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Background  miRNAs 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.    Results  We 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. Ca 2+ -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 Ca 2+  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 Ca 2+  concentration ( P  &lt; 0.05), while knockdown of  ATP2B4  accelerated intracellular Ca 2+  concentrations. We found similar results after  ATP2B4  knockdown by miR-449c-5p. Taken together, these results indicate that  ATP2B4  promotes uterine Ca 2+  trans-epithelial transport.    Conclusions  Clock-controlled miR-449c-5p regulates Ca 2+  transport in the chicken uterus by targeting  ATP2B4  during eggshell calcification.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12864-021-08074-3.

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