Application of SF3A2 in triple-negative breast cancer treatment and chemotherapy efficacy evaluation

Abstract

The application provides application of a selective splicing factor SF3A2 as a biomarker in preparation of a reagent for breast cancer diagnosis, treatment, prognosis evaluation and chemotherapy sensitivity evaluation. The application provides application of a detection reagent for expression level of a selective splicing factor SF3A2 gene or protein in preparation of a kit for breast cancer diagnosis, prognosis evaluation and chemotherapy sensitivity evaluation. Further, the application provides application of an expression inhibitor for a selective splicing factor SF3A2 gene or protein in preparation of a kit for breast cancer treatment and chemotherapy sensitivity improvement.

Description

Technical Field

[0001] This invention belongs to the field of breast cancer treatment technology, specifically relating to the application of selective splicing factor SF3A2 as a biomarker in the preparation of reagents for breast cancer diagnosis, treatment, prognostic assessment, and chemotherapy sensitivity assessment. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women. In recent years, a series of large-scale omics studies have shown that breast cancer is a highly heterogeneous malignant tumor in terms of molecular characteristics, including abnormal expression and / or mutations of a large number of key genes, ultimately leading to abnormal protein pathways. Based on the expression status of its estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), breast cancer is clinically classified into three main molecular subtypes: hormone receptor-positive, HER2-positive, and triple-negative.

[0003] Triple-negative breast cancer (TNC) is a subtype of breast cancer characterized by the absence of expression of ER, PR, and HER2, accounting for approximately 15% of all breast cancers. Compared to other breast cancer subtypes, TNC is characterized by high heterogeneity, aggressiveness, high rates of recurrence and distant metastasis, a lack of clearly defined therapeutic targets, and poor prognosis, making it a challenging area in breast cancer research and clinical practice. To date, the molecular mechanisms underlying the development and progression of TNC remain unclear, and there is a lack of suitable molecular targets and treatment strategies. Elucidating the molecular mechanisms of TNC development and progression and identifying new therapeutic targets are among the key scientific issues that urgently need to be addressed in breast cancer research.

[0004] Chemotherapy is currently one of the most effective treatments for triple-negative breast cancer. Chemotherapy, short for chemical drug therapy, uses chemical drugs to inhibit or kill tumor cells at different stages of tumor cell growth and reproduction, thereby achieving the therapeutic goal. Chemotherapy is a systemic treatment; the chemotherapeutic drugs circulate throughout most organs and tissues of the body via the bloodstream. Therefore, chemotherapy is a primary treatment for triple-negative breast cancer with high rates of recurrence and distant metastasis. However, in some patients, drug resistance during chemotherapy significantly limits its effectiveness.

[0005] Multiple studies have demonstrated that alternative splicing is tightly regulated across different tissues and developmental stages, and the absence or abnormal activation of splicing signaling pathways can lead to various human diseases, including malignant tumors. Therefore, research on alternative splicing is crucial not only for understanding individual development, disease susceptibility, and pathogenesis, but also for potentially providing new therapeutic targets for major human diseases.

[0006] Therefore, there is an urgent need in this field to find new biomarkers and therapeutic approaches for the diagnosis and treatment of breast cancer or breast cancer metastasis from molecules related to selective splicing. Summary of the Invention

[0007] To address the aforementioned issues, this invention investigated biomarkers of selective splicing factors in triple-negative breast cancer. It was found that the biomarker SF3A2 was highly expressed in triple-negative breast cancer tissues and was associated with patient prognosis and survival. Further verification revealed that targeting the selective splicing factor SF3A2 significantly inhibited the in vitro and in vivo growth of triple-negative breast cancer, and that its use as a combined target could enhance the sensitivity of triple-negative breast cancer to chemotherapy.

[0008] Specifically, the first aspect of the present invention provides the application of selective splicing factor SF3A2 as a biomarker in the preparation of reagents for the diagnosis, treatment, prognostic assessment, and chemotherapy sensitivity assessment of breast cancer.

[0009] The second aspect of this invention provides the application of a reagent for detecting the expression level of the selective splicing factor SF3A2 gene or protein in the preparation of kits for breast cancer diagnosis, prognostic assessment, and chemotherapy sensitivity assessment.

[0010] In some embodiments, the reagent for detecting the expression level of the alternative splicing factor SF3A2 gene is selected from reagents used for detecting mRNA expression levels by quantitative PCR, Northern-bot, or in situ hybridization.

[0011] In some embodiments, the reagent for detecting the expression level of the selective splicing factor SF3A2 protein is selected from reagents used for detecting protein expression levels using Western blotting, immunohistochemistry (IHC), or quantitative proteometry.

[0012] A third aspect of this invention provides the use of an inhibitor of selective splicing factor SF3A2 gene or protein expression in the preparation of a kit for breast cancer treatment and to improve chemotherapy sensitivity.

[0013] In some embodiments, the inhibitor is selected from shRNA, siRNA, and SF3A2 antibody.

[0014] In some embodiments, the shRNA comprises the nucleotide sequence shown in SEQ ID NO:5-6.

[0015] The fourth aspect of this invention provides the application of SF3A2 gene or protein expression inhibitors in combination with chemotherapeutic drugs in the preparation of breast cancer treatment drugs.

[0016] A fifth aspect of the present invention provides a breast cancer treatment composition comprising an SF3A2 gene or protein expression inhibitor and a chemotherapeutic agent.

[0017] In some embodiments, the SF3A2 gene or protein expression inhibitor and the chemotherapeutic drug are administered simultaneously.

[0018] In some embodiments, the SF3A2 gene or protein expression inhibitor and the chemotherapeutic drug are administered sequentially.

[0019] According to the application described in the fourth aspect or the composition described in the fifth aspect of the present invention, wherein the chemotherapeutic agent is selected from cisplatin.

[0020] According to the applications described in the first, second, third, and fourth aspects of the present invention or the compositions described in the fifth aspect, wherein the breast cancer is triple-negative breast cancer.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] (1) This invention demonstrates from our center’s cohort, public databases and studies of 20 cases of cancer and adjacent normal tissues that the biomarker SF3A2 is highly expressed in triple-negative breast cancer tissues and is associated with patient prognosis and survival, suggesting that it has the potential to be a biomarker for triple-negative breast cancer.

[0023] (2) This invention uses RNA interference technology to knock down SF3A2 molecules in triple-negative breast cancer. CCK8 and clonal growth experiments confirm that SF3A2 molecules can promote the proliferation of triple-negative breast cancer cells. Apoptosis experiments confirm that SF3A2 molecules can inhibit apoptosis in triple-negative breast cancer cells.

[0024] (3) The present invention uses an animal model, namely a mouse xenograft experiment of breast cancer cells, to confirm that SF3A2 molecules can promote the growth of triple-negative breast cancer cells in mice.

[0025] (4) This invention uses RNA interference technology to knock down SF3A2 molecules in triple-negative breast cancer, and then uses the chemotherapy drug cisplatin to test drug sensitivity. It confirms that SF3A2 molecules promote cisplatin resistance in triple-negative breast cancer cells. Therefore, targeting SF3A2 in triple-negative breast cancer cells can increase the sensitivity of cancer cells to cisplatin, which may have clinical application value for the treatment of triple-negative breast cancer. Attached Figure Description

[0026] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0027] Figure 1The study showed that the protein level of SF3A2 was significantly higher in TNBC tissue than in adjacent normal tissue. Figure 1 A: Quantitative proteomics analysis showed that the detection level of SF3A2 in TNBC was significantly higher than that in normal tissue; Figure 1 B: External CPTAC protein database analysis showed that the detection level of SF3A2 in breast cancer was significantly higher than that in normal tissue; Figure 1 C: Analysis of the CPTAC protein database showed that the detection level of SF3A2 in the TNB subtype was significantly higher than that in normal tissue.

[0028] Figure 2 Immunoblotting confirmed that SF3A2 was significantly elevated in the TNBC subtype. Figure 2 A: Immunoblotting assay showing the expression levels of SF3A2 protein in 20 pairs of TNBC tissues and adjacent normal tissues; Figure 2 B: Statistical analysis of immunoblotting experiments showed that SF3A2 protein was significantly elevated in the TNBC subtype.

[0029] Figure 3 Inhibition of SF3A2 showed that it significantly reduced the proliferation of TNBC cells. Figure 3 A: Immunoblotting experiments confirmed that small interfering RNAs shSF3A2#1 and shSF3A2#2 can significantly inhibit the protein expression level of SF3A2 in triple-negative breast cancer cells MDA-MB-231 and SUM159PT; Figure 3 B: CCK8 laboratory tests showed that the proliferation capacity of cells in the SF3A2 knockdown group (shSF3A2#1 and shSF3A2#2) was significantly lower than that of the control group (shNC); Figure 3 C: Representative images of the number of colonies formed in the SF3A2 knockdown group (shSF3A2#1 and shSF3A2#2) and the control group (shNC) in the colony formation assay; Figure 3 D: Statistical analysis of the colony formation experiment showed that the number of colonies formed in the SF3A2 knockdown group (shSF3A2#1 and shSF3A2#2) was significantly lower than that in the control group (shNC).

[0030] Figure 4 Inhibition of SF3A2 showed that it significantly increased the apoptosis rate of TNBC cells. Figure 4 A: Representative images of apoptosis levels in cells from the SF3A2 knockdown group (shSF3A2#1 and shSF3A2#2) and the control group (shNC) as detected by flow cytometry; Figure 4 B: Apoptosis assay analysis showed that the apoptosis level of cells in the SF3A2 knockdown group (shSF3A2#1 and shSF3A2#2) was significantly higher than that of the control group (shNC).

[0031] Figure 5Inhibition of SF3A2 significantly suppressed the growth of tumor cells in mouse xenografts. Figure 5 A: Mouse xenograft experiments showed that the tumor volume of cells in the SF3A2 knockdown group (shSF3A2#2) was significantly lower than that in the control group (shNC). Figure 5 B: Representative images of mouse xenograft tumors from the SF3A2 knockdown group (shSF3A2#2) and the control group (shNC). 5C: Mouse xenograft experiments showed that the tumor weight of cells in the SF3A2 knockdown group (shSF3A2#2) was significantly lower than that in the control group (shNC).

[0032] Figure 6 Inhibition of SF3A2 significantly suppressed the growth of tumor cells in mouse xenografts. Figure 6 A: IC50 was measured after cisplatin treatment of MDA-MB-231 cells in the SF3A2 knockdown group (shSF3A2#1 and shSF3A2#2) and the control group (shNC). The results showed that the IC50 value of the SF3A2 knockdown group was significantly lower than that of the control group. Figure 6 B: IC50 was measured after cisplatin treatment of SUM159PT cells in the SF3A2 knockdown groups (shSF3A2#1 and shSF3A2#2) and the control group (shNC). The results showed that the IC50 value of the SF3A2 knockdown group was significantly lower than that of the control group. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, not all, of the embodiments of the present invention. Based on the described 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.

[0034] Example 1: Experimental Materials and Methods

[0035] 1.1 RNA sequencing and clinical medical database analysis

[0036] RNA sequencing results analysis: After removing adaptor contamination, STAR2 was used to align polyA, polyC, and sequencing reads with human reference sequences. RSEM was used to quantify gene expression values ​​in the GENCODE transcript model, and the data were then standardized. RNA sequencing results were replicated across multiple batches, and batch effects were assessed using PCA analysis, with Combat correction applied in SVA. Finally, log2-transformed upper quartile normalized RSEM was used for subsequent analysis. Furthermore, to further explore SF3A2 expression in triple-negative breast cancer, this invention's protocol involved mining and analyzing the breast cancer clinical database in the CPTAC (Clinical Proteomic Tumor Analysis Consortium) database. The analysis primarily focused on the expression of SF3A2 in both cancerous and adjacent normal tissues in breast cancer.

[0037] 1.2 Real-time quantitative PCR

[0038] RNA extraction from tumor-derived cell samples was performed using the Trizol standard extraction method. Specifically, triple-negative cancer cells were collected, and an appropriate volume of Trizol was added. After thorough mixing and lysis, 1 / 5 volume of chloroform was added, mixed, and incubated at room temperature for 3 minutes. Centrifugation was then performed at 12000g, 4°C, for 15 minutes. The supernatant was transferred to an RNAase-free 1.5mL centrifuge tube. An equal volume of isopropanol was added, mixed, and incubated at room temperature for 10 minutes. Centrifugation was then performed at 12000g, 4°C, for 20 minutes. The supernatant was carefully discarded, retaining the RNA gel at the bottom of the centrifuge tube. The tube was washed twice with 75% ethanol, centrifuged at 7500g, 4°C, for 5 minutes. The supernatant was discarded, and the tube was incubated at room temperature for 3 minutes before dissolving in DEPC water. After concentration determination, total RNA was reverse transcribed into cDNA according to standard operating procedures. The cDNA and remaining RNA samples were stored at -20°C and -80°C, respectively. Primers required for real-time quantitative Q-PCR were synthesized by Shanghai Sangon Biotech Co., Ltd. Using cDNA synthesized by reverse transcription as a template and β-actin as an internal control, the target gene fragment was amplified using the SYBR Green PCR MasterMix kit. The reaction conditions are as follows:

[0039]

[0040] The above reactions constitute one group, with a total of 40 cycles.

[0041] Primer information is shown in Table 1 below:

[0042] Table 1 Primer Information

[0043] Gene name Sequence (5'-3') SF3A2-F 5'-GATTGACTACCCTGAGATCGCC-3'(SEQ ID NO:1) SF3A2-R 5'-CTCCCGGTTCCAGTGTGTC-3'(SEQ ID NO:2) β-actin-F 5'-CATGTACGTTGCTATCCAGGC-3'(SEQ ID NO:3) β-actin-R 5'-CTCCTTAATGTCACGCACGAT-3'(SEQ ID NO:4)

[0044] The relative mRNA levels of the target gene were calculated using β-actin as an internal reference and the ΔΔCT method. Then, the mRNA expression level of the control group was calibrated to 1, and the relative mRNA expression of the target gene in the experimental group was calculated.

[0045] 1.3 Immunoblotting method

[0046] The tissue samples to be tested were gently washed three times with pre-cooled PSB, and an appropriate amount of lysis buffer containing protease inhibitors and phosphatase inhibitors was added (RIPA lysis buffer was used if the purpose of protein extraction was for Western blotting). The tissue was homogenized and then placed on a low-speed shaker at 4°C for 20 min for lysis. The lysis buffer was then transferred to 1.5 mL centrifuge tubes and centrifuged at 15,000 rpm at 4°C for 20-30 min. The supernatant was transferred to new centrifuge tubes, and protein quantification was performed according to the BCA kit instructions, adjusting the protein concentration of each group of samples to be consistent. The lysis buffer formulation used for protein extraction is as follows:

[0047] RIPA lysis buffer: 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% NP-40, 1% Sodium deoxycholate, 0.1% SDS

[0048] Add 4°C SDS loading buffer to the prepared protein sample, mix well, boil in a 100°C metal bath for 5 minutes, centrifuge at room temperature, and then gently vortex to mix again. Prepare an appropriate concentration of SDS-PAGE gel based on the molecular weight of the target protein. Choose a 15-well or 10-well comb depending on the number of samples to be analyzed simultaneously. Load 10-40 μL of sample into each well. Use vertical constant voltage electrophoresis with a stacking gel voltage of 80V and a separating gel voltage of 110V. Electrophoresis is complete when bromophenol blue in the sample is about to or has completely escaped the gel. Proceed to the next step: membrane transfer. Before use, the PVDF membrane should be activated with methanol and then immersed in transfer buffer. Use low-temperature wet transfer with a constant voltage horizontal electrophoresis of 60-90V. Larger molecular weights require longer transfer times, generally 90-180 minutes.

[0049] Dilute the primary antibody (SF3A2 antibody: Proteintech, catalog number 15596-1-AP; Vinculin antibody: Sigma, catalog number V9131) according to the antibody instructions. Generally, dilute it 1:500 to 1:5000 into a 5% BSA solution prepared with PBST. Add the antibody dilution buffer to the antibody incubation chamber, place the blocked membrane in the antibody dilution buffer, and incubate overnight at 44°C on a shaker at 60 rpm. After incubation with the primary antibody, wash the membrane three times with PBST for 10 min each time. Select the appropriate secondary antibody according to the species of the primary antibody (rabbit secondary antibody: CST, catalog number 7074V; mouse secondary antibody: CST, catalog number 7076V), and incubate with the secondary antibody at room temperature for 1 h. Then wash three times with PBST for 10 min each time.

[0050] Prepare a fresh ECL developer, place the membrane in the ECL developer, gently shake and incubate for 10 seconds to 1 minute, and then expose the membrane to a multi-functional digital imaging system.

[0051] 1.4 Cell proliferation detection

[0052] Breast cancer cell lines in the logarithmic growth phase were selected, digested and counted using enzymes, and then passaged into 96-well cell culture plates. 1000 cells were cultured in 100 μL of medium per well. Each experimental group was repeated at least three times. The following day, after cell attachment, 10× Cell Counting Kit-8 reagent was added, and the reaction was carried out at 37°C for 2 hours. The absorbance at 450 nm (OD450) of each well was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The following day was counted as day one, and OD450 values ​​were measured and collected on days one, three, five, and seven. Cell growth curves were plotted to compare differences in cell proliferation capacity among different groups.

[0053] 1.5 Plate Cloning Experiment

[0054] The plate colony assay was used to examine the effect of SF3A2 knockdown on the clonal growth of triple-negative breast cancer cells. For triple-negative breast cancer cells in the control and SF3A2 knockdown groups, cells were digested with trypsin solution when they were in the logarithmic growth phase. Cell counts were collected, and cells were seeded into 6-well or 12-well plates at 500-1,500 cells per well. The culture medium was changed every three days. After approximately 14-20 days, cells grew into visible clones. Cells were fixed with methanol for 30 min, air-dried, stained with 0.5% violet crystal staining solution (prepared with 1% ammonium oxalate) for 1 h, rinsed with water, air-dried, photographed or scanned, and the number of clones in each group was counted to compare cell proliferation differences between groups.

[0055] 1.6 Flow Cytometry

[0056] Apoptosis assays were performed according to the instructions of the Yeasen apoptosis kit, and are briefly described below: Cell digestion and counting were performed, and one million cells were collected and resuspended in 1 ml of staining buffer. PBS was discarded, and 100 μL of 1× Binding Buffer was added to resuspend the cells; 5 μL of Annexin V-FITC and 10 μL of LPI Staining Solution were added, and the mixture was gently mixed; the mixture was incubated in the dark at room temperature for 10-15 min; 400 μL of 1× Binding Buffer was added, the mixture was mixed, and the sample was placed on ice. The samples were analyzed by flow cytometry within 1 hour.

[0057] 1.7 Tumor formation experiments in animals

[0058] To verify whether SF3A2 has the same function in vivo, the inventors established a knockdown-stable cell line from the triple-negative breast cancer cell line MDA-MB-231 for tumorigenesis experiments in nude mice. The inventors divided the two cell lines into two groups at a ratio of 2 × 10⁻⁶. 6 Cells were injected into the fat pads of nude mice to observe their tumorigenic ability. After 8 weeks of observation and measurement, the inventors dissected the cervical region of all nude mice and removed the tumors in situ for measurement. The experimental results showed that, in in vivo, the tumor growth ability of the SF3A2 protein knockdown group was significantly stronger than that of the control group.

[0059] 1.8 Drug sensitivity testing

[0060] SF3A2 knockdown and control triple-negative cells were seeded in 12-well plates at a density of 2,000 cells per well. Cisplatin at different concentrations was added 12 hours after seeding, with DMSO added as a control. The medium was changed every three days, with the specified concentration of cisplatin added each time. When cell clones were visible to the naked eye (approximately 20 days), the cells were fixed with methanol for 30 minutes. After drying, the cells were stained with 0.5% violet crystal staining solution (prepared with 1% ammonium oxalate) for 1 hour. Excess stain was rinsed with water and the cells were dried. The cells were photographed or scanned, and the number of clones in each group was counted to compare the differences in cell proliferation among the groups.

[0061] 1.9 Small interfering RNA inhibits the expression of SF3A2 molecules.

[0062] In a 6cm culture dish, approximately 0.5 × 10⁶ cells were seeded. 6Once the cells have adhered to the culture medium and reached approximately 50% confluence, the culture medium was aspirated, and 2 mL of crude virus (or an appropriate amount of high-titer concentrated virus) and 2 mL of fresh culture medium were added, along with 10 μg / mL of polybrene (Beyotime Biotech, catalog number C0351). Cells were cultured for another 24 hours, after which the virus-containing culture medium was replaced with fresh medium, and culture continued. 48-72 hours after viral infection, antibiotics containing the corresponding resistance gene of the target gene vector were added to the culture medium for positive cell selection. In this invention, the pLKO.1-shRNA vector is resistant to puromycin (Sangon Biotech, catalog number A610593-0025) in cells; the concentration of antibiotic used varies depending on the cell type. The inhibitory effect on intracellular SF3A2 was verified using Western blotting.

[0063] Example 2 Experimental Results

[0064] 2.1 The protein level of SF3A2 was significantly higher in TNBC tissues than in adjacent normal tissues.

[0065] Our center uses quantitative proteomics to screen for differentially expressed proteins in TNBC (tumor nephrotic syndrome) and adjacent normal tissues. Based on literature review and previous experimental results, SF3A2 was identified, and its expression level in our center's data was significantly higher in TNBC tissues than in adjacent normal tissues. Figure 1 A). To further verify that the protein level of SF3A2 was significantly higher in TNBC tissue than in adjacent normal tissue, the inventors used protein data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) to analyze the expression of SF3A2 in breast tumors. Figure 1 B). CPTAC analysis showed that SF3A2 protein levels were significantly elevated in breast cancer tissues, and SF3A2 was also significantly higher in TNBC subtypes than in adjacent normal tissues. Figure 1 C).

[0066] 2.2 Immunoblotting confirmed a significant increase in SF3A2 in the TNBC subtype.

[0067] The expression of SF3A2 protein in 20 paired TNBC tissues and adjacent normal tissues was analyzed using Western blotting (WB). The results showed that SF3A2 protein expression was significantly higher in TNBC tumor tissues compared to adjacent normal tissues. Figure 2 A and Figure 2 B).

[0068] 2.3 Inhibition of SF3A2 significantly reduced the proliferation of TNBC cells.

[0069] The MDA-MB-231 and BT-549 cell lines, which have high SF3A2 basal expression levels, were selected to knock down SF3A2 and examine the effect of SF3A2 inhibition on TNBC cell proliferation. First, the inventors constructed a specific shRNA to verify the knockdown of SF3A2, and used Western blotting to verify the SF3A2 knockdown status. Figure 3 A). CCK-8 proliferation experiment ( Figure 3 B) and clonal formation ( Figure 3 C and Figure 3 D) Experimental results showed that knockdown of shSF3A2 significantly inhibited the proliferation and clonal growth of MDA-MB-231 and BT-549 cells. These results indicate that inhibiting SF3A2 significantly suppresses the proliferation of TNBC breast cancer cells. shRNA information is shown in Table 2 below.

[0070] Table 2 shRNA sequence information table

[0071] Gene name Sequence (5'-3') shNC 5'-AGAAACCATGCAAAGTAAGG-3'(SEQ ID NO:7) shSF3A2#1 5'-CATCAACAAGGACCCGTACT-3'(SEQ ID NO:5) shSF3A2#2 5'-CAAAGTGACCAAGCCAGAGAGA-3'(SEQ ID NO:6)

[0072] 2.4 Inhibition of SF3A2 significantly increased the apoptosis rate of TNBC cells.

[0073] MDA-MB-231 and BT-549 cell lines, which have high SF3A2 basal expression levels, were selected to establish SF3A2 control and knockdown cell groups to examine the effect of SF3A2 inhibition on the apoptosis rate of TNBC cells. The apoptosis rate was detected using the Annexin V-FITC / PI apoptosis detection kit from Yeasen combined with flow cytometry. The results showed that SF3A2 inhibition significantly increased the apoptosis rate of MDA-MB-231 and BT-549 cells. Figure 4 A and Figure 4 B).

[0074] 2.5 Inhibition of SF3A2 significantly suppressed the growth of tumor cells in mouse xenografts.

[0075] MDA-MB-231 cells, which have a high SF3A2 basal expression level, were selected to establish SF3A2 control and knockdown cell groups. These cells were subcutaneously inoculated into nude mice to establish a mouse subcutaneous xenograft model, and tumor diameter was measured twice a week. Results showed that inhibiting SF3A2 significantly suppressed the growth of mouse xenografts. Figure 5 ).

[0076] 2.6 Inhibition of SF3A2 significantly suppressed the growth of tumor cells in mouse xenografts.

[0077] The above results show that inhibiting SF3A2 can significantly promote the proliferation of triple-negative breast cancer cells and inhibit the growth of tumor cell xenografts in mice. Therefore, the inventors hypothesize that inhibiting SF3A2 can sensitize triple-negative breast cancer cells to the chemotherapeutic drug cisplatin. Different concentrations of cisplatin were treated with SF3A2 control and knockdown cell groups established using MDA-MB-231 and BT-549 cells, and their IC50 (half-maximal inhibitory concentration) was measured. The results showed that inhibiting SF3A2 significantly reduced the IC50 of cisplatin in MDA-MB-231 and BT-549 triple-negative breast cancer cells. Figure 6 A and Figure 6 B) In other words, inhibiting SF3A2 can increase the sensitivity of MDA-MB-231 and BT-549 triple-negative breast cancer cells to cisplatin.

[0078] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the scope of the invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0079] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This description is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Application of reagents for detecting the expression level of the alternative splicing factor SF3A2 gene or protein in the preparation of diagnostic kits for triple-negative breast cancer.

2. The application according to claim 1, characterized in that, The reagent for detecting the expression level of the alternative splicing factor SF3A2 gene was selected from reagents used for detecting mRNA expression levels by quantitative PCR, Northern-bot, and in situ hybridization.

3. The application according to claim 1, characterized in that, The reagents for detecting the expression level of selective splicing factor SF3A2 protein are selected from those used for detecting protein expression levels by immunoblotting, immunohistochemistry (IHC), and quantitative proteometry.

4. Application of inhibitors of the expression of the alternative splicing factor SF3A2 gene or protein in the preparation of kits for the treatment of triple-negative breast cancer and for improving the chemosensitivity of triple-negative breast cancer; among which, The inhibitor is shRNA; the nucleotide sequence of the shRNA is shown in SEQ NO: 5 or SEQ ID NO: 6; the chemotherapy drug is cisplatin.

5. Application of SF3A2 gene or protein expression inhibitors in combination with chemotherapy drugs in the preparation of drugs for the treatment of triple-negative breast cancer; among which, The inhibitor is shRNA; the nucleotide sequence of the shRNA is shown in SEQ NO: 5 or SEQ ID NO: 6; the chemotherapeutic drug is cisplatin.

6. A therapeutic composition for triple-negative breast cancer, characterized in that, The composition comprises an SF3A2 gene or protein expression inhibitor and a chemotherapeutic agent; wherein the inhibitor is shRNA; the nucleotide sequence of the shRNA is shown in SEQ NO: 5 or SEQ ID NO: 6; and the chemotherapeutic agent is cisplatin.

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