Application of ESCO1 and NUFIP2 in diagnosis and treatment of pancreatic cancer

Abstract

The application discloses application of ESCO1 and NUFIP2 in diagnosis and treatment of pancreatic cancer, and relates to the technical field of biological medicine.The application provides application of ESCO1 and / or NUFIP2 as a pancreatic cancer marker in preparation of a pancreatic cancer diagnosis product or a prognosis evaluation product, and also provides application of ESCO1 and / or NUFIP2 as a drug target in screening or preparation of a drug for preventing and / or treating pancreatic cancer.The application finds that ESCO1 and NUFIP2 have important biological significance in pancreatic cancer, wherein the expression of NUFIP2 is regulated by ESCO1, and the expression levels of both are positively correlated with malignant progression of the pancreatic cancer.Therefore, ESCO1 and NUFIP2 can not only be used as potential diagnosis markers and treatment targets of the pancreatic cancer, but also can provide a new theoretical basis and research direction for optimization of an immunotherapy strategy of the pancreatic cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of ESCO1 and NUFIP2 in the diagnosis and treatment of pancreatic cancer. Background Technology

[0002] Pancreatic cancer is a highly malignant tumor primarily originating from the epithelial cells of the pancreatic ducts or acinar cells, and in recent years it has become one of the leading causes of cancer-related deaths. Due to its lack of specific early clinical manifestations, rapid progression, and high invasiveness, it is often clinically referred to as the "king of cancers" or the "silent killer." Most patients are diagnosed at an intermediate or advanced stage, resulting in a poor overall prognosis and a low 5-year survival rate. Currently, imaging examinations and serological tumor markers remain the main methods for pancreatic cancer screening and diagnosis, but their sensitivity and specificity for early, small lesions are limited, making it difficult to meet the needs of early screening in the context of precision medicine. Therefore, elucidating the molecular mechanisms of pancreatic cancer development and progression, and identifying more sensitive and specific biomarkers and potential therapeutic targets, is of great significance for improving early diagnosis rates, optimizing treatment strategies, and improving patient prognosis.

[0003] Tumor markers are biological indicators released by tumor cells or abnormally produced by the body in response to tumor-related stimuli during tumor formation and progression. Changes in their levels serve as important references for assessing the presence and development of tumors. In the clinical diagnosis of pancreatic cancer, CA19-9 is one of the most widely used serological markers, but it still has certain limitations. For example, some patients do not express this marker, and it can also be elevated in some benign diseases, thus affecting the accuracy of diagnosis. Therefore, a single marker is insufficient to comprehensively reflect the disease situation, and a comprehensive analysis combining multiple markers is usually necessary. Furthermore, current treatment options for pancreatic cancer remain relatively limited. In-depth research into its molecular mechanisms and screening for potential therapeutic targets is crucial for developing novel targeted drugs and immunotherapy strategies, and improving patient survival outcomes. Summary of the Invention

[0004] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide an application of ESCO1 and NUFIP2 in the diagnosis and treatment of pancreatic cancer.

[0005] The establishment of sister chromatid cohesion N-acetyltransferase 1 (ESCO1) is an important member of the ESCO family. ESCO1 (Gene ID: 114799) promotes stable binding between sister chromatids by acetylation of the cohesin complex, thereby ensuring accurate chromosome segregation during cell division and maintaining genomic stability. Previous research by the inventors revealed that ESCO1 is highly expressed in pancreatic cancer tissues and can positively regulate the expression of nuclear FMR1 interacting protein 2 (NUFIP2). NUFIP2 (Gene ID: 57532), initially identified for its binding to FMR1 (fragile X mental retardation 1 protein), is mainly located in the nucleus and cytoplasm and is an important component of the RNA-binding protein complex, playing a crucial role in RNA metabolism and post-transcriptional regulation. Therefore, the inventors systematically explored the expression characteristics and biological functions of ESCO1 and NUFIP2 in pancreatic cancer through bioinformatics analysis and in vitro experiments, aiming to provide new potential targets and theoretical basis for the clinical diagnosis and targeted therapy of pancreatic cancer.

[0006] This invention provides the application of ESCO1 and NUFIP2 in the diagnosis, targeted therapy, and immunotherapy of pancreatic cancer. This invention is the first to discover the significant biological significance of ESCO1 and NUFIP2 in pancreatic cancer. NUFIP2 expression is regulated by ESCO1, and the expression levels of both are positively correlated with the malignant progression of pancreatic cancer. Furthermore, the expression of ESCO1 and NUFIP2 is closely related to the immune microenvironment, specifically showing a negative correlation with the infiltration levels of follicular helper cells and activated natural killer cells (NK cells), and a significant correlation with the expression of various immune checkpoint molecules. Therefore, ESCO1 and NUFIP2 can not only serve as potential diagnostic biomarkers and therapeutic targets for pancreatic cancer, but also provide new theoretical basis and research directions for optimizing pancreatic cancer immunotherapy strategies.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides the application of ESCO1 and / or NUFIP2 as pancreatic cancer biomarkers in the preparation of pancreatic cancer diagnostic products or prognostic assessment products.

[0008] Preferably, the application includes the use of reagents for detecting ESCO1 and / or reagents for detecting NUFIP2 in the preparation of pancreatic cancer diagnostic products or prognostic assessment products.

[0009] Preferably, the pancreatic cancer diagnostic product or prognostic assessment product includes a test kit, which includes at least one of the following: reagents for detecting ESCO1 and / or NUFIP2, and reagents for detecting the expression products of the ESCO1 gene and / or NUFIP2 gene.

[0010] Preferably, the detection kit includes reagents for immunohistochemical analysis of ESCO1 and / or NUFIP2 expression, or includes reagents for immunoblotting detection of ESCO1 and / or NUFIP2 expression.

[0011] Secondly, this invention provides the application of the ESCO1 / NUFIP2 axis as a biomarker for pancreatic cancer in the preparation of pancreatic cancer diagnostic products or prognostic assessment products.

[0012] Preferably, the reagent for detecting the function of the ESCO1 / NUFIP2 axis is used in the preparation of pancreatic cancer diagnostic products or prognostic assessment products.

[0013] Preferably, the pancreatic cancer diagnostic product or prognostic assessment product includes a test kit that includes reagents for detecting the function of the ESCO1 / NUFIP2 axis.

[0014] Preferably, ESCO1 and NUFIP2 are biomarkers for poor prognosis of pancreatic cancer.

[0015] Thirdly, this invention provides the use of ESCO1 and / or NUFIP2 as drug targets in screening or preparing drugs for the prevention and / or treatment of pancreatic cancer.

[0016] Preferably, the application includes the use of reagents that inhibit ESCO1 expression levels and / or reagents that inhibit NUFIP2 expression levels in screening or preparing drugs for treating pancreatic cancer.

[0017] Fourthly, this invention provides the application of the ESCO1 / NUFIP2 axis as a drug target in screening or preparing drugs for the prevention and / or treatment of pancreatic cancer.

[0018] Preferably, the therapeutic target for pancreatic cancer is the ESCO1 / NUFIP2 pathway. Preferably, the application includes: the use of substances that regulate or inhibit or antagonize the function of the ESCO1 / NUFIP2 axis, and / or the expression or function of ESCO1, and / or the expression or function of NUFIP2 in screening or preparing drugs for treating pancreatic cancer.

[0019] This invention has at least one of the following beneficial effects: This invention analyzes the TCGA database to show that the expression levels of ESCO1 and NUFIP2 in human pancreatic cancer tissues are significantly increased compared to normal human pancreatic tissues. Analysis of the PAAD dataset from the TCGA database shows that pancreatic cancer patients with elevated ESCO1 and NUFIP2 expression levels have significantly reduced overall survival and disease-free survival. Western blot analysis reveals that, compared to normal human pancreatic ductal epithelial cells HPDE6-C7, the expression levels of ESCO1 and NUFIP2 are significantly increased in various pancreatic cancer cell lines (PANC-1, SW1990, ASPC-1, and MIAPaCa-2). This invention further constructs pancreatic cancer cell models with ESCO1 and NUFIP2 knockdown and NUFIP2 overexpression, and validates their function using plate colony assays, scratch assays, Transwell assays, flow cytometry, and immunofluorescence. The results showed that knocking down ESCO1 or NUFIP2 significantly inhibited the proliferation and invasion of pancreatic cancer cells and promoted their apoptosis; while reversing NUFIP2 overexpression partially reversed these effects, resulting in enhanced cell proliferation and invasion and reduced apoptosis. This invention systematically reveals the important roles of ESCO1 and NUFIP2 in the development and progression of pancreatic cancer, confirming that their expression levels are closely related to the occurrence and prognosis of pancreatic cancer. Therefore, ESCO1 and NUFIP2 can serve as potential diagnostic biomarkers and therapeutic targets for pancreatic cancer, providing new theoretical basis and research directions for the clinical detection, risk assessment, and targeted therapy of pancreatic cancer. Attached Figure Description

[0020] Figure 1 Example 1 integrates multiple public databases to analyze the expression level of ESCO1 in pan-cancer studies and assesses its correlation with immune cell infiltration (heatmap) and prognosis (forest plot), while comparing its expression differences between pancreatic cancer tissue and normal tissue. Figure 1 In this context, A represents the expression of ESCO1 in pan-cancer tissues in the TCGA database; Figure 1 B in the figure represents the expression of ESCO1 in pan-cancer tissues after integrating the TCGA and GTEx databases; Figure 1 In the diagram, C represents the forest plot for prognostic analysis of ESCO1 in pan-cancer. Figure 1 In the figure, D represents the analysis results of the correlation between ESCO1 and immune cell infiltration in pan-cancer data; Figure 1 In this context, E represents the protein expression level of ESCO1 in different tumor tissues and adjacent normal tissues in the CPTAC database.

[0021] Figure 2 Example 1 presents a systematic analysis of the expression characteristics of ESCO1 in pancreatic cancer and its clinical relevance. Figure 2 In the figure, A represents the differential expression analysis of ESCO1 in pancreatic cancer tissue and normal tissue; Figure 2 B in the figure represents the detection of ESCO1 protein expression in pancreatic cancer tissue and paired adjacent normal tissue; Figure 2 In this context, C represents the diagnostic value for pancreatic cancer as assessed by ROC curve analysis. Figure 2 D in the figure represents the survival (OS) curve analysis based on different ESCO1 expression levels; Figure 2 E in the figure represents the difference analysis of tumor microenvironment score (TME score) corresponding to different ESCO1 expression levels; Figure 2 F- Figure 2 In this context, G represents the correlation analysis between ESCO1 and the level of immune cell infiltration and immune checkpoint genes. Figure 2 H- Figure 2 In the figure, I represents the potential role of ESCO1 as an independent prognostic factor, which is evaluated by combining univariate and multivariate Cox regression analysis.

[0022] Figure 3 The results of Pearson correlation analysis of ESCO1 in Example 2 are shown, and the difference in overall survival between patients in the high-expression and low-expression groups of related genes was assessed by Kaplan-Meier survival analysis. Figure 3 In the A column, candidate genes significantly associated with ESCO1 are shown. Figure 3 B in the diagram is a Circos plot, which systematically shows the correlation between ESCO1 and pancreatic cancer-related genes (red shaded bars indicate positive correlation, and green shaded bars indicate negative correlation). Figure 3 The C-value in the figure shows the difference in overall survival between patients with high and low MIB1 expression. Figure 3 D in Figure 3 The F values ​​in the study compared the overall survival, progression-free survival, and disease-specific survival of patients in the high and low NUFIP2 expression groups, respectively. Figure 3 G in Figure 3 The N values ​​in the table represent the differences in overall survival between patients with high and low expression of ZNF12, ZMYM2, BRWD3, SF3B1, VKORC1, SIL1, CHID1, POLR2L, and PRDX4.

[0023] Figure 4 This is the result of a systematic analysis of NUFIP2 in Example 3, covering its expression level in pan-cancer studies and its expression in pancreatic cancer, as well as its clinical relevance. Figure 4 A in the figure shows the expression profile of NUFIP2 in pan-cancer tissues after integrating the TCGA and GTEx databases; Figure 4In the figure, B represents the differential expression analysis of NUFIP2 in pancreatic cancer tissue and normal tissue; Figure 4 In the figure, C represents the detection result of NUFIP2 protein expression level in pancreatic cancer tissue and paired adjacent normal tissue; Figure 4 The diagnostic value of NUFIP2 for pancreatic cancer was evaluated using ROC curves. Figure 4 E in Figure 4 The I in the figure shows the correlation analysis between NUFIP2 expression and clinicopathological features.

[0024] Figure 5 The system presents the prognostic value, immune microenvironment correlation, and interaction analysis results of NUFIP2 with ESCO1 in Example 3. Among them, Figure 5 A in Figure 5 B in the study evaluated the potential value of NUFIP2 as an independent prognostic factor for pancreatic cancer through univariate and multivariate Cox regression analysis. Figure 5 C in the figure represents the correlation analysis between NUFIP2 expression level and the infiltration ratio of 22 immune cells; Figure 5 D in Figure 5 Further analysis of F in the study revealed that NUFIP2 is associated with follicular helper T cells, activated natural killer cells, and resting memory CD4. + Significant correlation between T cell infiltration levels; Figure 5 The G group compared the differences in immune cell infiltration between the NUFIP2 high expression group and the low expression group. Figure 5 H in the figure represents the correlation analysis between NUFIP2 expression and multiple immune checkpoint genes; Figure 5 Figure I shows the GO and KEGG enrichment analysis results of NUFIP2-related genes in pancreatic cancer; Figure 5 J in the study used the HDOCK online tool to perform protein structure docking between ESCO1 and NUFIP2, and combined it with PyMOL for visualization analysis to evaluate their interaction mode.

[0025] Figure 6 This demonstrates the effect of ESCO1 on the malignant progression of pancreatic cancer in Embodiment 4 of the present invention. Figure 6 In section A, the expression levels of ESCO1 protein were compared between normal human pancreatic ductal epithelial cell lines and human pancreatic cancer cell lines. Figure 6 B- Figure 6 In the figure, C represents the change in ESCO1 protein expression level after knockdown in pancreatic cancer cell lines PANC-1 and SW1990; Figure 6 D- Figure 6 The effects of ESCO1 on the proliferation of pancreatic cancer cells were assessed using plate colony formation assays and EdU assays, respectively. Figure 6 H- Figure 6 K was further analyzed using scratch assay and Transwell assay to investigate the regulatory effect of ESCO1 on the migration ability of pancreatic cancer cells.

[0026] Figure 7 This demonstrates the effect of ESCO1 on pancreatic cancer cell apoptosis in Example 5, and the role of NUFIP2 in the malignant progression of pancreatic cancer. Among these, Figure 7 In the diagram, A–B represent the effect of ESCO1 on the apoptosis level of pancreatic cancer cells; Figure 7 C in Figure 7 In the figure, D represents the change in NUFIP2 protein expression level after knockdown in pancreatic cancer cell lines PANC-1 and SW1990; Figure 7 E in Figure 7 The effects of NUFIP2 on the proliferation of pancreatic cancer cells were evaluated using plate colony formation assays and EdU assays, respectively. Figure 7 I in Figure 7 The L in this study used scratch assay and Transwell assay to analyze the regulatory effect of NUFIP2 on the migration ability of pancreatic cancer cells; Figure 7 M in Figure 7 The N in the figure further demonstrates the effect of NUFIP2 on apoptosis in pancreatic cancer cells.

[0027] Figure 8 This demonstrates the regulatory role of the ESCO1 / NUFIP2 axis in the malignant progression of pancreatic cancer in Example 6. Figure 8 A in Figure 8 B in the figure represents the change in NUFIP2 protein expression level detected by Western blot after ESCO1 knockdown in PANC-1 and SW1990 cells; Figure 8 C in Figure 8 D in the figure represents the change in ESCO1 protein expression level detected by Western blot after knocking down NUFIP2 in PANC-1 and SW1990 cells; Figure 8 E in Figure 8 F in the figure represents the construction of a NUFIP2 reversion overexpression model in ESCO1 knockdown PANC-1 and SW1990 cells, and the detection of NUFIP2 protein expression level by Western blot. Figure 8 G in Figure 8 In this study, the effects of the ESCO1 / NUFIP2 axis on the proliferative capacity of pancreatic cancer cells were evaluated using plate colony formation assays and EdU assays, respectively. Figure 8 K in Figure 8 The role of the ESCO1 / NUFIP2 axis in regulating the migration ability of pancreatic cancer cells was analyzed using scratch assays and Transwell assays. Figure 8 O in Figure 8 The P-value further evaluated the effect of the ESCO1 / NUFIP2 axis on pancreatic cancer cell apoptosis. Detailed Implementation

[0028] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0029] The human pancreatic cancer cell lines (PANC-1, SW1990, ASPC-1 and MIAPaCa-2) and the human normal pancreatic ductal epithelial cell line HPDE6-C7 used in the various embodiments of the present invention were all purchased from Precytology Co., Ltd. Other reagents used are described in the corresponding embodiments. Example 1: Database analysis was conducted to study the expression patterns of ESCO1 in pan-cancer, with a focus on analyzing its expression characteristics in pancreatic cancer and its correlation with clinicopathological features.

[0030] 1.1 Analysis of ESCO1 expression characteristics, immune relevance, and prognostic value in pan-cancer We systematically analyzed the expression characteristics of ESCO1 in different tumor types using the Tumor Immunological Assessment Resource 2.0 (TIMER2.0, http: / / timer.cistrome.org). First, we compared the expression differences of ESCO1 in 33 tumor tissues with adjacent normal tissues using the Cancer Genome Atlas (TCGA) database. The results showed ( Figure 1 ESCO1 (A) was significantly overexpressed in breast cancer (BRCA), cholangiocarcinoma (CHOL), colonic adenocarcinoma (COAD), head and neck squamous cell carcinoma (HNSC), clear cell renal cell carcinoma (KIRC), hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), gastric adenocarcinoma (STAD), thyroid cancer (THCA), and endometrial cancer (UCEC).

[0031] Given the lack of normal tissue or limited sample size for some tumor types, we further integrated TCGA and the Genotype Tissue Expression Database (GTEx) into the GEPIA2 platform for validation and supplementary analysis, thereby expanding the sample size and improving the reliability of the results. The results showed ( Figure 1ESCO1 (B in the original text) also showed a high expression trend in pancreatic adenocarcinoma (PAAD), adrenocortical carcinoma (ACC), breast cancer (BRCA), cholangiocarcinoma (CHOL), esophageal cancer (ESCA), glioblastoma (GBM), head and neck squamous cell carcinoma (HNSC), renal clear cell carcinoma (KIRC), renal papillary cell carcinoma (KIRP), low-grade glioma (LGG), hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), sarcoma (SARC), cutaneous melanoma (SKCM), gastric adenocarcinoma (STAD), and endometrial cancer (UCEC).

[0032] Furthermore, we constructed a proportional hazards model based on a public database, performed Cox regression analysis using the `survival` package, and completed the proportional hazards hypothesis testing. We also used ggplot2 for forest plot visualization. The results show ( Figure 1 In the C-cell model, ESCO1 expression was significantly associated with the prognosis of patients with pancreatic cancer and other tumors. Furthermore, in terms of immune correlation analysis, we used the R language ggplot2 (v3.4.4) to perform correlation analysis between ESCO1 expression and immune infiltration levels in pan-cancer data. The results showed that ( Figure 1 ESCO1 (D) is significantly associated with immune cell infiltration in various tumors, suggesting that it may be involved in the regulation of the tumor immune microenvironment.

[0033] 1.2 ESCO1 is highly expressed in pancreatic cancer tissues Based on data from the CPTAC and TCGA databases, we first analyzed the protein expression level of ESCO1 in pancreatic cancer tissues. The results showed ( Figure 1 E and Figure 2 In the study, ESCO1 protein expression in primary pancreatic cancer tissue was significantly higher than in adjacent normal tissue.

[0034] To further validate the above bioinformatics analysis results, we randomly selected six pairs of pancreatic cancer tumor tissues and their corresponding adjacent normal tissues, and used Western blotting to detect the expression level of ESCO1 protein. The specific method is as follows: the tissues were minced and added to RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.), and thoroughly homogenized using a glass homogenizer. The mixture was centrifuged at 4℃ and 12000 rpm for 15 min, and the supernatant was collected as total protein. After determining the protein concentration using a BCA protein quantification kit (Beyotime, Shanghai), denaturing and reducing loading buffer (Yaxin, Cat: LT101L) was added, and the mixture was denatured in a 100℃ metal bath for 10 min.

[0035] Subsequently, 20 μg of total protein was taken from each well for 10% SDS-PAGE gel electrophoresis (Yaxin, Cat: 6PG112) under the following conditions: upper gel 80 V, lower gel 120 V. After electrophoresis, the membrane was transferred to a PVDF membrane at a constant current of 260 mA for 90 min. The membrane was blocked with 5% skim milk powder for 1 h–2 h, then incubated overnight at 4°C with rabbit anti-human ESCO1 primary antibody (1:1000, Affinity). GAPDH (1:5000, Proteintech) was used as an internal control. The next day, the membrane was washed three times with TBST (8 min–15 min each time), followed by incubation at room temperature for 1 h with HRP-labeled secondary antibody (1:3000–1:10000, Zenbio). After washing again, the membrane was developed using ECL chemiluminescence reagent (UElandy), and grayscale analysis was performed using Image Lab software (Bio-Rad, version 4.0).

[0036] Experimental results are as follows Figure 2 As shown in B, the expression level of ESCO1 protein in pancreatic cancer tissue (T group) was significantly higher than that in the corresponding adjacent normal tissue (NT group), which is consistent with the results of database analysis.

[0037] 1.3 Diagnostic value and immune correlation analysis of ESCO1 in pancreatic cancer To evaluate the discriminative power of ESCO1 expression on study outcomes, receiver operating characteristic (ROC) curve analysis was used. Sample status was used as the categorical variable, and ESCO1 expression level as the predictive variable; the area under the curve (AUC) was calculated to evaluate its diagnostic / predictive efficacy. Results showed that ESCO1 has high diagnostic / predictive power in pancreatic cancer. Figure 2 (C in the middle).

[0038] Transcriptome expression data and clinical information of pancreatic cancer were obtained from the TCGA database. After removing samples with incomplete survival time and status, the relationship between ESCO1 expression and overall survival (OS) was explored using the "survival" and "survminer" packages in R 4.3.0 through Kaplan-Meier survival analysis and univariate Cox regression analysis. The results showed that high ESCO1 expression was significantly associated with poorer OS. Figure 2 (D in the middle).

[0039] The samples were further divided into high-expression and low-expression groups based on the median ESCO1 expression level. Combined with tumor microenvironment scoring data, the Wilcoxon rank-sum test was used to compare the differences between the two groups, and the results were visualized using a violin plot. The results showed that ESCO1 expression level was significantly correlated with tumor immune cell infiltration. Figure 2(E in the text).

[0040] Subsequently, correlation analysis was used to assess the relationship between ESCO1 expression and different immune cell infiltration levels, and a lollipop plot was created using R language for visualization. The results showed that ESCO1 and CD8... + T cells, activated NK cells, and follicular helper cells were negatively correlated. Figure 2 (F in the text).

[0041] Furthermore, Pearson correlation analysis was used to assess the correlation between ESCO1 and immune checkpoint genes, with p < 0.001 as the screening criterion. A correlation matrix was constructed and visualized using the corrplot package. The results showed that ESCO1 was negatively correlated with immune checkpoint genes such as PDCD1, CD200, CD48, TNFRSF4, LAG3, TNFRSF8, ADORA2A, and CD276. Figure 2 (G in the middle).

[0042] Finally, univariate and multivariate analyses were performed using a Cox proportional hazards regression model to assess the independent prognostic value of ESCO1 in overall patient survival. Multivariate analysis was conducted after including clinical characteristic variables, and forest plots were generated for visualization. The results showed that ESCO1 was not an independent prognostic factor and could not be used as a sole prognostic indicator. Figure 2 H- Figure 2 (I in the middle).

[0043] Example 2: ESCO1 and NUFIP2 showed a significant correlation in pancreatic cancer.

[0044] To screen for potential downstream regulatory molecules of ESCO1, Pearson correlation analysis was performed on ESCO1 expression levels. The results showed that ESCO1 was significantly correlated with the expression of multiple genes, with the most prominent correlations observed with MIB1, NUFIP2, ZNF12, ZMYM2, BRWD3, SF3B1, VKORC1, SIL1, CHID1, POLR2L, and PRDX4. Figure 3 A in Figure 3 (B in the middle).

[0045] Based on this, to further evaluate the clinical significance of the above-mentioned related genes in pancreatic cancer, a Kaplan-Meier survival analysis was performed to examine the relationship between their expression levels and patient prognosis. The results showed that the expression of MIB1, ZNF12, ZMYM2, BRWD3, SF3B1, VKORC1, SIL1, CHID1, POLR2L, and PRDX4 was not significantly associated with overall survival (OS) in pancreatic cancer patients, while high expression of NUFIP2 was significantly associated with poorer OS, progression-free survival (PFI), and disease-specific survival (DSS). Figure 3 C in Figure 3 (N in the text).

[0046] Example 3: The systematic analysis of NUFIP2 covers its expression levels in pan-cancer, expression characteristics in different tissues and cells, and its expression in pancreatic cancer and its clinical relevance. 3.1 To systematically evaluate the expression characteristics of NUFIP2 in various tumors, data from TCGA and the Genotype Tissue Expression Database (GTEx) were integrated based on the GEPIA2 platform to analyze its expression level in pan-cancer.

[0047] The results show that ( Figure 4 NUFIP2 (A) showed an upregulation trend in various tumor tissues, including pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), cholangiocarcinoma (CHOL), esophageal cancer (ESCA), glioblastoma (GBM), head and neck squamous cell carcinoma (HNSC), renal clear cell carcinoma (KIRC), renal papillary cell carcinoma (KIRP), acute myeloid leukemia (LAML), low-grade glioma (LGG), rectal adenocarcinoma (READ), sarcoma (SARC), cutaneous melanoma (SKCM), and gastric adenocarcinoma (STAD).

[0048] 3.2 NUFIP2 is highly expressed in pancreatic cancer tissues Based on data from the TCGA database, we first analyzed the protein expression level of NUFIP2 in pancreatic cancer tissues. The results showed ( Figure 4 In the primary pancreatic cancer tumor tissue, NUFIP2 protein expression was significantly higher than that in adjacent normal tissue.

[0049] To further validate the above bioinformatics analysis results, we extracted proteins from six pairs of pancreatic cancer tumor tissues and their corresponding adjacent normal tissues, and detected NUFIP2 protein expression levels using Western blotting. After electrophoresis, the membranes were transferred to PVDF membranes at a constant current of 260 mA for 90 min. After blocking with 5% skim milk powder for 1–2 h, rabbit anti-human NUFIP2 primary antibody (1:1000, Cell Signaling Technology, Inc.) was added and incubated overnight at 4°C. GAPDH (1:5000, Proteintech) was used as an internal control. The next day, the membranes were washed three times with TBST (8–15 min each time), followed by incubation with HRP-labeled secondary antibody (1:3000–1:10000, Zenbio) at room temperature for 1 h. After washing again, the membranes were developed using ECL chemiluminescence reagent (UElandy), and grayscale analysis was performed using Image Lab software (Bio-Rad, version 4.0). The experimental results are as follows: Figure 4 As shown in C, the expression level of NUFIP2 protein in pancreatic cancer tissues was significantly higher than that in the corresponding adjacent normal tissues, consistent with the results of database analysis.

[0050] 3.3 Diagnostic value and immune correlation analysis of NUFIP2 in pancreatic cancer To evaluate the discriminative power of NUFIP2 expression in pancreatic cancer outcomes, receiver operating characteristic (ROC) curve analysis was used. Sample status was used as the categorical variable, and NUFIP2 expression level as the predictive variable. The area under the curve (AUC) was calculated to evaluate its diagnostic / predictive efficacy. Results showed that NUFIP2 has high diagnostic / predictive value in pancreatic cancer. Figure 4 (D in the middle).

[0051] To further investigate the differences in NUFIP2 expression and its potential prognostic significance among different clinical subgroups, Wilcoxon rank-sum test and Kruskal-Wallis test (with Dunn post-hoc test) were used for analysis. The results showed that NUFIP2 expression levels were significantly correlated with tumor stage (Stage I vs. Stage IV) and tumor grade (G1 vs. G4, G2 vs. G4, G3 vs. G4); however, no statistically significant differences were observed among T, N, and M stages. Figure 4 E in Figure 4 (I in the middle).

[0052] Subsequently, univariate and multivariate analyses were performed using a Cox proportional hazards regression model to assess the independent prognostic value of NUFIP2 in overall survival. Multivariate correction was performed after including relevant clinical characteristics, and the results were visualized using a forest plot. The results showed that NUFIP2 can serve as an independent prognostic factor for pancreatic cancer and has potential clinical predictive value. Figure 5 A in Figure 5 (B in the middle).

[0053] Regarding the tumor immune microenvironment, correlation analysis was used to assess the relationship between NUFIP2 expression and immune cell infiltration levels, and the results were visualized using a lollipop plot generated in R. Spearman correlation analysis was then used to further validate the correlation between gene expression and immune cell infiltration. The results showed that NUFIP2 expression was significantly negatively correlated with activated NK cells and T follicular helper cells (Tfh). Figure 5 C in Figure 5 (F in the text).

[0054] Further, the proportion of immune cell infiltration in tumor tissues was estimated using the CIBERSORT algorithm, and the samples were divided into high-expression and low-expression groups based on the median NUFIP2 expression. The Wilcoxon test was used to compare the differences in immune cell infiltration between the two groups. Results showed that, compared with the low-expression group, the NUFIP2 high-expression group had a higher proportion of resting memory CD4+. + The infiltration levels of T cells (CD4 memoryresting T cells) were significantly increased, while the infiltration levels of NK cells and follicular helper T cells were significantly decreased. Figure 5 (G in the middle).

[0055] Furthermore, Pearson correlation analysis was used to assess the relationship between NUFIP2 and immune checkpoint-related genes (screening criterion: p < 0.001), and a correlation matrix was constructed and visualized using the corrplot package. The results showed that NUFIP2 was significantly negatively correlated with TNFRSF4 and TNFRSF18. Figure 5 (H in the text).

[0056] At the functional level, differentially expressed genes were enriched using the clusterProfiler package using GO analysis, including biological processes (BP), cellular components (CC), and molecular functions (MF). The selection criteria were p < 0.05 and FDR < 0.05, and the results were presented using a bubble chart. The results showed that these genes were mainly enriched in multiple biological processes and functional pathways, such as the regulation of peptide transport, regulation of peptide secretion, regulation of insulin secretion, apoptosis-related mitochondrial changes, and cellular components such as endocytic vesicles. They were also involved in pathways related to platinum drug resistance. Figure 5 (I in the middle).

[0057] Finally, molecular docking analysis of the ESCO1 and NUFIP2 protein structures was performed using the HDOCK online platform. Potential binding conformations were searched using the Fast Fourier Transform algorithm, and docking modes were evaluated using a knowledge-based scoring function. The conformation with the lowest docking score (strongest binding ability) was selected and visualized using PyMOL. The results showed a stable binding between ESCO1 and NUFIP2. Figure 5 J in the middle.

[0058] Example 4: ESCO1 promotes the malignant progression of pancreatic cancer.

[0059] 4.1 Expression levels of ESCO1 protein in normal pancreatic ductal epithelial cells HPDE6-C7 and human pancreatic cancer cell lines (PANC-1, SW1990, ASPC-1 and MIAPaCa-2); Samples from human pancreatic cancer cell lines (PANC-1, SW1990, ASPC-1, and MIAPaCa-2) and human normal pancreatic ductal epithelial cell line HPDE6-C7 were selected. RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) was added, and the cells were lysed on ice for 30 min. After centrifugation at 12000 rpm for 15 min at 4℃, the supernatant was collected as total protein. Protein concentration was determined using a BCA protein quantification kit (Beyotime, Shanghai). Denaturing and reducing loading buffer (Yaxin, Cat: LT101L) was added, and the cells were denatured in a metal bath at 100℃ for 10 min.

[0060] Subsequently, 20 μg of total protein was taken from each well for 10% SDS-PAGE gel electrophoresis (Yaxin, Cat: 6PG112) under the following conditions: upper gel 80 V, lower gel 120 V. After electrophoresis, the membrane was transferred to a PVDF membrane at a constant current of 260 mA for 90 min. The membrane was blocked with 5% skim milk powder for 1–2 h, then incubated overnight at 4°C with rabbit anti-human ESCO1 primary antibody (1:1000, Affinity). GAPDH (1:5000, Proteintech) was used as an internal control. The next day, the membrane was washed three times with TBST (8–15 min each time), followed by incubation at room temperature for 1 h with HRP-labeled secondary antibody (1:3000–1:10000, Zenbio). After washing again, the membrane was developed using ECL chemiluminescence reagent (UElandy), and grayscale analysis was performed using Image Lab software (Bio-Rad, version 4.0).

[0061] Experimental results are as follows Figure 2 As shown in B, the expression level of ESCO1 protein in pancreatic cancer cell lines (PANC-1, SW1990, ASPC-1 and MIAPaCa-2) was significantly higher than that in normal pancreatic ductal epithelial cells HPDE6-C7.

[0062] 4.2 Construction of ESCO1 knockdown cell lines This invention utilizes lentivirus-mediated shRNA interference technology to construct an ESCO1 knockdown cell line. Using PLKO.1 as the lentiviral vector, the shRNA sequence targeting ESCO1 was cloned into the multiple cloning site of the vector through molecular biology methods such as PCR amplification, enzyme digestion, and ligation. After the recombinant plasmid was constructed, sequencing was used to verify the correctness and orientation of the inserted sequence. Lentiviral packaging was performed using 293T cells. 293T cells were seeded in 6-well plates and transfected after reaching a confluence of 80%–90%. Plasmid mixtures were prepared according to the transfection reagent (G1802, Servicebio) instructions. PMDL (1.16 µg), PVSVG (0.62 µg), PREV (0.45 µg), and the corresponding amount of target plasmid were added to each well. After thorough mixing, the mixture was added to the cells, gently shaken, and the final culture volume per well was 2 mL. The cells were then incubated at 37 ℃ in a 5% CO2 incubator. Forty-eight hours after transfection, the culture supernatant from each well was collected and centrifuged at 300–500 g for 5 min to remove cell debris. The supernatant was then filtered through a 0.45 µm membrane, and the resulting filtrate was the packaged lentiviral particles. Target cells (PANC-1 and SW1990) were digested and seeded into 6-well plates. After cell adhesion, the lentiviral supernatant was added for infection, maintaining a final culture volume of 2 mL per well. Forty-eight to seventy-two hours after infection, cells were collected, and ESCO1 expression levels were detected by Western blot to assess knockdown efficiency.

[0063] The oligonucleotide primer sequences used in this embodiment to construct the ESCO1-targeting shRNA expression vector are as follows: ShRNA ESCO1-1: Forward primer (SEQ ID NO: 1): 5'-CCGGCGGAGATCACAAAGGCTACAACTCGAGTTGTAGCCTTTGTGATCTCCGTTTTTG-3'; Reverse primer (SEQ ID NO: 2): 5'-AATTCAAAAACGGAGATCACAAAGGCTACAACTCGAGTTGTAGCCTTTGTGATCTCCG-3'; ShRNA ESCO1-2: Forward primer (SEQ ID NO: 3): 5'-CCGGCCAGCTAAATAGGAAGAATTACTCGAGTAATTCTTCCTATTTAGCTGGTTTTTG-3'; Reverse primer (SEQ ID NO: 4): 5'-AATTCAAAAACCAGCTAAATAGGAAGAATTACTCGAGTAATTCTTCCTATTTAGCTGG-3'; ShRNA ESCO1-3: Forward primer (SEQ ID NO: 5): 5'-CCGGGCCTAAGTGTAATTCCCATTTCTCGAGAAATGGGAATTACACTTAGGCTTTTTG-3'; Reverse primer (SEQ ID NO: 6): 5'-AATTCAAAAAGCCTAAGTGTAATTCCCATTTCTCGAGAAATGGGAATTACACTTAGGC-3'; Experimental results ( Figure 6 B in Figure 6 As shown in C), the expression level of ESCO1 protein was significantly reduced in both PANC-1 and SW1990 cell lines, indicating that a stable ESCO1 knockdown cell line was successfully constructed.

[0064] 4.3 ESCO1 promotes malignant progression of pancreatic cancer cells To evaluate the effect of ESCO1 on the malignant biological behavior of pancreatic cancer cells, this invention detected cell growth and proliferation using a plate colony assay and an EdU assay. PANC-1 and SW1990 cells from the ESCO1 knockdown group and control group were seeded at a density of 500 cells per well in 6-well plates and cultured. After 7 days of culture, the medium was changed and cultured for another 7 days. After culture, the cells were gently washed twice with PBS, then fixed with 4% paraformaldehyde for 30 min and stained with crystal violet for 30 min. After staining, the cells were washed with double-distilled water and dried at room temperature. Images were taken and the number of colonies formed was counted. Simultaneously, cell proliferation was detected using the YF® 488 Click-iT EdU staining kit (UElandy). After washing the cells three times with PBS, 100 µL of 1× Apollo reaction solution was added, and the cells were incubated in the dark for 1 h. Nuclear staining was then performed using Hoechst 33342 to assess DNA content. Images were acquired using an Olympus fluorescence microscope, and quantitative analysis was performed using ImageJ software. The results showed that ESCO1 knockdown significantly inhibited the growth and proliferation of pancreatic cancer cells. Figure 6 B in Figure 6 (G in the middle).

[0065] Cell migration ability was further evaluated using Transwell and scratch healing assays. In the Transwell assay, PANC-1 and SW1990 cells from the ESCO1 knockdown group and control group were resuspended in serum-free RPMI 1640 medium, with 2 × 10⁶ cells per well. 4 Cells were placed in a lower chamber with complete culture medium containing 15% fetal bovine serum. After incubation at 37 °C for 24 h, unmigrated cells were removed, and cells that had migrated to the lower surface were fixed with 3% paraformaldehyde for 30 min and stained with crystal violet. Images were taken using an inverted microscope, and cell counting analysis was performed using ImageJ software. In the scratch healing assay, 2 × 10⁶ cells were placed in the lower chamber. 5 Cells were seeded in 6-well plates and cultured until approximately 90% confluence. Wounds were then made using a 200 µL pipette tip. Cells were subsequently washed three times with PBS, added with serum-free medium, and incubated at 37 °C. Images were taken at 0 h, 24 h, and 48 h. Wound healing rate was calculated using ImageJ software. The results showed that ESCO1 knockdown significantly inhibited the migration ability of pancreatic cancer cells (…). Figure 6 H- Figure 6 (K in the middle).

[0066] In addition, Annexin V-FITC / PI double staining combined with flow cytometry was used to detect cell apoptosis levels. PANC-1 and SW1990 cells from the ESCO1 knockdown group and control group were resuspended in binding buffer and adjusted to a cell concentration of 1×10⁶. 6 Cells / mL were counted, with 100 µL of cell suspension taken from each group, and 5 µL of Annexin V-FITC and 10 µL of PI staining solution were added. The cells were incubated at room temperature in the dark for 15 min. Flow cytometry (FACScan; BD Biosciences) was then used for detection, and data analysis was performed using FlowJo software. The results showed that ESCO1 knockdown significantly promoted apoptosis in pancreatic cancer cells (…). Figure 7 A in Figure 7 (B in the middle).

[0067] Example 5: NUFIP2 promotes the malignant progression of pancreatic cancer.

[0068] 5.1 Construction of NUFIP2 knockdown cell lines This invention utilizes lentivirus-mediated shRNA interference technology to construct a NUFIP2 knockdown cell line. Using PLKO.1 as the lentiviral vector, the shRNA sequence targeting NUFIP2 was cloned into the multiple cloning site of the vector through molecular biology methods such as PCR amplification, enzyme digestion, and ligation. After the recombinant plasmid was constructed, sequencing was used to verify the correctness and orientation of the inserted sequence. Lentiviral packaging was performed using 293T cells. 293T cells were seeded in 6-well plates and transfected after reaching a confluence of 80%–90%. Plasmid mixtures were prepared according to the transfection reagent (G1802, Servicebio) instructions. PMDL (1.16 µg), PVSVG (0.62 µg), PREV (0.45 µg), and the corresponding amount of target plasmid were added to each well. After thorough mixing, the mixture was added to the cells, gently shaken, and the final culture volume per well was 2 mL. The cells were then incubated at 37 ℃ in a 5% CO2 incubator. Forty-eight hours after transfection, the culture supernatant from each well was collected and centrifuged at 300–500 g for 5 min to remove cell debris. The supernatant was then filtered through a 0.45 µm membrane, and the resulting filtrate was the packaged lentiviral particles. Target cells (PANC-1 and SW1990) were digested and seeded into 6-well plates. After cell adhesion, the lentiviral supernatant was added for infection, maintaining a final culture volume of 2 mL per well. Forty-eight to seventy-two hours after infection, cells were collected, and the expression level of NUFIP2 was detected by Western blot to assess the knockdown efficiency.

[0069] The oligonucleotide primer sequences used in this embodiment to construct the NUFIP2-targeting shRNA expression vector are as follows: ShRNA NUFIP2-1: Forward oligonucleotide (SEQ ID NO: 7): 5'-CCGGGAACAAATTAAGACTAGCCTTCTCGAGAAGGCTAGTCTTAATTTGTTCTTTTTG-3'; Reverse oligonucleotide (SEQ ID NO: 8): 5'-AATTCAAAAAGAACAAATTAAGACTAGCCTTCTCGAGAAGGCTAGTCTTAATTTGTTC-3'; ShRNA NUFIP2-2: Forward oligonucleotide (SEQ ID NO: 9): 5'-CCGGCGGTTACATATTGACTTTAACTCGAGTTAAAGTCAATATGTAACCGTTTTTG-3'; Reverse oligonucleotide (SEQ ID NO: 10): 5'-AATTCAAAAACGGTTACATATTGACTTTAACTCGAGTTAAAGTCAATATGTAACCG-3'; ShRNA NUFIP2-3: Forward oligonucleotide (SEQ ID NO: 11): 5'-CCGGGAAACTTTCAAGCCTGACTATCTCGAGATAGTCAGGCTTGAAAGTTTCTTTTTG-3'; Reverse oligonucleotide (SEQ ID NO: 12): 5'-AATTCAAAAAGAAACTTTCAAGCCTGACTATCTCGAGATAGTCAGGCTTGAAAGTTTC-3'; Experimental results ( Figure 7 C in Figure 7 As shown in D), the expression level of NUFIP2 protein was significantly reduced in both PANC-1 and SW1990 cell lines, indicating that a stable knockdown cell line of NUFIP2 was successfully constructed.

[0070] 5.2 NUFIP2 promotes malignant progression of pancreatic cancer cells To evaluate the effect of NUFIP2 on the malignant biological behavior of pancreatic cancer cells, this invention detected cell growth and proliferation using plate colony assays and EdU assays. PANC-1 and SW1990 cells from the NUFIP2 knockdown group and control group were seeded at a density of 500 cells per well in 6-well plates and cultured in an incubator. After 7 days of culture, the medium was changed and cultured for another 7 days. After culture, the cells were gently washed twice with PBS, then fixed with 4% paraformaldehyde for 30 min and stained with crystal violet for 30 min. After staining, the cells were washed with double-distilled water and dried at room temperature. Images were taken and the number of colonies formed was counted. Simultaneously, cell proliferation was detected using the YF® 488 Click-iT EdU staining kit (UElandy). After washing the cells three times with PBS, 100 µL of 1× Apollo reaction solution was added, and the cells were incubated in the dark for 1 h. Nuclear staining was then performed using Hoechst 33342 to assess DNA content. Images were acquired using an Olympus fluorescence microscope, and quantitative analysis was performed using ImageJ software. The results showed that NUFIP2 knockdown significantly inhibited the growth and proliferation of pancreatic cancer cells. Figure 7 E in Figure 7 (H in the text).

[0071] Cell migration ability was further evaluated using Transwell and scratch healing assays. In the Transwell assay, PANC-1 and SW1990 cells from the NUFIP2 knockout group and control group were resuspended in serum-free RPMI 1640 medium, with 2 × 10⁶ cells per well. 4 Cells were placed in a lower chamber with complete culture medium containing 15% fetal bovine serum. After incubation at 37 °C for 24 h, unmigrated cells were removed, and cells that had migrated to the lower surface were fixed with 3% paraformaldehyde for 30 min and stained with crystal violet. Images were taken using an inverted microscope, and cell counting analysis was performed using ImageJ software. In the scratch healing assay, 2 × 10⁶ cells were placed in the lower chamber. 5 Cells were seeded in 6-well plates and cultured until approximately 90% confluence. Wounds were then made using a 200 µL pipette tip. Cells were subsequently washed three times with PBS, added with serum-free medium, and incubated at 37 °C. Images were taken at 0 h, 24 h, and 48 h. Wound healing rate was calculated using ImageJ software. The results showed that NUFIP2 knockdown significantly inhibited the migration ability of pancreatic cancer cells. Figure 7 I in Figure 7 L in the middle.

[0072] In addition, Annexin V-FITC / PI double staining combined with flow cytometry was used to detect cell apoptosis levels. PANC-1 and SW1990 cells from the NUFIP2 knockdown group and control group were resuspended in binding buffer and adjusted to a cell concentration of 1×10⁶. 6 Cells / mL were counted, with 100 µL of cell suspension taken from each group, and 5 µL of Annexin V-FITC and 10 µL of PI staining solution were added. The cells were incubated at room temperature in the dark for 15 min. Flow cytometry (FACScan; BD Biosciences) was then used for detection, and data analysis was performed using FlowJo software. The results showed that NUFIP2 knockdown significantly promoted apoptosis in pancreatic cancer cells. Figure 7 M in Figure 7 (N in the text).

[0073] Example 6: ESCO1 regulates the malignant progression of pancreatic cancer through NUFIP2.

[0074] 6.1 ESCO1 positively regulates NUFIP2 expression in pancreatic cancer To investigate the regulatory relationship between ESCO1 and NUFIP2, ESCO1 and NUFIP2 were treated with knockdown methods (shNC refers to the negative control, shESCO1 refers to ESCO1 knockdown, and shNUFIP2 refers to NUFIP2 knockdown) in pancreatic cancer cell lines PANC-1 and SW1990, respectively. Western blot was used to detect changes in the expression levels of the other protein. The results showed that in the pancreatic cancer cell line PANC-1 (… Figure 8 (A) and SW1990 ( Figure 8 In cell line B), knocking down ESCO1 caused a corresponding change in NUFIP2 expression levels; however, knocking down NUFIP2 did not significantly alter ESCO1 expression in either of these two cell lines. Figure 8 C in Figure 8 (D in the text). The above results suggest that ESCO1 may be located upstream of NUFIP2 and play a positive regulatory role in NUFIP2 in pancreatic cancer.

[0075] 6.2 Construction of NUFIP2 overexpression cell lines This invention utilizes a pcDNA expression vector to construct a NUFIP2 overexpression cell line. First, the NUFIP2 coding sequence was obtained by PCR amplification, digested with enzymes, and cloned into a pcDNA vector to construct a recombinant overexpression plasmid. Sequencing was used to verify its correctness. ESCO1 knockdown PANC-1 and SW1990 cells were seeded in 6-well plates. When the cell confluence reached 70%–80%, the overexpression plasmid and transfection reagent (G1802, Servicebio) were incubated in serum-free medium to form a complex, which was then added to the cells. The mixture was gently mixed and cultured at 37 °C in a 5% CO2 incubator for 6–8 h. The culture medium was then replaced with complete medium, and the cells were cultured for another 48 h. Subsequently, cells were collected, and protein was extracted. Western blot analysis was used to detect the expression level of NUFIP2 to assess the overexpression efficiency. The primer sequences used to construct the NUFIP2 overexpression vector in this embodiment are as follows: forward primer (SEQ ID NO: 13): 5'-CTAGCTAGCATGGAGGAGAAGCCCGGCCA-3'; reverse primer (SEQ ID NO: 14): 5'-CGGGGTACCTCATTGATCTGGACTATCCA-3'. Experimental results ( Figure 8 E- Figure 8 The results (F) showed that NUFIP2 protein expression was significantly increased in ESCO1 knockdown PANC-1 and SW1990 cell lines.

[0076] 6.3 ESCO1 regulates pancreatic cancer progression via NUFIP2 To evaluate the role of the ESCO1 / NUFIP2 axis in the malignant biological behavior of pancreatic cancer, this invention constructed a NUFIP2 reversible overexpression cell model based on ESCO1 knockdown, and assessed cell growth and proliferation using plate colony assays and EdU assays. PANC-1 and SW1990 cells with ESCO1 knockdown and NUFIP2 reversible overexpression were seeded at a density of 500 cells per well in 6-well plates and cultured for 7 days. The culture medium was replaced, and the cells were cultured for another 7 days. After culture, cells were fixed with 4% paraformaldehyde for 30 min, stained with crystal violet, photographed, and colony formation counted. Simultaneously, cell proliferation was assessed using the YF® 488 Click-iT EdU staining kit, and quantitative analysis was performed using fluorescence microscopy and ImageJ software. The results showed that NUFIP2 reversible overexpression significantly reversed the inhibitory effect of ESCO1 knockdown on the growth and proliferation of pancreatic cancer cells. Figure 8 G in Figure 8 J in the middle.

[0077] Cell migration ability was further evaluated using Transwell and scratch healing assays. In the Transwell assay, the treated cells were resuspended in serum-free RPMI 1640 medium, and 2 × 10⁶ cells were seeded per well. 4 Cells were incubated in a complete culture medium containing 15% fetal bovine serum in the lower chamber for 24 h. After incubation, migrating cells were fixed, stained, and counted. In the scratch healing assay, cells were seeded in 6-well plates. After approximately 90% confluence, scratch treatment was performed, and cell migration was recorded at 0 h, 24 h, and 48 h to calculate the wound healing rate. The results showed that NUFIP2 reversion overexpression significantly reversed the inhibitory effect of ESCO1 knockdown on pancreatic cancer cell migration. Figure 8 I in Figure 8 (N in the text).

[0078] In addition, Annexin V-FITC / PI double staining combined with flow cytometry was used to detect apoptosis levels. Cells from different treatment groups were resuspended in binding buffer and the concentration was adjusted to 1×10⁻⁶. 6 Cells / mL were added with Annexin V-FITC and PI staining solution, incubated in the dark, and then analyzed by flow cytometry using FlowJo software. Results showed that NUFIP2 reversion overexpression significantly attenuated ESCO1 knockdown-induced apoptosis in pancreatic cancer cells. Figure 8 O in Figure 8 (P in the middle).

[0079] In summary, these results indicate that ESCO1 can participate in malignant biological processes such as growth, migration, and apoptosis of pancreatic cancer cells by regulating the expression of NUFIP2.

[0080] It should be noted that the experimental studies of this invention were mainly conducted based on the pancreatic cancer cell lines PANC-1 and SW1990, thereby confirming that ESCO1 and NUFIP2 can serve as potential novel biomarkers and therapeutic targets for pancreatic cancer. However, the technical solutions of this invention are not limited to the specific cell lines mentioned above. Based on the technical principles and experimental results disclosed in Examples 1 to 6 of this invention, it can be inferred that the ESCO1 / NUFIP2 axis is also applicable to other pancreatic cancer cell lines and can serve as biomarkers and therapeutic targets in related cell lines, and may participate in the process related to pancreatic cancer immunotherapy. Therefore, any application of the ESCO1 / NUFIP2 axis in other pancreatic cancer cell lines should be considered to fall within the protection scope of this invention. The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The use of reagents that inhibit ESCO1 expression and / or NUFIP2 expression in the preparation of drugs for treating pancreatic cancer, characterized in that, The reagent that inhibits ESCO1 expression is shRNA, specifically at least one of shRNA ESCO1-1, shRNA ESCO1-2, and shRNA ESCO1-3. The reagent used to inhibit NUFIP2 expression is shRNA, specifically at least one of shRNA NUFIP2-1, shRNA NUFIP2-2, and shRNA NUFIP2-3; The sequence of shRNA ESCO1-1 is shown in SEQ ID NO: 1~SEQ ID NO: 2; The sequences of shRNA ESCO1-2 are shown in SEQ ID NO: 3~SEQ ID NO: 4; The sequences of shRNA ESCO1-3 are shown in SEQ ID NO: 5~SEQ ID NO: 6; The sequence of shRNA NUFIP2-1 is shown in SEQ ID NO: 7~SEQ ID NO: 8; The sequence of shRNA NUFIP2-2 is shown in SEQ ID NO: 9~SEQ ID NO: 10; The sequences of shRNA NUFIP2-3 are shown in SEQ ID NO: 11~SEQ ID NO: 12.

Images