Abstract
BackgroundmiRNAs regulate circadian patterns by modulating the biological clocks of animals. In our previous study, we found that the clock gene exhibited a cosine expression pattern in the fallopian tube of chicken uterus. Clock-controlled miRNAs are present in mammals and Drosophila; however, whether there are clock-controlled miRNAs in the chicken uterus and, if so, how they regulate egg-laying rhythms is unclear. In this study, we selected 18 layer hens with similar ovipositional rhythmicity (each of three birds were sacrificed for study per 4 h throughout 24 h); their transcriptomes were scanned to identify the circadian miRNAs and to explore regulatory mechanisms within the uterus of chickens.ResultsWe identified six circadian miRNAs that are mainly associated with several biological processes including ion trans-membrane transportation, response to calcium ion, and enrichment of calcium signaling pathways. Verification of the experimental results revealed that miR-449c-5p exhibited a cosine expression pattern in the chicken uterus. Ca2+-transporting ATPase 4 (ATP2B4) in the plasma membrane is the predicted target gene of circadian miR-449c-5p and is highly enriched in the calcium signaling pathway. We speculated that clock-controlled miR-449c-5p regulated Ca2+ transportation during eggshell calcification in the chicken uterus by targeting ATP2B4. ATP2B4 mRNA and protein were rhythmically expressed in the chicken uterus, and dual-luciferase reporter gene assays confirmed that ATP2B4 was directly targeted by miR-449c-5p. The expression of miR-449c-5p showed an opposite trend to that of ATP2B4 within a 24 h cycle in the chicken uterus; it inhibited mRNA and protein expression of ATP2B4 in the uterine tubular gland cells. In addition, overexpression of ATP2B4 significantly decreased intracellular Ca2+ concentration (P < 0.05), while knockdown of ATP2B4 accelerated intracellular Ca2+ concentrations. We found similar results after ATP2B4 knockdown by miR-449c-5p. Taken together, these results indicate that ATP2B4 promotes uterine Ca2+ trans-epithelial transport.ConclusionsClock-controlled miR-449c-5p regulates Ca2+ transport in the chicken uterus by targeting ATP2B4 during eggshell calcification.
Highlights
MiRNAs regulate circadian patterns by modulating the biological clocks of animals
The CLOCK-BMAL1 complex is found in the mammalian circadian clock, where it binds to the CACGTG Ebox or its allied E-box-like sequence to promote rhythmic genes, regulate the transcription of those genes in peripheral tissues, and promote circadian oscillation [15,16,17,18,19,20]
Results of the morphological or physical observations of the oviducts showed that eggs were present in the oviduct ampulla and isthmus at ZT4 and ZT8, respectively; whereas we found that eggs were present in the uterus at ZT12, ZT16, ZT20 and ZT0
Summary
MiRNAs regulate circadian patterns by modulating the biological clocks of animals. We found that the clock gene exhibited a cosine expression pattern in the fallopian tube of chicken uterus. The CLOCK-BMAL1 complex is found in the mammalian circadian clock, where it binds to the CACGTG Ebox or its allied E-box-like sequence to promote rhythmic genes, regulate the transcription of those genes in peripheral tissues, and promote circadian oscillation [15,16,17,18,19,20]. Previous studies reported NPAS2 as a homolog of CLOCK; other vital clock homologous complexes such as CLOCKBMAL1 or NPAS2-BMAL1 facilitate E-box-dependent transcription [21, 22]. NPAS2 is reported to compensate CLOCK [23,24,25]; any alterations in the form of deletions or mutations of NPAS2 could directly cause a complete disruption of the biological rhythmical order [26]
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