Small interfering RNA for knocking down AURKA, recombinant vector and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses small interfering RNA for knocking down AURKA, a recombinant vector and application of the small interfering RNA. SiRNA is connected with pSIREN-RetroQ plasmids subjected to enzyme digestion to construct the recombinant interference vector, and a positive recombinant vector is obtained through transformation and screening; a transfection compound is formed by modified chitosan modified by (2-methyl-2-propenyl) succinic anhydride and (Z)-2-methyl-2-butenedioic anhydride, a positive recombinant vector and Lipofectamine3000, human breast cancer MCF-7 cells are transfected to obtain the knock-down cell strain, AURKA protein expression can be reduced, the proliferation rate of the MCF-7 cells is inhibited, meanwhile, the cell cycle is retarded in the G2 / M period, the cell apoptosis induction rate is increased, and the human breast cancer cell strain has the advantages of being capable of inhibiting the growth of breast cancer cells and the like. The transfection compound is high in stability, and an effective tool is provided for tumor targeted therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to small molecule interfering RNA, recombinant vectors, and their applications for knocking down AURKA. Background Technology

[0002] AURKA, a core member of the aurora kinase family, is a key signaling molecule regulating cell cycle progression and spindle dynamics. Its dysfunction is closely related to malignant cell proliferation, differentiation disorders, and tumor development. Clinical studies have shown that AURKA is highly expressed in various malignant tumors, including breast cancer, lung cancer, and ovarian cancer. Particularly in triple-negative breast cancer and HER2-positive breast cancer cell lines, AURKA expression levels are significantly positively correlated with tumor stage, lymph node metastasis, and poor patient prognosis. Furthermore, its high expression enhances tumor cell resistance to chemotherapy drugs, becoming a significant factor limiting the effectiveness of clinical anti-tumor treatment. Therefore, AURKA has become a core candidate target in the field of targeted cancer therapy.

[0003] Currently, targeted intervention strategies for AURKA mainly focus on two categories: small molecule inhibitors and gene silencing technologies. Small molecule inhibitors inhibit AURKA's kinase activity by competitively binding to its ATP-binding site, and several candidate drugs have entered clinical trials. However, these inhibitors have significant technical limitations: on the one hand, most inhibitors lack selectivity for AURKA and are prone to cross-interaction with other members of the Aurora kinase family or other kinases, leading to serious toxic side effects such as bone marrow suppression and gastrointestinal reactions; on the other hand, long-term use can easily induce drug resistance mutations in tumor cells, causing the drug's efficacy to rapidly decline and making it difficult to meet the needs of long-term clinical treatment.

[0004] The development of RNA interference technology has provided a new direction for AURKA targeted silencing. Among them, small interfering RNA, namely siRNA, has become an ideal tool for AURKA gene silencing because it can specifically recognize and degrade target gene mRNA, efficiently inhibit protein expression, and has a clear target and low off-target effect. However, siRNA faces multiple delivery bottlenecks in practical applications: siRNA has difficulty penetrating negatively charged cell membranes; and it is easily degraded by nucleases in vivo, has a short half-life, and extremely low bioavailability.

[0005] To address these issues, two types of delivery systems have been developed: viral vectors and non-viral vectors. However, existing vectors still have significant drawbacks. While viral vectors offer high transfection efficiency, they suffer from strong immunogenicity, the risk of insertional mutations, and limited vector capacity. In clinical applications, they are prone to triggering immune rejection and are difficult to deliver stably for long periods. Among non-viral vectors, traditional liposomes can encapsulate siRNA through charge-electric interactions, but they are prone to particle aggregation in the serum environment and clearance by the reticuloendothelial system, leading to decreased transfection efficiency.

[0006] In summary, current targeted therapies for AURKA suffer from drawbacks such as poor selectivity and easy drug resistance. While siRNA technology boasts high specificity, the lack of efficient, stable, and low-toxicity delivery vectors results in insufficient AURKA silencing efficiency and difficulty in effectively inhibiting tumor cell proliferation. Therefore, developing highly targeted AURKA siRNA sequences, combined with multi-component synergistically modified high-efficiency vector systems, to construct a technical solution capable of stable siRNA delivery and efficient silencing is crucial to overcoming the current bottlenecks in AURKA targeted therapy. Summary of the Invention

[0007] The purpose of this invention is to provide small molecule interfering RNA, recombinant vectors, and their applications for knocking down AURKA, achieving efficient knockdown of AURKA, inhibiting the proliferation of breast cancer MCF-7 cells, arresting the G2 / M phase, and inducing apoptosis. This addresses the shortcomings of existing AURKA-targeted therapies, such as insufficient selectivity of small molecule inhibitors for AURKA and easy induction of tumor cell resistance, delivery difficulties of siRNA (which can specifically silence AURKA but is difficult to penetrate cell membranes and is easily degraded by nucleases in vivo), and the limitations of viral vectors (strong immunogenicity) and non-viral vectors (poor stability) restricting the delivery efficiency of siRNA.

[0008] The technical solution adopted by the present invention to achieve the above objectives is as follows: Preferably, a recombinant vector includes a small interfering RNA and a linearized vector, wherein the small interfering RNA is used to knock down AURKA.

[0009] Preferably, the positive strand sequence of the small interfering RNA is as shown in SEQ ID No.1, which is 5'-GGAUUCUCCUUGAAGAUAUTT-3'.

[0010] Preferably, the antisense strand sequence of the small interfering RNA is as shown in SEQ ID No. 2, which is 5'-AUAUCUUCAAGGAGAAUCCTT-3'.

[0011] Preferably, the linearization vector is the enzyme-digested pSIREN-RetroQ plasmid.

[0012] Preferably, the enzyme digestion includes restriction endonucleases BamHI and HindIII.

[0013] Preferably, the small interfering RNA and the linearized vector are ligated in a ligation buffer using T4 DNA ligase.

[0014] Preferably, the volume ratio of T4 DNA ligase to ligation buffer is 0.2-1:1.

[0015] Preferably, the connection temperature is 15-20℃.

[0016] Preferably, the recombinant vector is used to verify the correct insertion of the small interfering RNA by RT-PCR.

[0017] Preferably, the RT-PCR reaction system includes a recombinant vector, a specific upstream primer of 8-12 μmol / L small interfering RNA, a specific downstream primer of 8-12 μmol / L small interfering RNA, 1-3×SYBR Green PCR Mix, and ultrapure water.

[0018] Preferably, the RT-PCR reaction conditions include 94-96℃ pre-denaturation for 12-18 min, 94-96℃ denaturation for 8-12 s, 58-62℃ annealing extension for 28-32 s, for a total of 35-45 cycles.

[0019] Preferably, the transfection complex comprises liposomes.

[0020] Preferably, the liposomes include Lipofectamine 3000.

[0021] Preferably, the transfection complex comprises anhydride-modified chitosan.

[0022] Preferably, the mass-to-volume ratio of anhydride-modified chitosan to the recombinant carrier is 5 μg: 1-5 μL.

[0023] Preferably, the volume ratio of the recombinant carrier to the liposomes is 1-2:4.

[0024] Preferably, the anhydride-modified chitosan is obtained by reacting a modifier with chitosan.

[0025] Preferably, the modifier includes (2-methyl-2-propenyl)succinic anhydride and (Z)-2-methyl-2-butenoic anhydride.

[0026] Preferably, the mass ratio of (2-methyl-2-propenyl)succinic anhydride to (Z)-2-methyl-2-butenoic anhydride is 0.5:0.5-1.5.

[0027] Preferably, the mass ratio of (2-methyl-2-propenyl)succinic anhydride to chitosan is 0.3-0.5:1.

[0028] 2-Methyl-2-propenyl succinic anhydride and (Z)-2-methyl-2-butenoic anhydride were used as chitosan modifiers to introduce hydrophilic carboxyl groups onto the chitosan backbone, significantly improving the water solubility and dispersion stability of the transfection complex. Optimizing the spatial conformation and charge distribution of chitosan enhanced its electrostatic interaction and tight encapsulation ability with the recombinant vector, effectively isolating extracellular nucleases from contact with small interfering RNA targeting AURKA within the positive recombinant vector, reducing degradation, and thus improving stability. This also reduced the probability of particle aggregation and clearance by the reticuloendothelial system in the serum environment, thereby improving transfection efficiency and enabling the efficient release and function of small interfering RNA targeting AURKA within cells, ultimately leading to a more significant induction of tumor cell apoptosis and inhibition of tumor cell proliferation. At a mass ratio of (2-methyl-2-propenyl)succinic anhydride to (Z)-2-methyl-2-butenoic anhydride of 0.5:1, the resulting anhydride-modified chitosan significantly improved the stability of the transfection complex.

[0029] Use of recombinant vectors or transfection complexes in the preparation of antitumor drugs.

[0030] Preferably, the antitumor drug can specifically knock down the expression of AURKA protein, thereby inhibiting tumor cell proliferation, arresting the tumor cell cycle, or inducing tumor cell apoptosis.

[0031] Preferably, the tumor cells include human breast cancer MCF-7 cells.

[0032] More preferably, the transfection complex includes cyclopentanone-modified chitosan, which is obtained by reacting anhydride-modified chitosan with 2-(1-hydroxypentyl)-1-cyclopentanone, with a mass ratio of anhydride-modified chitosan to 2-(1-hydroxypentyl)-1-cyclopentanone of 1:0.2-1.5. By introducing hydroxyl and ester functional groups into the chitosan molecular backbone, the spatial conformation and intermolecular forces of chitosan are optimized, enhancing the tightness of chitosan's encapsulation and binding stability of the recombinant vector. This effectively reduces the leakage of positive recombinant vectors during transfection, lowers the risk of degradation of small interfering RNA targeting AURKA within the vector by extracellular nucleases, reduces the non-specific binding of the transfection complex to protein components in serum, lowers the probability of recognition and clearance by the reticuloendothelial system, and improves the cyclic stability of the complex in the body fluid environment.

[0033] This invention also provides a method for preparing a targeted siRNA solution, comprising: Preparation of the targeted siRNA solution: The siRNA targeting AURKA was synthesized by solid-phase phosphoramide method and purified by high performance liquid chromatography. The siRNA targeting AURKA was dissolved in ultrapure water to obtain the targeted siRNA solution.

[0034] Preferably, the positive strand sequence of the siRNA targeting AURKA is as shown in SEQ ID No.1, which is 5'-GGAUUCUCCUUGAAGAUAUTT-3'.

[0035] Preferably, the antisense strand sequence of the siRNA targeting AURKA is as shown in SEQ ID No. 2, which is 5'-AUAUCUUCAAGGAGAAUCCTT-3'.

[0036] Preferably, the molar volume ratio of siRNA targeting AURKA to ultrapure water is 10-30 nmol: 1 mL.

[0037] This invention also provides a method for preparing a recombinant interference vector, comprising: Add 0.5-1.5 μg / μL of pSIREN-RetroQ plasmid, 0.5-1.5 U / μL of restriction endonuclease BamHI, and 0.5-1.5 U / μL of restriction endonuclease HindIII to 10× restriction buffer, along with the restriction solvent. Incubate at 36-38℃ for 1-3 hours, gently mixing every 20-40 minutes. Terminate the reaction by adding 0.4-0.6 mol / L EDTA solution. Separate the digested products by 0.5-1.5 wt% agarose gel electrophoresis. Observe a single linearized band using a gel imaging system. Recover the linearized vector using a gel recovery kit. Add the linearized vector and targeting siRNA solution to 8-12× ligation buffer, along with 4-6 U / μL of T4 DNA ligase and ligation solvent. Ligate at 15-17℃ for 15-20 hours to obtain the recombinant interference vector.

[0038] Preferably, the volume ratio of pSIREN-RetroQ plasmid to enzyme digestion buffer is 1:1-3.

[0039] Preferably, the volume ratio of restriction endonuclease BamHI to digestion buffer is 1:1-3.

[0040] Preferably, the volume ratio of restriction endonuclease HindIII to enzyme digestion buffer is 1:1-3.

[0041] Preferably, the enzyme digestion solvent is ultrapure water.

[0042] Preferably, the volume ratio of enzyme digestion solvent to enzyme digestion buffer is 15:1-3.

[0043] Preferably, the volume ratio of EDTA solution to enzyme digestion buffer is 1:1-3.

[0044] Preferably, the volume ratio of linearization vector to ligation buffer is 0.5-1.5:1μL.

[0045] Preferably, the volume ratio of the targeting siRNA solution to the ligation buffer is 0.2-1:1.

[0046] Preferably, the volume ratio of T4 DNA ligase to ligation buffer is 0.2-1:1.

[0047] Preferably, the connecting solvent is ultrapure water.

[0048] Preferably, the mass-to-volume ratio of the ligation solvent to the ligation buffer is 10-20:2.

[0049] Preferably, the pSIREN-RetroQ plasmid was purchased from Clontech.

[0050] Preferably, the gel recovery kit was purchased from Qiagen.

[0051] This invention also provides a method for preparing a positive recombinant vector, comprising: DH5α competent cells were added to the recombinant interference vector, incubated on ice for 20-40 min, heat-shocked at 40-44℃ for 80-100 s, transferred to ice bath and incubated for 1-3 min, then added to LB liquid medium and cultured at 36-38℃ with shaking at 150-250 rpm for 0.5-1.5 h to obtain the transformation culture. The transformation culture was evenly spread on LB solid medium containing 80-120 μg / mL ampicillin and cultured upside down at 36-38℃ for 15-20 h. Regularly shaped single colonies were inoculated into LB liquid medium containing 80-120 μg / mL ampicillin and cultured at 36-38℃ with shaking at 150-250 rpm for 10-15 h. The recombinant plasmid was extracted using a plasmid extraction kit and verified by quantitative real-time RT-PCR. siRNA insertion correctness; the RT-PCR reaction system includes a recombinant interference vector, 8-12 μmol / L of a specific upstream primer targeting siRNA, 8-12 μmol / L of a specific downstream primer targeting siRNA, 1-3×SYBR Green PCR Mix and ultrapure water; the RT-PCR reaction conditions include 94-96℃ pre-denaturation for 12-18 min, 94-96℃ denaturation for 8-12 s, 58-62℃ annealing extension for 28-32 s, for a total of 35-45 cycles; the sample that amplifies the target band is the positive recombinant vector.

[0052] Preferably, the volume ratio of the recombinant interference vector to DH5α competent cells is 1:5-15.

[0053] Preferably, the volume ratio of the recombinant interference vector to LB liquid culture medium is 1:50-150.

[0054] Preferably, the volume ratio of the transformation bacterial culture to LB liquid medium containing 100 μg / mL ampicillin is 10-30 μL:1 mL.

[0055] Preferably, the volume ratio of the recombinant interference vector to the specific upstream primer targeting the siRNA is 1:0.3-1.

[0056] Preferably, the volume ratio of the recombinant interference vector to the specific downstream primer targeting the siRNA is 1:0.3-1.

[0057] Preferably, the volume ratio of the recombinant interference vector to SYBR Green PCR Mix is ​​1:5-15.

[0058] Preferably, the volume ratio of the recombinant interference carrier to ultrapure water is 1:5-10.

[0059] Preferably, DH5α competent cells were purchased from Takara.

[0060] This invention also provides a method for preparing a transfection complex, comprising: The positive recombinant vector was added to liposome Lipofectamine 3000, diluted in serum-free DMEM medium, and mixed after standing at room temperature for 3-10 min. The mixture was then incubated at room temperature for 15-25 min to obtain the transfection complex.

[0061] Preferably, the volume ratio of the positive recombinant vector to Lipofectamine 3000 is 1-2:4.

[0062] Preferably, the volume ratio of the positive recombinant vector to serum-free DMEM medium is 1-2:50.

[0063] Preferably, Lipofectamine 3000 was purchased from Invitrogen.

[0064] This invention also provides a method for preparing a knockdown cell line, comprising: Human breast cancer MCF-7 cells were seeded in DMEM medium containing 8-12 v / v% fetal bovine serum, 80-120 U / mL penicillin, and 80-120 μg / mL streptomycin. The cells were cultured at 36-38℃ in a 4-6% CO2 incubator. When the cell confluence reached 75-85%, the cells were passaged using 0.2-0.3 wt% trypsin, and the cell density was adjusted to 0.5-1.5 × 10⁶ cells / mL. 6Cells / mL were used to obtain MCF-7 cell suspension; MCF-7 cell suspension was added to cell culture plates and cultured for 20-25 h until cell confluence reached 60-70%; old culture medium was discarded, and cells were washed 1-3 times with PBS buffer (pH 7.3-7.5); transfection medium and transfection complex were added, and the mixture was shaken well and cultured in a 36-38℃, 4-6% CO2 incubator for 45-50 h; empty vector control group and untransfected blank control group were set up simultaneously; transfection medium was discarded, and complete DMEM medium containing 600-1000 μg / mL G418 was added to each well; the medium was changed every 2-4 days, and screening was continued for 1-3 weeks until all control cells died to obtain knockdown cell lines.

[0065] Preferably, the transfection medium is serum-free DMEM medium.

[0066] Preferably, the volume ratio of MCF-7 cell suspension to transfection culture medium is 1:0.5-1.5.

[0067] Preferably, the volume ratio of MCF-7 cell suspension to transfection complex is 1:0.5-1.5.

[0068] Preferably, the volume ratio of MCF-7 cell suspension to complete DMEM culture medium is 1:0.5-1.5.

[0069] Preferably, the complete DMEM medium was purchased from Sigma.

[0070] This invention also provides a method for preparing anhydride-modified chitosan, comprising: (2-Methyl-2-propenyl)succinic anhydride and (Z)-2-methyl-2-butenoic anhydride were dissolved in acetone and stirred for 5-15 min to obtain a modifier solution. Acetic acid and chitosan were added to deionized water and stirred for 5-15 min at 40-50 °C. The modifier solution was added under nitrogen protection and reacted at 55-65 °C. After cooling the reaction solution to room temperature, it was transferred to a dialysis bag and dialyzed for 70-80 h, with the dialysate being replaced every 6-10 h. The solution was then frozen at -75-85 °C for 1-3 h and freeze-dried for 20-25 h to obtain anhydride-modified chitosan.

[0071] Preferably, in the modifier solution, the mass-to-volume ratio of (2-methyl-2-propenyl)succinic anhydride to acetone is 0.3-0.5 g: 5 mL.

[0072] Preferably, in the modifier solution, the mass-to-volume ratio of (Z)-2-methyl-2-butenoic anhydride to acetone is 0.5-1.5 g: 5 mL.

[0073] Preferably, the volume ratio of acetic acid to deionized water is 1:40-80.

[0074] Preferably, the mass-to-volume ratio of chitosan to deionized water is 1g:0.5-4mL.

[0075] Preferably, the mass of the modifier solution is measured by the mass of (2-methyl-2-propenyl)succinic anhydride therein, and the mass ratio of (2-methyl-2-propenyl)succinic anhydride to chitosan is 0.3-0.5:1.

[0076] Preferably, the molecular weight cutoff of the dialysis bag is 3000-4000 Da.

[0077] Preferably, the dialysate is deionized water.

[0078] Preferably, the freeze-drying conditions are a vacuum of 0.05-0.15 mbar and a cold trap temperature of -45-55°C.

[0079] This invention also provides a method for preparing cyclopentanone-modified chitosan, comprising: 2-(1-hydroxypentyl)-1-cyclopentanone was dissolved in anhydrous ethanol to obtain a 2-(1-hydroxypentyl)-1-cyclopentanone solution; anhydride-modified chitosan was dispersed in PBS solution at pH 5.4-5.6 and stirred for 1-2 h at 36-38℃ and 250-350 rpm; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added, and the mixture was stirred at room temperature and 150-250 rpm. Stir at 0 rpm for 20-40 min, add 2-(1-hydroxypentyl)-1-cyclopentanone solution, and stir at 40-50℃ and 250-350 rpm for 2-3 h. After cooling the reaction solution to room temperature, transfer it to a dialysis bag and dialyze for 70-75 h, changing the dialysis solution every 6-10 h. Freeze at -75-85℃ for 1-3 h and freeze-dry for 20-25 h to obtain cyclopentanone-modified chitosan.

[0080] Preferably, in the 2-(1-hydroxypentyl)-1-cyclopentanone solution, the mass-to-volume ratio of 2-(1-hydroxypentyl)-1-cyclopentanone to anhydrous ethanol is 0.5:5-15 mL.

[0081] Preferably, the mass-to-volume ratio of anhydride-modified chitosan to PBS solution is 1g:50-150mL.

[0082] Preferably, the mass ratio of anhydride-modified chitosan to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:0.5-1.5.

[0083] Preferably, the mass ratio of anhydride-modified chitosan to N-hydroxysuccinimide is 1:0.3-1.

[0084] Preferably, the mass of the 2-(1-hydroxypentyl)-1-cyclopentanone solution is measured by the mass of 2-(1-hydroxypentyl)-1-cyclopentanone therein, and the mass ratio of anhydride-modified chitosan to 2-(1-hydroxypentyl)-1-cyclopentanone is 1:0.2-1.5.

[0085] Preferably, the molecular weight cutoff of the dialysis bag is 3000-4000 Da.

[0086] Preferably, the dialysate is deionized water.

[0087] Preferably, the freeze-drying conditions are a vacuum of 0.05-0.15 mbar and a cold trap temperature of -45-55°C.

[0088] This invention also provides a method for preparing a transfection complex, comprising: The modified chitosan was dispersed in PBS buffer at pH 7.3-7.5, and the positive recombinant vector was added. The mixture was stirred at room temperature and 150-250 rpm for 10-20 min. Lipofectamine 3000 was added, and the mixture was diluted with serum-free DMEM medium. After standing at room temperature for 3-10 min, the mixture was mixed and incubated at room temperature for 15-25 min to obtain the transfection complex.

[0089] Preferably, the modified chitosan includes one of acid anhydride-modified chitosan and cyclopentanone-modified chitosan.

[0090] Preferably, the mass-to-volume ratio of modified chitosan to PBS buffer is 0.1 mg: 0.5-5 mL.

[0091] Preferably, the mass-to-volume ratio of modified chitosan to the positive recombinant vector is 5 μg: 1-5 μL.

[0092] Preferably, the volume ratio of the positive recombinant vector to Lipofectamine 3000 is 1-2:4.

[0093] Preferably, the volume ratio of the positive recombinant vector to serum-free DMEM medium is 1-2:50.

[0094] Preferably, Lipofectamine 3000 was purchased from Invitrogen.

[0095] This invention utilizes a small interfering RNA with a well-defined and specific target for AURKA. This RNA is combined with an enzyme-digested pSIREN-RetroQ plasmid to construct a recombinant vector using T4 DNA ligase. Simultaneously, chitosan is modified with 2-methyl-2-propenylsuccinic anhydride and Z-2-methyl-2-butenoic anhydride to obtain a modified chitosan, which, together with the recombinant vector and liposomes, forms a transfection complex. This complex is transfected into human breast cancer MCF-7 cells, and after G418 selection, a knockdown cell line is obtained. Therefore, this invention offers the following advantages: the knockdown cell line efficiently and specifically knocks down AURKA protein expression, significantly inhibits MCF-7 cell proliferation, effectively arrests the cell cycle at the G2 / M phase, and significantly increases the apoptosis rate. Furthermore, the transfection complex exhibits high stability, overcoming the problems of poor selectivity and drug resistance associated with existing small molecule inhibitors, as well as the low transfection efficiency and instability of traditional vectors. Therefore, this invention provides a technical solution that balances high efficiency, stability, and targeting, offering a key tool for targeted therapy of tumors with high AURKA expression and possessing significant clinical translational value. Attached Figure Description

[0096] Figure 1 This is a schematic diagram of the fluorescence expression of the transfection complex under a low magnification of ×100.

[0097] Figure 2 This is a schematic diagram of the fluorescence expression of the transfection complex under a high magnification of ×400.

[0098] Figure 3 This is a schematic diagram illustrating the knockdown of AURKA expression in cell lines.

[0099] Figure 4 This is a schematic diagram of AURKA expression in the empty control group. Detailed Implementation

[0100] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0101] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0102] Example 1: Preparation of the targeted siRNA solution: The siRNA targeting AURKA was synthesized using a solid-phase phosphorusamide method and purified by high-performance liquid chromatography (HPLC). The siRNA targeting AURKA was then dissolved in ultrapure water to obtain the targeted siRNA solution. The sense strand sequence of the siRNA targeting AURKA is shown in SEQ ID No. 1, which is 5'-GGAUUCUCCUUGAAGAUAUTT-3'; the antisense strand sequence of the siRNA targeting AURKA is shown in SEQ ID No. 2, which is 5'-AUAUCUUCAAGGAGAAUCCTT-3'; the molar ratio of the siRNA targeting AURKA to ultrapure water was 20 nmol:1 mL.

[0103] Preparation of recombinant interference vector: 1 μg / μL of pSIREN-RetroQ plasmid, 1 U / μL of restriction endonuclease BamHI and 1 U / μL of restriction endonuclease HindIII were added to 10× restriction enzyme buffer, along with the enzyme digestion solvent. The mixture was incubated at 37℃ for 2 h, with gentle mixing every 30 min. The reaction was terminated by adding 0.5 mol / L EDTA solution. The digestion products were separated by 1 wt% agarose gel electrophoresis. A single linearized band was observed using a gel imaging system, and the linearized vector was recovered using a gel extraction kit. The linearized vector and targeting siRNA solution were added to 10× ligation buffer, along with 5 U / μL of T4 DNA ligase and ligation solvent. The mixture was ligated at 16℃ for 16 h to obtain the recombinant interference vector. The volume ratio of pSIREN-RetroQ plasmid to digestion buffer was 1:2; the volume ratio of restriction endonuclease BamHI to digestion buffer was 1:2; the volume ratio of restriction endonuclease HindIII to digestion buffer was 1:2; the digestion solvent was ultrapure water; the volume ratio of digestion solvent to digestion buffer was 15:2; the volume ratio of EDTA solution to digestion buffer was 1:2; the volume ratio of linearization vector to ligation buffer was 1.1:1 μL; the volume ratio of targeting siRNA solution to ligation buffer was 0.4:1; the volume ratio of T4 DNA ligase to ligation buffer was 0.5:1; the ligation solvent was ultrapure water; the mass-to-volume ratio of ligation solvent to ligation buffer was 16:2; pSIREN-RetroQ plasmid was purchased from Clontech; and the gel extraction kit was purchased from Qiagen.

[0104] Preparation of positive recombinant vector: DH5α competent cells were added to the recombinant interference vector, incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, transferred to an ice bath and incubated for 2 min, then LB liquid medium was added and cultured at 37℃ with shaking at 200 rpm for 1 h to obtain the transformation culture. The transformation culture was evenly spread on LB solid medium containing 100 μg / mL ampicillin and incubated upside down at 37℃ for 18 h. The morphologically regular single colonies were inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured at 37℃ with shaking at 200 rpm for 12 h. The recombinant plasmid was extracted using a plasmid extraction kit, and the correct insertion of AURKA siRNA was verified by real-time RT-PCR. The RT-PCR reaction system included the recombinant interference vector, 10 μmol / L of specific upstream primer targeting siRNA, 10 μmol / L of specific downstream primer targeting siRNA, and 2×SYBR Green. PCRMix and ultrapure water were used. RT-PCR reaction conditions included 40 cycles of 95℃ pre-denaturation for 15 min, 95℃ denaturation for 10 s, and 60℃ annealing extension for 30 s. Samples amplifying the target band were used as positive recombinant vectors. The volume ratio of recombinant interference vector to DH5α competent cells was 1:10, the volume ratio of recombinant interference vector to LB broth was 1:100, and the volume ratio of transformation culture to LB broth containing 100 μg / mL ampicillin was 20 μL:1 mL. The volume ratio of recombinant interference vector to the specific upstream primer targeting siRNA was 1:0.5, the volume ratio of recombinant interference vector to the specific downstream primer targeting siRNA was 1:0.5, the volume ratio of recombinant interference vector to SYBR Green PCR Mix was 1:10, and the volume ratio of recombinant interference vector to ultrapure water was 1:8. DH5α competent cells were purchased from Takara.

[0105] Preparation of transfection complex: The positive recombinant vector was added to liposome Lipofectamine 3000, diluted with serum-free DMEM medium, and incubated at room temperature for 5 min before mixing and 20 min at room temperature to obtain the transfection complex. The volume ratio of positive recombinant vector to Lipofectamine 3000 was 1.8:4, and the volume ratio of positive recombinant vector to serum-free DMEM medium was 1.8:50. Lipofectamine 3000 was purchased from Invitrogen.

[0106] Preparation of knockdown cell lines: Human breast cancer MCF-7 cells were seeded in DMEM medium containing 10 v / v% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were cultured at 37°C in a 5% CO2 incubator. When the cell confluence reached 80%, the cells were passaged using 0.25 wt% trypsin, and the cell density was adjusted to 1 × 10⁻⁶ cells / mL. 6 Cells / mL were used to obtain MCF-7 cell suspension. The MCF-7 cell suspension was added to 6-well cell culture plates and cultured for 24 h until cell confluence reached 65%. The old culture medium was discarded, and the cells were washed twice with pH 7.4 PBS buffer. Transfection medium and transfection complex were added, and the mixture was shaken well. The plates were then incubated at 37°C with 5% CO2 for 48 h. Simultaneously, an empty vector control group and an untransfected blank control group were set up. The transfection medium was discarded, and each well was filled with complete DMEM medium containing 800 μg / mL G418. The medium was changed every 3 days, and selection was continued for 2 weeks until all control cells died, obtaining the knockdown cell line. The transfection medium was serum-free DMEM medium, with a 1:1 volume ratio of MCF-7 cell suspension to transfection medium, a 1:1 volume ratio of MCF-7 cell suspension to transfection complex, and a 1:1 volume ratio of MCF-7 cell suspension to complete DMEM medium. The complete DMEM medium was purchased from Sigma.

[0107] Example 2: The only difference between this example and Example 1 is the preparation of the transfection complex.

[0108] Preparation of anhydride-modified chitosan: (2-methyl-2-propenyl)succinic anhydride and (Z)-2-methyl-2-butenoic anhydride were dissolved in acetone and stirred for 10 min to obtain a modifier solution; acetic acid and chitosan were added to deionized water and stirred at 45 °C for 10 min; the modifier solution was added under nitrogen protection and reacted at 60 °C; the reaction solution was cooled to room temperature and transferred to a dialysis bag for dialysis for 72 h, with the dialysis solution being replaced every 8 h; the solution was frozen at -80 °C for 2 h and freeze-dried for 24 h to obtain anhydride-modified chitosan. In the modifier solution, the mass-to-volume ratio of (2-methyl-2-propenyl)succinic anhydride to acetone is 0.5 g:4 mL, and the mass-to-volume ratio of (Z)-2-methyl-2-butenoic anhydride to acetone is 0.5 g:4 mL; the volume ratio of acetic acid to deionized water is 1:49, and the mass-to-volume ratio of chitosan to deionized water is 1 g:1 mL; the mass of the modifier solution is measured by the mass of (2-methyl-2-propenyl)succinic anhydride, and the mass ratio of (2-methyl-2-propenyl)succinic anhydride to chitosan is 0.5:1; the molecular weight cutoff of the dialysis bag is 3500 Da, and the dialysate is deionized water; the freeze-drying conditions are a vacuum of 0.1 mbar and a cold trap temperature of -50 °C.

[0109] Preparation of the transfection complex: Anhydride-modified chitosan was dispersed in PBS buffer at pH 7.4, and the positive recombinant vector was added. The mixture was stirred at room temperature and 200 rpm for 15 min. Lipofectamine 3000 was added, and the mixture was diluted with serum-free DMEM medium. After standing at room temperature for 5 min, the mixture was mixed and incubated at room temperature for 20 min to obtain the transfection complex. The mass-to-volume ratio of anhydride-modified chitosan to PBS buffer was 0.1 mg:1 mL, the mass-to-volume ratio of anhydride-modified chitosan to positive recombinant vector was 5 μg:1.8 μL, the volume ratio of positive recombinant vector to Lipofectamine 3000 was 1.8:4, and the volume ratio of positive recombinant vector to serum-free DMEM medium was 1.8:50. Lipofectamine 3000 was purchased from Invitrogen.

[0110] Example 3: The only difference between this example and Example 2 is the preparation of anhydride-modified chitosan.

[0111] Preparation of anhydride-modified chitosan: (2-methyl-2-propenyl)succinic anhydride and (Z)-2-methyl-2-butenoic anhydride were dissolved in acetone and stirred for 10 min to obtain a modifier solution; acetic acid and chitosan were added to deionized water and stirred at 45 °C for 10 min; the modifier solution was added under nitrogen protection and reacted at 60 °C; the reaction solution was cooled to room temperature and transferred to a dialysis bag for dialysis for 72 h, with the dialysis solution being replaced every 8 h; the solution was frozen at -80 °C for 2 h and freeze-dried for 24 h to obtain anhydride-modified chitosan. In the modifier solution, the mass-to-volume ratio of (2-methyl-2-propenyl)succinic anhydride to acetone is 0.5 g:4 mL, and the mass-to-volume ratio of (Z)-2-methyl-2-butenoic anhydride to acetone is 1 g:4 mL; the volume ratio of acetic acid to deionized water is 1:49, and the mass-to-volume ratio of chitosan to deionized water is 1 g:1 mL; the mass of the modifier solution is measured by the mass of (2-methyl-2-propenyl)succinic anhydride, and the mass ratio of (2-methyl-2-propenyl)succinic anhydride to chitosan is 0.5:1; the molecular weight cutoff of the dialysis bag is 3500 Da, and the dialysate is deionized water; the freeze-drying conditions are a vacuum of 0.1 mbar and a cold trap temperature of -50 °C.

[0112] Example 4: The only difference between this example and Example 2 is the preparation of the transfection complex.

[0113] Preparation of anhydride-modified chitosan: (2-methyl-2-propenyl)succinic anhydride and (Z)-2-methyl-2-butenoic anhydride were dissolved in acetone and stirred for 10 min to obtain a modifier solution; acetic acid and chitosan were added to deionized water and stirred at 45 °C for 10 min; the modifier solution was added under nitrogen protection and reacted at 60 °C; the reaction solution was cooled to room temperature and transferred to a dialysis bag for dialysis for 72 h, with the dialysis solution being replaced every 8 h; the solution was frozen at -80 °C for 2 h and freeze-dried for 24 h to obtain anhydride-modified chitosan. In the modifier solution, the mass-to-volume ratio of (2-methyl-2-propenyl)succinic anhydride to acetone is 0.5 g:4 mL, and the mass-to-volume ratio of (Z)-2-methyl-2-butenoic anhydride to acetone is 0.5 g:4 mL; the volume ratio of acetic acid to deionized water is 1:49, and the mass-to-volume ratio of chitosan to deionized water is 1 g:1 mL; the mass of the modifier solution is measured by the mass of (2-methyl-2-propenyl)succinic anhydride, and the mass ratio of (2-methyl-2-propenyl)succinic anhydride to chitosan is 0.5:1; the molecular weight cutoff of the dialysis bag is 3500 Da, and the dialysate is deionized water; the freeze-drying conditions are a vacuum of 0.1 mbar and a cold trap temperature of -50 °C.

[0114] Preparation of cyclopentanone-modified chitosan: 2-(1-hydroxypentyl)-1-cyclopentanone was dissolved in anhydrous ethanol to obtain a 2-(1-hydroxypentyl)-1-cyclopentanone solution; anhydride-modified chitosan was dispersed in PBS solution at pH 5.5 and stirred at 37℃ and 300 rpm for 1.5 h; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added, and the mixture was stirred at room temperature and 200 rpm for 30 min; then the 2-(1-hydroxypentyl)-1-cyclopentanone solution was added, and the mixture was stirred at 45℃ and 300 rpm for 2.5 h; the reaction solution was cooled to room temperature and transferred to a dialysis bag for dialysis for 72 h, with the dialysis solution being replaced every 8 h; the mixture was then frozen at -80℃ for 2 h and freeze-dried for 24 h to obtain cyclopentanone-modified chitosan. In a 2-(1-hydroxypentyl)-1-cyclopentanone solution, the mass-to-volume ratio of 2-(1-hydroxypentyl)-1-cyclopentanone to anhydrous ethanol was 0.5:10 mL; the mass-to-volume ratio of anhydride-modified chitosan to PBS solution was 1 g:100 mL; the mass-to-volume ratio of anhydride-modified chitosan to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was 1:0.76; and the mass-to-volume ratio of anhydride-modified chitosan to N-hydroxysuccinimide... The mass ratio was 1:0.46; the mass of the 2-(1-hydroxypentyl)-1-cyclopentanone solution was measured by the mass of 2-(1-hydroxypentyl)-1-cyclopentanone in it, and the mass ratio of anhydride-modified chitosan to 2-(1-hydroxypentyl)-1-cyclopentanone was 1:0.5; the molecular weight cutoff of the dialysis bag was 3500 Da, and the dialysate was deionized water; the freeze-drying conditions were a vacuum of 0.1 mbar and a cold trap temperature of -50 °C.

[0115] Preparation of the transfection complex: Cyclopentanone-modified chitosan was dispersed in PBS buffer at pH 7.4, and the positive recombinant vector was added. The mixture was stirred at room temperature and 200 rpm for 15 min. Lipofectamine 3000 was added, and the mixture was diluted with serum-free DMEM medium. After standing at room temperature for 5 min, the mixture was mixed and incubated at room temperature for 20 min to obtain the transfection complex. The mass-to-volume ratio of cyclopentanone-modified chitosan to PBS buffer was 0.1 mg:1 mL, the mass-to-volume ratio of anhydride-modified chitosan to the positive recombinant vector was 5 μg:1.8 μL, the volume ratio of the positive recombinant vector to Lipofectamine 3000 was 1.8:4, and the volume ratio of the positive recombinant vector to serum-free DMEM medium was 1.8:50. Lipofectamine 3000 was purchased from Invitrogen.

[0116] Example 5: The only difference between this example and Example 4 is the preparation of cyclopentanone-modified chitosan.

[0117] Preparation of cyclopentanone-modified chitosan: 2-(1-hydroxypentyl)-1-cyclopentanone was dissolved in anhydrous ethanol to obtain a 2-(1-hydroxypentyl)-1-cyclopentanone solution; anhydride-modified chitosan was dispersed in PBS solution at pH 5.5 and stirred at 37℃ and 300 rpm for 1.5 h; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added, and the mixture was stirred at room temperature and 200 rpm for 30 min; then the 2-(1-hydroxypentyl)-1-cyclopentanone solution was added, and the mixture was stirred at 45℃ and 300 rpm for 2.5 h; the reaction solution was cooled to room temperature and transferred to a dialysis bag for dialysis for 72 h, with the dialysis solution being replaced every 8 h; the mixture was then frozen at -80℃ for 2 h and freeze-dried for 24 h to obtain cyclopentanone-modified chitosan. In a 2-(1-hydroxypentyl)-1-cyclopentanone solution, the mass-to-volume ratio of 2-(1-hydroxypentyl)-1-cyclopentanone to anhydrous ethanol was 1:10 mL; the mass-to-volume ratio of anhydride-modified chitosan to PBS solution was 1 g:100 mL; the mass-to-volume ratio of anhydride-modified chitosan to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was 1:0.76; and the mass-to-volume ratio of anhydride-modified chitosan to N-hydroxysuccinimide... The mass ratio is 1:0.46; the mass of the 2-(1-hydroxypentyl)-1-cyclopentanone solution is measured by the mass of 2-(1-hydroxypentyl)-1-cyclopentanone in it, and the mass ratio of anhydride-modified chitosan to 2-(1-hydroxypentyl)-1-cyclopentanone is 1:1; the molecular weight cutoff of the dialysis bag is 3500 Da, and the dialysate is deionized water; the freeze-drying conditions are a vacuum of 0.1 mbar and a cold trap temperature of -50°C.

[0118] Comparative Example 1: The only difference between this comparative example and Example 2 is that (2-methyl-2-propenyl) succinic anhydride was not used in the preparation of the anhydride-modified chitosan.

[0119] Comparative Example 2: The only difference between this comparative example and Example 2 is that (Z)-2-methyl-2-butenoic anhydride was not used in the preparation of the anhydride-modified chitosan.

[0120] Comparative Example 3: The only difference between this comparative example and Example 1 is the preparation of the transfection complex.

[0121] Preparation of the transfection complex: Chitosan was dispersed in PBS buffer at pH 7.4, and the positive recombinant vector was added. The mixture was stirred at room temperature and 200 rpm for 15 min. Lipofectamine 3000 was added, and the mixture was diluted with serum-free DMEM medium. After standing at room temperature for 5 min, the mixture was mixed and incubated at room temperature for 20 min to obtain the transfection complex. The mass-to-volume ratio of chitosan to PBS buffer was 0.1 mg:1 mL, the mass-to-volume ratio of chitosan to positive recombinant vector was 5 μg:1.8 μL, the volume ratio of positive recombinant vector to Lipofectamine 3000 was 1.8:4, and the volume ratio of positive recombinant vector to serum-free DMEM medium was 1.8:50. Lipofectamine 3000 was purchased from Invitrogen.

[0122] Experimental Example 1: Cellular fluorescence expression of the transfection complex.

[0123] Test sample: Transfection complex prepared in Example 1.

[0124] Test method: The transfection complex was added to a 6-well plate and cultured for 48 h. The fluorescence expression of cells was observed using an Olympus IX73 fluorescence microscope.

[0125] The fluorescence expression of the transfection complex under low magnification (×100) is as follows: Figure 1 As shown, most cells emit green fluorescence; the fluorescence expression of the transfection complex under high magnification (×400) is as follows. Figure 2 As shown, fluorescence can be clearly observed to be concentrated inside the cells. The proportion of fluorescent positive cells was greater than 80% when counted using ImageJ software, proving that the recombinant positive vector was successfully transfected.

[0126] Experimental Example 2: Verification test of knockdown efficiency of knockdown cell lines.

[0127] Test sample: Knockdown cell line prepared in Example 1.

[0128] Test method: Collect 1×10⁻⁶ cells from each of the knockdown cell line, empty vector control group, and blank control group. 6For each sample, add 100 μL of RIPA lysis buffer containing 1% protease inhibitor, lyse on ice for 30 min, centrifuge at 12000 rpm and 4℃ for 20 min, and collect the supernatant; determine the protein concentration using the BCA protein quantification kit, take 20 μg of protein sample, add 5 μL of 6× loading buffer, and boil at 95℃ for 5 min for denaturation; perform 10% SDS-PAGE electrophoresis, stacking gel voltage 80V, separating gel voltage 120V, time 90 min, transfer to PVDF membrane, 4℃, 120V, time 1 h; block with 5% skim milk at room temperature for 2 h, add 1:1000 diluted AURKA primary antibody and 1:5000 diluted β-actin primary antibody, incubate overnight at 4℃; wash 3 times with TBST buffer, 10 min each time, add 1:5000 diluted HRP-labeled secondary antibody, incubate at room temperature for 2 h, wash 3 times with TBST, and then develop with ECL chemiluminescence. RIPA lysis buffer was purchased from Beyotime, and AURKA primary antibody was purchased from CST.

[0129] The knockdown efficiency verification test results of the knockdown cell line prepared in this invention are shown in Table 1.

[0130] Table 1. Results of the knockdown efficiency verification test for the knockdown cell lines.

[0131] The AURKA protein expression levels in the blank control group and the empty vector control group were similar, indicating that transfection with the empty vector did not significantly affect intracellular AURKA protein expression. The AURKA protein expression level in the knockdown cell line was reduced by more than 50% compared to the empty vector control group, proving that AURKA knockdown was successful. This demonstrates that the specific sequence of small interfering RNA in this invention can effectively target AURKA and reduce its protein expression.

[0132] Experimental Example 3: Test on the ability of knockdown cell lines to promote tumor cell proliferation.

[0133] Test sample: Knockdown cell line prepared in Example 1.

[0134] Test method: Cells from the logarithmic growth phase knockdown cell line, empty vector control group, and blank control group were taken and their density adjusted to 5 × 10⁶ cells / year. 3Cells were seeded into 96-well plates with 5 replicates per group, and culture medium was added to each well to a final volume of 100 μL. The plates were incubated at 37°C in a 5% CO2 incubator for 0 h, 24 h, 48 h, and 72 h. At each time point, 20 μL of 5 mg / mL MTT solution was added to each well, and the plates were incubated at 37°C for 4 h. The liquid in the wells was discarded, and 150 μL of LDMSO was added to each well. The plates were shaken for 10 min to dissolve the crystals completely. The absorbance (OD) value at 490 nm was measured using a microplate reader, and the proliferation inhibition rate was calculated using the formula: proliferation inhibition rate (%) = (1 - OD value of knocked-down cell line / OD value of empty control group) × 100%.

[0135] The results of the test on the tumor cell proliferation ability of the knockdown cell line prepared in this invention are shown in Table 2.

[0136] Table 2 Results of the test on the proliferation ability of knockdown cell lines against tumor cells

[0137] The tumor cell proliferation inhibition rates of the blank control group and the empty vector control group were extremely low, indicating that AURKA knockdown treatment or only transfection with the empty vector had virtually no inhibitory effect on tumor cell proliferation, further demonstrating that the empty vector does not have a significant impact on the proliferation capacity of tumor cells; the cell line with knockdown showed a proliferation inhibition rate of more than 40%, proving that AURKA knockdown can inhibit the proliferation of MCF-7 cells.

[0138] Experiment 4: Effect of knockdown cell lines on tumor cell cycle.

[0139] Test sample: Knockdown cell line prepared in Example 1.

[0140] Test method: Collect 1×10⁻⁶ cells each from the knockdown cell line, empty vector control group, and blank control group after 48 h of culture. 6 Cells were washed twice with PBS, fixed overnight at 4°C with 75% pre-cooled ethanol, and then washed twice with PBS. 50 μL PPI staining solution was added and incubated in the dark for 30 min. Cell cycle distribution was analyzed by flow cytometry using ModFit software. PI staining solution was purchased from Beyotime and contained 50 μg / mL PI and 100 μg / mL RNase A.

[0141] The results of the test on the effect of the knockdown cell line prepared in this invention on the tumor cell cycle are shown in Table 3.

[0142] Table 3. Results of the test on the effect of knockdown cell lines on tumor cell cycle.

[0143] The proportions of tumor cell cycle stages were similar in the blank control group and the empty vector control group, with no significant difference in the proportions of cells in the G0 / G1, S, and G2 / M phases. This indicates that transfection with the empty vector alone does not significantly interfere with the normal cell cycle progression of tumor cells. However, the cell cycle distribution of the knockdown cell line in Example 1 showed significant changes. The proportion of cells in the G2 / M phase was significantly higher than that in the blank and empty vector control groups, while the proportion of cells in the G0 / G1 phase decreased accordingly, and the proportion of cells in the S phase also decreased slightly. The proportion of cells in the G2 / M phase in the knockdown cell line was ≥10% higher than that in the empty vector control group, proving that the cell cycle was arrested in the G2 / M phase. This demonstrates that the knockdown cell line can effectively achieve AURKA knockdown, thereby regulating the tumor cell cycle and arresting it in the G2 / M phase. This verifies the effectiveness of the recombinant vector and transfection complex in Example 1 in interfering with the tumor cell cycle progression. Experimental Example 5: Test on the expression level of related proteins by knocking down cell lines.

[0144] Test samples: Knockdown cell lines prepared in each example and comparative example.

[0145] Test method: Cell slides from knockdown cell lines and empty vector control groups were fixed with 4% paraformaldehyde and antigen retrieval was performed. The slides were then incubated with AURKA primary antibody diluted 1:500 and developed using DAB staining. Total protein was extracted from the knockdown cell lines, empty vector control group, and blank control group according to the method in Example 2. Proliferation markers Ki-67, invasion-related proteins MMP2 / MMP9, and apoptosis-related proteins BAX / P21 / CyclinB / Caspase3 were detected. Primary antibody dilution ratios: Ki-67 1:1000, MMP2 1:800, MMP9 1:800, BAX 1:1000, P21 1:800, CyclinB 1:800, and Caspase3 1:800.

[0146] The knockdown cell line prepared in this invention inhibits AURKA expression as follows: Figure 3 As shown, the empty vector control group prepared in this invention inhibits AURKA expression as follows. Figure 4 As shown in the figure, the AURKA positivity rate of the knockdown cell line was reduced by more than 60% compared with the empty vector control group, further confirming the success of AURKA knockdown.

[0147] The results of the test on the expression levels of related proteins by the knockdown cell lines prepared in this invention are shown in Table 4.

[0148] Table 4. Results of knockdown of cell lines on the expression levels of related proteins

[0149] In the blank control group and the empty vector control group, there was no significant difference in the relative expression levels of each detected protein. β-actin, used as an internal control, showed stable expression. The expression levels of proliferation markers, invasion-related proteins, and apoptosis-related proteins were also largely consistent, indicating that transfection with the empty vector alone does not interfere with the expression of these key functional proteins in tumor cells. In the knockdown cell line, the expression levels of proliferation markers and invasion-related proteins were significantly lower than those in the blank control group and the empty vector control group, while the expression levels of apoptosis-related proteins were significantly higher. The expression levels of Ki-67, MMP2, MMP9, and Cyclin B proteins were significantly higher than those in the empty vector control group. The cell line showed a greater than 30% reduction in AURKA expression, while the expression levels of BAX, P21, and activated Caspase 3 proteins increased by more than 30%. This indicates that the knockdown cell line can effectively achieve AURKA knockdown, thereby regulating the expression of proteins related to tumor cell proliferation, invasion, and apoptosis. This results in the inhibition of cell proliferation, reduction of cell invasion, and promotion of apoptosis. This verifies the effectiveness of the recombinant vector and transfection complex in Example 1 in targeting and regulating AURKA-related functional pathways, which is consistent with the technical goal of this invention to intervene in the biological behavior of tumor cells. AURKA knockdown can inhibit cell proliferation and invasion and promote apoptosis.

[0150] Experimental Example 6: Stability test of the transfection complex.

[0151] Test samples: Transfection complexes prepared in each example and comparative example.

[0152] Test method: Take 2 mL of each transfection complex prepared in each example and comparative example, place them in a clean sterile centrifuge tube, seal and store them at a constant temperature of 4℃, take samples once every day, store and test continuously for 7 days, observe whether the appearance of the transfection complex shows phenomena such as layering, precipitation, turbidity or color change, and use a dynamic light scattering instrument to determine its dispersion index.

[0153] The stability test results of the transfection complex prepared in this invention are shown in Table 5.

[0154] Table 5. Stability test results of the transfection complex

[0155] The transfection complex in Example 1 was formed by mixing a positive recombinant vector, liposomes Lipofectamine 3000, and serum-free DMEM medium. The positive recombinant vector lacked encapsulation and stabilization, leading to particle aggregation or uneven dispersion after 7 days, resulting in a low dispersion index and decreased stability. The transfection complex in Example 2 used chitosan modified with both (2-methyl-2-propenyl)succinic anhydride and (Z)-2-methyl-2-butenoic anhydride. The modified chitosan helped maintain particle dispersion, resulting in better dispersion uniformity and a higher dispersion index than Example 1 after 7 days. In Example 3, the modified chitosan used a higher amount of (Z)-2-methyl-2-butenoic anhydride than in Example 2. More (Z)-2-methyl-2-butenoic anhydride further enhanced the structural integrity and hydrophilicity of chitosan, improving particle dispersion stability and resulting in a higher dispersion index than Example 2. The transfection complex in Example 4 used... The modified chitosan was further modified with 2-(1-hydroxypentyl)-1-cyclopentanone on the basis of anhydride modification. This modification can enhance the binding stability of chitosan and positive recombinant carrier, reduce particle aggregation, and further improve stability and dispersion index. In Example 5, the amount of 2-(1-hydroxypentyl)-1-cyclopentanone was increased, resulting in a deeper degree of chitosan modification, a more significant improvement in particle dispersion, and optimal stability. The modified chitosan used in the transfection complex of Comparative Example 1 did not use (2-methyl-2-propenyl)succinic anhydride during preparation, and the modified chitosan used in the transfection complex of Comparative Example 2 did not use (Z)-2-methyl-2-butenoic anhydride during preparation, resulting in an incomplete modified chitosan structure, a weakened stabilizing effect on particle dispersion, and a lower stability than in Example 2. The transfection complex of Comparative Example 3 used unmodified chitosan, which had the basic encapsulation effect of chitosan, but the encapsulation and dispersion stabilization effect on the positive recombinant carrier was insufficient, indicating the necessity of modified chitosan.

[0156] Experiment 7: Apoptosis test of knockdown cell lines.

[0157] Test samples: Knockdown cell lines prepared in each example and comparative example.

[0158] Test method: 5 × 10⁵ cells were collected from each of the knockdown cell lines, the empty vector control group, and the blank control group after 48 h of culture for each example and comparative example. 5 Each sample was washed twice with PBS, and 100 μL of Annexin V-FITC binding solution was added, followed by 5 μL of Annexin V-FITC and 5 μL of LPI staining solution. The sample was incubated in the dark for 15 min. Then, 400 μL of Annexin V-FITC binding solution was added, and the sample was detected by flow cytometry.

[0159] The apoptosis test results of the knockdown cell lines prepared in this invention are shown in Table 6. Table 6. Results of apoptosis assay in knockdown cell lines

[0160] In Example 1, the positive recombinant vector lacked encapsulation and protection during the transfection complex preparation process, making it prone to degradation or aggregation during transfection and unable to effectively induce apoptosis, thus resulting in the lowest apoptosis rate. In Example 2, the transfection complex used chitosan co-modified with (2-methyl-2-propenyl)succinic anhydride and (Z)-2-methyl-2-butenoic anhydride. This modified chitosan enhanced the stability of the transfection complex, reduced the loss of the positive recombinant vector, improved transfection efficiency and AURKA knockdown effect, and showed better apoptosis induction than in Example 1. In Example 3, the amount of (Z)-2-methyl-2-butenoic anhydride was increased during the preparation of the modified chitosan. This component further optimized the structure of chitosan, enhanced its encapsulation ability for the positive recombinant vector, further improved the stability and transfection efficiency of the transfection complex, and resulted in a higher apoptosis rate than in Example 2. In Example 4, the modified chitosan, in addition to anhydride modification, was further modified with 2-(1-hydroxypentyl)-1-cyclopentanone. This modification further enhanced the... Example 5 enhanced the binding force between chitosan and the positive recombinant vector, reduced vector leakage during transfection, and achieved better transfection efficiency and AURKA knockdown effect than Example 3, with a stronger apoptosis-inducing effect. In Example 5, the amount of 2-(1-hydroxypentyl)-1-cyclopentanone used in the preparation of modified chitosan was higher than in Example 4, resulting in a deeper degree of chitosan modification, optimal transfection complex stability and efficiency, the best AURKA knockdown effect, and the strongest apoptosis-inducing effect, thus achieving the highest apoptosis rate. Comparative Example 1 did not use (2-methyl-2-propenyl)succinic anhydride in the preparation of modified chitosan, and Comparative Example 2 did not use (Z)-2-methyl-2-butenoic anhydride. The encapsulation and stabilization effect of the modified chitosan on the transfection complex was lower than in Example 2, resulting in worse transfection efficiency and AURKA knockdown effect, and a weaker apoptosis-inducing effect. Comparative Example 3 used unmodified chitosan, which had a weaker stabilizing effect on the transfection complex and a weaker apoptosis-inducing effect than Example 2.

[0161] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0162] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A recombinant vector, characterized in that: The recombinant vector comprises a small interfering RNA and a linearized vector. The small interfering RNA is used to knock down AURKA. The sense strand sequence of the small interfering RNA is shown in SEQ ID No. 1, which is 5'-GGAUUCUCCUUGAAGAUAUTT-3'. The antisense strand sequence of the small interfering RNA is shown in SEQ ID No. 2, which is 5'-AUAUCUUCAAGGAGAAUCCTT-3'.

2. A recombinant vector according to claim 1, characterized in that: The linearized vector is a pSIREN-RetroQ plasmid digested with enzymes, and the enzyme digestion includes restriction endonucleases BamHI and HindIII.

3. A recombinant vector according to claim 1, characterized in that: The small interfering RNA and the linearized vector are ligated in a ligation buffer using 4-6 U / μL of T4 DNA ligase, with the volume ratio of T4 DNA ligase to ligation buffer being 0.2-1:1, and the ligation temperature being 15-20℃.

4. A recombinant vector according to claim 1, characterized in that: The recombinant vector was verified by RT-PCR to confirm the correct insertion of small interfering RNA. The RT-PCR reaction system included the recombinant vector, 8-12 μmol / L of specific upstream primer for small interfering RNA, 8-12 μmol / L of specific downstream primer for small interfering RNA, 1-3×SYBRGreen PCR Mix and ultrapure water.

5. A recombinant vector according to claim 4, characterized in that: The RT-PCR reaction conditions include 94-96℃ pre-denaturation for 12-18 min, 94-96℃ denaturation for 8-12 s, and 58-62℃ annealing extension for 28-32 s, for a total of 35-45 cycles.

6. A transfection complex comprising the recombinant vector according to any one of claims 1-5, characterized in that: The transfection complex includes liposomes, and the liposomes include Lipofectamine 3000.

7. The transfection complex according to claim 6, characterized in that: The transfection complex includes anhydride-modified chitosan, wherein the mass-to-volume ratio of the anhydride-modified chitosan to the recombinant vector is 5 μg: 1-5 μL, and the volume ratio of the recombinant vector to the liposomes is 1-2:

4.

8. The transfection complex according to claim 7, characterized in that: The anhydride-modified chitosan is obtained by reacting chitosan with a modifier, the modifier including (2-methyl-2-propenyl)succinic anhydride and (Z)-2-methyl-2-butenoic anhydride.

9. The transfection complex according to claim 8, characterized in that: The mass ratio of (2-methyl-2-propenyl)succinic anhydride to (Z)-2-methyl-2-butenoic anhydride is 0.5:0.5-1.5, and the mass ratio of (2-methyl-2-propenyl)succinic anhydride to chitosan is 0.3-0.5:

1.

10. The use of the recombinant vector according to any one of claims 1-5 or the transfection complex according to any one of claims 6-9 in the preparation of antitumor drugs, characterized in that: The antitumor drug can specifically knock down the expression of AURKA protein.

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