Application of PPP1R3B as biomarker in preparation of pancreatic cancer diagnosis and prognosis preparation

Abstract

The invention is applicable to the technical field of tumor medicine, and particularly relates to application of PPP1R3B as a biomarker in preparation of a pancreatic cancer diagnosis and prognosis preparation. According to the invention, the specific high expression of PPP1R3B in pancreatic cancer tissues is found for the first time through transcriptome sequencing, and the expression level of PPP1R3B is significantly related to tumor staging, immune cell infiltration, immune checkpoints and poor prognosis of patients. Based on the discovery, the invention provides a diagnosis and prognosis evaluation kit containing a PPP1R3B detection reagent, and the kit can be used for early diagnosis, disease monitoring and prognosis judgment of pancreatic cancer. Compared with the prior art, the technical scheme provided by the invention has the advantages of high detection sensitivity, strong specificity, simplicity and convenience in operation, low cost and the like, and a new tool is provided for accurate diagnosis and treatment of pancreatic cancer.

Description

Technical Field

[0001] This invention belongs to the field of oncology medical technology, specifically relating to the application of PPP1R3B as a biomarker in the preparation of diagnostic and prognostic agents for pancreatic cancer. Technical Background

[0002] Pancreatic cancer is a highly malignant digestive system tumor with an increasing incidence rate, approximately 12.9 per 100,000 annually. Its 5-year survival rate is only about 7%, making it one of the malignant tumors with the worst prognosis. Early symptoms of pancreatic cancer are often subtle and lack specific clinical manifestations; approximately 80% of patients are diagnosed at an advanced stage, losing the opportunity for radical surgical treatment. Therefore, identifying biomarkers for early diagnosis and prognostic assessment is crucial for improving the prognosis of pancreatic cancer patients.

[0003] Currently, CA19-9 (carbohydrate antigen 19-9) is the commonly used diagnostic marker for pancreatic cancer in clinical practice. However, it has significant limitations: approximately 10% of pancreatic cancer patients do not express CA19-9 due to Lewis antigen negativity, leading to false negatives; at the same time, CA19-9 can also be elevated in benign diseases such as biliary tract diseases and pancreatitis, resulting in insufficient specificity (approximately 70-80%); furthermore, the diagnostic sensitivity of CA19-9 for early-stage pancreatic cancer (stage I-II) is only 40-60%, which is insufficient to meet clinical needs.

[0004] PPP1R3B (Protein phosphatase 1 regulatory subunit 3B) is the regulatory subunit of protein phosphatase 1, primarily involved in the regulation of glycogen metabolism and cell proliferation. Existing studies have found that PPP1R3B is aberrantly expressed in tumors such as gastric and ovarian cancer, possibly related to the reprogramming of energy metabolism in tumor cells. However, no research has yet reported on the expression characteristics and clinical significance of PPP1R3B in pancreatic cancer.

[0005] Therefore, this invention proposes the application of PPP1R3B as a biomarker in the diagnosis and prognostic assessment of pancreatic cancer, aiming to solve the problems of low sensitivity and poor specificity of existing diagnostic methods. Summary of the Invention

[0006] The purpose of this invention is to provide the application of PPP1R3B as a biomarker in the diagnosis and prognostic assessment of pancreatic cancer, so as to make up for the shortcomings of the prior art.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] The application of the PPP1R3B gene or its expression product as a biomarker in the preparation of diagnostic reagents for pancreatic cancer, wherein the nucleotide sequence of the PPP1R3B gene and the amino acid sequence of its expression product are disclosed.

[0009] The application of the PPP1R3B gene or its expression product as a biomarker in the preparation of a prognostic assessment reagent for pancreatic cancer, wherein the nucleotide sequence of the PPP1R3B gene and the amino acid sequence of its expression product are described.

[0010] 3. A pancreatic cancer diagnostic kit comprising a PPP1R3B specific detection reagent. Further, the detection reagent is an anti-PPP1R3B antibody (for detecting protein levels) or a PPP1R3B specific primer pair (for detecting mRNA levels).

[0011] 4. A prognostic assessment kit for pancreatic cancer, comprising a PPP1R3B specific detection reagent. Further, the detection reagent is an anti-PPP1R3B antibody or a PPP1R3B specific primer pair.

[0012] Compared with the prior art, the beneficial effects of the present invention are:

[0013] This study is the first to discover that PPP1R3B is specifically highly expressed in pancreatic cancer tissues, and its expression level is significantly correlated with tumor stage, immune cell infiltration, immune checkpoints, and poor patient prognosis, providing a new biomarker for the diagnosis and prognostic assessment of pancreatic cancer. The PPP1R3B-based detection kit is easy to operate (it can be implemented through conventional methods such as qPCR and immunohistochemistry), has a short detection cycle, and is inexpensive, making it suitable for clinical application. Compared with existing technologies, the technical solution of this invention has the advantages of simple detection, low cost, and reliable results, providing a new tool for the precision diagnosis and treatment of pancreatic cancer. Attached Figure Description

[0014] 1 Figure 1 The expression patterns of PPP1R3B in multiple independent datasets are shown.

[0015] Figure 1 Analysis of AC data based on the GEO database (GSE62165, GSE16515, GSE15471) showed that the expression level of PPP1R3B in pancreatic cancer tissues was significantly higher than that in normal control tissues (P<0.05).

[0016] Figure 1 Further validation using the TCGA-GTEx joint cohort and RNA-seq data analysis based on unpaired samples showed that the expression level of this gene in pancreatic cancer tissues was significantly higher than that in normal control samples from the GTEx database, suggesting that it may have a potential pro-cancer effect in pancreatic cancer.

[0017] 2 Figure 2 This study demonstrated the correlation between PPP1R3B expression levels and prognosis in pancreatic cancer patients.

[0018] Figure 2 AC Kaplan-Meier survival analysis showed that patients in the PPP1R3B high expression group had significantly shorter overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) than those in the low expression group (log-rank test), suggesting that PPP1R3B high expression may be an independent risk factor for poor prognosis in pancreatic cancer patients.

[0019] 3 Figure 3 This study demonstrates the association between PPP1R3B expression and clinicopathological features in pancreatic cancer patients.

[0020] Figure 3 The AF plots were analyzed for their association with pathological N stage (A: N0 vs N1), pathological stage (B: I-IV), residual tumor (C: R0 vs R1 & R2), initial treatment outcome (D: disease progression (PD), stable disease (SD), partial remission & complete remission (PR & CR), histological grade (E: G1-G4), and overall survival events (F: survival vs death). The results suggest that PPP1R3B is associated with disease progression in pancreatic cancer patients.

[0021] 4 Figure 4 This study demonstrates the diagnostic value of PPP1R3B gene expression in pancreatic cancer patients.

[0022] Figure 4 This chart shows the differential diagnostic efficacy of PPP1R3B expression levels in pancreatic cancer. The horizontal axis represents 1-specificity (false positive rate), and the vertical axis represents sensitivity (true positive rate). The ROC curve of PPP1R3B (solid blue line) shows an area under the curve (AUC) of 0.956 (95% CI: 0.931–0.981), significantly higher than the random reference line (dashed line, AUC = 0.5), indicating that this biomarker has excellent diagnostic discrimination ability. The narrow 95% confidence interval of the AUC value further validates the reliability of this result.

[0023] 5 Figure 5 This demonstrates the correlation between PPP1R3B gene expression and tumor immune checkpoints in pancreatic cancer.

[0024] Figure 5 A–F demonstrates the association between PPP1R3B and six immune checkpoint-related genes (CD274 / PD-L1, CD276 / B7-H3, CD40, CD47, CD80 / B7-1, and PVR / CD155) in pancreatic cancer samples.

[0025] 6 Figure 6 Immunohistochemistry of PPP1R3B in clinical tissue samples was demonstrated.

[0026] Figure 6 Image A is a representative IHC image of normal pancreatic tissue (35-year-old female), showing weak PPP1R3B staining (light brown), indicating low expression levels. The tissue structure is homogeneous. Figure 6 Image B is a representative IHC image of PAAD tissue (from a 63-year-old male), showing strong PPP1R3B staining (dark brown), indicating a high expression level. The tissue morphology is disordered, exhibiting malignant characteristics. Figure 6 C is a bar chart quantifying the relative changes in PPP1R3B expression. The results show that PPP1R3B expression is significantly upregulated in pancreatic adenocarcinoma (PAAD) compared to normal tissue. Detailed Implementation

[0027] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the specific implementation of the invention is described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0028] Example 1: Validation of differential expression of PPP1R3B in pancreatic cancer and normal tissues (corresponding to...) Figure 1 , Figure 6 )

[0029] Objective: To verify the differential expression of PPP1R3B in pancreatic cancer tissue and normal pancreatic tissue, and to provide experimental evidence for its use as a diagnostic biomarker.

[0030] Data sources: GEO, TCGA, GTEx databases

[0031] Sample source: Tumor tissue and corresponding adjacent normal pancreatic tissue (>5cm from the tumor margin, pathologically confirmed to be free of cancer cell infiltration) were collected from 5 patients who underwent pancreatic cancer surgery. All samples were approved by the ethics committee and the patients gave informed consent.

[0032] Immunohistochemical detection: The paraffin sections of the tissue were dewaxed to water, and after antigen retrieval, anti-PPP1R3B monoclonal antibody (1:500 dilution) was added and incubated overnight at 4°C; the next day, secondary antibody-HRP complex was added, DAB staining was performed, hematoxylin was counterstained, and the tissue was observed under a microscope.

[0033] Results: Analysis of the GEO database (GSE62165, GSE16515, GSE15471) and the TCGA-GTEx cohort showed that the mRNA expression level of PPP1R3B in pancreatic cancer tissues was significantly higher than that in normal pancreatic tissues (adjacent to cancer tissues). Figure 1 AD).

[0034] Immunohistochemical results showed that PPP1R3B staining was weak in normal pancreatic tissue, while it was significantly enhanced in pancreatic cancer tissue, with disordered tissue morphology. Figure 6 Quantitative analysis showed that the relative expression level of PPP1R3B in pancreatic cancer tissue was 5.2 times that in normal tissue (P<0.001). Figure 6 C).

[0035] Example 2: Correlation analysis of PPP1R3B expression and prognosis of pancreatic cancer patients (corresponding to...) Figure 2 )

[0036] Objective: To analyze the association between PPP1R3B expression level and survival in pancreatic cancer patients and to verify its prognostic value.

[0037] Data source: TCGA - Clinical Data Information

[0038] Survival analysis: Kaplan-Meier method was used to plot survival curves, log-rank test was used to compare differences between groups, and Cox regression model was used to analyze independent prognostic factors.

[0039] result:

[0040] Kaplan-Meier survival analysis showed that patients with high PPP1R3B expression had significantly shorter overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFI) than those with low expression (log-rank test), suggesting that high PPP1R3B expression may be an independent risk factor for poor prognosis in pancreatic cancer patients. Figure 2 AC).

[0041] Example 3: Association between PPP1R3B expression and clinicopathological features (corresponding to) Figure 3 )

[0042] Objective: To investigate the relationship between PPP1R3B expression level and clinical features of pancreatic cancer, such as pathological stage and lymph node metastasis.

[0043] Data source: TCGA - Clinical Data Information (records pathological N stage (N0 / N1), pathological stage (I-IV), residual tumor (R0 / R1 / R2), histological grade (G1-G4) and other clinicopathological features).

[0044] Statistics: Overall test (Kruskal-Wallis Test) + Multiple hypothesis test (Dunn's test).

[0045] result:

[0046] In pancreatic cancer patients, PPP1R3B expression was significantly associated with pathological N stage (A: N0 vs N1), pathological stage (B: I-II), residual tumor (C: R0 vs R1 & R2), initial treatment outcome (D: disease progression PD vs partial response & complete response PR & CR), histological grade (E: G1-G3), and overall survival events (F: survival vs death). These results suggest that PPP1R3B is associated with disease progression in pancreatic cancer patients. Figure 4 AF).

[0047] Example 4: Evaluation of the diagnostic efficacy of PPP1R3B for pancreatic cancer (corresponding to...) Figure 4 )

[0048] Objective: To evaluate the sensitivity and specificity of PPP1R3B as a diagnostic biomarker for pancreatic cancer using ROC curve analysis.

[0049] Data source: TCGA - Clinical Data Information

[0050] Statistical analysis: ROC curves were plotted using SPSS software, and the area under the curve (AUC), sensitivity (true positive rate), specificity (1-false positive rate), and 95% confidence interval (CI) were calculated.

[0051] result:

[0052] The ROC curve of PPP1R3B showed an AUC of 0.956 (95% CI: 0.931-0.981), indicating that PPP1R3B has excellent ability to differentiate pancreatic cancer. Figure 4 ).

[0053] Example 5: Correlation between PPP1R3B and immune checkpoint genes (corresponding to) Figure 5 )

[0054] Objective: To analyze the association between PPP1R3B expression and immune checkpoint genes, and to explore its potential role in tumor immunity.

[0055] Data source: TCGA database

[0056] Correlation analysis: The correlation coefficient (R) and p-value between genes were calculated using the Spearman rank correlation test.

[0057] result:

[0058] PPP1R3B showed significant positive correlations with CD274 (R = 0.447, P < 0.001), CD276 (R = 0.383, P < 0.001), CD40 (R = 0.275, P < 0.001), CD47 (R = 0.422, P < 0.001), CD80 (R = 0.396, P < 0.001), and PVR (R = 0.365, P < 0.001). Figure 5 AF) suggests that it may be involved in the regulation of the immune microenvironment in pancreatic cancer.

[0059] Example 6: Preparation of PPP1R3B Detection Kit

[0060] Prepare a diagnostic kit containing the following components:

[0061] Anti-PPP1R3B monoclonal antibody (1:500 dilution);

[0062] Secondary antibody-HRP complex;

[0063] DAB colorimetric solution;

[0064] Harris hematoxylin counterstaining solution;

[0065] Positive control section (pancreatic cancer tissue with high PPP1R3B expression);

[0066] Negative control section (normal pancreatic tissue);

[0067] Instruction manual.

[0068] The conventional operations in the operation steps of this invention are well known to those skilled in the art and will not be described in detail here.

[0069] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These modifications and improvements should also be considered within the scope of protection of the present invention and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. The application of the PPP1R3B gene or its expression product as a biomarker in the preparation of reagents for the diagnosis or prognostic assessment of pancreatic cancer, characterized in that: The diagnosis includes early diagnosis of pancreatic cancer, monitoring of disease progression, or differential diagnosis from benign pancreatic diseases; The prognostic assessment includes prediction of overall survival, disease-specific survival, progression-free interval, or risk assessment of disease progression in pancreatic cancer patients. The PPP1R3B gene includes variants (such as natural variants or alternative splice variants) with the same function, whose nucleotide sequence contains a sequence that is at least 90% homologous to and functionally equivalent to NM_001201329.2; Its expression product is the PPP1R3B protein, including its functionally equivalent variants, whose amino acid sequence contains a sequence that is at least 90% homologous to and functionally equivalent to NP_001188258.

1.

2. A kit for the diagnosis or prognostic assessment of pancreatic cancer, characterized in that, Includes reagents for the specific detection of the PPP1R3B gene or its expression product: The kit is used for early diagnosis of pancreatic cancer, monitoring of disease progression, differential diagnosis from benign pancreatic diseases, prediction of overall survival, prediction of disease-specific survival, prediction of progression-free interval, or assessment of disease progression risk. The detection reagents include: The reagents used to detect the mRNA level of the PPP1R3B gene are selected from PPP1R3B-specific primer pairs, probes, or nucleic acid chips, and the sequences of the primer pairs are as follows: Upstream primer (SEQ ID NO:1): 5'-TTCGATGACCCGCTAGATATGC-3' Downstream primer (SEQ ID NO:2): 5'-CGGCCTGAAGTCGATTTCTAAA-3' Alternatively, reagents for detecting PPP1R3B protein levels may be selected from anti-PPP1R3B monoclonal antibodies, polyclonal antibodies, or antibody chips.

3. The reagent kit according to claim 2, characterized in that, It also includes a detection buffer, a positive control, a negative control, and an instruction manual; the positive control is a solution containing a known concentration of recombinant PPP1R3B protein, and the negative control is a buffer that does not contain PPP1R3B protein.

4. The reagent kit according to claim 2, characterized in that, It also includes a standard curve or threshold reference for result interpretation. The threshold is determined by statistical analysis of a large number of samples from pancreatic cancer patients and normal controls. When the detected PPP1R3B expression level is higher than the threshold, it indicates an increased risk of poor prognosis for the patient.

5. The application of a detection method based on the PPP1R3B gene or its expression product in the diagnosis or prognostic assessment of pancreatic cancer, characterized in that, The method includes: The mRNA level of the PPP1R3B gene in the sample was specifically detected using qPCR, RT-PCR, or nucleic acid hybridization techniques. Alternatively, immunohistochemistry, ELISA, or Western blot techniques can be used to specifically detect the expression level of PPP1R3B protein in the sample; The specificity of the detection is achieved by the primer pairs, antibodies, or probes described in claim 2.

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