MiR-200-3p Is Potentially Involved in Cell Cycle Arrest by Regulating Cyclin A during Aestivation in Apostichopus japonicus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals
2.2. Prediction of the miR-200-3p Target
2.3. RNA Extraction, Cyclin A cDNA Cloning and Sequence Analysis
2.4. Expression Level Analysis of miR-200-3p and AjCA
2.5. Dual-Luciferase Reporter Assays
2.6. Loss and Gain-Functional Analysis of miR-200-3p In Vivo
2.7. Statistics
3. Results
3.1. Sequence Characterization of AjCA and Target Identification of miR-200-3p
3.2. The Expression of miR-200-3p and AjCA Gene and the Production of AjCA Protein during the Whole Period of Aestivation
3.3. Validation of the Interaction between the 3′UTR of AjCA and miR-200-3p by Dual-Luciferase Reporter Assays
3.4. Gain and Loss of Function Analysis of miR-200-3p In Vivo
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Name | Primer Sequences (5′–3′) | Location | |
---|---|---|---|
RACE | AjCA-F1 | AGCTGGTCGGAACGGCAAGTGCA | 1269–1291 |
AjCA-R1 | GATTGTCGGGACAGCCAGATCAAAGGAC | 1414–1441 | |
qRT-PCR | miR-200-3p-F | TAATACTGTCTGGTGATGATG | |
miR-200-3p-R | mRQ 3′primer | ||
5.8s-F | ATCACTCGGCTCGTGCGTC | ||
5.8s-R | GCCATTTGCGTTCGAATAAGT | ||
AjCA-RT-F | TATCAAGGCCAGCGACGAAGGAG | 1461–1448 | |
AjCA-RT-R | GGAGATGCAATGTGTGTCGAGCC | 1594–1616 | |
β-actin-F | AAGGTTATGCTCTTCCTCACGCT | ||
β-actin-R | GATGTCACGGACGATTTCACG | ||
β-Tubulin-F | GAAAGCCTTACGACGGAACA | ||
β-Tubulin-R | CACCACGTGGACTCAAAATG | ||
In vivo | miR-200-3p mimics-F | UAAUACUGUCUGGUGAUGAUG | |
miR-200-3p mimics-R | UCAUCACCAGACAGUAUUAUU | ||
miR-200-3p mimics-NC-F | GAGAUGUUCAAUCGGGUAUUU | ||
miR-200-3p mimics-NC-R | AUACCCGAUUGAACAUCUCUU | ||
miR-200-3p inhibitor | CAUCAUCACCAGACAGUAUUA | ||
miR-200-3p inhibitor-NC | GAAUUACAUGCACCACUCAAU | ||
Dual-luciferase | AjCA-WT-F | GCGGCTCGAGGACATCTCCAAATTAAAGGA | 1806–1836 |
AjCA-WT-R | AATGCGGCCGCGTAACACTTATGTACAAATG | 3010–3029 | |
AjCA-MUT-F | TGAATCTTGTCATAAACAAGAACGGGTTTTTAC | 2164–2197 | |
AjCA-MUT-R | GTTCTTGTTTATGACAAGATTCATTTTAAAAAC | 2155–2187 |
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Wang, S.; Chen, M.; Yin, Y.; Storey, K.B. MiR-200-3p Is Potentially Involved in Cell Cycle Arrest by Regulating Cyclin A during Aestivation in Apostichopus japonicus. Cells 2019, 8, 843. https://doi.org/10.3390/cells8080843
Wang S, Chen M, Yin Y, Storey KB. MiR-200-3p Is Potentially Involved in Cell Cycle Arrest by Regulating Cyclin A during Aestivation in Apostichopus japonicus. Cells. 2019; 8(8):843. https://doi.org/10.3390/cells8080843
Chicago/Turabian StyleWang, Shanshan, Muyan Chen, Yingchao Yin, and Kenneth B. Storey. 2019. "MiR-200-3p Is Potentially Involved in Cell Cycle Arrest by Regulating Cyclin A during Aestivation in Apostichopus japonicus" Cells 8, no. 8: 843. https://doi.org/10.3390/cells8080843