miRNA-10a-5p Targeting the BCL6 Gene Regulates Proliferation, Differentiation and Apoptosis of Chicken Myoblasts
Abstracts
1. Introduction
2. Results
2.1. miRNA-10a-5p Inhibits the Proliferation of Chicken Myoblasts
2.2. miRNA-10a-5p Inhibits the Differentiation of Chicken Myoblasts
2.3. miRNA-10a-5p Promotes the Apoptosis of Chicken Myoblasts
2.4. miRNA-10a-5p Has a Targeting Relationship with BCL6
2.5. BCL6 Gene Promotes the Proliferation of Chicken Myoblasts
2.6. BCL6 Gene Promotes the Differentiation of Chicken Myoblasts
2.7. BCL6 Gene Inhibits the Apoptosis of Chicken Myoblasts
3. Discussion
4. Materials and Methods
4.1. Ethics Statement
4.2. Cell Isolation, Culture, and Total RNA Extraction
4.3. Primer Design for Quantitative Real-Time PCR (qRT-PCR)
4.4. cDNA Synthesis and Quantitative Real-Time PCR (qRT-PCR)
4.5. Sequence Synthesis and Vector Construction
4.6. Cell Transfection and Dual-Luciferase Reporter Assay
4.7. CCK-8 Assay and Edu Assay
4.8. Cell Cycle Assay
4.9. Apoptosis Assay
4.10. Indirect Immunofluorescence Assay (IFA)
4.11. Western Blot Assay
4.12. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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Name | Primer Sequence (5′-3′) | Annealing Temperature (°C) |
---|---|---|
Gga-miRNA-10a-5p | F: CGCGTACCCTGTAGATCCGA R: AGTGCAGGGTCCGAGGTATT | 60 |
U6 | F: GTCACTTCTGGTGGCGGTAA R: GTTCAGTAGAGGGTCAAA | 60 |
Stem-loop primer | GTCGTATCCAGTGCAGGGTCC GAGGTATTCGCACTGGATACGAC |
Gene | Primer Sequence (5′-3′) | Product Size(bp) | Annealing Temperature (°C) | Accession Number |
---|---|---|---|---|
BCL6 | F: CTCTGGCTCAGCCTTTGGA R: GCACTTGTAGGGTTTGTCGC | 169 | 60 | NM_00102930.1 |
β-actin | F: CAGCCATCTTTCTTGGGTAT R: CTGTGATCTCCTTCTGCA TCC | 169 | 60 | NM_205518.1 |
MYOD 1 | F: GCTACTACACGGAATCACCAAAT R: CTGGGCTCCACTGTCACTCA | 200 | 60 | NM_204214.2 |
MYOG | F: CGGAGGCTGAAGAAGGTGAA R: CGGTCCTCTGCCTGGTCAT | 320 | 60 | NM_204184.1 |
Caspase3 | F: TGGCCCTCTTGAACTGAAAG R: TCCACTGTCTGCTTCAATACC | 139 | 60 | NM_204725.1 |
Fas | F: TCCACCTGCTCCTCGTCATT R: GTGCAGTGTGTGTGGGAACT | 78 | 60 | NM_001199487.1 |
Cyclin D1 | F: CTGCTCAATGACAGGGTGC R: TCGGGTCTGATGGAGTTG | 341 | 60 | NM_205381 |
CyclinE | F: ACCTAAAATGAGAACAATCC R: GGCAACAATACCTCCTAAA | 381 | 60 | NM_001031358.2 |
Fragment Name | Sequence(5′-3′) |
---|---|
miRNA-10a-5p mimic | UACCCUGUAGAUCCGAAUUUGU AAAUUCGGAUCUACAGGGUAUU |
mimic-NC | UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT |
miRNA-10a-5p inhibitor | ACAAAUUCGGAUCUACAGGGUA |
inhibitor-NC | CAGUACUUUUGUGUAGUACAA |
siRNA-1651 | GCGAGAACGGAGCCUUCUUTT AAGAAGGCUCCGUUCUCGCTT |
siRNA-1727 | GCAAGUCCACAGCGACAAATT UUUGUCGCUGUGGACUUGCTT |
siRNA-1968 | GCUCAUGUGCUCAUUCAUATT UAUGAAUGAGCACAUGAGCTT |
siRNA-NC | UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT |
Primer Name | Primer Sequence | Product Size (bp) | Annealing Temperature (°C) |
---|---|---|---|
BCL6-3′UTR-WT | F:AAAGATCCTTTATTAAGCTTTTTTCCCCAGTCTTATTT R:ATAGGCCGGCATAGACGCGTTTGGCGCTTCACTTTTCT | 143 | 62 |
BCL6-3′UTR-MT | F:CGGGAGACGTAGTTTGGGCTGTGTCTAAACTGCAT R:CAAACTACGTCTCCCGAGATTGTTTATAACACAAGC | 6581 | 65 |
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Zhang, G.; Zhang, X.; Zhou, K.; Ling, X.; Zhang, J.; Wu, P.; Zhang, T.; Xie, K.; Dai, G. miRNA-10a-5p Targeting the BCL6 Gene Regulates Proliferation, Differentiation and Apoptosis of Chicken Myoblasts. Int. J. Mol. Sci. 2022, 23, 9545. https://doi.org/10.3390/ijms23179545
Zhang G, Zhang X, Zhou K, Ling X, Zhang J, Wu P, Zhang T, Xie K, Dai G. miRNA-10a-5p Targeting the BCL6 Gene Regulates Proliferation, Differentiation and Apoptosis of Chicken Myoblasts. International Journal of Molecular Sciences. 2022; 23(17):9545. https://doi.org/10.3390/ijms23179545
Chicago/Turabian StyleZhang, Genxi, Xinchao Zhang, Kaizhi Zhou, Xuanze Ling, Jin Zhang, Pengfei Wu, Tao Zhang, Kaizhou Xie, and Guojun Dai. 2022. "miRNA-10a-5p Targeting the BCL6 Gene Regulates Proliferation, Differentiation and Apoptosis of Chicken Myoblasts" International Journal of Molecular Sciences 23, no. 17: 9545. https://doi.org/10.3390/ijms23179545