The Role Of E3 Ubiquitin Ligases Siah-1 Nuclear Aggregation In The Homocysteine Induced Cell Apoptosis

Objective To investigate the mechanism of homocysteine on cell apoptotic,and explore the role of siah-1 nuclear accumulation in Hcy-induced apoptosis.Methods C6 cells were treated with normal or Hcy of varied concentration for 48 h, the proliferation of the cells was analysed using the CCK-8, and to decide the intervention concentration of Hcy. TUNEL method was used to detect the level of apoptosis after giving Hcy. Quantitative real time PCR and Western blot technologies were used to measure the expression levels of siah-1 、GAPDH m RNA and protein in Hcy-induced cells. Laser confocal fluorescence microscopy was used to show the characteristic change of siah-1 and GAPDH. Siah-2 si RNA was transfected into C6 cells,q PCR and western blot analysis were used to detect to the interfering efficiency, Effects of siah-1 si RNA on the GAPDH nuclear protein expression in normal or Hcy-induced cells were detected using western blot analysis. Immunofluorescence staining were used to examine the location of GAPDH in the C6 cells after si RNA interfered. To observe the expression of P53 and caspase 3 protein after si RNA interfered.Results The results showed that The viability of C6 cells was significantly inhibited exposured to Hcy for 48 h, and the proliferation was declined in a dose-dependent manner.TUNEL staining showed that the apoptosis rate was increased in cells after exposured to Hcy,indicating that the Hcy increases the cell death. In the same time,we find that Hcy significantly increased siah-1 m RNA levels and protein expression compared with control cells cultured under normal,as well as GAPDH. Confocal fluorescence microscopy revealed that siah-1and GAPDH were all present nuclear accumulation in C6 cells with 8m M Hcy treatment for 48 h than the cells in the normal.Results from real-time PCR and western blot analysis showed that siah-1 si RNA efficiently inhibited the siah-1 expression. Down-regulation of siah-1 expression significantly decreased Hcy-induced GAPDH nuclear translocation,we also found that the increasing expression of apoptosis-related proteins P53 and caspase3 induced by Hcy were decreased too. It indicating that the siah- 1 is necessary in Hcy-induced GAPDH nuclear translocation, which binding GAPDH and transfering from cytoplasm to the nuclei which promoting apoptosis.Conclusion Hcy inhibited the viability of C6 cells,increased siah-1and GAPDH m RNA expression and protein levels.We finded that Hcy induced the association between GAPDH and siah-1, and transfering from cytoplasm to the nuclei which promoting apoptosis. In addition, siah-1 knockdown using siah-1 si RNA significantly decreased Hcy-induced GAPDH nuclear accumulation. Therefore, siah-1 may play an important role in Hcy-induced apoptosis in Rat C6 cells.