MiR-132 Modulates Circadian Rhythms Of Zebrafish Through The Input Pathway

A variety of organisms from bacteria to humans display circadian rhythms,which are an endogenously driven approximately 24- hour cycle in biochemical, physiological, or behavioral processes, including body temperature, feeding behavior, hormone secretion and cell-cycle progression. Disruption of circadian rhythms results in numerous disorders and diseases, such as sleep disorders, metabolic alteration, premature senescence and affective disorders. Thus, it is crucially important to reveal the circadian regulatory mechanisms to account for the physiological, biochemical activities of organisms and then to prevent some related diseases.micro RNAs(miRNAs) are a family of short, single-stranded, noncodingRNAs. They recognize and bind to the 3’UTR of their target mRNAs, and negatively regulate gene expression by inhibiting protein synthesis or degradating their mRNAs. miR-132 is a critical factor for light induced resetting of the SCN clock in mice. Its role in the zebrafish circadian clock, however, remains unclear.We found that the three copies of miR-132 display distinct oscillating patterns, as shown by in situ hybridization and q RT-PCR. We cloned the promoter of miR-132-1. Luciferase assays showed that miR-132-1 promoter activities is enhanced by Dbp-a and ROR-α. Light treatment experiments showed that the expression levels of miR-132 are significantly increased after t two-hour light exposure.To investigate miR-132’s roles in the zebrafish circadian clock, we use the CRISPR-Cas9 system to generate a miR-132 mutant fish line. q RT-PCR showed that both per2 and e4bp4-3 are up-regulated in miR-132 mutant fish. Behavior assays showed that the period of locomotor activities of miR-132 mutant fish is shortened a half hour under DD condition, and its amplitude is increased under LL condition. These results showed that miR-132 plays an important role in zebrafish circadian regulation. The expression of per2 and e4bp4-3 are down-regulated after injecting miR-132 mimic in one cell stage embryos. Luciferase assay indic ates that miR-132 can regulate the circadian timekeeping of zebrafish through combining the 3’UTR of per2. miRNA-132 can also effect chromatin remodling and translation by modulating Mecp2, Ep300 a and Jarid1 a.In our study, by suing cellular, molecular, genetic methods, we analyze the expression site, trends, and its regulation by the way of miR-132 in zebrafish and get the miR-132 mutant zebrafish to study its behavioral cycle. miR-132 mutant also can serve as an important model for studying of neurodevelopmental and many diseases. We also screened the target genes of miR-132. O ur research is important for clarifying how microRNA involved in the regulation of the circadian clock.