Effects of free fatty acids on apoptosis of human umbilical vein endothelial cell by RNA interference

Abstract

Objective To study the role of Wnt/β-catenin signal pathway in high free fatty acid (FFA)-induced apoptosis in human umbilical vein endothelial cell (HUVEC) by RNA interference (RNAi). Methods A recombinant adenovirus containing an RNAi cassette targeting the GSK-3β gene was produced and its silencing effect on GSK-3β gene was detected by Western blot analysis in HUVECs. The effect of the RNAi on the protein level of β-catenin was explored by transfecting the RNAi adenovirus to inhibit the expression of GSK-3β protein. The subsequent effect on the Wnt/GSK-3β/β-catenin signal pathway and on apoptosis of HUVECs cultured with FFAs was analyzed by the flow cytometer and Hoechst 33258 in order to explore the possible connection between the signaling pathway and FFA-induced apoptosis. The experimental cells were divided into four groups as followed: 0.75 mmol/L FFA group; RNAi adenovirus (Ad-640)+ FFA group; RNAi adenovirus without recombination (Ad-DEST)+ FFA group; normal cell group. Results The Western blot results showed that the expression of GSK-3β protein in HUVECs was inhibited by the RNAi adenovirus and the protein level of β-catenin was increased by RNAi adenovirus transfection. Apoptosis was observed in HUVECs exposed to 0.75 mmol/L FFAs for 72 h. The apoptotic rate of the Ad-640+ FFA group was significantly reduced with respect to that of the Ad-DEST+ FFA and FFA groups whereas there was no significant difference between the Ad-DEST+ FFA and FFA groups. Conclusion RNAi adenovirus specific to GSK-3β may partly protect HUVECs from apoptosis induced by FFAs. Key words: RNA interference; Human umbilical vein endothelial cells; Free fatty acids; Cell apoptosis; Wnt pathway