The Expression Pattern And Role Of CD147 In Gastroenteropancreatic Neuroendocrine Neoplasms

Backgroud&ObjectiveGastroenteropancreatic neuroendocrine neoplasms(GEP-NENs)are the most frequent NENs,and its incidence are increasing in recent years,but its mechanism is unclear,and are lack of effective markers for diagnosis and treatment.Our previous studies have found that CD 147 was not expressed in normal islets,whereas highly expressed in vast majority of pancreatic neuroendocrine neoplasms(pNENs),and correlated with the degree of its malignancy.CD 147 may be involved in the tumorogenesis of pNENs,but the expression of CD 147 in gastrointestinal NENs(GI-NENs),and its role and mechanism in pNENs,and the molecular mechanism of its absence in normal islets still needs further research.This study mainly focus on:(1)the validation test of CD 147 antibody;(2)to detect expression of CD 147 in GI-NENs,(3)to detect expression of CD 147,MCT-1,MCT-4 in chronic pancreatitis;(4)to test expression of MCT-1,MCT-4,PDX1,nkx6.1,Pax-6 in pNENs,then to analyze the correlation of expression level of CD 147 and MCT-1,MCT-4,PDX1,nkx6.1 expression levels in pNENs;(5)to detect the gene sequence of CD 147 in pNENs.Methods1.To construct eukaryotic overexpression vector of CD 147,transfect HEK293T cells,extract and deglycosylate cell proteins,western blot test by anti-CD 147 and anti-His antibodies.If get the same molecular weight fusion protein bands by anti-CD 147 and anti-His antibodies,then can prove that CD147 antibody specifically recognize CD 147 protein.2.Immunohistochemical staining was performed to detect the expression of CD 147 in 109 GI-NENs cases,the expression of CD147,MCT-1,MCT4 in 36 chronic pancreatitis cases,and the expression of MCT-1,MCT-4,PDX1,nkx6.1,Pax-6 in 42 pNENs cases,then to analyze the correlation of CD 147 expression with MCT-1,MCT-4,PDX1,nkx6.1 expression in pNENs.3.To test the gene sequence of CD 147 in tumor tissues and matched normal tissues of 29 pNENs cases.ResultsPart One:The validation test of CD 147 antibodyCD 147 monoclonal antibody used in previous experiments specifically recognize CD 147 protein.Part Two:Expression of CD 147 in GI-NENs1.CD 147 was expressed in GI-NENs,its immunophenotype can be characterized as cytoplasm type,membrane type and membrane-cytoplasm type,the positive expression rate of gastric NENs,duodenal NENs and colorectal NENs was 65.7%,76.5%and 19.3%respectively.2.The expression level CD 147 in GI-NENs was positively correlated with histological grade,its immunophenotype was positively correlated with its expression level.The higher CD 147 was expressed,the more membrane type exists.Part Three:Expression of CD 147,MCT-1,MCT-4 in pancreatic islets of chronic pancreatitis and peritumoral tissues in pNENsCD 147,MCT-1,MCT-4 was negative in islets of 36 chronic pancreatitis cases and islets of peritumoral tissues in 42 pNENs cases.Part Four:Expression of MCT-1,MCT-4,PDX1,nkx6.1,Pax-6 in pNENs and its correlation with the expression of CD 1471.MCT-1,MCT-4,Pax-6,nkx6.1,PDX1 were positively expressed in pNENs.2.The expression level of MCT-1,MCT-4 were positively correlated with tumor size,histological grading,pTNM stage;whereas the expression level of Pax-6,nkx6.1,PDXlwere negatively correlated with tumor size,histological grade,pTNM stage.3.The expression level of MCT-1,MCT-4 was higher in pNENs with lymph node metastasis or distant metastasis;the expression level of Pax-6 was higher in pNENs without local invasion and/or lymph node metastasis,the expression level of Pax-6 and PDX1 was higer in pNENs without distant metastasis.4.The expression level of CD 147 was positively correlated with MCT-1 expression level,whereas negatively correlated with PDX1,nkx6.1,Pax-6 expression level.Part Five:Sequence variation of CD 147 gene in pNENs47 variation sites of CD 147 gene were detected in 60 tissue samples of 29 pNENs cases.Heterozygous genotype of 19:580388C>G(rs2229664),homozygous genotype of 19:582775A>G,and homozygous genotype of 19:582927T>A(rs8259)were more in tumors>2cm than in tumors ?2cm.T homozygous genotype of 19:579627C>T(rsl 803202)was more than C/T heterozygous genotype in tumor tissues or normal-tumor tissues.The number of 19:582927T>A(rs8259)variation in normal tissues and tumor tissues were significantly correlated with histological grade,the higher histological grade is,the more rs8259 AA and AT genotype exists.Conclusions1.The expression of CD 147 in GEP-NENs vary in different locations of tumours.2.The expression level of CD147 in GI-NENs was positively correlated with histological grade.The high expression of CD 147 may indicate poor differentiation of tumour.CD 147 may be an indicator to assess the severity of GI-NENs.3.CD 147,MCT-1,MCT-4 were negatively expressed in islets of chronic pancreatitis and pNENs.4.The expression level of MCT-1,MCT-4 were positively correlated with tumor size,histological grading,pTNM stage,whereas the expression level of Pax-6,nkx6.1,PDXlwas negatively correlated with tumor size,histological grade,pTNM stage of pNENs.High expression levels of Pax-6,nkx6.1,PDX1 may indicate well differentiation and low malignancy degree of tumor.5.In pNENs,the expression level of CD 147 was positively correlated with MCT-1 expression level,whereas negatively correlated with expression levels of PDX1,nkx6.1,Pax-6.MCT-1,PDX1,nkx6.1,Pax-6 may participate in the role of CD147 in pNENs.6.Four variation sites of CD 147 gene significantly correlated with clinical pathological features of PNENs were detected,rs2229664 C>G,19:582775A>G,rs8259 T>A,rs 1803202 C>T,they may have clinical significance in PNENs but still needs further research.