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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 <jats:italic>Drosophila<\/jats:italic>; 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\u2009h throughout 24\u2009h); their transcriptomes were scanned to identify the circadian miRNAs and to explore regulatory mechanisms within the uterus of chickens.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>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<jats:sup>2+<\/jats:sup>-transporting ATPase 4 (<jats:italic>ATP2B4<\/jats:italic>) 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<jats:sup>2+<\/jats:sup> transportation during eggshell calcification in the chicken uterus by targeting <jats:italic>ATP2B4<\/jats:italic>. 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\u2009h\u2009cycle in the chicken uterus; it inhibited mRNA and protein expression of ATP2B4 in the uterine tubular gland cells. In addition, overexpression of <jats:italic>ATP2B4<\/jats:italic> significantly decreased intracellular Ca<jats:sup>2+<\/jats:sup> concentration (<jats:italic>P<\/jats:italic>\u2009&lt;\u20090.05), while knockdown of <jats:italic>ATP2B4<\/jats:italic> accelerated intracellular Ca<jats:sup>2+<\/jats:sup> concentrations. We found similar results after <jats:italic>ATP2B4<\/jats:italic> knockdown by miR-449c-5p. Taken together, these results indicate that <jats:italic>ATP2B4<\/jats:italic> promotes uterine Ca<jats:sup>2+<\/jats:sup> trans-epithelial transport.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>Clock-controlled miR-449c-5p regulates Ca<jats:sup>2+<\/jats:sup> transport in the chicken uterus by targeting <jats:italic>ATP2B4<\/jats:italic> during eggshell calcification.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12864-021-08074-3","type":"journal-article","created":{"date-parts":[[2021,10,26]],"date-time":"2021-10-26T06:03:05Z","timestamp":1635228185000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["Circadian miR-449c-5p regulates uterine Ca2+ transport during eggshell calcification in chickens"],"prefix":"10.1186","volume":"22","author":[{"given":"Zhifu","family":"Cui","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhichao","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Felix Kwame","family":"Amevor","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaxia","family":"Du","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liang","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yaofu","family":"Tian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xincheng","family":"Kang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gang","family":"Shu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qing","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diyan","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yao","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoling","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,10,26]]},"reference":[{"key":"8074_CR1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/S0065-2660(03)48000-0","volume":"48","author":"JC Hall","year":"2003","unstructured":"Hall JC. 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