Mechanism of miR-26a-5p/cAMP response element binding protein 1 molecular axis regulating osteogenic differentiation of adipose-derived mesenchymal stem cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIN Sanfu , CHEN Shoubo , FANG Kaibin , SHI Jinnan , WU Wenhua ,  WANG Wenhuai

Department of Orthopaedics, the Second Affiliated Hospital of Fujian Medical University, Quanzhou Fujian, 362000, P. R. China;

Corresponding?author：

WANG Wenhuai, Email: xiaodi50a@163.com

Keywords：

miR-26a-5p; adipose-derived mesechymal stem cells; osteogenic differentiation; cAMP response element binding protein 1; mouse

DOI：

10.7507/1002-1892.202211037

Video：

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Objective To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1). Methods The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture. Results The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased (P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day (P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points (P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity (P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased (P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups (P>0.05). Conclusion Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.

Citation： LIN Sanfu, CHEN Shoubo, FANG Kaibin, SHI Jinnan, WU Wenhua, WANG Wenhuai. Mechanism of miR-26a-5p/cAMP response element binding protein 1 molecular axis regulating osteogenic differentiation of adipose-derived mesenchymal stem cells. Chinese Journal of Reparative and Reconstructive Surgery, 2023, 37(5): 615-621. doi: 10.7507/1002-1892.202211037 Copy

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2.	Widera D. Recent advances in translational adipose-derived stem cell biology. Biomolecules, 2021, 11(11): 1660. doi: 10.3390/biom11111660.
3.	Correia de Sousa M, Gjorgjieva M, Dolicka D, et al. Deciphering miRNAs’ action through miRNA editing. Int J Mol Sci, 2019, 20(24): 6249. doi: 10.3390/ijms20246249.
4.	Zhou Y, Qiao H, Liu L, et al. miR-21 regulates osteogenic and adipogenic differentiation of BMSCs by targeting PTEN. J Musculoskelet Neuronal Interact, 2021, 21(4): 568-576.
5.	Ren LR, Yao RB, Wang SY, et al. MiR-27a-3p promotes the osteogenic differentiation by activating CRY2/ERK1/2 axis. Mol Med, 2021, 27(1): 43. doi: 10.1186/s10020-021-00303-5.
6.	Xing X, Guo S, Zhang G, et al. miR-26a-5p protects against myocardial ischemia/reperfusion injury by regulating the PTEN/PI3K/AKT signaling pathway. Braz J Med Biol Res, 2020, 53(2): e9106. doi: 10.1590/1414-431x20199106.
7.	Li Y, Wang P, Wu LL, et al. miR-26a-5p inhibit gastric cancer cell proliferation and invasion through mediated Wnt5a. Onco Targets Ther, 2020, 13: 2537-2550.
8.	Li H, Yang T, Fei Z. miR-26a-5p alleviates lipopolysaccharide-induced acute lung injury by targeting the connective tissue growth factor. Mol Med Rep, 2021, 23(1): 5. doi: 10.3892/mmr.2020.11643.
9.	Luzi E, Marini F, Tognarini I, et al. The regulatory network menin-microRNA 26a as a possible target for RNA-based therapy of bone diseases. Nucleic Acid Ther, 2012, 22(2): 103-108.
10.	Yao B, Zhang Q, Yang Z, et al. CircEZH2/miR-133b/IGF2BP2 aggravates colorectal cancer progression via enhancing the stability of m6A-modified CREB1 mRNA. Mol Cancer, 2022, 21(1): 140. doi: 10.1186/s12943-022-01608-7.
11.	Zhang H, Li L, Dong Q, et al. Activation of PKA/CREB signaling is involved in bmp9-induced osteogenic differentiation of mesenchymal stem cells. Cell Physiol Biochem, 2015, 37(2): 548-562.
12.	Wei S, Xie S, Yang Z, et al. Allogeneic adipose-derived stem cells suppress mTORC1 pathway in a murine model of systemic lupus erythematosus. Lupus, 2019, 28(2): 199-209.
13.	Iaquinta MR, Lanzillotti C, Mazziotta C, et al. The role of microRNAs in the osteogenic and chondrogenic differentiation of mesenchymal stem cells and bone pathologies. Theranostics, 2021, 11(13): 6573-6591.
14.	Ye Y, Liu Q, Li C, et al. miR-125a-5p regulates osteogenic differentiation of human adipose-derived mesenchymal stem cells under oxidative stress. Biomed Res Int, 2021, 2021: 6684709. doi: 10.1155/2021/6684709.
15.	Yang S, Guo S, Tong S, et al. Exosomal miR-130a-3p regulates osteogenic differentiation of human adipose-derived stem cells through mediating SIRT7/Wnt/β-catenin axis. Cell Prolif, 2020, 53(10): e12890. doi: 10.1111/cpr.12890.
16.	Shi H, Li H, Zhang F, et al. MiR-26a-5p alleviates cardiac hypertrophy and dysfunction via targeting ADAM17. Cell Biol Int, 2021, 45(11): 2357-2367.
17.	Yuan YL, Yu H, Mu SM, et al. MiR-26a-5p inhibits cell proliferation and enhances doxorubicin sensitivity in HCC cells via targeting AURKA. Technol Cancer Res Treat, 2019, 18: 1533033819851833. doi: 10.1177/1533033819851833.
18.	Liu Y, Wang L, Xie F, et al. Overexpression of miR-26a-5p suppresses Tau phosphorylation and Aβ accumulation in the Alzheimer's disease mice by targeting DYRK1A. Curr Neurovasc Res, 2020, 17(3): 241-248.
19.	Zhang Z, Guan B, Li Y, et al. Increased phosphorylated CREB1 protein correlates with poor prognosis in clear cell renal cell carcinoma. Transl Androl Urol, 2021, 10(8): 3348-3357.
20.	Chen S, Tao J, Zhong F, et al. Polydatin down-regulates the phosphorylation level of Creb and induces apoptosis in human breast cancer cell. PLoS One, 2017, 12(5): e0176501. doi: 10.1371/journal.pone.0176501.
21.	Zhang Y, Yuan Q, Wei Q, et al. Long noncoding RNA XIST modulates microRNA-135/CREB1 axis to influence osteogenic differentiation of osteoblast-like cells in mice with tibial fracture healing. Hum Cell, 2022, 35(1): 133-149.
22.	Huang X, Liu F, Jiang Z, et al. CREB1 suppresses transcription of microRNA-186 to promote growth, invasion and epithelial-mesenchymal transition of gastric cancer cells through the KRT8/HIF-1α axis. Cancer Manag Res, 2020, 12: 9097-9111.

1. Bunnell BA. Adipose tissue-derived mesenchymal stem cells. Cells, 2021, 10(12): 3433. doi: 10.3390/cells10123433.
2. Widera D. Recent advances in translational adipose-derived stem cell biology. Biomolecules, 2021, 11(11): 1660. doi: 10.3390/biom11111660.
3. Correia de Sousa M, Gjorgjieva M, Dolicka D, et al. Deciphering miRNAs’ action through miRNA editing. Int J Mol Sci, 2019, 20(24): 6249. doi: 10.3390/ijms20246249.
4. Zhou Y, Qiao H, Liu L, et al. miR-21 regulates osteogenic and adipogenic differentiation of BMSCs by targeting PTEN. J Musculoskelet Neuronal Interact, 2021, 21(4): 568-576.
5. Ren LR, Yao RB, Wang SY, et al. MiR-27a-3p promotes the osteogenic differentiation by activating CRY2/ERK1/2 axis. Mol Med, 2021, 27(1): 43. doi: 10.1186/s10020-021-00303-5.
6. Xing X, Guo S, Zhang G, et al. miR-26a-5p protects against myocardial ischemia/reperfusion injury by regulating the PTEN/PI3K/AKT signaling pathway. Braz J Med Biol Res, 2020, 53(2): e9106. doi: 10.1590/1414-431x20199106.
7. Li Y, Wang P, Wu LL, et al. miR-26a-5p inhibit gastric cancer cell proliferation and invasion through mediated Wnt5a. Onco Targets Ther, 2020, 13: 2537-2550.
8. Li H, Yang T, Fei Z. miR-26a-5p alleviates lipopolysaccharide-induced acute lung injury by targeting the connective tissue growth factor. Mol Med Rep, 2021, 23(1): 5. doi: 10.3892/mmr.2020.11643.
9. Luzi E, Marini F, Tognarini I, et al. The regulatory network menin-microRNA 26a as a possible target for RNA-based therapy of bone diseases. Nucleic Acid Ther, 2012, 22(2): 103-108.
10. Yao B, Zhang Q, Yang Z, et al. CircEZH2/miR-133b/IGF2BP2 aggravates colorectal cancer progression via enhancing the stability of m6A-modified CREB1 mRNA. Mol Cancer, 2022, 21(1): 140. doi: 10.1186/s12943-022-01608-7.
11. Zhang H, Li L, Dong Q, et al. Activation of PKA/CREB signaling is involved in bmp9-induced osteogenic differentiation of mesenchymal stem cells. Cell Physiol Biochem, 2015, 37(2): 548-562.
12. Wei S, Xie S, Yang Z, et al. Allogeneic adipose-derived stem cells suppress mTORC1 pathway in a murine model of systemic lupus erythematosus. Lupus, 2019, 28(2): 199-209.
13. Iaquinta MR, Lanzillotti C, Mazziotta C, et al. The role of microRNAs in the osteogenic and chondrogenic differentiation of mesenchymal stem cells and bone pathologies. Theranostics, 2021, 11(13): 6573-6591.
14. Ye Y, Liu Q, Li C, et al. miR-125a-5p regulates osteogenic differentiation of human adipose-derived mesenchymal stem cells under oxidative stress. Biomed Res Int, 2021, 2021: 6684709. doi: 10.1155/2021/6684709.
15. Yang S, Guo S, Tong S, et al. Exosomal miR-130a-3p regulates osteogenic differentiation of human adipose-derived stem cells through mediating SIRT7/Wnt/β-catenin axis. Cell Prolif, 2020, 53(10): e12890. doi: 10.1111/cpr.12890.
16. Shi H, Li H, Zhang F, et al. MiR-26a-5p alleviates cardiac hypertrophy and dysfunction via targeting ADAM17. Cell Biol Int, 2021, 45(11): 2357-2367.
17. Yuan YL, Yu H, Mu SM, et al. MiR-26a-5p inhibits cell proliferation and enhances doxorubicin sensitivity in HCC cells via targeting AURKA. Technol Cancer Res Treat, 2019, 18: 1533033819851833. doi: 10.1177/1533033819851833.
18. Liu Y, Wang L, Xie F, et al. Overexpression of miR-26a-5p suppresses Tau phosphorylation and Aβ accumulation in the Alzheimer's disease mice by targeting DYRK1A. Curr Neurovasc Res, 2020, 17(3): 241-248.
19. Zhang Z, Guan B, Li Y, et al. Increased phosphorylated CREB1 protein correlates with poor prognosis in clear cell renal cell carcinoma. Transl Androl Urol, 2021, 10(8): 3348-3357.
20. Chen S, Tao J, Zhong F, et al. Polydatin down-regulates the phosphorylation level of Creb and induces apoptosis in human breast cancer cell. PLoS One, 2017, 12(5): e0176501. doi: 10.1371/journal.pone.0176501.
21. Zhang Y, Yuan Q, Wei Q, et al. Long noncoding RNA XIST modulates microRNA-135/CREB1 axis to influence osteogenic differentiation of osteoblast-like cells in mice with tibial fracture healing. Hum Cell, 2022, 35(1): 133-149.
22. Huang X, Liu F, Jiang Z, et al. CREB1 suppresses transcription of microRNA-186 to promote growth, invasion and epithelial-mesenchymal transition of gastric cancer cells through the KRT8/HIF-1α axis. Cancer Manag Res, 2020, 12: 9097-9111.

Chinese Journal of Reparative and Reconstructive Surgery

Mechanism of miR-26a-5p/cAMP response element binding protein 1 molecular axis regulating osteogenic differentiation of adipose-derived mesenchymal stem cells

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