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Objective:To explore the relative genes that may influence kidney aging and verify the expression of clock gene Arntl in aging kidney. Methods:The differentially expressed genes between C57BL/6 male aging mice (24 months old) group and young mice (3 months old) group were identified by whole transcriptome sequencing, and the enriched biological pathways and key proteins were analyzed by bioinformatics methods. RT-qPCR and Western blotting were used to verify the mRNA and protein expression of Arntl.Results:(1) A total of 119 differentially expressed genes were screened between aging mice group and young mice group by whole transcriptome sequencing. Differentially expressed genes were mainly enriched in biological processes such as rhythmic process, circadian rhythm and circadian regulation of gene expression (all P<0.001). Protein-protein interaction analysis results showed that Nfil3, Hspa8, Arntl, Hlf, Rorc, Per3 and Npas2 and so on, were the key proteins in these differentially expressed genes. The results of RT-qPCR confirmed that the expression differences of clock genes Arntl, Nfil3, Npas2 and Per3 between aging mice group and young mice group were consistent with sequencing results (all P<0.05). (2) Compared with C57BL/6 young mice group and SAMR1 rapidly aging mice, the protein expression of Arntl in aging mice group and SAMP8 rapidly aging mice had downward trends. Conclusions:Clock genes and their circadian biological pathways may play an important role in the process of renal aging. The expression of Arntl in aging kidney has a downward trend.
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【Objective】 To evaluate the effect of Arntl on T cell development and T cell-mediated anti-viral immunity. 【Methods】 ArntlF/FCD4cre+(KO) in mice was constructed to delete Arntl gene specifically in T cells. We examined the percentage and number of T cell subsets in the thymus and spleen by flow cytometry (FCM). At day 8 after lymphocytic choriomeningitis virus (LCMV) infection, the proportions of T cell subsets, virus-specific CD8+ T cells and IFN-γ secreting T cells were analyzed. The viral load in the spleen was measured using qPCR. Naive CD4+ T cells (CD4+CD25-CD44-CD62L+) were sorted by flow cytometry to perform T helper cell differentiation in vitro. 【Results】 The percentage and number of T cells in the thymus and spleen of KO mice showed no significant change compared with those in the control group (ArntlF/FCD4cre- mice, WT) (P>0.05). Acute LCMV infection did not cause observable changes in effector T cell proportion in the spleen of KO mice compared to that in WT mice (P>0.05), but KO mice showed a higher proportion of IFN-γ secreting T cells (P<0.05) and better virus clearance (P<0.05). In addition, naive CD4+ T cells from KO mice were more prone to differentiate into Th1 cells in vitro (P<0.05). 【Conclusion】 Arntl deletion in T cells does not affect T cell development, but enhances their ability to defend against viral infection by promoting Th1 cell differentiation and response.
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Low density lipoprotein receptor (LDLR) plays an important role in the cholesterol homeostasis. We examined the possible circadian regulation of LDLR and mechanism(s) underlying it. In mice, blood glucose and plasma triglyceride, total and high density lipoprotein cholesterol varied distinctively throughout a day. In addition, LDLR mRNA oscillated in the liver in a functional clock-dependent manner. Accordingly, analysis of human LDLR promoter sequence revealed three putative E-boxes, raising the possible regulation of LDLR expression by E-box-binding transcription factors. To test this possibility, human LDLR promoter reporter constructs were transfected into HepG2 cells and the effects of CLOCK/BMAL1, Hes1, and Hes6 expression were analyzed. It was found that positive circadian transcription factor complex CLOCK/BMAL1 upregulated human LDLR promoter activity in a serum-independent manner, while Hes family members Hes1 and Hes6 downregulated it only under serum-depleted conditions. Both effects were mapped to proximal promoter region of human LDLR, where mutation or deletion of well-known sterol regulatory element (SRE) abolished only the repressive effect of Hes1. Interestingly, hes6 and hes1 mRNA oscillated in an anti-phasic manner in the wild-type but not in the per1-/-per2-/- mouse. Comparative analysis of mouse, rat and human hes6 genes revealed that three E-boxes are conserved among three species. Transfection and site-directed mutagenesis studies with hes6 reporter constructs confirmed that the third E-box in the exon IV is functionally induced by CLOCK/BMAL1. Taken together, these results suggest that LDLR expression is under circadian control involving CLOCK/BMAL1 and Hes family members Hes1 and Hes6.