Application of lysylation of lys592 site of stat1 protein in diagnosis, treatment and proliferation and metastasis of pancreatic cancer

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses application of lactic acidification of a STAT1 protein Lys592 site in diagnosis, treatment and proliferation and metastasis of pancreatic cancer, wherein it is first determined that lactic acidification modification of the STAT1 protein Lys592 site is closely related to occurrence and development of pancreatic cancer, the site is a specific target for diagnosis and treatment of pancreatic cancer, and a blank in the field of pancreatic cancer is filled in research on lactic acidification modification of the STAT1 protein; meanwhile, the expression level of STAT1-K592la is positively correlated with the malignant degree of pancreatic cancer, the pancreatic cancer patients with high expression of STAT1-K592la have a shorter survival period, the lactic acidification level of the site is negatively correlated with the survival rate in the pancreatic cancer patients receiving anti-PD-L1 treatment, and the site can be used as a biomarker for screening, diagnosis and curative effect monitoring of pancreatic cancer, and the accuracy of diagnosis is improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of lactation at the Lys592 site of the STAT1 protein in the diagnosis, treatment, proliferation, and metastasis of pancreatic cancer. Background Technology

[0002] Pancreatic cancer is a highly malignant solid tumor with an average overall survival of about 4 months and a 5-year survival rate of only 13%. Rapid disease progression, distant metastasis, and immune escape are the core causes of death. Anti-PD-L1 immunotherapy has shown some efficacy in the clinical application of pancreatic cancer, but most patients experience weak clinical responses due to immune escape and treatment resistance, and the underlying mechanisms remain unclear.

[0003] Hypoxia is a hallmark of the pancreatic cancer tumor microenvironment and is closely associated with the risk of metastasis and death in patients. Under hypoxic conditions, lactate produced by pancreatic cancer cells can act as a substrate to regulate the lactation (Kla) process of lysine residues in proteins. However, the role of protein lactation modification in the malignant transformation and tumor maintenance of pancreatic cancer has not been fully elucidated. Current research on the association between STAT1 protein and pancreatic cancer is significantly insufficient. No direct link has been found between lactation modification at specific sites of STAT1 protein and the occurrence, development, and immune escape of pancreatic cancer. Furthermore, there is a lack of diagnostic and therapeutic targets for pancreatic cancer targeting STAT1 protein lactation sites, as well as related diagnostic reagents and targeted drugs.

[0004] Therefore, identifying specific protein lactation targets for pancreatic cancer and developing corresponding diagnostic products and targeted therapies has become a key solution to the problems of low diagnostic efficiency and poor treatment outcomes in pancreatic cancer. Summary of the Invention

[0005] This invention provides an application of lactation at the Lys592 site of the STAT1 protein in the diagnosis, treatment, and proliferation and metastasis of pancreatic cancer.

[0006] The present invention solves its technical problem by adopting the following technical solution: The first aspect of this application provides the use of the STAT1 protein Lys592 site as a biomarker or target for the diagnosis, treatment, and prognosis of pancreatic cancer.

[0007] This application is the first to clearly demonstrate that lactation modification at the Lys592 site of the STAT1 protein is closely related to the occurrence and development of pancreatic cancer. This site is a specific target for the diagnosis and treatment of pancreatic cancer, filling a gap in research on lactation modification of the STAT1 protein in the field of pancreatic cancer. At the same time, the expression level of STAT1-K592la is positively correlated with the malignancy of pancreatic cancer, and pancreatic cancer patients with high expression of STAT1-K592la have shorter survival. In patients receiving anti-PD-L1 therapy, the lactation level at this site is negatively correlated with survival rate. It can be used as a biomarker for pancreatic cancer screening, diagnosis and efficacy monitoring, improving the accuracy of diagnosis.

[0008] This study found that targeting and inhibiting lactation at the Lys592 site of the STAT1 protein can effectively suppress immune escape, proliferation, and metastasis of pancreatic cancer cells. When this target is used in combination with anti-PD-L1 immunotherapy, it can significantly enhance the anti-tumor response of T cells in vivo and improve the efficacy of immunotherapy, providing a new direction for the combination therapy of pancreatic cancer.

[0009] The lactated antigen peptide and STAT1-K592la antibody provided by this invention can specifically recognize the lactation modification at the Lys592 site of the STAT1 protein. They can not only be used for the detection, diagnosis and prognosis of pancreatic cancer, but also provide an important tool for studying the biological mechanism of STAT1 protein lactation modification and exploring its role in other tumors or diseases. They have high scientific research value and application value.

[0010] The diagnostic and therapeutic products developed based on this invention are easy to operate, highly specific, and highly targeted, which can solve the problems of low diagnostic efficiency and drug resistance in existing pancreatic cancer treatments, and provide new technical support for precision medicine for pancreatic cancer.

[0011] The second aspect of this application provides the use of the STAT1 protein Lys592 site as a biomarker or target for pancreatic cancer growth, proliferation, and metastasis.

[0012] The third aspect of this application provides the use of a reagent for detecting the lactation level of STAT1 protein Lys592 in the preparation of products for the diagnosis, treatment and prognostic assessment of pancreatic cancer.

[0013] As an embodiment of this application, the product includes a chip, a reagent kit, and a test strip.

[0014] As an embodiment of this application, the reagent contains a nucleic acid probe, primer, or antibody that specifically recognizes the Lys592 lactation site of the STAT1 protein.

[0015] As an embodiment of this application, the lactation level of the STAT1 protein Lys592 is positively correlated with the malignancy of pancreatic cancer.

[0016] As an embodiment of this application, the lactation level of the STAT1 protein Lys592 is negatively correlated with the survival of pancreatic cancer patients.

[0017] The fourth aspect of this application provides the use of a reagent for detecting the lactation level of STAT1 protein Lys592 in the preparation of products that inhibit the growth, proliferation and metastasis of pancreatic cancer.

[0018] As an embodiment of this application, the product includes a chip, a reagent kit, and a test strip.

[0019] As an embodiment of this application, the reagent contains a nucleic acid probe, primer, or antibody that specifically recognizes the Lys592 lactation site of the STAT1 protein.

[0020] The fifth aspect of this application provides an antigenic peptide of the STAT1 protein at the Lys592 site that is lactated, with the amino acid sequence RALLK(kla)DQQPG, wherein the Lys amino acid residues are lactated.

[0021] The sixth aspect of this application provides the use of the antigenic peptide described above in the preparation of an antibody that specifically recognizes the Lys592 lactation site of the STAT1 protein.

[0022] The seventh aspect of this application provides a polyclonal antibody that specifically recognizes the Lys592 lactation site of the STAT1 protein. It is prepared by immunizing animals with an antigenic peptide that lactates and modifies the Lys592 site of the STAT1 protein. The amino acid sequence of the antigenic peptide is RALLK(kla)DQQPG, wherein the Lys amino acid residues are lactated.

[0023] The eighth aspect of this application provides the use of the antigenic peptide or the polyclonal antibody described above in the preparation of products for the diagnosis, treatment, and prognostic assessment of pancreatic cancer; or The ninth aspect of this application provides the use of the antigenic peptide or the polyclonal antibody described above in the preparation of a product that inhibits the growth, proliferation and metastasis of pancreatic cancer.

[0024] The beneficial effects of this invention are: (1) This application is the first to clearly demonstrate that lactation modification of the Lys592 site of STAT1 protein is closely related to the occurrence and development of pancreatic cancer. This site is a specific target for the diagnosis and treatment of pancreatic cancer, filling the gap in the research on lactation modification of STAT1 protein in the field of pancreatic cancer. At the same time, the expression level of STAT1-K592la is positively correlated with the malignancy of pancreatic cancer, and pancreatic cancer patients with high expression of STAT1-K592la have shorter survival time. In patients receiving anti-PD-L1 therapy, the lactation level of this site is negatively correlated with the survival rate. It can be used as a biomarker for pancreatic cancer screening, diagnosis and efficacy monitoring to improve the accuracy of diagnosis.

[0025] (2) The study found that targeting and inhibiting lactation at the Lys592 site of STAT1 protein can effectively inhibit immune escape, proliferation and metastasis of pancreatic cancer cells. When this target is used in combination with anti-PD-L1 immunotherapy, it can significantly enhance the anti-tumor response of T cells in vivo and improve the effect of immunotherapy, providing a new direction for the combined treatment of pancreatic cancer.

[0026] (3) The lactated antigen peptide and STAT1-K592la antibody provided by the present invention can specifically recognize the lactation modification of the Lys592 site of the STAT1 protein. They can not only be used for the detection, diagnosis and prognosis of pancreatic cancer, but also provide an important tool for studying the biological mechanism of lactation modification of the STAT1 protein and exploring its role in other tumors or diseases. They have high scientific research value and application value. Attached Figure Description

[0027] Figure 1 Hypoxia-induced STAT1 lactation. Figure 1 (A) Immunoprecipitation experiments demonstrated that hypoxia induces upregulation of STAT1 lactation. Figure 1 (B) Using pan-lactic acidification antibody, the co-localization of STAT1 and lactation under normoxic and hypoxic conditions was detected by co-localization assay.

[0028] Figure 2 The correlation diagram for hypoxia-induced STAT1-K592 lactation. Figure 2 (A) Table of results for quantitative analysis of STAT1 lactation modification sites by mass spectrometry. Figure 2 (B) Secondary mass spectrometry (MS / MS) spectrum of STAT1 lactation modification sites. Figure 2 (CD) Figure of the results of the co-immunoprecipitation (Co-IP) verification experiment in PANC-1 and MIA-PaCa-2 cells, confirming that K592 is a key lactation site induced by hypoxia on STAT1. Figure 2 (E) Species homology analysis results of different STAT1 sites. The results show that the K592 site is highly conserved in multiple species. Figure 2(F) Graph showing the results of dot blot assay to verify the specificity of STAT1-K592la antibody. Figure 2 (G) The results of the validation experiment on endogenous STAT1 K592 lactation show that hypoxia induces STAT1 K592 lactation. (H) The results of the validation of the STAT1-K592la antibody specificity with the K592R mutant show that the site specificity of the STAT1-K592la antibody is verified, and K592 is confirmed to be the lactation site of STAT1.

[0029] Figure 3 This is a diagram showing the correlation between STAT1-K592la and the proliferation and migration of pancreatic cancer cells. Figure 3 (AB) Colony formation assays were used to detect the clonogenic capacity of pancreatic cancer cells in different treatment groups: the clonogenic capacity of the sg-STAT1 group was significantly reduced; reintroduction of WT STAT1 completely restored clonogenic capacity, while reintroduction of the K592R mutant had no significant restorative effect; knockdown of AARS1 significantly inhibited WT STAT1-mediated clonogenic enhancement, but had no significant effect on the K592R mutant, confirming that AARS1 promotes pancreatic cancer cell proliferation by regulating STAT1-K592 lactation. The right side shows the statistical quantitative results of clonogenic number. Figure 3 (CD) Scratch healing assay to detect the migration ability of pancreatic cancer cells: reintroduction of WT STAT1 can significantly accelerate the cell scratch healing rate, while the K592R mutant cannot restore cell migration ability; knockdown of AARS1 significantly inhibits WT STAT1-induced cell migration, while the cell migration ability of the K592R mutant group has no significant change; the right side shows the statistical quantitative results of relative wound healing rate.

[0030] Figure 4 STAT1-K592 lactation promotes pancreatic cancer tumor growth in vivo and is associated with immunosuppression. Figure 4 (A) Actual image of the transplanted tumor tissue; Figure 4 (B) Tumor volume growth curve; Figure 4 (C) In vivo bioluminescence imaging of mice. Figure 4 (D) Quantitative bar chart of mean bioluminescence intensity in mice. The results showed that WT STAT1 promoted subcutaneous tumorigenesis, and the tumorigenesis ability of the K592R mutant was significantly weakened. Figure 4 (E) Representative images of mouse livers from each group; Figure 4 (F) Quantitative bar chart of liver metastatic lesion area percentage. The results confirmed that STAT1-K592 lactation is a key driver of liver metastasis in pancreatic cancer. Figure 4 (G) Representative images of liver metastatic nodules and HE-stained pathological sections. The results showed that the metastatic lesions in the liver tissue of the WT group were more extensive, while the metastatic lesions in the K592R group were significantly reduced. Figure 4(H) Flow cytometry analysis of tumor-infiltrating CD8 in mouse liver tissue + A representative scatter plot of the expression of T cell surface exhaustion markers PD-1 and LAG-3. Figure 4 (I)PD-1 + / LAG-3 + CD8 + A quantitative bar chart of T cell proportions confirms that STAT1-K592 lactation can induce CD8+. + T cell depletion promotes immunosuppression. Figure 4 (J) Flow cytometry determination of tumor-infiltrating CD8 in mouse liver tissue + T cells IFNγ + / GzmB + A representative scatter plot. Figure 4 (K)IFNγ + / GzmB + CD8 + A quantitative bar chart of T cell proportions indicates that STAT1-K592 lactation inhibits CD8+. + The anti-tumor activity of T cells promotes immune escape from pancreatic cancer.

[0031] Figure 5 Correlation diagram of STAT1-K592la with poor prognosis and immunotherapy efficacy in pancreatic cancer patients. Figure 5 (A) Representative images of pancreatic cancer tissue microarrays stained with IHC, detecting the expression level of STAT1-K592la in pancreatic cancer tissues and adjacent normal tissues. Figure 5 (B) Representative images of pancreatic cancer tissue microarrays stained with IHC, showing the expression level of total STAT1 in pancreatic cancer tissues and adjacent normal tissues. Figure 5 (CD) Quantitative statistical scatter plot of IHC scores for STAT1-K592la and STAT1 in pancreatic cancer tissue and adjacent normal tissue. These results indicate that STAT1-K592la is highly expressed in pancreatic cancer. Figure 5 (E) Representative images of pancreatic cancer tissue microarrays stained with IHC, showing the expression level of AARS1 in pancreatic cancer tissues and adjacent normal tissues. Figure 5 (F) Representative images of pancreatic cancer tissue microarrays stained with IHC, showing the expression level of PD-L1 in pancreatic cancer tissues and adjacent normal tissues. Figure 5 Quantitative statistical scatter plot of IHC scores for (GH)AARS1 and PD-L1 in pancreatic cancer tissue and adjacent normal tissue. These results indicate that AARS1 and PD-L1 are highly expressed in pancreatic cancer. Figure 5 (I) Representative images of pancreatic cancer tissue microarrays stained with IHC, detecting the expression level of CD8 in pancreatic cancer tissue and adjacent normal tissue. Figure 5(J) Quantitative statistical scatter plot of CD8 IHC score in pancreatic cancer tissue and adjacent normal tissue. (K) Representative IHC staining images of STAT1-K592la, STAT1, AARS1, PD-L1, and CD8 in pancreatic cancer tissue at different pathological stages. Figure 5 (L) Western blot results of STAT1-K592la, STAT1, PD-L1, and AARS1 in pancreatic cancer and adjacent normal tissue samples. Figure 5 (M) Kaplan-Meier survival curves for overall survival (OS) in pancreatic cancer patients. Figure 5 (N) Scatter plot of correlation analysis between STAT1-K592la and PD-L1 expression levels in pancreatic cancer tissues, showing a positive correlation between the two. Figure 5 (O) Scatter plot of correlation analysis between AARS1 and PD-L1 expression levels in pancreatic cancer tissues, showing a positive correlation between the two. Figure 5 (P) Scatter plot of correlation analysis between AARS1 and STAT1-K592la expression levels in pancreatic cancer tissue, showing a positive correlation between the two. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0034] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0035] In this invention, there are no particular limitations on the specific dispersion and stirring methods.

[0036] Unless otherwise specified, all reagents or instruments used in this invention are commercially available conventional products. Unless otherwise specified, the raw materials used in each comparative example and the parallel experiments of each embodiment are the same commercially available products.

[0037] Example 1 1. Antibodies and reagents The antibodies used in this invention are as follows: panKlac (PTM BIO, PTM-1401, 1:1000 dilution), anti-STAT1 (Proteintech, 66545-1-Ig, 1:3000 dilution), anti-PD-L1 (Proteintech, 28076-1-AP, 1:3000 dilution), anti-ACTIN (Proteintech, 20536-1-AP, 1:3000 dilution), anti-PD-L1 (Proteintech, 20960-1-AP, 1:3000), anti-AARS1 (Proteintech, 17394-1-AP, 1:3000 dilution), anti-MYC tag (Proteintech, 60003-2-Ig, 1:3000 dilution), anti-Flag M2 (Sigma-Aldrich, F1804, 1:5000 dilution), and anti-mouse IgG (H + L) / HRP (Proteintech, SA00001-1), anti-rabbit IgG (H + Anti-mouse IgG (Proteintech, SA00001-2), anti-mouse IgG (Proteintech, B900620, 1:200 dilution), anti-mouse IgG DyLight 488 (Abbkine, A23210, 1:150 dilution), and anti-rabbit IgG DyLight 594 (Abbkine, A23420, 1:150 dilution) were prepared. A STAT1 K592 specific lactation antibody was prepared at a 1:1000 dilution. This antibody was prepared by Abmart using the STAT1 peptide (RALLK(lac)DQQPG). PE / Cy7 Granzase B (QA16A02) monoclonal antibody (372213) and FITC-IFNγ (XMG1.2) monoclonal antibody (505806) were purchased from Biolegend and diluted 1:50.

[0038] The reagents used in this invention are as follows: a mixture of protease and phosphatase inhibitors (NCM, P002), an enhanced chemiluminescence assay kit (NCM, P10300), RIPA buffer lysis buffer (NCM, WB3100), a Pearson magnetic chromatin immunoprecipitation kit (Thermo Fisher Scientific, 26157X), protein A / G magnetic beads (Merck, 16-266), Pearson immunoprecipitation lysis buffer (Thermo Fisher Scientific, 87787), DMEM (Gibberellin, C11995500BT), 0.25% trichloroacetic acid-trypsin (NCM, C100C1), cell preservation solution (NCM, C40100), and Lipofectamine. TM 2000 (Invitrogen, 11668019), Opti-MEM I low serum medium (Gibberell, 31985062), bovine fetal serum (Prax Biotechnology, FSP500), dual-luciferase reporter assay system (Promega, E1910).

[0039] 2. Cell lines and plasmids Human pancreatic cancer cell lines (MIA PaCa-2, PANC-1) and HEK293T were provided by the Center for Type Culture Collection, Chinese Academy of Sciences (Shanghai, China). MIA PaCa-2 and PANC-1 cells were cultured in DMEM medium supplemented with 10% bovine fetal serum (FBS).

[0040] For transient overexpression in PC cells, STAT1, AARS1, and HDAC3 were cloned into the pCMV-3×Flag vector. For lentiviral vector-mediated overexpression, STAT1, STAT1 K592R, and AARS1 were cloned into the pCDH-puro vector. Mutant vectors for the genes of interest were generated using site-directed mutagenesis. For protein expression in *E. coli*, the full-length STAT1 protein and the HDAC3 fusion fragment were cloned into a modified pET28a vector with the corresponding tags. For STAT1 gene knockout experiments, oligonucleotide fragments were cloned into the LentiCRISPRv2 vector. The sgRNA sequence of STAT1 is as follows: UCU UGC UAC AGC AUA ACA UAG GUG GCA AAU GAA ACA UCAU.

[0041] 3. Experimental Methods 3.1. Immunoprecipitation, immunoblotting Cell lysate collection, centrifugation, and immunoprecipitation and immunoblotting procedures: PANC-1 and MIA-PaCa-2 cell lysates were collected and centrifuged at 12,000 × g at 4°C for 15 min. The lysates were then diluted with PBS at a concentration of 2 mg / mL for immunoprecipitation. 200 μg of the lysate was immunoprecipitated with the designated antibody. Immune complexes were captured by adding Protein G-sodium alginate beads to the lysate. Proteins bound to the beads were boiled in 2× Laemmli sample buffer. Samples were separated by 10% SDS-PAGE and then electrophoretically transferred to a polyethyleneimine (PVDF, Millipore, IPVH00010) membrane, which was blocked and incubated with the primary antibody. The secondary antibody used was an HRP-labeled anti-rabbit or anti-mouse antibody.

[0042] 3.2 Immunofluorescence assay: PC cells were cultured in six-well plates for 24 hours. Cells were then fixed with 4% formaldehyde and washed three times with PBS. Cells were infiltrated with PBS containing 0.2% Triton X-100 for 15 minutes and blocked with 3% BSA at room temperature for 1 hour. Subsequently, cells were incubated overnight at 4°C with anti-STAT1 and anti-AARS1 antibodies, followed by incubation for 1 hour with FITC-labeled goat anti-rabbit IgG or goat anti-mouse IgG. Cell nuclei were counterstained with DAPI. Fluorescence images were observed under a fluorescence microscope (Olympus).

[0043] 3.3. Tissue microarray (TMA) and immunohistochemistry (IHC) Tissue microarrays containing 48 pairs of pancreatic cancer samples were purchased from Shanghai Aode Biotechnology Co., Ltd. (Shanghai, China), and this application has been approved by the Ethics Committee of Shanghai General Hospital. Tissue samples were fixed and sectioned with 4% formaldehyde before paraffin embedding. Pathological sections were dewaxed in xylene and hydrated in a gradient of alcohols. The sections were then incubated overnight at 4°C with a primary antibody, followed by incubation with a secondary antibody. For histological analysis, sections were stained with DAB and hematoxylin.

[0044] 3.4. Transwell test, wound healing test, and colony formation test For the wound healing assay, PC cells were seeded in triplicate in six-well plates until 90% confluence was achieved. Then, RNase-free pipettes were used to make incisions at the wound site, followed by washing with PBS to remove any floating cells. Images were taken at 0, 12, 24, or 36 hours post-treatment.

[0045] Wound healing was assessed by measuring the remaining distance between the wound edges. For colony formation assays, PC cells were seeded in 6-well plates (500-1000 cells per well, 2 mL of DMEM medium containing 10% fetal bovine serum per well). After two weeks, cells were fixed with 4% paraformaldehyde for 15 minutes, stained with 1% crystal violet solution (#C0121, Beintop) for 20 minutes, and then washed three times with PBS.

[0046] 3.5. Animal Models For in vivo tumor growth studies, MIA PaCa-2 and PANC-1 cells (2 × 10⁶ cells) were subcutaneously injected into each BALB / c nude mouse (6 weeks old). Tumors were collected, photographed, and immunohistochemically analyzed upon mouse sacrifice. Tumor volume ((length × width) / 2) was measured weekly. Subcutaneous tumors were excised and weighed after 21 days. For an in vivo liver metastasis model, nude mice were also divided into four groups (n = 6 per group). Cells were injected via tail vein. In vivo tumor cell metastasis was observed every two weeks using the IVIS Illumina system (Caliper Life Sciences, USA). After 6 weeks, lung tissue was removed and stained with hematoxylin and eosin (HE) to observe metastatic nodules.

[0047] 3.6. Spontaneous pancreatic cancer model KPC (LSL-KrasG12D / +; LSL-Trp53R172H / +; Pdx-1-Cre, 16 weeks old, sex-matched) transgenic mice were purchased from MODEL ORGANISMS (Shanghai, China) and housed under pathogen-free conditions. AAV8-AARS1 virus was purchased from Hanbo Biotechnology (Shanghai, China). The virus was administered intraperitoneally at a dose of 100 µL per mouse, at a concentration of 6 × 10⁹ vector genomes per µL. Each group received anti-PD-L1 antibody treatment every 3 days at a dose of 100 µg / mouse for a total of three times. After 6 weeks, the mice were euthanized, and the pancreas was collected.

[0048] Flow cytometry was used to obtain single-cell suspensions from tumor samples from tumor-implanted mice through rapid and gentle centrifugation, physical grinding at 4°C, enzymatic digestion with collagenase IV at 37°C for 1 hour, and filtration. Tumor cells were collected using a tumor cell isolation kit, and other cells were then suspended in 40% Percoll reagent and centrifuged to obtain enriched immunosingle cells in the cell pellet and CD45-enriched non-tumor cells in the top layer. The enriched immunosingle cells were blocked with CD16 / CD32 antibodies and stained with specified fluorescently labeled antibodies at 4°C for 30 minutes. Cells were incubated with the following antibodies: anti-IFNγ, anti-TNFα, and anti-granzyme B, using a Cytofix / Cytoperm kit. Data were processed using FlowJo software.

[0049] 4. Experimental Results Specific lactation of STAT1 protein at Lys592 site under hypoxic conditions: Immunoblotting results showed that, compared with normoxic conditions, the overall lactation level of STAT1 protein in pancreatic cancer cells was significantly increased under hypoxic conditions. Furthermore, the STAT1-K592la antibody specifically detected lactation modification at the Lys592 site, and the lactation level at this site increased with prolonged hypoxia, confirming that Lys592 is a key lactation site for STAT1 protein under hypoxia. Figure 1 , Figure 2 ).

[0050] STAT1-K592la promotes the proliferation and metastasis of pancreatic cancer cells: Wound healing assays showed that pancreatic cancer cells overexpressing STAT1-K592la significantly increased migration distance; colony formation assays showed that pancreatic cancer cells overexpressing STAT1-K592la significantly increased the number of colonies, while knockdown of STAT1 or mutation at the Lys592 site (K592R) significantly inhibited the proliferation and migration of pancreatic cancer cells. Figure 3 ).

[0051] STAT1-K592la promotes immune escape from pancreatic cancer cells: Immunoblotting and quantitative PCR results showed that STAT1-K592la significantly upregulated the transcription and expression of PD-L1 in pancreatic cancer cells; flow cytometry results showed that knockdown of STAT1-K592la enhanced CD8+ T cell activation, increased the expression of IFNγ, TNFα and granzyme B, and inhibited tumor immune escape. Figure 4 ).

[0052] STAT1-K592la is associated with poor prognosis in pancreatic cancer patients: Immunohistochemical analysis of clinical samples showed that the expression level of STAT1-K592la in pancreatic cancer tissues was significantly higher than that in adjacent normal tissues, and was positively correlated with tumor stage and lymph node metastasis; survival analysis showed that the median survival of pancreatic cancer patients with high STAT1-K592la expression was significantly shorter than that of patients with low expression, and among patients receiving anti-PD-L1 therapy, those with high STAT1-K592la expression had a significantly lower survival rate. Figure 5 ).

[0053] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical spirit of the present invention. The technical scope of the present invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. Application of STAT1 protein Lys592 site as a biomarker or target for the diagnosis, treatment and prognosis of pancreatic cancer.

2. Application of the STAT1 protein Lys592 site as a biomarker or target for pancreatic cancer growth, proliferation and metastasis.

3. Application of reagents for detecting the lactation level of STAT1 protein Lys592 in the preparation of products for the diagnosis, treatment and prognostic assessment of pancreatic cancer.

4. The application according to claim 3, characterized in that, The products include chips, reagent kits, and test strips; and / or The reagent contains a nucleic acid probe, primer, or antibody that specifically recognizes the Lys592 lactation site of the STAT1 protein; and / or The lactation level of the STAT1 protein Lys592 was positively correlated with the malignancy of pancreatic cancer; and / or The lactation level of the STAT1 protein Lys592 was negatively correlated with the survival of pancreatic cancer patients.

5. Application of reagents for detecting the lactation level of STAT1 protein Lys592 in the preparation of products that inhibit the growth, proliferation and metastasis of pancreatic cancer.

6. The application according to claim 5, characterized in that, The products include chips, reagent kits, and test strips; and / or The reagent contains nucleic acid probes, primers, or antibodies that specifically recognize the Lys592 lactation site of the STAT1 protein.

7. An antigenic peptide at the Lys592 site of STAT1 protein that is lactated, characterized in that, The amino acid sequence is RALLK(kla)DQQPG, in which the Lys amino acid residues are lactated.

8. The use of the antigenic peptide of claim 7 in the preparation of antibodies that specifically recognize the Lys592 lactation site of the STAT1 protein.

9. A polyclonal antibody that specifically recognizes the Lys592 lactation site of the STAT1 protein, characterized in that, The antigen peptide was prepared by immunizing animals with an antigenic peptide at the Lys592 site of the STAT1 protein modified by lactation. The amino acid sequence of the antigenic peptide is RALLK(kla)DQQPG, wherein the Lys amino acid residues are modified by lactation.

10. The use of the antigenic peptide according to any one of claims 6-7 or the polyclonal antibody according to claim 9 in the preparation of products for the diagnosis, treatment, and prognostic assessment of pancreatic cancer; or The use of the antigenic peptide according to any one of claims 6 to 7 or the polyclonal antibody according to claim 9 in the preparation of products that inhibit the growth, proliferation and metastasis of pancreatic cancer.

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