CircRNA-006258 Sponge-Adsorbs miR-574-5p to Regulate Cell Growth and Milk Synthesis via EVI5L in Goat Mammary Epithelial Cells
Abstract
1. Introduction
2. Materials and Methods
2.1. Mammary Gland Sample Collection
2.2. Cell Culture
2.3. Vector Construction
2.4. Luciferase Assay
2.5. Transfection and RNA Extraction
2.6. Reverse Transcription and Real-Time PCR
2.7. Western Blot
2.8. Cell Cycle, Proliferation and Apoptosis Assay
2.9. Determination of β-Casein and Triglyceride Analysis
2.10. Statistical Analysis
3. Results
3.1. EVI5L Is One of Target Genes of miR-574-5p in GMECs
3.2. EVI5L Arrested G0/G1 Phase and Promoted S Phase in GMECs
3.3. EVI5L Promoted the Growth of GMECs
3.4. EVI5L Increased the Milk Synthesis via PI3K/AKT–mTOR Signaling Pathway
3.5. CircRNA-006258 Promoted EVI5L Expression in GMECs
3.6. CircRNA-006258 Promoted GMEC Proliferation in GMECs
3.7. CircRNA-006258 Promoted Milk Synthesis of GMECs
3.8. CircRNA-006258 Regulated Cell Growth and Milk Synthesis
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Gene | GenBank Accession No. | Primer Sequences (5’→3’) |
---|---|---|
miR-574-5p | FORWARD: CGGTATGAGTGTGTGTGTGTGAG | |
REVERSE: ATCCAGTGCAGGGTCCGAGG | ||
RT PRIMER: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCACACA | ||
U6 | FORWARD: CTCGCTTCGGCAGCACA | |
REVERSE: AACGCTTCACGAATTTGCGT | ||
EVI5L | XM_018050954.1 | FORWARD:TGTGTTCGTGCGGCTGATGC |
REVERSE:AGGTGGTGAGGAAGAGTGTGAGG | ||
circRNA-006258 | / | FORWARD:AGCGGCATCTCCACCATCTG |
REVERSE:GAAACACTTGGCTGGGCTGG | ||
β-actin | XM_018039831.1 | FORWARD: GATCTGGCACCACACCTTCT |
REVERSE: GGGTCATCTTCTCACGGTTG |
Name | Manufacturer | Product Number |
---|---|---|
β-actin | Beyotime, Shanghai, China | AA128 |
EVI5L | abcam, Cambridge, U.K. | ab243722 |
Rab 23 | ABclonal, Wuhan, China | A7979 |
ITGB 1 | Sangon Biotech, Shanghai, China | D120869 |
TIAM 1 | ABclonal, Wuhan, China | A10252 |
Rac 1 | ABclonal, Wuhan, China | A7720 |
Smad 3 | ABclonal, Wuhan, China | A11388 |
p-Smad 3 | ABclonal, Wuhan, China | Ao0554 |
CDK 4 | Sangon Biotech, Shanghai, China | D120396 |
p-CDK 4 | ABclonal, Wuhan, China | Ap0593 |
Cyclin D1 | ABclonal, Wuhan, China | A11310 |
CDK 2 | Sangon Biotech, Shanghai, China | D120395 |
p-CDK 2 | Sangon Biotech, Shanghai, China | D155352 |
Cyclin E1 | ABclonal, Wuhan, China | A14225 |
AKT | Cell Signaling, America | #9272 |
p-AKT | Cell Signaling, America | #9271 |
mTOR | Abway, Beijing, China | CY5306 |
p-mTOR | Boster, Wnhan, China | BM4840 |
Bcl 2 | Abway, Beijing, China | CY6717 |
BAX | Abway, Beijing, China | CY5059 |
S6K1 | BBI, Shanghai, China | D199437 |
p-S6K1 | BBI, Shanghai, China | D151520 |
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Zhang, M.; Ma, L.; Liu, Y.; He, Y.; Li, G.; An, X.; Cao, B. CircRNA-006258 Sponge-Adsorbs miR-574-5p to Regulate Cell Growth and Milk Synthesis via EVI5L in Goat Mammary Epithelial Cells. Genes 2020, 11, 718. https://doi.org/10.3390/genes11070718
Zhang M, Ma L, Liu Y, He Y, Li G, An X, Cao B. CircRNA-006258 Sponge-Adsorbs miR-574-5p to Regulate Cell Growth and Milk Synthesis via EVI5L in Goat Mammary Epithelial Cells. Genes. 2020; 11(7):718. https://doi.org/10.3390/genes11070718
Chicago/Turabian StyleZhang, Meng, Li Ma, Yuhan Liu, Yonglong He, Guang Li, Xiaopeng An, and Binyun Cao. 2020. "CircRNA-006258 Sponge-Adsorbs miR-574-5p to Regulate Cell Growth and Milk Synthesis via EVI5L in Goat Mammary Epithelial Cells" Genes 11, no. 7: 718. https://doi.org/10.3390/genes11070718
APA StyleZhang, M., Ma, L., Liu, Y., He, Y., Li, G., An, X., & Cao, B. (2020). CircRNA-006258 Sponge-Adsorbs miR-574-5p to Regulate Cell Growth and Milk Synthesis via EVI5L in Goat Mammary Epithelial Cells. Genes, 11(7), 718. https://doi.org/10.3390/genes11070718