SiRNA of targeted RPS4X gene, LAMB3-PI3K-AKT signaling pathway inhibitor, ovarian cancer drug and application of siRNA and LAMB3-PI3K-AKT signaling pathway inhibitor

Abstract

The invention relates to the technical field of RPS4X, in particular to siRNA of a targeted RPS4X gene, an LAMB3-PI3K-AKT signal channel inhibitor, an ovarian cancer drug and application of the siRNA, the LAMB3-PI3K-AKT signal channel inhibitor and the ovarian cancer drug. The siRNA interferes with the expression of the RPS4X gene in a targeted manner. The siRNA and the LAMB3-PI3K-AKT signal channel inhibitor act on in-vitro ovarian cancer cells and in-vivo ovarian cancer tissues, and play roles in inhibiting proliferation, migration and invasion of the in-vitro ovarian cancer cells and the in-vivo ovarian cancer tissues. The siRNA and LAMB3-PI3K-AKT signal channel inhibitors of multiple targeted RPS4X genes provided by the embodiment have the effects of promoting ovarian cancer cells and tissue apoptosis and inhibiting tumor angiogenesis, have obvious ovarian cancer prevention and treatment effects and have the application prospect of being developed as ovarian cancer drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of RPS4X, and particularly relates to siRNA targeting the RPS4X gene, an inhibitor of the LAMB3-PI3K-AKT signaling pathway, an ovarian cancer drug, and applications thereof. Background Art

[0002] The RPS4X gene is located on the X chromosome, specifically in the region of Xp22.31. The protein encoded by the RPS4X gene is the X-chromosome homolog of ribosomal protein S4 (X-linked ribosomal protein S4, RPS4X), which is a component of ribosomes and participates in the process of protein synthesis.

[0003] RPS4X is one of the components of the small ribosomal subunit and plays a key role in the assembly of ribosomes. It participates in the formation of specific ribosomal structures, provides a platform for the binding and translation of mRNA, and is crucial for all stages of protein synthesis, including initiation, elongation, and termination, ensuring the normal synthesis and renewal of proteins in cells.

[0004] The RPS4X gene indirectly affects cell growth and proliferation by participating in protein synthesis. Normal protein synthesis is the basis for cell growth, division, and proliferation. The normal expression and function of the RPS4X gene are necessary for maintaining the normal growth rate and proliferation ability of cells. At all stages of the cell cycle, proteins synthesized with the participation of RPS4X are required to provide the necessary material basis and regulatory signals.

[0005] Mutations or abnormal expressions of the RPS4X gene may be associated with certain neurodevelopmental diseases such as intellectual disabilities and developmental delays. Due to its important role in brain development and nerve cell function, gene alterations may affect protein synthesis and function in nerve cells, thereby leading to abnormal neurodevelopment, manifested as symptoms such as decreased cognitive ability, learning disabilities, and behavioral abnormalities. Summary of the Invention

[0006] The present invention discovers that RPS4X, as an RNA-binding protein, drives the invasion and metastasis of ovarian cancer cells by activating the LAMB3-PI3K-AKT signaling pathway.

[0007] The LAMB3-PI3K-AKT signaling pathway is a signaling pathway that plays an important role in cell biological processes and consists of LAMB3 (laminin β3), PI3K (phosphatidylinositol 3-kinase), and AKT (protein kinase B). LAMB3 participates in forming an important part of the basement membrane, can bind to receptors such as integrins on the cell surface, thereby mediating the adhesion between cells and the extracellular matrix, and can further activate the intracellular signaling pathway. PI3K is a lipid kinase and can be divided into type I, type II, and type III. In the LAMB3-PI3K-AKT signaling pathway, usually involved is type I PI3K. PI3K can be activated by a variety of cell surface receptors, such as integrins, growth factor receptors, etc. Activated PI3K can phosphorylate phosphatidylinositol-4,5-bisphosphate (PIP2) to generate phosphatidylinositol-3,4,5-trisphosphate (PIP3). AKT is a serine / threonine protein kinase, also known as PKB. AKT is an important downstream effector molecule of PI3K. PIP3 can recruit AKT to the cell membrane and make it phosphorylated and activated under the action of phosphoinositide-dependent kinase 1 (PDK1), etc.

[0008] The present invention discovers that RPS4X can not only bind a large amount of oncogene mRNA, but also bind LAMB3 mRNA and activate its expression, thereby recruiting and activating PI3K. Activated PI3K catalyzes the conversion of PIP2 into PIP3, and PIP3 accumulates on the cell membrane and plays a role as a second messenger. PIP3 binds to the PH domain of AKT, causing AKT to transfer from the cytoplasm to the cell membrane. Under the action of kinases such as PDK1 and mTORC2, the Thr308 and Ser473 sites of AKT are phosphorylated, and thus AKT is fully activated. As the LAMB3-PI3K-AKT signaling pathway is activated, it can promote the invasion and migration of ovarian cancer cells.

[0009] Therefore, the present invention provides siRNAs targeting the RPS4X gene, inhibitors of the LAMB3-PI3K-AKT signaling pathway, ovarian cancer drugs, and their applications. The siRNAs target and interfere with the expression of the RPS4X gene. When the siRNAs and inhibitors of the LAMB3-PI3K-AKT signaling pathway act on ovarian cancer cells in vitro and ovarian cancer tissues in vivo, they both play a role in inhibiting their proliferation, migration, and invasion. The various siRNAs targeting the RPS4X gene and inhibitors of the LAMB3-PI3K-AKT signaling pathway provided by the examples have the effects of promoting apoptosis of ovarian cancer cells and tissues and inhibiting their tumor angiogenesis, have obvious preventive and therapeutic effects on ovarian cancer, and have the application prospect of developing into ovarian cancer drugs.

[0010] The first aspect of the present invention provides a biomarker associated with the prognosis of ovarian cancer, including RPS4X.

[0011] In a second aspect of the present invention, a kit for predicting the prognosis of ovarian cancer is provided, which comprises at least one of RT-PCR reagents for detecting the relative expression level of RPS4X mRNA and anti-RPS4X monoclonal antibodies. Among them, the RT-PCR reagents for detecting the relative expression level of RPS4X mRNA include primer pairs shown in SEQ ID NO:13 and SEQ ID NO:14, and primer pairs shown in SEQ ID NO:15 and SEQ ID NO:16.

[0012] In a third aspect of the present invention, provided is the use of at least one of the RT-PCR reagents for detecting the relative expression level of RPS4X mRNA and anti-RPS4X monoclonal antibodies in the second aspect in the preparation of a kit for detecting the prognosis of ovarian cancer.

[0013] In a fourth aspect of the present invention, an RPS4X inhibitor is provided, which comprises at least one of siRNA targeting the RPS4X gene, a recombinant lentivirus containing the siRNA, and a recombinant lentiviral vector containing the siRNA.

[0014] In a fifth aspect of the present invention, an LAMB3-PI3K-AKT signaling pathway inhibitor is provided, which comprises at least one of siRNA targeting the RPS4X gene, a recombinant lentivirus containing the siRNA, and a recombinant lentiviral vector containing the siRNA.

[0015] In a sixth aspect of the present invention, an ovarian cancer drug is provided, which uses at least one of siRNA targeting the RPS4X gene, a recombinant lentivirus containing the siRNA, and a recombinant lentiviral vector containing the siRNA as an active ingredient.

[0016] In a specific embodiment, the siRNA targeting the RPS4X gene is at least one of the siRNAs shown in SEQ ID NO:1 and 2, the siRNAs shown in SEQ ID NO:3 and 4, the siRNAs shown in SEQ ID NO:5 and 6, or the siRNAs shown in SEQ ID NO:7 and 8.

[0017] In a specific embodiment, the recombinant lentiviral vector containing the siRNA is the GV493 plasmid (GeneChem) carrying the nucleotide sequence shown in at least one of the siRNAs shown in SEQ ID NO:1 and 2, the siRNAs shown in SEQ ID NO:3 and 4, the siRNAs shown in SEQ ID NO:5 and 6, or the siRNAs shown in SEQ ID NO:7 and 8.

[0018] In a specific embodiment, the recombinant lentiviral vector containing the siRNA is a lentivirus carrying the nucleotide sequence shown in at least one of the siRNAs shown in SEQ ID NO: 1 and 2, the siRNAs shown in SEQ ID NO: 3 and 4, the siRNAs shown in SEQ ID NO: 5 and 6, or the siRNAs shown in SEQ ID NO: 7 and 8.

[0019] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present invention.

[0020] Advantageous technical effects of the present invention:

[0021] The present invention provides interfering RNAs capable of interfering with the expression of RPS4X in ovarian cancer cells, and through detection, it is found that these interfering RNAs can also inhibit the LAMB3-PI3K-AKT signaling pathway in ovarian cancer cells, thereby inhibiting the migration and invasion of ovarian cancer cells.

[0022] The present invention finds through comparison that the interfering RNAs provided in the examples have significantly better interfering effects on ovarian cancer cells than the comparative examples. Moreover, the interfering RNAs provided in the examples have better inhibitory effects on the LAMB3-PI3K-AKT signaling pathway, migration, and invasion of ovarian cancer cells than the comparative examples.

[0023] In addition, the present invention finds through in vivo experiments that the lentivirus carrying the interfering RNA sequence provided in the examples can inhibit the growth of tumor volume in ovarian cancer tumor-bearing mice, and can inhibit the expression of NRP-2, RAD51C, and VEGF in ovarian cancer tissues, inhibit genomic instability in tumor tissues, inhibit angiogenesis, and inhibit lymphatic metastasis of tumor cells, thereby inhibiting the growth, metastasis, and invasion of ovarian cancer cells. Moreover, the lentivirus carrying the interfering RNA sequence provided in the examples has obvious inhibitory effects on NRP-2, RAD51C, and VEGF in ovarian cancer in vivo tissues, and has the application prospect of developing into an ovarian cancer drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A Analysis of the expression levels of RPS4X in ovarian cancer tissues and adjacent tissues (red represents the ovarian cancer group, and blue represents the control group).

[0025] Figure 1B Grade analysis chart of RPS4X in TCGA ovarian cancer data (p-value < 0.001).

[0026] Figure 1C Stage analysis chart of RPS4X in TCGA ovarian cancer data (p-value < 0.001).

[0027] Figure 1D Prognostic graph of RPS4X expression in ovarian cancer.

[0028] Figure 2 Graph showing the results of the relative expression levels of RPS4X mRNA in SKOV3 cells and Anglne cells interfered with by siRNA-1, siRNA-2, siRNA-3, siRNA-4, siNC1, siNC2, and siNC3 respectively. "*" indicates p-value < 0.05, "**" indicates p-value < 0.01, "***" indicates p-value < 0.001, and "ns" indicates no significant difference.

[0029] Figure 3 Graph showing the results of RPS4X protein expression in SKOV3 cells and Anglne cells interfered with by siRNA-1, siRNA-2, siRNA-3, siRNA-4, siNC1, siNC2, and siNC3 respectively.

[0030] Figure 4 Graph showing the results of the protein expression of PI3K, P-PI3K, AKT, and P-AKT in SKOV3 cells interfered with by siRNA-1, siRNA-2, siRNA-3, siRNA-4, siNC1, siNC2, and siNC3 respectively.

[0031] Figure 5 Graph showing the migration results of SKOV3 cells and Anglne cells interfered with by siRNA-1, siRNA-2, siRNA-3, siRNA-4, siNC1, siNC2, and siNC3 respectively. "*" indicates p-value < 0.05, "**" indicates p-value < 0.01, "***" indicates p-value < 0.001, and "ns" indicates no significant difference.

[0032] Figure 6 Graph showing the invasion results of SKOV3 cells and Anglne cells interfered with by siRNA-1, siRNA-2, siRNA-3, siRNA-4, siNC1, siNC2, and siNC3 respectively. "*" indicates p-value < 0.05, "**" indicates p-value < 0.01, and "ns" indicates no significant difference.

[0033] Figure 7 Graph showing the results of the protein expression of NRP-2, RAD51C, and VEGF in the ovarian cancer tissues of ovarian cancer mice intervened with lentiviruses carrying siRNA-1, siRNA-2, siRNA-3, siRNA-4, siNC1, siNC2, and siNC3 respectively.

[0034] Figure 8For Figure 7 Statistical chart. "**" indicates p-value < 0.01, "***" indicates p-value < 0.05, and "ns" indicates no significant difference. Detailed implementation mode

[0035] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Reagents not specifically described in detail in the present invention are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0036] 1. Analysis of RPS4X expression in ovarian cancer tissues and normal tissues using the TCGA public database

[0037] The proteomic data of ovarian cancer in the TCGA public database was systematically analyzed, and the research group analyzed ovarian cancer and control group samples. The results showed that in the ovarian cancer data in the TCGA database, the protein expression level of RPS4X was analyzed in terms of expression level, disease stage and grade, and it was found that RPS4X was highly expressed in ovarian cancer tissues ( Figure 1A ); the results of clinical staging and grading of ovarian cancer showed that the expression level of RPS4X was significantly up-regulated in patients with stage 1, 2, and 3 ovarian cancer / grade ( Figure 1B and 1C ), and it was found that the prognosis of ovarian cancer patients with high expression of RPS4X was poor ( Figure 1D ).

[0038] 2. Targeted interference with RPS4X expression

[0039] (1) siRNA

[0040] Based on the siRNA method, the present invention realizes the inhibition of RPS4X expression in ovarian cancer cells. For this purpose, the present invention provides siRNAs that interfere with RPS4X. These siRNAs can be synthesized by chemical or biological methods.

[0041] Among them, the sense strand of siRNA-1 that interferes with RPS4X is: 5'-GAGGAACAGACUUAAGUAUTT-3', SEQ ID NO: 1. The antisense strand is: 5'-AUACUUAAGUCUGUUCCUCTT-3', SEQ ID NO: 2.

[0042] The sense strand of siRNA-2 targeting RPS4X is: 5’-GUGCUAACCUAGGAAGAAUTT-3’, SEQ ID NO:3. The antisense strand is: 5’-AUUCUUCCUAGGUUAGCACTT-3’, SEQ ID NO:4.

[0043] The sense strand of siRNA-3 targeting RPS4X is: 5’-CCACUCGACUUUCCAACAUTT-3’, SEQ ID NO:5. The antisense strand is: 5’-AUGUUGGAAAGUCGAGUGGTT-3’, SEQ ID NO:6.

[0044] The sense strand of siRNA-4 targeting RPS4X is: 5’-AGCAUCUGAAGCGGGUGGCUUCAAGAGA-3’, SEQID NO:7. The antisense strand is: 5’-GCCACCCGCUUCAGAUGCUUUUUU-3’, SEQ ID NO:8.

[0045] As a control, the present invention also provides that the sense strand of siRNA (siNC1) targeting RPS4X is: 5’-UUCUCCGAACGUGUCACGUTT-3’, SEQ ID NO:9. The antisense strand is: 5’-ACGUGACACGUUCGGAGAATT-3’, SEQID NO:10.

[0046] The present invention also provides that the sense strand of siRNA (siNC2) targeting RPS4X is: 5’-CAGATCTTTGTACGTAATTAA-3’, SEQ ID NO:11. The antisense strand is: 5’-CGGGAGAGAATTTCCGTCTGA-3’; SEQ ID NO:12.

[0047] The present invention also provides siRNA (siNC3) targeting RPS4X: Catalog#AM16708, Thermo Fisher.

[0048] (2) Transfecting ovarian cancer cells with siRNA

[0049] The present invention transfects the above double-stranded siRNA into ovarian cancer cells, which can not only interfere with the expression of RPS4X in ovarian cancer cells, but also inhibit the LAMB3-PI3K-AKT signaling pathway, thereby inhibiting their proliferation, migration and invasion, promoting apoptosis of ovarian cancer cells and tissues and inhibiting their tumor angiogenesis, having an obvious preventive and therapeutic effect on ovarian cancer, and having an application prospect of developing as an ovarian cancer drug.

[0050] The present invention provides a method for interfering with the expression of RPS4X in ovarian cancer cells or inhibiting the LAMB3-PI3K-AKT signaling pathway. The method includes: mixing at least one of the siRNA interfering with RPS4X and the siRNA interfering with KRAS with a transfection reagent to form a complex of siRNA and the transfection reagent; co-culturing the complex with ovarian cancer cells; harvesting and screening positive cells from the co-culture, and the positive cells are ovarian cancer cells in which the LAMB3-PI3K-AKT signaling pathway is inhibited.

[0051] In some embodiments, the method for interfering with the expression of RPS4X in ovarian cancer cells or inhibiting the LAMB3-PI3K-AKT signaling pathway specifically includes:

[0052] 1) Chemically synthesize the above-mentioned siRNA interfering with RPS4X or siRNA interfering with KRAS, and dissolve it in RNase-free water to 20 μM.

[0053] 2) Inoculate SKOV3 cells (human ovarian cancer cells, CC-Y1473, Shanghai Enzyme Research Biotechnology) or Anglne cells (human ovarian cancer cells, BTCC-1255) in the logarithmic growth phase at 2×10 6 cells / well into a 6-well plate, add 2 ml of complete medium containing 10% fetal bovine serum to each well, and culture at 37 °C and 5% CO2 until the cells adhere and reach 50% confluence.

[0054] 3) Preparation of transfection complex: Operate according to the instructions of Lipofectamine RNAiMAX transfection reagent. In a sterile centrifuge tube, incubate 100 μL of the serum-free medium solution of 50 pmol siRNA for 5 min. Take 5 μL of the transfection reagent and dilute it to 100 μL with serum-free medium, mix gently, and incubate at room temperature for 5 min. Then mix the diluted siRNA and the transfection reagent, mix gently, and incubate at room temperature for 20 min to form a complex of siRNA and the transfection reagent.

[0055] 4) Cell transfection: Aspirate the original medium in the 6-well plate, and add 800 μL of Opti-MEM medium to each well. Then add the complex of siRNA and the transfection reagent dropwise to the cell culture wells, gently shake the culture plate to make the complex evenly distributed. Put the culture plate back into the incubator and continue to culture at 37 °C and 5% CO2 for 6 h, then replace it with complete medium and continue to culture for 48 h. Screen positive cells from the culture, and the positive cells are ovarian cancer cells in which RPS4X is interfered and / or the LAMB3-PI3K-AKT signaling pathway is inhibited.

[0056] (3) Transfect ovarian cancer cells with lentivirus or vector carrying siRNA

[0057] The embodiment also provides a lentiviral vector carrying the above siRNA, and uses this vector as a mediator to interfere with RPS4X. Specifically, this recombinant lentiviral vector is the GV493 plasmid (GeneChem) carrying the above siRNA cloning sequence.

[0058] Specifically, the preparation method of this recombinant lentiviral vector includes: obtaining a linearized GV493 fragment by enzymatic digestion, ligating a DNA molecule of any one of SEQ ID NOs: 1-6 with the linearized GV493 fragment to obtain a recombinant fragment; transferring the recombinant fragment into Escherichia coli; screening positive clones from the culture of the transformants; and extracting the recombinant lentiviral vector from the culture of the positive clones.

[0059] Among them, the step of obtaining the linearized GV493 fragment includes: preparing a plasmid digestion reaction solution, digesting the plasmid digestion reaction solution at 37 °C for 3 h, and performing agarose gel recovery on the digested solution to obtain the linearized GV493 fragment. Among them, the plasmid digestion reaction solution contains 2 μg of lenti-CRISPRv2, 1 μL of 10 U / μL AgeI, 1 μL of 10 U / μL EcoRI, 5 μL of 10×CutSmart Buffer and the balance of water in a volume of 50 μL.

[0060] Among them, the step of ligating a DNA molecule of any one of SEQ ID NOs: 1-8 with the linearized GV493 fragment to obtain a recombinant fragment includes: preparing a ligation reaction solution, and reacting the ligation reaction solution at 20 °C for 1 h. Among them, the ligation reaction solution contains 100 ng of the linearized GV493 fragment, 100 ng of a DNA molecule of any one of SEQ ID NOs: 1-8, 2 μL of 10×T4 DNA Ligation Buffer, 1 μL of T4 DNA ligase and the balance of water in a volume of 20 μL.

[0061] Based on the recombinant lentiviral vector obtained by the above method, when it is transfected into cells, it can synthesize recombinant lentivirus carrying any one of the nucleotide sequences of SEQ ID NOs: 1-8 in vivo.

[0062] Among them, the method for preparing the recombinant lentivirus carrying any one of the nucleotide sequences of SEQ ID NOs: 1-8 specifically includes:

[0063] 1) Seed 4×10 6 HEK293T cells in a 10 cm dish until they reach 80% confluence;

[0064] 2) Mix 30 μg of the recombinant lentiviral vector carrying any one of the nucleotide sequences of SEQ ID NO: 1-8, 15 μg of pHelper1.0, and 10 μg of pHelper2.0 and add them to 1.5 mL of Opti-MEM to obtain a plasmid dilution;

[0065] 3) Add 80 μL of Lipo-2000 to 1.5 mL of Opti-MEM and incubate at room temperature for 10 min to obtain a Lipo-2000 dilution;

[0066] 4) Mix the plasmid dilution and the Lipo-2000 dilution and incubate at room temperature for 30 min to obtain a complex formed by the full binding of the plasmid and Lipo-2000.

[0067] 5) Remove the medium from the 10-cm dish, add the plasmid-liposome complex dropwise to the dish, gently shake the dish to evenly distribute the complex on the cell surface, add an appropriate amount of complete medium to 10 mL, and place it back in the incubator for continued culture.

[0068] 6) 72 hours after transfection, collect the supernatant containing virus particles. Transfer the supernatant to a sterile centrifuge tube, centrifuge at 3000 rpm for 15 min to remove cell debris, and concentrate the virus by ultracentrifugation.

[0069] 7) The collected virus solution needs to be assayed for its titer. Commonly used methods include fluorescence quantitative method (for lentivirus with fluorescent label), TCID 50 method, etc., to determine the infectivity of the virus and the amount of virus required for subsequent experiments.

[0070] Through the above examples, recombinant lentivirus carrying any one of the nucleotide sequences of SEQ ID NO: 1-8 was obtained. Transferring the recombinant lentivirus into ovarian cancer cells can obtain ovarian cancer cells with RPS4X knockdown.

[0071] Therefore, the present invention discloses a method for preparing ovarian cancer cells with RPS4X knockdown or inhibiting the LAMB3-PI3K-AKT signaling pathway. The method includes: obtaining ovarian cancer cells and recombinant lentivirus carrying any one of the nucleotide sequences of SEQ ID NO: 1-8; mixing and transfecting the recombinant lentivirus solution with the culture solution of ovarian cancer cells, culturing the transfected solution, and screening ovarian cancer cells with RPS4X knockdown.

[0072] In some embodiments, the method for preparing ovarian cancer cells with RPS4X knockdown or inhibiting the LAMB3-PI3K-AKT signaling pathway specifically includes:

[0073] 1) One day before transfection, in a 24-well plate, inoculate about 2-5×104 Cells were added with an appropriate amount of complete medium to reach a confluence of 40% at the time of transfection, and then the culture plates were returned to the incubator for further culture.

[0074] 2) With an MOI of 10 and 5×10 4 cells per well and a lentivirus titer of 1×10 8 TU / mL, 5 μL of the lentivirus stock solution was added to each well and then diluted to 200 μL with serum-free medium.

[0075] 3) 2 μL of 8 mg / mL polybrene solution was added to the 200 μL virus dilution, gently mixed, and incubated at room temperature for 20 min to improve the infection efficiency of the lentivirus.

[0076] 4) The original medium in the cell culture plates was aspirated, the cells were gently washed once with PBS, and then the virus dilution containing polybrene was added to the cell culture wells. The culture plates were gently shaken to evenly distribute the virus solution. The culture plates were returned to the incubator for further culture.

[0077] 5) 18 hours after transfection, the medium containing the virus was aspirated, and an appropriate amount of fresh complete medium was added to continue culturing the cells to reduce the toxicity to the cells.

[0078] 6) After 48 h of infection, puromycin was added for screening to kill the cells that were not successfully infected, and the successfully infected cells would survive. Puromycin needed to be added to the subsequent medium all the time.

[0079] 7) After 2 days of puromycin screening, WB or Real-time PCR could be used to verify the knockdown or reduction of RPS4X.

[0080] 3. RT-PCR detection

[0081] Total RNA in the fresh ovarian cancer cells before and after the above transfection was extracted using Trizol reagent. The concentration of the total RNA was measured using NANODROP2000, and the quality of the RNA was analyzed by agarose gel electrophoresis. The total RNA with determined concentration and quality was reverse-transcribed into cDNA. The reverse transcription system was calculated based on 10 μL and included 2 μL of RT Master Mix (5×), 100 ng - 1 μg of total RNA, and the remaining volume of Nuclease-free water.

[0082] Reverse transcription was performed to obtain cDNA samples for qPCR. The qPCR reaction system (20 μL in total) contained 1 μL of cDNA, 0.8 μL of 10 μM forward primer, 0.8 μL of 10 μM reverse primer, 10 μL of 1×TSING Master SYBR Green I qPCR Mix-UDG (Without ROX), and the balance of double-distilled water. The qPCR reaction steps included: 50 °C for 2 min; 95 °C for 2 min; 95 °C for 15 s; 40 cycles; 60 °C for 1 min. The expression of RNA was normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Data analysis was performed using Bio-Rad CFX96 Manager software.

[0083] Among them, the forward primer for detecting RPS4X mRNA was: F1: shown as SEQ ID NO:13, CAAGGTCCGAACTGATATAAC. R1: shown as SEQ ID NO:14, CCTCAGGTGTAATACGATGT.

[0084] The forward primer for detecting GAPDH mRNA was: F2: shown as SEQ ID NO:15, GGTCGGAGTCAACGGATTTG. R2: shown as SEQ ID NO:16, GGAAGATGGTGATGGGATTTC.

[0085] As Figure 2 shown, the relative expression levels of RPS4X mRNA in SKOV3 cells or Anglne cells transfected with siRNA-1, siRNA-2, siRNA-3, and siRNA-4 were significantly lower than those in the control group (NC), which were SKOV3 cells not transfected with siRNA. However, there was no significant difference in the relative expression level of RPS4X mRNA in SKOV3 cells transfected with siNC1 and siNC3 compared with the control group. This indicates that the siRNA-1, siRNA-2, siRNA-3, and siRNA-4 provided by the present invention have an interfering effect on the expression of RPS4X in SKOV3 cells or Anglne cells, and siRNA-4 has the best interfering effect on RPS4X in SKOV3 cells or Anglne cells.

[0086] 4. WB detection

[0087] Ovarian cancer cells before and after transfection were lysed with RIPA lysis buffer, and total proteins were extracted. The protein concentration was detected by the BCA method. SDS-PAGE electrophoresis was performed, and the gel was transferred to a PVDF membrane using a membrane transfer instrument and blocked at room temperature for 1-2 h. The primary antibody was added and incubated at 4 °C for 12 h. After washing, the secondary antibody (goat anti-rabbit IgG labeled with horseradish peroxidase, diluted with TBST) was added and incubated at room temperature for 2 h, and then developed. The protein expression level was analyzed using ImageJ software with β-Actin as a control. Among them, the primary antibody was the RPS4X antibody, 14799-1-AP, Proteintech, diluted at a ratio of 1:1000; or the PI3K antibody, sc-365290, Santa Cruz Biotechnology, Inc., diluted at a ratio of 1:1000; or the AKT antibody, 60203-2-Ig, diluted at a ratio of 1:5000; or the Phospho-PI3 Kinase p85(Tyr458) / p55(Tyr199) antibody, #4228, Cell Signaling Technology, Inc., diluted at a ratio of 1:1000; or the Phospho-Akt(Ser473) antibody, #9271, Cell Signaling Technology, Inc., diluted at a ratio of 1:1000.

[0088] As Figure 3 shown, the relative expression levels of RPS4X protein in Anglne cells transfected with siRNA-1, siRNA-2, siRNA-3, and siRNA-4 were significantly lower than those in the control group (NC) of Anglne cells not transfected with siRNA, while the relative expression levels of RPS4X protein in Anglne cells transfected with siNC1, siNC2, and siNC3 were not significantly different from those in the control group. This indicates that the siRNA-1, siRNA-2, siRNA-3, and siRNA-4 provided by the present invention have an interfering effect on the expression of RPS4X in SKOV3 cells, and the interfering effect of siRNA-4 on RPS4X in SKOV3 cells is the best.

[0089] As Figure 4As shown, the expression levels of PI3K protein, AKT protein, p-PI3K protein, and p-AKT protein in SKOV3 cells transfected with siRNA-1, siRNA-2, siRNA-3, and siRNA-4 were significantly lower than those in the control group (NC) of SKOV3 cells not transfected with siRNA. However, the expression levels of PI3K protein, AKT protein, p-PI3K protein, and p-AKT protein in SKOV3 cells transfected with siNC1, siNC2, and siNC3 were not significantly different from those in the control group or only a few proteins showed significant differences. This indicates that the siRNA-1, siRNA-2, siRNA-3, and siRNA-4 provided by the present invention have an interfering effect on the expression of PI3K protein, AKT protein, p-PI3K protein, and p-AKT protein in SKOV3 cells, and siRNA-4 has the best interfering effect on the PI3K protein, AKT protein, p-PI3K protein, and p-AKT protein in SKOV3 cells.

[0090] 5. Cell phenotype detection

[0091] (1) Detection steps

[0092] The ovarian cancer cells SKOV3 cells or Anglne cells before and after transfection were cultured in a complete medium containing 10% fetal bovine serum at 37°C and 5% CO2 until the logarithmic phase. After removing the medium, they were washed with PBS, digested with trypsin for 1 - 3 minutes, and pipetted into single cells. Then, 15 μL of the cell suspension was taken for counting and waiting for testing.

[0093] After diluting the Matrigel in the Transwell chamber at a ratio of 1:8, it was incubated at 37°C for 2 hours until the Matrigel solidified. After removing the excess supernatant, subsequent experiments were carried out without treating the transferred Transwell chamber. The above-mentioned cells to be tested were centrifuged to discard the culture medium, washed 1 - 2 times with PBS, and resuspended in serum-free medium.

[0094] Take 200 μL of resuspended SK-OV-3 cells (invasion: 150,000 cells / chamber; metastasis: 50,000 cells / chamber) or take 200 μL of resuspended Anglne cells (invasion: 200,000 cells / chamber; metastasis: 100,000 cells / chamber) and add them to the Transwell chamber respectively. Add 600 μL of medium containing 10% FBS to the lower chamber of the 24-well plate. After culturing at 37°C for 48 hours, take out the Transwell chamber, discard the culture medium in the well, wash 2 times with PBS; fix with 4% PFA for 20 minutes, wash 2 times with PBS; stain with 0.1% crystal violet for 10 minutes, wash 2 times with PBS, and wipe off the cells in the upper chamber with a cotton swab. Observe the transferred cells under a microscope, randomly select 3 fields of view that can represent the cell distribution in the whole chamber for photographing, and count the number of migrated and invaded cells.

[0095] AsFigure 5 As shown, the migration numbers of SKOV3 cells or Anglne cells transfected with siRNA-1, siRNA-2, siRNA-3, and siRNA-4 were significantly lower than those of the control group, while the migration numbers of SKOV3 cells or Anglne cells transfected with siNC1, siNC2, and siNC3 were slightly lower than those of the control group and significantly higher than those of SKOV3 cells or Anglne cells transfected with siRNA-1, siRNA-2, siRNA-3, and siRNA-4. Moreover, the migration number of SKOV3 cells or Anglne cells transfected with siRNA-4 was the lowest. This indicates that the siRNA provided by the present invention can inhibit the migration of ovarian cancer cells, especially siRNA-4.

[0096] As Figure 6 shown, the invasion numbers of SKOV3 cells or Anglne cells transfected with siRNA-1, siRNA-2, siRNA-3, and siRNA-4 were significantly lower than those of the control group, while the invasion numbers of SKOV3 cells or Anglne cells transfected with siNC1, siNC2, and siNC3 were slightly lower than those of the control group and significantly higher than those of SKOV3 cells or Anglne cells transfected with siRNA-1, siRNA-2, siRNA-3, and siRNA-4. Moreover, the invasion number of SKOV3 cells or Anglne cells transfected with siRNA-4 was the lowest. This indicates that the siRNA provided by the present invention can inhibit the invasion of ovarian cancer cells, especially siRNA-4.

[0097] 6. In vivo experiments

[0098] (1) Experimental animals

[0099] Female Balb / c nude mice (4 weeks old, SPF breeding conditions, purchased from Jiangsu Jicui Yakang Biotech Co., Ltd.). This animal experiment research was approved by the Ethics Committee of the Affiliated Tumor Hospital of Xinjiang Medical University (approval number: K-2024179), and all experiments complied with the requirements of the Declaration of Helsinki. SKOV3 cells (human ovarian cancer cells, CC-Y1473, Shanghai Meiyan Biotech).

[0100] (2) Group experiments

[0101] The female Balb / c nude mice were raised at a room temperature of 20 - 26 °C, a humidity of 40% - 70%, and a 12h / 12h light / dark condition. Take SKOV3 cells in the logarithmic growth phase and adjust the cell density to 1×10 8cells / mL. Inoculate 0.1 mL in the right dorsal area of nude mice, and divide them into a model group (NC), a control group and an interference group. Among them, after injecting SKOV3 cells into the mice in the interference group, inject 50 μL of lentivirus containing the above siRNA-1 to 4 with a titer of 1×10 8 TU / mL every other day, and inject three times in total. After injecting SKOV3 cells into the mice in the control group, inject 50 μL of lentivirus containing the above siNC1-3 with a titer of 1×108 TU / mL every other day, and inject three times in total. Inject the same volume of normal saline into the mice in the model group. Measure the long and short diameters of the tumor once every 3 days, observe for 1 month, and calculate the tumor volume.

[0102] The tumor volume of the nude mice in the model group on the 10th day was 715.2 mm 3 . In the interference group, the tumor volumes of the mice interfered with by lentivirus of siRNA-1 to 4 were 674.3 mm 3 , 519.8 mm 3 , 551.8 mm 3 and 258.5 mm 3 respectively on the 10th day. In the control group, the tumor volumes of the mice interfered with by lentivirus of siNC1 to 3 were 674.2 mm 3 , 694.3 mm 3 and 701.3 mm 3 respectively on the 10th day. This shows that the ovarian cancer model was successfully constructed by implanting SKOV3 cells into mice. And lentivirus of siRNA-1 to 4 can interfere with tumor growth to a certain extent, and the infection effect of lentivirus of siRNA-4 is the best.

[0103] (3) WB detection of nude mouse tumor tissues

[0104] Take 50 mg of tumor tissue, add RIPA lysis buffer, grind with a grinder, and extract total protein. Centrifuge at 4°C in a high-speed centrifuge (12,000 rpm) for 10 min, take the supernatant, and quantify the total protein using a BCA protein quantification kit. Denature the protein sample and perform (SDS-PAGE) gel electrophoresis for 1.5 h. Block the PVDF membrane (Millipore) with skim milk powder, incubate with the primary antibody overnight at 4°C, the next day incubate the PVDF membrane with the secondary antibody at room temperature for 2 h, soak the PVDF membrane with luminescent solution, and develop using an ultra-high sensitivity chemiluminescence imaging system. The antibodies used and their corresponding dilution factors are: 1:1000 (Rabbit Anti-RAD51C, Rabbit Anti-VEGF, Rabbit Anti-NRP-2) and 1:1200 (HRP conjugated Goat Anti-Mouse IgG(H+L), HRP conjugated Goat Anti-Mouse IgG(H+L), Mouse Anti-β-Actin).

[0105] Figure 7 WB images of tumor tissues of mice in each group Figure 8 are Figure 7 statistical charts of the expression levels of each protein in Figure 7 and Figure 8 It can be seen from

[0106] that in the interference group, the lentiviruses of siRNA-1 to 3 did not have inhibitory effects on NRP-2 and RAD51C of tumor cells, but only had interference inhibitory effects on VEGF, while the lentivirus of siRNA-4 had interference inhibitory effects on NRP-2, RAD51C and VEGF of tumor cells. The lentiviruses of siNC1 to 3 did not have inhibitory effects on NRP-2 and RAD51C of tumor cells, but only had interference inhibitory effects on VEGF. This shows that the lentivirus of siRNA-4 provided by the present invention has interference inhibitory effects on NRP-2, RAD51C and VEGF of ovarian cancer cells.

[0107] RAD51C, VEGF, and NRP-2 all play key roles in regulating processes such as tumor cell proliferation, migration, and apoptosis. They are all associated with tumor infiltration, invasion, and metastasis, and are also potential targets for targeted therapy of malignant tumors.

[0108] From this, it can be seen that the siRNA, related lentiviral vectors, and lentiviruses provided by the present invention can interfere with the expression of RAD51C, VEGF, and NRP-2 genes in ovarian cancer tissues, inhibit genomic instability in tumor tissues, inhibit angiogenesis, and inhibit lymphatic metastasis of tumor cells, thereby inhibiting the growth, metastasis, and invasion of ovarian cancer cells.

[0109] As described above, the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A biomarker associated with the prognosis of ovarian cancer, characterized in that, It includes RPS4X.

2. A kit for predicting the prognosis of ovarian cancer, characterized in that, It includes at least one of the RT-PCR reagent for detecting the relative expression level of RPS4X mRNA and the anti-RPS4X monoclonal antibody; wherein, the RT-PCR reagent for detecting the relative expression level of RPS4X mRNA includes the primer pairs shown in SEQ ID NO:13 and SEQ ID NO:14, and the primer pairs shown in SEQ ID NO:15 and SEQ ID NO:

16.

3. Application of at least one of the RT-PCR reagent for detecting the relative expression level of RPS4X mRNA and the anti-RPS4X monoclonal antibody according to Claim 2 in the preparation of a kit for detecting the prognosis of ovarian cancer.

4. An RPS4X inhibitor, characterized in that, It includes at least one of the siRNA targeting the RPS4X gene, the recombinant lentivirus containing the siRNA, and the recombinant lentiviral vector containing the siRNA.

5. Inhibitor of the LAMB3 - PI3K - AKT signaling pathway, characterized in that, It includes at least one of the siRNA targeting the RPS4X gene, the recombinant lentivirus containing the siRNA, and the recombinant lentiviral vector containing the siRNA.

6. An ovarian cancer drug which uses at least one of the siRNA targeting the RPS4X gene, the recombinant lentivirus containing the siRNA, and the recombinant lentiviral vector containing the siRNA as the active ingredient.

7. The ovarian cancer drug according to claim 6, wherein The siRNA targeting the RPS4X gene is at least one of the siRNA shown in SEQ ID NO:1 and 2, the siRNA shown in SEQ ID NO:3 and 4, the siRNA shown in SEQ ID NO:5 and 6, or the siRNA shown in SEQ ID NO:7 and 8.

8. The ovarian cancer drug according to claim 6, wherein, The recombinant lentiviral vector containing the siRNA is the GV493 plasmid carrying the nucleotide sequence shown in at least one of the siRNA shown in SEQ ID NO:1 and 2, the siRNA shown in SEQ ID NO:3 and 4, the siRNA shown in SEQ ID NO:5 and 6, or the siRNA shown in SEQ ID NO:7 and 8.

9. The ovarian cancer drug according to claim 6, characterized in that, The recombinant lentivirus containing the siRNA is a lentivirus carrying the nucleotide sequence shown in at least one of the siRNA shown in SEQ ID NO:1 and 2, the siRNA shown in SEQ ID NO:3 and 4, the siRNA shown in SEQ ID NO:5 and 6, or the siRNA shown in SEQ ID NO:7 and 8.

10. Application of at least one of the siRNA targeting the RPS4X gene, the recombinant lentivirus containing the siRNA, and the recombinant lentiviral vector containing the siRNA in the preparation of at least one of an RPS4X inhibitor, a LAMB3-PI3K-AKT signaling pathway inhibitor, or an ovarian cancer drug.

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