Autophagy Induced By Starvation Results In Degradation Of UVRAG

Objective :To observe changes of UVRAG levels in the cell after cell autophagy induced by starvation and to determine whether or not autophagy was affected by degradation of UVRAG.Methods:LC3-RFP was transfected into RD-A cells,then the cells were grown to logarithmic growth phase in MEM complete medium at 37 ℃,5% CO2 conditions,then cultured in the eagle’s solution for 6h. LC3-RFP punctate were detected by fluorescence microscope. RD-A cell which was grown to logarithmic growth phase in MEM complete medium at 37 ℃,5% CO2 conditions, then treated by a eagle’s solution for 2h and 6h.Western blots were performed to examine the conversion of LC3-Ⅰ to LC3-Ⅱ,the degradation of P62 and UVRAG in the initial period and autophagosome formation stage of the cell autophagy induced by starvation. RD-A cell which was grown to logarithmic growth phase in MEM complete medium at 37℃,5% CO2 conditions and treated by autophagy inhibitor 3-MA at 37 ℃,5% CO2 conditions for 6h.Western blots were performed to examine the conversion of LC3-Ⅰ to LC3-Ⅱ,the degradation of P62 and UVRAG after the action of 3-MA.RD-A cell which was grown to logarithmic growth phase in MEM complete medium then cultured in the eagle’s solution as well as autophagy inhibitor E64 d andpepstatin A,at 37 ℃,5% CO2 conditions for 6h.Western blots were performed to examine the change of the conversion of LC3 and the degradation of P62 and UVRAGafter cells autophagy were inhibited by autophagy inhibitor 3-MA or E64 d and pepstatin.Results:1.Autophagy in RD-A cell can be induced by the starvation and accompanied by the degradation of UVRAG;2.The conversion of LC3 of cells has occurred by starved for 2h,and indicated that Autophagy was induced, but no change of the level of UVRAG molecule be observed. Whether UVRAG was involved in the formation of autophagy body remains to be proved;3.The degradation of UVRAG is associated with cell autophagy;4.After using of E64 d and pepstatin A to inhibit autophagy induced by the starvation, autophagic lysosomal degradation of the material is suppressed, while the degradation of UVRAG was reduced. It showed that the degradation of UVRAG occurs in the effector phase of autophagy and indicated that UVRAG was degrated by autophagy lysosomes.Conclusions:Autophagy might be induced by starvation; Autophagy might contribute to degradation of UVRAG.