The Influences Of Circadian CLOCK Protein On The Transcriptional Regulation And The Function Of The Atherosclerosis-related Genes

Part I:The role of circadian CLOCK protein in the transcriptional regulation of the atherosclerosis-related genesObjective: To identify which were the binding targets from the atherosclerosis-related genes for circadian CLOCK protein via the canonical E-box or E-box-like motifs in the promoters, and to confirm which were the possible ccgs (clock-controlled genes).Methods:1. We analyzed the E-box or E-box-like motifs within the-2kb promoter regions of the atherosclerosis-related genes in PubMed. ChIP (chromatin immunoprecipitation) assays were used to identify which were the targets of CLOCK protein, and to confirm that which the possible ccgs were. The infusion method with collagen IV was adopted to cuture the primary hepatocytes of C57BL/6J mice.2. We synthesized the siRNA (small interference RNA, siRNA) of mouse clock gene and constructed the clock over-expressed adenovirus.48h after transfection of clock-siRNA or infection of clock over-expressed adenovirus into primary hepatocytes, the mRNA levels of the atherosclerosis-related genes (genes identified to be regulated by CLOCK via ChIP assays) were measured by Realtime PCR.3. We cultured the primary hepatocytes of the CLOCK mutant mice and C57BL/6J mice. Then Realtime PCR method was used to detect the mRNA levels of the atherosclerosis-related genes (genes identified to be regulated by CLOCK via ChIP assays).Results:1. The binding capacities between CLOCK protein and LIF (leukemia inhibitory factor)、ICAM-1、ADFP(adipose differentiation-related protein), NF-κB1(encoding the NF-κB p105) genes were proved to be2.58、3.48、2.45、2.27-fold that of the negative control group via the chromatin immunoprecipitation assays.. The CLOCK protein could regulate the transcriptions of these genes by binding to the E-box or E-box-like motifs in their promoters in vivo. LIF、ICAM-1、ADFP、NF-κB1may be the clock-controlled genes.2.48hours after transfection with clock-siRNA, the mRNA level of clock gene was down-regulated by81%in primary hepatocytes of C57BL/6J mice. The mRNA levels of LIF, NF-κB1、ADFP, ICAM-1genes were reduced by31.6%、40.5%、10%、41.8%respectively.48hours after infection with clock over-expressed adenoviruses in hepatocytes of C57BL/6J mice, the mRNA level of clock gene is201-fold that of GFP-adenovirus group, while ADFP、ICAM-1showed a2.03-fold and2.81-fold increase that of GFP-adenovirus group group respectively. The changes in mRNA levels of LIF and NF-κB1genes were not significant.3. Compared to C57BL/6J mice, the mRNA levels of LIF、NF-κB1、ADFP、 ICAM-1genes in primary hepatocytes of homozygous CLOCK mutant mice decreased significantly by46.9%、52%、16.7%and45.7%respectively.Conclusion:1. We identified that CLOCK could bind to the E-box or E-box-like motifs in the promoters of LIF、ICAM-1、ADFP、ICAM-1genes in vivo for the first time.2. Either inhibiting the expression of clock gene of the C57BL/6J mice or in primary hepatocytes of CLOCK mutant mice, the mRNA levels of LIF、NF-κB1、ADFP、 ICAM-1genes attenuated significantly.3. Our research hinted that LIF、NF-κB1、ADFP、ICAM-1might be the possible circadian clock-controlled genes.Part Ⅱ:The circadian CLOCK protein may regulate the transcription of ICAM-1gene and modulate the endothelial cell adhesive ability of mouseObjective:The study tried to clarify the transcriptional regulation role of CLOCK protein on the ICAM-1gene and to implore that how the CLOCK protein influences the endothelial cell adhesive ability.Methods:1. Using pGL3-Promoter Luciferase Reporter Vector, we constructed the promoter reporter of mouse ICAM-1gene, amplifying the specific promoter region (from-729to-329) of ICAM-1which includes the E-box-like motif (CAAGTG, from-584to-579) The mICAMl-pGL3-LUC reporter construct and the clock-siRNA or the negative control siRNA were co-transfected into the hepatocytes of C57BL/6J mice. The luciferase activities were measured48h after transfection.2. The clock-siRNA or negative control-siRNA was introduced into the bEnd.3cells, and the mRNA levels of the atherosclerosis-related genes (genes identified to be transcriptional regulated by CLOCK protein via ChIP assays) were measured by Realtime PCR24h after transfection.3. The cell adhesive ability of bEnd.3cells and the mRNA expression levels of cell-adhesion-related genes were measured48h after transfection with clock-siRNA or negative control siRNA in bEnd.3cells.Results:1. We successfully constructed the promoter reporter of mouse ICAM-1gene which includes the E-box-like enhancer, the mICAM1-pGL3-LUC reporter construct. Compared to the negative control group, the luciferase activity was down-regulated by61.5%48h after co-transfection with the mICAMl-pGL3-LUC reporter construct and clock-siRNA in the hepatocytes of C57BL/6J mice.2. Compared to the negative control group, the mRNA level of clock gene was down-regulated by54.33%24hours after transfection with clock-siRNA in bEnd.3cells. The mRNA levels of ICAM-1、LIF、NF-κB1、ADFP attenuated by34.43%、38%、26%and8%respectively.3. Compared to the negative control group, the cell adhesive ability of bEnd.3cells was down-regulated by41.7%48h after transfection with clock-siRNA. The mRNA levels of cell-adhesion-related genes, such as VCAM-1、CCL-2、CCL-5attenuated by21.7%、27.4%、19%respectively. Conclusions:1. The circadian clock gene could regulate the transcriptional activity and level of ICAM-1gene.2. The endothelial cell adhesive ability and the mRNA levels of cell-adhesion-related genes attenuated after down-regulation of clock gene. Our present study showed the possible mechanism that the circadian clock gene may influence the occurrence and development of atherosclerosis through regulating the cell adhesive ability.