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Effect of CpG island methylation on microRNA expression in the k-562 cell line
Yang Yang1, Li-Li Wang, Yong-Hui Li
1Department of Hematology and BMT Center, Chinese PLA General Hospital, 28 Fuxing Road, Beijing, China.
Abstract:
To test the hypothesis that methylation of a CpG island is associated with regulation of microRNA expression, we investigated CpG islands in the upstream sequences of microRNA precursors (pre-miRNAs) through bioinformatic analysis and determined whether the CpG islands were methylated by methylation-specific PCR in the k-562 cell line. We used 5-azacytidine for DNA demethylation, and changes in microRNA expression were detected by microarray assay, RT-PCR, and real-time PCR after 5-azacytidine induction. We showed that the CpG islands in the upstream regions of 18 pre-miRNAs were methylated, including miR-663, miR-369, miR-615, and miR-410, and promoter activity was detected in the upstream region of pre-miR-663. We found that a decrease in methylation of a CpG island could up-regulate the expression of miR-663, suggesting that miR-663 could be regulated by DNA methylation. Expression levels of miR-369, miR-615, and miR-410 were not regulated by DNA methylation in this cell line.
Insights
DNA methylation regulates microRNA expression. CpG island methylation in pre-miRNA upstream regions was investigated, revealing miR-663 expression is controlled by DNA methylation.
Area of Science:
- Epigenetics
- Molecular Biology
- Genomics
Background:
- MicroRNA (miRNA) dysregulation is implicated in various diseases.
- CpG islands in gene promoter regions are often targets of DNA methylation, influencing gene expression.
- The role of CpG island methylation in regulating miRNA expression remains incompletely understood.
Purpose of the Study:
- To investigate the association between CpG island methylation and microRNA expression.
- To determine if specific CpG islands in microRNA precursor (pre-miRNA) upstream sequences are methylated.
- To assess the impact of DNA demethylation on miRNA expression.
Main Methods:
- Bioinformatic analysis to identify CpG islands in pre-miRNA upstream sequences.
- Methylation-specific PCR to detect CpG island methylation in K-562 cells.
- DNA demethylation using 5-azacytidine.
- Microarray assay, RT-PCR, and real-time PCR to quantify miRNA expression changes.
Main Results:
- CpG islands in the upstream regions of 18 pre-miRNAs, including miR-663, were found to be methylated.
- Promoter activity was detected in the upstream region of pre-miR-663.
- Demethylation led to up-regulation of miR-663 expression, indicating DNA methylation-mediated regulation.
- Expression of miR-369, miR-615, and miR-410 was not significantly affected by DNA methylation in this cell line.
Conclusions:
- CpG island methylation in pre-miRNA upstream regions can regulate miRNA expression.
- miR-663 expression is subject to epigenetic control via DNA methylation.
- The regulatory role of DNA methylation varies among different miRNAs.
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