Ox-LDL-Induced MicroRNA-155 Promotes Autophagy in Human Endothelial Cells via Repressing the Rheb/ mTOR Pathway

Abstract

Background/aims: Autophagy, an evolutionary conserved biological process, is activated in cells to cope with various types of stress. MicroRNAs control several activities related to autophagy. However, the role of autophagy-related microRNAs during atherosclerosis is far from known. MicroRNA-155 was identified to be a crucial regulator of atherosclerosis. The objectives of the study were to analyze the effect of microRNA-155 on autophagic signaling and explore its mechanism in human endothelial cells under ox-LDL stress.

Methods: The study included human endothelial cells surrogate EA.hy926 lines (EA.hy926 cells). The expression of microRNA-155 was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The effect of microRNA-155 on endothelial autophagy was observed along with the expression levels of Rheb, LC3B, Beclin1, and P62/SQSTM1 by western blotting (WB) and immunofluorescence through microRNA-155 overexpression or inhibition. Bioinformatics analysis and Luciferase reporter assay were used to explore the target gene of microRNA-155. Cell viability and apoptosis were examined by 3-[4,5-dimethylthiazol-2-yl]-5- [3-carboxy-methoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium inner salt (MTS) assay and TdT-mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay.

Results: MicroRNA-155 expression was significantly increased under ox-LDL stress. MicroRNA-155 increased autophagic activity, while inhibition of it alleviated ox-LDL-induced autophagy in EA.hy926 endothelial cells. In addition, dual-luciferase reporter assays showed that microRNA-155 suppressed Rheb transcription. MicroRNA-155 increased autophagic activity in EA.hy926 cells via inhibition of Rheb-mediated mTOR/P70S6kinase/4EBP signaling pathway. Furthermore, we demonstrated that microRNA-155 could regulate not only autophagy but also apoptosis in EA.hy926 cells.

Conclusions: MicroRNA-155 works as a regulator of endothelial function under ox-LDL stress, making it a potential candidate for the novel therapeutic strategies against atherosclerotic diseases.

Keywords: Atherosclerosis; Autophagy; Endothelial cells; Microrna-155; Rheb; mTOR.