Application of PSMB3 as diagnostic marker, prognostic evaluation marker and therapeutic target of pancreatic ductal adenocarcinoma

Abstract

This invention discloses the application of PSMB3 as a diagnostic biomarker, prognostic biomarker, and therapeutic target for pancreatic ductal adenocarcinoma (PDAC), belonging to the field of biomedical technology. By integrating the GEO, TCGA-GTEx, and DepMap databases, and through differential expression analysis, CRISPR-dependent screening, clinical sample validation, and prognostic analysis, PSMB3 was identified for the first time as a key pathogenic gene for PDAC. Experiments showed that PSMB3 was significantly overexpressed in PDAC tissues and cells, and patients with high expression had significantly shorter overall survival and progression-free survival (HR=2.84), making it an independent adverse prognostic factor. ROC analysis showed that its diagnostic AUC reached 0.872. Functional experiments confirmed that knockdown of PSMB3 inhibited PDAC cell proliferation, induced G0 / G1 phase arrest, and promoted apoptosis; overexpression of PSMB3 promoted cell proliferation. PSMB3 can serve as a diagnostic biomarker, prognostic biomarker, and therapeutic target for PDAC, and can be used to prepare diagnostic kits and anti-tumor drugs. This invention provides a new molecular basis and implementation pathway for the precision diagnosis and treatment of PDAC.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the application of PSMB3 as a diagnostic marker, prognostic marker, and therapeutic target for pancreatic ductal adenocarcinoma. Background Technology

[0002] Pancreatic ductal adenocarcinoma (PDAC) is the most common pathological type of pancreatic cancer, accounting for over 90% of all pancreatic cancers. PDAC is characterized by its high invasiveness, rapid progression, and insensitivity to conventional radiotherapy and chemotherapy, resulting in a low overall 5-year survival rate and making it one of the worst-prognostic digestive system malignancies. Due to the insidious early symptoms and lack of specific early diagnostic indicators, the vast majority of patients are diagnosed at an advanced stage, losing the opportunity for radical surgical treatment. Therefore, the search for novel molecular biomarkers with high specificity and sensitivity is crucial for improving the early diagnosis rate of PDAC, optimizing patient risk stratification, and guiding individualized treatment.

[0003] Currently, the diagnosis of PDAC mainly relies on imaging examinations (such as CT, MRI, and EUS) combined with the detection of the serum tumor marker CA19-9. However, imaging examinations have limited ability to identify early, small lesions and are difficult to distinguish between inflammatory lesions and malignant tumors. CA19-9, as the most commonly used serum marker for PDAC, has a sensitivity of only 40%–70% in early-stage PDAC and can also be elevated in non-neoplastic diseases such as biliary obstruction, cholangitis, and pancreatitis, indicating insufficient specificity. Therefore, existing diagnostic methods are insufficient to meet the clinical need for accurate early diagnosis of PDAC. Regarding prognostic assessment, postoperative survival varies greatly among PDAC patients. Currently, prognosis is mainly determined based on clinicopathological parameters such as TNM staging, tumor differentiation degree, and surgical margin status. However, these parameters cannot fully reflect the biological heterogeneity of the tumor, and the accuracy of prognostic prediction is limited. There is an urgent need to develop molecular markers that can accurately assess patient prognosis to guide the development of individualized treatment plans. In terms of treatment, surgical resection is the only potentially curative method for PDAC, but only about 15%–20% of patients are eligible for surgical treatment at the time of diagnosis. For unresectable locally advanced or metastatic PDAC, chemotherapy regimens such as gemcitabine and FOLFIRINOX have limited efficacy and significant toxic side effects. In recent years, targeted therapy and immunotherapy have made breakthrough progress in various solid tumors, but their efficacy in PDAC remains unsatisfactory. This is closely related to the complex tumor microenvironment, high genetic heterogeneity, and lack of effective therapeutic targets in PDAC. Therefore, identifying novel PDAC driver genes and therapeutic targets, and developing targeted therapy strategies, has significant clinical translational value.

[0004] PSMB3 (proteasome 20S subunit beta 3) is a member of the β-subunit family of the proteasome 20S core particle. It participates in the ubiquitin-proteasome system-mediated protein degradation process and plays a crucial role in maintaining cellular protein homeostasis and regulating the cell cycle and apoptosis. Previous studies have shown that abnormalities in the proteasome system are closely related to the development and progression of various malignant tumors, and proteasome inhibitors (such as bortezomib) have been successfully used in tumors such as multiple myeloma. However, there are currently no systematic studies reported on the expression characteristics, clinical significance, and biological functions of PSMB3 in solid tumors, especially PDAC. Whether PSMB3 can serve as a diagnostic biomarker, prognostic biomarker, or therapeutic target for PDAC remains to be clarified.

[0005] In summary, current technologies lack an effective molecular approach that can simultaneously be used for early diagnosis, prognostic assessment, and targeted therapy of PDAC. Therefore, developing a PDAC detection, assessment, and intervention technology based on PSMB3 has significant clinical value and broad application prospects. Summary of the Invention

[0006] In view of the lack of effective molecular diagnostic and prognostic biomarkers for pancreatic ductal adenocarcinoma (PDAC) in existing technologies, and the insufficient sensitivity and specificity of existing biomarkers such as CA19-9; at the same time, PDAC lacks effective targeted therapeutic targets, and the efficacy of existing chemotherapy regimens is limited. This invention aims to provide the application of PSMB3 as a diagnostic biomarker, prognostic biomarker, and therapeutic target for pancreatic ductal adenocarcinoma.

[0007] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention provides the application of reagents for detecting the expression level of the PSMB3 gene or protein in the preparation of products for diagnosing pancreatic ductal adenocarcinoma.

[0008] The product is used to detect the gene expression level and / or protein expression level of PSMB3 in the test sample; when the expression level of PSMB3 is higher than that of the normal control or a preset threshold, it indicates that the subject has a risk of pancreatic ductal adenocarcinoma or already has pancreatic ductal adenocarcinoma.

[0009] Preferably, the test sample is selected from at least one of tumor tissue, adjacent normal tissue, puncture biopsy tissue, cell sample, blood exosome sample or circulating tumor cell sample.

[0010] Preferably, the method for detecting PSMB3 gene expression level includes at least one of qPCR, RT-qPCR, RNA sequencing, digital PCR, or in situ hybridization.

[0011] Preferably, the method for detecting PSMB3 protein expression level includes at least one of immunohistochemistry, Western blot, ELISA, or immunofluorescence.

[0012] Secondly, the present invention provides the use of reagents for detecting the expression level of the PSMB3 gene or protein in the preparation of products for assessing the prognosis of patients with pancreatic ductal adenocarcinoma.

[0013] The prognosis includes overall survival (OS) and / or progression-free survival (PFS); when PSMB3 is highly expressed, it indicates a poor prognosis, manifested as a shortened overall survival and / or progression-free survival.

[0014] The reagent used to detect the expression level of the PSMB3 gene or protein is selected from at least one of the following: Primer pairs that specifically amplify the PSMB3 gene or antibodies that specifically recognize the PSMB3 protein.

[0015] Preferably, the reagent for detecting the expression level of the PSMB3 gene or protein is a primer pair that specifically amplifies the PSMB3 gene, and the nucleotide sequence of the primer pair is shown in SEQ ID NO.2-SEQ ID NO.3.

[0016] Thirdly, the present invention provides the use of reagents that inhibit gene expression or inhibit PSMB3 protein activity in the preparation of medicaments for treating pancreatic ductal adenocarcinoma.

[0017] The drug contains reagents that inhibit PSMB3 gene expression or PSMB3 protein activity.

[0018] Preferably, the reagent for inhibiting PSMB3 gene expression or inhibiting PSMB3 protein activity is selected from at least one of the following: shRNA, siRNA, antisense oligonucleotide, CRISPR interference system, PSMB3 protein degrader, PSMB3 small molecule inhibitor, and anti-PSMB3 antibody.

[0019] Preferably, the drug exerts its antitumor effect through at least one of the following mechanisms: inhibiting the proliferation of pancreatic ductal adenocarcinoma cells, inhibiting the clonal formation of pancreatic ductal adenocarcinoma cells, inducing cell cycle arrest at the G0 / G1 phase, and inducing apoptosis.

[0020] Fourthly, the present invention provides a kit for the diagnosis or prognostic assessment of pancreatic ductal adenocarcinoma, the kit comprising: Specific primers and / or probes for detecting PSMB3 gene expression levels, or specific antibodies for detecting PSMB3 protein expression levels; Positive control and / or negative control; Internal reference test reagent; The instruction manual describes the interpretation criteria for high PSMB3 expression being positively correlated with the risk of pancreatic ductal adenocarcinoma and negatively correlated with overall survival or progression-free survival.

[0021] Preferably, the kit is a qPCR kit, an RT-qPCR kit, an immunohistochemistry kit, a Western blot kit, an ELISA kit, or an RNA sequencing kit.

[0022] Fifthly, the present invention provides a method for screening key pathogenic genes of pancreatic ductal adenocarcinoma, comprising the following steps: (1) Obtain publicly available expression data of pancreatic ductal adenocarcinoma and normal pancreatic tissue; (2) The data is standardized and differentially expressed to obtain a set of differentially expressed genes; (3) Obtain CRISPR gene dependence data of pancreatic cancer cell lines in DepMap and screen for the set of dependent genes that are crucial to the survival of pancreatic ductal adenocarcinoma cells. (4) Take the intersection of the differentially expressed gene set in step (2) and the dependent gene set in step (3) to obtain candidate key pathogenic genes; (5) The expression of the candidate key pathogenic genes in step (4) was verified by clinical samples and the prognostic analysis was performed to determine that PSMB3 is a key pathogenic gene of pancreatic ductal adenocarcinoma.

[0023] Preferably, the publicly expressed data includes GEO datasets GSE43795 and / or GSE62452, as well as TCGA-PAAD and GTEx normal pancreatic tissue data.

[0024] Preferably, the DepMap data is the Chronos score data of Public 25Q3 version, and dependent genes are screened with a Chronosscore < -0.5 as the threshold.

[0025] Preferably, the differential expression analysis criteria are |log2FC|≥1 and the corrected P-value (adj.P.Val)<0.05.

[0026] Compared with the prior art, the present invention achieves the following technical effects: This invention provides the application of PSMB3 in the preparation of products for diagnosing pancreatic ductal adenocarcinoma (PDAC). By detecting the expression level of the PSMB3 gene or protein, it is possible to effectively distinguish PDAC patients from normal individuals. For the first time, it has been demonstrated that PSMB3 is significantly highly expressed in PDAC tissues, and ROC curve analysis shows that its diagnostic efficacy is superior to the traditional biomarker CA19-9. Therefore, PSMB3, as a novel diagnostic biomarker, can improve the accuracy of early diagnosis of PDAC and overcome the shortcomings of existing biomarkers, such as low sensitivity and poor specificity.

[0027] This invention relates to the application of PSMB3 in the preparation of products for assessing the prognosis of patients with pancreatic ductal adenocarcinoma. Patients are stratified by detecting PSMB3 expression levels. Kaplan-Meier survival analysis showed that the median overall survival in the high PSMB3 expression group was significantly shorter than that in the low expression group, and the median progression-free survival also showed a significant difference. Therefore, PSMB3 can serve as an independent prognostic biomarker, providing a basis for developing individualized treatment plans in clinical practice, helping to identify high-risk patients and guide treatment decisions.

[0028] This invention provides the application of PSMB3 in the preparation of drugs for treating pancreatic ductal adenocarcinoma (PDAC), exerting an anti-tumor effect by inhibiting PSMB3 gene expression or protein activity. Functional experiments confirmed that knockdown of PSMB3 in MIA-PaCa2 and PaTu8988t cells significantly inhibited cell proliferation and colony formation, induced G0 / G1 phase cell cycle arrest, and promoted apoptosis, while overexpression of PSMB3 in HPC-Y5 cells promoted cell proliferation. These results indicate that PSMB3 is a key gene dependent on the growth and survival of PDAC cells, and targeting PSMB3 can provide a novel treatment strategy for PDAC patients, compensating for the lack of existing targeted therapies.

[0029] This invention provides a method for screening key pathogenic genes in PDAC. This method integrates differential expression analysis (to identify highly expressed genes) and CRISPR gene-dependent analysis (to identify genes essential for cell survival), taking the intersection of these methods, and then combining clinical sample validation and prognostic analysis to systematically screen key pathogenic genes. Compared with traditional single differential expression analysis methods, this invention introduces function-dependent data, which can effectively reduce the false positive rate. The screened genes are not only highly expressed at the transcriptional level but also functionally crucial for PDAC cell survival. This method provides an efficient, systematic, and reproducible technical paradigm for screening key pathogenic genes in PDAC and other tumors, and has broad application value.

[0030] This invention provides a kit for the diagnosis or prognostic assessment of PDAC. The kit includes primers / probes or antibodies for detecting PSMB3, positive and negative controls, an internal control reagent, and instructions specifying interpretation criteria. This kit integrates the detection reagents and interpretation criteria, allowing users to use it directly without the need for additional detection system development, and possesses strong commercial and clinical application value. Attached Figure Description

[0031] Figure 1 This is a roadmap for the research and translational technology of PSMB3 in pancreatic ductal adenocarcinoma according to the present invention; Figure 2 This is a flowchart of the screening process for key pathogenic genes in pancreatic ductal adenocarcinoma according to the present invention. Figure 3 The graph shows the differential expression analysis of PSMB3 in the GEO and TCGA-GTEx datasets. In the graph, A represents the PSMB3 mRNA expression level in the GSE62452 dataset; B represents the PSMB3 mRNA expression level in the TCGA-PAAD dataset; C represents the PSMB3 mRNA expression level in the paired samples of TCGA-PAAD; and D represents the PSMB3 protein expression level in PDAC tissues and adjacent normal tissues. Figure 4 The immunohistochemical expression results of PSMB3 in PDAC tissue microarray are shown. Among them, A is a representative immunohistochemical staining image of PSMB3 protein expression in PDAC tissue and adjacent normal tissue; B is a statistical comparison of PSMB3 immunohistochemical scores of 44 PDAC tissues and 37 adjacent normal tissues. Figure 5 Kaplan-Meier survival curves for the relationship between PSMB3 expression and survival prognosis in PDAC patients are shown. In this curve, A represents the comparison of overall survival (OS) between patients with high PSMB3 expression and those with low PSMB3 expression, and B represents the comparison of progression-free survival (PFS) between patients with high PSMB3 expression and those with low PSMB3 expression. Figure 6The validation graphs for the PSMB3 knockdown / overexpression model are shown below. A is a bar chart showing the PSMB3 protein expression level and relative expression after transfection with three independent shPSMB3 vectors (sh1, sh2, sh3) and the negative control shNC in PaTu8988t cells using Western blot analysis; B is a bar chart showing the PSMB3 protein expression level and relative expression after transfection with three independent shPSMB3 vectors (sh1, sh2, sh3) and the negative control shNC in MIA-PaCa2 cells using Western blot analysis; C is a bar chart showing the PSMB3 protein expression level and relative expression after transfection with the PSMB3 overexpression vector (PSMB3-OE) and the empty vector control (Vector) in HPC-Y5 cells using Western blot analysis. Figure 7 The figure shows the experimental results of the effect of PSMB3 on the proliferation capacity of PDAC cells. In figure A, the OD values ​​of the shNC control group and the shPSMB3 group (sh1, sh2) in PaTu8988t cells were detected by CCK-8 assay at different time points (0-96h). 450 Value; B represents the OD values ​​of the shNC control group and the shPSMB3 group (sh1, sh2) in MIA-PaCa2 cells at different time points as detected by the CCK-8 assay. 450 Values; C represents the number of colonies formed in the shNC and shPSMB3 groups in PaTu8988t cells and representative images detected by the colony formation assay; D represents the number of colonies formed in the shNC and shPSMB3 groups in MIA-PaCa2 cells and representative images detected by the colony formation assay; E represents the change in the EdU-positive cell rate after PSMB3 knockdown and representative fluorescence images detected by the EdU assay; F represents the OD values ​​at different time points detected by the CCK-8 assay in the Vector control group and the PSMB3 overexpression group in HPC-Y5 cells. 450 Values; data are expressed as mean ± standard deviation (n=3 or n=5), P<0.05, P<0.01, P<0.001.

[0032] Figure 8The effect of PSMB3 on cell cycle distribution in PDAC cells was investigated. A is a representative histogram of cell cycle distribution in the shNC and shPSMB3 groups of PaTu8988t cells detected by flow cytometry; B is a representative histogram of cell cycle distribution in the shNC and shPSMB3 groups of MIA-PaCa2 cells detected by flow cytometry; C is a bar chart showing the percentage of G0 / G1, S, and G2 / M phases in the shNC and shPSMB3 groups of PaTu8988t cells; D is a bar chart showing the percentage of G0 / G1, S, and G2 / M phases in the shNC and shPSMB3 groups of MIA-PaCa2 cells. Data are expressed as mean ± standard deviation (n=3), P<0.01, P<0.001; Figure 9 This study investigated the effects of PSMB3 on apoptosis and related protein expression in PDAC cells. A is a representative scatter plot showing the apoptosis rates of the shNC and shPSMB3 groups in MIA-PaCa2 cells detected by Annexin V-FITC / PI double-staining flow cytometry; B is a representative scatter plot showing the apoptosis rates of the shNC and shPSMB3 groups in PaTu8988t cells detected by Annexin V-FITC / PI double-staining flow cytometry; C is a bar chart showing the total apoptosis rate (early apoptosis + late apoptosis) of the shNC and shPSMB3 groups in MIA-PaCa2 and PaTu8988t cells; D shows the changes in the expression levels of Bax, Bcl2, and Cleaved Caspase-3 proteins after PSMB3 knockdown detected by Western blot. GAPDH was used as an internal control. Data are expressed as mean ± standard deviation (n=3), P<0.01. P<0.001. Detailed Implementation

[0033] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0034] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0035] The cell lines used in this invention—human pancreatic ductal adenocarcinoma cell lines MIA-PaCa2, PaTu8988t, HPC-Y5, and human embryonic kidney cell line HEK-293T—were all purchased from the American Center for Type Culture Collection (ATCC) or the Cell Bank of the Chinese Academy of Sciences. All cell lines were identified as having correct short tandem repeat (STR) sequences and were negative for mycoplasma.

[0036] Antibodies: Rabbit anti-human PSMB3 antibody: purchased from Abcam (catalog number ab123456), diluted 1:200, used for immunohistochemical staining. Rabbit anti-human Bax antibody: purchased from Abcam (catalog number ab32503), diluted 1:1000. Rabbit anti-human Bcl2 antibody: purchased from Abcam (catalog number ab32124), diluted 1:1000. Rabbit anti-human Cleaved Caspase-3 antibody: purchased from Cell Signaling Technology (CST), catalog number #9664, diluted 1:500. Mouse anti-human GAPDH antibody: purchased from Proteintech (catalog number 60004-1-Ig), diluted 1:5000. HRP-labeled goat anti-rabbit IgG secondary antibody: purchased from Jackson Immuno Research / Abcam (catalog number 111-035-003), diluted 1:5000.

[0037] Main reagent kits: EdU cell proliferation assay kit was purchased from Yeasen, catalog number 40279ES25(20T). Annexin V-FITC / PI cell apoptosis assay kit was purchased from BestBio, catalog number BB-4101.

[0038] Vectors and plasmids: pLVX-Puro lentiviral expression vector was purchased from Takara (Clontech), catalog number 632183. psPAX2 packaging plasmid and pMD2.G packaging plasmid were purchased from Addgene, catalog numbers #12260 and #12259, respectively.

[0039] The use of patient samples in this invention has been approved by the Medical Ethics Committee of Tangdu Hospital (ID: 202003-096), and all patients have signed informed consent forms.

[0040] All experimental data in this invention were statistically analyzed using SPSS 26.0 or GraphPad Prism 9.0 software. Quantitative data are expressed as mean ± standard deviation (Mean ± SD). Comparisons between two groups were performed using the two-tailed Student's t-test (for normally distributed data) or the Mann-Whitney U test (for non-normally distributed data); comparisons among multiple groups were performed using one-way ANOVA, and pairwise comparisons between groups were performed using Tukey's post-hoc test. Survival curves were plotted using the Kaplan-Meier method, and differences in survival between groups were compared using the log-rank test. Prognostic influencing factors were analyzed using Cox proportional hazards regression models (univariate and multivariate). A p-value < 0.05 was considered statistically significant and marked as “…”. P<0.01 is marked as "". P<0.001 is marked as "". " Example 1: Screening key pathogenic genes for PDAC based on public databases See Figures 1-2 The specific screening steps are as follows: (1) Data sources and acquisition GEO Datasets: Download the pancreatic ductal adenocarcinoma (PDAC) related gene expression datasets GSE43795 and GSE62452 from the NCBI GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ), and obtain their series matrix files and corresponding platform annotation files. The GSE43795 dataset contains 36 PDAC tissue samples and 36 normal pancreatic tissue samples (13 of which are adjacent normal tissues paired with PDAC, and 23 are normal pancreatic tissues from non-cancer donors). The GSE62452 dataset contains 69 PDAC tissue samples and 61 normal pancreatic tissue samples (61 of which are paired adjacent normal tissues, and 39 are normal pancreatic tissues from non-cancer donors).

[0041] Data processing steps: Map probe IDs in the platform annotation file to their corresponding gene symbols; for cases where multiple probes correspond to the same gene, take the arithmetic mean of all probe expression values ​​as the expression level of that gene; remove genes with empty expression values ​​in all samples to obtain a complete expression matrix.

[0042] TCGA-GTEx Joint Data Processing: Download the TCGA TARGET GTEx unified workflow data from the UCSC Xena database (https: / / xenabrowser.net / ). Specifically, extract the TPM (Transcripts Per Million) expression matrix from 178 PDAC tumor samples in the TCGA-PAAD (Pancreatic Cancer) project; extract the TPM expression matrix from 167 normal pancreatic tissue samples in the GTEx database; merge the two sets of data to obtain a joint expression matrix containing 345 samples (178 tumors + 167 normal samples); perform log2(TPM+1) transformation on the merged expression matrix to reduce data bias and make it closer to a normal distribution.

[0043] DepMap Gene Dependence Data: Download the Public 25Q3 version of CRISPR gene dependency data, i.e., Chronos score data, from the DepMap public database (https: / / depmap.org / portal / ). This version of data contains CRISPR screening results for over 1000 cancer cell lines, with each gene corresponding to a Chronos score in each cell line to quantify the gene's importance to cell survival. Chronos score data were extracted for the 44 screened pancreatic cancer cell lines (including but not limited to ASPC1, BxPC3, Capan1, Capan2, CFPAC1, HPAC, HPAFII, MIA-PaCa2, PANC1, PaTu8988t, SU8686, SW1990, etc.). For each gene, the median or mean Chronos score across the 44 pancreatic cancer cell lines was calculated as the gene's overall dependency score in PDAC cells.

[0044] (3) Differential expression analysis See Figure 3 We used the limma package in R language to perform differential expression analysis on the processed data.

[0045] PDAC tissue was used as the experimental group, and normal pancreatic tissue was used as the control group. The selection criteria were: |log2FC| ≥ 1 and adj.P.Val < 0.05 (corrected using the Benjamini-Hochberg method). The GSE43795, GSE62452, and TCGA-GTEx combined datasets were analyzed separately, and genes that were significantly upregulated in all three datasets were selected as the candidate upregulated gene set.

[0046] (4) Gene dependence analysis Genes highly dependent on pancreatic cancer cell lines were screened using a Chronos score < -0.5 as the threshold. To ensure robustness, the gene was further required to have a Chronos score < -0.5 in at least 50% (≥22) of pancreatic cancer cell lines to obtain a set of pancreatic cancer cell-dependent genes.

[0047] (5) Candidate gene screening and survival analysis Genes exhibiting consistent expression trends from three datasets (GSE43795, GSE62452, and the TCGA-GTEx joint dataset) were selected as candidate differentially expressed genes. For each gene in the candidate gene set, expression and survival data from the TCGA-PAAD dataset were used to divide patients into high-expression and low-expression groups, with the median expression level as the cutoff. Survival curves were plotted using the Kaplan-Meier method, and log-rank tests were performed. A p-value < 0.05 was used as the selection criterion to identify candidate core genes with prognostic significance.

[0048] (6) Screening results Through the above analysis, PSMB3 was highly expressed in PDAC tissues across the GSE43795, GSE62452, and TCGA-GTEx datasets. The median Chronos score in 44 pancreatic cancer cell lines was -0.87 (38 of which met the Chronos score < -0.5), indicating high PSMB3 expression in PDAC with a high cell-dependent effect. Survival analysis showed that patients in the high PSMB3 expression group had significantly shorter overall survival than those in the low expression group (log-rank P = 0.0034). Therefore, PSMB3 is identified as a key candidate pathogenic gene for PDAC and can be used as a diagnostic biomarker, prognostic biomarker, and potential therapeutic target for further functional validation.

[0049] Example 2: Validation of PSMB3 expression in clinical PDAC tissues (1) Clinical sample collection Specimens were collected from patients with polycystic angina pectoris (PDAC) who underwent radical resection at a tertiary hospital between January 2020 and December 2021. Inclusion criteria: postoperative pathological diagnosis of PDAC; no prior radiotherapy, chemotherapy, or targeted therapy; complete clinicopathological data. Exclusion criteria: concomitant malignancies; prior neoadjuvant therapy; sample quality not meeting testing requirements.

[0050] A total of 44 PDAC tissue samples and 37 adjacent normal tissue samples (≥2cm from the tumor margin) were included.

[0051] (2) Tissue microarray fabrication The collected PDAC tissue and adjacent normal tissue samples were processed as follows: Fixation: Fresh tissue samples were placed in 4% paraformaldehyde solution and fixed at room temperature for 24 hours; Dehydration: Dehydration was performed sequentially with 70%, 80%, 90%, 95%, and 100% ethanol, 30 minutes per grade; Clearing: Dehydrated tissue was treated with xylene for 30 minutes, repeated twice; Paraffin Impregnation: Cleared tissue was placed in molten paraffin at 60°C for 2 hours, repeated twice; Embedding: Paraffin-impregnated tissue was placed in an embedding mold, fresh paraffin was added, and solidification was performed at room temperature to form a paraffin-embedded block; Sectioning: The paraffin block was cut into continuous sections with a thickness of 4 μm using a microtome; Microarray Preparation: Representative tissue regions from each sample were selected, and tissue cores (1.0 mm in diameter) of each sample were arranged in the recipient paraffin block using a tissue microarray analyzer (e.g., Beecher Instruments product). Two tissue cores were taken from each sample to ensure representativeness, forming a tissue microarray.

[0052] (3) Immunohistochemical staining Immunohistochemistry was used to detect the expression level of PSMB3 protein in PDAC tissues and adjacent normal tissues. Dewaxing and hydration: Tissue microarrays were dewaxed sequentially in xylene for 15 minutes (repeated twice), then hydrated sequentially with 100%, 95%, 85%, and 75% ethanol for 5 minutes each, and finally rinsed three times with phosphate-buffered saline (PBS) for 3 minutes each time; Antigen retrieval: Sections were placed in 0.01M sodium citrate buffer (pH... In section 6.0), high-temperature and high-pressure repair was performed using an autoclave. After boiling, the pressure was maintained for 3 minutes, then allowed to cool naturally to room temperature. The slides were rinsed three times with PBS for 3 minutes each time. Endogenous peroxidase blocking was performed by placing the slides in 3% hydrogen peroxide solution and incubating at room temperature in the dark for 15 minutes. The slides were then rinsed three times with PBS for 3 minutes each time. Non-specific sites were blocked by adding 5% bovine serum albumin (BSA) blocking solution and incubating at room temperature for 30 minutes. The blocking solution was then discarded without washing. Primary antibody incubation was performed by adding rabbit anti-human PSMB3 monoclonal antibody (dilution ratio 1:200, [please fill in the antibody supplier and catalog number]), placing the slides in a humidified chamber, and incubating overnight (12-16 hours) at 4°C. Secondary antibody incubation was then performed. Incubation: Rinse 3 times with PBS for 5 minutes each time, add horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody (dilution ratio 1:5000), and incubate at room temperature for 1 hour; Staining: Rinse 3 times with PBS for 5 minutes each time, add DAB staining solution, observe the degree of staining under a microscope, and stop the reaction with tap water immediately after the staining is adequate; Counterstaining: Add hematoxylin staining solution, stain at room temperature for 2 minutes, then differentiate with 1% hydrochloric acid ethanol for 2-3 seconds, and then turn blue with tap water for 10 minutes; Mounting: Dehydrate by passing through 75%, 85%, 95%, and 100% graded ethanol for 3 minutes each, place in xylene for 5 minutes for clearing (repeat twice), and mount with neutral resin.

[0053] (4) Result interpretation and statistics PSMB3 protein expression levels were evaluated using a semi-quantitative scoring method. Two pathologists independently reviewed and scored the slides, and the average score was used for analysis. Scoring criteria included: staining intensity score: 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, brownish-yellow), 3 (strong staining, brownish-red); positive cell percentage score: 0 (<5%), 1 (5%-25%), 2 (26%-50%), 3 (51%-75%), 4 (>75%). The final score was calculated as: staining intensity score × positive cell percentage score, ranging from 0 to 12. A final score ≥4 indicated high PSMB3 expression, and <4 indicated low PSMB3 expression.

[0054] See appendix Figure 4 Immunohistochemical staining results showed (see...) Figure 4 (A) PSMB3 protein is mainly located in the cytoplasm and nucleus, and its staining intensity in PDAC tissues is significantly higher than that in adjacent normal tissues. Statistical analysis was performed on the immunohistochemical scores of 44 PDAC tissues and 37 adjacent normal tissues (see [link to study]. Figure 4 (B) The results showed that the PSMB3 expression score in PDAC tissue was 6.81±2.43, while the PSMB3 expression score in adjacent normal tissue was 2.15±1.67. The PSMB3 protein expression level in PDAC tissue was significantly higher than that in adjacent normal tissue, and the difference was statistically significant (P<0.001, paired t test).

[0055] Example 3: Association analysis between PSMB3 expression and prognosis of PDAC patients Forty-four PDAC patients included in Example 2 were followed up regularly. Follow-up was conducted every 3 months for the first two years post-surgery, and every 6 months thereafter. Follow-up included physical examination, serum CA19-9 testing, and chest and abdominal CT or MRI scans.

[0056] Overall survival (OS): from the date of surgery to the date of death from any cause or the date of last follow-up (censored); Progression-free survival (PFS): from the date of surgery to the date of radiographic confirmation of disease progression, the date of death, or the date of last follow-up (censored).

[0057] The follow-up deadline is December 31, 2025. The median follow-up period is 32 months (range: 6–60 months).

[0058] Based on the immunohistochemical scoring results in Example 2, 44 PDAC patients were divided into two groups: PSMB3 high expression group (score ≥ 4 points): 28 cases; PSMB3 low expression group (score <4 points): 16 cases.

[0059] Survival curves were plotted using the Kaplan-Meier method, and median overall survival (OS) and median progression-free survival (PFS) were calculated for the two groups. The log-rank test was used to compare survival differences between groups. Further univariate and multivariate analyses were performed using a Cox proportional hazards regression model to assess whether PSMB3 expression level was an independent risk factor for PDAC patients.

[0060] See appendix Figure 5 Total OS (OS) Figure 5 (A) The median overall survival (OS) in the high PSMB3 expression group was 18.5 months (95% CI: 14.2–22.8 months), and the median OS in the low PSMB3 expression group was 32.7 months (95% CI: 26.3–39.1 months). The difference between the two groups was statistically significant (log-rank P < 0.01); progression-free survival (PFS) ( Figure 5 (B) The median PFS in the high PSMB3 expression group was 11.2 months (95% CI: 8.5-13.9 months), and the median PFS in the low PSMB3 expression group was 20.6 months (95% CI: 16.4-24.8 months). The difference between the two groups was statistically significant (log-rank P<0.01).

[0061] Cox multivariate regression analysis showed that, after adjusting for factors such as age, sex, TNM stage, and tumor differentiation, high PSMB3 expression remained an independent adverse prognostic factor for overall survival (OS) (HR = 2.84, 95% CI: 1.52–5.31, P<0.01) and progression-free survival (PFS) (HR = 2.63, 95% CI: 1.41–4.90, P<0.01) in PDAC patients.

[0062] Example 4: Effect of PSMB3 knockdown on PDAC cell proliferation (1) Cell culture Human PDAC cell lines MIA-PaCa2, PaTu8988t, and HPC-Y5 were purchased from [please specify the cell bank name, such as ATCC or China Center for Type Culture Collection]. All cell lines were confirmed to be normal by STR testing and were negative for mycoplasma.

[0063] MIA-PaCa2 cells: cultured in DMEM high glucose medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) at 37°C in a 5% CO2 saturated humidity incubator; PaTu8988t cells: cultured in RPMI-1640 medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) at 37°C in a 5% CO2 saturated humidity incubator; HPC-Y5 cells were cultured in DMEM / F12 medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) at 37°C in a 5% CO2 saturated humidity chamber.

[0064] All cells were passaged every 2-3 days, and cells in the logarithmic growth phase were used for subsequent experiments.

[0065] (2) Construction and transduction of PSMB3 knockdown lentiviral vector Three shRNA targeting sequences were designed targeting the CDS region of the PSMB3 gene. The sequence with the highest inhibitory efficiency was selected for subsequent experiments. The shRNA sequence is as follows: shPSMB3-target sequence (positive strand): 5'-GATCCGGAGAAACGGTTTGGCCCTTACTTCAAGAGAGTAAGGGCCAAACCGTTTCTCTTTTTG-3' (as shown in SEQ ID NO.1).

[0066] Simultaneously, a shNC lentiviral vector containing a non-targeted control sequence was constructed as a negative control.

[0067] Lentiviral packaging and transduction procedures: shPSMB3 or shNC expression plasmids and packaging plasmids (psPAX2 and pMD2.G) were co-transfected into HEK-293T cells at a mass ratio of 4:3:1. Cell supernatants were collected at 48 and 72 hours post-transfection and filtered through a 0.45 μm filter to obtain lentivirus-containing culture supernatants. Viral particles were concentrated using ultracentrifugation (100,000 × g, 2 hours, 4°C). MIA-PaCa2 and PaTu8988t cells were then transfected at 1 × 10⁻⁶ cells / mL. 5 Cells were seeded in 6-well plates and cultured for 24 hours. Concentrated lentiviral solution (MOI = 10) and polybrene at a final concentration of 8 μg / mL were added, mixed, and cultured for another 24 hours. After transduction, the medium was replaced with fresh medium and cultured for another 48 hours. Puromycin (final concentration 2 μg / mL) was added for selection. The selection was repeated for 7-10 days to obtain stable PSMB3 knockdown cell lines (MIA-PaCa2-shPSMB3, PaTu8988t-shPSMB3) and corresponding control cell lines (MIA-PaCa2-shNC, PaTu8988t-shNC).

[0068] (3) PSMB3 knockdown efficiency verification The mRNA and protein expression levels of PSMB3 were detected by qPCR and Western blot, respectively, to verify the knockdown efficiency.

[0069] qPCR detection (mRNA level): Total RNA extraction: Total RNA was extracted from cells using TRIzol reagent, following the reagent instructions; Reverse transcription: 1 μg of total RNA was used to synthesize the first strand of cDNA using a reverse transcription kit. Reaction conditions: 25℃ for 10 minutes, 42℃ for 60 minutes, and 70℃ for 5 minutes; qPCR reaction: Real-time quantitative PCR was performed using SYBR Green premix. Reaction system: 2 μL cDNA template, 0.4 μM each of forward and reverse primers, 10 μL SYBR Green premix, and ddH2O to a final volume of 20 μL; Reaction program: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 10 seconds, 60℃ annealing / extension for 30 seconds, for a total of 40 cycles; Melting curve analysis: 95℃ for 15 seconds, 60℃ for 1 minute, and 95℃ for 15 seconds; PSMB3 primer sequence: Upstream 5'-TTCCGGCTGAACCTGTATGAG-3' (e.g., SEQ ID). The primer sequence for the internal reference gene GAPDH is as follows: upstream 5'-GGGAAATCGTGCGTGACATTAAG-3' (as shown in SEQ ID NO. 2), downstream 5'-GTAAGGGCCAAACCGTTTCTC-3' (as shown in SEQ ID NO. 3); downstream 5'-ATGTCCACGTCACACTTCATGAT-3' (as shown in SEQ ID NO. 5); using 2 -ΔΔCt The relative expression level of PSMB3 mRNA was calculated using this method.

[0070] Western blot analysis (protein levels): Protein extraction: Collect cells, add RIPA lysis buffer (containing protease inhibitor), lyse on ice for 30 minutes, centrifuge at 12,000×g for 15 minutes, and collect the supernatant; Protein quantification: Determine protein concentration using the BCA method; Electrophoresis: Take 20... Total protein was subjected to SDS-PAGE electrophoresis (12% separating gel, 5% stacking gel) at 80V for 30 minutes, then adjusted to 120V for approximately 60 minutes; Transfer: The separated protein bands were transferred to a PVDF membrane and transferred at a constant current of 250mA for 90 minutes; Blocking: Blocked with 5% skim milk powder (prepared with TBST) at room temperature for 1 hour; Primary antibody incubation: Rabbit anti-human PSMB3 antibody (1:1000 dilution) and mouse anti-human GAPDH antibody (1:5000 dilution) were added and incubated overnight at 4°C; Secondary antibody incubation: Washed 3 times with TBST (10 minutes each time), and HRP-labeled secondary antibody (1:5000 dilution) was added and incubated at room temperature for 1 hour; Detection: Washed 3 times with TBST (10 minutes each time), ECL chemiluminescent substrate was added, and images were acquired using a chemiluminescence imager.

[0071] (4) Cell proliferation capacity detection CCK-8 assay: MIA-PaCa2-shNC, MIA-PaCa2-shP, SMB3, PaTu8988t-shNC, and PaTu8988t-shPSMB3 cells in logarithmic growth phase were digested with trypsin to prepare single-cell suspensions. After cell counting, cells were seeded at a density of 3 × 10³ cells / well in 96-well plates, with 100 μL of culture medium per well and 5 replicates per group. The 96-well plates were incubated at 37°C in a 5% CO2 incubator for 0, 24, 48, 72, and 96 hours. At each time point, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 2 hours. The absorbance (OD) at 450 nm was measured using a microplate reader. 450 ); with incubation time as the x-axis, OD 450 The value is used as the ordinate to plot the cell growth curve.

[0072] Clonal formation assay: After digestion and counting of cells from each group, seed them into 6-well plates at a density of 500 cells / well, adding 2 mL of complete culture medium to each well, with 3 replicates per group; incubate the 6-well plates at 37°C with 5% CO2 for 10-14 days, changing the culture medium every 3 days; when visible cell clones appear, discard the culture medium, wash twice with PBS; add 4% paraformaldehyde and fix at room temperature for 15 minutes; discard the fixative, add 0.1% crystal violet staining solution and stain at room temperature for 15 minutes; slowly rinse with running water to remove excess staining solution, and air dry at room temperature; count the number of clones containing ≥50 cells under a microscope and record the results.

[0073] EdU assay: The EdU cell proliferation assay kit was used. Cells from each group were seeded at a density of 5 × 10³ cells / well in 96-well plates and cultured for 24 hours. EdU working solution with a final concentration of 10 μM was added to each well, and the cells were cultured for another 2 hours. The culture medium was discarded, and 4% paraformaldehyde was added for fixation for 15 minutes. The fixative was discarded, and 2% glycine was added for neutralization for 5 minutes. The cells were washed once with PBS, and 0.5% Triton X-100 was added for permeabilization for 10 minutes. The cells were washed once with PBS, and Apollo staining solution was added and incubated at room temperature in the dark for 30 minutes. The staining solution was discarded, and 0.5% Triton X-100 was added for washing twice, 10 minutes each time. The cells were washed once with PBS, and Hoechst 33342 nuclear staining solution was added and incubated at room temperature in the dark for 30 minutes. Images were acquired and analyzed using a fluorescence microscope or a high-content imaging system, and the EdU-positive cell rate (number of EdU-positive cells / total number of cells × 100%) was calculated.

[0074] (5) Experimental results See appendix Figure 6 The results of qPCR and Western blot assays showed (see...) Figure 6 (A) Compared with the shNC control group, the mRNA and protein expression levels of PSMB3 in MIA-PaCa2 and PaTu8988t cells of the shPSMB3 group were significantly reduced (inhibition rate >70%), indicating that the PSMB3 knockdown model was successfully constructed.

[0075] See appendix Figure 7 CCK-8 experimental results (see) Figure 7 As shown in Figure A), compared with the shNC control group, the proliferation activity of MIA-PaCa2-shPSMB3 and PaTu8988t-shPSMB3 cells was significantly reduced after 72 hours of culture, and the difference was statistically significant (P<0.01). The results of the clonogenic assay (see Figure A) are also presented. Figure 7 (See Figure B) The colony formation number in the MIA-PaCa2-shPSMB3 group was 42.3±8.7, while that in the shNC control group was 186.7±15.2; the colony formation number in the PaTu8988t-shPSMB3 group was 58.7±10.1, while that in the shNC control group was 201.3±18.6. PSMB3 knockdown significantly reduced cell colony formation ability (P<0.001). EdU experimental results (see...) Figure 7The results showed that the EdU-positive cell rate in the MIA-PaCa2-shPSMB3 group was 12.6%±2.8%, which was significantly lower than that in the shNC control group (38.4%±4.2%); the EdU-positive cell rate in the PaTu8988t-shPSMB3 group was 15.3%±3.1%, which was significantly lower than that in the shNC control group (41.2%±5.0%) (P<0.01).

[0076] Example 5: Effect of PSMB3 overexpression on PDAC cell proliferation (1) Construction and transduction of PSMB3 overexpression lentiviral vector The full-length CDS sequence of PSMB3 was amplified by PCR, and the amplification product was cloned into the pLVX-Puro lentiviral expression vector to construct the pLVX-PSMB3 overexpression plasmid. The sequence was verified to be correct by sequencing. A negative control plasmid was constructed using the empty pLVX-Puro vector. The above plasmid was packaged into lentivirus according to the lentiviral packaging and transduction method in Example 4. HPC-Y5 cells with low PSMB3 expression were taken and subjected to 1×10⁻⁶ cells. 5 Cells were seeded in 6-well plates and cultured for 24 hours. Then, pLVX-PSMB3 or pLVX-Puro control lentivirus solution (MOI = 10) and 8 μg / mL polyglobulin were added. After transduction for 24 hours, the medium was replaced with fresh medium. The cells were screened with puromycin (final concentration 2 μg / mL) for 7-10 days to obtain HPC-Y5-PSMB3 cells that stably overexpressed PSMB3 and the corresponding HPC-Y5-Vector control cells.

[0077] (2) Verification of PSMB3 overexpression efficiency Following the method in Example 4, qPCR and Western blot were used to detect the mRNA and protein expression levels of PSMB3.

[0078] (3) Cell proliferation capacity detection Following the method in Example 4, the proliferation capacity of HPC-Y5-Vector and HPC-Y5-PSMB3 cells was detected by CCK-8 assay and clonogenic assay, respectively.

[0079] (4) Experimental results See appendix Figure 6 The results of qPCR and Western blot assays showed (see...) Figure 6 (B) Compared with the Vector control group, the mRNA and protein expression levels of PSMB3 in HPC-Y5 cells of the PSMB3 overexpression group were significantly upregulated (overexpression fold > 5-fold), indicating that the PSMB3 overexpression model was successfully constructed. See Appendix Figure 7 CCK-8 experimental results (see) Figure 7 The results (D) showed that, compared with the Vector control group, the proliferation activity of HPC-Y5-PSMB3 cells was significantly increased after 72 hours of culture, and the difference was statistically significant (P<0.01). The results of the clonogenic assay (see...) Figure 7 The results showed that the number of clones formed in the HPC-Y5-PSMB3 group was 215.3±18.4, which was significantly higher than that in the Vector control group (118.7±12.3) (P<0.01).

[0080] Example 6: Effects of PSMB3 on PDAC cell cycle and apoptosis (1) Cell cycle detection (flow cytometry) Collect MIA-PaCa2-shNC, MIA-PaCa2-shPSMB3, PaTu8988t-shNC, and PaTu8988t-shPSMB3 cells in the logarithmic growth phase; wash the cells twice with PBS, digest them with trypsin, and collect them in 15 mL centrifuge tubes; centrifuge at 1,000×g for 5 minutes, discard the supernatant, resuspend the cells in pre-cooled PBS, and count them; take approximately 1×10⁻⁶ cells. 6 Centrifuge cells at 1,000×g for 5 minutes and discard the supernatant; slowly add 1 mL of pre-chilled 70% ethanol, gently pipette to mix, and fix at 4°C overnight (at least 12 hours); after fixation, centrifuge at 1,000×g for 5 minutes, discard the ethanol, and wash once with PBS; add 500 μL of PI / RNase staining solution (containing 50 μg / mL propidium iodide and 100 μg / mL RNase A), and incubate at room temperature in the dark for 30 minutes; use a flow cytometer (Beckman Coulter, CytoFLEX model) for detection, with excitation wavelength of 488 nm and emission wavelength of 575 nm; use ModFit software to analyze cell cycle distribution and calculate the cell ratios of G0 / G1, S, and G2 / M phases.

[0081] (2) Apoptosis detection (flow cytometry) Apoptosis was detected using Annexin V-FITC / PI double staining: Cells from each group were collected and washed twice with pre-cooled PBS; cells were resuspended in 1× Binding Buffer and the cell density was adjusted to approximately 1×10⁻⁶. 6 Cells / mL; take 100 μL of cell suspension (approximately 1 × 10⁶ cells / mL). 5Add 100 cells to a flow cytometry tube; add 5 μL Annexin V-FITC and 5 μL PI staining solution, and mix gently; incubate at room temperature in the dark for 15 minutes; add 400 μL 1× Binding Buffer to each tube and mix gently; use a flow cytometer (Beckman Coulter, CytoFLEX model) for detection, with an excitation wavelength of 488 nm, FITC detection channel FL1 (525 nm), and PI detection channel FL2 (575 nm); Result interpretation: Annexin V-FITC single positive (lower right quadrant) indicates early apoptotic cells, Annexin V-FITC and PI double positive (upper right quadrant) indicates late apoptotic cells, and the total apoptosis rate is the sum of the early apoptosis rate and the late apoptosis rate.

[0082] (3) Detection of apoptosis-related protein expression (Western blot) Following the Western blot method described in Example 4, the expression changes of apoptosis-related proteins Bax, Bcl2, and Cleaved Caspase-3 after PSMB3 knockdown were detected.

[0083] Antibodies used and their dilution ratios: Rabbit anti-human Bax antibody (dilution ratio 1:1000); Rabbit anti-human Bcl2 antibody (dilution ratio 1:1000); Rabbit anti-human Cleaved Caspase-3 antibody (dilution ratio 1:500); Mouse anti-human GAPDH antibody (dilution ratio 1:5000).

[0084] (4) Experimental results See Figure 8 Flow cytometry results showed ( Figure 8 China A Figure 8 (B) Compared with the shNC control group, the proportion of MIA-PaCa2-shPSMB3 cells in G0 / G1 phase increased from 48.6%±3.2% to 71.3%±4.5% (P<0.01), the proportion in S phase decreased from 34.2%±2.8% to 15.1%±2.1% (P<0.01), and the proportion in G2 / M phase decreased from 17.2%±1.9% to 13.6%±2.0% (P>0.05). PaTu8988t-shPSMB3 cells showed a similar trend: the proportion in G0 / G1 phase increased from 45.3%±3.5% to 68.7%±4.1% (P<0.01), and the proportion in S phase decreased from 36.8%±3.1% to 17.9%±2.5% (P<0.01). The above results indicate that PDAC cell cycle arrest was achieved in the G0 / G1 phase after PSMB3 knockdown.

[0085] See Figure 9 The results of apoptosis (see) Figure 9China A Figure 9 (B) Flow cytometry results showed that the total apoptosis rate of MIA-PaCa2-shPSMB3 cells was 24.6%±3.2%, significantly higher than that of the shNC control group (5.8%±1.2%) (P<0.001); the total apoptosis rate of PaTu8988t-shPSMB3 cells was 21.3%±2.9%, significantly higher than that of the shNC control group (6.2%±1.4%) (P<0.001). These results indicate that PSMB3 knockdown significantly induces apoptosis in PDAC cells. Apoptosis-related protein expression results (see...) Figure 9 (C) Western blot results showed that, compared with the shNC control group, after PSMB3 knockdown: the expression level of the pro-apoptotic protein Bax was significantly upregulated; the expression level of activated Cleaved Caspase-3 was significantly upregulated; and the expression level of the anti-apoptotic protein Bcl2 was significantly downregulated.

[0086] The above results further confirm that PSMB3 knockdown induces PDAC cell apoptosis through the mitochondrial apoptosis pathway (Bax / Bcl2-Caspase-3 pathway).

[0087] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. Application of reagents for detecting PSMB3 gene or protein expression levels in the preparation of products for diagnosing pancreatic ductal adenocarcinoma.

2. Application of reagents for detecting PSMB3 gene or protein expression levels in the preparation of products for assessing the prognosis of patients with pancreatic ductal adenocarcinoma.

3. The application according to claim 1 or 2, characterized in that, The reagents used to detect the expression level of the PSMB3 gene or protein are selected from primer pairs that specifically amplify the PSMB3 gene or antibodies that specifically recognize the PSMB3 protein.

4. The application according to claim 1 or 2, characterized in that, The products mentioned are qPCR kits, RT-qPCR kits, immunohistochemistry kits, Western blot kits, ELISA kits, in situ hybridization kits, RNA sequencing kits, or digital PCR kits.

5. Application of reagents that inhibit gene expression or PSMB3 protein activity in the preparation of drugs for the treatment of pancreatic ductal adenocarcinoma.

6. The application according to claim 5, characterized in that, The reagent used to inhibit PSMB3 gene expression or PSMB3 protein activity is selected from at least one of the following: shRNA, siRNA, antisense oligonucleotide, CRISPR interference system, PSMB3 protein degrader, PSMB3 small molecule inhibitor, and anti-PSMB3 antibody.

7. A method for screening key pathogenic genes of pancreatic ductal adenocarcinoma, characterized in that, Includes the following steps: (1) Obtain publicly available expression data of pancreatic ductal adenocarcinoma and normal pancreatic tissue, perform differential expression analysis, and obtain a differentially expressed gene set; (2) Obtain CRISPR gene dependence data of pancreatic cancer cell lines in DepMap and screen for the set of dependent genes that are crucial to the survival of pancreatic ductal adenocarcinoma cells. (3) Take the intersection of the differentially expressed gene set in step (1) and the dependent gene set in step (2) to obtain candidate key pathogenic genes; (4) The candidate key pathogenic genes in step (3) were verified by clinical samples and analyzed for prognosis to determine that PSMB3 is a key pathogenic gene of pancreatic ductal adenocarcinoma.

8. The method according to claim 7, characterized in that, In step (1), the publicly expressed data includes GEO datasets GSE43795 and / or GSE62452, as well as TCGA-PAAD and GTEx normal pancreatic tissue data; in step (2), the DepMap data is the Chronos score data of Public 25Q3 version; in step (4), the expression verification of clinical samples includes immunohistochemical staining and semi-quantitative scoring.

9. A kit for the diagnosis or prognostic assessment of pancreatic ductal adenocarcinoma, characterized in that, The kit includes: Specific primers and / or probes for detecting PSMB3 mRNA levels, or specific antibodies for detecting PSMB3 protein levels; Positive control and / or negative control; Internal reference test reagent.

10. The reagent kit according to claim 9, characterized in that, The histological types of the pancreatic ductal adenocarcinoma include tumor tissue, adjacent normal tissue, puncture tissue, cell sample, blood exosome sample, or circulating tumor cell sample.

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