Circadian miR-449c-5p regulates uterine Ca2+ transport during eggshell calcification in chickens

Abstract

Background: miRNAs regulate circadian patterns by modulating animal biological clock. Clock genes exhibited a cosine expression pattern in the fallopian tube of chicken uterus in our previous study. Clock-controlled miRNAs are present in mammals and Drosophila; however, whether there are clock-controlled miRNAs in chicken uterus and, if so, how they regulate egg-laying rhythms are not clear. Here, we selected 18 layer hens with similar ovipositional rhythmicity (three birds were sacrificed for study per at 4 h intervals throughout 24 h); their transcriptomes were scanned to identify the circadian miRNAs and to explore regulatory mechanisms within the uterus of chickens. Results: We identified six circadian miRNAs mainly associated with several biological processes including ion trans-membrane transportation, response to calcium ion, and enrichment of calcium signaling pathways. Verification of experimental results revealed that miR-449c-5p exhibited a cosine expression pattern in 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 chicken uterus by targeting ATP2B4. ATP2B4 mRNA and protein were rhythmically expressed in chicken uterus, and dual-luciferase reporter gene assays confirmed that ATP2B4 was directly targeted by miR-449c-5p. miR-449c-5p showed an opposite expression profile with ATP2B4 within a 24h cycle in chicken uterus; it inhibited mRNA and protein expressions of ATP2B4 in uterine tubular gland cells. Additionally, overexpression of ATP2B4 significantly decreased intracellular Ca2+ concentration (P < 0.05), while knockdown of ATP2B4 accelerated intracellular Ca2+ concentrations. Similar results were found after ATP2B4 knockdown by miR-449c-5p. These results indicated that ATP2B4 promoted uterine Ca2+ trans-epithelial transport. Conclusions: Clock-controlled miR-449c-5p regulates Ca2+ transport in chicken uterus by targeting ATP2B4 during eggshell calcification.