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Research Article
Xiaoling Zhao
Sichuan Agricultural University
Corresponding Author
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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.
Keywords
Chicken uterine, Circadian miRNAs, Tubular gland cells, Ca 2+ transport
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No competing interests reported.
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CURRENT STATUS: Under Review
Version 2
Posted 15 Mar, 2021
No community comments so far
Editorial decision: Major revision
On 06 May, 2021
Reviews received
Received 25 Apr, 2021
Reviewers agreed
On 11 Apr, 2021
Reviews received
Received 25 Mar, 2021
Reviewers agreed
On 15 Mar, 2021
Reviewers invited
Invitations sent on 15 Mar, 2021
Editor assigned
On 12 Mar, 2021
Editor invited
On 11 Mar, 2021
Submission checks complete
On 11 Mar, 2021
First submitted
On 22 Feb, 2021
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This preprint is under consideration at BMC Genomics. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research Article
Xiaoling Zhao
Sichuan Agricultural University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 2
Posted 15 Mar, 2021
No community comments so far
Editorial decision: Major revision
On 06 May, 2021
Reviews received
Received 25 Apr, 2021
Reviewers agreed
On 11 Apr, 2021
Reviews received
Received 25 Mar, 2021
Reviewers agreed
On 15 Mar, 2021
Reviewers invited
Invitations sent on 15 Mar, 2021
Editor assigned
On 12 Mar, 2021
Editor invited
On 11 Mar, 2021
Submission checks complete
On 11 Mar, 2021
First submitted
On 22 Feb, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Epigenetics & Genomics
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
No competing interests reported.
This is a list of supplementary files associated with this preprint. Click to download.
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