| Objective:To investigated the effect on apoptosis of AXâ…¡R in cultured choroidal melanoma cells and whether autophagy plays a role in this process.Methods:First we constructed and indentify the recombinant expression plasmid pcDNA3.1-AXâ…¡R. Transfecting recombinant plasmid pcDNA3.1-AXâ…¡R to Mum-2B Cell mediated by LipofectamineTM 2000, cell morphological observation was conducted by using reverse-microscope.CCK-8 assay was used to detect cell viability. Besides, Flow cytometry with annexin â…¤ FITC/PI double staining and Hoechst 33258 staining were applied to determine the apoptosis process. Further, the expression of apoptosis-related gene were analyzed by Western Blotting. To explore whether over-expression of AXâ…¡R can induce autophagy, Western Blotting and laser confocal microscopy were used to detect autophagosome. Then, autophagy flux was tested by the expression of p62.The expression of LC3 (â… , â…¡) was also detected after exposure of autophagy inhibitor chloroquine to further identify the autophagy flux. Finally, using chloroquine to block autophagy procession, cell viability assay, as well as the expression of apoptosis-related gene was applied to figure out the role of autophagy.Results:Gene sequencing proved that we succeeded in constructing the recombinant expression plasmid pcDNA3.1-AXâ…¡R. After the transfection of plasmid, Mum-2B cells suffered bad condition by cell morphological observation. CCK-8 assay revealed that OD value was declined and Hoechst 33258 staining showed the obvious dense bright blue fluorescent light in nucleus in the experiment group. Flow Cytometry told that the apoptosis rates of cells were higher.Caspase-3, caspase-8, caspase-9 and PARP were also activated.Treatment of over-expressing AXâ…¡R strongly induced the formation of GFP-LC3 dots, the conversion from LC3-1 to LC3-â…¡, as well as the decrease of p62 protein levels. These results indicated that over-expression of AX â…¡R also induced autophagy activation. Moreover, Autophagy inhibition through chloroquine pretreatment led to decrease of cell viability and the increase of caspase-3 and PARP activation.Conclusions:These results demonstrated that over-expression of AX â…¡ R induced apoptosis and autophagy activation in Mum-2B cells, and autophagy played a protective role in AX â…¡ R-induced apoptosis. |
