Inhibition of miR-143-3p Restores Blood–Testis Barrier Function and Ameliorates Sertoli Cell Senescence
Abstract
1. Introduction
2. Materials and Methods
2.1. Ex Vivo Culture of Mouse SCs
2.2. Animals
2.3. Cell Transfection
2.4. Quantitative Real-Time PCR (qRT-PCR)
2.5. Western Blot Assay
2.6. Treatment of SCs with SB431542
2.7. Transepithelial Electrical Resistance (TER) Measurements
2.8. Luciferase Reporter Assay
2.9. β-Gal Staining
2.10. Oil Red O Staining
2.11. EdU Staining
2.12. Biotin-NHS Ester Labeling Assay
2.13. Testicular Injection of miR-143-3p
2.14. Treatment of Mice with SB431542
2.15. Statistical Analysis
3. Results
3.1. Role of miR-143-3p Overexpression in SC Senescence and BTB Dysfunction
3.2. miR-143-3p Promoted the Senescence of SCs by Targets UBE2E3
3.3. miR-143-3p Overexpression in Testis Induced SCs Dysfunction
3.4. Loss of UBE2E3 Induce SCs Senescence
3.5. Loss of UBE2E3 Induced SC Dysfunction
3.6. SB431542 Attenuated Age-Related SC Dysfunction by Inhibiting miR-143-3p and Upregulating UBE2E3
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Name | Sequences (5′-3′) |
---|---|
miR-143-3p-mimic | UGAGAUGAAGCACUGUAGCUC |
NC-mimic | UUUGUACUACACAAAAGUACUG |
miR-143-3p-inhibitor | GAGCUACAGUGCUUCAUCUCA |
NC-inhibitor | CAGUACUUUUGUGUAGUACAAA |
si-Ube2e3 | GCATAGCCACTCAGTATTT |
Name | Sense Primers (5′-3′) | Anti-Sense Primers |
---|---|---|
β-actin | GAGCGCAAGTACTCTGTGTG | AACGCAGCTCAGTAACAGTC |
Ube2e3 | TGCAACATCAACAGTCAGGGA | GAGTGGCTATGCTTCCGACC |
Il-1β | GCCACCTTTTGACAGTGATGAG | GACAGCCCAGGTCAAAGGTT |
Il-6 | CACTTCACAAGTCGGAGGCT | CTGCAAGTGCATCATCGTTGT |
Il-10 | GGAGGGGTTCTTCCTTGGGA | TGAGCTGCTGCAGGAATGAT |
Cxcl-1 | TGCACCCAAACCGAAGTCAT | CTCCGTTACTTGGGGACACC |
Cxcl-2 | TCATAGCCACTCTCAAGGGC | TCAGGTACGATCCAGGCTTC |
Tnf-α | ATGTCTCAGCCTCTTCTCATTC | GCTTGTCACTCGAATTTTGAGA |
Antibody | Source | Item Number | Working Dilution |
---|---|---|---|
p53 | Proteintech (Wuhan, China) | 10442 | 1:1000 (WB) |
p38 MAPK | Cell Signaling Technology (Boston, MA, USA) | 8690T | 1:1000 (WB) |
p16INK4a | Abcam (Cambridge, MA, USA) | Ab211542 | 1:1000 (WB) |
β-actin | Fude BioTECH (Hangzhou, China) | FD0060 | 1:5000 (WB) |
UBE2E3 | Proteintech | 15488 | 1:1000 (WB) |
ZO-1 | Abcam | Ab221547 | 1:100 (IF)–1:1000 (WB) |
Occludin | Abcam | Ab216327 | 1:1000 (WB) |
Claudin-11 | Invitrogen (Carlsbad, CA, USA) | 364500 | 1:100 (IF)–1:500 (WB) |
PCNA | Abcam | Ab29 | 1:100 (IF) |
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Xiao, Z.; Liang, J.; Huang, R.; Chen, D.; Mei, J.; Deng, J.; Wang, Z.; Li, L.; Li, Z.; Xia, H.; et al. Inhibition of miR-143-3p Restores Blood–Testis Barrier Function and Ameliorates Sertoli Cell Senescence. Cells 2024, 13, 313. https://doi.org/10.3390/cells13040313
Xiao Z, Liang J, Huang R, Chen D, Mei J, Deng J, Wang Z, Li L, Li Z, Xia H, et al. Inhibition of miR-143-3p Restores Blood–Testis Barrier Function and Ameliorates Sertoli Cell Senescence. Cells. 2024; 13(4):313. https://doi.org/10.3390/cells13040313
Chicago/Turabian StyleXiao, Ziyan, Jinlian Liang, Rufei Huang, Derong Chen, Jiaxin Mei, Jingxian Deng, Zhaoyang Wang, Lu Li, Ziyi Li, Huan Xia, and et al. 2024. "Inhibition of miR-143-3p Restores Blood–Testis Barrier Function and Ameliorates Sertoli Cell Senescence" Cells 13, no. 4: 313. https://doi.org/10.3390/cells13040313
APA StyleXiao, Z., Liang, J., Huang, R., Chen, D., Mei, J., Deng, J., Wang, Z., Li, L., Li, Z., Xia, H., Yang, Y., & Huang, Y. (2024). Inhibition of miR-143-3p Restores Blood–Testis Barrier Function and Ameliorates Sertoli Cell Senescence. Cells, 13(4), 313. https://doi.org/10.3390/cells13040313