The Experimental Research Of Bufalin On The Apoptosis Of Human Osteosarcoma Saos-2and U-2Os Cell Lines

Objective:Aimed to discussion about Bufalin inducing apoptosis and suppressing proliferation of cells in human osteosarcoma Saos-2and U-2os cell lines.Methods: Bufalin of different concentration was added on Saos-2and U-2os cells. CCK-8assay was used to detect the cell proliferation rate, and flow cytometry with Anexin/PI double dye was to test the cell death and apoptosis, Hoechst33258fluorescent dye to observe the morphological changes with light microscope, JC-1staining was used to observe the changes of mitochondrial membrane potential,2,7-double hydrogen chloride fluorescent yellow dye detect the cellular reactive oxygen species(ROS) content. Micro-arry analysis the microRNAs expression differences betweet the control group and0.25umol/L Bufalin group. Real-Time polymerase chain reation(PCR) was used to evaluate the expression of miRNA-29a in human osteosarcoma Saos-2and U-2os cells. The software prediction of Targetscan was used to gain targets of miRNA-29a, and luciferase activity was used to verification regulated relationshio between miRNA-29a and targeting proteion BBC3(PUMA). Apoptosis-related proteins PUMAwere detected by using Western blotting assay.Results:The cells proliferation dealed with bufalin were inhibited and the percentage increased with the dosage increased,10nmol/L above the concentration of bufalin could induce20%inhibition rate after24hours on Saos-2and U-2os cells. Cellular reactive oxygen species(ROS) increased with the bufalin concentration increased, at the same time the mitochondrial membrane potential reduced oppositely, which indicated that bufalin induced cell apoptosis and inhibited proliferation of cells in human osteosarcoma Saos-2and U-2os cell lines by activation of the mitochondrial pathway. Bufalin induced apoptosis of osteosarcoma cells by down-regulation of miRNA-29a(p<0.05), and luciferase assay demonstrated that miRNA-29a targeting regulation of BBC3, which both showed a concentration-dependence. An apoptosis-related BBC3protein was found upregulation with drug congcentration increased.Conclusions Bufalin can increase ROS leval and activate the mitochondrial pathway of apoptosis in a dose-dependent induction in human osteosarcoma Saos-2and U-2os cells by inhibiting miRNA-29a expression and targeting BBC3protein upregulation.