Melatonin Regulates Osteoblast Differentiation through the m6A Reader hnRNPA2B1 under Simulated Microgravity
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals and Experimental Design
2.2. Micro-CT Scanning
2.3. Immunohistochemistry (IHC)
2.4. Enzyme-Linked Immunosorbent Assay (ELISA)
2.5. Cell Culture and In Vitro Differentiation
2.6. Two-Dimensional Clinorotation
2.7. Cell Transfection
2.8. Quantitative Real-Time PCR (qRT-PCR) and Analysis
2.9. Western Blotting and Analysis
2.10. Alkaline Phosphatase Staining (ALP Staining)
2.11. ALP Activity Assay
2.12. Rescue Experiments
2.13. Statistical Analysis
3. Results
3.1. Expression of hnRNPA2B1 in Osteoblasts under Simulated Microgravity
3.2. hnRNPA2B1 Regulates Osteoblast Differentiation
3.3. Overexpression of hnRNPA2B1 Attenuates the Inhibitory Effect of Simulated Microgravity on Osteoblast Differentiation in MC3T3-E1 Cells
3.4. Melatonin Promotes hnRNPA2B1 Expression in Osteoblasts under Simulated Microgravity and Improves Bone Architecture in HLU Mice
3.5. Melatonin Regulates Osteoblast Differentiation by Targeting hnRNPA2B1
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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RNA Oligo | Sequences (5′-3′) |
---|---|
si-hnRNPA2B1 sense | 5′-GGUGGCUUAAGCUUUGAAATT-3′ |
si-hnRNPA2B1 antisense | 5′-UUUCAAAGCUUAAGCCACCTT-3′ |
si-NC sense | 5′-UUCUCCGAACGUGUCACGUTT-3′ |
si-NC antisense | 5′-ACGUGACACGUUCGGAGAATT-3′ |
Name of Primers | Sequences (5′-3′) |
---|---|
hnRNPA2B1-F | 5′-GCGGAGGAAGAGGCGGTTAC-3′ |
hnRNPA2B1-R | 5′-GTTAGAAGGCTGCTGGTTGTAGTTG-3′ |
Runx2-F | 5′-GAACCAAGAAGGCACAGACAGA-3′ |
Runx2-R | 5′-GGCGGGACACCTACTCTCATAC-3′ |
Col1a1-F | 5′-GGCGGGACACCTACTCTCATAC-3′ |
Col1a1-R | 5′-GGGACCCTTAGGCCATTGTGTA-3′ |
ALP-F | 5′-GCAGTATGAATTGAATCGGAACAAC-3′ |
ALP-R | 5′-ATGGCCTGGTCCATCTCCAC-3′ |
GAPDH-F | 5′-TGTGTCCGTCGTGGATCTGA-3′ |
GAPDH-R | 5′-TTGCTGTTGAAGTCGCAGGAG-3′ |
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Sun, Q.; Xu, L.; Hu, Z.; Liu, J.; Yu, T.; Li, M.; Zhang, S.; Shi, F. Melatonin Regulates Osteoblast Differentiation through the m6A Reader hnRNPA2B1 under Simulated Microgravity. Curr. Issues Mol. Biol. 2024, 46, 9624-9638. https://doi.org/10.3390/cimb46090572
Sun Q, Xu L, Hu Z, Liu J, Yu T, Li M, Zhang S, Shi F. Melatonin Regulates Osteoblast Differentiation through the m6A Reader hnRNPA2B1 under Simulated Microgravity. Current Issues in Molecular Biology. 2024; 46(9):9624-9638. https://doi.org/10.3390/cimb46090572
Chicago/Turabian StyleSun, Quan, Liqun Xu, Zebing Hu, Jingchun Liu, Tingfei Yu, Meng Li, Shu Zhang, and Fei Shi. 2024. "Melatonin Regulates Osteoblast Differentiation through the m6A Reader hnRNPA2B1 under Simulated Microgravity" Current Issues in Molecular Biology 46, no. 9: 9624-9638. https://doi.org/10.3390/cimb46090572