The Epigenetics Regulation Of NF-κB-Jmjd3 Downstream Genes’ Expression In Endothelial Cells Induced By Lipopolysaccharide

ObjectiveChronic inflammation communed closely with cancer, of which vascular endothelial cells play a key role in the pathological process. The research was designed to observe the inductive effect of inflammatory mediators and adhesion molecules in LPS induced vascular endothelial cells, and to explpore the epigenetic mechanism of NF-κB- Jmjd3 signaling pathway, which will provide reliable experimental basis for effective inhibition of inflammation in vessels and prommising target therapies of related diseases.Methods1. The primary human umbilical vein endothelial cells were thawed and cultured routinely, then divided into differrent LPS groups randomly by processing with LPS for certain time course according to the demand of each experiment, control group was set simultanously.2. The concentration of IL-6, MMP-9 and ICAM-1 in supernatants were detected by ELISA kits to identify the release of inflammatory mediators and adhesion molecules induced by LPS in endothelial cells.3. The expression and location of NF-κB/p65 and Jmjd3 were observed by immuno-fluorescence technique, and RNA levels of Jmjd3 was determined by RT-PCR,then the expression of NF-κB/p65, IκBα, and Jmjd3 were verified by Western blotting.4. Activity of transcription factor NF-κB in nuclear extraction was detected by Transcription Factor Assay Kit of Millipore.5. Recruitment of NF-κB/p65, Jmjd3 and H3K27me3 along transcription start binding sites of target genes was analyzed by chromatin immunprecipitation technique: the targeted DNA library was build by Chromatin Immuno-precipitation kit of Millipore, following Ch IP-q PCR to analyze NF-κB/p65, Jmjd3 and H3K27me3 recruitment along transcription start binding sites of target genesincluding IL-6, MMP-9 and ICAM-1.Results1. The release of inflammatory mediators and adhesion molecules in LPS induced HUVECs turned out to be that the relative concentration of IL-6, MMP-9 and ICAM-1 were up-regulated significantly(P<0.01)compared with their control group respectively.2. The activation of NF-κB-Jmjd3 signaling pathways was found as follows:(1) LPS groups showed increasing fluorescence intensity of NF-κB/p65 compared with control group(0 h), indicated the up-regulation of NF-κB/p65. Meanwhile, the translocation of NF-κB/p65 was found remarkable in LPS groups and peaked at 2h.(2) Relative activity of nuclear NF-κB/p65 was remarkblely increased in LPS 2h gorup compared with control group(P<0.01), but little changes were found in the rest LPS groups( P>0.05).(3) The RNA level of Jmjd3 was strikingly increased in LPS 2h gorup compared with the control group(0h)(P<0.01), however, there was no statistically significant difference between the rest groups and control group( P>0.05).(4) LPS groups showed increased Jmjd3 fluorescence intensity when compared with that of control group in immuno-fluorescence assay, the highest intensity emergedat 1to 2h after LPS stimulation.(5) Total NF-κB/p65 level increased with the LPS stimulating time, which was verified by Western Blot, nuclear NF-κB/p65 in 2h and 6h LPS groups was found up-regulated and IκBα was found down-regulation in 2h and 4h LPS groups, Jmjd3 was also found increased in LPS groups especially at 1 and 2h.(6) Ch IP-qPCR technique was employed to analysisthe recruitmentof NF-κB/p65 and Jmjd3 with transcription start binding sites of target genes: the results indicated that the relative amplification of targeted genes such as IL-6 and MMP-9 from the NF-κB/p65 related DNA library was prominently higher in LPS group than that of the corresponding control group( P<0.01), while ICAM-1 showed higher amplification in LPS group but proved no significant difference compared with its control group(P>0.05). The results of Jmjd3 and H3K27me3 related DNA library was worked out to be the same as that of NF-κB/p65.ConclusionNF-κB-Jmjd3 signaling pathways was activated in LPS-induced endothelial cells and reached a peak at 2h. Nuclear translocation of the transcription factor leaded to NF-κB activation, followed by combination to Jmjd3 promoter region to regulate its expression. The up-regulated Jmjd3 was recruited to target gene regions and worked as a demethylase on H3K27me3, which resulted in demethylation of H3K27me3, conformational change and exposure of transcription start sequence of targeted genes, then finally the expression of targe genes. The activated NFκB-Jmjd3 pathways drives the expression of inflammatory mediators and adhesion molecules which act on microenvironment around endothelial cells and participates in inflammatory pathological process.