CN119318708B

CN119318708B - Application of EYA3 as a target in the treatment of cervical cancer

Info

Publication number: CN119318708B
Application number: CN202411435790.6A
Authority: CN
Inventors: 郭丹玉; 钱东; 周培杰
Original assignee: Anhui Provincial Hospital First Affiliated Hospital Of Ustc; University of Science and Technology of China USTC
Current assignee: Anhui Provincial Hospital First Affiliated Hospital Of Ustc; University of Science and Technology of China USTC
Priority date: 2024-10-15
Filing date: 2024-10-15
Publication date: 2025-10-10
Anticipated expiration: 2044-10-15
Also published as: CN119318708A

Abstract

The present invention discloses the use of EYA3 as a target in the treatment of cervical cancer. The present invention determines the key role of EYA3 expression in cervical cancer. Knocking down EYA3 can significantly slow or reduce the growth of cervical cancer tumor tissue or the progression of the disease, thereby providing a new drug target for the treatment of cervical cancer. At the same time, the present invention finds that EYA3 is also a key target for the response to immunotherapy after radiotherapy of cervical cancer. By knocking down EYA3, the responsiveness of immunotherapy after radiotherapy of cervical cancer can be significantly improved, the effect of immunotherapy can be improved, and a more accurate and better solution for the treatment of cervical cancer can be provided to improve the treatment effect of cervical cancer.

Description

Application of EYA3 as target in cervical cancer treatment

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of EYA3 serving as a target spot in cervical cancer treatment.

Background

Cervical cancer is the fourth biggest malignancy of women worldwide, with new cases of 57 tens of thousands per year and death numbers of more than 31 tens of thousands. Wherein, the new occurrence rate of China is close to 13 ten thousand per year, and the new occurrence rate of China accounts for about 1/4 of the number of new occurrence rates worldwide. For early cervical cancer, surgery is mainly used, and for local and late cervical cancer, radiotherapy and contemporaneous chemotherapy are mainly recommended. In China, more than half of cervical cancer patients are advanced (IB 3, IIA2, IIB-IVA stage), and radical radiotherapy is the most important treatment means. The overall curative effect of patients with cervical cancer at the advanced stage is poor, and 30% of patients after first-line radical radiotherapy have recurrent metastasis within 2 years.

To improve the second-line therapeutic effect of cervical cancer, the latest NCCN guidelines recommend immunotherapy as the most promising complementary treatment regimen. However, cervical cancer immunotherapy has a lower overall response rate compared to other solid tumors. Of particular note, the response rate of immunotherapy following radiation therapy is particularly low in most cervical cancer patients. It has been shown that immunotherapy mainly eliminates cancer cells by stimulating an anti-tumor immune response, which is mainly dependent on activating CD8 ⁺ T cells or utilized immune cells, and thus radiation therapy may reduce the therapeutic effect of immunotherapy by up-regulating infiltration of inhibitory immune cells and directly damaging circulating lymphocytes, including CD8 ⁺ T cells. Searching for a key target for influencing the growth or proliferation of tumor tissues of cervical cancer, even a key target for influencing the immune response of the tumor tissues after radiotherapy, can provide a new thought for the treatment of cervical cancer and improve the treatment effect of cervical cancer.

Eye-deficiency protein 3 (EYA 3) is one of the members of the eye-deficiency (EYA) protein family, belonging to the transcriptional coactivators and phosphatases in humans. The EYA3 gene is located in human at position 1p35.3 of chromosome 1, and the protein it encodes functions 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 that abnormal expression of the EYA3 gene is associated with tumor formation, proliferation, invasion and survival. In particular EYA3 is associated with protein phosphatase 2A (PP 2A), a major Ser/Thr phosphatase that plays a key role in regulating a variety of cellular processes. EYA3 regulates PP2A activity and substrate specificity by interacting with the B55 alpha subunit of PP2A, thereby affecting tumor progression.

There is no report on the application of EYA3 in cervical cancer treatment.

Disclosure of Invention

In view of the above, the primary object of the present invention is to provide application of EYA3 in cervical cancer treatment, and the present invention finds that the expression level of EYA3 affects the growth and progress of cervical cancer, and knocking down EYA3 can significantly reduce the growth or proliferation rate of cervical cancer tumor tissue and increase the survival period. In addition, the invention discovers that EYA3 expression in residual cancer tissues after radiotherapy is obviously increased compared with that before radiotherapy, which affects the effect of subsequent immunotherapy, and can obviously improve the responsiveness of the cervical cancer patient after radiotherapy and the effect of immunotherapy by knocking down EYA3, thereby providing a better, more accurate and better clinical scheme for the treatment of cervical cancer.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the first aspect of the invention provides application of EYA3 as a target in preparing a medicament for treating cervical cancer or a medicament for improving the response of immunotherapy after radiotherapy of cervical cancer.

The second aspect of the invention provides the use of an agent that inhibits EYA3 expression levels in the manufacture of a medicament for the treatment of cervical cancer or a medicament for improving responsiveness of immunotherapy after radiotherapy of cervical cancer.

In a third aspect, the invention provides a medicament for the treatment of cervical cancer comprising an agent that inhibits the expression level of EYA 3.

In a fourth aspect, the invention provides a medicament for improving responsiveness of immunotherapy after radiotherapy of cervical cancer, comprising an agent that inhibits the expression level of EYA 3.

In the invention, the expression level of EYA3 is found to affect the growth or proliferation of cervical cancer cells by obtaining tissue specimens from cervical cancer patients before and after radiotherapy and detecting the expression level of EYA3 therein, and then the EYA3 is knocked down or overexpressed in cervical cancer cell lines by animal experiments, so that the growth of cervical cancer cells is found to be remarkably slowed down. In addition, the expression level of EYA3 in residual cervical cancer tissues after radiotherapy is also found to be significantly increased in the invention, which can be a key target for influencing subsequent immunotherapy. In the invention, EYA3 in cancer cells after radiotherapy is knocked down, so that the responsiveness of immunotherapy is remarkably improved, and the effect of the immunotherapy is improved.

In this context, cervical cancer patients refer to patients who have been confirmed to suffer from cervical cancer through clinical diagnosis. The tissue sample refers to cervical cancer tissue or a beside cancer tissue of a cervical cancer patient, and it is understood that the beside cancer tissue has a definition commonly used in clinical research, namely, refers to a tissue at a certain distance from the cervical cancer tissue, and specifically, refers to a tissue at a distance of <3cm from the cervical cancer tissue.

Cervical cancer tissue or paracancerous tissue of a cervical cancer patient is obtained in the present invention by means known or well known in the art, such as biopsy or the like, and will not be described in detail herein. .

In the present invention, the agent for inhibiting the expression level of EYA3 is an agent for inhibiting the expression level of EYA3 gene or an agent for inhibiting the expression level of EYA3 protein, and in some specific embodiments, the agent for inhibiting the expression level of EYA3 is an agent for inhibiting the expression level of EYA3 protein.

In this context, the agent that inhibits the expression level of EYA3 refers to a substance that is capable of targeted inhibition of the activity or expression of EYA3, or targeted blocking of EYA3, or targeted degradation of EYA 3. In particular, it may be a gene interference, gene editing, gene silencing or gene knockout material, for example, in some embodiments of the present invention, the agent that inhibits the expression level of EYA3 is shRNA, siRNA or sgRNA designed based on EYA3, but not limited thereto, and any agent or agent capable of targeted inhibition of EYA3 in the art is within the scope of the present invention. In some embodiments of the invention, the agent that inhibits the expression level of EYA3 is an shRNA designed based on EYA3, the shRNA comprising a nucleotide sequence as shown in SEQ ID NO.1 or 2, more preferably the shRNA comprising a nucleotide sequence as shown in SEQ ID NO. 2.

In the invention, the medicament for treating cervical cancer contains an effective amount of the agent for inhibiting EYA3 expression level and any pharmaceutically acceptable carrier and/or auxiliary materials.

Wherein, in some embodiments of the invention, an effective amount as described herein refers to the minimum dose that inhibits EYA3 expression levels and results in an improvement or even cure in the disease in cervical cancer patients, where improvement refers primarily to a reduction in tumor growth or volume.

The carrier and/or adjuvant may be designed according to the specific pharmaceutical dosage form and the like, and is not particularly limited, and those skilled in the art may select or adjust accordingly according to pharmacology and the like, and will not be specifically described herein.

The drug for improving the response of the immunotherapy after the cervical cancer radiotherapy contains an effective amount of the agent for inhibiting the EYA3 expression level and any pharmaceutically acceptable carrier and/or auxiliary materials.

Wherein, the effective amount as described herein refers to the minimum dose capable of inhibiting the expression level of EYA3 and allowing the effect of immunotherapy to be improved.

It is understood that in the present invention, the immunotherapy refers to a therapeutic method that uses the autoimmune system of the human body to recognize, attack and destroy cancer cells, thereby enhancing or directly activating the immune response of the human body to combat tumors, mainly by some immune checkpoint inhibitors, therapeutic antibodies or cancer vaccines, etc. In the invention, the immunotherapeutic agent is an immune checkpoint inhibitor, and any immune checkpoint inhibitor used for cervical cancer treatment in the art is applicable, in particular an alpha PD-1 antibody.

The invention has the beneficial effects that:

according to the invention, through database belief analysis, the significant difference of mRNA expression of EYA3 in cancer tissues and beside cancer tissues of cervical cancer is determined, and the growth or development of tumor tissues of cervical cancer patients is influenced by the expression level of EYA3 through a mouse test. The knockdown EYA3 obviously slows down the growth or progress of cervical cancer tumor tissues, so that the compound can be used as a drug target for cervical cancer treatment, and corresponding drugs for treating cervical cancer can be developed based on the compound.

The invention simultaneously carries out negative screening through proteomics sequencing and CRISPR-sgRNA library, and determines EYA3 to be a key target for regulating and controlling cervical cancer radiotherapy resistance through experiments. Treatment of cervical cancer is often followed by radiation and/or chemotherapy, and combination immunotherapy is also required to further enhance the therapeutic effect. In the invention, EYA3 is determined by a mouse test to be a key target of immune tolerance after cervical cancer radiotherapy, and by knocking down EYA3, the responsiveness of the cervical cancer patient to the immune treatment after radiotherapy can be remarkably improved, the immune treatment of the cervical cancer patient can be effectively guided, and the cervical cancer treatment effect of the patient can be improved. The invention has good application prospect and value.

Drawings

Fig. 1 shows the results of the TCGA database of example 1, P <0.05.

FIG. 2 shows the proteomics analysis and CRISPR-sgRNA library negative selection results in example 2, wherein FIG. 2A shows the KEGG analysis results of the proteomics analysis before and after cervical cancer radiotherapy, FIG. 2B shows the CRISPR-Cas9 sgRNA whole genome selection diagram, 4-6 candidate interference sequences are selected for each gene, and FIG. 2C shows the first 22 candidate genes with significant differences obtained from human SiHa (left) and HeLa (right) cell line-gene library databases through cross-screening.

Fig. 3 shows the results of statistical analysis of clinical relevance of Eya expression levels in cervical cancer side and tumor tissues, wherein fig. 3A shows the statistical results of EYA3 expression in IHC staining results of 41 cervical cancer patients in example 3, data are mean+ -SD, P <0.001, and fig. 3B shows IHC staining results before and after radiotherapy of cancer tissues of 4 cervical cancer patients in example 2, with a scale of 50 μm.

Fig. 4 shows the efficiency of Eya3 expression in Eya knockdown and over-expressed U14 cells constructed in example 4, P <0.05, P <0.01, P <0.001.

FIG. 5 shows the results of the subcutaneous tumor growth of C57 mice implanted with U14 cells with different Eya expression levels in example 4, wherein FIG. 5A shows the line graph of the subcutaneous tumor growth of C57 mice implanted with U14 cells with different Eya3 expression levels, including Eya3-CON control (Eya 3 expression normal group), eya3-KD1, eya3-KD2 (Eya 3 knock-down group), eya-OE (Eya gene over-expression group), P <0.05, P <0.01, P <0.001, and FIG. 5B shows the line graph of the subcutaneous mouse growth of C57 mice implanted with U14 cells with different Eya3 expression levels, including Eya3-CON control (Eya expression normal group), eya-KD 1, eya-KD 2 (Eya 3 knock-down group), and Eya-OE (Eya gene over-expression group).

FIG. 6 shows the results of the ratio of apoptosis of U14 and U14-R cells of example 5 at different irradiation doses.

FIG. 7 shows the results of Western blotting assays for Eya in U14 and U14-R cells in example 5.

FIG. 8 shows the results of the immune response test of Eya in example 5 after radiotherapy, wherein FIG. 8A shows the tumor growth curve of C57 mice after injection of U14-NC and U14-R into mice, FIG. 8B shows the expression efficiency of Eya3 in U14-R-KD1 and U14-R-KD2, and FIG. 8C shows the tumor growth curve and survival curve of C57 mice after injection of U14-R and U14-KD2 into mice, respectively. * P <0.05, < P <0.01, < P <0.001.

Detailed Description

The following detailed description of embodiments of the invention is exemplary and is provided merely to illustrate the invention and is not to be construed as limiting the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, unless otherwise specified, the conditions or procedures are not specifically described and the reagents and materials employed are commercially available.

It will be understood that "EYA3", "Eya3", "ocular delectant 3", etc. as described herein have the same meaning and are merely different representations of the same protein or gene without distinction.

The tissue samples of cervical cancer patients referred to in the following examples were all from the affiliated first hospital of the university of chinese science and technology, and were approved by the ethics committee, and the patients signed informed consent.

Example 1 bioinformatics analysis

In this example, by performing a belief analysis by downloading cervical squamous cell carcinoma dataset TCGA-CESC (data type is transcriptome data) in cancer gene expression database TCGA (website: https:// cancerargenome. Nih. Gov /), and by analyzing differential mRNA in the paracancerous and cancerous tissues of cervical cancer, it was determined that Eya3 had significant differences in mRNA expression in the paracancerous and cancerous tissues of cervical cancer (FIG. 1), suggesting Eya3 may be a potential target for treating cervical cancer.

EXAMPLE 2 proteomic sequencing and negative selection of CRISPR-sgRNA library

In the embodiment, through proteomics sequencing of cervical cancer radiotherapy and immune tolerance tissues and negative screening of a CRISPR-sgRNA library, eya genes for simultaneously regulating and controlling cervical cancer radiotherapy resistance are identified and obtained, and the specific steps are as follows:

1. the cervical cancer patients with 4 exception-irradiated tumor residues were subjected to proteomic analysis by cervical cancer tissue or beside cancer tissue commission Jing Jie biotechnology, inc., and their resistance to subsequent Immune Checkpoint Inhibitor (ICIs) treatment was analyzed again to determine the biological mechanism of residual cervical cancer after radiotherapy, wherein 4 exception-irradiated tumor residues of cervical cancer patients information is shown in Table 1.

TABLE 1 external illumination of cervical cancer patient information of tumor residues

	Age of	Diagnosis of	Stage by stage	Radiotherapy conditions
					Patient A	66	Cervical squamous carcinoma	FIGO IIIB	25 Times, 2 Gy/time
Patient B	56	Cervical squamous carcinoma	FIGO IIB	25 Times, 2 Gy/time
					Patient C	46	Cervical squamous carcinoma	FIGO IIIC1	25 Times, 2 Gy/time
Patient D	64	Cervical squamous carcinoma	FIGO IIA2	25 Times, 2 Gy/time

Through proteomics analysis, most peptides are distributed among 7-20 amino acids, which accords with the general rules of enzymolysis and mass spectrometry fragmentation. Further, 7072 functional proteins were identified in total by mass spectrometry detection, of which 523 proteins were up-regulated and 258 proteins were down-regulated, and changes exceeding 1.5-fold were defined as significant change thresholds. To determine if the differentially expressed proteins have a significant tendency to enrich in certain biological processes, all proteins of differential interest were further annotated by KEGG pathway classification. In comparison to current maximum-scale cervical cancer tissue whole genome and exome sequencing (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 enrichment of the Hippo pathway, PI3K-Akt pathway and EMT process is similar to the study described above. In particular, the nascent processes including carbohydrate metabolism and base excision repair are elevated, while the leukocyte-mediated immune and apoptosis signaling pathways are down-regulated (fig. 2A), suggesting that this may be responsible for the lower efficiency of ICIs after radiotherapy.

2. Whole genome CRISPR SGRNA library customized by Hongsu biotechnology Co., ltd (STCRI 211101)Human whole genome, CRISPR knockout library Human Genome CRISPR Knockout Libraries) was screened for whole genome RNAi, exploring how residual cervical tumors after radiation treatment escape immune clearance or resist ICIs.

Cervical cancer cells were transfected with low abundance lentiviruses for 48h, respectively, ensuring that after each cell was transfected with at most one lentivirus, cancer cells were selected with puromycin, treated with 6gy 6 mv-X-rays, and PBS treated as a control group. The sgRNA sequence and copy number changes in surviving cells were amplified and sequenced for negative selection (see fig. 2B for a schematic flow chart, specific steps and conditions were performed according to the instructions provided by the sohong biotechnology, inc. Of su state).

In order to explore the adaptive changes caused by radiation therapy specificity, the importance of radiotherapy-induced biological effects, i.e., the importance of gene sequences related to DNA damage repair and cell cycle shift for cell survival, was evaluated with emphasis in this example. Since knockout of important genes related to cell survival leads to increased apoptosis under radiotherapy conditions, negative selection was performed in this example, i.e., the portion of the gene sequence with reduced expression abundance was of major concern.

As a result, this example identifies 22 genes that differ significantly by taking intersections of negative selection data obtained from the screening of the SiHa and HeLa cell gene library, with most significantly altered genes being interconnected and elevated in residual cervical tissue after radiotherapy, such as Eya, eya, RCF4, MDC1, and RAD50. (FIG. 2C)

According to the results in the embodiment, eya genes for regulating and controlling the cervical cancer radiotherapy resistance are identified and obtained.

Example 3Eya clinical relevance study of expression in cervical cancer microarrays

In this example, 41 cervical cancer patients (CC) included in the cervical cancer microarray, which are pre-radiotherapy tissue samples, are cervical squamous cell carcinoma, and stage II-III or stage III, and the age distribution of the patients is 9 cases less than or equal to 45 years old, and 32 cases less than or equal to 45 years old. Of these tissue samples, 19 cases of paracancerous tissue and 22 cases of cancerous tissue.

In this example, immunohistochemical (IHC) staining is performed on the tissue samples, wherein the IHC staining step is that the sections are dewaxed to water (Servicebio, china), antigen retrieval (Servicebio, china), 3% hydrogen peroxide (G0115 Servicebio) treatment, 3% BSA blocking (G5001 Servicebio), overnight incubation of primary antibody (ab 95876, abcam) at 4 ℃, incubation of secondary antibody at room temperature (GB 23301, servicebio), DAB coloration (DA 1016, solebao), hematoxylin counterstain (Servicebio, china) and microscopic examination. Eya3 expression was found to be significantly higher in the queued cancer region in this example than in the paracancerous tissue by IHC staining (fig. 3A).

In addition, in this example, the tumor tissue before radiotherapy and the residual tumor tissue after radiotherapy in the clinical tissue sample of 4 cervical cancer patients in example 2 were stained with IHC, and the amount of Eya expression in the residual cancer tissue after Radiotherapy (RT) was found to be higher than that in the biopsy specimen before Radiotherapy (RT) (FIG. 3B).

The result further suggests that Eya gene is a key target for regulating and controlling the resistance of cervical cancer radiotherapy.

Example 4Eya relation of different expression levels to tumor growth and mouse survival

In this embodiment, eya genes in U14 cells are knocked down and overexpressed by shRNA lentiviral transfection technique and overexpressed lentiviral transfection technique to realize in vivo observation of the relationship between different expression amounts of Eya3 to growth of mouse tumor and survival period of mouse, specifically comprising the following steps:

1. U14 cell treatment on day one

(1) Culturing U14 cells (Shanghai cell bank) in a 10cm dish complete medium until the cell density is about 80%, and placing the culture medium in a biosafety cabinet for passaging, wherein the complete medium comprises DMEM basal medium (Cat.No SH30022.01, cytiva) +10% serum (F0193, sigma) +1% penicillin streptomycin (15140122, gibco);

(2) The medium was then discarded, washed 2 times with PBS, and digested with 1mL of 0.25% pancreatin (accession No. 25200056, gibco) at 37℃for 2min;

(3) Terminating digestion with 5mL of the same complete medium as in step (1);

(4) Transferring the U14 cells after digestion termination into a 15mL centrifuge tube, blowing uniformly, and taking 20 mu L for cell counting;

(5) Centrifugation at 1000rpm for 3 min, supernatant was discarded, cells were resuspended in complete medium, and plated into six well plates containing complete medium, 8 ten thousand U14 cells per well.

2. Lentiviral transfection was performed the next day:

(1) The lentivirus was removed from the refrigerator and slowly thawed on ice, and the lentivirus information involved in this step was as follows:

<EYA3-RNAi1>

CCGGAGTGAATTGGAACGGGTATTTCTCGAGAAATACCCGTTCCAATTCACTTTTTTG (SEQ ID NO. 1), wherein the target sequence is AGTGAATTGGAACGGGTATTT.

<EYA3-RNAi2>

CCGGCCACAACATTAGCAGCTACAACTCGAGTTGTAGCTGCTAATGTTGTGGTTTTTG (SEQ ID NO. 2), wherein the target sequence is CCACAACATTAGCAGCTACAA.

< Negative control Virus >

Control number CON207

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

< Overexpressed lentivirus >

Gene name Eya (NM-010166), the sequence of the over-expressed lentivirus is shown in SEQ ID NO. 4.

All of the above lentiviruses were assigned to Shanghai Ji Kai Genech medical science and technology Co.Ltd.

(2) The supernatant from each well of the six-well plate was aspirated, and 2mL of complete medium +30. Mu.L of virus solution (MOI=100) +5. Mu.L of lentivirus infection enhancer HITRANSG P (cat# REVG005, ji Kai gene) was added to the wells;

(3) After 48h of infection, the whole medium was changed, and after 24h of change, the infected cells were screened starting from 0.5. Mu.M with puromymin (P8230-100 mg, soy).

Target cells are obtained through screening, so that Eya-CON (control) cell lines, eya3 knockdown cell lines U14-KD1 (shRNA 1) and U14-KD2 (shRNA 2) and Eya3 over-expression cell lines U14-OE are constructed.

In this example, the above cells were collected after puromycin screening, and the expression efficiency of Eya shRNA 1#, shRNA2# and Eya-OE in U14 were verified by qPCR analysis. Wherein, the total RNA of the cells is extracted by using a total RNA extraction kit (19221ES50, YESEN) of the cells/tissues, mRNA is reversely transcribed by using a Takara reverse transcription kit (RR 047A, takara), cDNA amplification and quantitative analysis are carried out by using GS ANTIQ QPCR SYBR GREEN FAST Mix (SQ 410, genesand), the specific operations are carried out according to the specification of the kit, and the related primer information is as follows:

the results are shown in FIG. 4, which illustrates the successful construction of Eya knockdown and over-expression cell lines in this example.

3. The U14 cell lines constructed above were uniformly mixed with matrigel (Cultrex Basement Membrane Extract, PATHCLEAR,3432-010-01, R & D Systems) and then injected subcutaneously into the back of 6-week-old C57 female mice (Shanghai Srile) at an injection rate of 2X 10 ⁵ cells/mouse.

The tumor size of the mice was measured from day 9, the mice were sacrificed when the maximum tumor diameter of the mice was not less than 2cm, and the survival period of the mice was recorded. Throughout the experiment, mice were fed normally at SPF level, see figure 5 for results.

From FIG. 5, it can be seen that the tumors of the Eya knockdown group (Eya 3-KD 1# and Eya3-KD 2#) mice grew more slowly, had longer survival times, and had significant differences compared to the Eya3 expression normal group (Eya-CON), while the tumors of the Eya3 overexpressing group (Eya 3-OE) mice grew more rapidly, with shorter overall survival times (FIGS. 5A and 5B). The results show that the different expression levels of Eya < 3 > can influence the growth of tumors and the survival time of mice, and the knock-down of Eya < 3 > can delay the growth or development of cervical cancer tumors.

Example 5 in vivo animal experiment-Eya study of responsiveness of 3 to immunotherapy after cervical cancer radiation therapy

1. In vitro construction of U14 radiotherapy tolerance strain (U14-R), wherein the tolerance strain is induced by intermittently inducing from low to high dosage, and the specific steps are as follows:

(1) Taking U14 cells (Shanghai cell bank), subculturing the complete culture medium until the cell density is 60-80%, irradiating an X-ray (6 mV), carrying out Varinan C/D linear accelerator with the dosage rate of 200cGy/min and the radiation dosage of 2Gy, immediately replacing the culture medium to prevent pollution, and subsequently observing the growth condition of the cells until the cell density is regrown to 80%, and carrying out 1-2 transmission, wherein the mark is U14-2Gy;

(2) Culturing U14-2Gy cells to a density of 60-80%, irradiating with X-ray with radiation dose of 4Gy, changing culture medium to prevent pollution, observing cell growth condition, and marking as U14-4Gy when cell density is regrown to 80%, and 1-2;

(3) Similarly, the radiation dose is eventually increased to 12Gy, labeled U14-12Gy. The final cumulative bioequivalent dose (EQD 2) was approximately 65Gy.

The construction effect of the U14-R cells is judged by using an apoptosis experiment, and the specific steps are that the U14-R cells which grow stably after each irradiation and U14 parent cells irradiated under the same dose are respectively planted in a 24-hole plate, when the cell density is 80%, cell culture solution is sucked into a centrifuge tube, the adherent cells are washed once by PBS, and proper amount of 0.25% pancreatin is added to digest the cells, so that the digestion time is prevented from being too short or too long. Adding a proper amount of cell complete culture medium, lightly blowing off the cells, transferring the cells into a corresponding centrifuge tube added with cell culture solution, centrifuging for 5 minutes at 1000g, discarding the supernatant, collecting the cells, lightly suspending the cells with PBS and counting. Taking 5-10 ten thousand cells, centrifuging for 5 minutes at 1000g, discarding supernatant, adding detection reagent according to an Annexin V-FITC apoptosis detection kit (Biyun, china) instruction book, and performing apoptosis detection.

The results show that the apoptosis proportion of U14-R is significantly lower than that of U14 normal cells under the same dose irradiation condition, which indicates successful construction of the radiotherapy-tolerant strain (FIG. 6)

2. Western blot experiment

The U14 cells and the constructed U14-R cells are respectively subjected to protein quantification through Western blot experiments, and the specific steps are as follows:

1X 10 ⁶ cancer cells (U14 or U14-R) were lysed with RIPA buffer to give total protein, and protein concentration levels were determined by BCA protein concentration assay kit (P0010, beyotime). Protein samples with addition of loading buffer (P0015F, beyotime) were boiled for 10min, 20-40 μg of each sample was separated for 1.5h at constant pressure (80V 30min first, 100V 60min second) in 4-20% protein pre-Gel (ET 12420Gel, ACE), and the separated protein constant flow (260 mA,90 min) was transferred to PVDF membrane (IPVH 00010, millipore) for 1.5h. After blocking with 5% BSA for 1h at Room Temperature (RT), the protein strips were incubated with primary antibodies (EYA 3 Polyclonal antibody,21196-1-AP-50ul, proteintech; GAPDH, AB0037-100. Mu.l, abways) overnight at 4 ℃. Finally, protein bands were incubated with HRP conjugated secondary antibody (a 0208, beyotime) for 1h at RT, interacted with HRP substrate and imaged with a chemiluminescent imager (Thermo FISHER SCIENTIFIC, USA).

As shown in FIG. 7, it can be seen that EYA3 protein was expressed in U14-R cells in higher amounts, indicating a correlation between immune tolerance and Eya over-expression.

3. In vivo injection

(1) After uniformly mixing the U14 radiation-resistant strain constructed in the foregoing, normal untreated U14 cells with matrigel, respectively, the cells were injected subcutaneously into the back of 6-week-old C57 female mice (Shanghai Srilk) at an injection rate of 1.3X10 ⁵/mouse, and half of the NC group (normal untreated U14 cells) and U14-R group each received a treatment with an αPD-1 antibody (InVivoMAb anti-mouse PD-1 (CD 279) BE0146-50MG, clone RMP1-14, bioxcell) at day 10, 200 μg per mouse was injected once every four days, and the other half received an equivalent amount of IgG2a isotype control treatment (InVivoPlus rat IgG a isotype control, anti-trinitrophenol, catalog#BP0089, clone 2A3, bioxcell). During the whole experiment, mice are fed according to SPF grade. The grouping is specifically U14-NC+IgG (normal group combined with IgG2a isotype control), U14-NC+alpha PD-1 (normal group combined with PD-1 inhibitor), U14-R+lgG (radiation resistant group combined with IgG2a isotype control), U14-R+alpha PD-1 (radiation resistant group combined with PD-1 inhibitor). The results were similar to those of the clinical treatment, and the immunotherapeutic effect was poorer in the U14-R mice (FIG. 8A).

(2) Eya3 genes in U14-R cell lines constructed in the previous way are knocked down by adopting the shRNA lentiviral transfection technology which is the same as that of example 4 and adopting lentiviruses EYA3-RNAi1 and EYA3-RNAi2 with sequences shown as SEQ ID NO.1 and SEQ ID NO. 2, so that Eya knockdown radiotherapy-resistant cell lines U14-R-KD1 and U14-R-KD2 are obtained. And the expression efficiencies of Eya3 in U14-R, U14-R-KD1 and U14-R-KD2 were verified by qPCR analysis using the same method as in example 4. The results showed that Eya was knockdown successfully in both U14-R-KD1 and U14-R-KD2, and that the knockdown effect of U14-R-KD2 was better (FIG. 8B), so U14-R-KD2 was selected for subsequent experiments.

(3) The U-14R and U14-R-KD2 cells constructed in step (2) were subcutaneously injected into C57 mice in the same number and manner as in example 4, i.e., 2X 10 ⁵ cells/mouse, and half of the mice were also treated with the alpha PD-1 antibody (200. Mu.g/mouse, injected once every four days, 3 times total injection), and the other half with the same amount of IgG2a isotype control treatment (InVivoPlus rat IgG2a isotype control, anti-trinitrophenol, catalyst#BP 0089, clone 2A3, bioxcell), and tumor growth of the mice was observed. During the whole experiment, mice are fed according to SPF grade. The specific grouping conditions are U14-R+lgG (IgG 2a isotype control of the radiotherapy tolerance group), U14-R+alpha PD-1 (combined PD-1 inhibitor of the radiotherapy tolerance group), U14-R-KD2+lgG (knock-down Eya gene IgG2a isotype control of the radiotherapy tolerance strain), U14-R-KD 2+alpha PD-1 (knock-down Eya gene combined PD-1 inhibitor of the radiotherapy tolerance strain).

Experimental results showed that tumor growth was slower, survival longer, and therapeutic response to αpd-1 antibodies was reversed in mice of the U14-R-KD2 group relative to mice of the U14-R group (fig. 8C and 8D).

The results show that the increase of Eya < 3 > indicates the radiotherapy tolerance of cervical cancer, if the EYA3 gene expression is not reduced, the subsequent combined immunotherapy effect is poor, and the responsiveness of the cervical cancer immunotherapy after radiotherapy can be improved by reducing the EYA3 gene expression, so that the treatment effect of the cervical cancer is improved.

From the above examples, it can be seen that EYA3 expression 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. And EYA3 is also a key target for influencing the response of the immunotherapy after the radiotherapy of cervical cancer, if EYA3 expression in cervical cancer tissues after the radiotherapy is obviously increased, the effect of the subsequent immunotherapy is poor, and the response of the immunotherapy after the radiotherapy of cervical cancer can be obviously improved by knocking down EYA3, so that more accurate guidance is provided for the treatment of cervical cancer patients, and the treatment effect of cervical cancer is improved.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. Application of an agent for inhibiting EYA3 expression level in preparing a medicament for treating cervical cancer, wherein the agent for inhibiting EYA3 expression level is shRNA designed based on EYA3, and the nucleotide sequence of the shRNA is shown as SEQ ID NO.1 or 2.

2. The use of claim 1, wherein the medicament is a medicament for enhancing responsiveness of immunotherapy after radiation therapy of cervical cancer.

3. The use of claim 2, wherein the medicament for immunotherapy is an αpd-1 antibody.