in an incubator for culturing for 48 hours. Cell fixation: the chamber was removed, the medium was aspirated, and the Matrigel and cells in the upper chamber were gently rubbed with a cotton swab. A new 24-well plate was added with 600. Mu.L of 4% paraformaldehyde, and the cell was fixed for 30 minutes after being placed. The fixative was discarded, stained with 0.1% crystal violet for 5 minutes, washed 3 times with PBS to remove crystal violet not bound to cells, and the upper side of the chamber was gently rubbed with a cotton swab to wipe off dye not specifically bound to the upper surface of the chamber. After air-drying, cells were randomly selected under a high power microscope for 5 fields of view and counted. The results showed (FIG. 10) that sh-U2AF2 cells had significantly lower invading cell numbers than the control, the difference was statistically significant (P < 0.001), suggesting that U2AF2 interference was evidentInhibiting the invasive capacity of triple negative breast cancer cells.
9. Effect of sh-U2AF2 on migration ability of triple negative breast cancer drug-resistant cells
First, a marker pen was used to traverse the via hole at about every 0.5cm, followed by a straight line to evenly scribe the cross line, at the back of the 6-well plate. At 5X 10 5 cell/well plating was performed after 24 hours with a gun head to scratch the cell layer along a line drawn on the back of the plate on the first day. After the completion of the scratch, the cells were washed 3 times with sterile PBS and photographed, then the L15 medium was changed, and the cells were placed in an air incubator at 37℃for culturing. After 24 hours, microscopic line observations were photographed and the width of the scratches was measured. The results are shown in fig. 11, which shows that the migration distance of sh-U2AF2 cells is significantly smaller than that of the control group, and that the two groups have statistical differences, indicating that U2AF2 interference significantly inhibits the migration ability of triple negative breast cancer drug-resistant cells.
Sequence listing
<110> Nantong tumor Hospital
<120> shRNA interfering U2AF2 gene and application thereof in preparation of anti-triple negative breast cancer drugs
<130> 100
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Homo sapiens
<400> 1
cgccttctgt gagtacgtgg a 21
<210> 2
<211> 58
<212> DNA
<213> sh-U2AF2-F(Artificial)
<400> 2
ccggcgcctt ctgtgagtac gtggactcga gtccacgtac tcacagaagg cgtttttg 58
<210> 3
<211> 58
<212> DNA
<213> sh-U2AF2-R(Artificial)
<400> 3
gatccaaaaa cgccttctgt gagtacgtgg actcgagtcc acgtactcac agaaggcg 58
Claims (4)
1. Application of shRNA interfering U2AF2 gene or vector containing shRNA interfering U2AF2 gene or recombinant lentivirus in preparation of anti-triple negative breast cancer drugs;
the shRNA interfering with the U2AF2 gene contains a target sequence 5'-CGCCTTCTGTGAGTACGTGGA-3';
the shRNA interfering the U2AF2 gene has the nucleotide sequence as follows:
sense strand:
5’-CcggcgCCTTCTGTGAGTACGTGGACTCGAGTCCACGTACTCACAGAAGGCGTTTTTg-3’，
antisense strand:
5’-GATCCAAAAAcgCCTTCTGTGAGTACGTGGACTCGAGTCCACGTACTCACAGAAGGCG-3’；
the vector containing shRNA interfering with the U2AF2 gene is GV298;
the recombinant lentivirus contains a nucleic acid sequence encoding a shRNA that interferes with the U2AF2 gene.
2. The use according to claim 1, wherein the anti-triple negative breast cancer drug is a drug that increases sensitivity of triple negative breast cancer cells to chemotherapeutic agents.
3. The use according to claim 2, wherein said anti-triple negative breast cancer drug is a drug that inhibits the invasive capacity of triple negative breast cancer cells.
4. The use according to claim 2, wherein said anti-triple negative breast cancer drug is a drug which inhibits the migration ability of triple negative breast cancer cells.
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