Mechanism of lncRNA NEAT1 promoting hepatocyte injury and inflammatory reaction induced by sepsis / 局解手术学杂志

ABSTRACT

Objective To explore the mechanism of hepatocyte injury and inflammatory reaction induced by sepsis based on miR-129-5p/TLR4 signaling axis regulated by lncRNA NEAT1.Methods Peripheral blood from 15 sepsis patients(the sepsis group)and 15 healthy individuals(the healthy group)were collected for gene expression detection.The mouse normal liver cell line NCTC1469 was used in in vitro experiments,and an in vitro sepsis model(the LPS group)was established by lipopolysaccharide(LPS).The cells stimulated with LPS and transfected with mimic NC,mimic,siNC or siNEAT1 plasmid vectors were named as the LPS+mimic NC group,the LPS+mimic group,the LPS+siNC group or the LPS+siNEAT1 group,respectively.The cells treated with a combination of LPS,siNEAT1,and TLR4 recombinant protein or LPS,mimic,TLR4 recombinant protein were named as the LPS+siNEAT1+TLR4 group or the LPS+mimic+TLR4 group,while the control group was not treated.The mRNA and protein expression levels of TNF-α,IL-6,and IL-1β were detected by qPCR and ELISA,respectively.The cell apoptosis rate was detected by TUNEL assay.The binding of lncRNA NEAT1/miR-129-5p and miR-129-5p/TLR4 was detected by dual-luciferase reporter gene analysis.The TLR4 expression in cells was detected by Western blot.Results In the in vivo experi-ment,compared with the healthy group,the expression of TNF-α,IL-6,IL-1β,lncRNA NEAT1,and TLR4 of peripheral blood in the sepsis group was significantly increased(P<0.05),while the expression of miR-129-5p was decreased(P<0.05).In the in vitro experiment,compared with the control group,the expression of lncRNA NEAT1 in the LPS group was increased(P<0.05).Compared with the LPS group and the LPS+siNC group,the expression of lncRNA NEAT1 in the LPS+siNEAT1 group was decreased(P<0.05),and the expression of miR-129-5p was increased(P<0.05).Compared with the control group,the cell apoptosis rate and the expression of TLR4,TNF-α,IL-6,and IL-1β in the LPS+siNC group were up-regulated(P<0.05).Compared with the LPS+siNC group,the cell apoptosis rate and the expression of TLR4,TNF-α,IL-6,and IL-1β in the LPS+siNEAT1 group were down-regulated(P<0.05).Compared with the LPS+siNEAT1 group,the cell apoptosis rate and the expression of TLR4,TNF-α,IL-6,and IL-1β in the LPS+siNEAT1+TLR4 group were up-regulated(P<0.05).Dual-luciferase reporter gene assay revealed an interaction between lncRNA NEAT1 and miR-129-5p,and TLR4 was the target gene of miR-129-5p.Compared with the control group,the expression of TNF-α,IL-6,and IL-1β as well as cell apoptosis rate in the LPS+ mimic NC group were increased(P<0.05).Compared with the LPS+mimic NC group,the expression of TNF-α,IL-6,and IL-1β as well as cell apoptosis rate in the LPS+mimic group were down-regulated(P<0.05).Compared with the LPS+mimic group,the expression of TNF-α,IL-6,and IL-1β as well as cell apoptosis rate in the LPS+mimic+TLR4 group were up-regulated(P<0.05).Conclusion lncRNA NEAT1 interacts with miR-129-5p and directly targets TLR4 to promote LPS-induced hepatocyte injury and inflammatory reaction.This study can provide a new therapeutic target for hepatocyte injury caused by sepsis.