Application of epidermal regulatory protein

Abstract

The invention relates to the technical field of biological medicine, in particular to application of epidermal regulatory protein, and provides application of a reagent for detecting expression of the epidermal regulatory protein in preparation of a product for detecting pancreatic ductal adenocarcinoma and application of a reagent for silencing the epidermal regulatory protein in preparation of a product for treating the pancreatic ductal adenocarcinoma. The invention finds that the expression of the EREG in the PDAC is up-regulated and is related to the prognosis of a PDAC patient, and the silent EREG can effectively inhibit the in-vivo and in-vitro cell proliferation and progress of the PDAC, so that the application of the epidermal regulatory protein is provided.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, and particularly to the application of epiregulin. Background Art

[0002] Pancreatic cancer is the seventh leading cause of cancer death globally. Among them, pancreatic ductal adenocarcinoma, abbreviated as PDAC, is the most important pathological type. It is generally believed that PDAC is highly invasive and has extremely poor prognosis. Finding effective treatment and prognostic markers is crucial for improving the prognosis of PDAC.

[0003] Epiregulin is abbreviated as EREG, and epidermal growth factor is abbreviated as EGF. EREG is a member of the EGF family, mainly binding to the epidermal growth factor receptor family of EGFR / ErbB1 and ErbB4 receptors, inducing receptor tyrosine phosphorylation, and participating in intracellular signal transduction. The human EREG gene is located on chromosome 4q13.3, where the epidermal growth factor receptor is abbreviated as ErbB. Like other members of the EGF family, the EREG protein is initially expressed in a transmembrane form, and the mature soluble form contains the extracellular region of the former form. EREG includes an N-terminal signal peptide, a propeptide region, a short proximal membrane stalk of EGF-like, a hydrophobic transmembrane domain, and a cytoplasmic domain. In addition, EREG also contains an additional heparin-binding domain. Under physiological conditions, the expression of EREG in normal tissues is extremely low. It mainly participates in many biological processes, including cell regeneration, skin inflammation, and wound healing. Emerging research in recent years has clarified the differential expression of EREG in various tumor types, highlighting its close relationship with prognosis. In 2000, a study found that EREG was significantly upregulated in some pancreatic cancer cells and could promote cell growth in vitro. In addition, in cholangiocarcinoma, there is a strong correlation between the elevated expression level of EREG and the rapid progression of tumors and poor tumor prognosis. However, the expression of EREG in clinical PDAC tissues and the relationship between EREG and the progression and prognosis of PDAC are still unclear. In addition, through bioinformatics analysis, researchers found that EREG plays an important role in the immune response and cell activation of lung adenocarcinoma. It was also found to be co-expressed with programmed death ligand 1, indicating its potential as a promising biomarker for immunotherapy response, where programmed death ligand 1 is abbreviated as PD-L1. However, the biological role, clinical significance of EREG in PDAC, and its relationship with immunotherapy response are still unclear.

[0004] Tumorigenesis is a complex process involving multiple genes and multiple steps. Among them, mitogen-activated protein kinase, abbreviated as MAPK, and the MAPK signaling pathway are closely related to tumorigenesis. The MAPK signaling pathway is a complex network system that plays a crucial role in cell proliferation, apoptosis, invasion, metastasis, and angiogenesis. In recent years, a large number of studies have confirmed that the MAPK pathway is involved in the process of tumorigenesis and progression. Studies have found that the activated MAPK-Nrf2-GCLC pathway is involved in regulating the level of cellular reactive oxygen species by myosin heavy chain 9, promoting cell invasion and radiation resistance in head and neck tumors, where myosin heavy chain 9 is abbreviated as MHC9 and cellular reactive oxygen species is abbreviated as ROS. Another study on glioblastoma showed that EREG promotes tumorigenicity by activating the ERK / MAPK pathway. However, the role of EREG in PDAC tumorigenesis is currently unclear, and its use cannot be determined. Summary of the Invention

[0005] To solve the above problems, the present invention provides the application of epiregulin.

[0006] Any of the following applications:

[0007] The application of a reagent for detecting the expression of epiregulin in the preparation of a product for detecting pancreatic ductal adenocarcinoma;

[0008] The application of a reagent for silencing epiregulin in the preparation of a product for treating pancreatic ductal adenocarcinoma;

[0009] Epiregulin is abbreviated as EREG, and the accession number of EREG in NCBI is NM_001432.3.

[0010] Preferably, the product for detecting pancreatic ductal adenocarcinoma includes a reagent for detecting epiregulin.

[0011] Preferably, the reagent for detecting epiregulin includes a reagent for detecting the expression level of epiregulin in a sample by sequencing technology, probe hybridization technology, gene chip technology, or fluorescence quantitative PCR technology.

[0012] Preferably, the reagent for detecting epiregulin is an amplification primer for epiregulin, and the amplification primer is shown as SEQ ID NO.4-5.

[0013] Preferably, the product for detecting pancreatic ductal adenocarcinoma is a kit, and the kit includes a reagent for detecting the expression level of epiregulin in a test sample.

[0014] Preferably, the test sample is from a patient with pancreatic ductal adenocarcinoma, a suspected patient with pancreatic ductal adenocarcinoma, a susceptible population of pancreatic ductal adenocarcinoma, a high-risk population of pancreatic ductal adenocarcinoma, or a healthy person.

[0015] Preferably, the sample to be tested is tissue.

[0016] Preferably, the product for treating pancreatic ductal adenocarcinoma comprises an agent for silencing epiregulin.

[0017] Preferably, the agent for silencing epiregulin comprises shRNA, and the sequence of the shRNA is any one of the sequences shown in SEQ ID NO.1-3.

[0018] Preferably, the agent for silencing epiregulin further comprises a lentiviral vector.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The present invention discovers that EREG is up-regulated in PDAC and is related to the prognosis of PDAC patients. Silencing EREG can effectively inhibit the in vitro and in vivo cell proliferation and progression of PDAC. Therefore, the present invention proposes the application of an agent for detecting the expression of epiregulin in the preparation of a product for detecting pancreatic ductal adenocarcinoma, and the application of an agent for silencing epiregulin in the preparation of a product for treating pancreatic ductal adenocarcinoma.

[0021] In addition, the ERK1 / 2 agonist senkyunolide I effectively reverses the reduction in cell proliferation, migration and invasion induced by EREG silencing. EREG mediates PDAC tumorigenesis in a manner dependent on the ERK1 / 2 and p38 MAPK signaling pathways. Finally, the present invention discovers a significant association between EREG and PD-L1. The research results of the present invention further understand the pathogenesis of PDAC and provide a promising idea for the diagnosis of pancreatic cancer and the development of new drugs.

[0022] The present invention discovers that EREG is highly expressed in PDAC and can be used as an independent prognostic indicator. This confirms the anti-tumor activity of EREG blockers and emphasizes the important role of the ERK / p38MAPK signaling pathway in PDAC tumorigenesis, and clarifies the relationship between EREG and immune therapy response. Brief Description of the Drawings

[0023] Figure 1To show the expression of EREG in PDAC and its relationship with clinicopathological features; among them, A is the comparison of EREG expression, A(1) is the expression of EREG in the TCGA database, A(2) is the expression of EREG in the TCGA+GTEx database; B is the EREG mRNA expression between PDAC tissues and ANCT in 62 clinical samples, C is the EREG protein expression between PDAC tissues and ANCT in clinical samples (n = 8), C(1) is the Western Blot band diagram of EREG protein expression, C(2) is the statistical chart of Western Blot results, D shows the representative image of EREG immunohistochemical staining, E is the related results of EREG in 30 PDAC tissues, E(1) is the positive area, E(2) is the area density, E(3) is the proportion of the positive rate, F shows the relationship of EREG expression, F(1) is the relationship between EREG expression and lymph node metastasis, F(2) is the relationship between EREG expression and microvascular invasion, F(3) is the relationship between EREG expression and tissue grade, **p<0.01, ***p<0.001.

[0024] Figure 2 To show that high expression of EREG is an independent prognostic factor for poor prognosis in PDAC patients; among them, A is the survival analysis, A(1) is the survival analysis of EREG expression and overall survival (OS) in the TCGA database, A(2) is the survival analysis of EREG expression and recurrence-free survival (RFS) in the TCGA database, B is the survival analysis, B(1) is the survival analysis of EREG expression and OS in the TCGA+GTEx database, B(2) is the survival analysis of EREG expression and RFS in the TCGA+GTEx database, C is the survival analysis, C(1) is the survival analysis of EREG expression and OS in clinical samples, C(2) is the survival analysis of EREG expression and RFS in clinical samples, D is the univariate Cox regression analysis of the expression of EREG and clinical factors, E is the multivariate Cox regression analysis of the expression of EREG and clinical factors, F is the nomogram based on COX regression analysis, G is the calibration curve for predicting 1- and 3-year survival probabilities, H is the ROC curve for EREG predicting survival.

[0025] Figure 3Show the effects of EREG knockdown on the functions of pancreatic cancer cells; among them, A is the mRNA expression of EREG in pancreatic cancer cell lines, B is the silencing efficiency of lentivirus-mediated EREG mRNA interference, C is the silencing efficiency, C(1) is the EREG protein silencing efficiency in PANC-1 cells, C(2) is the EREG protein silencing efficiency in BxPC-3 cells, D is cell proliferation, D(1) is the effect of EREG silencing on the proliferation of PANC-1 cells evaluated by CCK-8 assay, D(2) is the effect of EREG silencing on the proliferation of BxPC-3 cells evaluated by CCK-8 assay, E is cell proliferation, E(1) is the effect of EREG silencing on the proliferation of PANC-1 cells evaluated by colony formation assay, E(2) is the statistical chart, E(3) is the effect of EREG silencing on the proliferation of BxPC-3 cells, E(4) is the statistical chart, F shows the cell cycle, F(1) is the change in the cell cycle after EREG silencing, F(2) is the statistical chart of the cell levels in each stage of the cell cycle in PANC-1 cells, F(3) is the statistical chart of the cell levels in each stage of the cell cycle in BxPC-3 cells, G shows cell apoptosis, G(1) is the change in apoptosis of pancreatic cancer cells after EREG silencing, G(2) is the statistical chart of apoptosis in PANC-1 cells, G(3) is the statistical chart of apoptosis in BxPC-3 cells, H shows the expression of cell cycle- and apoptosis-related proteins, H(1) is the Western Blot band diagram of the expression of cell cycle- and apoptosis-related proteins in PANC-1 cells, H(2) is the Western Blot band diagram of the expression of cell cycle- and apoptosis-related proteins in BxPC-3 cells, H(3) is the statistical chart of Western Blot results, I shows the cell migration diagram, I(1) is the cell migration diagram of PANC-1 cells, I(2) is the cell migration diagram of BxPC-3 cells, I(3) is the statistical chart of cell migration in PANC-1 cells, I(4) is the statistical chart of cell migration in BxPC-3 cells, J shows cell invasion, J(1) is the Transwell cell invasion diagram, J(2) is the statistical chart of cell invasion in PANC-1 cells, J(3) is the statistical chart of cell invasion in BxPC-3 cells.

[0026] Figure 4 Show the inhibition of the growth of subcutaneous tumors in mice after EREG silencing; among them, A shows the macroscopic view of the subcutaneous transplanted tumor mice after anesthesia at 27 days, B is the fluorescence intensity, B(1) is the measurement of fluorescence intensity using an in vivo imager at 27 days, B(2) is the comparison of the fluorescence intensity of subcutaneous tumors between the EREG silencing group and the control group, C shows the tumors, C(1) is the macroscopic view after taking samples of subcutaneous tumors, C(2) is the comparison of the weights of subcutaneous tumors between the EREG silencing group and the control group, D is the comparison of the volumes of subcutaneous tumors between the EREG silencing group and the control group, E shows the positive rate, E(1) is the representative images of HE staining and Ki67 immunohistochemical staining of subcutaneous tumor tissues, E(2) is the comparison of the Ki67 positive rates between the EREG silencing group and the control group.

[0027] Figure 5 To show the effects of EREG overexpression on the functions of pancreatic cancer cells; among them, A(1) is the number of cells quantitatively expressing green fluorescent protein during 5 consecutive days after infection with the overexpression virus, A(2) is the statistical result graph of fluorescence intensity, B is the qRT-PCR verification of the EREG overexpression efficiency in PaTu-8988t cells, C(1) is the Western Blot band graph for verifying the EREG overexpression efficiency in PaTu-8988t cells, C(2) is the statistical graph of Western Blot results, D is the CCK-8 experiment to evaluate the effect of EREG overexpression on the proliferation of PaTu-8988t cells, E shows the late changes of cells, E(1) is the change of cell cycle after EREG overexpression - EREG-OE group, E(2) is the change of cell cycle after EREG overexpression - Vetor group, E(3) is the statistical graph of cell levels at each stage of the cell cycle of PaTu-8988t cells, F shows cell proliferation, F(1) is the plate clone experiment to evaluate the effect of EREG overexpression on the proliferation of PaTu-8988t cells, F(2) is the statistical graph, G shows cell migration, G(1) is the migration graph of PaTu-8988t cells, G(2) is the statistical graph of PaTu-8988t cell migration, H shows cell invasion, H(1) is the Transwell cell invasion graph, H(2) is the statistical graph of PaTu-8988t cell invasion.

[0028] Figure 6Show the effect of EREG on the MAPK signaling pathway; among them, A is a volcano plot drawn based on all differentially expressed genes after silencing EREG cells, B is an enriched pathway map, B(1) is an enriched pathway map of GO analysis after silencing EREG cells, B(2) is an enriched pathway map of KEGG analysis after silencing EREG cells, C shows protein expression, C(1) is the effect of EREG on the protein expression of ERK1 / 2, p38 MAPK and JNK, C(2) is a statistical chart of protein expression in PANC-1 cells, C(3) is a statistical chart of protein expression in PaTu-8988t cells, D is the working concentration of Senkyunolide I, E shows phosphorylation, E(1) is a Western Blot band diagram of the effect of Senkyunolide I on the phosphorylation levels of ERK1 / 2 and p38 MAPK in EREG-silenced PANC-1 cells, E(2) is a statistical chart of the phosphorylation level of ERK1 / 2, E(3) is a statistical chart of the phosphorylation level of p38 MAPK, F is the effect of Senkyunolide I on cell viability after silencing EREG detected by CCK-8, G shows cell migration, G(1) is the effect of Senkyunolide I on cell migration after silencing EREG, G(2) is a statistical chart of cell migration, H shows cell proliferation, H(1) is the effect of Senkyunolide I on cell proliferation after silencing EREG detected by the plate cloning experiment, H(2) is a statistical chart of the plate cloning experiment, I shows cell invasion, I(1) is the effect of Senkyunolide I on the cell invasion ability after silencing EREG, I(2) is a statistical chart of cell invasion.

[0029] Figure 7 Show the relationship between EREG and immunotherapy responsiveness; among them, A is a circular Sankey diagram of the correlation analysis between EREG expression and ICB-related markers, B is a box plot of the correlation analysis between EREG expression and ICB-related markers, C(1) is the correlation analysis between EREG expression and IPS score, C(2) is the correlation analysis between EREG expression and the IPS score of CTLA4_pos_PD1_neg, D(1) is the correlation analysis between EREG expression and MSI in clinical samples, D(2) is the correlation analysis between EREG expression and TMB in clinical samples, E is the correlation analysis between EREG expression and TPS score, F is the comparison of EREG and PD-L1 expression by HE staining and immunohistochemical staining, G is the comparison of EREG and PD-L1 expression by immunofluorescence staining.

[0030] Figure 8Show the correlation between EREG and MAPK signaling pathways and PD-L / CTLA-4 expression; among them, A is the ROC curve, A(1) is the ROC curve for analyzing the prognosis of pancreatic cancer by TNM staging in the TCGA database, A(2) is the ROC curve for analyzing the prognosis of pancreatic cancer by major blood vessel invasion in the TCGA database, A(3) is the ROC curve for analyzing the prognosis of pancreatic cancer by postoperative liver metastasis in the TCGA database, B is the correlation analysis of the expression of EREG and related proteins in the MAPK signaling pathway in the database, B(1) is the correlation between EREG and ERK1 expression in the TIMER database, B(2) is the correlation between EREG and ERK2 expression in the TIMER database, B(3) is the correlation between EREG and JNK1 expression in the TIMER database, B(4) is the correlation between EREG and JNK2 expression in the TIMER database, B(5) is the correlation between EREG and ERK1 expression in the GEPIA database, B(6) is the correlation between EREG and ERK2 expression in the GEPIA database, B(7) is the correlation between EREG and JNK1 expression in the GEPIA database, B(8) is the correlation between EREG and JNK2 expression in the GEPIA database, C shows the correlation analysis, C(1) is the correlation analysis of the IPS score between EREG expression and CTLA4_pos_PD1_pos, C(2) is the correlation analysis of the IPS score between EREG expression and CTLA4_neg_PD1_neg, C(3) is the correlation analysis of the IPS score between EREG expression and CTLA4_neg_PD1_pos, D is the comparison of EREG and PD-1 expression by immunofluorescence staining, and E is the comparison of EREG and CTLA4 expression by immunofluorescence staining. Detailed implementation manners

[0031] The following is a detailed description of the specific implementation manners of the present invention, but it should be understood that the protection scope of the present invention is not limited by the specific implementation manners. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0032] The transcriptomic and prognostic data of PDAC and normal pancreatic tissues were obtained using the TCGA database (https: / / portal.gdc.cancer.gov / ) and the GTEx database (https: / / gtexportal.org / ). These datasets were used for the expression analysis and survival analysis of EREG. To predict the clinical outcomes of responses to immune checkpoint blockade (ICB), the TIDE algorithm from the website (http: / / tide.dfci.harvard.edu / ) was used in the present invention. The Cancer Immunome Atlas (TCIA, https: / / tcia.at / home) database provided the IPS scores of PDAC patients for the present invention.

[0033] A total of 62 patients with PDAC were enrolled in this study from December 2018 to December 2020. Clinical tumor tissues and corresponding adjacent non-cancerous tissues, with a distance from the lesion margin as the non-cancerous tissues, were collected. Exclusion criteria included: (1) previous radiotherapy, chemotherapy, or immunotherapy; (2) no clinical samples; (3) patients without PDAC; (4) refusal to participate in the follow-up. The enrolled patients had relevant records of follow-up results and clinicopathological data. The determination and classification of PDAC were based on the guidelines provided by the seventh edition of the American Joint Committee on Cancer (AJCC)

[18] . The Ethics Committee of Xijing Hospital, the First Affiliated Hospital of Air Force Medical University, China, has approved the conduct of this study.

[0034] Human malignant PANC-1, BxPC-3, SW1990, PaTu-8988t, and Capan-2 cells were provided by the Cell Bank of the Chinese Academy of Medical Sciences (Shanghai, China). The cells were cultured in DMEM or 1640 medium supplemented with 10% (v / v) fetal bovine serum (Gibco), 1% (v / v) penicillin (Gibco), and streptomycin (China), and the cells were cultured in a cell culture chamber at 37 °C and 5% (v / v) CO2.

[0035] The lentiviral vectors of EREG shRNA and NC shRNA were provided by HANBIO Biotechnology Co., Ltd. (China). The shRNA sequences targeting EREG were as follows: sh1EREG: 5’-CATCTTCTACAG GCAGTCCTCAGTA-3’, denoted as SEQ ID NO.1, sh2EREG: 5’-CGTGTGGCT CAAGTGTCAATA-3’, denoted as SEQ ID NO.2, sh3EREG: 5’-TGAATGGCTATTGTTTGCATGGACA-3’, denoted as SEQ ID NO.3. The lentivirus for stable overexpression of EREG (EREG-OE) was produced by BioToxin Biotechnology Co., Ltd. (China). A non-filled vector was used as the control group.

[0036] The ERK agonist senkyunolide I (S327502) was provided by Selleck. Puromycin (HY-B1743A) was provided by MedChemExpress.

[0037] Table 1 Antibodies and their specific information for the experiment

[0038] Antibody Target Company Company Address Catalog Number Elegant Abessa Cambridge, UK abx100508 Elegant CST Boston, USA D405I CTLA4 CST Boston, USA 53560S p38MAPK Proteintech Chicago, USA 14064-1-AP p-p38MAPK Proteintech Chicago, USA 28796-1-AP ERK1 / 2 Proteintech Chicago, USA 11257-1-AP p-ERK1 / 2 Proteintech Chicago, USA 28733-1-AP Amino-Terminal Kinase Proteintech Chicago, USA 66210-1-Ig p-JNK Proteintech Chicago, USA 80024-1-RR CyclinB1 Proteintech Chicago, USA 55004-1-AP Cyclin D1 Proteintech Chicago, USA 26939-1-AP β-Actin Proteintech Chicago, USA 20536-1-AP α-Tubulin Proteintech Chicago, USA 11224-1-AP PD-L1 / CD274 Proteintech Chicago, USA 66248-1-Ig CDC20 BBI Life Sciences Shanghai, China D220392-0025 BCL-2 Youpin Biotechnology Shenzhen, China YP-Ab-00107 PD-1 abcam Cambridge, UK ab52587 Ki67 Saiweixibiao Wuhan, China GB121499-100

[0039] The experiments involved in the present invention were conducted as follows:

[0040] 1. IHC and IF staining

[0041] Immunohistochemistry (IHC) and immunofluorescence (IF) staining were performed as described previously. Briefly, the samples were immersed in paraffin, then treated with xylene to remove affinity, and then rehydrated with ethanol at different concentrations.

[0042] Antigen was extracted with sodium citrate (10 mM, pH 6.0). Subsequently, the samples were incubated successively with anti-EREG I antibody (4 °C, overnight) and II antibody (37 °C, 1 h). Then, visualization was performed using a DAB color development kit (Servicebio, China). The cell nuclei were immunofluorescently stained with 4′,6-diamidino-2-phenylindole (DAPI). Finally, hematoxylin was used for retention. Finally, 5 random fields were evaluated under a microscope (Olympus, Japan). The data analysis formulas are as follows: surface density = integrated optical density (IOD) / measured area; positive rate (%) = number of positive cells / total number of cells; positive area ratio (%) = positive area / tissue area of the measured area.

[0043] 2. Virus infection

[0044] Lentiviral vectors containing shEREG (EREG shRNA) and shCRrl (NC shRNA) were transduced into PANC-1 and BxPC-3 cells, and the multiplicity of infection (MOI) was 20 and 40, respectively. The infection was carried out using the 1 / 2 volume method, and the other half of the medium was supplemented at 6 h, and the medium was renewed at 24 h. Stable clone cells were screened with puromycin 2 weeks after 72 h.

[0045] PaTu-8988t cells with an MOI of 50 were infected with lentiviral vectors for EREG overexpression (EREG-OE) and NC overexpression (vector). The determination of the infection rate included detecting the expression of green fluorescent protein (GFP) using a fluorescence microscope (Olympus, Japan) 2 - 3 days after virus infection. To establish stable clone cells, puromycin treatment was performed for 2 weeks 72 hours after infection.

[0046] 3. Real-time fluorescence quantitative PCR (qRT-PCR)

[0047] Total RNA was extracted from cells using Trizol reagent (Accurate Biology, China). The RNA concentration was measured using an ultraviolet spectrophotometer (Bio-rad, USA). Reverse transcription and PCR were performed using an RNA reverse transcription kit (Accurate Biology, China) and a qRT-PCR SYBR Green kit (Accurate Biology, China), respectively. The PCR protocol was established as follows: 95 °C for 3 min, followed by 94 °C for 10 s and 58 °C for 30 s, repeated for a total of 40 cycles. To ensure internal control, GAPDH was used, and the relative quantity was determined using formula 2. -ΔΔCT 。

[0048] The primer sequences of the present invention are as follows: EREG-F: 5'-CTGCAGGTGTGAAGTGGGGTTATA-3', denoted as SEQ ID NO.4, EREG-R: 5'-GAATCACGGTCAAAGCCACATATT-3', denoted as SEQ ID NO.5, GAPDH-F: 5'-GGGTGTGAACCATGAGAAGTATG-3', denoted as SEQ ID NO.6, GAPDH-R: 5'-GAGTCCTTCCACGATACCAAAGT-3', denoted as SEQ ID NO.7.

[0049] 4. Protein extraction and Western blotting (WB)

[0050] Proteins were extracted using RIPA lysis buffer (Beyotime, China), and the protein concentration was quantified using the BCA method (Solarbio, China). Then, the protein samples were fixed to an equal concentration and denatured in a metal bath at 100 °C for 15 min. Then, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 30 min and 120 V for 60 min. Then, proteins of different molecular weights were transferred to a polyvinylidene fluoride (PVDF) membrane using a transfer program of 170 mA for 60 min. Subsequently, the PVDF membrane was blocked with a solution containing 5% bovine serum albumin (BSA) for 60 min. The primary antibody was incubated overnight at 4 °C, and the secondary antibody was incubated for 1 h at room temperature. Proteins were observed using a chemiluminescence imager. Finally, semi-quantification was performed using Image Lab software (Bio-Rad, USA).

[0051] 5. Cell proliferation and colony formation assays

[0052] To evaluate cell proliferation, the treated cells were diluted to 1×10 4The density of cells was adjusted to cells / mL, and 200 μL of the cell suspension was added to each well of a 96-well plate, which was then returned to the cell incubator for culturing. After 24 h, 10 μL of Cell Counting Kit-8 (CCK8, TargetMol, China) was used to replace the culture medium, and the plate was incubated for 2 h. Then, the absorbance at a wavelength of 450 nm was measured using an ultraviolet spectrophotometer (BioTek, USA). The absorbance values at 48, 72, 96, and 120 h were measured using the same method.

[0053] In the colony formation assay, the treated cells were diluted to a concentration of 1×10 3 / mL. Subsequently, 500 μL of the cell suspension was added to each well of a 6-well plate and incubated for two weeks. Subsequently, the cells were fixed with a solution containing 4% paraformaldehyde (Servicebio, China) for 20 min. Subsequently, they were stained with a solution containing 0.1% crystal violet (Servicebio, China) for approximately 30 min. Finally, the visible colonies in each well were captured by photography and quantified.

[0054] 6. Migration and invasion assays

[0055] The migration ability of the cells was evaluated using a scratch assay. The cells were cultured in a 6-well plate until they reached approximately 90% confluence, and then the serum was replaced with serum-free medium for 24 h. A scratch was made on the confluent cells using a 10-μL pipette tip. Subsequently, images were taken at two time points under a microscope: immediately after scratching (0 h) and 48 h after scratching. The distance covered by cell migration was quantified using Image J software.

[0056] The cell invasion was evaluated using a transwell assay. Briefly, the upper part of the transwell chamber was filled with 80 μL / well of Matrigel (Corning, USA) and incubated at 37 °C for 4 h. Subsequently, the upper part of the chamber received 200 μL of serum-free medium containing 5×10 4 while the lower chamber contained 500 μL of medium containing 10% fetal bovine serum. After 48 h, the cells on the upper surface of the chamber were carefully wiped off first. Then the cells were fixed with a 4% paraformaldehyde solution (China) for 20 min and then stained with crystal violet (Saiweier Bio, China) for 30 min. The stained cells under each chamber were observed under a microscope, and images were taken from 5 random fields.

[0057] 7. Cell cycle and apoptosis

[0058] To study the distribution of cell cycle phases, 1 mL of DNA staining solution (China) and 10 μL of permeabilization solution (China) were added to the cell pellet, and the mixture was incubated at room temperature for 30 min and then analyzed on a flow cytometer using flow cytometry software.

[0059] For apoptosis detection, cells prepared for analysis were first washed three times with ice-cold PBS and then resuspended at a concentration of 10 6 / ml. Subsequently, flow cytometry was used to analyze the cell suspension after adding FITC-conjugated Annexin V and PI (5 μl, BD) in a 500 μl volume.

[0060] 8. Tumorigenic model in nude mice

[0061] Male BALB / c nude mice (6 weeks old, weighing 18 - 21 g) were purchased from KEAO Company in Chengdu, China. These mice were divided into two groups, with 10 mice in each group. Then, stably infected PANC-1 cells were injected subcutaneously into the subcutaneous tissue of the axillary region of the mice. Subsequently, starting from the 7th day, the size of the subcutaneous tumor was measured every 5 days, and the calculation formula was as follows: Tumor volume V (mm 3 ) = 0.5 × length (mm) × width (mm). On the 27th day, under isoflurane anesthesia (concentration of 1%), small animal imaging was performed to capture images of the subcutaneous tumors in vivo and record the total fluorescence emitted by these tumors. Subsequently, all mice were euthanized, and tumor samples were collected for measurement. All procedures involving animals were strictly in accordance with the guidelines established by the Animal Ethics Committee of Xijing Hospital, the First Affiliated Hospital of Air Force Medical University.

[0062] Statistical analysis

[0063] Data were analyzed using SPSS 25 (Chicago, IL, USA), and graphs were generated using GraphPad Prism 8.0 (La Jolla, CA, USA). Paired sample statistical tests were used to compare the EREG levels of independent samples between PDAC tissues and their corresponding adjacent non-cancerous tissues (ANCT). The relationship between EREG expression and clinicopathological features was evaluated using the Pearson chi-square test to assess the correlation. The Kaplan-Meier method and log-rank test were used to analyze the survival of PDAC patients. Survival analysis was performed and visualized using the R package "survival". Multivariate analysis was performed using the Cox proportional hazards model (positive: LR). The Wilcoxon test was used to evaluate the correlation between EREG and immunotherapy. A two-tailed p-value of p < 0.05 was considered statistically significant.

[0064] Example 1

[0065] EREG is highly expressed in PDAC and is related to clinicopathological features

[0066] First, the TCGA database was used to evaluate the expression of EREG in PDAC. Compared with normal pancreatic tissues, the expression of EREG in PDAC was significantly increased ( Figure 1A) in. To verify this finding, the expression of EREG was detected in tissue samples obtained from clinical PDAC patients. The results showed that the expression of EREG in tumor tissues was significantly increased at the mRNA level (n = 62) and protein level (n = 8) compared with the corresponding adjacent non-cancerous tissues (ANCT). Figure 1 B and 1C) in. Then, 30 pairs of paraffin blocks of PDAC tissues and ANCT were selected for IHC analysis. The results also showed that EREG was highly expressed in tumor tissues. Figure 1 D and E) in. In addition, the relationship between the expression of EREG and clinicopathological features was analyzed. Chi-square test showed that there were significant differences in EREG expression between histological grade, lymph node metastasis, and large vessel invasion, but there were no significant differences in EREG expression between other clinicopathological features such as age, gender, tumor location, and perineural invasion. Figure 1 F in; Table 2).

[0067] The above results indicate that EREG is highly expressed in PDAC.

[0068] Table 2 Relationship between EREG expression and clinicopathological features in PDAC patients (n = 62)

[0069]

[0070]

[0071]

[0072] Example 2

[0073] High EREG expression is an independent poor prognostic factor for PDAC patients.

[0074] The study examined the correlation between EREG expression and the prognostic outcomes of PDAC patients. The results showed that in the TCGA and GTEx databases, the expression of EREG was significantly negatively correlated with overall survival (OS) and recurrence-free survival (RFS). Figure 2 A and B) in. The high EREG expression group showed 2.29 times the likelihood of PDAC-related mortality compared with the low EREG expression group, with an average OS duration of 12.17 and 23.17 months observed in the low expression group, respectively. Figure 2 B) in. The high EREG expression group had a 3.2-fold increased risk of recurrence compared with the low EREG expression group, while the upper quartile RFS of the low expression group was 20.67 months, while that of the high expression group was only 12.13 months. Figure 2in B). Similarly, the cohort of the present invention also confirmed that, compared with the low EREG expression group in PDAC, the high EREG expression group was closely associated with shorter OS and RFS (OS: p = 0.0018, RFS: p = 0.0105)( Figure 2 in C). In addition, univariate analysis showed that the expression of EREG, histological grade, pTNM stage, major vascular invasion, R0 / R1 resection, and postoperative liver metastasis were closely associated with the prognosis of PDAC patients( Figure 2 in D). COX proportional hazards analysis was performed, and the results showed that EREG expression (p = 0.016), pTNM stage (p = 0.012), vascular invasion (p = 0.023), postoperative liver metastasis (p = 0.008), and EREG expression (p = 0.016) were independent prognostic factors for PDAC patients( Figure 2 in E). Next, COX regression analysis was used to construct a nomogram based on the identified important factors. Higher values of these four factors were associated with reduced 1-year and 3-year survival chances. The nomogram was evaluated using a calibration curve, and the 1-year calibration curve was mainly in a diagonal pattern, indicating high reliability of the nomogram( Figure 2 in G). The ROC curve showed that EREG, pTNM stage, major vascular invasion, and postoperative liver metastasis had high accuracy in predicting the survival of PDACS patients. The area under the curve (AUC) of EREG for predicting 1-year survival was 0.67, and the AUC for predicting 3-year survival of PDAC patients was 0.82( Figure 2 in H; Figure 8 in A).

[0075] In summary, high EREG expression indicates poor prognosis, and its expression can be used as an independent prognostic indicator for PDAC patients.

[0076] Example 3

[0077] Silencing EREG can inhibit the in vitro proliferation and progression of PDAC cells

[0078] To investigate the functional role of EREG in the malignant progression of PDAC, the expression of EREG in 5 PDAC cell lines was detected. Compared with human pancreatic duct epithelial cells HPDE6-7C, the EREG mRNA levels were upregulated in PANC-1, BxPC-3, and SW1990 cells, while the EREG mRNA levels were downregulated in Capan-2 and PaTu-8988t cells.( Figure 3 in A). Two cells with relatively high EREG expression, including PANC-1 and BxPC-3, were selected and three different short hairpin RNA (shRNA) lentiviral sequences were used to silence EREG Figure 3B) in the following experiments, the shEREG group represents sh1EREG. Meanwhile, after silencing EREG, the protein level of EREG was significantly downregulated ( Figure 3 C) in the following. Then, CCK8 and colony formation assays showed that in PANC-1 and BxPC-3 cells, silencing EREG significantly inhibited the cell proliferation ability ( Figure 3 D and E) in the following. In particular, the present invention observed the effects of cell cycle and apoptosis on cell proliferation. Flow cytometry detection results showed that inhibiting EREG led to G0 / G1 phase arrest, a decrease in the proportion of S-phase cells, and an increase in apoptosis ( Figure 3 F and G) in the following. In addition, western blot analysis showed that after silencing EREG, the expression levels of cell cycle-related proteins CyclinB1, CyclinD1, CDC20 and apoptosis-related protein Bcl-2 were significantly decreased ( Figure 3 H) in the following. Next, the present invention studied the effects of EREG inhibition on the migration and invasion characteristics of PDAC cells. Scratch assays showed that in PANC-1 and BxPC-3 cell lines, after silencing EREG, the migration ability was significantly reduced by 1.4 - 2.0 times ( Figure 3 I) in the following. Similarly, transwell cell invasion assays showed that in PANC-1 and BxPC-3 cells, after knocking down EREG, the invasion ability of the cells was significantly reduced by 2.3 times ( Figure 3 J) in the following.

[0079] In summary, silencing EREG can significantly inhibit the proliferation, migration and invasion of PDAC.

[0080] Example 4

[0081] Silencing EREG can inhibit tumor growth in vivo

[0082] To further study the effect of EREG silencing on tumor growth in vivo, the present invention injected a suspension of PANC-1 cells subcutaneously into the right axilla of nude mice and regularly monitored tumor growth. After 27 days, compared with the control group (shCtrl), the fluorescence intensity of the shEREG group was significantly decreased. Specifically, silencing EREG led to a 1.96-fold decrease in fluorescence intensity ( Figure 4 A and B) in the following. After euthanizing the subjects, the present invention continued to extract subcutaneous tumors. The research results of the present invention showed that compared with the control group (shCtrl), the volume and weight of subcutaneous tumors in the shEREG group were significantly reduced. The tumor volume was 2.6 times lower, while the tumor weight was 2.2 times lower in the shEREG group ( Figure 4 C and D) in the following. IHC staining of subcutaneous tumors showed that the positive rate of Ki67 in the shEREG group was lower than that in the shCtrl control group ( Figure 4in E). These results further suggest that silencing EREG can inhibit the growth of tumors in vivo.

[0083] Example 5

[0084] Overexpression of EREG promotes the in vitro proliferation and progression of PDAC cells

[0085] Next, the present invention explored the effect of EREG overexpression (EREG-OE) on PDAC cells. First, the present invention selected PaTu-8988t cells with relatively low expression levels compared to HPDE6-7C cells and stably overexpressed EREG using a lentivirus containing the green fluorescent protein (GFP) expression sequence, which helped to evaluate cell proliferation based on the presence of GFP-expressing fluorescent cells. As Figure 5 shown in A of, compared with the vector group, the cell proliferation ability in the EREG-OE group increased significantly with the prolongation of the virus infection duration. The present invention began to collect cells on the 5th day to confirm the expression of EREG. Notably, there was a significant increase in both the mRNA and protein levels in the EREG-OE group ([[]] Figure 5 B and C in). Similarly, the CCK-8 and colony formation assays also showed that overexpression of EREG in PaTu-8988t cells significantly increased the proliferation ability ([[]] Figure 5 D and F in). In addition, flow cytometry results showed that overexpression of EREG led to a short resting phase G0 / G1 and a long S phase compared to the vector group ([[]] Figure 5 E in). In addition, the scratch and transwell assays showed that the migration and invasion abilities of the EREG-oe group increased significantly, by 2.1-fold and 1.9-fold respectively ([[]] Figure 5 G and H in). These results together indicate that overexpression of EREG promotes the proliferation and progression of PDAC in vitro.

[0086] Example 6

[0087] EREG regulates the tumorigenesis of PDAC through the ERK / p38 MAPK pathway

[0088] To study the precise molecular pathway by which EREG promotes the development of PDAC, the present invention performed transcriptome sequencing on EREG-silenced PDAC cell lines and control cells, screening for p values < 0.05 and Log2|fold change (FC)| ≥ 1, and finally obtaining 285 different genes, including 160 upregulated genes and 125 downregulated genes ([[]] Figure 6 ) At the same time, based on the expression profile, the present invention analyzed the GSEA differential signaling pathways and then found some cell proliferation and inflammation-related signaling pathways, such as the TNF signaling pathway, the PI3K-AKT signaling pathway, and the MAPK signaling pathway, enriched in the EREG-silenced group ([[]] Figure 6in B). It is generally believed that the MAPK pathway is a well-established signaling pathway during tumorigenesis and is significantly associated with the proliferation, differentiation, and apoptosis of tumor cells. A study on glioblastoma showed that EREG can enhance tumorigenicity through the ERK / MAPK pathway. Interestingly, the present invention found that EREG is closely related to the malignant processes of proliferation, apoptosis, and progression of PDAC. Therefore, the present invention speculates that EREG may play its tumorigenic role by regulating the MAPK signaling pathway.

[0089] To verify this, the present invention first investigated the correlation between EREG and related genes at the transcriptional level of the MAPK pathway in the public data databases TCGA and TIMER, showing the correlation between EREG, MAPK, ERK, and JNK in PDAC (p < 0.05, R > 0) Figure 8 in B). Then, the present invention used Western blot analysis to evaluate the levels of ERK1 / 2, p38 MAPK, and JNK proteins involved in the MAPK signaling pathway. The results showed that inhibiting EREG led to a decrease in the phosphorylation levels of ERK and p38MAPK in PANC-1 cells, while there was no significant change in the total amounts of phosphorylated JNK and ERK1 / 2, p38MAPK, and JNK compared with the control group. Conversely, overexpression of EREG led to an increase in the phosphorylation levels of ERK1 / 2 and p38 MAPK in PaTu-8988t cells. However, no significant changes were observed in the total amounts of phosphorylated JNK and ERK1 / 2, p38MAPK, and JNK Figure 6 in C). In addition, the present invention also conducted a rescue experiment using the ERK1 / 2 agonist I to evaluate the regulatory association between EREG and the MAPK pathway. Selenolactone I (optimal concentration: 5 μl) effectively increased the activity of ERK1 / 2 Figure 6 in D). The rescue experiment showed that selenolactone I significantly reversed the decreased ERK1 / 2 activity after EREG silencing. At the same time, the present invention also noted that the activation of p38MAPK increased synchronously with the activation of ERK1 / 2 Figure 6 in E). Subsequently, the present invention continued to confirm the effect of selenolactone I on cell behavior. The results of CCK8 and colony formation showed that selenolactone I reversed the reduction in cell proliferation induced by EREG silencing Figure 6 in F and 6H). In addition, the scratch and transwell invasion experiments also showed that selenolactone I reversed the reduction in cell migration and invasion caused by EREG silencing Figure 6 in G and I). These results indicate that EREG silencing inhibits tumorigenesis by inactivating the ERK1 / 2 and p38 MAPK signaling pathways in PDAC.

[0090] Example 7

[0091] The expression of EREG may be closely related to the immunotherapy response of clinical PDAC patients

[0092] As a malignant tumor with poor prognosis, PDAC usually does not respond to immunotherapy. Objective: To investigate the role of EREG in the immunotherapy response of PDAC. First, the present invention used the TIDE algorithm to predict the efficacy of immune checkpoint blockade (ICB) treatment in 178 PDAC patients from the TCGA database. Correlation analysis showed that PDL1 (CD274) (p = 0.001), myeloid-derived suppressor cells (MDSC) (p = 0.017), cancer-associated fibroblasts (CAF) (p < 0.001), and T cell exclusion (p = 0.0027) were elevated in the high EREG expression group, while tumor-associated macrophage M2 (TAM_M2) (p = 1e-04) was decreased. However, there was no difference in the TIDE score (p = 0.8317), MSI_score (p = 0.2555), interferon gamma (IFNG) (p = 0.0946), CD8 (p = 0.5465), and T cell dysfunction (p = 0.7043) between the high and low EREG expression groups ( Figure 7 A and B in). In addition, IPS score analysis showed that the total IPS score and IPS fraction were significantly reduced in the high EREG expression group compared with the low expression group (total IPS score: 29.92 vs 30.80, p = 0.0224; CTLA4_pos_PD1_neg: 7.85 vs 8.20, p = 0.0025), while there was no significant difference in other scores between the two groups ( Figure 7 C in; Figure 8 C in).

[0093] In addition, the present invention collected 65 tumor tissues and corresponding next-generation sequencing (NGS) data from clinical PDAC patients, and performed IHC and IF staining on the specimens to verify the correlation between EREG expression and immunotherapy-related genes. The results showed that the expression of PDL-1 in the high EREG expression group was significantly increased compared with the low expression group ( Figure 7 F and G in). However, there was no significant difference in the expression levels of PD-1 and CTLA4 between the low EREG expression groups ( Figure 8 D and E in). Correlation analysis further showed that EREG expression was positively correlated with the TPS score (Spearman correlation, p = 0.008, r = 0.327) ( Figure 7 E in). In addition, the MSI_score (p = 0.0018) and tumor mutation burden (TMB) were lower in the high EREG expression group compared with the low expression group (p < 0.001) ( Figure 7in D). In short, these results indicate that the expression of EREG is closely related to the expression of PD-L1, and it may become a promising biomarker for predicting the response to immunotherapy.

[0094] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To avoid redundancy, the preferred embodiments of the present invention are described.

[0095] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0096] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Any of the following applications: Use of a reagent for detecting epiregulin expression in the preparation of a product for detecting pancreatic ductal adenocarcinoma; Use of an agent for silencing epidermal regulin in the preparation of a product for treating pancreatic ductal adenocarcinoma.

2. The use according to claim 1, characterized in that: The product for detecting pancreatic ductal adenocarcinoma comprises a reagent for detecting epiregulin.

3. The use according to claim 2, characterized in that: The reagent for detecting epidermal regulin includes a reagent for detecting the expression level of epidermal regulin in a sample using sequencing technology, probe hybridization technology, gene chip technology or fluorescent quantitative PCR technology.

4. The use according to claim 3, characterized in that: The reagent for detecting epiregulin is an amplification primer for epiregulin, and the amplification primer is shown in SEQ ID NO. 4-5.

5. The use according to claim 4, characterized in that: The product for detecting pancreatic ductal adenocarcinoma is a kit, which includes a reagent for detecting the expression level of epiregulin in a sample to be tested.

6. The use according to claim 1, characterized in that: The sample to be tested comes from a patient with pancreatic ductal adenocarcinoma, a patient suspected of pancreatic ductal adenocarcinoma, a population susceptible to pancreatic ductal adenocarcinoma, a population at high risk of pancreatic ductal adenocarcinoma, or a healthy population.

7. The use according to claim 1, characterized in that: The sample to be tested is tissue.

8. The use according to claim 1, characterized in that: The product for treating pancreatic ductal adenocarcinoma includes an agent that silences epiregulin.

9. The use according to claim 1, characterized in that: The agent for silencing epidermal regulin includes shRNA, and the sequence of shRNA is any one of the sequences shown in SEQ ID NO. 1-3.

10. The use according to claim 9, characterized in that: The agent for silencing epidermal regulin also includes a lentiviral vector.

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