Application of ICAT gene as treatment target in cervical cancer

Abstract

The invention discloses application of an ICAT gene as a treatment target in cervical cancer, and relates to the technical field of biomedicine and oncology. The prognosis of the patient is evaluated by detecting the expression level of ICAT in cervical cancer tissues; the detection method comprises the following steps: S1, analyzing correlation between ICAT and CC immune infiltration by a clinical sample; s2, identifying that ICAT promotes the malignant progression of cervical cancer through glycolysis; s3, determining a molecular mechanism for regulating and controlling ENO1 expression by ICAT to remodel cervical cancer glycolysis reprogramming; s4, determining a molecular mechanism of ICAT-dependent lactic acid accumulation for triggering polarization of tumor-associated macrophages; and S5, evaluating a result. According to the invention, ICAT remodeling cervical cancer metabolism reprogramming is taken as an entry point, and ICAT is revealed from the aspects of cells, animals and tissues to promote the activity of cervical cancer tumors, and has clinical value.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomedicine and oncology, and in particular to the application of ICAT gene as a therapeutic target in cervical cancer. Background Art

[0002] Cervical cancer (CC) is a common malignant tumor in women. Most early-stage CC patients can achieve long-term survival after standard treatment. However, the treatment of patients with late-stage recurrent CC is difficult and ineffective, resulting in a high mortality rate and high morbidity and mortality. During the inflammation-carcinogenesis transformation process, the reprogramming of the tumor microenvironment (reprogram tumor microenvironment) caused by changes in the number and types of immune cells in the local microenvironment of the cervix is ​​an important factor in promoting tumor occurrence, development, invasion and metastasis. Gene-targeted therapy and immunotherapy have become research hotspots. However, the overall response rate of immune checkpoint blockade therapy is low (4-26%). Therefore, elucidating the molecular mechanism of tumor microenvironment remodeling in cervical cancer will provide new ideas for finding new therapeutic targets and improving the immunotherapy effect of tumors.

[0003] Although some studies have revealed some molecules associated with cervical cancer progression, molecular targets that can effectively intervene and improve treatment outcomes have yet to be found, limiting the development and implementation of personalized treatment options. Summary of the Invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art and to propose the application of ICAT gene as a therapeutic target in cervical cancer.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] Application of ICAT gene as a therapeutic target in cervical cancer, such as Figure 1 As shown, the prognosis of patients is evaluated by detecting the expression level of ICAT in cervical cancer tissues; the detection method includes the following steps:

[0007] S1: Correlation between ICAT and CC immune infiltration in clinical sample analysis;

[0008] S2: Identification of ICAT promoting cervical cancer malignant progression through glycolysis;

[0009] S3: Clarify the molecular mechanism by which ICAT regulates ENO1 expression to reshape glycolytic reprogramming in cervical cancer;

[0010] S4: Elucidate the molecular mechanisms by which ICAT-dependent lactate accumulation triggers tumor-associated macrophage polarization;

[0011] S5: Result evaluation.

[0012] Preferably, the S1 specifically includes:

[0013] S11: Determine the relationship between ICAT and the clinicopathological characteristics of CC and patient prognosis: Select CC samples for immunohistochemical detection of ICAT expression levels and collect clinical information, including tumor TNM stage, Ki-67 level, histological grade, and treatment status; collect survival prognosis information for patients receiving immunotherapy, and perform univariate and multivariate Cox regression analysis to analyze the relationship between ICAT and overall survival and disease-free survival after immunotherapy;

[0014] S12: Verify the relationship between ICAT and immune cell infiltration in clinical samples: Detect the expression of immune cell markers CD4, CD8, POXP3, CD206, PD-1, and TIM3 in tumor tissues by immunohistochemistry, analyze the correlation, and compare the expression results with those obtained from public database analysis.

[0015] Preferably, the S2 specifically includes:

[0016] S21: Construction of subcutaneous xenograft model;

[0017] S22: RNA-Seq analysis;

[0018] S23: In vitro detection of the effects of altered ICAT expression on glycolysis;

[0019] S24: ICAT regulates immune infiltration in cervical cancer;

[0020] S25: Construct a co-culture system.

[0021] Preferably, the step S3 specifically includes:

[0022] S31: Protein profiling analysis of ICAT-binding protein profiles;

[0023] S32: It is clear that ICAT regulates ENO1 expression by binding to c-Myc;

[0024] S33: Extract nuclear protein of cells with different ICAT expression levels and detect the expression level of c-Myc in the nucleus; detect the expression level of c-Myc in the nucleus by immunofluorescence;

[0025] S34: Verification of c-Myc binding to the ENO1 promoter;

[0026] S35: Designed binding sequence segmented mutation plasmids and transformed them into HeLa cells. Luciferase assay was used to verify the specific binding region between c-Myc and ENO1 promoter.

[0027] Preferably, in said S31, specifically: extracting ICAT-overexpressing HeLa cell protein, using anti-ICAT monoclonal antibody for immunoprecipitation and purification, identifying the ICAT-binding protein map using protein spectrum technology, and predicting ENO1 transcriptional regulatory factors in the HumanTFDB database, drawing a wenn diagram to analyze the intersection area between positive binding proteins and ENO1 transcriptional regulatory factors, and determining c-Myc as the only intersection protein.

[0028] Preferably, in said S32, specifically: extracting ICAT-overexpressing HeLa cell protein, using Anti-ICAT / c-Myc monoclonal antibody for immunoprecipitation and purification, and detecting whether there is binding between ICAT and c-Myc by immunoblotting; treating HeLa cells with a c-Myc-specific inhibitor and then detecting the ENO1 expression level by qPCR and WB.

[0029] Preferably, in said S34, specifically: searching the ENO1 promoter sequence on the NCBI website, constructing a pGL3-basic reporter gene plasmid connected to the promoter sequence, and constructing an overexpression plasmid of an ICAT-related transcription factor in the same manner; co-transfecting the overexpression plasmid and the promoter reporter gene plasmid into HK293T cells, adding a specific luciferase substrate, and measuring the luciferase activity by detecting the fluorescence intensity to determine whether the ENO1 promoter sequence can be targeted and bound; and further performing a ChIP experiment on the promoter sequence to verify the binding specificity of PRKDC.

[0030] Preferably, the step S5 specifically includes:

[0031] S51: Identification of the role of ICAT in establishing a suppressive immune microenvironment;

[0032] S52: ICAT regulates immune infiltration in cervical cancer;

[0033] S53: Demonstrate the dependence of macrophages in ICAT-mediated carcinogenesis;

[0034] S54: Lactate promotes tumor-associated macrophage polarization;

[0035] S55: Analysis of the role of histone lactylation in tumor-associated macrophages.

[0036] The application of ICAT gene as a therapeutic target in cervical cancer. The Seahorse experiment and kit were used to detect the effect of changes in ICAT expression on glycolysis in cervical cancer cells HeLa and SiHa, which served as the basis for analyzing the prognosis of cervical cancer.

[0037] The application of ICAT gene as a therapeutic target in cervical cancer. CRISP-CAS9 technology was used to perform gene editing on THP-1, PBMCs and BMDMs to obtain macrophages with stable ICAT knockdown or overexpression. These macrophages were then co-cultured with cervical cancer cells with different ICAT expression levels to form tumor-associated macrophages. Flow cytometry, qRT-PCR, Western Blot and immunofluorescence methods were used to detect the expression of macrophage markers iNOS, TNF-α, ARG1, TGF-β and IL-10 in TAMs, which served as the basis for analyzing the prognosis of cervical cancer.

[0038] The beneficial effects of the present invention are:

[0039] 1. This invention takes ICAT as the starting point to reshape the metabolic reprogramming of cervical cancer, and reveals that ICAT promotes cervical cancer tumor activity from the cellular, animal and tissue levels, which has clinical value.

[0040] 2. The present invention clarifies the specific molecular mechanism by which ICAT can promote the expression of ENO1 in tumor cells, triggering hyperglycolysis and thus causing lactic acid accumulation to participate in the construction of an inhibitory immune microenvironment.

[0041] 3. This invention proposes "ICAT-promoting glycolysis-lactic acid accumulation-reshaping the immune microenvironment", which has clear logic and reasonable experimental design. It is expected to provide a new strategy for the immunotherapy of cervical cancer and has high clinical translation value. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a research flow chart for the application of the ICAT gene as a therapeutic target in cervical cancer proposed by the present invention;

[0043] Figure 2 Schematic diagram of overexpression of ICAT promoting nuclear translocation of c-Myc in the present invention;

[0044] Figure 3 Schematic diagram showing that pan-lactylation modification and H3K18la expression in macrophages increase in a lactate concentration-dependent manner in the present invention;

[0045] Figure 4 Schematic diagram of the present invention showing that the addition of 2-DG inhibits the increase in pantolactation caused by ICAT;

[0046] Figure 5 Schematic diagram of immunohistochemical staining of ICAT expression in cervical cancer tissue and adjacent normal tissue in the present invention;

[0047] Figure 6 Schematic diagram of qRT-PCR verification of the mRNA level of ICAT knockout or overexpression in HeLa cells in the present invention;

[0048] Figure 7 Schematic diagram of qRT-PCR verification of the mRNA level of ICAT knockout or overexpression in SiHa cells of the present invention;

[0049] Figure 8 Schematic diagram showing changes in cell migration ability in the cell scratch assay of the present invention;

[0050] Figure 9 Schematic diagram of the Transwell assay used in the present invention to evaluate changes in cell migration and invasion abilities;

[0051] Figure 10 Schematic diagram of the detection of lactic acid content in HeLa cells in the present invention;

[0052] Figure 11 Schematic diagram of HeLa cell glucose uptake detection in the present invention;

[0053] Figure 12 Schematic diagram of the detection of ATP production in HeLa cells in the present invention;

[0054] Figure 13 Schematic diagram of the schematic diagram of the detection of lactic acid content in SiHa cells in the present invention;

[0055] Figure 14 Schematic diagram of SiHa cell glucose uptake detection in the present invention;

[0056] Figure 15 Schematic diagram of the SiHa cell ATP production detection method in the present invention;

[0057] Figure 16 Schematic diagram of the scratch assay for evaluating the migration ability of HeLa cells after 2-DG treatment in the present invention;

[0058] Figure 17 Schematic diagram of the scratch assay for evaluating the migration ability of SiHa cells after 2-DG treatment in the present invention. DETAILED DESCRIPTION

[0059] The technical solution of the present invention will be further described in detail below in conjunction with specific implementation methods.

[0060] Example 1:

[0061] The application of ICAT gene as a therapeutic target in cervical cancer needs to be verified through experiments. The specific methods are as follows:

[0062] Part I: Correlation between ICAT and CC immune infiltration in clinical samples

[0063] (1) Clarify the relationship between ICAT and the clinicopathological characteristics of CC and the prognosis of patients: (1) Clarify the relationship between ICAT and the clinicopathological characteristics of TNBC and the efficacy of immunotherapy in patients: With the approval of the hospital ethics committee, 100 CC samples were selected to detect the expression level of ICAT by immunohistochemistry, and the clinical information of the patients was collected, including the tumor TNM stage, Ki-67 level, histological grade, treatment status and other clinical characteristics. The prognosis of patients receiving immunotherapy was followed up, and the survival prognosis information of the patients was collected. Univariate and multivariate COX regression analysis was performed to analyze the relationship between ICAT and the overall survival rate and disease-free survival rate after immunotherapy.

[0064] (2) Verify the relationship between ICAT and immune cell infiltration in clinical samples: Detect the expression of immune cell markers such as CD4, CD8, POXP3, CD206, PD-1, and TIM3 in tumor tissues by immunohistochemistry, analyze their correlation, and compare the expression results with those obtained from public databases such as GEO.

[0065] Part II: Identification of ICAT as a driver of cervical cancer progression through glycolysis

[0066] Construction of subcutaneous xenograft model in mice: A subcutaneous xenograft model was established in 5-week-old nude mice using control and ICAT knockdown Hela cell lines. Tumors were measured with a caliper one week after tumor formation, and changes in mouse body weight and tumor volume were monitored. Tumor volume is calculated as length × width. 2 ×0.5 was used to assess subcutaneous tumor growth and generate tumor growth curves. At the end of the experiment, mice were sacrificed by cervical dislocation, and tumors, heart, liver, lungs, and kidneys were harvested. Heart, liver, lung, and kidney function were assessed by HE sections. Tumor levels of glucose, ATP, ECAR, and lactate were measured using test kits.

[0067] RNA-Seq analysis: RNA-Seq analysis was performed after ICAT knockdown to explore the cell biological processes involved in ICAT.

[0068] In vitro testing of the effects of altered ICAT expression on glycolysis: The effects of altered ICAT expression on glycolysis in HeLa and SiHa cervical cancer cells were assessed using a seahorse assay and a kit. Following treatment with the glycolysis inhibitor 2-DG, changes in proliferation, migration, and invasion were analyzed using CCK-8, Transwell, and wound wound assays.

[0069] ICAT regulates immune infiltration in cervical cancer: Tumors were minced and digested with collagenase at 38°C for 1 hour. Single-cell suspensions were prepared after filtration through a cell strainer. Flow cytometry was used to examine the expression of CD4, CD8, CD25, FOXP3, CD11b, CD206, F4 / 80, and T cell exhaustion markers PD-1, LAG3, and TIM-3 in the tumor single-cell suspensions. The abundance and immune status of CD4+ T cells, CD8+ T cells, Tregs, M1 cells, and M2 cells in the tumor microenvironment were assessed. ICAT was found to have the most significant effect on M2 infiltration.

[0070] (5) Construction of a co-culture system: THP-1, PBMCs, and BMDMs that knocked down / overexpressed ICAT using CRISP-CAS9 technology were co-cultured with different cervical cancer cells to form TAMs. Macrophage markers such as iNOS, TNF-α, ARG1, TGF-β, and IL-10 in TAMs after co-culture were detected by flow cytometry, qRT-PCR, Western Blot, and immunofluorescence.

[0071] Part III: Elucidating the molecular mechanism by which ICAT regulates ENO1 expression to reshape glycolytic reprogramming in cervical cancer

[0072] (1) Protein spectrum analysis of ICAT binding protein map: ICAT overexpression HeLa cell protein was extracted and immunoprecipitated and purified using anti-ICAT monoclonal antibody. Protein spectrum technology was used to identify the ICAT binding protein map, and ENO1 transcriptional regulatory factors were predicted in the HumanTFDB database. Wenn diagrams were drawn to analyze the intersection areas between positive binding proteins and ENO1 transcriptional regulatory factors, and c-Myc was found to be the only intersection protein.

[0073] (2) To clarify that ICAT regulates ENO1 expression by binding to c-Myc: ICAT-overexpressing HeLa cell proteins were extracted and purified by immunoprecipitation using anti-ICAT / c-Myc monoclonal antibodies, and the binding between ICAT and c-Myc was detected by immunoblotting; HeLa cells were treated with a c-Myc-specific inhibitor and the ENO1 expression level was detected by qPCR and WB.

[0074] (3) Detection of the nuclear expression level of c-Myc: Extract nuclear protein of cells with different expression levels of ICAT and detect the nuclear expression level of c-Myc; immunofluorescence detection of the nuclear expression level of c-Myc.

[0075] (4) Verify that c-Myc binds to the ENO1 promoter: Use the NCBI website to search for the ENO1 promoter sequence, construct a pGL3-basic reporter gene plasmid connected to the promoter sequence, and construct an overexpression plasmid for ICAT-related transcription factors in the same way. The overexpression plasmid and the promoter reporter gene plasmid are co-transfected into HK293T cells, and a specific luciferase substrate is added. The luciferase activity can be measured by detecting the fluorescence intensity to determine whether the ENO1 promoter sequence can be targeted. ChIP experiments are further performed on the promoter sequence to verify the binding specificity of PRKDC.

[0076] (5) Explore the binding region between c-Myc and ENO1 promoter: Design binding sequence segment mutation plasmids and transfer them into HeLa cells, and verify the specific binding region between c-Myc and ENO1 promoter through luciferase assay.

[0077] Part IV: Clarifying the molecular mechanisms of ICAT-dependent lactate accumulation in triggering tumor-associated macrophage polarization

[0078] (1) Identification of the role of ICAT in constructing an inhibitory immune microenvironment in mice: 8-week-old BALB / c mice were treated with control and ICAT knockdown U14 cell lines at 3×10 6 A subcutaneous injection model was constructed with a concentration of 10 cells / mouse. One week after tumor formation, the tumor was measured with a caliper to monitor changes in mouse weight and tumor volume. Tumor volume is calculated as length × width. 2 ×0.5 was used to assess subcutaneous tumor growth and plot tumor growth curves. At the end of the experiment, mice were sacrificed by cervical dislocation, and tumors, heart, liver, lung, and kidneys were harvested. Heart, liver, lung, and kidney function were assessed using HE sections.

[0079] (2) ICAT regulates immune infiltration in cervical cancer: The tumor was minced and digested with collagenase at 38°C for 1 hour, filtered through a cell strainer to prepare a single-cell suspension, and flow cytometry was used to detect the expression of CD4, CD8, CD25, FOXP3, CD11b, CD206, F4 / 80 and T cell exhaustion markers PD-1, LAG3, TIM-3 in the tumor single-cell suspension to identify the infiltration abundance and immune status of CD4+T, CD8+T, Tregs, M1, and M2 cells in the tumor microenvironment.

[0080] (3) Further demonstrating the dependence of macrophages in ICAT-mediated carcinogenesis: macrophages were depleted by intraperitoneal injection of clodronate liposomes and tumor volume and weight in shICAT tumors were observed after macrophage depletion.

[0081] (4) Lactic acid promotes tumor-associated macrophage polarization: Lactic acid was introduced into SiICATCC cells for co-culture experiments. PCR and Western blotting were used to detect the expression of M2 macrophage markers in macrophages co-cultured with SiICAT tumor cells.

[0082] (5) Exploring the role of histone lactylation in tumor-associated macrophages: Histones play a crucial role in gene expression as transcriptional cofactors. ChIP was used to examine the role of H3K18la modification in the transcriptional expression of genes in M2 macrophages, including CD206, ARG1, IL10, and ADM.

[0083] To explore the tumor functional significance of ICAT in cervical cancer (CC), we first analyzed 13 normal cervical tissues and 306 cervical tumor tissues in the GEPIA2 database. The results showed that the expression of ICAT in cervical tumor tissues was significantly upregulated compared with normal tissues. We further performed a survival analysis on the GSE44001 dataset in the GEO database and found that patients with high ICAT expression had a poor prognosis. In addition, the expression level of ICAT was detected in tumors and adjacent tissues of clinical CC patients. The immunohistochemistry (IHC) results showed that ICAT was highly expressed in tumor tissues, such as Figure 5 shown.

[0084] like Figure 6 、 Figure 7 As shown in Figure 2, to further study the function of ICAT, ICAT overexpression and knockdown models were successfully constructed in HeLa and SiHa cell lines. Subsequently, the effects of ICAT on the migration and invasion of CC cells were evaluated by scratch assay and Transwell assay. The results showed that knockdown of ICAT significantly inhibited the migration and invasion of CC cells. Figure 8 、 Figure 9 The above results indicate that ICAT may play a carcinogenic role in cervical cancer, and its high expression is closely related to tumor progression and poor prognosis.

[0085] like Figure 10-15As shown, in order to explore the biological significance of ICAT in cervical cancer, transcriptomic analysis was performed on HeLa cells to identify the expression profiles of ICAT-related genes. For the differentially expressed genes related to ICAT, KEGG enrichment was further used to identify the cellular pathways involved in ICAT. The results showed that ICAT was significantly involved in the glycolysis pathway. At the same time, GSEA showed that ICAT was positively correlated with glycolysis. Subsequently, the glycolysis-related indicators ATP, lactate, and glucose content in HeLa and SiHa cells were detected, showing that ICAT upregulated the glycolysis level in cervical cancer. In order to further explore the biological significance of ICAT in cervical cancer, the addition of the glycolysis inhibitor 2-DG to HeLa and SiHa cells can reverse the cancer-promoting ability of ICAT, as shown in Figure 2. Figure 16 , Figure 17 shown.

[0086] Based on the above experimental results, it is believed that ICAT can exert its tumor-promoting effect by upregulating glycolysis in cervical cancer.

[0087] Next, we explored ICAT's downstream target molecules and mapped glycolysis expression using RNAseq. The results showed that ICAT could upregulate the expression of glycolysis-related molecules in tumor cells. We screened four glycolysis-related molecules with the most significant expression differences (ENO1, HK1, PFKL, and PGK1) and further validated them with qPCR. The analysis revealed that ENO1 expression was the most significant. Further validation in HeLa and SiHa cell lines revealed that ENO1 expression was most pronounced after overexpression and knockdown of ICAT. TCGA data showed a positive correlation between ICAT and ENO1. Next, we further validated the relationship between ICAT and ENO1, and Western blot results showed that ICAT upregulated ENO1. Subsequently, we performed a response experiment to test glycolysis indicators, and the results showed that knockdown of ENO1 reversed ICAT's promoting effect on glycolysis. This suggests that ICAT upregulates glycolysis in cervical cancer through ENO1.

[0088] like Figure 2 To further explore the key molecules involved in ICAT's impact on ENO1 expression, the intersection of five bioinformatics databases revealed the ENO1 transcription factor c-Myc, which was positively correlated with ENO1 expression. Subsequent nuclear-cytoplasmic fractionation experiments revealed that overexpression of ICAT promoted c-Myc nuclear translocation. Overall, it is believed that ICAT promotes c-Myc nuclear translocation, thereby upregulating ENO1 transcription and translation, ultimately leading to CC progression.

[0089] In the early stages of the study, a mouse model with ICAT knockdown was constructed to analyze the significance of ICAT in immune resistance. U14 cells were injected subcutaneously into BALB / c cells to construct a mouse model with ICAT knockdown. The nude mice were finally killed by cervical dislocation on the 21st day and the tumors were collected. The tumor size of the ICAT knockdown group was significantly reduced. IHC showed that knocking down ICAT could significantly inhibit the expression of M2 macrophage markers CD206, IL-10, and TGFβ1. An analysis of 306 cervical cancer tissue samples using the TCGA database (https: / / portal.gdc.com) found that the ICAT expression level in cervical cancer tissue was negatively correlated with the number of M1 TAMs and positively correlated with the number of M2 TAMs. The GSE9750 dataset included 21 normal cervical tissues and 33 cervical cancer tissues for analysis, and the results showed that the expression level of ICAT was positively correlated with the M2 macrophage markers Arg-1 and TGF-β; in the GSE122697 dataset, 5 normal cervical tissues and 11 cervical cancer tissues were analyzed, and it was found that ICAT expression was positively correlated with the M2 macrophage marker CD163.

[0090] like Figure 3 、 Figure 4 To further validate these results in vitro, THP-1-induced macrophages were successfully constructed using PMA. To better mimic the effects of tumor cells on macrophages within the TME, CM containing ICAT-overexpressing and ICAT-knockdown cells from human cervical cancer cells, SiHa, was co-cultured with THP-1 macrophages to induce them into TAMs. Western blot and interferon (IF) analysis revealed that treatment with (SiHa / AdICAT)-CM downregulated the expression of M1 macrophage markers IL-1β and CD86, while upregulated the expression of M2 macrophage markers Arg-1 and TGF-β. Furthermore, ICAT-stimulated glycolysis to produce lactate induced TAM polarization toward M2 macrophages. Furthermore, a lactate-concentration-dependent increase in pan-lactation and H3K18la expression was observed in macrophages. Furthermore, treatment with (SiHa / AdICAT)-CM increased pan-lactation in TAMs, but the addition of 2-DG inhibited the ICAT-induced increase in pan-lactation.

[0091] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. The use of ICAT gene as a therapeutic target in cervical cancer, characterized in that: By detecting the expression level of ICAT in cervical cancer tissues, the prognosis of patients can be evaluated; The detection method includes the following steps: S1: Correlation between ICAT and CC immune infiltration in clinical sample analysis; S2: Identification of ICAT promoting cervical cancer malignant progression through glycolysis; S3: Clarify the molecular mechanism by which ICAT regulates ENO1 expression to reshape glycolytic reprogramming in cervical cancer; S4: Elucidate the molecular mechanisms by which ICAT-dependent lactate accumulation triggers tumor-associated macrophage polarization; S5: Result evaluation.

2. The use of the ICAT gene as a therapeutic target in cervical cancer according to claim 1, characterized in that: Said S1 specifically includes: S11: Determine the relationship between ICAT and the clinicopathological characteristics of CC and patient prognosis: Select CC samples for immunohistochemical detection of ICAT expression levels and collect clinical information, including tumor TNM stage, Ki-67 level, histological grade, and treatment status; collect survival prognosis information for patients receiving immunotherapy, and perform univariate and multivariate Cox regression analysis to analyze the relationship between ICAT and overall survival and disease-free survival after immunotherapy; S12: Verify the relationship between ICAT and immune cell infiltration in clinical samples: Detect the expression of immune cell markers CD4, CD8, POXP3, CD206, PD-1, and TIM3 in tumor tissues by immunohistochemistry, analyze the correlation, and compare the expression results with those obtained from public database analysis.

3. The use of the ICAT gene as a therapeutic target in cervical cancer according to claim 1, characterized in that: Said S2 specifically includes: S21: Construction of subcutaneous xenograft model; S22: RNA-Seq analysis; S23: In vitro detection of the effects of altered ICAT expression on glycolysis; S24: ICAT regulates immune infiltration in cervical cancer; S25: Construct a co-culture system.

4. The use of the ICAT gene as a therapeutic target in cervical cancer according to claim 1, characterized in that: Said S3 specifically includes: S31: Protein profiling analysis of ICAT-binding protein profiles; S32: It is clear that ICAT regulates ENO1 expression by binding to c-Myc; S33: Extract nuclear protein of cells with different expression levels of ICAT and detect the expression level of c-Myc in the nucleus; detect the expression level of PRKDC in the nucleus by immunofluorescence; S34: Verification of c-Myc binding to the ENO1 promoter; S35: Designed binding sequence segmented mutation plasmids and transformed them into HeLa cells. Luciferase assay was used to verify the specific binding region between c-Myc and ENO1 promoter.

5. The use of the ICAT gene as a therapeutic target in cervical cancer according to claim 4, characterized in that: In the S31, specifically: extract ICAT-overexpressing HeLa cell protein, use Anti-ICAT monoclonal antibody for immunoprecipitation and purification, use protein spectrum technology to identify the ICAT binding protein map, and predict ENO1 transcription regulatory factors in the HumanTFDB database, draw a wenn diagram to analyze the intersection area between positive binding proteins and ENO1 transcription regulatory factors, and determine c-Myc as the only intersection protein.

6. The use of the ICAT gene as a therapeutic target in cervical cancer according to claim 4, characterized in that: In the above S32, specifically: extracting ICAT-overexpressing HeLa cell protein, using Anti-ICAT / c-Myc monoclonal antibody for immunoprecipitation and purification, and detecting whether there is binding between ICAT and c-Myc by immunoblotting; treating HeLa cells with a c-Myc-specific inhibitor and then detecting the ENO1 expression level by qPCR and WB.

7. The use of the ICAT gene as a therapeutic target in cervical cancer according to claim 4, characterized in that: In the S34, specifically: using the NCBI website to search for the ENO1 promoter sequence, constructing a pGL3-basic reporter gene plasmid connected to the promoter sequence, and constructing an overexpression plasmid of an ICAT-related transcription factor by the same method; co-transfecting the overexpression plasmid and the promoter reporter gene plasmid into HK293T cells, adding a specific luciferase substrate, and measuring the activity of luciferase by detecting the intensity of fluorescence to determine whether the ENO1 promoter sequence can be targeted and bound; further performing a ChIP experiment on the promoter sequence to verify the binding specificity of c-Myc.

8. The use of the ICAT gene as a therapeutic target in cervical cancer according to claim 1, characterized in that: Said S5 specifically includes: S51: Identification of the role of ICAT in establishing a suppressive immune microenvironment; S52: ICAT regulates immune infiltration in cervical cancer; S53: Demonstrate the dependence of macrophages in ICAT-mediated carcinogenesis; S54: Lactate promotes tumor-associated macrophage polarization; S55: Analysis of the role of histone lactylation in tumor-associated macrophages.

9. Use of ICAT gene as a therapeutic target in cervical cancer, characterized in that: The Seahorse assay and kit were used to detect the effect of ICAT expression changes on glycolysis in cervical cancer cells HeLa and SiHa, which served as the basis for analyzing the prognosis of cervical cancer.

10. Use of ICAT gene as a therapeutic target in cervical cancer, characterized in that: Methods THP-1, PBMCs, and BMDMs were gene-edited using CRISP-CAS9 technology to obtain macrophages with stable ICAT knockdown or overexpression. These macrophages were then co-cultured with cervical cancer cells with different ICAT expression levels to form tumor-associated macrophages. Flow cytometry, qRT-PCR, Western Blot, and immunofluorescence were used to detect the expression of macrophage markers iNOS, TNF-α, ARG1, TGF-β, and IL-10 in TAMs, which served as the basis for analyzing the prognosis of cervical cancer.

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