Use of STAT5A inhibitor in preparation of a drug for improving sensitivity of cervical cancer to platinum-based chemotherapy drugs

Abstract

The application relates to the technical field of tumor treatment, in particular to application of a STAT5A inhibitor in preparation of a drug for improving sensitivity of cervical cancer to platinum-based chemotherapy drugs. The application overcomes the problems of limited cisplatin efficacy and high drug resistance rate in the prior art by inhibiting a STAT5A-mediated RNF8-dependent DNA damage repair process, weakening the repair capacity of cervical cancer cells to cisplatin-induced DNA damage, making DNA double-strand breaks continuously accumulate, thereby significantly enhancing the cytotoxic effect of cisplatin, and achieving the technical effect of reversing or reducing platinum-based chemotherapy drug resistance of cervical cancer.

Description

Technical Field

[0001] This application relates to the field of tumor treatment technology, and in particular to the use of STAT5A inhibitors in the preparation of drugs for improving the sensitivity of cervical cancer to platinum-based chemotherapy drugs. Background Technology

[0002] Cervical cancer, one of the most common malignant tumors of the female reproductive system, still relies on concurrent chemoradiotherapy as the standard treatment strategy recommended by current clinical guidelines for locally advanced and recurrent / metastatic patients. Cisplatin is widely used as a radiosensitizer and a base chemotherapy drug. Existing research and clinical practice indicate that cisplatin-containing treatment regimens can improve objective response rates and survival benefits to some extent. However, in actual clinical application, whether cisplatin monotherapy or in combination with taxanes or anti-angiogenic drugs, its efficacy is often limited by the development of cisplatin resistance in tumor cells. Cisplatin resistance not only leads to treatment failure and disease recurrence but also significantly shortens patient survival, becoming one of the key bottlenecks restricting the effectiveness of cervical cancer treatment.

[0003] Cisplatin primarily inhibits tumor growth by inducing intra- or inter-strand cross-linking of DNA, leading to DNA double-strand breaks and activating apoptosis pathways. However, tumor cells generally possess a highly active DNA damage response system, capable of rapidly repairing cisplatin-induced DNA damage through mechanisms such as homologous recombination repair and non-homologous end joining. Studies have confirmed that enhanced DNA damage repair capacity is one of the important molecular bases for cisplatin acquired resistance. If tumor cells can efficiently repair cisplatin-induced DNA double-strand breaks, they will exhibit significant resistance to drug-induced cytotoxicity, thereby weakening the efficacy of chemotherapy. Therefore, selectively inhibiting the DNA damage repair function of tumor cells has become one of the key strategies for reversing cisplatin resistance and improving chemosensitivity.

[0004] Currently, intervention strategies targeting DNA damage response pathways mainly focus on key kinase inhibitors such as ATR and CHK1 / 2. Although these drugs have shown some potential in enhancing cisplatin cytotoxicity, their clinical application is limited by insufficient target selectivity, significant systemic toxicity, and poor patient tolerability. Therefore, there is an urgent need to discover novel, more tumor-selective DNA damage repair regulatory targets to achieve precise chemotherapy sensitization and reduce damage to normal tissues.

[0005] The signal transduction and activating transcription (STAT) protein family plays an important regulatory role in tumorigenesis and development. Previous studies have shown that STAT proteins participate in DNA repair by regulating the transcription of genes related to DNA damage responses. However, current research mainly focuses on members such as STAT3, and systematic and in-depth studies on the role of STAT5A in DNA damage repair, especially its function and mechanism in cisplatin resistance in cervical cancer, are still lacking. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this application provides the use of STAT5A inhibitors in the preparation of drugs for improving the sensitivity of cervical cancer to platinum-based chemotherapy drugs. By inhibiting the STAT5A-mediated RNF8-dependent DNA damage repair process, it overcomes the problems of limited efficacy and high drug resistance rate of cisplatin in existing technologies, weakens the ability of cervical cancer cells to repair DNA damage induced by cisplatin, and causes the continuous accumulation of DNA double-strand breaks, thereby significantly enhancing the cytotoxic effect of cisplatin and achieving the technical effect of reversing or reducing platinum-based chemotherapy resistance in cervical cancer.

[0007] The technical solution adopted by this application to solve its technical problem is: A first aspect of this application provides the use of a STAT5A inhibitor in the preparation of a medicament for improving the sensitivity of cervical cancer to platinum-based chemotherapy drugs.

[0008] In some specific embodiments, the STAT5A inhibitor is selected from at least one of the following: nucleic acid molecules capable of downregulating the expression level of STAT5A gene mRNA, small molecule inhibitors capable of blocking the phosphorylation activation of STAT5A protein, neutralizing antibodies or targeting peptides capable of specifically binding to STAT5A protein; And / or, the STAT5A inhibitor weakens the ability of cervical cancer cells to repair DNA damage induced by the drug by inhibiting the STAT5A-mediated RNF8-dependent DNA damage repair process, causing the continuous accumulation of DNA double-strand breaks, thereby significantly enhancing the cytotoxic effect of cisplatin and achieving the reversal or reduction of cervical cancer resistance to platinum-based chemotherapy drugs.

[0009] In some specific embodiments, the platinum-based chemotherapy drug is selected from at least one of cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin.

[0010] A second aspect of this application provides a pharmaceutical composition for improving the sensitivity of cervical cancer to platinum-based chemotherapy drugs, the pharmaceutical composition comprising a pharmaceutically effective amount of an active ingredient, and a pharmaceutically acceptable carrier, diluent, or excipient; the active ingredient comprising a STAT5A inhibitor.

[0011] In some specific embodiments, the active ingredient further includes a platinum-based chemotherapy drug, wherein the mass ratio of the STAT5A inhibitor to the platinum-based chemotherapy drug is (0.1~10):1.

[0012] A third aspect of this application provides a combination drug kit for platinum-based chemotherapy for cervical cancer, comprising a first vial, a second vial, and instructions for use. The first vial contains a first pharmaceutical preparation containing a STAT5A inhibitor; The second vial contains a second pharmaceutical preparation containing platinum-based chemotherapy drugs; The instructions for use describe how the first and second drug formulations can be used in combination to improve the sensitivity of cervical cancer to platinum-based chemotherapy.

[0013] A fourth aspect of this application provides the use of a STAT5A inhibitor in the preparation of a drug that synergistically enhances the sensitivity of cervical cancer to platinum-based chemotherapy drugs.

[0014] The fifth aspect of this application provides the use of a STAT5A inhibitor in the preparation of a drug that reverses platinum-based chemotherapy resistance in cervical cancer cells.

[0015] In a sixth aspect of this application, a reagent for inhibiting DNA damage repair in cervical cancer cells in vitro is provided, wherein the reagent is a STAT5A inhibitor.

[0016] A seventh aspect of this application provides a method for in vitro screening of candidate compounds that improve the sensitivity of cervical cancer to platinum-based chemotherapy drugs, comprising the following steps: S1. Cervical cancer cells in the logarithmic growth phase were divided into an experimental group and a blank control group. The test compound was added to the experimental group, and an equal amount of solvent was added to the blank control group. They were cultured in vitro under the same conditions. S2. After the culture was completed, the expression levels of STAT5A, phosphorylation levels, RNF8 expression levels, and E3 ubiquitin ligase activity in cervical cancer cells of the experimental group and the blank control group were detected respectively. S3. Compared with the blank control group, if the test compound in the experimental group can significantly downregulate the expression level and phosphorylation level of STAT5A, significantly downregulate the expression level of RNF8, and increase the activity of E3 ubiquitin ligase, then the test compound is determined to be a candidate compound for improving the sensitivity of cervical cancer to platinum-based chemotherapy drugs.

[0017] The beneficial effects achieved by this application are as follows: This application targets the STAT5A-RNF8 axis to selectively inhibit the DNA damage repair capacity of tumor cells, avoiding the systemic toxicity caused by broad-spectrum DDR inhibition; it significantly enhances the accumulation of cisplatin-induced DNA double-strand breaks and improves the sensitivity of tumor cells to platinum-based chemotherapy drugs; it provides a new molecular target and combination therapy strategy for platinum-based drug resistance in cervical cancer, and has clear clinical translational value. The application of this application has good reproducibility and scalability, and can be extended to other tumor types that rely on DNA repair to develop drug resistance. Attached Figure Description

[0018] The present application will be further described below with reference to the accompanying drawings and embodiments.

[0019] Figure 1 The figure shows the experimental results of the effect of STAT5A on the cisplatin sensitivity of cervical cancer cells; Figure 2 : Effect of STAT5A overexpression on cisplatin resistance in a cervical cancer xenograft model; Figure 3 A diagram illustrating the mechanism by which STAT5A regulates cisplatin-induced DNA damage repair; Figure 4 A diagram illustrating the molecular mechanism of DNA damage repair mediated by STAT5A via RNF8; Figure 5 An experimental diagram illustrating how STAT5A promotes DNA damage repair in cervical cancer cells; Figure 6 A diagram illustrating an experiment to inhibit STAT5A, which enhances the sensitivity of cervical cancer cells to cisplatin. Figure 7 The image shows the effect of STAT5A inhibitor combined with cisplatin in treating xenograft tumors in mice. Detailed Implementation

[0020] To facilitate understanding by those skilled in the art, the present application will be further described below with reference to embodiments. The content mentioned in the embodiments is not intended to limit the present application.

[0021] As used herein, “and / or” includes all combinations of any and one or more of the associated listed items. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. As used herein, the singular forms “a,” “an,” “an,” and “the” are also intended to include the plural forms unless the context clearly indicates otherwise. Further understanding is needed; when used in this specification, “comprising” designates the stated features, integers, steps, operations, elements, and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or combinations thereof.

[0022] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Furthermore, terms, as defined in commonly used dictionaries, are interpreted in accordance with their meaning in the context of the relevant field and are not idealized or overly formal, unless explicitly defined herein.

[0023] The exemplary invention described herein may suitably omit any one or more limiting elements, which are not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” “containing,” etc., should be interpreted broadly and non-limitingly. Furthermore, the terminology used herein is for descriptive purposes without limitation, and it is unintentional to use terms that do not include any equivalent characteristics, but only to describe a subset of their characteristics; however, various modifications are possible within the scope of this application according to the claims. Therefore, while this application has been specifically disclosed through preferred embodiments and optional features, modifications disclosed herein to embody variations of this application may be noted by those skilled in the art, and such modifications and variations are considered to be within the scope of this application.

[0024] All raw materials or reagents used in the embodiments and comparative examples of this application were purchased from mainstream manufacturers on the market. Those without specified manufacturers or concentrations are all analytical grade raw materials or reagents that are routinely available. There are no particular restrictions as long as they achieve the expected effect. The instruments and equipment used in this embodiment were all purchased from major manufacturers on the market. There are no particular limitations as long as they achieve the expected effect. Where specific techniques or conditions are not specified in this embodiment, they should be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions.

[0025] To date, no publicly available technology has explicitly revealed the following: Does STAT5A enhance the DNA damage repair capacity of cervical cancer cells by regulating specific DNA repair factors? Could STAT5A serve as a potential molecular target for cisplatin combination therapy? Can intervention in the STAT5A-mediated DNA repair axis effectively improve the sensitivity of cervical cancer to cisplatin treatment?

[0026] The aforementioned problems are precisely the key scientific challenges that urgently need to be overcome in the current technological system.

[0027] Therefore, the first aspect of this application provides the use of STAT5A inhibitors in the preparation of drugs for improving the sensitivity of cervical cancer to platinum-based chemotherapy drugs. Thus, the use of STAT5A inhibitors in the preparation of drugs for improving the sensitivity of cervical cancer to platinum-based chemotherapy drugs can weaken the DNA damage repair capacity of cervical cancer cells to platinum-induced chemotherapy drugs by inhibiting the STAT5A-mediated RNF8-dependent DNA damage repair process, causing the continuous accumulation of DNA double-strand breaks, enhancing the cytotoxicity of platinum-based drugs, thereby reversing or reducing the resistance of cervical cancer to platinum-based chemotherapy drugs, solving the problems of limited efficacy and high resistance rate of cisplatin, providing a new strategy for the treatment of cervical cancer, and possessing clinical translational value.

[0028] In some embodiments, the STAT5A inhibitor is selected from at least one of the following: nucleic acid molecules capable of downregulating the expression level of STAT5A gene mRNA, small molecule inhibitors capable of blocking the phosphorylation activation of STAT5A protein, neutralizing antibodies or targeting peptides capable of specifically binding to STAT5A protein. In some embodiments, the STAT5A inhibitor weakens the ability of cervical cancer cells to repair DNA damage induced by the drug by inhibiting the STAT5A-mediated RNF8-dependent DNA damage repair process, causing the continuous accumulation of DNA double-strand breaks, thereby significantly enhancing the cytotoxic effect of cisplatin and achieving the purpose of reversing or reducing the resistance of cervical cancer cells to platinum-based chemotherapy drugs.

[0029] The above technical solutions clarify the specific types and mechanisms of action of STAT5A inhibitors. By downregulating STAT5A expression, blocking its activation or binding to proteins, they can inhibit RNF8-dependent DNA damage repair, weaken the ability of cervical cancer cells to repair DNA damage induced by platinum-based drugs, accumulate DNA double-strand breaks, and enhance drug cytotoxicity, thereby reversing or reducing the resistance of cervical cancer to platinum-based chemotherapy drugs and providing a feasible strategy for treatment.

[0030] In some embodiments, the platinum-based chemotherapy drugs are selected from at least one of cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin, which clarifies the range of platinum-based chemotherapy drugs that can synergize with STAT5A inhibitors. This range covers commonly used clinical types such as cisplatin and carboplatin, including both traditional first-line drugs (such as cisplatin) and drugs with lower toxicity or suitable for special populations (such as carboplatin and nedaplatin). This can meet the treatment needs of different patients, expand the application scenarios, and enhance the clinical applicability and translational value of the regimen.

[0031] A second aspect of this application provides a pharmaceutical composition for enhancing the sensitivity of cervical cancer to platinum-based chemotherapy drugs. The pharmaceutical composition comprises a pharmaceutically effective amount of an active ingredient, and a pharmaceutically acceptable carrier, diluent, or excipient; the active ingredient includes a STAT5A inhibitor. By containing a STAT5A inhibitor, the active ingredient can inhibit RNF8-dependent DNA damage repair, enhance the cytotoxicity of platinum-based drugs, provide a practical administration method for enhancing the sensitization of cervical cancer chemotherapy, and improve the feasibility of clinical translation. In some embodiments, the active ingredient further includes a platinum-based chemotherapy drug, wherein the mass ratio of the STAT5A inhibitor to the platinum-based chemotherapy drug is (0.1~10):1. For example, the mass ratio of the STAT5A inhibitor to the platinum-based chemotherapy drug can be 0.1:1, 0.5:1, 1:1, 2:1, 4:1, 6:1, 8:1, 10:1, or any combination thereof. This ensures that the STAT5A inhibitor effectively inhibits RNF8-dependent DNA damage repair, enhances the cytotoxicity of platinum-based drugs, and avoids dose imbalance leading to reduced efficacy or increased toxicity. This provides a standardized basis for clinical drug use and improves the stability and safety of treatment regimens.

[0032] A third aspect of this application provides a combination drug kit for platinum-based chemotherapy for cervical cancer, comprising a first vial, a second vial, and instructions for use. The first vial contains a first pharmaceutical preparation containing a STAT5A inhibitor; The second vial contains a second pharmaceutical preparation containing platinum-based chemotherapy drugs; The instructions for use describe how the first and second drug formulations can be used in combination to improve the sensitivity of cervical cancer to platinum-based chemotherapy.

[0033] The aforementioned technical solution utilizes a standardized dosing regimen by separately loading STAT5A inhibitors and platinum-based chemotherapy drugs into individual vials, in accordance with the instructions for combined use. This ensures the synergistic effect of the two drugs, inhibiting RNF8-dependent DNA damage repair and enhancing the cytotoxicity of platinum-based drugs. Simultaneously, it facilitates clinical operation and dosage control, improving treatment stability and safety, and providing a practical tool for chemosensitizing cervical cancer.

[0034] A fourth aspect of this application provides the use of a STAT5A inhibitor in the preparation of a drug that synergistically enhances the sensitivity of cervical cancer to platinum-based chemotherapy drugs.

[0035] The fifth aspect of this application provides the use of a STAT5A inhibitor in the preparation of a drug that reverses platinum-based chemotherapy resistance in cervical cancer cells.

[0036] In a sixth aspect of this application, a reagent for inhibiting DNA damage repair in cervical cancer cells in vitro is provided, wherein the reagent is a STAT5A inhibitor.

[0037] A seventh aspect of this application provides a method for in vitro screening of candidate compounds that improve the sensitivity of cervical cancer to platinum-based chemotherapy drugs, comprising the following steps: S1. Cervical cancer cells in the logarithmic growth phase were divided into an experimental group and a blank control group. The test compound was added to the experimental group, and an equal amount of solvent was added to the blank control group. They were cultured in vitro under the same conditions. S2. After the culture was completed, the expression levels of STAT5A, phosphorylation levels, RNF8 expression levels, and E3 ubiquitin ligase activity in cervical cancer cells of the experimental group and the blank control group were detected respectively. S3. Compared with the blank control group, if the test compound in the experimental group can significantly downregulate the expression level and phosphorylation level of STAT5A, significantly downregulate the expression level of RNF8, and the activity of E3 ubiquitin ligase, then the test compound is determined to be a candidate compound for improving the sensitivity of cervical cancer to platinum-based chemotherapy drugs.

[0038] In the above method, cervical cancer cells are cultured in vitro, and the expression and phosphorylation levels of STAT5A, RNF8 expression, and E3 ubiquitin ligase activity are compared and detected. Compounds that can downregulate the activity of the STAT5A-RNF8 axis are specifically screened. This provides a standardized method for developing drugs that improve the sensitivity of cervical cancer to platinum-based chemotherapy, can efficiently identify potential sensitizing candidates, and accelerate the process of therapeutic drug development.

[0039] The following experimental procedure further illustrates the application of STAT5A inhibitors in the preparation of drugs for improving the sensitivity of cervical cancer to platinum-based chemotherapy drugs. The specific experimental procedure is as follows: Example 1 CCK-8 cell viability assay: Wild-type cell lines in logarithmic growth phase, STAT5A-overexpressing cell lines, and STAT5A-knockdown cell lines (1x10^3) ​​were compared. 5 Cells were seeded with cisplatin ( / mL) into 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator (Likang HF100). Different concentrations of cisplatin were added to the cells (n=8) for treatment. After 48 h, the culture medium was removed, and CCK-8 stock solution was added at a ratio of 1:100 (i.e., 10 μL of CCK-8 stock solution was added to 100 μL of fresh culture medium). The cells were incubated at 37°C in a 5% CO2 incubator for 2 h. The absorbance was then read using a microplate reader (BioTek) at a wavelength of 450 nm. Eight replicates were set for each assay, and growth curves were plotted.

[0040] Cell scratch test: Wild-type cell lines in logarithmic growth phase, STAT5A-overexpressing cell lines, and STAT5A-knockdown cell lines (1x10^3) ​​were compared. 6 Cells were seeded into 6-well plates with 10 μL of sterile pipette tip and incubated overnight at 37°C in a 5% CO2 incubator (Likang HF100). Straight scratches were made using the tip of a 10 μL sterile pipette. After scratching, cell debris was washed away with sterile PBS, followed by treatment with 5 μM cisplatin (CDDP). Cells were then incubated at 37°C. Images were acquired at 0 h, 24 h, and 48 h using a standard optical microscope (Leica DMi1). The scratch healing area was calculated using ImageJ software, and graphs were plotted using Graphad Prism 8.0 software.

[0041] Cloning assay: 1000 cells were seeded into each well of a 6-well plate and cultured at 37°C in a 5% CO2 incubator. After 14 days of culture, the cells were observed to determine if typical colonies had formed. The cells were then fixed with 4% paraformaldehyde for 10 min, washed three times with PBS (5 min each time), stained with crystal violet for 15 min, washed three times with PBS (5 min each time), and the cell colonies were observed and counted using a standard optical microscope (Leica DMi1). The number of cell colonies was calculated using ImageJ software, and graphs were created using Graphad Prism 8.0 software.

[0042] Cell viability was measured using CCK-8 assays, cell scratch assays, and colony formation assays to verify that STAT5A overexpression increased cell viability after cisplatin treatment, while STAT5A depletion enhanced sensitivity to cisplatin.

[0043] Combination Figure 1 It is known that STAT5A depletion increases the sensitivity of cervical cancer cells to cisplatin. Among these factors, Figure 1 Figure A shows the results of the CCK-8 assay for cell viability. Figure 1 B is a photograph of the cell scratch test; Figure 1 C represents the results of the cell scratch assay; Figure 1 Figure D shows the results of the clonogenic assay. The data shown represent the mean ± SD of at least three independent assays, *P<0.05, **P<0.01.

[0044] Figure 1 A. Wild-type, stably STAT5A-carrying, and STAT5A-knockout HeLa cells were treated with cisplatin for 48 hours, and cell viability was measured by CCK-8 assay. Figure 1 A to Figure 1As can be seen from D, overexpression of STAT5A increased cell viability after cisplatin (CDDP) treatment, while STAT5A depletion increased sensitivity to cisplatin.

[0045] Example 2 STAT5A overexpression leads to cisplatin resistance in a cervical cancer xenograft model. Animal grouping: Group A: STAT5A overexpression; Group B: Control group; Group C: STAT5A overexpression + cisplatin; Group D: Control + cisplatin.

[0046] Five BALB / c-Nude nude mice, weighing 20 g, were housed in an SPF-grade barrier environment. 1 × 10^ 7 Tumor cells in the logarithmic growth phase were suspended in 100 μL of PBS containing matrix gel and subcutaneously injected into the axillary region of the right forelimb of nude mice. The tumors were allowed to grow to approximately 80 mm in diameter. 3 Treatment commencement: Groups A and B: 200 μL of physiological saline injected intraperitoneally; Groups C and D: 200 μL of CDDP (5 mg / kg) injected intraperitoneally; injections were administered every 3 days for 3 weeks. During this period, the mice's diet, activity, urination and defecation, and tumor weight were observed daily; the long and short diameters of the tumor were measured every three days using calipers. Tumor volume calculation method: Tumor volume (mm3) = (short diameter^ 2 × major diameter) / 2.

[0047] Combination Figure 2 It is known that STAT5A overexpression leads to cisplatin resistance in a cervical cancer xenograft model. Among other things, Figure 2 A represents the tumor growth curve determined by tumor volume measured twice weekly. Figure 2 B shows photographs of representative tumors in each group. Figure 2 C represents the tumor volume at the start and end of cisplatin treatment. Figure 2 D represents the weight of the xenograft tumor. The data shown represent mean ± SD. * P <0.05、** P <0.01.

[0048] Example 3 Assay of STAT5A on cisplatin-induced DNA damage repair Wild-type cell lines in logarithmic growth phase and STAT5A knockdown cell lines (1x10^ 6Cells were seeded into 6-well plates at 37°C and incubated overnight in a 5% CO2 incubator (Likang HF100). The next day, cells were treated with 0, 5, and 10 μM cisplatin (CDDP) for 48 h. The culture medium was then discarded, and 1 mL of pre-chilled PBS was added to scrape off the cells and transfer them to 1.5 mL centrifuge tubes. The tubes were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. An appropriate volume of 5 × SDS loading buffer was added, and the mixture was vortexed, heated in a water bath for 10 min, and centrifuged at 13000 rpm for 5 min. 10 µL of the sample was added to an SDS-PAGE gel, and the protein was transferred to a nitrocellulose membrane at a constant flow rate of 200 mA for 2 h. The membrane was placed in pre-prepared 5% skim milk blocking buffer and incubated at room temperature for 1 h. The membrane was washed three times with TBST for 5 min each time. Primary antibody was prepared according to the Western blot dilution provided in the antibody manufacturer's instructions and incubated at room temperature for 2 h. Subsequently, the samples were washed three times with TBST for 5 minutes each time; Anti-mouse IgG, HRP-linked Antibody (Promega, cat. no. W4021) and Anti-biotin, HRP-linked Antibody (Promega, cat. no. W4011) were diluted at a ratio of 1:5000 and incubated at room temperature for 1 hour; the samples were washed three times with TBST for 5 minutes each time, and then detected using a chemiluminescence analyzer (ChemiScope 6100).

[0049] Wild-type cell lines in logarithmic growth phase, STAT5A-overexpressing cell lines, and STAT5A-knockdown cell lines (1x10^3) ​​were compared. 6 Cells were seeded into 6-well plates at 37°C and incubated overnight in a 5% CO2 incubator (Likang HF100). The next day, cells were treated with 0, 5, and 10 μM cisplatin (CDDP) for 48 h. The culture medium was then discarded, and the cells were digested with trypsin into single cells and transferred to flow cytometry tubes. After centrifugation at 500g for 5 min, the cells were washed once with PBS and incubated with Anti-γH2AX-APC antibody for 30 min. After washing once with PBS and centrifugation at 500g for 5 min, the expression of APC channels was detected by flow cytometry (BD FACSCelesta) to assess DNA damage.

[0050] Immunofluorescence detection of the colocalization of STAT5A and the DNA damage marker protein rH2AX: Logarithmic growth phase cells were seeded into confocal culture dishes. After cell attachment, 5 μM cisplatin (CDDP) was added. After 48 h, the culture medium was discarded, the cells were washed once with PBS, and fixed with 4% paraformaldehyde for 10 min. Cells were permeabilized with 0.2% Triton X-100 for 5 min, blocked with 5% BSA for 1 h, and incubated with anti-STAT5A and anti-rH2AX at room temperature for 2 h. Cells were washed three times with PBST. The secondary antibody conjugated with fluorescein was then diluted with PBST and incubated at room temperature for 45 min. Finally, images were captured using a fluorescence confocal microscope (Nikon). The colocalization signals of the two fluorescence molecules were calculated using ImageJ software, and graphs were plotted using Graphad Prism 8.0 software.

[0051] Combination Figure 3 This indicates the regulatory role of STAT5A in cisplatin-induced DNA damage repair. Figure 3 A represents the Western blot detection of rH2AX expression in wild-type and STAT5A-deficient cervical cancer cells. rH2AX is a marker of DNA damage. Figure 3 B represents the detection of DNA damage after CDDP treatment of different types of cancer cells by flow cytometry. Figure 3 C represents the immunofluorescence assay for STAT5A and rH2AX expression; Figure 3 D is the co-localization of STAT5A and rH2AX in ImageJ quantitative analysis.

[0052] Example 4 STAT5A enrichment at DNA double-strand break sites was detected using a chromatin segregation assay. Cancer cells from the control and treatment groups were collected and lysed on ice for 5 minutes with Buffer I (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.05% NP40, protease and phosphatase inhibitors). The cells were then centrifuged at 3500 rpm for 5 minutes at 4°C. The supernatant was discarded, and the precipitate was dispersed with Buffer I. The supernatant was collected after centrifugation and was the nucleoprotein. The remaining precipitate was lysed on ice for 20 minutes with Buffer II (50 mM Tris, 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS, protease and phosphatase inhibitors). The supernatant was collected as the chromatin enrichment fraction. The chromatin supernatant was transferred to a 1.5 mL centrifuge tube, and the corresponding volume of 5 × SDS loading buffer was added. The mixture was vortexed, heated in a water bath for 10 minutes, and centrifuged at 13000 rpm for 5 minutes. 10 µL of the sample was added to an SDS-PAGE gel, and the protein was transferred to a nitrocellulose membrane at a constant flow rate of 200 mA for 2 hours. The membrane was placed in a pre-prepared 5% skim milk blocking solution and incubated at room temperature for 1 h; washed three times with TBST for 5 min each time; the primary antibody was prepared according to the WB dilution provided in the antibody instructions and incubated at room temperature for 2 h. Subsequently, the membrane was washed three times with TBST for 5 min each time; Anti-mouse IgG and HRP-linked Antibody were diluted at a ratio of 1:5000 and incubated at room temperature for 1 h; washed three times with TBST for 5 min each time, and then detected using a chemiluminescence analyzer (ChemiScope 6100).

[0053] The JASPAR website (http: / / jaspar2016.genereg.net / cgi-bin / jaspar_db.pl) predicts the presence of a STAT-binding motif on the RNF8 promoter and finds a strong binding motif for STAT5A, CCTCCAAGTA.

[0054] The RNF8 promoter sequence was constructed into the PGL3-Basic vector via double enzyme digestion. Positive clones were selected by colony PCR and then sequenced for identification. HeLa cells were seeded into 24-well plates with a confluence of 80%. 0.5 μg pGL3-RNF8, 0.5 μg pCMV-STAT5A expression plasmid, and 0.1 μg pRL-TK (pGL3-RNF8:pCMV-STAT5A:pRL-TK=5:5:1) were co-transfected into the cells using the Lip2000 liposome transfection reagent. After 48 h, cells were collected, and the activities of firefly luciferase and Renilla luciferase were measured using a dual-luciferase reporter gene assay kit. The luciferase activity of STAT5A regulating the RNF8 promoter was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity.

[0055] Immunofluorescence detection of co-localization of STAT5A and RNF8: Logarithmic growth phase cells were seeded into confocal culture dishes. After cell adhesion, 5 μM cisplatin (CDDP) was added. After 48 h, the culture medium was discarded, the cells were washed once with PBS, and fixed with 4% paraformaldehyde for 10 min. The cells were then permeated with 0.2% Triton X-100 for 5 min, blocked with 5% BSA for 1 h, and incubated with anti-STAT5A and anti-RNF8 at room temperature for 2 h. The cells were then washed three times with PBST. Subsequently, the secondary antibody conjugated with fluorescein was diluted with PBST and incubated at room temperature for 45 min. Finally, images were captured using a fluorescence confocal microscope (Nikon). The colocalization signals of the two fluorescence molecules were calculated using ImageJ software, and graphs were plotted using Graphad Prism 8.0 software.

[0056] Figure 4 It is known that STAT5A mediates the molecular mechanism of DNA damage repair by regulating RNF8 expression. Figure 4 A represents the detection of DNA damage repair pathway proteins by Western blot after isolating chromatin proteins. Figure 4 B represents the RNF8 promoter map; Figure 4 C represents the results of a dual-luciferase reporter gene assay, which showed that STAT5A can regulate the expression of RNF8. The data were analyzed using a two-tailed t-test (*p<0.05, **p<0.01). Figure 4 D represents the expression of STAT5A and RNF8 detected by immunofluorescence. Example 5 To directly assess whether STAT5A regulates cisplatin-induced DNA damage by promoting DNA repair pathways, we used the Sce-GFP assay. This laboratory utilizes the recombinant reporter gene pHR donor and pDRReporter plasmids. The homologous recombination plasmid pHRdonor contains an inactivated GFP gene (Sce-GFP) and a GFP gene fragment as a donor for homologous repair, while the non-homologous end-joining plasmid pDRReporter contains an inactivated GFP gene (Sce-GFP). The Sce-GFP cassette contains an inactivated insert consisting of two STOP codons and a restriction site for the rare endonuclease I-SceI. When I-SceI is expressed in cells expressing either pHRdonor or pDRReporter, it causes a double-strand break in the DNA within the Sce-GFP fragment and signals DNA repair to mimic cisplatin-induced DNA damage. This is followed by the reconstruction of functional GFP, which is easily detected by flow cytometry. Therefore, we used the above method to detect the levels of homologous recombination or non-homologous end-joining DNA repair in different groups of cells.

[0057] Wild-type HeLa cell lines were seeded into 24-well plates and transfected with empty vector, STAT5A overexpression plasmid, and RNF8 overexpression plasmid using Lip2000. After 24 h, cells were transfected with I-SceI and HR-GFP or I-SceI and DRReporter-GFP vectors. Cells were collected after 28 h, and GFP expression was detected by flow cytometry (BD FACSCelesta) to assess the cells' repair capacity.

[0058] WT group, STAT5A - / - Group, RNF8 - / - Cells were seeded into 24-well plates and transfected with I-SceI and HR-GFP or I-SceI and DRReporter-GFP vectors using Lip2000. Cells were collected after 48 h, and GFP expression was detected by flow cytometry (BD FACSCelesta) to assess the cells' repair capacity. Results showed that the loss of RNF8 or STAT5A significantly reduced the level of homologous recombination repair in cells (RNF8 loss led to a decrease in homologous recombination from 3.35% to 0.94%, and STAT5A loss led to a decrease in homologous recombination from 3.35% to 1.7%).

[0059] from Figure 5 It was found that STAT5A and RNF8 co-localize, and STAT5A can regulate RNF8 expression. Therefore, we further explored whether STAT5A regulates homologous recombination repair through RNF8. We compared the WT group and RNF8... - / -Cells were seeded into 24-well plates and transfected with I-SceI, HR-GFP, and STAT5A overexpression vectors using Lip2000. Cells were collected after 48 h, and GFP expression was detected by flow cytometry (BDFACSCelesta). Results showed that overexpression of STAT5A in the wild-type cell group increased homologous recombination repair from 1.97% to 3.35%; while overexpression of STAT5A in the RNF8-deficient group did not increase DNA repair levels. These results indicate that STAT5A regulates DNA repair through RNF8.

[0060] Quantitative DNA damage assay using comet assay: Six groups of cells (wild-type cells, RNF8 overexpressing cells, STAT5A overexpressing cells, RNF8 knockout cells, STAT5A knockout cells, and RNF8 knockout cells + STAT5A overexpressing cells) were collected and mixed with 0.5% low-melting-point agarose, then spread evenly on CometSlide microscope slides. After the agarose solidified, the slides were immersed in lysis buffer for cell lysis. Electrophoresis was then performed at 0.8 V / cm (Tianneng EPS-100). After 30 min, the cells were stained with ethidium bromide for 20 min and observed under a fluorescence microscope (Olympus). The amount of unrepaired DNA released by the cells was quantified by parameters such as tail DNA percentage (tail fluorescence intensity / total fluorescence intensity), tail distance (the product of tail length and tail DNA percentage), and olive tail moment ((distance from the comet center to the tail centroid) × tail DNA%). The results showed that compared with wild-type cells, cells overexpressing RNF8 and STAT5A had reduced DNA damage rates, while cells knocking out STAT5A or RNF8 showed significantly increased DNA damage rates. However, overexpression of STAT5A in the RNF8-deficient group did not reduce DNA damage. These results indicate that STAT5A regulates DNA repair through RNF8. The obtained images were analyzed using CASP comet analysis software to calculate the percentage of tail DNA, tail distance, and olive tail moment parameters to quantify the amount of unrepaired DNA released by the cells, and graphs were created using Graphad Prism 8.0 software.

[0061] Combination Figure 5 It is known that STAT5A regulates cisplatin-induced DNA damage repair through RNF8. Figure 5 A- Figure 5 B represents the level of homologous recombination or non-homologous end joining repair after DNA damage repair following STAT5A or RNF8 overexpression, as detected by flow cytometry. Figure 5 C- Figure 5 D is STAT5A, which regulates DNA repair levels through RNF8. Figure 5 E- Figure 5F represents the comet assay showing that STAT5A regulates DNA repair levels via RNF8. Data shown represent the mean ± SD of at least three independent assays. * P <0.05、** P <0.01.

[0062] Example 6 Wild-type HeLa cell lines in logarithmic growth phase were seeded into 24-well plates and incubated overnight at 37°C in a 5% CO2 incubator (Likang HF100). The cells were divided into 6 groups: blank control, 5 μM CDDP, 10 μM CDDP, 20 μM STAT5-IN-1, 20 μM STAT5-IN-1+5 μM CDDP, and 20 μM STAT5-IN-1+10 μM CDDP. The 24-well plates were placed in a live-cell imaging system (Zeiss CellDiscoverer 7, Germany) to observe cell proliferation in real time. After 96 h, the cell proliferation data for each group were imported into an Excel spreadsheet and plotted using Graphad Prism 8.0 software.

[0063] Wild-type HeLa cell lines in logarithmic growth phase were seeded into 24-well plates and incubated overnight at 37°C in a 5% CO2 incubator (Likang HF100). Straight scratches were made using the tip of a 10 μL sterile pipette. After scratching, cell debris was washed away with sterile PBS. The cells were then divided into 6 groups and treated with the following drugs: blank control, 5 μM CDDP, 10 μM CDDP, 20 μM STAT5-IN-1, 20 μM STAT5-IN-1 + 5 μM CDDP, and 20 μM STAT5-IN-1 + 10 μM CDDP. Cells were incubated at 37°C. Images were acquired at 0 h, 24 h, 36 h, and 48 h using a standard optical microscope (Leica DMi1). The scratch healing area was calculated using ImageJ software, and graphs were plotted using Graphad Prism 8.0 software.

[0064] Immunofluorescence detection of the DNA damage marker γH2AX expression: Logarithmic growth phase cells were seeded into confocal culture dishes. After cell adhesion, cisplatin or STAT5A inhibitors were added according to the groups. After 48 h, the culture medium was discarded, the cells were washed once with PBS, fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, blocked with 5% BSA for 1 h, incubated with anti-γH2AX at room temperature for 2 h, and washed three times with PBST. Subsequently, the secondary antibody conjugated with fluorescein was diluted with PBST and incubated at room temperature for 45 min. Finally, images were captured using a fluorescence confocal microscope (Nikon). The obtained images were used to calculate the γH2AX fluorescence foci using ImageJ software and plotted using Graphad Prism 8.0 software.

[0065] Combination Figure 6 It is known that inhibiting STAT5A makes cervical cancer cells sensitive to cisplatin-induced cell death. Figure 6 A- Figure 6 C was STAT5-IN-1-inhibited HeLa cells, which were then treated with 5 µM and 10 µM cisplatin. After 48 hours, A) cell growth and C migration were measured; B) cell migration level was quantitatively analyzed using ImageJ. The data were statistically analyzed using a two-tailed t-test (*p<0.05, **p<0.01). Figure 6 D represents the expression of the DNA damage marker rH2AX detected by immunofluorescence. Figure 6 E represents the quantitative analysis of rH2AX levels in cell nuclei using ImageJ. The data shown represent the mean ± SD of at least three independent trials. P <0.05、** P <0.01.

[0066] Example 7 Animal groups: Group A: STAT5A overexpression; Group B: STAT5A overexpression + STAT5A inhibitor; Group C: STAT5A overexpression + cisplatin; Group D: STAT5A overexpression + STAT5A inhibitor + cisplatin. Five BALB / c-Nude nude mice, weighing 20 g, were used in each group and housed in an SPF-grade barrier environment. 1 × 10^ 7 Tumor cells in the logarithmic growth phase were suspended in 100 μL of PBS containing matrix gel and subcutaneously injected into the axillary region of the right forelimb of nude mice. The tumors were allowed to grow to approximately 80 mm in diameter. 3Treatment commencement: Group A: Intraperitoneal injection of 200 μL of normal saline; Group B: Intraperitoneal injection of STAT5-IN-1 (10 mg / kg); Group C: Intraperitoneal injection of 200 μL of CDDP (5 mg / kg); Group D: Intraperitoneal injection of 200 μL of CDDP (5 mg / kg) combined with STAT5-IN-1 (10 mg / kg); injections were administered every 3 days for 3 weeks. During this period, the mice's diet, activity, urination and defecation, and tumor weight were observed daily; the long and short diameters of the tumor were measured every three days using calipers. Tumor volume calculation method: Tumor volume (mm3) = (short diameter^ 2 × major diameter) / 2.

[0067] Combination Figure 7 It can be seen that STAT5A inhibitors combined with cisplatin treatment lead to tumor shrinkage in mice. Figure 7 A represents the tumor growth curve determined by tumor volume measured twice weekly. Figure 7 B shows photographs of representative tumors in each group. Figure 7 C represents the tumor volume at the start and end of monotherapy or combination therapy. Figure 7 D represents the weight of the xenograft tumor. The data shown represent mean ± SD, *P<0.05, **P<0.01.

[0068] The above embodiments are preferred implementations of this application. In addition, this application can be implemented in other ways. Any obvious substitutions without departing from the concept of this application are within the protection scope of this application.

Claims

1. The application of STAT5A inhibitors in the preparation of drugs for improving the sensitivity of cervical cancer to platinum-based chemotherapy drugs.

2. The application according to claim 1, characterized in that, The STAT5A inhibitor is selected from at least one of the following: nucleic acid molecules that can downregulate the expression level of STAT5A gene mRNA, small molecule inhibitors that can block the phosphorylation and activation of STAT5A protein, neutralizing antibodies or targeting peptides that can specifically bind to STAT5A protein. And / or, the STAT5A inhibitor weakens the ability of cervical cancer cells to repair DNA damage induced by the drug by inhibiting the STAT5A-mediated RNF8-dependent DNA damage repair process, causing the continuous accumulation of DNA double-strand breaks, thereby significantly enhancing the cytotoxic effect of cisplatin and achieving the reversal or reduction of cervical cancer resistance to platinum-based chemotherapy drugs.

3. The application according to claim 1 or 2, characterized in that, The platinum-based chemotherapy drugs are selected from at least one of cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin.

4. A pharmaceutical composition for improving the sensitivity of cervical cancer to platinum-based chemotherapy drugs, characterized in that, The pharmaceutical composition comprises a pharmaceutically effective amount of an active ingredient, and a pharmaceutically acceptable carrier, diluent, or excipient; the active ingredient includes a STAT5A inhibitor.

5. The pharmaceutical composition according to claim 4, characterized in that, The active ingredient also includes platinum-based chemotherapy drugs, and the mass ratio of the STAT5A inhibitor to the platinum-based chemotherapy drugs is (0.1~10):

1.

6. A combination drug kit for platinum-based chemotherapy in cervical cancer, characterized in that, Includes the first medicine bottle, the second medicine bottle, and the instruction manual; The first vial contains a first pharmaceutical preparation containing a STAT5A inhibitor; The second vial contains a second pharmaceutical preparation containing platinum-based chemotherapy drugs; The instructions for use describe how the first and second drug formulations can be used in combination to improve the sensitivity of cervical cancer to platinum-based chemotherapy.

7. Application of STAT5A inhibitors in the preparation of drugs that synergistically enhance the sensitivity of cervical cancer to platinum-based chemotherapy drugs.

8. Application of STAT5A inhibitors in the preparation of drugs to reverse platinum-based chemotherapy resistance in cervical cancer cells.

9. A reagent for inhibiting DNA damage repair in cervical cancer cells in vitro, characterized in that, The reagent is a STAT5A inhibitor.

10. A method for in vitro screening of candidate compounds that improve the sensitivity of cervical cancer to platinum-based chemotherapy drugs, characterized in that, Includes the following steps: S1. Cervical cancer cells in the logarithmic growth phase were divided into an experimental group and a blank control group. The test compound was added to the experimental group, and an equal amount of solvent was added to the blank control group. They were cultured in vitro under the same conditions. S2. After the culture was completed, the expression levels of STAT5A, phosphorylation levels, RNF8 expression levels, and E3 ubiquitin ligase activity in cervical cancer cells of the experimental group and the blank control group were detected respectively. S3. Compared with the blank control group, if the test compound in the experimental group can significantly downregulate the expression level and phosphorylation level of STAT5A, significantly downregulate the expression level of RNF8, and the activity of E3 ubiquitin ligase, then the test compound is determined to be a candidate compound for improving the sensitivity of cervical cancer to platinum-based chemotherapy drugs.

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