ARHGAP42 marker for diagnosis and prognosis evaluation of pancreatic cancer and application of ARHGAP42 marker

Abstract

The invention relates to the technical field of biomedicine, and particularly discloses an ARHGAP42 marker for pancreatic cancer diagnosis and prognosis evaluation and application of the ARHGAP42 marker. Experiments prove that the ARHGAP42 is remarkably and highly expressed in pancreatic cancer tissues, and the expression level of the ARHGAP42 is remarkably related to clinical histological grading, lifetime and immunity of a patient. The invention provides application of a reagent for detecting the expression level of ARHGAP42 in preparation of a pancreatic cancer diagnosis or prognosis evaluation tool, including but not limited to qPCR primers, antibodies, ELISA kits or gene chips. In addition, the invention also covers the application of the drug for targeted inhibition of ARHGAP42 in treatment of pancreatic cancer.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medical diagnostics, specifically relating to the ARHGAP42 biomarker for the diagnosis and prognostic assessment of pancreatic cancer and its applications. Technical Background

[0002] Pancreatic cancer is a highly malignant digestive system tumor characterized by insidious onset, rapid progression, and extremely poor prognosis.

[0003] Current clinical status of pancreatic cancer

[0004] Early diagnosis of pancreatic cancer remains a major clinical challenge. Due to the deep location of the pancreas, early-stage pancreatic cancer often lacks obvious specific symptoms, and most patients are diagnosed at an advanced stage, missing the optimal window for surgical resection. Statistics show that only about 15%-20% of pancreatic cancer patients are eligible for surgical resection at the time of diagnosis, and even with radical resection, the 5-year survival rate remains below 20%. For patients with unresectable advanced pancreatic cancer, the prognosis is even more dire, with a median survival of only 6-10 months.

[0005] Limitations of existing diagnostic methods

[0006] Currently, the main methods used in clinical diagnosis of pancreatic cancer include imaging examinations and serological marker detection.

[0007] Imaging examinations, such as ultrasound, CT, and MRI, are valuable in detecting pancreatic space-occupying lesions. However, their sensitivity and specificity for diagnosing early-stage pancreatic cancer are relatively low, making it difficult to detect tiny tumors less than 1 cm in diameter. Furthermore, imaging examinations cannot accurately determine the benign or malignant nature of a tumor or its biological behavior.

[0008] Serological biomarker testing: Currently, commonly used serological biomarkers, such as carbohydrate antigen 19-9 (CA19-9), are the most widely used diagnostic biomarkers for pancreatic cancer. However, the specificity of CA19-9 is not ideal, and it can also be elevated in patients with other digestive system diseases such as cholangitis, cholecystitis, and pancreatitis. Furthermore, approximately 10% of pancreatic cancer patients are Lewis antigen negative and do not express CA19-9, making early diagnosis impossible through CA19-9 testing. In addition, CA19-9 has limited value in assessing the prognosis of pancreatic cancer and cannot accurately predict patient survival.

[0009] The Importance of Prognostic Assessment and the Limitations of Existing Methods

[0010] Accurate prognostic assessment is crucial for developing individualized treatment plans and determining the survival of pancreatic cancer patients. Currently, clinical prognosis is primarily assessed based on tumor stage, pathological grade, and the patient's overall health. However, these traditional methods have limitations. Tumor stage and pathological grade only reflect the morphological characteristics of the tumor at a specific point in time and cannot comprehensively reflect the tumor's biological behavior and individual patient differences. Therefore, there is an urgent clinical need to find more sensitive and specific biomarkers for the early diagnosis and prognostic assessment of pancreatic cancer.

[0011] Research progress on biomarkers in tumor diagnosis and prognostic assessment

[0012] In recent years, with the rapid development of molecular biology techniques, an increasing number of studies have focused on finding new biomarkers for tumor diagnosis and prognostic assessment. Biomarkers are substances that reflect the physiological and pathological states of an organism, such as nucleic acids, proteins, and metabolites. In the field of oncology, ideal biomarkers should possess high sensitivity, high specificity, and ease of detection, exhibiting abnormal changes in the early stages of tumor development and being closely related to tumor occurrence, development, metastasis, and prognosis. Currently, some biomarkers associated with pancreatic cancer have been discovered, such as carcinoembryonic antigen (CEA) and carbohydrate antigen 125 (CA125), but the clinical application value of these biomarkers still needs further improvement. Therefore, continuing to search for new and more effective pancreatic cancer biomarkers is a current hot topic and focus in the field of pancreatic cancer research.

[0013] ARHGAP42 (also known as GRAF3) is a member of the RhoGAP family, and previous studies have mainly focused on its role in vascular smooth muscle contraction (such as hypertension). Existing research has found that ARHGAP42 is aberrantly expressed in nasopharyngeal carcinoma and can promote cell migration and invasion through the PI3K / Akt signaling pathway. However, there are currently no reports on the expression characteristics and clinical significance of ARHGAP42 in pancreatic cancer. Therefore, this invention proposes the application of ARHGAP42 as a biomarker in the diagnosis and prognostic assessment of pancreatic cancer, aiming to address the problems of low sensitivity and poor specificity of existing diagnostic methods. Summary of the Invention

[0014] The purpose of this invention is to provide an ARHGAP42 biomarker for the diagnosis and prognostic assessment of pancreatic cancer and its application, so as to overcome the limitations of existing methods for the diagnosis and prognostic assessment of pancreatic cancer.

[0015] 1. This invention, through extensive experimental research, has discovered that ARHGAP42 is highly expressed in pancreatic cancer tissues and cells, and its expression level is closely related to the occurrence, development, and prognosis of pancreatic cancer. Based on this finding, this invention provides the following technical solution:

[0016] A biomarker for the diagnosis of pancreatic cancer: the biomarker is ARHGAP42. Detecting the expression level of ARHGAP42 in a sample can aid in the diagnosis of pancreatic cancer.

[0017] 2. Application of reagents for detecting ARHGAP42 expression levels in samples in the preparation of pancreatic cancer diagnostic kits: The samples include, but are not limited to, pancreatic cancer tissue, blood, serum, and plasma. The detection reagents can be primers and / or probes for detecting ARHGAP42 nucleic acid levels, or antibodies for detecting ARHGAP42 protein levels.

[0018] 3. Application of reagents for detecting ARHGAP42 expression levels in samples in the preparation of pancreatic cancer prognostic assessment kits: Similarly, samples include, but are not limited to, pancreatic cancer tissue, blood, serum, and plasma. Detection reagents include primers and / or probes for detecting ARHGAP42 nucleic acid levels, or antibodies for detecting ARHGAP42 protein levels.

[0019] 4. A method for diagnosing pancreatic cancer:

[0020] Obtain the sample to be tested, such as pancreatic cancer tissue, blood, serum, plasma, etc.

[0021] The expression level of ARHGAP42 in the sample was detected using appropriate methods, such as real-time quantitative PCR to detect nucleic acid levels, and Western blotting and enzyme-linked immunosorbent assay (ELISA) to detect protein levels.

[0022] The expression level of ARHGAP42 was compared with that of normal control samples. If the expression level of ARHGAP42 in the test sample was significantly higher than that in the normal control sample, it would suggest that the patient may have pancreatic cancer.

[0023] 5. A method for prognostic assessment of pancreatic cancer:

[0024] Obtain samples from pancreatic cancer patients, such as pancreatic cancer tissue, blood, serum, and plasma.

[0025] The expression level of ARHGAP42 in the sample was detected.

[0026] Patient prognosis is assessed based on ARHGAP42 expression levels; higher ARHGAP42 expression levels indicate a worse prognosis.

[0027] 6. A diagnostic and prognostic kit for pancreatic cancer: comprising reagents for detecting the expression level of ARHGAP42 in a sample, said reagents including primers and / or probes for detecting the nucleic acid level of ARHGAP42, or antibodies for detecting the protein level of ARHGAP42.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] This invention is the first to discover that ARHGAP42 can serve as a biomarker for the diagnosis and prognostic assessment of pancreatic cancer, providing a new indicator for the early diagnosis and prognosis of pancreatic cancer.

[0030] The diagnostic and prognostic assessment method provided by this invention is simple to operate, highly sensitive, and highly specific, which helps to improve the early diagnosis rate and prognostic accuracy of pancreatic cancer, and provides an important basis for the formulation of clinical treatment plans. Attached Figure Description

[0031] 1 Figure 1 ARHGAP42 is highly expressed in pancreatic cancer.

[0032] Figure 1 Validated by the TCGA-GTEx joint cohort, RNA-seq data analysis based on unpaired samples showed that the expression level of ARHGAP42 in pancreatic cancer tissues was significantly higher than that in normal control samples from the GTEx database. Figure 1 Analysis based on the GEO database (GSE16515) showed that the expression level of ARHGAP42 in pancreatic cancer tissues was significantly higher than that in normal control tissues (P<0.05), suggesting that it may have a potential pro-cancer effect in pancreatic cancer.

[0033] 2 Figure 2 The correlation between ARHGAP42 expression level and prognosis in pancreatic cancer patients.

[0034] Figure 2 AC Kaplan-Meier survival analysis showed that patients in the high ARHGAP42 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 high ARHGAP42 expression may be an independent risk factor for poor prognosis in pancreatic cancer patients.

[0035] 3 Figure 3 Association between ARHGAP42 expression and clinicopathological features in pancreatic cancer patients.

[0036] Figure 3AC plots were analyzed to determine their association with histological grade (A: G1-G4), patient age (<=60 vs>60), and overall survival events (F: survival vs. death). The results suggest that ARHGAP42 is associated with disease progression in pancreatic cancer patients.

[0037] 4 Figure 4 Diagnostic value assessment of ARHGAP42 gene expression in pancreatic cancer patients

[0038] Figure 4 This chart shows the differential diagnostic efficacy of ARHGAP42 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 ARHGAP42 (solid blue line) shows an area under the curve (AUC) of 0.961 (95% CI: 0.938–0.984), 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.

[0039] 5 Figure 5 Correlation between ARHGAP42 gene expression and tumor immune checkpoints in pancreatic cancer

[0040] Figure 5 A–H demonstrates the association between ARHGAP42 and six immune checkpoint-related genes (CD47, CD40, CD276, CD274, HAVCR2, CTLA4, PVR, and CD80) in pancreatic cancer samples.

[0041] 6 Figure 6 Assess the expression of ARHGAP42 in clinical tissue samples

[0042] Figure 6 Image A is a representative IHC image of normal pancreatic tissue (from a 74-year-old female), showing weak ARHGAP42 staining (light blue), indicating low expression levels. The tissue structure is homogeneous. Figure 6 Image B is a representative IHC image of PAAD tissue (from a 65-year-old male), showing strong ARHGAP42 staining (dark brown), indicating high expression levels. The tissue morphology is disordered, exhibiting malignant characteristics. Figure 6 C is a bar chart quantifying the relative changes in ARHGAP42 expression. The results show that ARHGAP42 expression is significantly upregulated in pancreatic cancer (PAAD) compared to normal tissues.

[0043] 7 Figure 7 Effects of ARHGAP42 gene knockdown on the proliferation and invasion of PAAD cells (ASPC-1)

[0044] Figure 7 The results showed that the proliferation rate of PAAD cells (ASPC-1) slowed down as the expression of ARHGAP42 was inhibited. Detailed Implementation

[0045] 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.

[0046] Example 1: Validation of differential expression of ARHGAP42 in pancreatic cancer and normal tissues. Data source: GEO, TCGA, GTEx databases.

[0047] 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 6 patients who underwent pancreatic cancer surgery. All samples were approved by the ethics committee and the patients gave informed consent.

[0048] Immunohistochemical detection: The paraffin sections of the tissue were dewaxed to water, and after antigen retrieval, anti-ARHGAP42 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.

[0049] Results: Analysis of the GEO database (GSE16515) and the TCGA-GTEx cohort showed that the mRNA expression level of ARHGAP42 in pancreatic cancer tissues was significantly higher than that in normal pancreatic tissues (adjacent to cancer tissues). Figure 1 AB).

[0050] Immunohistochemical results showed that ARHGAP42 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 ARHGAP42 in pancreatic cancer tissue was 2.5 times that in normal tissue (P<0.001). Figure 6 C).

[0051] Example 2: Correlation analysis of ARHGAP42 expression and prognosis in pancreatic cancer patients

[0052] Data source: TCGA - Clinical Data Information

[0053] 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.

[0054] result:

[0055] Kaplan-Meier survival analysis showed that patients in the high ARHGAP42 expression group had significantly shorter overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFI) than those in the low expression group (log-rank test), suggesting that high ARHGAP42 expression may be an independent risk factor for poor prognosis in pancreatic cancer patients. Figure 2 AC).

[0056] Example 3: Association between ARHGAP42 expression and clinicopathological features

[0057] Data source: TCGA - Clinical data information (historical grade (G1-G4), patient age (<=60 vs>60), and overall survival events (F: survival vs. death), etc., clinicopathological features).

[0058] Statistics: Overall test (Kruskal-Wallis Test) + Multiple hypothesis test (Dunn's test).

[0059] result:

[0060] In pancreatic cancer patients, ARHGAP42 expression was significantly associated with histological grade (E: G1-G3), patient age (<=60 vs >60), and overall survival events (F: survival vs death). These results suggest that ARHGAP42 is associated with disease progression in pancreatic cancer patients. Figure 3 AC.

[0061] Example 4: Evaluation of the diagnostic efficacy of ARHGAP42 in pancreatic cancer

[0062] Data source: TCGA - Clinical Data Information

[0063] 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.

[0064] result:

[0065] The ROC curve of ARHGAP42 showed an AUC of 0.961 (95% CI: 0.938-0.984), indicating that ARHGAP42 has excellent ability to differentiate pancreatic cancer. Figure 4 ).

[0066] Example 5: Correlation between ARHGAP42 and immune checkpoint genes

[0067] Data source: TCGA database

[0068] Correlation analysis: The correlation coefficient (R) and p-value between genes were calculated using the Spearman rank correlation test.

[0069] result:

[0070] ARHGAP42 was significantly positively correlated with CD47 (R = 0.586, P < 0.001), CD40 (R = 0.283, P < 0.001), CD276 (R = 0.432, P < 0.001), CD274 (R = 0.477, P < 0.001), HAVCR2 (R = 0.354, P < 0.001), CTLA4 (R = 0.206, P < 0.001), PVR (R = 0.343, P < 0.001), and CD80 (R = 0.373, P < 0.001). Figure 5 The presence of AH suggests that it may be involved in the regulation of the immune microenvironment in pancreatic cancer.

[0071] The study evaluated the effects of ARHGAP42 gene knockdown on the proliferation and invasion of PAAD cells (ASPC-1).

[0072] Materials source: The relevant cell culture medium, cells, siRNA, and CCK8 reagent can all be purchased from biotechnology companies.

[0073] Statistical analysis: Student's T test (two-tailed test) and analysis of variance (ANOVA) were used for statistical comparisons between groups.

[0074] result:

[0075] like Figure 7 As shown, the proliferation rate of PAAD cells (ASPC-1) with suppressed ARHGAP42 expression was slowed compared to the control group.

[0076] Example 7: Preparation of ARHGAP42 Detection Kit

[0077] Prepare a diagnostic kit containing the following components:

[0078] Anti-ARHGAP42 monoclonal antibody (1:500 dilution);

[0079] Secondary antibody-HRP complex;

[0080] DAB colorimetric solution;

[0081] Harris hematoxylin counterstaining solution;

[0082] Positive control section (pancreatic cancer tissue with high ARHGAP42 expression);

[0083] Negative control section (normal pancreatic tissue);

[0084] Instruction manual.

[0085] 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.

[0086] 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 ARHGAP42 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 ARHGAP42 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_001367945.1; Its expression product is the ARHGAP42 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_001354874.

1.

2. A kit for the diagnosis or prognostic assessment of pancreatic cancer, characterized in that, Includes reagents for the specific detection of the ARHGAP42 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 ARHGAP42 gene are selected from ARHGAP42-specific primer pairs, probes, or nucleic acid chips. The sequences of the primer pairs are as follows: Upstream primer (SEQ ID NO:1): 5'-TGCTAACCAGGACTACAGACCACC-3'; Downstream primer (SEQ ID NO:2): 5'-CATTCATTTTCCCACTGGATTTCA-3' Alternatively, reagents for detecting ARHGAP42 protein levels may be selected from anti-ARHGAP42 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 ARHGAP42 protein, and the negative control is a buffer that does not contain ARHGAP42 protein.

4. The reagent kit according to claim 2, characterized in that, It also includes a standard curve or threshold reference for interpreting the results. The threshold is determined by statistical analysis of a large number of samples from pancreatic cancer patients and normal controls. When the detected ARHGAP42 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 ARHGAP42 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 ARHGAP42 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 ARHGAP42 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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