Application of EYA3 as target spot in cervical cancer treatment

Abstract

The invention discloses application of EYA3 as a target spot in cervical cancer treatment, the key effect of the expression quantity of EYA3 in cervical cancer is determined, and the growth or disease course development of cervical cancer tumor tissues can be obviously slowed down or reduced by knocking down EYA3, so that a new drug target spot is provided for cervical cancer treatment. Meanwhile, it is found that EYA3 is also a key target spot for immunotherapy response after cervical cancer radiotherapy, the responsiveness of immunotherapy after cervical cancer radiotherapy can be remarkably improved by knocking down EYA3, the immunotherapy effect is improved, a more accurate and better scheme is provided for cervical cancer treatment, and the cervical cancer treatment effect is improved.

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, there are nearly 130,000 new cases in my country each year, accounting for about 1 / 4 of the new cases in the world. For early 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 therapeutic effect 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 are activated; therefore, radiation therapy may upregulate the infiltration of suppressive immune cells and directly damage circulating lymphocytes, including CD8 + T cells, thereby reducing the efficacy of immunotherapy. Finding key targets that affect the growth or proliferation of cervical cancer tumor tissue, or even key targets that affect its immune response after radiotherapy, can provide new ideas for the treatment of cervical cancer and improve the treatment effect of cervical cancer.

[0004] Eyes Absent 3 (EYA3) is a member of the Eyes Absent (EYA) protein family and is a transcriptional coactivator and phosphatase in humans. The EYA3 gene is located at 1p35.3 on chromosome 1 in humans. The protein it encodes acts as a transcriptional activator during development and is expressed in a variety of tissues. Studies have shown that EYA3 is associated with the development of a variety of 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 a variety of cellular processes. EYA3 affects tumor progression by interacting with the B55α subunit of PP2A, regulating the activity and substrate specificity of PP2A.

[0005] There is currently no report 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 application of EYA3 in the treatment of cervical cancer. The present invention finds that the expression of EYA3 affects the growth and progression of cervical cancer, and knocking down EYA3 can significantly reduce the growth or proliferation rate of cervical cancer tumor tissue and improve survival. In addition, the present invention finds that the expression of EYA3 in residual cancer tissue after radiotherapy is significantly higher than that before radiotherapy, which affects the effect of subsequent immunotherapy. By knocking down EYA3, the responsiveness of immunotherapy in patients with cervical cancer after radiotherapy can be significantly improved, the effect of immunotherapy can be improved, and a better, more accurate and more effective clinical solution can be provided for the treatment of cervical cancer.

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

[0008] The first aspect of 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 immunotherapy after radiotherapy of cervical cancer.

[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 immunotherapy after radiotherapy of cervical cancer.

[0010] The third aspect of the present invention provides a drug for treating cervical cancer, which contains 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 immunotherapy after radiotherapy of cervical cancer, which contains 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, by detecting the expression level of EYA3 therein, it is found that the expression level of EYA3 affects the growth or proliferation of cervical cancer cells; then, by animal experiments, EYA3 is knocked down or overexpressed in cervical cancer cell lines, and it is found that knocking down EYA3 significantly slows the growth of cervical cancer cells. In addition, the present invention also finds that the expression level of EYA3 in residual cervical cancer tissue after radiotherapy is 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 is significantly improved, and the effect of immunotherapy is improved.

[0013] In this article, the cervical cancer patient refers to a patient who has been confirmed to have cervical cancer through clinical diagnosis. The tissue sample refers to the cervical cancer tissue or paracancerous tissue of the cervical cancer patient. It can be understood that the paracancerous tissue here has a common definition in clinical research, that is, it refers to the tissue at a certain distance from the cervical cancer tissue, specifically, the tissue <3 cm away from the cervical cancer tissue.

[0014] In the present invention, cervical cancer tissue or paracancerous 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 the expression level of EYA3 is a reagent containing the expression level of EYA3 gene or the expression amount of EYA3 protein. In some specific embodiments, the reagent for inhibiting the expression level of EYA3 is a reagent containing the expression amount of EYA3 protein.

[0016] In this article, the agent that inhibits the expression level of EYA3 refers to a substance that can target the inhibition of the activity or expression of EYA3, or target the blocking of EYA3, or target the degradation of 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 a shRNA, siRNA or sgRNA designed based on EYA3, but it is not limited to this. Any substance and agent that can target the inhibition of EYA3 in the art belongs to the protection scope of the present invention. In some specific embodiments of the present invention, the agent that inhibits the expression level of EYA3 is a shRNA designed based on EYA3, and the shRNA contains a nucleotide sequence as shown in SEQ ID NO.1 or 2. More preferably, the shRNA contains a nucleotide sequence as 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 also 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 one by one here.

[0020] The drug for improving the responsiveness of immunotherapy after radiotherapy of cervical cancer in the present invention contains an effective amount of the above-mentioned agent for inhibiting the expression level of EYA3, and also 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, the immunotherapy refers to a treatment 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, mainly through some immune checkpoint inhibitors, therapeutic antibodies or cancer vaccines, etc. In the present invention, the immunotherapy drug is an immune checkpoint inhibitor, and any immune checkpoint inhibitor used for the treatment of cervical cancer in the art is applicable, especially αPD-1 antibodies.

[0023] Beneficial effects of the present invention:

[0024] The present invention determined that the mRNA expression of EYA3 in cervical cancer tissue and adjacent tissue was significantly different through database bioinformatics analysis, and determined through mouse experiments that the expression level of EYA3 affects the growth or development of tumor tissue in cervical cancer patients. Knocking down EYA3 significantly slowed the growth or progression of cervical cancer tumor tissue, and thus can be used as a drug target for the treatment of cervical cancer, based on which corresponding drugs for the treatment of cervical cancer can be developed.

[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 immunotherapy after radiotherapy in cervical cancer patients can be significantly improved, and the immunotherapy of cervical cancer patients can be effectively guided, and the treatment effect of cervical cancer in patients can be improved. 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; wherein, Figure 2 A is the KEGG analysis result of proteomic analysis of cervical cancer before and after radiotherapy; Figure 2B is a schematic diagram of the whole genome 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 databases 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; Figure 3 A is the statistical result of EYA3 expression in the IHC staining results of tumor tissues and adjacent tissues of 41 cervical cancer patients in Example 3, the 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; wherein, Figure 5 A is a line graph of tumor growth size of U14 cells implanted subcutaneously in C57 mice with different Eya3 expression levels, including Eya3-CON control (normal Eya3 expression group), Eya3-KD1, Eya3-KD2 (Eya3 gene knockdown groups), 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 are for the apoptosis ratio of U14 and U14-R cells in Example 5 at different irradiation doses.

[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: 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 growth curve and survival curve of C57 mouse tumor after U14-R and U14-KD2 were injected into mice. *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0034] 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 should not 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 that do not specifically record conditions or steps are conventional methods, and the reagents and materials used can be obtained from commercial sources.

[0036] It can be understood 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 University of Science and Technology of China, and passed the review of the Ethics Committee, and the patients signed informed consent.

[0038] Example 1 Bioinformatics Analysis

[0039] In this example, the cervical squamous cell carcinoma dataset TCGA-CESC (data type is transcriptome data) was downloaded from the cancer gene expression database TCGA (URL: https: / / cancergenome.nih.gov / ) for bioinformatics analysis. By analyzing the differential mRNA in the paracancerous tissue and cancerous tissue of cervical cancer, it was determined that the mRNA expression of Eya3 in the paracancerous tissue and cancerous tissue 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 library

[0041] In this example, the Eya3 gene that simultaneously regulates cervical cancer radiotherapy resistance was identified and obtained through proteomic sequencing of cervical cancer radiotherapy and immune tolerance tissues and negative screening of CRISPR-sgRNA libraries, as follows:

[0042] 1. Cervical cancer tissues or adjacent tissues were obtained from 4 patients with residual cervical cancer after external irradiation and were commissioned to Hangzhou Jingjie Biotechnology Co., Ltd. for proteomic analysis. The focus was on analyzing the resistance of these patients to subsequent immune checkpoint inhibitors (ICIs) treatment to determine the biological mechanism of residual cervical cancer after radiotherapy. Table 1 shows the information of 4 patients with residual cervical cancer after external 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 II 25 fractions, 2 Gy / fraction Patient C 46 Adenosquamous carcinoma of the cervix FIGO IIIC1 25 fractions, 2 Gy / fraction Patient D 64 Cervical squamous cell carcinoma FIGO IIA2 25 fractions, 2 Gy / fraction

[0045] After proteomic analysis, most peptides were distributed between 7 and 20 amino acids, which is consistent with the general rules of enzymatic hydrolysis and mass spectrometry fragmentation. Further, a total of 7072 functional proteins were identified by mass spectrometry detection, of which 523 proteins were upregulated and 258 proteins were downregulated, and changes of more than 1.5 times were defined as significant change thresholds. In order to determine whether differentially expressed proteins have a significant enrichment trend in certain biological processes, all proteins of interest were further annotated by KEGG pathway classification. Compared with the current largest-scale whole genome and exome sequencing of cervical cancer tissue (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), it can be seen that the enrichment of the Hippo pathway, PI3K-Akt pathway and EMT process is similar to the above studies. In particular, neoplastic processes including carbohydrate metabolism and base excision repair were elevated, whereas leukocyte-mediated immunity 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. Human Genome CRISPR Knockout Libraries) performed genome-wide RNAi screening to explore how cervical tumors that survived radiotherapy escaped immune clearance or resisted ICIs.

[0047] Cervical cancer cells were transfected with low-abundance lentivirus for 48 hours to ensure that each cell was transfected with at most one lentivirus. Cancer cells were selected with puromycin and treated with 6Gy 6MV-X-rays. PBS was used as a control group. The sgRNA sequences and copy number changes in surviving cells were amplified and sequenced for negative screening (see the process diagram for details). Figure 2 B, the specific steps and conditions were carried out according to the operating instructions provided by Suzhou Hongxun Biotechnology Co., Ltd.).

[0048] In order to explore the adaptive changes caused by radiotherapy specificity, this example focuses on evaluating the biological effects induced by radiotherapy, that is, the importance of gene sequences related to DNA damage repair and cell cycle transition to cell survival. Since the knockout of important genes related to cell survival will lead to increased cell apoptosis under radiotherapy conditions, this example performs negative screening, that is, focusing on the 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 example identified 22 genes with significant differences. Most of the significantly changed genes were interconnected and increased in the residual cervical tissue after radiotherapy, such as Eya3, Eya4, RCF4, MDC1 and RAD50. Figure 2 C)

[0050] Through the results of this example, the Eya3 gene that regulates the radiotherapy resistance of cervical cancer was identified and obtained.

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

[0052] In this example, 41 tissue samples of cervical cancer patients (CC) before radiotherapy were included in the cervical cancer microarray. All of these cervical cancer patients had cervical squamous cell carcinoma, and the stages were all II-III or III. The age distribution of the patients was 9 cases ≤45 years old and 32 cases >45 years old. Among these tissue samples, 19 were paracancerous tissues and 22 were cancerous 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 repair (Servicebio, China) → 3% hydrogen peroxide (G0115Servicebio) treatment → 3% BSA blocking (G5001 Servicebio) → primary antibody (ab95876, Abcam) overnight incubation at 4°C → secondary antibody incubation at room temperature (GB23301, Servicebio) → DAB color development (DA1016, Solebol) → hematoxylin counterstaining of the nucleus (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 tissue ( 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, and 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 technology and overexpression lentiviral transfection technology to observe the relationship between different expression levels of Eya3 and the growth of mouse tumors and the survival of mice 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 dish was placed in a biosafety cabinet for subculturing. The complete culture medium consisted of: DMEM basal culture medium (Cat. No SH30022.01, Cytiva) + 10% serum (F0193, Sigma) + 1% penicillin-streptomycin (15140122, Gibco);

[0060] (2) The culture medium was then discarded, the cells were washed twice with PBS, and 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) After the digestion was terminated, the U14 cells were transferred to a 15 mL centrifuge tube, pipetted evenly, and 20 μL was taken for cell counting;

[0063] (5) Centrifuge at 1000 rpm for 3 minutes, discard the supernatant, resuspend the cells in complete medium, and plate the cells into a six-well plate containing complete 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 all commissioned to be 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, GeneCare Gene) into each well;

[0077] (3) 48 h after infection, the medium was replaced with complete culture medium. 24 h after the medium 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 above cells after puromycin selection were collected, and the knockdown efficiency of Eya3 shRNA1# and shRNA2# and the expression efficiency of Eya3-OE in U14 were verified by qPCR analysis. Among them, the total RNA of cells was extracted using a cell / tissue total RNA extraction kit (19221ES50, YESEN), and the mRNA was reverse transcribed using a Takara reverse transcription kit (RR047A, Takara); GS AntiQ qPCR SYBR Green Fast Mix (SQ410, Genesand) was used for cDNA amplification and quantitative analysis. The specific operations were carried out according to the instructions of the kit, and 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 matrix gel (Cultrex Basement Membrane Extract, PathClear, 3432-010-01, R&D Systems) and injected subcutaneously into the back of 6-week-old C57 female mice (Shanghai Slake) at a volume of 2×10 5 Piece / piece.

[0083] The size of the mouse tumor was measured from the 9th day. The mice were killed when the maximum diameter of the mouse tumor was ≥ 2 cm, and the survival time of the mice was recorded. During the whole experiment, the mice were fed with normal feed according to the 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, which was significantly different; 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). The above results indicate that different expression levels of Eya3 affect tumor growth and mouse survival, and knocking down Eya3 can delay the growth or development 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 U14 radiotherapy resistant strain (U14-R), the resistant strain was induced by intermittent induction from low to high doses. The specific steps are as follows:

[0087] (1) U14 cells (Shanghai Cell Bank) were obtained and subcultured with 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 and a radiation dose of 2 Gy. The medium was then replaced to prevent contamination. The cell growth was subsequently observed. When the cell density grew to 80% again, the cells were subcultured twice and labeled as U14-2Gy.

[0088] (2) Take U14-2Gy cells, culture them until the density reaches 60-80%, irradiate them with X-rays, and use a radiation dose of 4Gy. Then replace the culture medium to prevent contamination. Observe the cell growth status later. When the cell density grows to 80% again, pass 1 to 2 and mark them as U14-4Gy.

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

[0090] Among them, the construction effect of U14-R cells was judged by a cell apoptosis experiment. The specific steps are as follows: take the U14-R cells that grow stably after each radiation and the U14 parent cells irradiated at the same dose and plant them in a 24-well plate. When the cell density is 80%, aspirate the cell culture medium into a centrifuge tube, wash the adherent cells once with PBS, add an appropriate amount of 0.25% trypsin to digest the cells, and avoid digestion time being too short or too long. Add an appropriate amount of complete cell culture medium, gently blow down the cells, transfer them to the corresponding centrifuge tube that has been added with cell culture medium, centrifuge at 1000g for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells with PBS and count them. Take 50,000-100,000 cells, centrifuge at 1000g for 5 minutes, discard the supernatant, add the detection reagent according to the instructions of Annexin V-FITC Cell Apoptosis Detection Kit (Biyuntian, China) and perform cell apoptosis detection.

[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 the radiotherapy-resistant strain ( Figure 6 )

[0092] 2. Western blot experiment

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

[0094] 1×10 6 The total protein of each cancer cell (U14 or U14-R) was lysed with RIPA buffer, and the protein concentration level was determined by BCA protein concentration determination kit (P0010, Beyotime). The protein samples with loading buffer (P0015F, Beyotime) were boiled for 10 minutes, and 20-40 μg of each sample was separated in 4-20% protein precast gel (ET12420Gel, ACE) at constant voltage (first 80V 30min, then 100V 60min) for 1.5h, and the separated protein was transferred to PVDF membrane (IPVH00010, Millipore) at constant current (260mA, 90min) for 1.5h. 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-50ul, Proteintech; GAPDH, AB0037-100μl, 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 U14 radiotherapy-resistant strain constructed in the previous article and normal untreated U14 cells were mixed with matrix gel and injected subcutaneously into the back of 6-week-old female C57 mice (Shanghai Slake) at a volume of 1.3×10 5 / mouse, half of the mice in the NC group (normal untreated U14 cells) and the U14-R group were treated with αPD-1 antibody (InVivoMAb anti-mouse PD-1 (CD279) BE0146-50MG, Clone RMP1-14, Bioxcell) starting on day 10, intraperitoneal injection, 200 μg per mouse, once every four days, a total of 3 intraperitoneal drug injections, and the other half received an equal amount of IgG2a isotype control treatment (InVivoPlus rat IgG2a isotype control, anti-trinitrophenol, Catalog # BP0089, Clone 2A3, Bioxcell). During the entire experiment, the mice were fed normally according to the SPF level. 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+lgG (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. The same method as in Example 4 was used for qPCR analysis 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 number and manner as in Example 4, i.e., 2×10 5 / mouse, and half of the mice were treated with αPD-1 antibody (200μg / mouse, injected once every four days, for a total of 3 injections), and the other half were treated with an equal amount of IgG2a isotype control (InVivoPlus ratIgG2a isotype control, anti-trinitrophenol, Catalog#BP0089, Clone 2A3, Bioxcell), and the tumor growth of the mice was observed. Throughout the experiment, the mice were fed normally according to the SPF level. The specific groupings were: U14-R+lgG (IgG2a isotype control in the radiotherapy tolerant group); U14-R+αPD-1 (radiotherapy tolerant group combined with PD-1 inhibitor); U14-R-KD2+lgG (radiotherapy tolerant strain knocking down Eya3 gene IgG2a isotype control); U14-R-KD2+αPD-1 (radiotherapy tolerant strain knocking down Eya3 gene 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, survived longer, and responded more strongly to the αPD-1 antibody treatment ( 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 directly performed without reducing the expression of the EYA3 gene, the subsequent combined immunotherapy will be ineffective. 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] Through the above examples, it can be seen that the expression of EYA3 affects the growth or development of cervical cancer, and knocking down EYA3 can significantly slow down the growth or development of cervical cancer tumor tissue. In addition, EYA3 is also a key target that affects the responsiveness of immunotherapy after radiotherapy of cervical cancer. If the expression of EYA3 in cervical cancer tissue after radiotherapy is significantly increased, this will make the subsequent immunotherapy less effective. By knocking down EYA3, the responsiveness of immunotherapy after radiotherapy of cervical cancer can be significantly improved, thereby providing more accurate guidance for the treatment of cervical cancer patients to improve the treatment effect of cervical cancer.

[0103] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of EYA3 as a target in the preparation of drugs for treating cervical cancer or for improving the responsiveness of immunotherapy after radiotherapy of cervical cancer.

2. Use of an agent that inhibits the expression level of EYA3 in the preparation of a drug for treating cervical cancer or a drug for improving the responsiveness of immunotherapy after radiotherapy of cervical cancer.

3. The use according to claim 2, characterized in that The reagent for inhibiting the expression level of EYA3 is a reagent containing an amount of EYA3 protein that is inhibited from being expressed.

4. The use according to claim 2 or 3, characterized in that The reagent for inhibiting the expression level of EYA3 is a shRNA designed based on EYA3; Preferably, the shRNA contains the nucleotide sequence shown in SEQ ID NO.1 or 2.

5. A drug for treating cervical cancer, characterized in that: It contains an agent that inhibits the expression level of EYA3; Preferably, the reagent for inhibiting the expression level of EYA3 is a reagent containing an amount of EYA3 protein that is inhibited.

6. The drug according to claim 5, characterized in that The reagent for inhibiting the expression level of EYA3 is a shRNA designed based on EYA3; Preferably, the shRNA contains the nucleotide sequence shown in SEQ ID NO.1 or 2.

7. A drug for improving the responsiveness of immunotherapy after radiotherapy of cervical cancer, characterized in that: It contains an agent that inhibits the expression level of EYA3.

8. The drug according to claim 7, characterized in that The reagent for inhibiting the expression level of EYA3 is a reagent containing an amount of EYA3 protein that is inhibited from being expressed.

9. The drug according to claim 7, characterized in that The reagent for inhibiting the expression level of EYA3 is a shRNA designed based on EYA3; Preferably, the shRNA contains the nucleotide sequence shown in SEQ ID NO.1 or 2.

10. The drug according to claim 7, characterized in that The drug used in the immunotherapy is αPD-1 antibody.

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