chi-miR-487b-3p Inhibits Goat Myoblast Proliferation and Differentiation by Targeting IRS1 through the IRS1/PI3K/Akt Signaling Pathway
Abstract
:1. Introduction
2. Results
2.1. miR-487b-3p Acts as a Candidate Regulator in Goat Skeletal Myogenesis
2.2. miR-487b-3p Inhibits Goat Myoblast Proliferation
2.3. miR-487b-3p Inhibits Goat Myoblast Differentiation
2.4. IRS1 Is a Direct Target of miR-487b-3p
2.5. IRS1 Knockdown Suppresses Goat Myoblast Proliferation and Differentiation
2.6. miR-487b-3p Affects the IRS1/PI3K/Akt Signaling Pathway
3. Discussion
4. Materials and Methods
4.1. Animal Tissue Sample Collection
4.2. Cells Culture
4.3. RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
4.4. RNA Oligonucleotides
4.5. Cell Transfection
4.6. Cell Counting Kit-8 (CCK-8) Assays
4.7. 5-Ethynyl-2-deoxyuridine (EdU) Imaging Assays
4.8. Flow Cytometric Analysis
4.9. Bioinformatics Analysis for Target Genes Prediction
4.10. Dual-Luciferase Reporter Assay
4.11. Protein Extraction and Western Blot Analysis
4.12. Immunofluorescence
4.13. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Primer Name | Primer Sequence (5′ to 3′) | Reference |
---|---|---|---|
miR-487b-3p | Stem-loop | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAGTGG | [18] |
miR-487b-3p-F | CGGGCAATCGTACAGGGT | This manuscript | |
miR-487b-3p-R | CAGTGCAGGGTCCGAGGTAT | ||
18S-rRNA | 18S-rRNA-F | GTGGTGTTGAGGAAAGCAGACA | [18] |
18S-rRNA-R | TGATCACACGTTCCACCTCATC | ||
IRS1 | IRS1-F | GTAGTGGCAAACTCCTGTCTTGT | This manuscript |
IRS1-R | GAGTAGTAGGAGAGGACGGGCT | ||
PCNA | PCNA-F | CGCTTAAGGATCTCATCAATGAG | This manuscript |
PCNA-R | GTTACGGTCGCAGCGGTAAG | ||
CDK2 | CDK2-F | TCATGGATGCCTCTGCACTC | [42] |
CDK2-R | CTCTGGCTAGTCCGAAGTCTG | ||
p27 | p27-F | CGGCGGTGCCTTTACTT | [42] |
p27-R | GCAGGTCGCTTCCTTATCC | ||
MyoG | MyoG-F | GGACCCTACAGATGCCCACAA | This manuscript |
MyoG-R | TTGGTATGGTTTCATCTGGG | ||
MyHC | MyHC-F | GTGAAGGAGGACCAGGTGTTG G | [42] |
MyHC-R | GTTGATGGTGACGCAGAAGAG | ||
GAPDH | GAPDH-F | CCACGCCATCACTGCCACCC | [42] |
GAPDH-R | CAGCCTTGGCAGCGCCAGTA |
Name | Sense Sequence (5′-3′) |
---|---|
Mimics | AAUCGUACAGGGUCAUCCACUU |
Mimics NC | UUCUCCGAACGUGUCACGUTT |
Inhibitors | AAGUGGAUGACCCUGUACGAUU |
Inhibitors NC | CAGUACUUUUGUGUAGUACAA |
siIRS1 | GCAUGCACAAGCGAUUCUUTT |
AAGAAUCGCUUGUGCAUGCTT | |
siNC | UUCUCCGAACGUGUCACGUTT |
ACGUGACACGUUCGGAGAATT |
Name | Primer Name | Primer Sequence (5′ to 3′) |
---|---|---|
Wild-IRS1-3′UTR | Wild-IRS1-F | cggCTCGAGCAGCAAATCCTCCTTTAACTC |
Wild-IRS1-R | aatGCGGCCGCGCACGATATACAACGTGCAG | |
Mutant-IRS1-3′UTR | Mutant-IRS1-F | CTCAGTAGATGGGCTAATGCACCC |
Mutant-IRS1-R | GAAATGGGTGCCGATTACCATCTAC |
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Lyu, M.; Wang, X.; Meng, X.; Qian, H.; Li, Q.; Ma, B.; Zhang, Z.; Xu, K. chi-miR-487b-3p Inhibits Goat Myoblast Proliferation and Differentiation by Targeting IRS1 through the IRS1/PI3K/Akt Signaling Pathway. Int. J. Mol. Sci. 2022, 23, 115. https://doi.org/10.3390/ijms23010115
Lyu M, Wang X, Meng X, Qian H, Li Q, Ma B, Zhang Z, Xu K. chi-miR-487b-3p Inhibits Goat Myoblast Proliferation and Differentiation by Targeting IRS1 through the IRS1/PI3K/Akt Signaling Pathway. International Journal of Molecular Sciences. 2022; 23(1):115. https://doi.org/10.3390/ijms23010115
Chicago/Turabian StyleLyu, Ming, Xu Wang, Xiangyu Meng, Hongrun Qian, Qian Li, Baoxia Ma, Zhiying Zhang, and Kun Xu. 2022. "chi-miR-487b-3p Inhibits Goat Myoblast Proliferation and Differentiation by Targeting IRS1 through the IRS1/PI3K/Akt Signaling Pathway" International Journal of Molecular Sciences 23, no. 1: 115. https://doi.org/10.3390/ijms23010115