miR-182 is a strong negative regulator of osteoblast proliferation, differentiation and skeletogenesis through targeting FoxO1
- miR-182 is a strong negative regulator of osteoblast proliferation, differentiation and skeletogenesis through targeting FoxO1=FoxO1을 억제함으로써 조골세포의 증식, 분화 및 골형성의 부정적 조절인자로 작용하는 miR-182/Kyoung Min Kim.
- Seoul : Graduate School, Yonsei University, 2012.
- v, 36 p. : ill ; 26 cm.
- 학위논문(박사) --Graduate School, Yonsei University :Dept. of Medicine,2012.2
Uncontrolled oxidative stress impairs bone formation and induces age-related bone loss in humans. The FoxO family is widely accepted to play an important role in protecting diverse cells from reactive oxygen species (ROS). Activation of FoxO1, the main FoxO in bone, stimulates proliferation and differentiation and inhibits apoptosis of osteoblast lineage cells. Despite the important role of FoxO1, little is known about how FoxO1 expression in bone is regulated. Meanwhile, several recent studies reported that microRNAs (miRNAs) could play a role in osteoblast differentiation and bone formation by targeting various transcriptional factors. Here, one additional crucial miRNA was identified, miR-182, which regulates osteoblastogenesis by repressing FoxO1 and thereby negatively affecting osteogenesis. Overexpression of miR-182 in osteoblast lineage cells increased cell apoptosis and inhibited osteogenic differentiation, while in vivo overexpression of miR-182 in zebrafish impaired bone formation. From in silico analysis and validation experiments, FoxO1 was identified as the target of miR-182, and restoration of FoxO1 expression in miR-182 overexpressing osteoblasts rescued them from the inhibitory effects of miR-182. These results indicate that miR-182 functions as FoxO1 inhibitor to antagonize osteoblast proliferation and differentiation, with a subsequent negative effect on osteogenesis. To treat bone aging, an antisense approach targeting miR-182 could be of therapeutic value.
II. MATERIALS AND METHODS
1. Cell cultures and alkaline phosphatase staining
2. Transfection of miRNAs
3. Cell proliferation and viability assays
4. Cell death assay and flow cytometry
5. Real time quantitative PCR for miRNA
7. Western blot analysis
8. Plasmid constructs and transfection of plasmid
9. Luciferase constructs and reporter assay
10. Zebrafish experiments
11. Statistical analysis
1. miR-182 is expressed in bone and MC3T3E1 preosteoblasts and its expression increases with osteoblast differentiation
2. Effect of miR-182 on the proliferation of C3H10T1/2 mesenchymal stem cells, MC3T3E1 preosteoblasts and primary calvaria cells
3. Effect of miR-182 on the death of C3H10T1/2 mesenchymal stem cells, MC3T3E1 preosteoblasts and primary calvaria cells
4. Effect of miR-182 on the differentiation of C3H10T1/2 mesenchymal stem cells, MC3T3E1 preosteoblasts
5. FoxO1 is a target of miR-182 in C3H10T1/2 mesenchymal stem cells and MC3T3E1 preosteoblasts
6. In vivo effect of miR-182 in bone and zebrafish model