The Expression Of CPNE8 In Pancreatic Cancer And Its Role In Proliferation,Migration And Invasion

BackgroundPancreatic cancer is a malignant tumor with poor prognosis.It has high proliferation capacity and high ability to invade and metastasize.Most pancreatic cancer patients are often in advanced disease with local or lymph node metastasis at the time of consultation,which is difficult to treat.Less than 20%of patients can be treated with radical resection.Radical resection is the most basic treatment for pancreatic cancer,but studies have shown that it would not improve the median survival time of patients.Therefore,exploring the causes and molecular mechanisms of pancreatic cancer with high proliferation,invasion and metastasis ability can provide new theoretical basis for the development of drugs and provide technical support for effective treatment of patients with advanced pancreatic cancer.Different gene expression patterns between tumor cells and normal cells may be one of the reasons for the malignant phenotype of tumor cells.Different expressions of oncogenes or tumor suppressor genes will produce cancer-promoting or tumor-suppressing effects,which will affect the function of tumor cells and can change the process of tumor progression.The high proliferation,invasion and metastasis ability of pancreatic cancer may be related to the high expression of some oncogenes and the low expression of tumor suppressor genes in cancer cells.The discovery and verification of characteristic genes in pancreatic cancer is of great significance for elucidating the molecular mechanism of the occurrence and development of the disease,and can provide an important basis for the early diagnosis of pancreatic cancer and the development of targeted therapeutic drugs.CPNE is a Ca²⁺dependent cell membrane-binding protein found in paramecium,whose family proteins are closely related to the development of tumors,including CPNE1,CPNE3,CPNE7,etc.However,there are few studies on CPNE8 and tumors.CPNE8 has been confirmed to bind to Runx1 and play a role in acute myeloid leukemia.It is also a potential target gene of mi R-375,which may affect the development of breast cancer.At the same time,CPNE8 is a characteristic gene of ovarian clear cell carcinoma.However,CPNE8 has not been investigated in pancreatic cancer.ObjectiveTo investigate the expression of CPNE8 in pancreatic cancer and its role in promoting proliferation,invasion and metastasis of pancreatic cancer cells.Methods1.Identification of characteristic genes for pancreatic cancer1.1 High-throughput gene chip was used to estimate gene expression in pancreatic cancer and normal tissues,and the differential genes were analyzed（fold change>1.2,P<0.01）.1.2 Analyzing gene expression in 39 pancreatic cancer samples and 39 matched pancreas normal tissue samples in the GSE15471 data set and the differential genes（fold change>1.2,P<0.01）.1.3 Jointly analyzing gene expression data and differential genes of the gene chip data and GSE15471 data set.1.4 Survival analysis of the effect of CPNE8 expression on the prognosis of pancreatic cancer.2.Clinical correlation analysis and in vitro cell function experiments2.1 The gene chip results,GSE15471,GSE19650,GSE16515,GSE28735,TCGA,and GTEx databases were analyzed for the expression of CPNE8 in pancreatic cancer tissues and normal pancreatic tissues.2.2 Immunohistochemical analysis of CPNE8 protein expression in pancreatic cancer tissues and normal pancreatic tissues.2.3 Immunofluorescence analysis of the localization of CPNE8 protein in pancreatic cancer cells.2.4 Pancreatic cancer patients were divided into CPNE8 high expression group（n=88）and CPNE8 low expression group（n=88）,and the differences in gender,survival status,and age distribution between the two groups were analyzed.2.5 Western blot was used to detect the expression of CPNE8 in pancreatic cancer cell lines and normal pancreatic cell lines.2.6 siRNA was used to interfere with the expression of CPNE8 in pancreatic cancer cell lines CFPAC-1 and AsPC-1,and CCK8 and clone formation assay were utilized for estimating proliferation activity.Transwell was utilized for estimating migration and invasion ability,as well as scratch test.2.7 siRNA was used to interfere with the expression of CPNE8 in pancreatic cancer cell line CFPAC-1.The proportion of apoptosis was detected by flow cytometry and cell cycle changes were detected by flow cytometry.3.In vivo experiments and preliminary exploration of mechanisms3.1 Western blot was used to detect the changes of Bmi1 and Vimentin expression after interfering with the expression of CPNE8 in pancreatic cancer cell line CFPAC-1.3.2 The effects of knockdown of CPNE8 expression on the ability of pancreatic cancer cell lines to form subcutaneous tumors in nude mice were tested in vivo.Immunohistochemical detection of ki67 and PCNA expression were performed.3.3 Pancreatic cancer patients were divided into CPNE8 high expression and low expression groups,and differentially expressed genes were compared.3.4 GSEA was used to analyze the effects of CPNE8 expression on pathway enrichment.3.5 Western blot was used to detect changes in MAPK and p-MAPK expression after interference with CPNE8 expression.3.6 The correlation between CPNE8 expression and the expression of key molecules in Hippo pathway was analyzed.3.7 Construction of CPNE8 protein interaction network.Results1.Identification of characteristic genes for pancreatic cancer1.1 High-throughput gene chip results showed that some genes in pancreatic cancer tissues were significantly overexpressed,including CEACAM6,TCN1,GPRC5A,SLPI.Some genes were significantly under expressed including SERPINI2、ALB、CEL、ERP27.1.2 Analysis of the GSE15471 data set revealed that SLC6A14,GJB2,COL11A1,and MATN3 were significantly higher expressed in pancreatic cancer tissues,while DPP10,GUCA1C,PM20D1,and GNMT were significantly lower expressed in pancreatic cancer tissues.1.3 The gene chip data and GSE15471 data set were jointly analyzed and found that a total of 267 genes were common differential genes（fold change>1.2,P<0.01）,such as CXCR4,KLF5,S100A4,S100A6,CPNE8,and so on.1.4 CPNE8 had a significant effect on the survival time of patients with pancreatic cancer（P=0.033）.2 Clinical correlation analysis and in vitro cell function experiments2.1 The gene chip results in this study,GSE15471,GSE19650,GSE16515,GSE28735,TCGA and GTEx databases showed that CPNE8 expressed stronger in pancreatic cancer.2.2 Immunohistochemistry confirmed that the protein level of CPNE8 was highly expressed in pancreatic cancer tissues and lowly in normal pancreatic tissues adjacent to the cancer.2.3 Immunofluorescence showed that CPNE8 was mainly expressed in the cytoplasm of pancreatic cancer cells.2.4 The expression of CPNE8 was significantly correlated with the age distribution of patients with pancreatic cancer（P=0.032）,and had a significant relationship with disease grade（P=0.035）.2.5 Western blot showed that CPNE8 was highly expressed in the pancreatic cancer cells.2.6 After knocking down CPNE8 expression in CFPAC-1 and As PC-1 cells with siRNA,CCK8 experiments and clone formation experiments showed that proliferation ability decreased.Transwell experiments showed that the cell migration and invasion capacity were significantly reduced（P<0.05）,and scratch experiments showed that CFPAC-1 cells with low expression of CPNE8 had weaker migration capacity.2.7 After the expression of CPNE8 in CFPAC-1 cells was knocked down by si RNA,flow cytometry analysis showed that the proportion of apoptosis increased in CPNE8 low expressed cancer cell.Cell cycle analysis showed that cells in the G0/G1 phase was higher in CPNE8 low expressed cancer cell than CPNE8 high expressed cancer cell.3 In vivo experiments and preliminary exploration of mechanisms3.1 Western blot showed that the expression of stem molecule Bmi1 and epithelial-mesenchymal transition-related molecule Vimentin were down-regulated after interference with CPNE8 expression.3.2 In vivo experiments showed that reduced expression of CPNE8 significantly inhibited the ability of pancreatic cancer cells to form subcutaneous tumors in nude mice（P<0.05）,and it could reduce the expression of ki67 and PCNA.3.3 Analysis of the transcriptome data of pancreatic cancer patients revealed that changes in the expression of CPNE8 can affect signal pathway activities such as Hippo,mTOR and Erb B.3.4 GSEA analysis found signal pathways such as Hippo（NES=2.02,FDR=0.000）,MAPK/ERK（NES=1.82,FDR=0.001;NES=1.63,FDR=0.011）,WNT（NES=1.51,FDR=0.021）significantly enriched in patients with high expression of CPNE8.3.5 Western blot showed that the expression of MAPK and p-MAPK decreased after interfering with the expression of CPNE8.3.6 Correlation analysis showed that CPNE8 expression was positively related to key molecules of Hippo pathway including YAP1（R=0.82,P=0）,STK3（R=0.8,P=0）,STK4（R=0.73,P=0）and SGMS1（R=0.7,P=0）.3.7 The construction of protein interaction network shows that CPNE8 and other members of the CPNE protein family may form a protein interaction network to affect the occurrence and development of pancreatic cancer.ConclusionCPNE8 expression is significantly higher in pancreatic cancer tissues,and it has significant effects on the proliferation,invasion,metastasis,apoptosis and cell cycle of pancreatic cancer cells.CPNE8 is an important cancer-promoting gene.