Pancreatic cancer diagnosis marker and application thereof

Abstract

The invention discloses a pancreatic cancer diagnosis marker and application thereof, and belongs to the technical field of biological medicines, a serological marker for pancreatic cancer diagnosis is thymosin beta-10, namely Tbeta10, a biomarker for pancreatic cancer diagnosis is a gene TMSB10 for coding the Tbeta10, the gene is 9168 in an NCBI database, a qRT-PCR upstream primer of the TMSB10 is shown as SEQ ID NO.1, a qRT-PCR downstream primer of the TMSB10 is shown as SEQ ID NO.2, the qRT-PCR downstream primer of the TMSB10 is shown as SEQ ID NO.1, the qRT-PCR upstream primer of the TMSB10 is shown as SEQ ID NO.1, and the qRT-PCR downstream primer of the TMSB10 is shown as SEQ ID NO.2. Tbeta10 protein or TMSB10 gene high expression serves as a biomarker for diagnosis and poor prognosis of a pancreatic cancer patient, it is determined that the content of Tbeta10 in serum of the pancreatic cancer patient is remarkably increased, the Tbeta10 protein or TMSB10 gene high expression serves as a serum marker for diagnosis and curative effect and prognosis monitoring of the pancreatic cancer, and the total survival rate of the pancreatic cancer patient is reduced; the TMSB10 is used as a biomarker for poor prognosis of pancreatic cancer patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a diagnostic biomarker for pancreatic cancer and its application. Background Technology

[0002] Pancreatic cancer is a highly malignant tumor with an extremely poor prognosis, often referred to as the "king of cancers." The number of pancreatic cancer cases continues to rise, and the mortality rate remains high, making it a pressing challenge for the global medical community.

[0003] Pancreatic cancer can originate from exocrine pancreatic cells that secrete digestive juices or endocrine pancreatic cells that secrete hormones. Approximately 95% of pancreatic cancers originate from exocrine cells, namely pancreatic ductal adenocarcinoma (PDAC) (hereinafter referred to as pancreatic cancer). Pancreatic cancer has an insidious onset, with atypical early symptoms. Tumor cells exhibit invasive and metastatic characteristics in the early stages of the disease, and 85% of patients already have local or distant metastases at the time of diagnosis. Often, when surgery is no longer an option, the 5-year survival rate is only about 12%. Although there are many factors contributing to the lethality of PDAC, the lack of diagnostic biomarkers for early tumors and the difficulty in early diagnosis are important factors contributing to the high mortality rate of pancreatic cancer. Current traditional pancreatic cancer screening programs include contrast-enhanced computed tomography (CT), magnetic resonance imaging (MRI), and endoscopic ultrasound, but these methods have certain limitations due to the unique anatomical location of the pancreas. The serum biochemical marker CA19-9 is currently widely used in clinical practice, but its specificity and sensitivity are not satisfactory. With the increasing obese population and the accelerating aging process, further elucidating the mechanisms of pancreatic cancer development and progression, and developing molecular markers for early diagnosis and prognosis, are of great significance for improving the early diagnosis rate, survival rate, and mortality rate of pancreatic cancer patients.

[0004] Thymosin is a polypeptide initially isolated from the thymus of calves, mainly comprising three families: α, β, and γ. The thymosin β (TMSB) family includes TMSB4, TMSB10, and TMSB15, whose main function is to inhibit actin polymerization and disrupt the formation of F-actin. TMSB10 is a 44-amino acid polypeptide with various physiological functions, such as early organ development, apoptosis, proliferation, migration, and angiogenesis (Safer D, Golla R, Nachmias VT: Isolation of a 5-kilodalton actin-sequestering peptide from human blood plates. Proceedings of the National Academy of Sciences of the United States of America 1990, 87(7):2536-2540.). Recent studies have shown that TMSB10 is highly expressed in various solid tumors, including breast cancer, ovarian cancer, glioma, and lung adenocarcinoma, and is closely associated with the progression of many solid tumors (Lee SH, Son MJ, Oh SH, Rho SB, Park K, Kim YJ, Park MS, Lee JH: Thymosin beta10 inhibits angiogenesis and tumor growth by interfering with Ras function. Cancer Research 2005, 65(1):137-148.). However, no studies have reported on the role of TMSB10 in pancreatic cancer. Summary of the Invention

[0005] Technical problem solved: Addressing the severe lack of molecular biomarkers for early diagnosis and prognosis assessment of pancreatic cancer in existing technologies, this invention provides a diagnostic biomarker for pancreatic cancer and its application. A polypeptide molecule with increased expression in pancreatic cancer tissue and elevated serum levels in pancreatic cancer patients is used as a serum biomarker, providing a new indicator for the diagnosis, detection, and prognostic assessment of pancreatic cancer. The invention also describes the role of this molecule as a novel target in the treatment of pancreatic cancer, obtaining a biomarker for pancreatic cancer diagnosis and prognostic assessment, as well as an effective therapeutic target. Furthermore, the invention provides the application of shRNA targeting the gene expression of this polypeptide molecule in the preparation of biological agents for the treatment of pancreatic cancer.

[0006] Technical solution:

[0007] A diagnostic biomarker for pancreatic cancer, wherein the biomarker is thymosin β-10, a serological marker for the diagnosis of pancreatic cancer, and the thymosin β-10 protein UniProt ID: P63313.

[0008] Preferably, the biomarker is a biomarker for the diagnosis of pancreatic cancer, specifically the gene TMSB10 encoding Tβ10, which has a Gene ID of 9168 in the NCBI database.

[0009] Preferably, the upstream primer for qRT-PCR of TMSB10 is shown in SEQ ID NO.1, and the downstream primer for qRT-PCR is shown in SEQ ID NO.2.

[0010] Application of a reagent for detecting diagnostic markers of pancreatic cancer in the preparation of a kit for the diagnosis of pancreatic cancer.

[0011] Application of a reagent for detecting diagnostic markers of pancreatic cancer in the preparation of a kit for assessing the prognosis of pancreatic cancer.

[0012] Application of a reagent targeting a diagnostic biomarker for pancreatic cancer in the preparation of a biological agent for the treatment of pancreatic cancer.

[0013] Application of a reagent targeting a diagnostic biomarker for pancreatic cancer in the preparation of drugs for treating pancreatic cancer.

[0014] The application of a reagent targeting a diagnostic biomarker for pancreatic cancer in the preparation of a drug for immunotherapy of pancreatic cancer.

[0015] Preferably, the drug comprises one or more of Tβ10, its active fragment, its derivative, siRNA, and a pharmaceutically acceptable carrier.

[0016] Preferably, the pharmaceutical composition is one or more of the following: tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, lyophilized preparations, inhalers, and sprays.

[0017] Beneficial effects:

[0018] 1. Compared with the prior art, the polypeptide molecule Tβ10 of the present invention is significantly increased in the serum of pancreatic cancer patients, and can be used as a serum marker for the diagnosis of pancreatic cancer;

[0019] 2. This invention clarifies that high expression of Tβ10 protein or TMSB10 gene can serve as a biomarker for the diagnosis and poor prognosis of pancreatic cancer patients;

[0020] 3. The TMSB10-shRNA achieves the therapeutic effect of pancreatic cancer by inhibiting the proliferation, colony formation, and cell migration of pancreatic cancer cells;

[0021] 4. This invention clarifies that serum Tβ10 levels are significantly elevated in pancreatic cancer patients (p<0.0001, t=5.125, df=76), which has diagnostic significance (AUC=0.8763, 95%CI: 0.803 to 0.950, p<0.0001), and can be used as a serum biomarker for the diagnosis of pancreatic cancer and monitoring of treatment efficacy and prognosis;

[0022] 5. Pancreatic cancer patients with high TMSB10 gene expression have a reduced overall survival rate (p<0.05), and TMSB10 can serve as a biomarker for poor prognosis in pancreatic cancer patients;

[0023] 6. This invention provides a new target source for diagnostic kits, therapeutic drugs, and pancreatic cancer monitoring. It is easy to promote and has significant clinical application prospects, and can generate huge clinical application prospects and social benefits in a short period of time. Attached Figure Description

[0024] Figure 1 This is a diagram showing the expression of Tβ10 protein in pancreatic cancer cells in Example 1 of the present invention.

[0025] Figure 2 This is a diagram showing the separation of low molecular weight proteins from cancer cell secretion and quality control in Example 2 of the present invention, where A is a diagram of the low molecular weight protein separation method and B is a diagram of the low molecular weight protein separation result;

[0026] Figure 3 The image shows the results of Tβ10 detection in the serum of human pancreatic cancer patients in Example 3 of the present invention. In the image, A is a comparison of serum Tβ10 protein levels between the pancreatic cancer patient group and the healthy control group, and B is a diagnostic value graph of serum Tβ10 protein levels in the pancreatic cancer patient group analyzed by receiver operating characteristic curve (ROC).

[0027] Figure 4 The graph shows the expression of TMSB10 in pancreatic cancer tissue and the prognosis and survival of patients in Example 4 of the present invention. A is a comparison graph of TMSB10 expression in pancreatic cancer tissue and adjacent tissue, and B is a graph analyzing the overall survival difference between pancreatic cancer patients with high and low TMSB10 expression.

[0028] Figure 5 This diagram illustrates the role of TMSB10-shRNA in the proliferation, colony formation, and cell metastasis of pancreatic cancer cells in Example 5 of this invention. A represents the TMSB10-shRNA knockdown efficiency detection diagram, B represents the effect of TMSB10 knockdown on the proliferation ability of pancreatic cancer cells, C represents the effect of TMSB10 knockdown on the colony formation ability of pancreatic cancer cells, and D represents the effect of TMSB10 knockdown on the migration ability of pancreatic cancer cells. Detailed Implementation

[0029] The specific steps of the present invention are described below with reference to the accompanying drawings and embodiments. It should be understood that the embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0030] Unless otherwise specified, the terms used in this invention generally refer to the meanings understood by those skilled in the art in ordinary circumstances.

[0031] In the following embodiments, experimental conditions and methods not described in detail are generally in accordance with conventional experimental conditions and methods known in the art; materials, reagents, etc. not specifically mentioned are all available commercially.

[0032] All quantitative experiments in the following examples were performed in at least three replicates.

[0033] The present invention will be further described below with reference to specific embodiments.

[0034] The thymosin β-10 mentioned in this application is Thymosinbeta-10, Tβ10, and the thymosin β-10 protein UniProtID: P63313.

[0035] Example 1

[0036] Expression of Tβ10 in pancreatic cancer cells:

[0037] 1. Pancreatic cancer cell culture:

[0038] Cells were cultured in high-glucose DMEM or PM1640 medium containing 10% FBS, with 1% penicillin-streptomycin added, and placed in a 5% CO2, saturated humidity, 37°C CO2 incubator. Cells were digested with 0.25% trypsin and passaged every two days.

[0039] 2. Western Blot analysis:

[0040] Total protein was extracted from cells using RIPA lysis buffer, mixed with loading buffer, and heated at 95°C for 10 min. Equal volumes of protein sample were separated by SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane. After incubation with 5% skim milk at room temperature for 1 h, the membrane was incubated with primary antibody overnight at 4°C. The membrane was rinsed thoroughly and then incubated with secondary antibody at room temperature for 1 h. After rinsing, the membrane was incubated with ECL chemiluminescence buffer and analyzed using ChemiDoc XRS. + The imaging system develops the strips.

[0041] 3. Data Analysis:

[0042] Quantitative data are expressed as mean ± SEM. GraphPad Prism 8.0 software was used to generate bar charts, and one-way ANOVA was used for statistical analysis. p < 0.05 was considered statistically significant.

[0043] 4. Experimental Results:

[0044] like Figure 1 As shown, among the five human pancreatic cancer cell lines, Tβ10 protein was most abundantly expressed in AsPC-1 cells, followed by CFPAC-1, Capan-2 and BxPC-3 cells, while Panc-1 cells showed almost no expression of Tβ10 protein.

[0045] Example 2

[0046] Isolation and quality control of low molecular weight proteins in the secretomic genome of pancreatic cancer cells:

[0047] 1. Collection of conditioned media (CM) for pancreatic cancer cells:

[0048] Three cell lines with high Tβ10 protein expression were selected for culture. When the cell confluence in the cell culture flask reached 80%, the culture supernatant was discarded, and the cells were washed with serum-free medium and cultured for another 24 hours. CM was then collected. 2. Isolation and extraction of low molecular weight proteins from CM:

[0049] The collected CM was centrifuged at 2000g for 5 minutes at 4°C, and the supernatant was collected. Further separation and concentration were achieved by centrifugation at 3000g at 4°C using 10kDa and 3kDa Centricon Mini-Preps, respectively, until the retained buffer was approximately 50μL. The buffer was then stored at -80°C.

[0050] Take 30 μL of concentrated CM and separate it using a 17.5% Tris gel electrophoresis. Stain the separating gel with Colloidal Brilliant Blue for 24 hours, wash with water, image using a gel imaging system, and cut off the leading edge of the gel. Figure 2 As shown.

[0051] The excised gel slices were placed in 50 mM ammonium bicarbonate and cleaned with 50% acetonitrile. They were then reduced in 50 mM DTT for 1 hour, followed by alkylation in 30 mM iodoacetate for 1 hour, and then vacuum dried. The alkylated trypsin was then digested overnight in ammonium bicarbonate at 37°C. The resulting peptide mixture was dried under vacuum, and the protein was extracted with a solution containing 20% ​​acetonitrile and 0.1% trifluoroacetic acid.

[0052] 3. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis:

[0053] All experiments were performed using a Scientific Easy-nanoLC II nanofluid chromatograph in tandem with an LTQ OrbitrapVelos mass spectrometer. Lyophilized digested peptides were redissolved in 0.1% formic acid solution and loaded onto a peptide column at a flow rate of 10 μL / min for separation. Subsequently, ion trap CID / mass spectrometry scans were performed on the 20 most abundant precursor ions (charge state greater than 1). The target value was set at 10⁶ ions with a fill time of 500 ms; for ion trap / mass spectrometry scans, the target value was set at 5000 ions with a fill time of 100 ms. The normalized collision energy was set to 30; the minimum signal intensity requirement was set to 500; the activation time was 10 ms; and the activation Q was 0.250. A dynamic exclusion procedure was implemented to avoid duplicate analysis of highly abundant peptide ions.

[0054] 4. Experimental Results:

[0055] LC-MS / MS yielded 68 common proteins from three cell lines, with Tβ10 protein ranking third in abundance.

[0056] Example 3

[0057] Serum Tβ10 detection in pancreatic cancer patients:

[0058] 1. Clinical sample collection:

[0059] Serum and surgically removed pancreatic cancer tissue (including cancerous and adjacent tissue) were collected from patients before surgical treatment at the hospital. Patients had not received anti-tumor treatment before surgery. Inclusion criteria: (1) preliminary pathological diagnosis of pancreatic cancer; (2) informed consent from patients after a detailed explanation of the study objectives. Serum and surgically removed pathological tissue were collected from the above patients. After meeting the clinical pathological diagnosis, the following experiments were conducted.

[0060] 2. Serum sample collection and storage:

[0061] Draw 2 mL of venous blood from the subject, allow it to stand at room temperature for 2 hours, then centrifuge at 1000×g for 15 minutes at 4°C. Transfer the supernatant serum to cryovials, aliquot and store at -80°C, avoiding repeated freeze-thaw cycles.

[0062] 3. ELISA kit detection:

[0063] ELISA assays were performed strictly in accordance with the kit instructions. Collected serum samples were diluted 1:100, and three wells each were used for positive control, negative control, and blank control in each assay. ELISA results were interpreted by plotting a standard curve with the standard protein concentration in each well as the average absorbance of each well and its replicates on the y-axis. The average concentration of the sample in each well and its replicates was calculated from the standard curve and multiplied by the dilution factor to obtain the actual concentration of serum added to each well.

[0064] 4. Data Analysis:

[0065] Quantitative data are expressed as mean ± SEM. The t-test was used to compare the quantitative data between the two groups. Receiver operating characteristic curves (ROC) were plotted using GraphPadPrism 8.0 software to analyze the diagnostic value of serum biomarkers; p < 0.05 was considered statistically significant.

[0066] 5. Experimental Results:

[0067] like Figure 3 As shown in Figure A, the serum Tβ10 level in the pancreatic cancer group (PDAC) was significantly higher than that in the healthy control group (Control) (p<0.0001, t=5.125, df=76); the ROC analysis results are as follows. Figure 3 As shown in Figure B, the area under the curve for serum Tβ10 was 0.8763 ± 0.0374, with a confidence interval of 0.8031–0.9496 (p < 0.0001). This result indicates that serum Tβ10 is a reliable serum biomarker for the diagnosis of pancreatic cancer.

[0068] Example 4

[0069] Analysis of TMSB10 expression and clinical characteristics in pancreatic cancer patients and adjacent tissues:

[0070] 1. Tissue RNA extraction and qRT-PCR detection:

[0071] RNA extraction from tissues was performed according to the Reagent (Invitrogen) instructions. After reverse transcription, qRT-PCR was performed according to the kit instructions. A Bio-rad CFX96 PCR instrument was used, with the following reaction program: Stage 1: 95℃ for 3 min; Stage 2 (Cycle: 40): 95℃ for 15 s, 60℃ for 30 s; Stage 3: 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s. All experiments were repeated at least three times, and results were presented as quadratically. －△△CTCalculations were performed (using GAPDH as an internal reference), and statistical analysis was conducted. The upstream primer for qRT-PCR using TMSB10 was SEQ ID NO.1: 5'-TGGCAGACAAACCAGACATG-3', and the downstream primer for qRT-PCR was SEQ ID NO.2: 5'-CAATGGTCTCTTTGGTCGGC-3'.

[0072] 2. Data Analysis:

[0073] Quantitative data are expressed as mean ± SEM. Paired t-tests were used to compare the two groups of quantitative data; survival curves were plotted using GraphPadPrism 8.0 software, and p < 0.05 was considered statistically significant.

[0074] 3. Experimental Results:

[0075] like Figure 4 As shown in Figure A, qRT-PCR analysis revealed a significant increase in TMSB10 expression in pancreatic cancer (PDAC) tumor tissue compared to adjacent normal tissue (paraTumor, with an expression level set at 1), a statistically significant difference (p<0.0001, t=10.53, df=41). Based on the TMSB10 mRNA expression levels detected by qRT-PCR in tumor tissue, pancreatic cancer patients were divided into two groups: high TMSB10 expression and low TMSB10 expression. Overall survival (OS) was then analyzed in conjunction with clinical data. Figure 4 As shown in Figure B, patients with high TMSB10 expression had significantly lower overall survival (OS) (p<0.05).

[0076] Example 5

[0077] Effects of TMSB10 on pancreatic cancer cell proliferation:

[0078] 1. shRNA design and synthesis:

[0079] Based on Gene ID:9168, specific targeting shRNAs 1 to shRNA2 were designed using BLAST to target the specific sequence region of this gene. The sequences are shown in SEQ ID NO.3 to SEQ ID NO.6.

[0080] The positive strand of shRNA1 is as shown in SEQ ID NO.3: 5'-GCCGACCAAAGAGACCATTGA-3';

[0081] shRNA1 antisense strand, such as SEQ ID NO.4: 5'-TCAATGGTCTCTTTGGTCGGC-3';

[0082] The positive strand of shRNA2 is as shown in SEQ ID NO.5: 5'-CAGGAGAAGCGGAGTGAAATT-3';

[0083] The antisense strand of shRNA2 is as follows: SEQ ID NO.6: 5'-AATTTCACTCCGCTTCTCCTG-3';

[0084] The shRNA was synthesized by Shanghai Jikai Gene Chemical Technology Co., Ltd., which also provided a negative control shRNA (shRNA-NC).

[0085] 2. Cell culture and transfection:

[0086] Cell culture: Human pancreatic cancer cell line AsPc-1 was cultured in 1640 medium containing 10% FBS in a 5% CO2, saturated humidity, 37°C CO2 incubator. Cells were digested with 0.25% trypsin and passaged every two days.

[0087] shRNA transfection: Before transfection, seed cells in 6-well plates and incubate overnight. Transfection is performed when cells reach 30-50% confluence. Using Lipofectamine™ RNAiMAX (Thermo Fisher Scientific), adjust the final concentration of shRNA1 and shRNA2 to 100 nM and transfect them into AsPc-1 cells. Alternatively, mix shRNA1 and shRNA2 at a 1:1 ratio to prepare shRNA1+2, and transfect the cells at a final concentration of 100 nM. Transfection is performed strictly according to the manufacturer's instructions. The medium is replaced with complete medium the next day, and subsequent experiments are conducted as needed.

[0088] 3. Western Blot interference detection efficiency:

[0089] After transfection and replacement with complete culture medium, the cells were cultured for another 48 hours, and pancreatic cancer cells were harvested. Total protein was extracted from the cells using RIPA lysis buffer, and Western blotting was performed as in Example 1. The results are as follows: Figure 5 As shown in Figure A, the preferred shRNA1+2 significantly inhibits intracellular TMSB10 expression.

[0090] 4. Cell proliferation experiment:

[0091] AsPc-1 cells in the logarithmic growth phase were seeded into 96-well culture plates at a cell number of 1.5 × 10⁻⁶. 3Each well was transfected with shRNA-NC and shRNA1+2, respectively. The cells were then cultured in 1640 medium containing 10% FBS the next day. Cells cultured for 0, 12, 24, 36, 48, and 60 hours were added to each well with 10 μL of CCK8 (Dojindo) working solution and incubated for 2 hours. The absorbance at 450 nm was measured using a microplate reader. Each experimental group was configured with 5 replicates, and the experiment was repeated 3 times. Cell growth curves were plotted. Results are shown below. Figure 5 As shown in Figure B, knockdown of TMSB10 inhibited the proliferation of pancreatic cancer cells. The difference was statistically significant (***p<0.001).

[0092] 5. Cloning experiment:

[0093] AsPc-1 cells in logarithmic growth phase were seeded into 12-well plates at a density of 100 cells per well. They were transfected with shRNA-NC and shRNA1+2, respectively. The culture medium was replaced with 1640 medium containing 10% FBS the next day, and cultured every 4 days. After 3 weeks, cells were stained with 0.1% crystal violet (prepared with 75% ethanol) for 5 minutes, and excess staining solution was washed away with PBS. Cell colony formation was observed and counted. The experiment was repeated at least three times, and statistical analysis was performed. The results are as follows: Figure 5 As shown in Figure C, knockdown of TMSB10 inhibited the colony formation of pancreatic cancer cells. The difference was statistically significant (***p<0.001, ****p<0.0001).

[0094] 6. Transwell migration experiment:

[0095] AsPc-1 cells in logarithmic growth phase were seeded in 6-well plates and cultured overnight. When cells reached 30-50% confluence, they were transfected with shRNA-NC and shRNA1+2, respectively. The next day, the plates were replaced with 1640 culture medium containing 10% FBS and cultured for another 48 hours. After trypsin digestion, the cells were resuspended, counted, and the cell concentration was adjusted to 1×10⁻⁶ cells / well. 5 / mL. Take 8μm pore size Transwell chambers (BD Biosciences) from 24-well plates and place them into 24-well plates pre-filled with 500μl of complete culture medium. Add 100μl of cells from different groups to the upper layer of the chamber and incubate for 24 hours. Remove the Transwell chambers, stain with 0.1% crystal violet (prepared with 75% ethanol) for 5 minutes, rinse with tap water to remove excess staining solution, and gently wipe away any cells that have not penetrated the membrane from the upper layer of the chamber with a cotton swab. Place the chambers in PBS. Observe under an inverted microscope, randomly select at least 5 fields of view, take pictures, and count the cells, taking the average value. Repeat the experiment at least 3 times and perform statistical analysis. Results are as follows: Figure 5As shown in Figure D, knockdown of TMSB10 inhibited the migration of pancreatic cancer cells. The difference was statistically significant (***p<0.001).

Claims

1. A diagnostic biomarker for pancreatic cancer, characterized in that, The biomarker is thymosin β-10, a serological marker for the diagnosis of pancreatic cancer, and the thymosin β-10 protein is UniProt ID: P63313.

2. The pancreatic cancer diagnostic marker according to claim 1, characterized in that, The biomarker is a biomarker for the diagnosis of pancreatic cancer. The biomarker is the gene TMSB10 encoding Tβ10, which has a Gene ID of 9168 in the NCBI database.

3. The pancreatic cancer diagnostic marker according to claim 2, characterized in that, The upstream primer for qRT-PCR of TMSB10 is shown in SEQ ID NO.1, and the downstream primer for qRT-PCR is shown in SEQ ID NO.

2.

4. The use of a reagent for detecting diagnostic markers of pancreatic cancer in the preparation of a kit for the diagnosis of pancreatic cancer.

5. The use of a reagent for detecting diagnostic markers of pancreatic cancer in the preparation of a kit for assessing the prognosis of pancreatic cancer.

6. The application of a reagent targeting a diagnostic biomarker for pancreatic cancer in the preparation of a biological agent for the treatment of pancreatic cancer.

7. The use of a reagent targeting a diagnostic biomarker for pancreatic cancer in the preparation of a pharmaceutical composition for treating pancreatic cancer.

8. The use of a reagent targeting a diagnostic biomarker for pancreatic cancer in the preparation of a pharmaceutical composition for immunotherapy of pancreatic cancer.

9. The application according to claim 7 or 8, characterized in that: The pharmaceutical composition comprises one or more of Tβ10, its active fragment, its derivatives, siRNA, and a pharmaceutically acceptable carrier.

10. The application according to claim 9, characterized in that: The pharmaceutical composition is one or more of the following: tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, lyophilized preparations, inhalers, and sprays.

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