Lymphotoxin beta receptor is associated with regulation of microRNAs expression and nuclear factor-kappa B activation in lipopolysaccharides (LPS)-stimulated vascular smooth muscle cells

Abstract

Background: The aim of present study is to investigate the role of lymphotoxin beta receptor (Ltßr) in lipopolysaccharides (LPS)-induced inflammation in vascular smooth muscle cells (VSMCs) and whether its effects are mediated by modulating microRNAs (miRNAs) and nuclear factor-kappa B (NF-κB). Methods: Mouse aortic smooth muscle cell (SMC) line (MOVAS cells) were transduced with short hairpin Ltßr (shLtßr) and mRNA and protein expression level of Ltßr were measured by qPCR and Western blot in shLtßr-transduced cells. Lentiviral vector-transduced (control) and lentiviral vector/shLtßr-transduced MOVAS cells were stimulated with LPS (1 μg/mL) for 0, 16, or 24 h. Then the mRNA and protein levels of Ltßr, interleukin-18 (IL-18), p-p65, p65 and vascular cell adhesion molecule 1 (VCAM-1) were measured by real-time quantitative polymerase chain reaction (qPCR), Western blot and enzyme-linked immunosorbent assay (ELISA). Different miRNAs expression in LPS-stimulated normal and shLtßr-transduced cells were detected by small RNA sequencing (smRNA-seq). Results: The mRNA and protein expression of Ltßr was significantly downregulated in shLtßr-transduced cells. LPS-increased the mRNA and protein levels of Ltßr, IL-18, p-p65 and VCAM-1 in were attenuated by shLtßr transducing compared with LPS-stimulated control group. Moreover, LPS treatment induced 10 upregulated and 64 downregulated miRNAs in shLtßr-transduced cells compared with control cells. Moreover, miR-146b-5p and miR-27a-5p levels were significantly decreased in shLtßr-transduced cells. Conclusions: Our results show for the first time that the role of Ltßr in regulating inflammatory response in LPS-stimulated VSMCs via modulating miRNAs and NF-κB pathway. Our findings might provide valuable information with respect to better understanding in the treatment of cardiovascular diseases, such as atherosclerosis.