Application of eya3 as a target in treatment of cervical cancer

Abstract

The application discloses application of EYA3 as a target point in treatment of cervical cancer, determines a key role of an expression amount of EYA3 in the cervical cancer, and finds that knocking down EYA3 can significantly slow down or reduce growth or disease development of cervical cancer tumor tissues, thereby providing a new drug target point for treatment of the cervical cancer. Meanwhile, the application finds that EYA3 is also a key target point for immunotherapy response after radiotherapy of the cervical cancer, and knocking down EYA3 can significantly improve the response of the immunotherapy after radiotherapy of the cervical cancer, improve the effect of the immunotherapy, and provide a more accurate and better scheme for treatment of the cervical cancer, so as to improve the treatment effect of the cervical cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of EYA3 as a target in the treatment of cervical cancer. Background Art

[0002] Cervical cancer is the fourth most common malignant tumor in women worldwide, with 570,000 new cases and more than 310,000 deaths each year. Among them, my country has nearly 130,000 new cases each year, accounting for about 1 / 4 of the new cases worldwide. For early-stage cervical cancer, surgery is the main treatment, while for locally advanced cervical cancer, radiotherapy combined with concurrent chemotherapy is the main recommended option. In China, more than half of cervical cancer patients are in the locally advanced stage (IB3, IIA2, IIB-IVA stage), and radical radiotherapy has become the most important treatment method. The overall efficacy of patients with locally advanced cervical cancer is poor, and 30% of patients will experience recurrence and metastasis within 2 years after first-line radical radiotherapy.

[0003] To improve the second-line treatment effect of cervical cancer, the latest NCCN guidelines recommend immunotherapy as the most promising supplementary treatment option. However, compared with other solid tumors, the overall response rate of cervical cancer immunotherapy is low. It is particularly noteworthy that the response rate of most cervical cancer patients to immunotherapy after radiotherapy is particularly low. Studies have shown that immunotherapy mainly eliminates cancer cells by stimulating anti-tumor immune responses, and anti-tumor immune responses mainly rely on activating CD8 + T cells or immune cells that utilize them; therefore, radiation therapy may upregulate the infiltration of suppressive immune cells and directly damage circulating lymphocytes, including CD8 + Finding key targets that affect the growth or proliferation of cervical cancer tumor tissue, or even key targets that affect the immune response after radiotherapy, can provide new ideas for the treatment of cervical cancer and improve its treatment effect.

[0004] Eyes Absent 3 (EYA3) is a member of the Eyes Absent (EYA) protein family and, in humans, a transcriptional coactivator and phosphatase. The EYA3 gene is located on chromosome 1p35.3 in humans. The protein it encodes acts as a transcriptional activator during development and is expressed in multiple tissues. Studies have shown that EYA3 is associated with the development of various cancers, and abnormal expression of the EYA3 gene is associated with tumor formation, proliferation, invasion, and survival. Specifically, EYA3 is associated with protein phosphatase 2A (PP2A), a major Ser / Thr phosphatase that plays a key role in regulating various cellular processes. EYA3 interacts with the B55α subunit of PP2A, regulating PP2A activity and substrate specificity, thereby affecting tumor progression.

[0005] There are currently no reports on the application of EYA3 in the treatment of cervical cancer. Summary of the Invention

[0006] In view of this, the primary purpose of the present invention is to provide the use of EYA3 in the treatment of cervical cancer. The present invention found that the expression level of EYA3 affects the growth and progression of cervical cancer. Knockdown of EYA3 can significantly reduce the growth or proliferation rate of cervical cancer tumor tissue and improve survival. In addition, the present invention found that the expression of EYA3 in residual cancer tissue after radiotherapy is significantly increased compared with that before radiotherapy, which affects the effectiveness of subsequent immunotherapy. By knocking down EYA3, the responsiveness of cervical cancer patients to immunotherapy after radiotherapy can be significantly improved, the effect of immunotherapy can be improved, and a more optimal, accurate and effective clinical solution for the treatment of cervical cancer can be provided.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides the use of EYA3 as a target in the preparation of a drug for treating cervical cancer or a drug for improving the responsiveness of cervical cancer to immunotherapy after radiotherapy.

[0009] The second aspect of the present invention provides the use of an agent for inhibiting the expression level of EYA3 in the preparation of a drug for treating cervical cancer or a drug for improving the responsiveness of cervical cancer to immunotherapy after radiotherapy.

[0010] A third aspect of the present invention provides a drug for treating cervical cancer, comprising an agent that inhibits the expression level of EYA3.

[0011] A fourth aspect of the present invention provides a drug for improving the responsiveness of cervical cancer to immunotherapy after radiotherapy, comprising an agent that inhibits the expression level of EYA3.

[0012] In the present invention, by obtaining tissue specimens from cervical cancer patients before and after radiotherapy and detecting the expression level of EYA3 in them, it was found that the expression level of EYA3 affects the growth or proliferation of cervical cancer cells. Subsequently, through animal experiments, EYA3 was knocked down or overexpressed in cervical cancer cell lines, and it was found that knocking down EYA3 significantly slowed the growth of cervical cancer cells. In addition, the present invention also found that the expression level of EYA3 in residual cervical cancer tissue after radiotherapy was significantly increased, which may be a key target affecting subsequent immunotherapy. In the present invention, by knocking down EYA3 in cancer cells after radiotherapy, the responsiveness of immunotherapy was significantly improved, and the effect of immunotherapy was improved.

[0013] In this article, the term "cervical cancer patient" refers to a patient clinically diagnosed with cervical cancer. The term "tissue sample" refers to cervical cancer tissue or paracancerous tissue from a cervical cancer patient. Paracancerous tissue has the common definition used in clinical research, meaning it refers to tissue located a certain distance from the cervical cancer tissue, specifically, less than 3 cm from the cervical cancer tissue.

[0014] In the present invention, cervical cancer tissue or adjacent tissue of cervical cancer patients is obtained by means known or familiar in the art, such as biopsy, etc., which will not be described in detail here.

[0015] In the present invention, the reagent for inhibiting EYA3 expression level is a reagent containing an agent for inhibiting EYA3 gene expression level or an agent for inhibiting EYA3 protein expression. In some specific embodiments, the reagent for inhibiting EYA3 expression level is a reagent containing an agent for inhibiting EYA3 protein expression.

[0016] Herein, the agent that inhibits the expression level of EYA3 refers to a substance that can target and inhibit the activity or expression of EYA3, or target and block EYA3, or target and degrade EYA3. Specifically, it can be a gene interference, gene editing, gene silencing or gene knockout material. For example, in some specific embodiments of the present invention, the agent that inhibits the expression level of EYA3 is an shRNA, siRNA or sgRNA designed based on EYA3, but is not limited thereto. Any substance and agent that can target and inhibit EYA3 in the art falls within the scope of protection of the present invention. In some specific embodiments of the present invention, the agent that inhibits the expression level of EYA3 is an shRNA designed based on EYA3, and the shRNA contains the nucleotide sequence shown in SEQ ID NO.1 or 2. More preferably, the shRNA contains the nucleotide sequence shown in SEQ ID NO.2.

[0017] In the present invention, the drug for treating cervical cancer contains an effective amount of the above-mentioned agent for inhibiting the expression level of EYA3, and further contains any pharmaceutically acceptable carrier and / or excipient.

[0018] Among them, in some specific embodiments of the present invention, the effective amount mentioned here refers to the minimum dose that can inhibit the expression level of EYA3 and improve or even cure the disease of cervical cancer patients. The improvement here mainly refers to the slowing down of tumor growth or the reduction of tumor volume.

[0019] The carriers and / or excipients described herein can be designed according to specific pharmaceutical dosage forms, etc., without particular limitation. Those skilled in the art can make corresponding selections or adjustments based on pharmacology, etc., and will not be elaborated on in detail here.

[0020] The drug for improving the responsiveness of cervical cancer to immunotherapy after radiotherapy in the present invention contains an effective amount of the above-mentioned agent for inhibiting the expression level of EYA3, and further contains any pharmaceutically acceptable carrier and / or excipient.

[0021] The effective amount mentioned here refers to the minimum dose that can inhibit the expression level of EYA3 and improve the effect of immunotherapy.

[0022] It is understood that, in the present invention, immunotherapy refers to a therapeutic method that uses the body's own immune system to identify, attack, and eliminate cancer cells, thereby enhancing or directly activating the body's immune response to fight tumors. This is primarily achieved through immune checkpoint inhibitors, therapeutic antibodies, or cancer vaccines. In the present invention, the immunotherapy drug is an immune checkpoint inhibitor, and any immune checkpoint inhibitor used in the art for the treatment of cervical cancer is applicable, particularly αPD-1 antibodies.

[0023] Beneficial effects of the present invention:

[0024] This study, using bioinformatics analysis from a database, confirmed significant differences in EYA3 mRNA expression between cervical cancer tissue and adjacent adjacent tissues. Furthermore, mouse studies confirmed that EYA3 expression levels influence the growth or progression of tumor tissue in cervical cancer patients. Knockdown of EYA3 significantly slowed the growth or progression of cervical cancer tumor tissue, suggesting that it could serve as a drug target for the treatment of cervical cancer and could be used as a basis for the development of corresponding therapeutic agents.

[0025] The present invention also uses proteomic sequencing and CRISPR-sgRNA library negative screening, and through experiments, it is determined that EYA3 is also a key target for regulating cervical cancer radiotherapy resistance. The treatment of cervical cancer usually requires combined immunotherapy after radiotherapy and / or chemotherapy to further improve the treatment effect. In the present invention, EYA3 was determined to be a key target for immune tolerance after cervical cancer radiotherapy through mouse experiments. By knocking down EYA3, the responsiveness of cervical cancer patients to immunotherapy after radiotherapy can be significantly improved, effectively guiding the immunotherapy of cervical cancer patients and improving the treatment effect of cervical cancer in patients. The present invention has good application prospects and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the bioinformatics analysis result of the TCGA database in Example 1, *P<0.05.

[0027] Figure 2 The results of proteomic analysis and negative screening of CRISPR-sgRNA library in Example 2 are shown; wherein, Figure 2 A is the KEGG analysis results of proteomic analysis of cervical cancer before and after radiotherapy; Figure 2B is a schematic diagram of genome-wide screening of CRISPR-Cas9 sgRNA, where 4 to 6 candidate interference sequences are screened for each gene; Figure 2 C shows the top 22 candidate genes with significant differences obtained from the human SiHa (left) and HeLa (right) cell line-gene library database after cross-screening of differentially expressed genes.

[0028] Figure 3 The results of the statistical analysis of the clinical correlation of Eya3 expression levels in cervical cancer adjacent tissues and tumor tissues are shown in Figure 2. Figure 3 A is the statistical results of EYA3 expression in the IHC staining results of tumor tissues and adjacent tissues of 41 cervical cancer patients in Example 3. Data are mean ± SD, ***P < 0.001; Figure 3 B is the IHC staining results of cancer tissues of 4 cervical cancer patients before and after radiotherapy in Example 2, scale bar: 50 μm.

[0029] Figure 4 The expression efficiency of Eya3 in the Eya3 knockdown and overexpression U14 cells constructed in Example 4, *P<0.05, **P<0.01, ***P<0.001.

[0030] Figure 5 The results of tumor growth in C57 mice subcutaneously implanted with U14 cells expressing different Eya3 levels in Example 4 are shown; Figure 5 A is a line graph of tumor growth size after subcutaneous implantation of U14 cells with different Eya3 expression levels in C57 mice, including Eya3-CON control (normal Eya3 expression group), Eya3-KD1, Eya3-KD2 (Eya3 gene knockdown groups), and Eya3-OE (Eya3 gene overexpression group), *P<0.05, **P<0.01, ***P<0.001; Figure 5 B is a line graph of mouse growth after subcutaneous implantation of U14 cells with different Eya3 expression levels in C57 mice, including Eya3-CON control (normal Eya3 expression group), Eya3-KD1, Eya3-KD2 (Eya3 gene knockdown groups), and Eya3-OE (Eya3 gene overexpression group).

[0031] Figure 6 The results show the apoptosis ratio of U14 and U14-R cells under different irradiation doses in Example 5.

[0032] Figure 7 These are the results of Western blotting of Eya3 in U14 and U14-R cells in Example 5.

[0033] Figure 8The experimental results of Eya3 immune response after radiotherapy in Example 5 are as follows: Figure 8 A is the tumor growth curve of C57 mice after U14-NC and U14-R were injected into mice; Figure 8 B is the expression efficiency of Eya3 in U14-R, U14-R-KD1, and U14-R-KD2; Figure 8 C and Figure 8 D shows the tumor growth curve and survival curve of C57 mice after injection of U14-R and U14-KD2, respectively. *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0034] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In addition, unless otherwise specified, methods without specific conditions or steps are conventional methods, and the reagents and materials used can be obtained from commercial sources.

[0036] It is understandable that the "EYA3", "Eya3", "eye deficiency protein 3" and the like mentioned in this article all have the same meaning. They are just different ways of expressing the same protein or gene, and there is no difference.

[0037] The tissue samples of cervical cancer patients involved in the following examples were all obtained from the First Affiliated Hospital of the University of Science and Technology of China, and were reviewed by the Ethics Committee, and the patients signed informed consent forms.

[0038] Example 1 Bioinformatics Analysis

[0039] In this example, the TCGA-CESC dataset (data type: transcriptome data) of cervical squamous cell carcinoma was downloaded from the Cancer Gene Expression Database (website: https: / / cancergenome.nih.gov / ) for bioinformatics analysis. By analyzing the differential mRNA expression between para-cancer tissues and cancerous tissues of cervical cancer, it was determined that the mRNA expression of Eya3 in para-cancer tissues and cancerous tissues of cervical cancer was significantly different ( Figure 1 ), which suggests that Eya3 may be a potential target for the treatment of cervical cancer.

[0040] Example 2 Proteomic Sequencing and Negative Screening of CRISPR-sgRNA Libraries

[0041] In this example, proteomic sequencing of cervical cancer radiotherapy and immune tolerance tissues and negative screening of CRISPR-sgRNA libraries were used to identify and obtain the Eya3 gene that simultaneously regulates cervical cancer radiotherapy resistance, as follows:

[0042] 1. Cervical cancer tissue or adjacent tissue was obtained from four patients with residual cervical cancer after irradiation and was commissioned to Hangzhou Jingjie Biotechnology Co., Ltd. for proteomic analysis. The analysis focused on the resistance of these patients to subsequent treatment with immune checkpoint inhibitors (ICIs) to determine the biological mechanism of residual cervical cancer after radiotherapy. Table 1 shows the information of the four patients with residual cervical cancer after irradiation.

[0043] Table 1 Information of patients with cervical cancer who underwent external irradiation for residual tumor

[0044] age diagnosis Staging Radiotherapy Patient A 66 Cervical squamous cell carcinoma FIGO IIIB 25 fractions, 2 Gy / fraction Patient B 56 Cervical squamous cell carcinoma FIGO IIB 25 fractions, 2 Gy / fraction Patient C 46 Cervical adenosquamous carcinoma FIGO IIIC1 25 fractions, 2 Gy / fraction Patient D 64 Cervical squamous cell carcinoma FIGO IIA2 25 fractions, 2 Gy / fraction

[0045] Proteomic analysis revealed that the majority of peptides were distributed between 7 and 20 amino acids, consistent with the general patterns of enzymatic hydrolysis and mass spectrometry fragmentation. Furthermore, mass spectrometry analysis identified a total of 7,072 functional proteins, of which 523 were upregulated and 258 were downregulated. A change exceeding 1.5-fold was defined as a significant change threshold. To determine whether the differentially expressed proteins showed significant enrichment trends in certain biological processes, all differentially expressed proteins of interest were further annotated using the KEGG pathway classification. Compared with the largest-scale whole-genome and exome sequencing of cervical cancer tissue to date (Cancer Genome Atlas Research Network et al. “Integrated genomic and molecular characterization of cervical cancer.” Nature vol. 543, 7645 (2017): 378-384. doi:10.1038 / nature21386), enrichment of the Hippo pathway, PI3K-Akt pathway, and EMT was similar to that observed in the aforementioned studies. In particular, neoplastic processes including carbohydrate metabolism and base excision repair were elevated, whereas leukocyte-mediated immune and apoptosis signaling pathways were downregulated ( Figure 2 A), suggesting that this may be the reason for the lower efficiency of ICIs after radiotherapy.

[0046] 2. The whole genome CRISPR sgRNA library (STCRI211101) customized by Suzhou Hongxun Biotechnology Co., Ltd. A genome-wide RNAi screen was performed in human whole genome, CRISPR knockout libraries to explore how residual cervical tumors escape immune clearance or resist ICIs after radiotherapy.

[0047] Cervical cancer cells were transfected with low-abundance lentivirus for 48 h, respectively, and after ensuring that at most one lentivirus was transfected in each cell, the cancer cells were selected with puromycin, and were treated with 6Gy 6MV-X rays, while PBS treatment was used as a control group. Amplification and sequencing were performed on sgRNA sequences and copy number changes in surviving cells, and negative screening was performed (see the flowchart in Figure 2 B, and the specific steps and conditions were performed according to the operation manual provided by Suzhou Hongxun Biotechnology Co., Ltd.).

[0048] In order to explore the adaptive changes specifically caused by radiotherapy, the biological effects induced by radiotherapy, i.e., the importance of gene sequences related to DNA damage repair and cell cycle transition to cell survival, were evaluated in this embodiment. Since the knockout of important genes related to cell survival will lead to an increase in apoptosis under radiotherapy conditions, this embodiment performed negative screening, i.e., focusing on the part of gene sequences with reduced expression abundance.

[0049] As a result, by taking the intersection of the negative screening data obtained by screening the SiHa and HeLa cell gene libraries, this embodiment identified 22 significantly different genes, most of which were significantly changed in the residual cervical tissue after radiotherapy, such as Eya3, Eya4, RCF4, MDC1 and RAD50. Figure 2 C)

[0050] Through the results in this embodiment, the Eya3 gene regulating radiotherapy resistance of cervical cancer was identified and obtained.

[0051] Example 3 Clinical relevance study of expression of Eya3 in cervical cancer microarray

[0052] In this embodiment, a total of 41 pre-radiotherapy tissue samples of cervical cancer patients (CC) were included in the cervical cancer microarray, and these cervical cancer patients were all cervical squamous cell carcinomas, and the stages were all II-III or III, and the age distribution of the patients was 9 cases of ≤45 years old and 32 cases of >45 years old. Among these tissue samples, 19 were paracancerous tissues and 22 were cancer tissues.

[0053] In this embodiment, immunohistochemistry (IHC) staining was performed on the above tissue samples, wherein the IHC staining steps were: dewaxing the sections to water (Servicebio, China) → antigen retrieval (Servicebio, China) → treatment with 3% hydrogen peroxide (G0115Servicebio) → blocking with 3% BSA (G5001Servicebio) → incubation with primary antibody (ab95876, Abcam) at 4°C overnight → incubation with secondary antibody at room temperature (GB23301, Servicebio) → DAB color development (DA1016, Solebol) → counterstaining the nucleus with hematoxylin (Servicebio, China) → microscopic examination. Through IHC staining, it was found that the expression of Eya3 in the cancer area of ​​the cohort in this embodiment was significantly higher than that in the adjacent cancer tissues ( Figure 3 A).

[0054] In addition, in this example, IHC staining was performed on the tumor tissues before radiotherapy and the residual tumor tissues after radiotherapy in the clinical tissue samples of the four cervical cancer patients in Example 2. It was found that the expression level of Eya3 in the residual cancer tissues after radiotherapy (RT) was higher than that in the biopsy specimens before radiotherapy (RT). Figure 3 B).

[0055] The above results further suggest that the Eya3 gene is a key target for regulating cervical cancer radioresistance.

[0056] Example 4 Relationship between different Eya3 expression levels and tumor growth and mouse survival

[0057] In this example, the Eya3 gene in U14 cells was knocked down and overexpressed by shRNA lentiviral transfection and overexpression lentiviral transfection techniques to observe the relationship between different Eya3 expression levels and mouse tumor growth and mouse survival in vivo. The specific steps are as follows:

[0058] 1. U14 cell treatment on the first day

[0059] (1) U14 cells (Shanghai Cell Bank) were cultured in a 10 cm dish with complete culture medium. When the cell density reached about 80%, the cells were placed in a biosafety cabinet for passage. The complete culture medium consisted of: DMEM basal medium (Cat. No. SH30022.01, Cytiva) + 10% serum (F0193, Sigma) + 1% penicillin-streptomycin (15140122, Gibco);

[0060] (2) The culture medium was then discarded, and the cells were washed twice with PBS. The cells were then digested with 1 mL of 0.25% trypsin (Cat. No. 25200056, Gibco) at 37°C for 2 min.

[0061] (3) Terminate the digestion with 5 mL of the same complete medium as in step (1);

[0062] (4) Transfer the U14 cells after digestion to a 15 mL centrifuge tube, pipette evenly, and take 20 μL for cell counting;

[0063] (5) Centrifuge at 1000 rpm for 3 minutes, discard the supernatant, resuspend the cells in complete culture medium, and plate the cells into a six-well plate containing complete culture medium, with 80,000 U14 cells per well.

[0064] 2. Perform lentiviral transfection on the second day:

[0065] (1) Take out the lentivirus from the refrigerator and slowly thaw it on ice. The lentivirus information involved in this step is as follows:

[0066] <eya3-rnai1>

[0067] ccggAGTGAATTGGAACGGGTATTTctcgagAAATACCCGTTCCAATTCACTtttttg (SEQ ID NO. 1), wherein the target sequence is AGTGAATTGGAACGGGTATTT.

[0068] <eya3-rnai2>

[0069] ccggCCACAACATTAGCAGCTACAActcgagTTGTAGCTGCTAATGTTGTGGtttttg (SEQ ID NO. 2), wherein the target sequence is CCACAACATTAGCAGCTACAA.

[0070] <Negative Control Virus>

[0071] Reference number: CON207

[0072] GV298 vector (U6-MCS-Ubiquitin-Cherry-IRES-puromycin), control insert sequence: TTCTCCGAACGTGTCACGT (SEQ ID NO. 3).

[0073] Overexpression lentivirus

[0074] Gene name: Eya3 (NM_010166), the sequence of the overexpression lentivirus is shown in SEQ ID NO.4.

[0075] The above lentiviruses were synthesized by Shanghai GeneCare Gene Medicine Technology Co., Ltd.

[0076] (2) Aspirate the supernatant from each well of the six-well plate and add 2 mL of complete medium + 30 μL of virus solution (MOI = 100) + 5 μL of lentiviral infection enhancement solution Hitrans GP (Cat. No.: REVG005, GeneChip Gene) to each well;

[0077] (3) After 48 h of infection, the medium was replaced with complete culture medium. After 24 h of replacement, infected cells were screened with puromymin (P8230-100 mg, Solebol) starting from 0.5 μM.

[0078] After screening, the target cells were obtained, and the Eya3-CON (control) cell line, Eya3 knockdown cell lines U14-KD1 (shRNA1) and U14-KD2 (shRNA2), and Eya3 overexpression cell line U14-OE were constructed.

[0079] In this example, the cells were collected after puromycin selection, and the knockdown efficiency of Eya3 shRNA1# and shRNA2# and the expression efficiency of Eya3-OE in U14 cells were verified by qPCR analysis. Total RNA was extracted from cells using a cell / tissue total RNA extraction kit (19221ES50, YESEN), and mRNA was reverse transcribed using a Takara reverse transcription kit (RR047A, Takara). cDNA was amplified and quantitatively analyzed using GS AntiQ qPCR SYBR Green Fast Mix (SQ410, Genesand). Specific operations were performed according to the kit instructions. The primer information involved is as follows:

[0080]

[0081] The results are as follows Figure 4 As shown, it illustrates the successful construction of Eya3 knockdown cell lines and overexpression cell lines in this example.

[0082] 3. The U14 cell line constructed above was evenly mixed with Matrigel (Cultrex Basement Membrane Extract, PathClear, 3432-010-01, R&D Systems) and injected subcutaneously into the back of 6-week-old female C57 mice (Shanghai Slake) at an injection volume of 2×10 5 Pieces / pieces.

[0083] The size of the mouse tumor was measured from the 9th day. The mice were killed when the maximum diameter of the tumor was ≥2cm, and the survival time of the mice was recorded. During the whole experiment, the mice were fed with normal SPF level. The results are shown in Figure 5 .

[0084] pass Figure 5 , it can be seen that compared with the Eya3 normal expression group (Eya3-CON), the tumor growth of mice in the Eya3 knockdown group (Eya3-KD 1# and Eya3-KD 2#) was slower and the survival period was longer, with significant differences; while the tumor growth of mice in the Eya3 overexpression group (Eya3-OE) was faster and the overall survival period was shorter ( Figure 5 A and Figure 5 B) These results indicate that different levels of Eya3 expression affect tumor growth and mouse survival, and knocking down Eya3 can delay the growth or progression of cervical cancer tumors.

[0085] Example 5 In vivo animal experiment - Study on the responsiveness of Eya3 to immunotherapy after radiotherapy of cervical cancer

[0086] 1. In vitro construction of the U14 radiotherapy-resistant strain (U14-R). The resistant strain was induced by intermittent induction with a low-to-high dose. The specific steps are as follows:

[0087] (1) U14 cells (Shanghai Cell Bank) were obtained and subcultured in complete medium until the cell density reached 60-80%. The cells were then irradiated with X-rays (6 mV) using a Varinan 2300C / D linear accelerator at a dose rate of 200 cGy / min, resulting in a radiation dose of 2 Gy. The culture medium was then replaced to prevent contamination. The cell growth was subsequently observed. When the cell density grew back to 80%, the cells were subcultured twice, and the cells were labeled as U14-2 Gy.

[0088] (2) Take U14-2Gy cells, culture them to a density of 60-80%, irradiate them with X-rays at a dose of 4Gy, and then replace the culture medium to prevent contamination. Observe the cell growth status and wait until the cell density grows back to 80%. Passage 1 to 2 and label them as U14-4Gy.

[0089] (3) By analogy, the radiation dose is eventually increased to 12 Gy, labeled U14-12 Gy. The final cumulative biological equivalent dose (EQD2) is approximately 65 Gy.

[0090] Among them, the effect of U14-R cell construction was judged by cell apoptosis experiment. The specific steps are as follows: U14-R cells that have grown stably after each radiation and U14 parent cells irradiated at the same dose were respectively taken and planted in 24-well plates. When the cell density reached 80%, the cell culture medium was aspirated into a centrifuge tube, the adherent cells were washed once with PBS, and an appropriate amount of 0.25% trypsin was added to digest the cells, avoiding digestion time that was too short or too long. An appropriate amount of complete cell culture medium was added, the cells were gently pipetted off, and transferred to the corresponding centrifuge tube that had been added with cell culture medium. Centrifuge at 1000g for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells in PBS and count them. Take 50,000-100,000 cells, centrifuge at 1000g for 5 minutes, discard the supernatant, add detection reagents and perform cell apoptosis detection according to the instructions of Annexin V-FITC Cell Apoptosis Detection Kit (Biyuntian, China).

[0091] The results showed that under the same irradiation dose, the apoptosis rate of U14-R cells was significantly lower than that of U14 normal cells, indicating the successful construction of radiotherapy-resistant strains ( Figure 6 )

[0092] 2. Western blot experiment

[0093] The aforementioned U14 cells and constructed U14-R cells were subjected to protein quantification by Western blot assay. The specific steps are as follows:

[0094] 1×10 6 Individual cancer cells (U14 or U14-R) were lysed with RIPA buffer to obtain total protein, which was then measured using a BCA protein assay kit (P0010, Beyotime). Protein samples were added with loading buffer (P0015F, Beyotime) and boiled for 10 minutes. A 20-40 μg portion of each sample was separated on a 4-20% precast protein gel (ET12420Gel, ACE) at constant voltage (80 V for 30 minutes, then 100 V for 60 minutes) for 1.5 hours. The separated proteins were then transferred to a PVDF membrane (IPVH00010, Millipore) at a constant current (260 mA for 90 minutes) for 1.5 hours. After blocking with 5% BSA for 1 h at room temperature (RT), the protein strips were incubated with primary antibodies (EYA3 Polyclonal antibody, 21196-1-AP-50 μl, Proteintech; GAPDH, AB0037-100 μl, Abways) overnight at 4°C. Finally, the protein strips were incubated with HRP-conjugated secondary antibodies (A0208, Beyotime) for 1 h at RT to interact with HRP substrate and imaged using a chemiluminescent imager (Thermo Fisher Scientific, USA).

[0095] The results are as follows Figure 7 As shown, it can be seen that the expression level of EYA3 protein in U14-R cells is higher, indicating that there is a correlation between immune tolerance and Eya3 overexpression.

[0096] 3. In vivo injection

[0097] (1) The radiotherapy-resistant U14 cell line constructed above and normal untreated U14 cells were mixed with matrigel and injected subcutaneously into the back of 6-week-old female C57 mice (Shanghai Slake) at a volume of 1.3×10 5 Half of the mice in the NC group (normal untreated U14 cells) and the U14-R group received αPD-1 antibody (InVivoMAb anti-mouse PD-1 (CD279) BE0146-50MG, Clone RMP1-14, Bioxcell) starting on day 10. Each mouse received 200 μg of the drug via intraperitoneal injection every four days for a total of three injections. The other half received an equal amount of IgG2a isotype control (InVivoPlus rat IgG2a isotype control, anti-trinitrophenol, Catalog #BP0089, Clone 2A3, Bioxcell). Mice were maintained on a standard SPF chow diet throughout the experiment. The groups were as follows: U14-NC+IgG (normal group combined with IgG2a isotype control); U14-NC+αPD-1 (normal group combined with PD-1 inhibitor); U14-R+IgG (radiotherapy resistant group combined with IgG2a isotype control); U14-R+αPD-1 (radiotherapy resistant group combined with PD-1 inhibitor). The results were similar to those of clinical treatment, and the immunotherapy effect of U14-R mice was worse ( Figure 8 A).

[0098] (2) The same shRNA lentiviral transfection technology as in Example 4 was used to knock down the Eya3 gene in the U14-R cell line constructed above using lentiviral EYA3-RNAi1 and EYA3-RNAi2 with sequences such as SEQ ID NO.1 and 2, to obtain Eya3-knocked-down radiotherapy-resistant cell lines U14-R-KD1 and U14-R-KD2. qPCR analysis was performed using the same method as in Example 4 to verify the expression efficiency of Eya3 in U14-R, U14-R-KD1 and U14-R-KD2. The results showed that Eya3 was successfully knocked down in both U14-R-KD1 and U14-R-KD2, and the knockdown effect of U14-R-KD2 was better ( Figure 8 B), so U14-R-KD2 was selected for subsequent experiments.

[0099] (3) The U-14R and U14-R-KD2 cells constructed in step (2) were injected subcutaneously into C57 mice in the same amount and manner as in Example 4, i.e., 2×10 5 Half of the mice were treated with αPD-1 antibody (200 μg / mouse, injected once every four days for a total of three injections), while the other half received an equal amount of IgG2a isotype control (InVivoPlus rat IgG2a isotype control, anti-trinitrophenol, Catalog #BP0089, Clone 2A3, Bioxcell). The tumor growth of the mice was observed. Throughout the experiment, the mice were fed a normal diet according to the SPF level. The specific groups were: U14-R + IgG (IgG2a isotype control for the radiotherapy-resistant group); U14-R + αPD-1 (radiotherapy-resistant group combined with PD-1 inhibitor); U14-R-KD2 + IgG (IgG2a isotype control for the radiotherapy-resistant strain with Eya3 gene knockdown); U14-R-KD2 + αPD-1 (radiotherapy-resistant strain with Eya3 gene knockdown combined with PD-1 inhibitor).

[0100] The experimental results showed that compared with the mice in the U14-R group, the tumors in the U14-R-KD2 group grew more slowly, the survival time was longer, and the response to αPD-1 antibody treatment was improved ( Figure 8 C and 8D).

[0101] The above results show that the increase in Eya3 expression indicates radiotherapy resistance of cervical cancer. If immunotherapy is performed directly without reducing the expression of the EYA3 gene, the subsequent combined immunotherapy effect will be poor. By reducing the expression of the EYA3 gene, the responsiveness of cervical cancer immunotherapy after radiotherapy can be improved, thereby improving the treatment effect of cervical cancer.

[0102] The above examples show that EYA3 expression affects the growth or development of cervical cancer, and knocking down EYA3 can significantly slow the growth or development of cervical cancer tumor tissue. Furthermore, EYA3 is a key target that affects the responsiveness of cervical cancer to post-radiotherapy immunotherapy. If EYA3 expression in cervical cancer tissue increases significantly after radiotherapy, this will result in poor efficacy of subsequent immunotherapy. By knocking down EYA3, the responsiveness of cervical cancer to post-radiotherapy immunotherapy can be significantly improved, thereby providing more precise guidance for the treatment of cervical cancer patients and improving the therapeutic efficacy of cervical cancer.

[0103] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0104] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. Use of an agent for inhibiting EYA3 expression in the preparation of a drug for treating cervical cancer, wherein the agent for inhibiting EYA3 expression is a shRNA designed based on EYA3, and the nucleotide sequence of the shRNA is shown in SEQ ID NO. 1 or 2.

2. The use according to claim 1, characterized in that The medicine is a medicine for improving the responsiveness of immunotherapy after radiotherapy of cervical cancer.

3. The use according to claim 2, characterized in that The drug used in the immunotherapy is αPD-1 antibody.

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