| Tight junctions(TJs) are made of three kinds of integral transmembrane protein occludins, CLDNs, junction adhesion molecules, and some periphery plasmosins as ZO-1,-2,-3. CLDNs and occludins are important components of TJs function, especially CLDNs. We can say that CLDNs are the most important backbone protein of TJs. CLDN, which contains at least 24 members, is a kind of integral transmembrane protein. They play essential roles in maintain fence and barrier functions of TJs. CLDN6 gene is located at chromosome 16p13.3 and encodes a protein of 219-amino acid with a molecular weight of～23-kDa. CLDN6 protein, similar to other members of the CLDN tight junctional family, contains four transmembrane domain. At present, it is thought that TJs highly associate with cell proliferation, polarity, adhesion mobility and invasive phenotype properties of tumor. It is easy to see changed tight junction structures and functions in human epithelium origin tumors. And this kind of changes can destroy cell adhesion and disturb cell differentiation causing tumor development. The downregulation or loss expression of CLDN and other kind of TJs proteins are thought to be important steps for tumor cells development and metastasis. These steps then develop significance effects on decreasing cohesion, worsening differentiation and increasing invasion of cancer cells.In previous work we have identified CLDN6 as a potential mammary cancer suppressor gene, which may contribute to the mammary cancer resistant phenotype observed in Copenhagen rats. In addition, we found that the expression of CLDN6 was undetectable or at low levels in human and rat mammary cancer cell lines, suggesting that CLDN6 may play a role in suppressing the development of mammary cancer. However, there is little knowledge about CLDN6 on biological behaviors of cells. In order to determine if CLDN6 play any roles on junctional function, proliferation and migration of cells, we constructed CLDN6 small interfering RNA (siRNA) and transfected it into human mammary epithelium cell line HBL-100. There are two parts:First, we designed four siRNA fragment according to CLDN6 gene and then inserted to eukaryotic expression vector which contain green fluorescent protein respectively. The vector which didn't silence any gene acted as negative control. Each plasmid was transfected into cells using lipidosome . G418 -resistant cells were expanded in culture as relative stable expression population. Cells were selected to examine the expression of introduced genes by quantitative reverse transcription–polymerase chain reaction (RT-PCR).Second, determine the effects of CLDN6 gene silencing on cell biological phenotype. Examine the cell appearance by inverted microscope; measure the cell monolayer transepithelial resistance which reflect TJ function; detect the cell proliferative ability by cell counting; check out the cell apoptosis by Annexin V-FITC; determine the cell migration ability by wound healing assay.Our results clearly showed that transfection of CLDN6 shRNA into HBL-100 cells resulted in an altered phenotype. The appearance of cells transfected siRNA changed a lot when compared with control groups. The growth rate and migration ability of the CLDN6 silencing cells was higher than that of control cells. The cell monolayer transepithelial resistance of the CLDN6 silencing cells was lower than other groups. These findings further support the hypothesis that CLDN6 is cancer suppressor, decreased expression of which contribute to malignant progression of cancer, at least for breast cancers. |
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