Application of OCIAD2 as pancreatic cancer marker, therapeutic target and pancreatic cancer immunotherapy

Abstract

The invention relates to the field of biological medicine, and provides application of OCIAD2 as a pancreatic cancer marker and a treatment target and in pancreatic cancer immunotherapy. The invention provides an application of OCIAD2 as a pancreatic cancer marker in pancreatic cancer detection products, an application of OCIAD2 as a pancreatic cancer treatment target in preparation of pancreatic cancer treatment drugs and an application of OCIAD2 in pancreatic cancer immunotherapy products. According to the application of the OCIAD2 as the pancreatic cancer marker and the treatment target and the application of the OCIAD2 in pancreatic cancer immunotherapy, it is found that the OCIAD2 is the pancreatic cancer marker and the treatment target for the first time, expression of the OCIAD2 is in negative correlation with infiltration of pancreatic cancer immune cells and is closely related to various immune checkpoint genes, a new target is provided for clinical detection and clinical treatment of pancreatic cancer, and the application of the OCIAD2 in pancreatic cancer immunotherapy is developed. And a new direction is provided for pancreatic cancer immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an application of OCIAD2 as a pancreatic cancer marker, a therapeutic target and in pancreatic cancer immunotherapy. Background Art

[0002] Pancreatic cancer is a malignant tumor originating from the pancreatic ductal epithelium and acinar cells. It is highly malignant, easily metastatic, and has a poor prognosis. Worldwide, pancreatic cancer has always been at the forefront of the mortality rate of malignant tumors. The incidence and death of pancreatic tumors have increased year by year, while the incidence and mortality of other common cancers have decreased. Despite the progress in the diagnosis and treatment of pancreatic cancer, the 5-year survival rate of pancreatic cancer patients is only about 4%, and the survival rate of pancreatic cancer patients without metastasis is higher. Currently, surgical resection is the only cure for pancreatic cancer. However, 80-85% of pancreatic cancer patients have already developed to the advanced stage without surgical indications when diagnosed, and pancreatic cancer does not respond well to most chemotherapy drugs. Among them, pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic cancer tissue type, accounting for 85%-90% of all pancreatic tumors. It is known for its high malignancy and difficulty in diagnosis. It is characterized by rapid local progression and easy metastasis to distant sites. Therefore, we need to understand the biological mechanisms of pancreatic tumor occurrence and development and find new targets for the diagnosis and treatment of pancreatic cancer.

[0003] Tumor markers refer to a class of substances that are synthesized and secreted by the gene expression of tumor cells or abnormally produced or increased by the body's response to tumors during the occurrence and proliferation of malignant tumors, reflecting the existence and growth of tumors. Commonly used tumor markers related to pancreatic cancer diagnosis in clinical practice include CA19-9, CEA, CA125, etc. Among them, CA19-9 is the most commonly used, but about 10% of pancreatic cancer patients do not express CA19-9, and CA19-9 may be falsely positive in other cases of biliary diseases, so it is necessary to combine other tumor markers to assist in diagnosis. At the same time, pancreatic cancer is also in urgent need of new targets for the development of molecular targeted drugs to develop inhibitors, monoclonal antibodies, etc. to improve the survival of patients.

[0004] Ovarian Cancer Immunoreactive Antigen Domain Containing 2 (OCIAD2) and its homolog OCIAD1 are human cancer-associated proteins associated with ovarian cancer, thyroid cancer, lung cancer, and various blood tumors including multiple myeloma and neutrophilia. Studies have found that OCIAD2 (GeneID: 132299) is abnormally expressed in a variety of cancers and neurodegenerative diseases. In addition, this protein is involved in endocytosis, regulating hematopoietic stem cell homeostasis, and positively regulating receptor signaling pathways through JAK-STAT, but its role in pancreatic cancer has not been studied and reported.

[0005] Therefore, we explored the expression and function of OCIAD2 in pancreatic cancer through bioinformatics analysis and in vitro experiments, aiming to provide new research targets and treatment methods for the clinical detection and treatment of pancreatic cancer. Summary of the invention

[0006] The purpose of the present invention is to provide an OCIAD2 as a pancreatic cancer marker, a therapeutic target and an application in pancreatic cancer immunotherapy. The present invention first discovered that OCIAD2 is a marker and therapeutic target for pancreatic cancer, and that the expression of OCIAD2 is negatively correlated with the infiltration of pancreatic cancer immune cells and is closely related to a variety of immune checkpoint genes, providing a new target for clinical detection and clinical treatment of pancreatic cancer and a new direction for pancreatic cancer immunotherapy.

[0007] In order to solve the above technical problems, the present invention provides an application of OCIAD2 as a pancreatic cancer marker, a therapeutic target and in pancreatic cancer immunotherapy as follows: An application of OCIAD2 as a pancreatic cancer marker in detecting pancreatic cancer products.

[0008] Optionally, the pancreatic cancer detection product includes a detection reagent and a detection kit.

[0009] Optionally, the detection kit includes: a reagent for detecting OCIAD2, a reagent for detecting the OCIAD2 gene, a reagent for detecting an expression product of the OCIAD2 gene, or / and a reagent for detecting the OCIAD2 antigen.

[0010] Optionally, the detection kit includes reagents used for analyzing the expression of OCIAD2 by immunohistochemistry, or includes reagents used for detecting the expression of OCIAD2 by immunoblotting.

[0011] Optionally, the detection kit includes reagents for detecting OCIAD2 mRNA by real-time quantitative polymerase chain reaction.

[0012] Optionally, OCIAD2 is a marker for poor prognosis of pancreatic cancer.

[0013] A use of OCIAD2 as a pancreatic cancer therapeutic target in the preparation of a drug for treating pancreatic cancer.

[0014] Optionally, the pancreatic cancer therapeutic target is the AKT pathway.

[0015] Optionally, the pancreatic cancer therapeutic target is AKT phosphorylation, or the expression of Cyclin D1 and Cyclin E1.

[0016] An application of OCIAD2 in a pancreatic cancer immunotherapy product.

[0017] The present invention analyzes the expression level of OCIAD2 in human pancreatic cancer tissues significantly increased relative to human normal pancreatic tissues through the TCGA database; the overall survival and disease-free survival of pancreatic cancer patients with OCIAD2 expression levels analyzed by the TCGA database and the GSE183795 data set are significantly reduced; the present invention performs real-time fluorescence quantitative polymerase chain reaction (qRT-PCR, Quantitative Real-Time Polymerase Chain Reaction,) and Western Blot (protein immunoblotting) experiments to confirm that compared with human normal pancreatic ductal cells HPNE, the expression of OCIAD2 gene and its expression products in pancreatic cancer cell lines PANC-1, AsPC-1, MIAPACA-2, CFPAC-1, CAPAN-1 is significantly increased; compared with normal tissues, the protein expression level of OCIAD2 in pancreatic cancer tissues is significantly increased by immunohistochemistry. The present invention further constructs a pancreatic cancer cell model with knockdown of OCIAD2, and through CCK8 experiments, flow cytometry and cell immunofluorescence experiments, it is found that low expression of OCIAD2 gene can effectively inhibit the proliferation of pancreatic cancer cells. The present invention discovers and verifies the relationship between the expression level of the OCIAD2 gene and the onset of pancreatic cancer. The OCIAD2 gene or its expression product can be used as a pancreatic tumor marker to accurately determine whether an individual has pancreatic cancer or assess the risk of pancreatic cancer, providing a new research target for clinical detection and therapeutic drugs for pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The database of Example 1 analyzes the expression level of OCIAD2 in human pancreatic cancer tissue and human normal pancreatic tissue; Figure 2The following are the disease-free survival curve, overall survival, clinical staging diagram and cox regression analysis result diagram of pancreatic cancer patients with different OCIAD2 expression levels analyzed by the database in Example 1: Figure 2 A is a curve of disease-free survival (Disease Free Survival) of pancreatic cancer patients with different OCIAD2 expression levels. Figure 2 B is the overall survival curve of pancreatic cancer patients with OCIAD2 expression levels analyzed by TCGA database. Figure 2 C is the overall survival curve of pancreatic cancer patients with OCIAD2 expression levels analyzed from the GSE183795 dataset. Figure 2 D is the OCIAD2 expression level in pancreatic cancer patients at different clinical stages. Figure 2 E is the result graph of cox regression analysis; Figure 3 The expression of OCIAD2 mRNA and protein in pancreatic cancer cells was verified by Western Blot, qPCR and IHC in Example 2: Figure 3 A is a comparison of the expression levels of OCIAD2 protein in normal human pancreatic ductal epithelial cell lines and human pancreatic cancer cell lines. Figure 3 B is a comparison of the mRNA expression levels of OCIAD2 in normal human pancreatic ductal epithelial cell lines and human pancreatic cancer cell lines. Figure 3 C is a comparison of the expression of OCIAD2 in pancreatic cancer tissues and normal tissues analyzed by immunohistochemistry; Figure 4 This is a diagram showing the construction of a pancreatic cancer cell line model with OCIAD2 knockdown in Example 3 of the present invention, as well as a comparison of Western Blot and qPCR verification: Figure 4 A is a comparison of the expression of OCIAD2 protein and ACTB protein in the human pancreatic cancer cell line PANC-1 with OCIAD2 knockdown. Figure 4 B is a comparison of the expression of OCIAD2 protein and ACTB protein in the human pancreatic cancer cell line AsPC-1 with OCIAD2 knockdown. Figure 4 C is a quantitative analysis comparison of OCIAD2 protein expression levels in the human pancreatic cancer cell line PANC-1 with OCIAD2 knockdown. Figure 4 D is a quantitative analysis comparison of OCIAD2 protein expression levels in the human pancreatic cancer cell line AsPC-1 with OCIAD2 knockdown. Figure 4 E is a comparison of OCIAD2 mRNA expression in the human pancreatic cancer cell line PANC-1 with OCIAD2 knockdown. Figure 4F is a comparison of OCIAD2 mRNA expression in the human pancreatic cancer cell line AsPC-1 with OCIAD2 knockdown; Figure 5 This is a comparison chart of the proliferation of human pancreatic cancer cell lines with OCIAD2 knockdown detected by CCK8 method in Example 4 of the present invention: Figure 5 A is the proliferation curve of the human pancreatic cancer cell line PANC-1 and the control cell line detected by CCK8 method with OCIAD2 knockdown. Figure 5 B is a graph showing the proliferation curves of the human pancreatic cancer cell line AsPC-1 with OCIAD2 knockdown and the control cell line; Figure 6 This is a diagram of Ki67 and BrdU signals of human pancreatic cancer cell lines PANC-1 and AsPC-1 with OCIAD2 knockdown detected by flow cytometry in Example 4 of the present invention: Figure 6 A is the Ki67 signal diagram and quantitative analysis diagram of the human pancreatic cancer cell line PANC-1 with OCIAD2 knockdown. Figure 6 B is the Ki67 signal diagram and quantitative analysis diagram of the human pancreatic cancer cell line AsPC-1 with OCIAD2 knockdown. Figure 6 C is the BrdU signal diagram and quantitative analysis diagram of the human pancreatic cancer cell line PANC-1 with OCIAD2 knockdown. Figure 6 D is a diagram of BrdU signal and quantitative analysis of the human pancreatic cancer cell line AsPC-1 with OCIAD2 knockdown; Figure 7 This is a comparison diagram of Ki67 and BrdU fluorescence signals of OCIAD2 knockdown human pancreatic cancer cell lines PANC-1 and AsPC-1 detected by cell immunofluorescence method in Example 4. Figure 7 A is the Ki67 fluorescence signal diagram of the human pancreatic cancer cell line PANC-1 with OCIAD2 knockdown. Figure 7 B is the Ki67 fluorescence signal diagram of the human pancreatic cancer cell line AsPC-1 with OCIAD2 knockdown. Figure 7 C is the BrdU fluorescence signal of the human pancreatic cancer cell line PANC-1 with OCIAD2 knockdown. Figure 7 D is a graph of BrdU fluorescence signal of human pancreatic cancer cell line AsPC-1 with OCIAD2 knockdown; Figure 8 This is a validation diagram of the regulatory pathway of OCIAD2 overexpression in Example 5, including the expression levels of p-AKT, AKT, Cyclin D1, Cyclin E1, and ACTB proteins in OCIAD2 knockdown human pancreatic cancer cell lines PANC-1, AsPC-1, and control cell lines: Figure 8 A is the protein expression of OCIAD2 knockdown in the human pancreatic cancer cell line PANC-1. Figure 8B is the protein expression of OCIAD2 knockdown in the human pancreatic cancer cell line AsPC-1. Figure 8 C is a relative quantitative analysis of the expression of various proteins in the human pancreatic cancer cell line PANC-1 with OCIAD2 knockdown. Figure 8 D is a relative quantitative analysis of the expression of various proteins in the human pancreatic cancer cell line AsPC-1 with OCIAD2 knockdown; Fig. 9 This is a correlation analysis diagram of OCIAD2 and immune cell infiltration in PAAD according to Example 6 of the present invention: Fig. 9 A is the correlation between OCIAD2 expression level and various immune cell infiltrations. Fig. 9 B is the correlation analysis diagram between OCIAD2 expression level and immune checkpoint-related genes. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail in the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0020] The human pancreatic cancer cell lines (PANC-1, AsPC-1, MIAPACA-2, CFPAC-1, CAPAN-1) and human normal pancreatic ductal epithelial cell line HPNE used in the embodiments of the present invention were purchased from the American Type Culture Collection (ATCC) cell bank. Other reagents used are described in the corresponding embodiments.

[0021] Example 1: Database analysis of OCIAD2 expression in pancreatic cancer tissue and its relationship with patient prognosis Ovarian cancer immune response antigen domain protein 2 (OCIAD2) was studied using the BEST (Biomarker Exploration of Solid Tumors) database for its expression in pancreatic cancer tissues. The GEPIA2 (Gene Expression Profiling Interactive Analysis 2) was used to study the expression of OCIAD2 in pancreatic cancer tissues in the TCGA database. The difference in the expression of OCIAD2 in pancreatic cancer tissues and adjacent tissues was compared, and it was found that OCIAD2 mRNA was significantly overexpressed in pancreatic cancer tissues, such as Figure 1 As shown in Figure 2, the expression of OCIAD2 in pancreatic cancer tissues is significantly higher than that in adjacent tissues ( P<0.05); the relationship between OCIAD2 expression and disease-free survival was predicted using the TCGA database, and it was found that OCIAD2 expression was closely related to the poor prognosis of patients, such as Figure 2 As shown in A, the disease-free survival rate of pancreatic cancer patients with high expression of OCIAD2 was significantly reduced ( P =0.033); TCGA database and GSE183795 dataset were used to analyze the relationship between OCIAD2 expression and overall survival of pancreatic cancer patients. Figure 2 As shown in B and C, the overall survival rate of pancreatic cancer patients with high expression of OCIAD2 was significantly reduced ( P =0.0061, P =0.0119). The relationship between the expression level of OCIAD2 and clinical stage was analyzed using the TCGA database. Figure 2 As shown in Figure D, it was found that the expression level of OCIAD2 gradually increased with the advancement of tumor development. To further prove the relationship between the expression level of OCIAD2 and the poor prognosis of pancreatic cancer patients, univariate and multivariate analyses were performed, as shown in Figure 4. Figure 2 As shown in E, univariate analysis showed that poor prognosis was related to factors such as age and gender, while multivariate analysis showed that poor prognosis was significantly correlated with OCIAD2 ( P =0.001).

[0022] Example 2: Comparison of OCIAD2 protein expression level and OCIAD2 gene expression level in human pancreatic cancer cell line and OCIAD2 protein expression level and OCIAD2 gene expression level in normal human pancreatic ductal epithelial cell line, and immunohistochemical analysis of OCIAD2 expression in pancreatic cancer tissue 2.1 Expression levels of OCIAD2 protein in normal human pancreatic ductal epithelial cell line HPNE and human pancreatic cancer cell lines PANC-1, AsPC-1, MIAPACA-2, CFPAC-1, and CAPAN-1; Samples of human pancreatic cancer cell lines (PANC-1, AsPC-1, MIAPACA-2, CFPAC-1, CAPAN-1) and human normal pancreatic ductal epithelial cell line HPNE were selected and the following operations were performed: lysed on ice for 60 minutes with medium-efficiency RIPA lysis buffer (Biyuntian cat#P0013C) containing protease inhibitors and phosphatase inhibitors, the cells were scraped and collected in a 1.5mL EP tube, and centrifuged at 4℃ 12000rpm for 15 minutes. The supernatant was aspirated, and the protein concentration was measured by BCA (Thermo Fisher Cat: 23227), and then the denaturing reduction loading buffer (Yazyme Cat: LT101L) was added to adjust the sample protein concentration to be consistent. The protein sample was boiled in a 100℃ metal bath for 10 minutes, and the obtained protein sample was loaded on a 10% polyacrylamide gel (Yazyme Cat: PG112), and the SDS-PAGE gel electrophoresis conditions were 80V for the upper gel and 120V for the lower gel. Then, the membrane was transferred to a 0.45 μm cellulose acetate membrane (millpore Cat: HATF00010) at 130 V for 50 min, blocked with 5% skim milk powder at room temperature for 1-2 h at low speed, and then incubated with primary antibodies ACTB (Anti-beta Actin Rabbit pAb, Seville Cat: GB11001, 1:3000) and OCIAD2 (OCIAD2Rabbit pAb, Ibotek Cat: A14962, 1:1000) at 4 ° C overnight at low speed on a shaker. The next day, after recovering the primary antibody, the bands were washed three times at high speed in TBST (Seville Cat: T1085) buffer for 8 minutes each time. Then, the secondary antibody (HRPGoat Anti-Rabbit IgG (H+L), Ibotek Cat: AS014, 1:3000) was added and incubated at room temperature for 1 h at low speed, and then washed three times at high speed in TBST for 8 minutes each time. Finally, protein bands were visualized using ECL ultrasensitive luminescent liquid (Kermei Cat: M0101) on a BioRad gel imaging system.

[0023] WB reagent: medium-effect RIPA (Biyuntian Cat: P0013C), lyse on ice for 60 minutes, centrifuge at 4℃ 12000rpm for 15 minutes, discard cell debris and other precipitates, and collect protein supernatant. Then add BCA (Thermo Fisher Cat: 23227) colorimetric reagent for protein quantification, and add denaturing reduction loading buffer (Yazyme Cat: LT101L) metal bath 100℃ to cook the sample for 10 minutes. Electrophoresis was performed according to 20ug total protein per well, and the gel concentrations were 12.5% ​​(Yazyme Cat: LT101L) and 10% (Yazyme Cat: PG112). Skim milk powder (Biyuntian Cat: P0216), 10*TBS (Sevier Cat: G0015). The electrophoresis tank, glass plate, and electrophoresis instrument are all Bio-Rad series. Protein marker (Yamei Cat: WJ103), 0.45μm cellulose acetate membrane (Millipore Cat: HATF00010), ECL ultra-sensitive luminescent liquid (Keermei Cat: M0101); Experimental conditions: electrophoresis upper gel 80V, lower gel 120V, constant voltage 130V, 50min transfer. 5% skim milk powder was blocked at room temperature at low speed for 1-2h, primary antibody 1:1000 (with 3% BSA) was incubated at 4℃ shaker at low speed overnight. After the primary antibody incubation, the membrane was washed three times at high speed in TBST, each time for 8min. Then, the secondary antibody 1:3000 (with 5% skim milk powder) was added and incubated at room temperature at low speed for 1h. After the secondary antibody incubation, the membrane was washed three times at high speed in TBST, each time for 8min, and finally developed and exposed.

[0024] The experimental results are as follows Figure 3 As shown in A: The expression of ACTB (42KD) protein in the normal pancreatic ductal epithelial cell line HPNE and the human pancreatic cancer cell lines PANC-1, AsPC-1, MIAPACA-2, CFPAC-1, and CAPAN-1 is basically the same; however, compared with the normal pancreatic ductal epithelial cell line HPNE, the OCIAD2 (17KD) protein is significantly overexpressed in the human pancreatic cancer cell lines PANC-1, AsPC-1, MIAPACA-2, CFPAC-1, and CAPAN-1.

[0025] 2.2 Comparison of OCIAD2 mRNA expression levels in normal human pancreatic ductal epithelial cell line HPNE and human pancreatic cancer cell lines PANC-1, AsPC-1, MIAPACA-2, CFPAC-1, and CAPAN-1; Description of the experimental samples and reagents used in this example: RNA extraction kit Novizan FastPure® Cell / Tissue Total RNA Isolation Kit V2#RC112-01, Buffer RL, Buffer RW1Buffer RW2RNase-free ddH2O, FastPure gDNA-FilterColumns III (each in a 2 mL Collection Tube), FastPure RNA Columns III (each in a 2 mL Collection Tube), RNase-free Collection Tubes 1.5 mL; RT-PCR reagent 5×Primescript Rt Master Mix 2uL: Takara#RR036Q PrimeScript™ RT Master Mix (Perfect Real Time); qPCR reagent Takara#CN830S TB Green® Premix Ex Taq™ II FAST qPCR; Takara#CN830A TB Green® Premix Ex Taq™ II FAST qPCR; Total RNA was extracted from the samples using a column-based kit (NoviZan FastPure® Cell / Tissue Total RNA Isolation Kit V2#RC112-01). After trypsin digestion, cells were collected by centrifugation and added to Buffer RL. 6Add 500 μL Buffer RL to each cell and vortex until there is no obvious cell cluster; after lysis, transfer the sample to FastPure gDNA-Filter Columns III (FastPure gDNA-Filter Columns III has been placed in the collection tube), centrifuge at 12000 rpm for 30 sec, discard FastPure gDNA-Filter Columns III, and collect the filtrate; add 0.5 times the volume of filtrate anhydrous ethanol to the filtrate, mix thoroughly, transfer all the mixed solution to FastPure RNA Columns III (FastPure RNA Columns III has been placed in the collection tube), centrifuge at 12000 rpm for 30 sec, and discard the filtrate; add 700 μL Buffer RW1 to FastPure RNA Columns III, centrifuge at 12000 rpm for 30 sec, and discard the filtrate; add 700 μL Buffer RW2 to FastPure RNA Columns III (anhydrous ethanol has been added), centrifuge at 12000 rpm for 30 sec, discard the filtrate; add 500 μL Buffer RW2 (with anhydrous ethanol added) to FastPure RNA Columns III, centrifuge at 12,000 rpm for 2 min, remove the adsorption column from the collection tube; transfer the adsorption column to a new RNase-free Collection Tubes 1.5 mL centrifuge tube, drop it in the middle of the adsorption column, add 50 μL RNase-free ddH2O, let it stand at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min to elute the RNA; use NanoDrop2000 to determine the RNA concentration; prepare the reverse transcription system according to 2 μL of 5× Primescript Rt Master Mix (Takara#RR036Q PrimeScript™ RTMaster Mix (Perfect Real Time)) 4.0 μL, 1.0 μg of Total RNA, and 20 μL of RNase-Free dH2O for each system, and use a PCR instrument (Veriti™ Dx 96-well Thermal Cycler, 0.2 mL#4452330) was reverse transcribed by one-step method (37℃15min, 85℃5s, 4℃) to obtain cDNA; 2μL of cDNA, 7.2μL of enzyme-free H2O, 10μL of SYBgreen, 0.8μL, prepare the system, add the sample to the PCR plate, seal it with a sealing film (Biyuntian fluorescent quantitative PCR sealing film (ABI subpackage) #FSF002), centrifuge at 4℃, 3000rpm for 2min, and use a PCR instrument (VeritiPro™ Thermal Cycler, 96 well#A48141) to amplify the cDNA using a two-step method (2 stepPCR*4, Cycles: 40, 95℃ 5s, 60℃ 10s). .

[0026] The upstream and downstream primer sequences of the OCIAD2 gene in this example are: Forward 5 '-TAGTCCCTGGGTGACAAGCATG-3 '; Reverse 5 '-GCACATCCACAGAGCAGAGATC-3 '.

[0027] The internal reference GAPDH gene primer sequence in the PCR of this example is: Forward 5 '-GTCTCCTCTGACTTCAACAGCG-3 '; Reverse 5'-ACCACCCTGTTGCTGTAGCCAA-3'.

[0028] The experimental results are as follows Figure 3 As shown in B: Compared with the normal pancreatic ductal epithelial cell line HPNE, the mRNA level of OCIAD2 was significantly overexpressed in PANC-1, AsPC-1, MIAPACA-2, CFPAC-1, and CAPAN-1 cell lines.

[0029] 2.3 The human pancreatic cancer tissue and normal tissue samples in this example were obtained from Shanghai Xinchao Technology Co., Ltd. (Cat. No.: HPanA020PG03; Ethics No.: SHYJS-CP-1810012). The OCIAD2 protein expression levels of pancreatic cancer tissue and normal tissue were analyzed by immunohistochemical staining results.

[0030] Description of reagents used in this example experiment: PBST (Cat: P1031), 10*PBS (Cat: G4207-500ML), Tween 20 (Cat: T8220); SP Rabbit & Mouse HRP Kit (DAB) (Cat: CW2069S): Solution A (Endogenous Peroxydase Enzymes Blocking Buffer, White Solution), Solution B (Normal Goat Serum For Blocking, White Solution), Solution C (Goat anti-Rabbit / Mouse IgG, Biotin, Ready to Use, Yellow Solution), Solution D (Streptavidin-HRP, Ready to Use, Red Solution), DAB-A (20×), DAB-B (1×); In the experiment, six paraffin sections of human pancreatic cancer tissue and six paraffin sections of normal tissue were prepared and processed for staining comparison; The section processing and staining process is as follows: paraffin sections were placed in a 60°C oven for 30 min, then immersed in xylene for 10 min, replaced with xylene and immersed for another 10 min for dewaxing; sections were immersed in anhydrous ethanol for 5 min, replaced with anhydrous ethanol and immersed for another 5 min, then immersed in 90% ethanol, 80% ethanol, 70% ethanol, and 60% ethanol for 5 min each, and finally immersed in ddH2O for 2 min, then washed 3 times with PBS, 3 min / time, for hydration; sections were placed in a container containing citrate buffer (pH=6.0), and the container was placed in a microwave oven and heated at high speed until the repair solution boiled, and the microwave oven was adjusted to low speed to maintain heating for 10 min, and the container, repair solution and sections were removed and cooled to room temperature; sections were immersed in ddH2O for 3 min; then washed in a liquid tank with PBST (PBS+0.1% Tween 20) for 3 min; a drop of endogenous peroxidase blocking solution (SP Rabbit & Mouse HRP Kit was incubated at room temperature for 10 min, rinsed thoroughly with PBST, and blocked with sheep serum for 1 h at room temperature; after rinsing thoroughly with PBST, OCIAD2 primary antibody (OCIAD2 Polyclonal Antibody, Thermo Fisher Scientific Cat: PA5-59421, 1:200) was added, incubated at 4°C overnight, and then recovered at room temperature for 15 min, rinsed thoroughly with PBST, and secondary antibody (SP Rabbit & Mouse HRP Kit, Kangwei Century Cat: CW2069S) was added and incubated at room temperature for 30 min, rinsed thoroughly with PBST, and then DAB colorimetric solution (SP Rabbit & Mouse HRP Kit, Kangwei Century, Cat: CW2069S, DAB-A: DAB-B = 1:19) was added and stained in the dark for 3 min, and washed with ddH2O for 1 min; counterstained with hematoxylin for 30 s, and differentiation solution (1% HCl in The sections were immersed in 70%, 80%, and 90% ethanol for 20 seconds each after rinsing with tap water to blue, and finally immersed in 100% ethanol for 30 seconds, replaced with 100% ethanol and immersed for another 30 seconds, immersed in xylene for 2 minutes, replaced with xylene and immersed for another 2 minutes; then the sections were taken out, sealed with neutral gum, and observed and photographed with a slide scanner SQS-1000 of Shengqiang Technology.

[0031] The results are as follows Figure 3 C, The expression level of OCIAD2 protein in pancreatic cancer tissue (PAAD group) was significantly higher than that in normal tissue (Normal group).

[0032] Example 3: In this example, human pancreatic cancer cell lines PANC-1 and AsPC-1 that highly expressed OCIAD2 were transfected with siRNA-OCIAD2 sequences to construct an OCIAD2 knockdown model, and the knockdown effect was verified by Western Blot and qPCR.

[0033] 3.1 OCIAD2 mRNA expression level in OCIAD2 knockdown human pancreatic cancer cell lines PANC-1 and AsPC-1; Experimental reagents: siRNA transfection reagent: Gene siRNA-mate plus transfection kit (universal siRNA / miRNA); RNA extraction kit: Novezan FastPure® Cell / Tissue Total RNA Isolation Kit V2#RC112-01; RT-PCR reagent: 5×Primescript RT Master Mix, Takara#RR036); qPCR reagent: Takara#CN830STB Green® Premix Ex Taq™ II FAST qPCR; Takara#CN830A TB Green® Premix ExTaq™ II FAST qPCR.

[0034] The specific experimental process is as follows: dilute 22pmol siRNA with 200μL jetPRIME solution, vortex for 10s, add 4μL jetPRIME transfection reagent, vortex for 10s, incubate at room temperature for 10min, add the transfection solution to the cells in serum-containing medium, replace the culture medium after 6h, and collect the cells after 48h. Use the column method kit (Novizan FastPure® Cell / TissueTotal RNA Isolation Kit V2#RC112-01) to extract total RNA from the sample. The specific operation is: after trypsin digestion of cells, centrifuge to collect the cell precipitate and add Buffer RL, each <5×10 6Add 500 μL Buffer RL to each cell, vortex until there is no obvious cell cluster; transfer the lysed sample to FastPure gDNA-Filter Columns III (FastPuregDNA-Filter Columns III has been placed in a collection tube), centrifuge at 12000 rpm for 30 sec, discard FastPure gDNA-Filter Columns III, and collect the filtrate; add 0.5 times the volume of filtrate anhydrous ethanol to the filtrate, mix thoroughly, transfer all the mixed solution to FastPure RNA Columns III (FastPure RNA Columns III has been placed in a collection tube), centrifuge at 12000 rpm for 30 sec, and discard the filtrate; add 700 μL BufferRW1 to FastPure RNA Columns III, centrifuge at 12000 rpm for 30 sec, and discard the filtrate; add 700 μL BufferRW2 to FastPure RNA Columns III (anhydrous ethanol has been added), centrifuge at 12000 rpm for 30 sec, discard the filtrate; add 500 μL Buffer RW2 (with anhydrous ethanol added) to FastPure RNA Columns III, centrifuge at 12000 rpm for 2 min, remove the adsorption column from the collection tube; transfer the adsorption column to a new RNase-free Collection Tubes 1.5 mL centrifuge tube, drop it in the middle of the adsorption column, add 50 μL RNase-free ddH2 O, let it stand at room temperature for 1 min, centrifuge at 12000 rpm for 1 min to elute RNA; use NanoDrop2000 to determine the RNA concentration; prepare the reverse transcription system according to 5× Primescript Rt Master Mix 2uL (Takara#RR036Q PrimeScript™ RT Master Mix (Perfect Real Time)) 4.0μL, Total RNA 1.0μg, RNase-Free dH2O to supplement the system to 20μL, and use a PCR instrument (Veriti™ Dx 96-well Thermal Cycler, 0.2 mL#4452330) was reverse transcribed by one-step method (37℃15min, 85℃5s, 4℃) to obtain cDNA; the system was prepared according to the following steps: 2μL of cDNA, 7.2μL of enzyme-free H2O, 10μL of SYBgreen, and 0.8μL of upstream and downstream primer mixture (10μm) per well of a 96-well plate.The sample was added to the PCR plate, sealed with a sealing film (Biyuntian Cat: FSF002), centrifuged at 3000 rpm for 2 min at 4°C, and amplified using a PCR instrument (VeritiPro™ Thermal Cycler, 96 well#A48141) using a two-step method (2 step PCR*4, Cycles: 40, 95°C 5s, 60°C 10s) to obtain cDNA.

[0035] The sequence of siRNA-OCIAD2 in this example is shown in Table 1:

[0036] Table 1 The primer sequence of OCIAD2 in the qRT-PCR experiment of this example is: Forward 5 '-TAGTCCCTGGGTGACAAGCATG-3 '; Reverse 5 '-GCACATCCACAGAGCAGAGATC-3 '.

[0037] The GAPDH primer sequences in the qPCR test of this example are: forward 5 '-GTCTCCTCTGACTTCAACAGCG-3 '; reverse 5'-ACCACCCTGTTGCTGTAGCCAA-3'.

[0038] The experimental results are as follows Figure 4 As shown in E and F, human pancreatic cancer cell lines PANC-1 and AsPC-1 that highly expressed OCIAD2 were transfected with siRNA-OCIAD2 sequence, and the expression of OCIAD2 mRNA was significantly reduced.

[0039] 3.2 OCIAD2 protein expression levels in OCIAD2 knockdown human pancreatic cancer cell lines PANC-1, AsPC-1 and control cell lines In this example, human pancreatic cancer cell lines PANC-1 and AsPC-1 with high expression of OCIAD2 were selected, 22 pmol siRNA was diluted with 200 μL jetPRIME solution, vortexed for 10 s, 4 μL jetPRIME was added, vortexed for 10 s, incubated at room temperature for 10 min, and the transfection solution was added to the cells in serum-containing medium for incubation. Fresh medium was replaced after 6 h, and cells were collected after 48 h. The cells were lysed on ice for 60 min using a medium-efficiency RIPA lysis buffer (Biyuntian Cat: P0013) containing protease inhibitors and phosphatase inhibitors, and then centrifuged at 4°C 12000 rpm for 15 min. The supernatant was aspirated and the protein concentration was measured by BCA (Thermo Cat: 23227). Then, the denaturing reducing loading buffer (Yazyme Cat: LT101) was added to adjust the sample protein concentration to be consistent. The sample tube was boiled in a 100°C metal bath for 10 min, and the obtained protein sample was loaded onto an SDS-PAGE gel for electrophoresis. The upper gel was 80V, the lower gel was 120V, and then transferred to a nitrocellulose membrane at 130V for 50 min. The membrane was blocked at room temperature for 2 h with 5% skim milk powder (TBST). Then, the primary antibodies ACTB (Anti-beta Actin Rabbit pAb Servicebio, Cat: GB11001, 1:3000) and OCIAD2 (OCIAD2 Rabbit pAb (A14962), Aibocan Cat: A14962, 1: 1000) was incubated overnight at 4°C on a shaker at low speed. The next day, the membrane was washed three times at high speed with TBST buffer, and then incubated at low speed for 1 hour at room temperature with secondary antibody (HRP Goat Anti-Rabbit IgG (H+L) abclonal (AS014) 1: 3000). After the secondary antibody was finished, the membrane was washed three times at high speed with TBST, each time for 8 minutes, and the protein bands were developed on the BioRad ChemiDoc XRS+ imaging system with ECL supersensitive luminescent liquid (Ke Ermei Cat: M010).

[0040] WB reagents: medium-acting RIPA (Biyuntian Cat: P0013C), BCA (Thermo Fisher Scientific Cat: 23227), loading buffer (Yazyme Cat: LT101L), protein gel (Yazyme Cat: PG112), marker (Yazyme Cat: WJ103), 0.45μm NC membrane (Millipore Cat: HATF0001), skimmed milk powder (Biyuntian Cat: P021), ECL ultra-sensitive luminescent liquid (Keermei Cat: M0101), 10*TBS (Sevier Cat: G0015).

[0041] Experimental conditions: protein electrophoresis upper gel 80V, lower gel 120V, transfer membrane 130V constant voltage 50min. 5% skim milk powder (TBST) blocked at room temperature at low speed for 2h, primary antibody 1:1000 (3% BSA) incubated at 4℃ shaker at low speed overnight, washed three times at medium and high speed in TBST, 8min each time, added secondary antibody 1:3000 (5% skim milk powder) incubated at room temperature at low speed for 1h, washed three times at medium and high speed in TBST, 8min each time, and finally exposed to light.

[0042] The experimental results are as follows Figure 4 As shown in A and B, the expression of OCIAD2 protein was significantly reduced after the human pancreatic cancer cell lines PANC-1 and AsPC-1, which highly expressed OCIAD2, were transfected with the siRNA-OCIAD2 sequence. Figure 4 A and Figure 4 B shows that the expression of ACTB protein remains unchanged, indicating that the siRNA transfection cell experiment of the present invention did not affect the expression of other proteins, and the pancreatic cancer cell line model with knockdown of OCIAD2 was successful. The quantitative results of the three experiments are shown in Figure 4 C and 4D, after the human pancreatic cancer cell lines PANC-1 and AsPC-1 that highly expressed OCIAD2 were transfected with siRNA-OCIAD2 sequence, the expression of OCIAD2 protein was significantly reduced; Figure 4 E and 4F After the human pancreatic cancer cell lines PANC-1 and AsPC-1 that highly expressed OCIAD2 were transfected with siRNA-OCIAD2 sequences, the expression of OCIAD2 mRNA was significantly reduced.

[0043] Example 4: Effect of OCIAD2 on the proliferation of pancreatic cancer cells In this example, the proliferation of OCIAD2 knockdown human pancreatic cancer cell lines PANC-1 and AsPC-1 cells was detected by CCK8 method, cell proliferation signals Ki67 and BrdU were detected by flow cytometry, and cell immunofluorescence was used to detect cell proliferation signals Ki67 and BrdU. The specific experiments are as follows: 4.1 CCK8 assay to detect proliferation of OCIAD2 knockdown human pancreatic cancer cell lines PANC-1, AsPC-1 and control cell lines; Experimental steps: In the PANC-1 and AsPC-1 cell lines with OCIAD2 knockdown and the control cell line, the cells were digested with trypsin, and the cell suspension was plated in a 96-well plate with 3000 cells per well and 4 replicates per group. After continued culture for 24, 48, 72, and 96 h, the culture medium containing CCK8 reagent (Cat: CK04, Japan Tongjin) was replaced. After incubation for 1 h, the absorbance value of the supernatant was detected at 450 nm using a microplate reader (SpectraMax i3x Multi-Mode Microplate Reader) to detect cell proliferation.

[0044] Experimental reagents: Trypsin (Gibco™ Trypsin-EDTA (0.25%), containing phenol red #25200072); CCK8 (Japan Tongren Cat: CK04); The experimental results are as follows Figure 5 A and 5B, CCK8 experiments showed that compared with the control cell line si-NC, the proliferation of cells in the OCIAD2 knockdown human pancreatic cancer cell lines PANC-1 si-1, si-2 and si-3, and OCIAD2 knockdown human pancreatic cancer cell lines AsPC-1 si-1, si-2 and si-3 was significantly inhibited after continued culture for 48 h.

[0045] 4.2 Comparison of Ki67 and BrdU signals in OCIAD2 knockdown human pancreatic cancer cell lines PANC-1 and AsPC-1 by flow cytometry; Experimental steps: Ki67 (antigen identified by monoclonal antibody Ki67) OCIAD2 knockdown PANC-1, AsPC-1 cell lines and control cell lines were digested with trypsin (Gibco™ Trypsin-EDTA (0.25%), containing phenol red #25200072), centrifuged at 1000rpm for 5 minutes, discarded the supernatant, and permeabilized with membrane permeabilization buffer for 15-20 minutes; an appropriate amount of 1× washing buffer (10× washing buffer: ddH2O=1:9) was added, centrifuged at 3000rpm for 5 minutes, and the supernatant was discarded; Ki67 antibody (Brilliant Violet 605™ anti-human Ki-67 Antibody, biolegendCat:350521) was prepared with 1× washing buffer, and stained at 4℃ in the dark for 30 minutes; after washing the cells twice with 1× washing buffer, the cells were resuspended with 500μL washing buffer and detected using BD FACSCanto™ 10-Color Configuration#657338.

[0046] BrdU (5-Bromo-2′-deoxyuridine, an analog of thymidine, with a bromine atom replacing the hydrogen atom at the fifth carbon of thymidine) OCIAD2 knockdown PANC-1, AsPC-1 cell lines and control cell lines were taken, and BrdU dye BrdU 5-bromo-2′-deoxyuridine, Solarbio Cat: B801, was added at a concentration of 10ug / mL. After 24 hours, trypsin (Gibco™ Trypsin-EDTA (0.25%), containing phenol red #25200072) was used for digestion, centrifuged at 1000rpm for 5 minutes, and the supernatant was discarded. The membrane was permeabilized with membrane permeabilization buffer for 15-20 minutes; an appropriate amount of 1× washing buffer (10× washing buffer: ddH2O=1:9) was added, and the cells were centrifuged at 3000rpm for 5 minutes, and the supernatant was discarded; 1× washing buffer was used to prepare BrdU antibody (BrdU (Rabbit): Anti-BrdU Recombinant Rabbit MonoclonalAntibody [PSH0-18], Huaan Bio Cat:HA721283, 1:400), stained at 4℃ in the dark for 30min; washed cells twice with 1× washing solution, resuspended cells with 500μL washing solution, and detected using BD FACSCanto™ 10-ColorConfiguration#657338.

[0047] Experimental reagents: Trypsin (Gibco™ Trypsin-EDTA (0.25%), containing phenol red #25200072); Brilliant Violet605™ anti-human Ki-67 Antibody, biolegend Cat: 350521); BrdU (Rabbit): Anti-BrdU Recombinant Rabbit Monoclonal Antibody [PSH0-18] Huaan Biotechnology, Cat: HA721283, 1: 400; Foxp3 / transcription factor flow fixation permeabilization buffer #00-5523-00; The experimental results are as follows Figure 6 Shown: Flow cytometry showed that the Ki67 and BrdU signals of OCIAD2 knockdown human pancreatic cancer cell lines PANC-1 and AsPC-1 were significantly reduced compared with the control cell lines.

[0048] 4.3. Comparison of Ki67 and BrdU fluorescence signals of OCIAD2 knockdown human pancreatic cancer cell lines PANC-1 and AsPC-1 detected by cell immunofluorescence assay; Experimental steps: Ki67: The culture plates of PANC-1, AsPC-1 cell lines with OCIAD2 knockdown and control cell lines were taken out of the culture medium, fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 1% Triton (Thermo Fisher Triton X-100 Cat: HFH10) for 15 minutes, blocked with 3% BSA for 30 minutes, and added with Ki67 primary antibody: Ki-67 Monoclonal Antibody (SolA15), eBioscience™(14-5698-82) 1:300), incubated at 4°C overnight, and then added with fluorescent secondary antibodies (Donkey anti-Rat IgG (H+L) HighlyCross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 555, Thermo Fisher: A48270, 1:200; Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488, Thermo Fisher: A-11008, 1:200), followed by the addition of DAPI (Thermo Fisher DAPI and Hoechst Nucleic Acid Stains #D1306) to stain the nucleus for 10 min in the dark, and the staining results were observed using the PerkinElmer UltraVIEW VoX high-speed laser confocal real-time imaging analysis system.

[0049] BrdU: OCIAD2 knockdown PANC-1, AsPC-1 cell lines and control cell lines were taken, and BrdU dye (BrdU 5-bromo-2′-deoxyuridine, Solarbio Cat: B8010, 10ug / ml) was added. After 48h of transfection, the culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 15min, permeabilized with Triton (Thermo Fisher Triton X-100 (1%) #HFH10) for 15min, blocked with 3% BSA for 30min, and incubated with BrdU primary antibody (Anti-BrdU Recombinant Rabbit Monoclonal Antibody [PSH0-18], Huaan Bio Cat: HA721283) at 4℃ overnight, followed by the addition of fluorescent secondary antibodies (Donkey anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 555, Thermo Fisher Cat: A48270, 1:200; Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, AlexaFluor™ 488, Thermo Fisher Scientific Cat: A11008, 1:200) was incubated at room temperature in the dark for 30 min. After the secondary antibody reaction, DAPI (Thermo Fisher Scientific DAPI and Hoechst Nucleic Acid Stains #D1306) was added to stain the nucleus in the dark for 10 min, and the staining results were observed using the PerkinElmer UltraVIEW VoX high-speed laser confocal real-time imaging analysis system.

[0050] Experimental reagents: Trypsin (Gibco™ Trypsin-EDTA (0.25%), containing phenol red #25200072); Triton (Thermo Fisher Triton X-100 (1%) #HFH10), Ki-67 Monoclonal Antibody (SolA15) (eBioscience™ (14-5698-82) 1:300); BrdU dye (BrdU 5-bromo-2′-deoxyuridine Solarbio (B8010), used at a concentration of 10ug / ml); BrdU (Anti-BrdU Recombinant Rabbit Monoclonal Antibody [PSH0-18] Huaan Bio (HA721283)); fluorescent secondary antibody (Donkey anti-Rat IgG (H+L) Highly Cross-AdsorbedSecondary Antibody, Alexa Fluor™ Plus 555, ThermoFisher (A48270) 1:200; Goatanti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488, ThermoFisher (A-11008) 1:200); DAPI (ThermoFisher DAPI and Hoechst Nucleic AcidStains#D1306)) The experimental results are as follows Figure 8 As shown: Cell immunofluorescence analysis showed that the Ki67 and BrdU fluorescence signals of OCIAD2 knockdown human pancreatic cancer cell lines PANC-1 and AsPC-1 were significantly reduced compared with the control cell lines.

[0051] Example 4: By transfecting the human pancreatic cancer cell lines PANC-1 and AsPC-1 that highly express OCIAD2 with the sequence of siRNA-OCIAD2, an OCIAD2 knockdown model was constructed, and it was found that the expression of OCIAD2 mRNA was significantly reduced, and the expression of OCIAD2 protein was significantly reduced; the growth of the human pancreatic cancer cell lines PANC-1 and AsPC-1 with OCIAD2 knockdown was significantly inhibited by continuing to culture them for 48 hours, and the Ki67 and BrdU signals of the human pancreatic cancer cell lines PANC-1 and AsPC-1 with OCIAD2 knockdown were significantly reduced; the Ki67 and BrdU fluorescence signals of the human pancreatic cancer cell lines PANC-1 and AsPC-1 with OCIAD2 knockdown were significantly reduced, which shows that knocking down OCIAD2 can significantly inhibit the proliferation of pancreatic cancer cells.

[0052] Example 5: Regulatory pathways for overexpression of OCIAD2 p-AKT, AKT, Cyclin D1, Cyclin E1, and ACTB protein expression levels in OCIAD2-knockdown human pancreatic cancer cell lines PANC-1, AsPC-1, and control cell lines.

[0053] Experimental steps: The OCIAD2 knockdown human pancreatic cancer cell lines PANC-1, AsPC-1 and control cell lines were lysed on ice for 60 min using a medium-efficiency RIPA lysis buffer (Biyuntian Cat: P0013C) containing protease inhibitors and phosphatase inhibitors, centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was aspirated. The protein concentration was measured by the BCA (Thermo Cat: 23227) method, and then the denaturing reducing loading buffer (Yazyme Cat: LT101L) was added and boiled in a 100°C metal bath for 10 min. The obtained protein samples were electrophoresed using 10% SDS-PAGE gel (Yazyme Cat: PG112). The electrophoresis conditions of the upper gel were 80V, the lower gel was 120V, and the transfer conditions were 130V constant voltage for 50min. Then, the membrane was blocked with 5% skim milk powder (prepared with TBST) at room temperature for 2h at a low speed. Then, the primary antibodies ACTB (Anti-betaActin Rabbit pAb, Sewell Cat: GB11001, 1:3000), Cyclin D1 (Aibon Cat: A19038, 1:1000), Cyclin E1 (Aibon Cat: A14225, 1:100), p-AKT (Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060, Cell Signaling Cat: 4060S, 1:600), AKT (akt (pan) (C67E7) Rabbit mAb #4060, Cell Signaling Cat: 4060S, 1:600), and AKT (p-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT (P-AKT mAb#4691 Cellin Cat:4691S, 1:1000) was incubated overnight at 4°C in a shaker at low speed. The next day, the membrane was washed three times at high speed with TBST buffer, and then incubated with secondary antibody (HRP Goat Anti-Rabbit IgG (H+L) Aibo Anti Cat:AS014, 1:3000) at room temperature at low speed for 1h, washed three times at high speed with TBST (Solebo Cat:T1085), each time for 8min, and protein bands were displayed on the BioRad ChemiDoc XRS+ imaging system using ECL supersensitive luminescent solution (Ke Ermei Cat:M0101).

[0054] WB reagents: Medium-effect RIPA (Biyuntian Cat: P0013C), BCA (Thermo Fisher Scientific Cat: 23227), loading buffer (Yazyme Cat: LT101L), protein gel (Yazyme Cat: PG112), marker (Yazyme Cat: WJ103), 0.45μm NC membrane (Millipore Cat: HATF0001), skim milk powder (Biyuntian Cat: P021), ECL ultra-sensitive luminescent solution (Keermei Cat: M0101) Experimental conditions: Electrophoresis upper gel 80V, lower gel 120V, transfer constant voltage 130V 50min. 5% skim milk powder (TBST) blocked at room temperature at low speed for 2h, primary antibody 1:1000 (3% BSA) incubated at 4℃ shaker at low speed overnight, washed three times at medium and high speed in TBST, each time for 8min, added secondary antibody 1:3000 (5% skim milk powder) incubated at room temperature at low speed for 1h, washed three times at medium and high speed in TBST, each time for 8min, exposed and developed.

[0055] The experimental results are as follows Figure 8 As shown: Compared with the control cell lines, the AKT phosphorylation level in the human pancreatic cancer cell lines PANC-1 and AsPC-1 with OCIAD2 knockdown was significantly reduced, and the expression levels of Cyclin D1 and Cyclin E1 were significantly reduced. This shows that the overexpression of OCIAD2 affects the cell cycle by regulating the AKT pathway, thereby promoting the proliferation of pancreatic cancer cells.

[0056] Example 6: Relationship between OCIAD2 and immune infiltration The TIMER (Tumor Immune Estimation Resource) database was used to analyze the relationship between OCIAD2 expression level and immune cell infiltration level, and the correlation between OCIAD2 expression level and immune checkpoint-related genes was analyzed. Fig. 9 A is the relationship between OCIAD2 expression level and immune cell infiltration level, where OCIAD2 expression level is related to CD4+T cells ( cor =-0.203, P <0.05), macrophages ( cor =-0.086, P <0.05). In addition, the correlation analysis between OCIAD2 and immune checkpoint-related genes showed that OCIAD2 was closely related to multiple immune checkpoint genes (SIGLEC15, PDCD1, PDCD1LG2, HAVCR2, CTLA4, TIGIT, CD274, LAG3), such as Fig. 9As shown in Figure B. It can be seen that the expression of OCIAD2 in pancreatic cancer is not only related to the infiltration of immune cells, but also to the immunotherapy of PAAD patients. PAAD patients with high expression of OCIAD2 may have a better effect on related immunotherapy and can choose immunotherapy for treatment.

[0057] It should be noted that: the present invention is conducted using pancreatic cancer cell lines PANC-1, AsPC-1, MIAPACA-2, CFPAC-1 and CAPAN-1 to determine that OCIAD2 is a new marker and therapeutic target for pancreatic cancer, but the present invention does not limit OCIAD2 to be a new marker and therapeutic target for other pancreatic cancer cell lines. When conducting the experiments of the above-mentioned Examples 1 to 7 of the present invention, it is determined that OCIAD2 is a marker and therapeutic target for other pancreatic cancer cell lines and is related to pancreatic cancer immunotherapy, and other pancreatic cancer cell lines are also within the protection scope of the present invention.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of OCIAD2 as a pancreatic cancer marker in the detection of pancreatic cancer products.

2. The use according to claim 1, characterized in that: The pancreatic cancer detection product includes a detection reagent and a detection kit.

3. The use according to claim 2, characterized in that: The detection kit comprises: a reagent for detecting OCIAD2, a reagent for detecting the OCIAD2 gene, a reagent for detecting the OCIAD2 gene expression product, and / or a reagent for detecting the OCIAD2 antigen.

4. The use according to claim 3, characterized in that: The detection kit comprises reagents used for analyzing the expression of OCIAD2 by immunohistochemistry, or reagents used for detecting the expression of OCIAD2 by immunoblotting.

5. The use according to claim 3, characterized in that: The detection kit comprises reagents used for detecting OCIAD2 mRNA by real-time quantitative polymerase chain reaction.

6. The use according to any one of claims 1 to 5, characterized in that: The OCIAD2 is a marker for poor prognosis in pancreatic cancer.

7. An application of OCIAD2 as a pancreatic cancer therapeutic target in the preparation of a drug for treating pancreatic cancer.

8. The use according to claim 7, characterized in that: The pancreatic cancer treatment target is the AKT pathway.

9. The use according to claim 8, characterized in that: The pancreatic cancer therapeutic target is AKT phosphorylation, or the expression of Cyclin D1 and Cyclin E1.

10. An application of OCIAD2 in a pancreatic cancer immunotherapy product.

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