Expression Of Tight Junctions Protein Claudin-6 In Different Tumors And Its Eukaryon Expression Vector Construction

Tight junctions exist in the junction complex in epithelial and endothelial cells,which play important roles in cell adhesion, maintaining cell polarity and permeability,assisting signal transducin to regulate cell proliferation and differentiation.Mammary cancer is a kind of epithelial malignant tumor,which comes from mammary gland terminal duct lobular unit epithelial.These cells may gain the property of stroma and abnormal structure and function of tight junctions,such as losing typical adhesion and polarity of epithelial cells,thus to obtain the capability of migration.Tight junctions are made from claudins,occluding and junctional adhesion molecules. Claudin-6 is a member of the claudin tight junction family. Claudin-6 locates at chromosome 16p13.3,encoding 219-amino acid and synthesising of a～23-kDa protein,which was identical to the members of the Claudin tight junction family,with four transmembrane domains. Claudins are often abnormal regulated in human tumors.In our prework,we cloned and identified claudin-6 as mammary cancer phenotype suppressor related gene from Copenhagen resistant to mammary cancer,in addition we found the level of claudin-6 was undetectable or hypo-expression in human and rat mammary cancer cell lines ,supposing that claudin-6 may play essensial roles in suppressing occurrence and development of mammary cancer.Up to now,there is little knowledge about the relativity between claudin-6 and mammary cancer.In order to approaching the effectiveness of claudin-6 abnormal regulated in generate and development of mammary cancer, we use immunohistochemistry and RT-PCR analysis to detecting claudin-6 expression in human mammary cancer tissues and various kinds of tumor strains in protein and mRNA level respectively,then analyzed the relation between claudin-6 and mammary cancer clinical index. Simultaneously, we construct claudin-6 eukaryotic expression vector,which is the experiment evidence of further studying claudin-6 as tight junction protein effecting the invasion and metastasis phenotype of mammary cancer. Experimental result as follows:1. Results of RT-PCR:In examined many cell lines ,the level of claudin-6 mRNA gene was expressed in ovarian cancer strain cocⅠand mammary gland nomal strain HBL-100,and was undetectable in other tumor strains,indicating differential expression in distinct tumor cell may relate to tissue specificity of claudin-6.2. Results of immunohistochemistry: Comparing to mammary gland benign fibroadenoma,the level of claudin-6 was down regulation in breast cancer, ( P<0.05 ) .Furthermore,this down regulation had negative correlation evident with mammary tumor pathology ranking,and the level of claudin-6 in lymphode metastasis group was lower than control group, (P<0.05).These results suggest that the expression of claudin-6 may involve in invasiving and metastasizing of mammary tumor.3.Results in constructing laudin-6 eukaryotic expression vector: We gained whole cDNA fragment of human claudin-6 gene,constructed claudin-6 protocaryon cloning vector and eukaryotic expression vector.In the research,we found that Chinese claudin-6 gene base group of 461 site A was different from Spaniards claudin-6 gene base group of 461 site G,elucidating that the base group of 461 site of claudin-6 may exhibit gene site polymorphism.Above-mentioned results indicating that the down regulation of claudin-6 in mammary gland may contribute to invasiving and metastasizing of mammary tumor by destroying integrity of tight junction. The construction of claudin-6 eukaryotic expression vector provide experiment foundation for further investigating the effectiveness and mechanism of phenotype changed which was induced by claudin-6 in the tumorigenesis and development in mammary cancer cells.