| Lipopolysaccharides (LPS), as a component of the cell wall of Gram-negative bacteria, such as Escherichia coli and Brucella melitensis, activate monocyte/macroph-age, stimulate monocytes/macrophages to produce and release large amounts of inflammatory factor, such as nitric oxide (NO), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) and cause inflammatory injury, which might induce sepsis, septic shock, multiple organ dysfunction, even septic death. MicroRNAs (miRNAs) are a class of non-coding single-stranded small molecule RNA about18to24nucleotide molecule, which match with the3’untranslated region (3’UTR) of mRNA of the target gene, resulting in the mRNA cut or translation inhibition of target gene. Currently, the study of LPS is focused on signal transduction pathway, cytokine secretion, organ injure and apoptosis induced by LPS. In the study, we analyzed the differentially expressed miRNAs and its function mechanism in macrophage stimulated by E.coli LPS and B. melitensis LPS, which provide some important informations for the study of miRNAs in various LPS-induced diseases in epigenetic. Methods1) MicroRNA expression profiling analysis of E.coli LPS treated RAW264.7macrophage cells were performed with TaqMan MicroRNA Array and further qRT-PCR validation;2) Bioinformatics approaches were used to analyze the target genes of the differentially expressed miRNAs and then Gene Ontology were used for classifying;3) The expression of miR-27a-5p in RAW264.7cells stimulated with E.coli LPS and B.melitensis LPS were analyzed by qRT-PCR. The expression of predict target genes of miR-27a-5p which is involved in the macrophage response to E.coli LPS and B.melitensis LPS stimulation were confirmed by western blot;4) The3’UTR of monocyte chemotactic protein induced protein1(MCPIP1) which validated by qRT-PCR and Western blot and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were cloned, and then inserted into pmirGLO luciferase reporter plasmid to generate pmir-luciferase-MCPIP1-3’UTR and pmir-luciferase-GAPDH-3’UTR, We transfected HEK293cells with these constructs in the presence of miR-27-5p mimic, and the luciferase activities were determined with Dual-Glo Luciferase Assay System;5) When MCPIP1was overexpressed in RAW264.7cells stimulated by E.coli LPS and B.melitensis LPS, the secretion of IL-6, IL-10, and IL-1β of RAW264.7cells were analyzed by ELISA. Results1) By comparing RNA samples obtained from RAW264.7cells treated and untreated with E.coli LPS, the microarray data were analyzed. We found that the number of the differentially expressed genes was7. Among them,5miRNAs (miR-196b, miR-196c, miR-146a, miR-155, and miR-222) were up-regulated and2miRNAs (miR-27a-5p, miR-532-5p) were down-regulated in E.coli LPS group compared with NC group. Down-regulation of miR-27a-5p and miR-532-5p and up-regulation of miR-146a and miR-155were confirmed by qRT-PCR. In RAW264.7cells stimulated with B.melitensis LPS, the expression of miR-27a-5p was down-regulated and miR-532-5p, miR-146a and miR-155were up-regulated, and which were validated by qRT-PCR;2) More than1000candidate target genes of differentially expressed miRNAs were predicted by at least of one of four different algorithms (TargetScan, PicTar, miRDB, and microRNA.org), with Gene Ontology classification, we were able to detect39inflammation-related candidate target gene;3) miR-27a-5p were down-regulated with E.coli LPS and B.melitensis LPS stimulation. At6h post-incubation with E.coli LPS, the mRNA level of MCPIP1, which was predicted to be potential targets for miR-27-5p, was up-regulated, and which was determined by qRT-PCR. The protein expression of MCPIP1was increased gradually after3h with E.coli LPS stimulation, and highly expressed during6-12h. The protein expression levels returned to the base level compared to no stimulation at24h. Under B.melitensis LPS stimulation, the expression levels of MCPIP1were unchanged;4) Luciferase reporter assay results showed that compared to the NC group, co-transfection of miR-27-5p mimic with the pmir-luciferase-MCPIP1-3’UTR constructs results in the significant decrease of luciferase activities in HEK293, indecated that miR--27-5p can inhibite the expression of MCPIP1by matched to its3’UTR;5) ELISA.was used to analyze the cytokine secretion in RAW264.7stimulated with E.coli LPS and B.melitensis LPS after over-expressing MCPIP1. The secretion of IL-6, IL-10, and IL-1β,which were induced by E.coli LPS and B.melitensis LPS were significantly reduced.ConclusionsIn this study, we analyzed the miRNAs expression profile of E.coli LPS-stimulated RAW264.7cells and obtained4differentially expressed miRNAs, in which miR-27a-5p were down-regulated, miR-155and miR-146a were all up-regulated. Under E.coli LPS stimulation, miR-532-5p were down-regulated; Under B.melitensis LPS stimulation, miR-532-5p were up-regulated. More than1000target genes of differentially expressed miRNAs were predicted by bioinformatics software (TargetScan, PicTar, miRDB and microRNA.org). Because LPS caused inflammation reaction of macrophage, we used bioinformatics software and focused on the gene that involved in apoptosis, cytokine-mediated signaling pathway, immune response, inflammation response, cytokine production, and the innate immune response,39inflammation-related genes were obtained. qRT-PCR were performed to confirm the down-gulation of miR-27a-5p in RAW264.7cells stimulated by E.coli LPS and B.melitensis LPS, respectively. The expression of MCPIP1, which is the potential target gene of miR-27a-5p was up-regulated in RAW264.7cells stimulated by E.coli LPS, which were analyzed by western blot. Luciferase reporter gene experiment results indicated that miR-27a-5p inhibited the exprerssion of MCPIP1by bind to its3’UTR specifically. Over-expression of MCPIP1decreased the secretion of IL-6, IL-10and IL-1β from RAW264.7stimulated with E.coli LPS and B.melitensis LPS. These findings suggested that miR-27a-5p might play an important role in E.coli LPS-induced immune response of macrophage via regulating the expression of MCPIP1. |
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