Expression changes of canine transporter targeting-related genes during epithelial cells differentiation determined by microarray analysis and RT-PCR.

摘要

The establishment and continuous renewal of differentiated apical and basolateral plasma membrane domains with distinct transporters play important roles in maintaining functions of epithelial cells. This process is initiated during differentiation of epithelial cells, which indicates that genes taking part in targeting transporters to different domains of polarized epithelial cells may change their expression significantly during epithelial cells differentiation. However, little is known about the genes that take part in this process and the mechanism of targeting. In this thesis, we performed analysis of Caco-2 and mouse microarrays to look for genes that changed their expression levels significantly during epithelial cell differentiation, especially KIF and RAB genes that have been proven important in transporting cellular cargo. Based on microarray analysis and known or predicted functions of their encoded proteins, six non-metabolic genes, eight KIF and ten RAB genes were chosen to further test their expression changes during MDCK cells differentiation by performing semi-quantitative RT-PCR. Based on the PCR results of MDCK cells, genes shown to have higher expression levels in differentiated MDCK cells were selected to further investigate their specific roles in polarized epithelial cells by doing RT-PCR with skeletal muscle cells (C2C12) and non-differentiating epithelial cells (IEC-6). While comparing PCR results of selected genes in three types of cells, four genes (SDCBP2, KIF12, KIF27 and KIFAP3) that only increased their expression levels significantly during epithelial cell differentiation. Based on the known or predicted functions of their encoded proteins, all the four genes play roles in transporting cellular cargo, which also indicates that they may play roles in targeting transporters to different domains of polarized cells.