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A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro
Published on: May 17, 2017
DNA Methylation Profiling in Chondrocyte Dedifferentiation In Vitro
Li Duan1,2, Yujie Liang3,4, Bin Ma5
1Shenzhen Key Laboratory of Tissue Engineering, Department of Sports Medicine, Shenzhen Second People's Hospital (The First Hospital Affiliated to Shenzhen University), Shenzhen, Guangdong Province, China.
Abstract:
DNA methylation has emerged as a crucial regulator of chondrocyte dedifferentiation, which severely compromises the outcome of autologous chondrocyte implantation (ACI) treatment for cartilage defects. However, the full-scale DNA methylation profiling in chondrocyte dedifferentiation remains to be determined. Here, we performed a genome-wide DNA methylation profiling of dedifferentiated chondrocytes in monolayer culture and chondrocytes treated with DNA methylation inhibitor 5-azacytidine (5-AzaC). This research revealed that the general methylation level of CpG was increased while the COL-1A1 promoter methylation level was decreased during the chondrocyte dedifferentiation. 5-AzaC could reduce general methylation levels and reverse the chondrocyte dedifferentiation. Surprisingly, the DNA methylation level of COL-1A1 promoter was increased after 5-AzaC treatment. The COL-1A1 expression level was increased while that of SOX-9 was decreased during the chondrocyte dedifferentiation. 5-AzaC treatment up-regulated the SOX-9 expression while down-regulated the COL-1A1 promoter activity and gene expression. Taken together, these results suggested that differential regulation of the DNA methylation level of cartilage-specific genes might contribute to the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation. J. Cell. Physiol. 232: 1708-1716, 2017. © 2016 Wiley Periodicals, Inc.
Insights
DNA methylation influences chondrocyte dedifferentiation, impacting cartilage repair. Inhibiting DNA methylation reversed dedifferentiation and altered specific gene expression, offering potential epigenetic strategies for cartilage defects.
Area of Science:
- Epigenetics
- Cell Biology
- Biochemistry
Background:
- Chondrocyte dedifferentiation is a key challenge in autologous chondrocyte implantation (ACI) for cartilage defects.
- DNA methylation plays a critical role in regulating chondrocyte dedifferentiation, but comprehensive profiling is lacking.
Purpose of the Study:
- To perform genome-wide DNA methylation profiling in dedifferentiated chondrocytes.
- To investigate the effect of DNA methylation inhibition on chondrocyte dedifferentiation and gene expression.
Main Methods:
- Genome-wide DNA methylation profiling of chondrocytes in monolayer culture.
- Treatment of chondrocytes with the DNA methylation inhibitor 5-azacytidine (5-AzaC).
- Analysis of gene expression and promoter activity for COL1A1 and SOX9.
Main Results:
- Chondrocyte dedifferentiation showed increased general CpG methylation and decreased COL1A1 promoter methylation.
- 5-AzaC treatment reduced general methylation, reversed dedifferentiation, and increased COL1A1 promoter methylation.
- SOX9 expression decreased during dedifferentiation and was upregulated by 5-AzaC, while COL1A1 expression and promoter activity were downregulated.
Conclusions:
- Differential DNA methylation of cartilage-specific genes contributes to chondrocyte dedifferentiation.
- Epigenetic manipulation targeting these genes presents a potential strategy to counteract dedifferentiation during in vitro propagation for cartilage repair.
