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.
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This preprint is available for download as a PDF.
No competing interests reported.
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Posted 15 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
Invitations sent on 15 Mar, 2021
On 12 Mar, 2021
On 11 Mar, 2021
On 11 Mar, 2021
On 22 Feb, 2021
Posted 15 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
Received 25 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
On 15 Mar, 2021
Invitations sent on 15 Mar, 2021
On 12 Mar, 2021
On 11 Mar, 2021
On 11 Mar, 2021
On 22 Feb, 2021
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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This preprint is available for download as a PDF.
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