The TGF-β/SMAD Signaling Pathway Prevents Follicular Atresia by Upregulating MORC2
Abstract
:1. Introduction
2. Results
2.1. MORC2 Inhibits Apoptosis of Porcine Follicular Granulosa Cells
2.2. MORC2 Inhibits Apoptosis of Porcine Granulosa Cells Independently of the DNA Repair Pathway
2.3. MORC2 Is a Downstream Target of the TGF-β/SMAD Signaling Pathway
2.4. The Transcription Factor SMAD4 Directly Binds to the MORC2Promoter
3. Discussion
4. Materials and Methods
4.1. Reagents
4.2. Isolation and Culture of Porcine Granulosa Cells
4.3. Plasmid Construction
4.4. Cell Transfection
4.5. Total RNA Extraction and Quantitative PCR
4.6. WB Analysis
4.7. Tunel
4.8. Luciferase Reporter Gene Assay
4.9. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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siRNA | Sequence(5′ to 3′) |
---|---|
NC-siRNA | F: UUCUCCGAACGUGUCACGUTT R: UUCUCCGAACGUGUCACGUTT |
SMAD4-siRNA | F: CACCAGGAAUUGAUCUCUCAGGAUU R: CACCAGGAAUUGAUCUCUCAGGAUU |
MORC2-294 | F: GCAAUACGGGAAUGGGUUATT R: UAACCCAUUCCCGUAUUGCTT |
MORC2-584 | F: GCGGAACGCUGGUCAUCAUTT R: AUGAUGACCAGCGUUCCGCTT |
MORC2-1164 | F: CCGCCUGAUCAAGAUGUAUTT R: AUACAUCUUGAUCAGGCGGTT |
Name | Primer Sequence | Anneal T (°C) | Product Size (bp) | Usage |
---|---|---|---|---|
MORC2 | F: TCACCAAGAAGGAAGACACTA R: AGATGATGACCAGCGTTCC | 58.9 | 252 | qRT-PCR |
Bax | F: GCCGAAATGTTTGCTGAC R: GCCGATCTCGAAGGAAGT | 58 | 154 | qRT-PCR |
Bcl2 | F: TCAGGGATGGGGTGAACT R: TCAGAGACAGCCAGGAGAAAT | 60 | 240 | qRT-PCR |
GAPDH | F: GGACTCATGACCACGGTCCAT R: TCAGATCCACAACCGACACGT | 60 | 220 | qRT-PCR |
SMAD4 | F: ATTGGTGTTCCATTGCCTAC R: TGGTCACTAAGGCACCTGAC | 58 | 250 | qRT-PCR |
ATR | F: TATCTGGGCTCCCTCCTC R: CTCCTTTCTGTATTCCTGTAGAACT | 58.6 | 204 | qRT-PCR |
ATM | F: CTGTGAGATTGGCGTTAGT R: AACATGCAAACTTGGTGAT | 59 | 154 | qRT-PCR |
P53 | F: TGACTGTACCACCATCCACTAC R: AGGCACAAACACGCACCT | 60 | 149 | qRT-PCR |
Rad51 | F: GGTGGAGGTGAAGGAAAG R: CTGGGTCTGGTGGTCTGT | 60 | 156 | qRT-PCR |
PARP-1 | F: AGCACCTGTGACGGGCTAC R: CTTGCTGATGTGCGAAGC | 61 | 171 | qRT-PCR |
Caspase-3 | F: TTGGACTGTGGGATTGAGACG R: CGCTGCACAAAGTGACTGGA | 59 | 165 | qRT-PCR |
pcDNA3.1-his-v5-MORC2 | F: CCCAAGCTTGCCACCATGGCTTTCACAAATTACAGCAGTCTGAATC R: CCGCTCGAGGTCCCCCTTGGTGATCAGGTCC | 68 | 3102 | Over-expression |
pcDNA3.1-SMAD4 | F: CCCAAGCTTGCCACCATGGACAATATGTCTATTACAAATACACCAACAAG R: CCGCTCGAGTCAGTCTAAAGGCTGTGGGTCGGC | 68 | 1659 | Over-expression |
pGL3-MORC2-1347 | F: CGGGTACCGGTCAAGAAAGCAGGAAGAGAG | 68 | 1347 | promoter |
pGL3-MORC2-1479 | F: CGGGTACCGCTTCTTCGAGTTAGGGGCCTGACT | 68 | 1479 | promoter |
pGL3-MORC2-R | R: CCGCTCGAGTGCAGTGAGGTCTCCAGTTCTTT | - | promoter | |
pGL3-SBE1-mut | F: TGACTCCCCGGCGGATGGCTCCTCCGGTCCGC R: CATCTCGCCGGGGATCAGGCCCCTAACTCGAA | SEB mutation |
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Liu, J.; Qi, N.; Xing, W.; Li, M.; Qian, Y.; Luo, G.; Yu, S. The TGF-β/SMAD Signaling Pathway Prevents Follicular Atresia by Upregulating MORC2. Int. J. Mol. Sci. 2022, 23, 10657. https://doi.org/10.3390/ijms231810657
Liu J, Qi N, Xing W, Li M, Qian Y, Luo G, Yu S. The TGF-β/SMAD Signaling Pathway Prevents Follicular Atresia by Upregulating MORC2. International Journal of Molecular Sciences. 2022; 23(18):10657. https://doi.org/10.3390/ijms231810657
Chicago/Turabian StyleLiu, Jiying, Nannan Qi, Wenwen Xing, Mengxuan Li, Yonghang Qian, Gang Luo, and Shali Yu. 2022. "The TGF-β/SMAD Signaling Pathway Prevents Follicular Atresia by Upregulating MORC2" International Journal of Molecular Sciences 23, no. 18: 10657. https://doi.org/10.3390/ijms231810657