A viral vector targeting KIF4A to inhibit angiogenesis, its preparation method and application

Abstract

This invention discloses a viral vector targeting KIF4A to inhibit angiogenesis, its preparation method, and its applications. The viral vector contains three shRNA gene fragments targeting the KIF4 protein-coding region, as shown in SEQ ID NO.1-SEQ ID NO.3. Using a lentiviral packaging system, a stable KIF4A knockdown cell line can be constructed, effectively inhibiting endothelial cell proliferation and angiogenesis, and also inhibiting the paracrine promotion of angiogenesis by glioma cells, providing a new strategy for selecting targets for anti-angiogenic tumor therapy.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a viral vector that targets KIF4A to inhibit angiogenesis, its preparation method, and its application. Background Technology

[0002] Glioblastoma (GBM) is a common primary malignant brain tumor in adults, characterized by its aggressive nature, poor prognosis, and high recurrence rate; most patients survive less than 15 months. Complete surgical removal of the tumor is often impossible without impairing brain function, and the primary tumor is prone to metastasis, ultimately leading to recurrence and patient death. Surgery combined with radiotherapy or chemotherapy is the standard clinical treatment for gliomas. Currently, FDA-approved drugs for GBM treatment include temozolomide (TMZ), carmustine, lomustine, and bevacizumab. Temozolomide is a first-line treatment for gliomas, but it offers limited help in improving progression-free survival and overall survival in patients with recurrent gliomas, and long-term use can lead to drug resistance. Bevacizumab, as the first approved anti-angiogenic drug, has shown significant clinical benefits in the treatment of various cancers. Although anti-angiogenic therapy has been successful in the treatment of some cancers, its efficacy is often limited to specific cancer types and faces challenges in its widespread clinical application. These challenges include drug resistance, treatment side effects, and insufficient understanding of tumor vascular heterogeneity. Future research needs to further explore how to optimize these therapies and how to combine them with other treatment strategies (such as immunotherapy) to improve efficacy and expand their application in different types of cancer. Targeted therapy for gliomas and the elucidation of drug resistance mechanisms are important issues and research hotspots in biomedicine that urgently need to be addressed.

[0003] Recent research has revealed that the KIF4A gene encodes a molecular motor protein that primarily participates in chromosome movement and separation during cell division, influencing the normal cell cycle. Studies have shown that KIF4A is highly expressed in various tumor tissues and promotes malignant biological behaviors such as tumor cell proliferation and migration. In gliomas, KIF4A promotes malignant tumor cell proliferation by regulating HIF1 / VEGFA. Previous studies have shown that KIF4A regulates vascular endothelial cell proliferation, migration, and angiogenesis. KIF4A knockdown inhibits vascular endothelial cell proliferation, migration, and angiogenesis. Therefore, we employed genetic engineering techniques to prepare a highly efficient lentiviral shRNA vector targeting the KIF4A gene; and used a lentiviral packaging system to prepare a high-purity, high-titer viral solution targeting KIF4A. This viral preparation can be used to infect tumor cells that highly express KIF4A, inhibiting glioma angiogenesis and hindering the malignant development of glioma cells by targeting KIF4A. This method provides a novel viral preparation that can replace bevacizumab in inhibiting angiogenesis, offering a new option for clinical treatment. Because the lentiviral vector has been engineered, the prepared virus does not contain self-replicating structural units, but only has infective properties, and has high safety. Summary of the Invention

[0004] The purpose of this invention is to provide a viral vector that targets KIF4A to inhibit angiogenesis, as well as its preparation method and application.

[0005] A viral vector that targets KIF4A to inhibit angiogenesis, the viral vector containing three shRNA gene fragments targeting the KIF4 protein coding region.

[0006] The three shRNA gene fragment sequences targeting the KIF4 protein coding region are shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

[0007] The sequence of an artificially fused gene fragment containing three shRNA gene fragments targeting the KIF4 protein coding region is shown in SEQ ID NO.4.

[0008] The method for preparing the viral vector that targets KIF4A to inhibit angiogenesis is carried out according to the following steps: (1) The artificial fusion gene fragment shown in SEQ ID NO.4 was cloned into the lentiviral backbone plasmid pLKO.1-GFP to obtain the KIF4A knockdown recombinant lentiviral plasmid pLKO.1-KIF4AshRNA; (2) pLKO.1-KIF4AshRNA, packaging plasmid pSPAX2 and pMD2G were co-transfected into 293T cells to prepare pLKO.1-KIF4AshRNA lentivirus; (3) The filtered virus supernatant was concentrated and purified by ultracentrifugation; then it was stored in virus preservation solution; after the virus solution was aliquoted, it was used directly or stored at -80℃ for later use. (4) Use the pLKO.1-KIF4AshRNA virus solution obtained in step (3) to directly infect HUVEC or U87 cells, add 2-6 μg / mL puromycin for screening for 5-9 days until a stable KIF4A knockdown cell line is obtained.

[0009] The virus preservation solution in step (3) is a HEPES buffer containing 10-50% glycerol, 0.1-1.0M trehalose and 1-10% DMSO, with a pH of 7.0-8.0.

[0010] The application of the viral vector in the preparation of drugs that inhibit endothelial cell proliferation and angiogenesis.

[0011] The application of the viral vector in the preparation of drugs that inhibit tumor cell angiogenesis.

[0012] The beneficial effects of this invention are as follows: This invention utilizes a lentiviral vector constructed using genetic engineering technology, containing three shRNA sequences targeting different protein-coding regions of KIF4. This allows for the simultaneous synthesis of three shRNA nucleotide sequences targeting different regions, significantly improving the specificity of targeting the KIF4A gene, effectively preventing off-target effects, and achieving highly efficient and specific knockdown of the target gene. The KIF4A-targeting lentiviral shRNA sequences prepared by this invention do not contain self-replicating structural units and lack self-replication ability; they require co-transfection with packaging plasmids into recipient cells before self-assembling into viral particles and gaining the ability to infect host cells. Therefore, the lentiviral solution prepared using this invention has high biosafety. This invention employs a lentiviral packaging system to prepare a viral solution targeting the KIF4 gene. After enrichment and concentration, the viral titer reaches above 10E+10, greatly improving viral infection efficiency. Using the viral preservation solution of this invention effectively avoids the practical problems of decreased viral titer and weakened infectivity due to freeze-thaw cycles, providing a new method for long-term stable preservation of viral solutions. This invention also designs a qRT-PCR method for detecting lentiviral titers. This method quantifies viral expression levels by detecting lentiviral gene sequences. It is also applicable to detecting lentiviral titers targeting other genes, providing a detection method for assessing viral infectivity. The KIF4A-targeting viral solution prepared in this invention has the ability to inhibit endothelial cell proliferation and angiogenesis; simultaneously, it can indirectly inhibit endothelial cell angiogenesis by regulating paracrine activity in glioma cells. Therefore, the KIF4A-targeting viral solution can serve as an alternative drug regimen for inhibiting tumor cell angiogenesis. Attached Figure Description

[0013] Figure 1 A schematic diagram for constructing a lentiviral vector targeting KIF4A.

[0014] Figure 2 The image shows the expression of green fluorescent protein in 293T cells transfected with KIF4A lentiviral plasmid; Figure A shows the growth status of 293T cells under bright field microscopy (4x magnification), and Figure B shows the expression of green fluorescent protein in the viral vector under fluorescence microscopy (4x magnification).

[0015] Figure 3 The amplification curve of KIF4AshRNA lentiviral plasmid was detected using the qRT-PCR method.

[0016] Figure 4 The expression level of KIF4A protein in HUVEC cells was detected using Western blot.

[0017] Figure 5 To investigate the effect of KIF4A gene knockout on HUVEC cell proliferation.

[0018] Figure 6 To reduce the effect of KIF4A gene knockout on HUVEC cell invasion, Figure A shows the cell scratch assay, with images taken at 0 and 24 hours to show cell scratch healing; Figure B shows the data analysis of cell scratch healing.

[0019] Figure 7 To illustrate the effect of KIF4A gene knockout on HUVEC cell migration; Figure A shows the Transwell cell migration experiment, and Figure B shows the data analysis of cell migration.

[0020] Figure 8 To investigate the effect of KIF4A gene knockdown on angiogenesis in HUVEC cells; in the figure, A represents the HUVEC cell tubule formation experiment; B is the data analysis graph of the tubule formation experiment.

[0021] Figure 9 To detect the KIF4A knockdown U87 cell line using Western blot.

[0022] Figure 10 To investigate the regulatory effect of KIF4A knockdown U87 cell conditioned medium on HUVEC cell proliferation.

[0023] Figure 11 To investigate the regulatory effect of KIF4A knockdown U87 cell conditioned medium on angiogenesis in HUVEC cells. Detailed Implementation

[0024] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0025] Example 1: Preparation method of highly efficient and specific viral vector targeting KIF4A to inhibit angiogenesis Construct a KIF4A knockdown lentiviral expression vector, such as Figure 1 As shown, the specific steps are as follows: (1) Synthesized fusion gene SEQ ID NO.4, which targets different protein coding regions of KIF4A (NM_012310) with nucleotide sequences SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. Among them, the 5' end has a SpeI restriction site and the 3' end has an EcoRV restriction site.

[0026] (2) The lentiviral backbone plasmid pLKO.1-GFP was digested with SpeI and EcoRV restriction endonucleases from Takara Bio Inc. The digestion reaction system is as follows: SpeI 1μL EcoRV 1 mL 10XH buffer 1μL 10XM buffer 1μL pLKO.1-GFP 1μg dH2O up to 20 μL Enzyme digestion reaction conditions: 37℃ water bath, reaction for 1 h; after the reaction, the above enzyme digestion products were recovered by 1% agarose gel electrophoresis to prepare linear vectors.

[0027] (3) Fragment ligation: T4 DNA Ligase from Takara Bio was used to ligate the linear vector to the artificially synthesized gene fragment. The mass ratio of the linear vector to the gene fragment was 1 / 4. The ligation reaction system is as follows: T4 DNA Ligase 2 μL 10X T4 DNA Ligase Buffer 1 μL Linear pLKO.1-GFP vector 10 ng 40 ng fusion gene fragment dH2O up to 20 μL Ligation reaction conditions: Place in a 16℃ water bath and react for 30 min. After the reaction is complete, place the ligation product on ice for later use or store directly at -20℃.

[0028] (4) Heat transformation of Escherichia coli: Take 10 μL of ligation reaction solution, add it to 100 μL of competent Escherichia coli cells, mix well, and place on ice for 0.5 h; then, heat shock at 42℃ for 45 s, cool on ice for 2 min; add 1 mL of antibiotic-free LB medium, and incubate at 37℃ on a shaker for 1 h; finally, centrifuge at 12000 rpm for 1 min, retain 200 μL of supernatant, and resuspend the cells. Spread evenly on LB agar containing ampicillin, and incubate overnight at 37℃.

[0029] (5) Plasmid extraction: Take out the LB plate from the incubator and observe the bacterial growth; pick out a single colony and put it into LB liquid medium containing ampicillin, and incubate overnight in a shaker at 37°C.

[0030] The following day, plasmids were extracted using the TaKaRa MiniBEST Plasmid Purification Kit (9760), following the instructions. The extracted plasmids were then sent for gene sequencing identification.

[0031] Example 2: Preparation of highly efficient and specific lentivirus targeting KIF4A knockdown A highly efficient and specific shRNA viral solution targeting KIF4A knockdown was prepared using a lentiviral packaging system; further, the viral solution was enriched and purified. The specific operational steps are as follows: 1. Using a lentiviral packaging system, prepare KIF4A-targeted knockdown shRNA viral fluid. (1) Day 0: 293T cells were pre-seeded into T75cm culture dishes, with 3 x 10 cells per dish. 6 cells.

[0032] (2) Day 1: Preparation of transfection system: Mix lentiviral vector, packaging plasmid pSPAX2 and pMD2G in a mass ratio of 10:9:1; take 0.5 mL of serum-free Opti-MEM®I culture medium into a 1.5 mL centrifuge tube, add 4 μg pLKO.1-shCon or pLKO.1-KIF4AshRNA, 3.6 μg pSPAX2 and 0.4 μg pMD2G plasmid in sequence, and mix gently; then add 16 μL Lipofectamine 2000 transfection reagent, mix well and place at room temperature for 15 min; slowly add the above transfection reagent mixture dropwise to the culture dish pre-seeded with 293T cells, and mix gently; finally, incubate in a 37℃ incubator for 48 h.

[0033] (3) Day 3: Collect the culture supernatant containing the virus solution, filter it using a 0.45 μM filter membrane to obtain pLKO.1-shCon or pLKO.1-KIF4AshRNA virus solution ( Figure 2 ).

[0034] 2. Collect the supernatant of the culture medium containing the virus solution, and enrich and concentrate the virus particles. The specific steps are as follows: (1) Centrifuge the collected virus supernatant at 4°C and 1000 g for 5 min to remove cell debris; (2) Then, filtration was performed using a 0.45μm PES filter membrane; (3) Transfer the filtered virus supernatant to a sterile ultracentrifuge tube and centrifuge at 4°C and 25,000 rpm for 2 hours; (4) Remove the supernatant, add virus preservation solution containing glycerol and protectant to the precipitate, and slowly dissolve at 4°C; (5) Finally, the dissolved virus solution is dispensed into 50 μL portions and used directly or stored in a -80℃ refrigerator for later use.

[0035] Example 3: Preparation method of virus preservation solution To maintain viral stability and reduce the decrease in viral titer and weakened infectivity caused by freeze-thaw cycles, the virus preservation solution prepared in this invention contains the following components: 15% glycerol, 0.1M trehalose, and 2% DMSO in HEPES buffer, adjusted to pH 7.4 ± 0.2; the specific operating method is as follows: (1) Prepare 1 liter of HEPES buffer: Weigh 238.3 g of HEPES (N-hydroxyethylpiperazine-2-ethanesulfonic acid) powder, add it to about 400 mL of distilled water, and stir with a magnetic stirrer until completely dissolved; then, add 0.5 M NaOH solution dropwise to adjust the pH to 7.4 ± 0.2; bring the solution volume to 1 liter and mix evenly; finally, filter the solution through a 0.22 μm PES filter membrane for sterilization, store it in a sterile reagent bottle, and store it at 4 °C.

[0036] (2) Preparation of 1M trehalose solution: Prepare a 500mL sterile beaker, add 150mL HEPES solution, weigh 68.46 g of trehalose (molecular weight 342.30), and dissolve it completely; then, continue to add HEPES solution to make up the volume to 200mL and mix evenly; finally, filter it with a 0.45μm PES filter membrane for sterilization, store it in a sterile reagent bottle, and store it at 4℃.

[0037] (3) Prepare 100mL virus preservation solution: Prepare a 200mL sterile beaker, add 50mL HEPES buffer, 15mL glycerol, 2mL DMSO solution and 10mL 1M trehalose solution in sequence, and mix well; then, continue to add HEPES solution to make up the volume to 200mL, and mix well; finally, filter sterilize with a 0.45μm PES filter membrane, store in a sterile reagent bottle, and store at 4℃.

[0038] Example 4: Determination of Virus Titer Viral RNA was extracted, and the viral titer was determined using qRT-PCR; the specific method is as follows: 1. Viral nucleic acid extraction method. Viral nucleic acid was extracted using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit.

[0039] (1) Virus lysis: Take 100 μl of virus stock solution. If the initial amount is less than 200 μl, add PBS solution or RNase-free dH2O to make up to 200 μl. Add 200 μl of Buffer VGB, 20 μl of Proteinase K and 1.0 μl of Carrier RNA, mix thoroughly and incubate at 56°C for 10 min. Add 200 μl of 98% ethanol to the lysis buffer and mix thoroughly by pipetting.

[0040] (2) Place the Spin Column on the Collection Tube, transfer the solution into the Spin Column, centrifuge at 12,000 rpm for 2 min, and discard the filtrate.

[0041] (3) Add 500 μl of Buffer RWA to the Spin Column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0042] (4) Add 700 μl of Buffer RWB to the Spin Column, centrifuge at 12,000 rpm for 1 min, discard the filtrate, and repeat the operation once.

[0043] (5) Place the Spin Column on the Collection Tube and centrifuge at 12,000 rpm for 2 min.

[0044] (6) Place the Spin Column on a new 1.5 ml RNase-free collection tube, add 40 μl of RNase-free dH2O to the center of the Spin Column membrane, and let it stand at room temperature for 5 min.

[0045] (7) Elute the nucleic acid and centrifuge at 12,000 rpm for 2 min; finally, use Qubit to quantify the nucleic acid.

[0046] 2. DNase I digestion reaction to remove any remaining plasmid DNA; RecombinantDNase I reagent from Takara Bio was used. The reaction system is as follows: RNA Sample 0.1~1μg 10X DNase I Buffer 5.0 μL Recombinant DNase I (5 U / µl) 2.0µL RNase Inhibitor 20 U RNase-Free Water up to 50.0μL Reaction conditions: 37°C for 30 min, 70°C for 5 min, 4°C.

[0047] 3. Template dilution method Using lentiviral plasmids as standards (10E+8 copies / μL), standard curves were prepared by serially diluting them 10-fold using Takara Bio's EASY Dilution (for Real-Time PCR) solution. The viral samples to be tested were also serially diluted in the same manner. Specific procedures are shown in Table 1.

[0048] Table 1. qRT-PCR template dilution methods

[0049] The formula for calculating viral RNA copy number is as follows: Copies / mL = Measured copy number (copies / µL) × Template dilution factor × DNase I digestion dilution factor.

[0050] Converting viral RNA copy number to viral infection titer can predict the infectivity of the viral fluid. The specific method is as follows: To determine the infection titer, 293T cells were infected with supernatant containing a lentivirus carrying KIF4A expression, titrated by qRT-PCR. Cells were harvested 48 h post-infection and analyzed using a BD FACSCalibur flow cytometer. RNA copy number was determined by qRT-PCR titer, and IFU value was determined by FACS to establish the relationship between the two. Based on previous experimental results in our laboratory, the correlation between qRT-PCR-detected RNA copy number (copies / ml) and viral titer (IFU / ml) is: Titration Ratio (copies / IFU) = 90. Based on this, the viral RNA copy number was converted into viral infection titer to predict the infectivity of the viral fluid.

[0051] 4. qRT-PCR method for detecting viral titer. Viral titer was detected using the One Step TB Green® PrimeScript™ RT-PCR Kit (Perfect Real Time) from Takara Bio Inc. The forward PCR primer sequences are shown in SEQ ID NO. 5, and the reverse PCR primer sequences are shown in SEQ ID NO. 6.

[0052] qRT-PCR reaction system: 2X One Step TB Green RT-PCR Buffer III 12.5μL TaKaRa Ex Taq HS (5 U / μl) 12.5μL PrimeScript RT enzyme Mix Ⅱ 0.5μL PCR Forward Primer (10 µM) 0.5µL PCR Reverse Primer (10 µM) 0.5µL Total RNA 2.0 μL RNase-free dH2O up to 25 μL qRT-PCR reaction conditions: RT reaction conditions: 42°C for 5 min; 95°C for 10 sec.

[0053] qPCR reaction conditions: 95°C for 5 seconds; 60°C for 30 seconds; 40 cycles.

[0054] 5. Virus titer test results Three batches of prepared virus solutions were subjected to qRT-PCR detection. The viral RNA copy number and corresponding viral titer results are as follows: Figure 3 As shown in Table 2, the copy number of viral RNA exceeded 10. 10 copies / μL, viral titer reaches 10 9 IFU / ml or higher.

[0055] Table 2. Experimental results of virus titer detection using qRT-PCR method.

[0056] Example 4: Evaluation of the protective effect of virus preservation solution on virus titer 1. Experimental setup: The experiment consisted of a control group and an experimental group. The specific procedures are as follows: (1) Referring to the method of Example 2, a lentivirus packaging system was used to prepare 20 mL of culture medium supernatant containing the virus; the supernatant was divided into two equal parts, each 10 mL; referring to the method of Example 3, the virus solution was enriched and concentrated respectively.

[0057] (2) In the control group, the virus solution was enriched and concentrated, dissolved in PBS solution, aliquoted into 50 μL portions, and stored in a -80℃ freezer; in the experimental group, the virus solution was enriched and concentrated, dissolved in virus preservation solution, aliquoted into 50 μL portions, and stored in a -80℃ freezer.

[0058] 2. Following the method described in Example 3, qRT-PCR was used to detect changes in viral RNA copy number after different storage times. Experimental results showed that the viral copy number decreased with prolonged storage time. Compared to the control PBS solution, the viral preservation solution demonstrated better preservation performance and a longer storage time.

[0059] Table 3. Changes in viral copy number at different storage times detected by qRT-PCR.

[0060] Example 5: Construction of a stable HUVEC cell line expressing KIF4A knockdown, and evaluation of the regulatory effect of KIF4A knockdown on endothelial cell function. 1. A stable HUVEC cell line expressing KIF4A knockdown was constructed using lentiviral transfection. The specific procedures are as follows: (1) Day 0: HUVEC cells were seeded in 6-well plates at a density of 2 x 10⁻⁶ cells per well. 5 cells.

[0061] (2) Day 1: Take out the pre-allotted virus solution (KIF4AshRNA and shCon virus solution are handled in the same way) from the -80℃ freezer, 50μL / tube, and place it on ice to thaw slowly; then, add 200μL of complete endothelial cell culture medium and mix gently; slowly add dropwise to a 6-well plate pre-inoculated with HUVEC cells, and co-culture for 8h; then, remove the virus solution, replace with fresh culture medium, and continue culturing in a 37℃, 5% CO2 incubator for 2 days. The control virus solution is handled in the same way.

[0062] (3) Screening stable cell lines: Take out the cell line from the incubator, add 4 μg / mL puromycin to screen positive cell lines, continue for one week until positive clones are screened out, at which point green fluorescent protein expression can be observed under a microscope.

[0063] (4) Detection of KIF4A gene knockdown effect: Cell proteins were extracted using RIPA lysis buffer, and BCA protein was quantified. KIF4A protein expression was detected by Western blot.

[0064] Experimental results are as follows Figure 4 As shown, the expression level of KIF4A protein was significantly decreased compared with the control cells, indicating that the stable HUVEC cell line with KIF4A knockdown was successfully constructed.

[0065] 2. The CCK-8 assay was used to detect the regulatory effect of KIF4A gene knockdown on HUVEC cell proliferation.

[0066] Experimental Methods: Cells in the logarithmic growth phase were collected, a cell suspension was prepared, and the cells were counted. Approximately 2000 cells per well were seeded into 96-well plates and incubated at 37°C with 5% CO2 for 1, 2, 3, 4, or 5 days to ensure cell adhesion. At the end of the incubation period, 10 μL of CCK-8 solution (1 / 10 of the total culture medium volume) was added to each well, and the plates were incubated at 37°C with 5% CO2 for another 1.5 hours. After incubation, the plates were removed, and the absorbance was measured at 450 nm using a microplate reader. The data were recorded and statistically analyzed. P < 0.05 was marked with "*", P < 0.01 with "**", and P < 0.001 with "***".

[0067] Experimental results are as follows Figure 5 As shown, compared with the control group, the viability of HUVEC cells was significantly reduced after KIF4A gene knockdown (P < 0.01).

[0068] 3. The Transwell assay was used to detect the regulatory effect of KIF4A gene knockdown on HUVEC cell invasion.

[0069] Experimental Methods: Transwell chambers pre-coated with matrix gel were placed in culture plates. 250 μL of serum-free culture medium was added to the upper chamber, and the plates were pre-incubated at 37°C with 5% CO2 for 30 minutes to reduce air bubble interference. Control cells and HUVEC cells expressing KIF4AshRNA were removed from the incubator. After trypsin digestion, the cells were resuspended and counted, and the cell concentration was adjusted to 1 × 10⁻⁶. 5 Cells / mL. Remove the liquid culture medium from the Transwell chamber, add 250 μl of cell suspension to the chamber, and add 300 μL of complete culture medium containing serum to the lower chamber. Incubate at 37°C, 5% CO2 for 16 h. After culture, remove the cells. Gently rinse the upper chamber of the Transwell with PBS to remove residual cells, repeating this process three times. Fix the cells with 4% paraformaldehyde for 30 min, then stain with 0.1% crystal violet for 30 min, and finally wash with deionized water. After the Transwell chamber is air-dried, observe and photograph it under a microscope. Count the number of cells stained with crystal violet and perform statistical analysis.

[0070] Experimental results are as follows Figure 6 As shown, compared with the control group, the invasive ability of HUVEC cells was significantly reduced after KIF4A gene knockdown (P < 0.05).

[0071] 4. The scratch assay was used to detect the regulatory effect of KIF4A gene knockdown on HUVEC cell migration.

[0072] Experimental method: HUVEC cells were seeded in 6-well plates, approximately 8 × 10⁸ cells per well. 5 Cells were collected, and the cell confluence reached over 90% the following day. Horizontal scratches were made on the cell surface using a 10 μL sterile pipette tip, ensuring uniform scratches. 1 mL of PBS solution was added to wash the cells, removing any detached cells; this process was repeated three times. Serum-free medium was then used. Cells were incubated at 37°C with 5% CO2. Scratch healing was recorded using microscopy at the beginning of cell culture (0 h) and at 24 h. ImageJ software was used to measure and statistically analyze the cell scratch healing process.

[0073] Experimental results are as follows Figure 7 As shown, compared with the control group, the migration ability of HUVEC cells was significantly reduced after KIF4A gene knockdown (P < 0.01).

[0074] 5. The tube formation assay was used to detect the regulatory effect of KIF4A gene knockdown on angiogenesis in HUVEC cells.

[0075] Experimental Methods: Beyotime's Matrix-Gel™ matrix gel was used for in vitro angiogenesis experiments. The Matrix-Gel matrix gel was thawed overnight at 4°C and mixed thoroughly with a pre-cooled pipette tip, avoiding air bubbles. The Matrix-Gel™ matrix gel and ECM culture medium were diluted at a ratio of 2:1 (66.7%). 50 μl of the diluted matrix gel was evenly spread at the bottom of a 96-well plate and incubated at 37°C for 30 min to 1 h to allow the matrix gel to solidify. When HUVEC cells reached 85% confluence, they were digested, counted, and resuspended in medium containing an appropriate concentration of FBS, adjusting the cell density to 1 × 10⁶ cells / well. 4 Cells / wells. Add 100 μL of cell suspension to each well of a 96-well plate containing matrix gel, avoiding air bubbles. Return the 96-well plate to a 37°C cell culture incubator for further culture. After 6 hours of culture, observe tube formation under a microscope and photograph the results. Measure and perform statistical analysis using ImageJ software.

[0076] Experimental results are as follows Figure 8 As shown, compared with the control group, the tube formation ability of HUVEC cells was significantly reduced after KIF4A gene knockdown (P < 0.01).

[0077] Example 6: KIF4A knockdown inhibits glioma cells from promoting endothelial cell proliferation and angiogenesis through paracrine effects. 1. A stable U87 cell line expressing KIF4A knockdown was constructed using lentiviral transfection.

[0078] (1) Day 0: U87 cells were seeded in 6-well plates at a seeding density of 2 x 10⁶ cells per well. 5 cells.

[0079] (2) Day 1: Take out a 50 μL virus solution from the -80℃ freezer and place it on ice to thaw slowly; then, add 200 μL of DMEM complete medium and mix gently; slowly add it dropwise to a 6-well plate pre-inoculated with U87 cells and culture for 8 hours; then, remove the virus solution, replace with fresh medium, and place in a 37℃, 5% CO2 incubator to continue culturing for 2 days.

[0080] (3) Screening stable cell lines: Take out the cell line from the incubator, add 4 μg / mL puromycin to screen positive cell lines, continue for one week until positive clones are screened out, at which point green fluorescent protein expression can be observed under a microscope.

[0081] (4) Detection of KIF4A gene knockdown effect. Cell proteins were extracted using RIPA lysis buffer, and BCA protein was quantified. KIF4A protein expression was detected using Western blot.

[0082] Experimental results are as follows Figure 9 As shown, the expression level of KIF4A protein decreased significantly, indicating that the stable KIF4A knockdown U87 cell line was successfully constructed.

[0083] 2. KIF4A knockout U87 cell conditioned medium has an inhibitory effect on endothelial cell proliferation.

[0084] (1) Collection of conditioned medium: U87 cells stably expressing shCon and KIF4AshRNA were seeded in 10cm culture dishes, and cultured at a ratio of 2×10 6 Cells / plates were incubated in a 37°C, 5% CO2 incubator for 3 days. When the cell confluence reached more than 90%, the culture supernatant was collected and filtered through a 0.45μM filter membrane to remove cell debris.

[0085] (2) HUVEC cells were seeded in a 10cm culture dish. When the cell coverage reached more than 70%, shCon and KIF4AshRNA conditioned medium was added.

[0086] (3) HUVEC cells were seeded in 96-well plates at a rate of 2000 cells / well; and incubated in a 37°C, 5% CO2 incubator.

[0087] (4) Add the filtered shCon and KIF4AshRNA conditioned medium to a 96-well plate pre-inoculated with HUVEC cells at a rate of 100 μL / well and incubate at 37°C and 5% CO2 for 24 h.

[0088] (5) Take out the 96-well plate and use the CCK-8 method to detect cell viability.

[0089] Experimental results are as follows Figure 10 As shown, compared with the control group, the viability of HUVEC cells was significantly reduced after adding KIF4A knockdown U87 cell conditioned medium (P < 0.01), indicating that KIF4A knockdown U87 cell conditioned medium has the effect of inhibiting endothelial cell proliferation.

[0090] 3. KIF4A knockout U87 cell conditioned medium has the effect of inhibiting endothelial cell angiogenesis.

[0091] (1) Collection of conditioned medium: U87 cells stably expressing shCon and KIF4AshRNA were seeded in 10cm culture dishes, and cultured at a ratio of 2×10 6 Cells / plates were incubated in a 37°C, 5% CO2 incubator for 3 days. When the cell confluence reached more than 90%, the culture supernatant was collected and filtered through a 0.45μM filter membrane to remove cell debris.

[0092] (2) Seed HUVEC cells in 10cm culture dishes at a ratio of 2 x 10 6 Cells / plate; when cell coverage reaches 60%, replace the original culture medium with 10 mL of shCon or KIF4AshRNA U87 conditioned medium. Incubate at 37°C, 5% CO2 for 2 days.

[0093] (3) Tube formation experiment: referring to the experimental method in (5) of Example 5, the diluted Matrix-Gel™ matrix gel was evenly spread on the bottom of the 96-well plate and placed in a 37°C curing plate; then, the HUVEC cell suspension was added to the 96-well plate containing matrix gel, 100 μL per well, and placed in a 37°C cell culture incubator for further culture; after 6 h of culture, the tube formation was observed under a microscope and photographed and recorded.

[0094] (4) ImageJ software was used to measure and perform statistical analysis.

[0095] Experimental results are as follows Figure 11 As shown, compared with the control group, the addition of KIF4A knockdown U87 cell conditioned medium significantly reduced HUVEC cell tubule formation (P < 0.01), indicating that KIF4A knockdown U87 cell conditioned medium has the effect of inhibiting endothelial cell angiogenesis.

[0096] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A viral vector that targets KIF4A to inhibit angiogenesis, characterized in that, The viral vector contains three shRNA gene fragments that target the KIF4 protein coding region.

2. The viral vector targeting KIF4A to inhibit angiogenesis according to claim 1, characterized in that, The three shRNA gene fragment sequences targeting the KIF4 protein coding region are shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

3. The viral vector targeting KIF4A to inhibit angiogenesis according to claim 1, characterized in that, The sequence of an artificially fused gene fragment containing three shRNA gene fragments targeting the KIF4 protein coding region is shown in SEQ ID NO.

4.

4. The method for preparing the viral vector targeting KIF4A to inhibit angiogenesis as described in claim 1, characterized in that, Follow these steps: (1) The artificial fusion gene fragment shown in SEQ ID NO.4 was cloned into the lentiviral backbone plasmid pLKO.1-GFP to obtain the KIF4A knockdown recombinant lentiviral plasmid pLKO.1-KIF4AshRNA; (2) pLKO.1-KIF4AshRNA, packaging plasmid pSPAX2 and pMD2G were co-transfected into 293T cells to prepare pLKO.1-KIF4AshRNA lentivirus; (3) The filtered virus supernatant was concentrated and purified by ultracentrifugation; then it was stored in virus preservation solution. After aliquoting the virus solution, use it directly or store it at -80℃ for later use; (4) Use the pLKO.1-KIF4AshRNA virus solution obtained in step (3) to directly infect HUVEC or U87 cells, add 2-6 μg / mL puromycin for screening for 5-9 days until a stable KIF4A knockdown cell line is obtained.

5. The method for preparing the viral vector targeting KIF4A to inhibit angiogenesis according to claim 4, characterized in that, The virus preservation solution in step (3) is a HEPES buffer containing 10-50% glycerol, 0.1-1.0M trehalose and 1-10% DMSO, with a pH of 7.0-8.

0.

6. The use of the viral vector of claim 1 in the preparation of a drug that inhibits endothelial cell proliferation and angiogenesis.

7. The use of the viral vector of claim 1 in the preparation of a drug that inhibits tumor cell angiogenesis.

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