Related Experiment Video
Updated: Jul 19, 2025
The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression
Published on: July 24, 2018
miR34a-5p impedes CLOCK expression in chronodisruptive C57BL/6J mice and potentiates pro-atherogenic manifestations
Hitarthi Vyas1, Aliasgar Vohra1, Kapil Upadhyay2
1Division of Metabolic Endocrinology, Department of Zoology, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, India.
Introduction:
Altered circadian rhythms underlie manifestation of several cardiovascular disorders, however a little is known about the mediating biomolecules. Multiple transcriptional-translational feedback loops control circadian-clockwork wherein; micro RNAs (miRNAs) are known to manifest post transcriptional regulation. This study assesses miR34a-5p as a mediating biomolecule.
Method:
8-10-week-old male C57BL/6J mice (n = 6/group) were subjected to photoperiodic manipulation induced chronodisruption and thoracic aortae were examined for miRNA, gene (qPCR) and protein (Immunoblot) expression studies. Histomorphological changes were assessed for pro-atherogenic manifestations (fibrillar arrangement, collagen/elastin ratio, intima-media thickening). Computational studies for miRNA-mRNA target prediction were done using TargetScan and miRDB. Correlative in vitro studies were done in serum synchronized HUVEC cells. Time point based studies were done at five time points (ZT 0, 6, 12, 18, 24) in 24h.
Results:
Chronodisruption induced hypomethylation in the promoter region of miR34a-5p, in the thoracic aortae, culminating in elevated miRNA titers. In a software-based detection of circadian-clock-associated targets of miR34a-5p, Clock and Sirt1 genes were identified. Moreover, miR34a-5p exhibited antagonist circadian oscillations to that of its target genes CLOCK and SIRT1 in endothelial cells. Luciferase reporter gene assay further showed that miR34a-5p interacts with the 3'UTR of the Clock gene to lower its expression, disturbing the operation of positive arm of circadian clock system. Elevated miR34a-5p and impeded SIRT1 expression in a chronodisruptive aortae exhibited pro-atherogenic changes observed in form of gene expression, increased collagen/elastin ratio, fibrillar derangement and intimal-media thickening.
Conclusion:
The study reports for the first time chronodisruption mediated miR34a-5p elevation, its circadian expression and interaction with the 3'UTR of Clock gene to impede its expression. Moreover, elevated miR34a-5p and lowered SIRT1 expression in the chronodisruptive aortae lead off cause-consequence relationship of chronodisruption mediated proatherogenic changes.
Insights
Chronodisruption elevates miR34a-5p, impacting the Clock gene and leading to pro-atherogenic changes in the aorta. This study identifies miR34a-5p as a key mediator in circadian rhythm disruption-induced cardiovascular disease.
Area of Science:
- Cardiovascular Biology
- Chronobiology
- Molecular Biology
Background:
- Altered circadian rhythms are linked to cardiovascular disorders, but the specific biomolecules involved remain unclear.
- MicroRNAs (miRNAs) play a role in post-transcriptional regulation within circadian feedback loops.
- This research investigates miR34a-5p as a potential mediator in circadian rhythm disruption and cardiovascular disease.
Purpose of the Study:
- To assess the role of miR34a-5p in chronodisruption-induced cardiovascular changes.
- To investigate the interaction between miR34a-5p, Clock, and Sirt1 genes under conditions of altered circadian rhythms.
- To establish a cause-consequence relationship between miR34a-5p dysregulation and pro-atherogenic manifestations.
Main Methods:
- Mice were subjected to photoperiodic manipulation to induce chronodisruption.
- Thoracic aortae were analyzed for miRNA, gene (qPCR), and protein (Immunoblot) expression.
- Computational and in vitro studies, including luciferase reporter gene assays, were performed to elucidate miRNA-mRNA interactions.
Main Results:
- Chronodisruption led to hypomethylation and elevated miR34a-5p levels in thoracic aortae.
- miR34a-5p was found to have antagonist circadian oscillations with its target genes, Clock and Sirt1.
- Elevated miR34a-5p and reduced SIRT1 expression correlated with pro-atherogenic changes, including intima-media thickening and altered collagen/elastin ratios.
Conclusions:
- This study demonstrates that chronodisruption elevates miR34a-5p, which interacts with the Clock gene's 3'UTR, impeding its expression.
- The findings establish a direct link between elevated miR34a-5p, reduced SIRT1, and the development of pro-atherogenic changes in the context of circadian disruption.
- miR34a-5p is identified as a novel mediator linking circadian disruption to cardiovascular pathology.
