The. ¦¢4galt1 Transfected The Impact On The Behavior Of Pc12 Cells And Its Mechanism

β1 , 4-galactosyltransferase(β4GalT1) is an amazing enzyme which is best-investigated extensively for many years. The vast majority of long and shortβ4GalT1 has been viewed as a biosynthetic enzyme of the Golgi apparatus, where it transfers galactose from uridine diphosphate-galactose (UDP-Gal) donors to terminal N-acetylglucosamine (GlcNAc) residues on elongating oligosaccharide chains of membranebound and secretory glycoconjugates. However, a portion ofβ4GalT I is also found on the cell surface where it functions as a cell adhesion molecule associated with a variety of biological functions. Early work has shown that a number of highly metastatic murine and human cell lines, such as melanoma, astrocytic glioma, lung carcinoma, are characterized by the elevated levels of cell surfaceβ4GalT1. The initiation of neurite outgrowth and the process extension from neuronal cells is mediated by the cell surfaceβ4GalT1 as well.For the expression level of cell surfaceβ4GalT1 is promoted remarkably in serum of many kinds of tumor patients and cell surfaceβ4GalT plays an important part in neurite outgrowth, this study focuses onβ4GalT, aims to explore the differences on cell behavior of two cell lines which has distinct level ofβ4GalT1 and its underlying mechanisms.1. Construction of PC12-GalT cell line which has a high level ofβ4GalT1 and assessment of the transfection efficiency.The plasmid pcDNA3.1-cmyc-β4GalT1 and the vector pcDNA3.1-cmyc was transfected in PC12 cell to construct two cell lines GalT and mock which have distinct levels ofβ4GalT1 expression. RT-PCR, western blotting and several kinds of empirical methods were used to assess the efficiency of transfection. All the results showed that the expression of target mRNA, protein and the cell surface glycans changed remarkably.2. Cell behavior of three cell lines-PC12, mock, GalT We observed the different behavior of PC12, mock and GalT with the stimulation of FN, LN and CollagenⅣ. We found out that the expression ofβ4GalT1 significantly enhanced the migration with FN, especially with CollagenⅣcompared with the negative control cells and PC12 cells, but there is an opposite effect with LN. We also found out that the higher level ofβ4GalT1 with a higher number of neurite outgrowth with LN and CollagenⅣ, and there is no significant changes with FN.3. Research on initial mechanisms of disparity of PC12, mock and GalTWe chose LN and CollagenⅣwhich cause the significant effect of cell behavior to examine the initial mechanism. Firstly we detected the activation of the key signaling regulator molecular ERK1/2 and FAK. The result showed that the ERK1/2 and FAK on GalT cell is more sensitive to the stimulation of LN and CollagenⅣ, but no impact on P38 signaling. Then we detected the the cytoskeleton protein associated withβ4GalT1 stimulating by CollagenⅣ. The level of phosphorylation of the RhoA is no obviously disparity, but decreased in the phosphorylation of cofilin in GalT cells.Conclusion:1. PC12-GalT cell line was contructed, which has a high level ofβ4GalT1. We found out that the expression ofβ4GalT1 significantly enhanced the migration with CollagenⅣcompared with the negative control cells and PC12 cells, but there is an opposite effect with LN. We also found out that the higher level ofβ4GalT1 with a higher number of neurite outgrowth with LN and CollagenⅣ.2. The ERK1/2 and FAK on GalT cell is more sensitive to the stimulation of LN and CollagenⅣ. The level of phosphorylation of cofilin in GalT cells was reduced by . stimulation of CollagenⅣ.