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MiR‐599 regulates LPS‐mediated apoptosis and inflammatory responses through the JAK2/STAT3 signalling pathway via targeting ROCK1 in human umbilical vein endothelial cells
Author(s) -
Wang Jia,
Du Aolin,
Wang Hexilin,
Li Yang
Publication year - 2020
Publication title -
clinical and experimental pharmacology and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.752
H-Index - 103
eISSN - 1440-1681
pISSN - 0305-1870
DOI - 10.1111/1440-1681.13316
Subject(s) - apoptosis , umbilical vein , viability assay , microbiology and biotechnology , biology , transfection , human umbilical vein endothelial cell , tunel assay , chemistry , cancer research , cell culture , biochemistry , in vitro , genetics
MicroRNA plays an integral role in the development of atherosclerosis. Our study aimed to investigate the roles of miR‐599 in lipopolysaccharide (LPS)‐induced endothelial damage in human umbilical vein endothelial cells (HUVECs). HUVECs were transfected with a miR‐599 mimic and negative control, and then exposed to LPS. The expression of miR‐599 was detected by quantitative real time‐polymerase chain reaction (RT‐qPCR). Cell viability was analyzed by CCK‐8 assay and trypan blue exclusion assay; the formation of DNA fragments was tested by Cell Death Detection ELISA Plus kit; the incidence of apoptosis was detected by flow cytometry; the expression of p53 and cleaved‐caspase 3 (c‐caspase 3) was evaluated by western blot. Moreover, the mRNA levels and concentrations of tumour necrosis factor (TNF)‐α, interleukin (IL)‐6, ICAM‐1 and VCAM‐1 were assayed by RT‐qPCR and ELISA. The results showed that overexpression of miR‐599 increased cell viability, reduced DNA fragments, the incidence of apoptosis, as well as the protein levels of p53 and c‐caspase 3 in the presence of LPS. TNF‐α, IL‐6, ICAM‐1 and VCAM‐1 mRNA levels and concentrations were also decreased upon miR‐599 upregulation. In addition, the dual luciferase reporter assay demonstrated that ROCK1 is a direct target of miR‐599. MiR‐599 overexpression inhibited ROCK1 expression. Induced expression of ROCK1 reversed the roles of miR‐599 in apoptosis and inflammation. The gain function of miR‐599 function inhibited activation of the JAK2/STAT3 signalling pathway, which was abrogated by overexpression of ROCK1. Taken together, our results indicate that miR‐599 attenuates LPS‐caused cell apoptosis and inflammatory responses through the JAK2/STAT3 signalling pathway via targeting ROCK1.