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Published on: November 23, 2017
Increased CXCR4 expression in AsPC1 pancreatic carcinoma cells with RNA interference-mediated knockdown of DNMT1 and
M Przybylski1, A Kozłowska, P P Pietkiewicz
1Department of Biochemistry and Molecular Biology, Poznań University of Medical Sciences, 6, Swiecickiego Street, 60-781 Poznań, Poland.
Abstract:
The effect of DNA methylation on CXCR4 expression has been demonstrated in pancreatic cancer and melanoma cells, but little is known about the effect of DNA methyltransferases 1 and 3 (DNMT1 and DNMT3B) on CXCR4 expression. Employing lentiviral vectors, we created stable RNA interference-mediated knockdown of DNMT1 and DNMT3B in AsPC1 pancreatic cancer cells. Using reverse transcription real-time quantitative PCR and flow cytometric analysis, we evaluated the increase in the expression of CXCR4 transcript and protein levels in these cells. Bisulfite sequencing analysis showed that the level of promoter demethylation appeared more effective in cells with knockdown of DNMT1 than in those with DNMT3B knockdown. Furthermore, the combined RNA interference knockdown of both DNMT1 and DNMT3B increased promoter demethylation, leading to a slight increase in CXCR4 expression. However, the demethylating agent 5-Aza-2'-deoxycytidine exhibited the strongest effect on promoter demethylation, which correlated with the highest production of CXCR4 transcript and protein in AsPC1 cells. Our results indicate that DNMT1 plays the main role in maintenance of methylation of CXCR4 promoter, while DNMT3B may function as an accessory DNA methyltransferase to modulate CXCR4 expression in AsPC1 cells.
Insights
DNA methyltransferases 1 and 3B (DNMT1 and DNMT3B) impact CXCR4 expression in pancreatic cancer. DNMT1 is key in maintaining CXCR4 promoter methylation, influencing cancer cell behavior.
Area of Science:
- Molecular Biology
- Cancer Research
- Epigenetics
Background:
- DNA methylation regulates gene expression, impacting cancer development.
- CXCR4 (C-X-C motif chemokine receptor 4) is implicated in pancreatic cancer progression.
- The roles of DNMT1 and DNMT3B in modulating CXCR4 expression remain unclear.
Purpose of the Study:
- To investigate the influence of DNA methyltransferases 1 and 3B (DNMT1 and DNMT3B) on CXCR4 expression in pancreatic cancer cells.
- To elucidate the specific roles of DNMT1 and DNMT3B in regulating CXCR4 promoter methylation and expression.
Main Methods:
- Stable RNA interference-mediated knockdown of DNMT1 and DNMT3B in AsPC1 pancreatic cancer cells.
- Quantitative PCR and flow cytometry to assess CXCR4 transcript and protein levels.
- Bisulfite sequencing to analyze CXCR4 promoter methylation status.
Main Results:
- Knockdown of DNMT1 led to greater promoter demethylation and increased CXCR4 expression compared to DNMT3B knockdown.
- Combined knockdown of DNMT1 and DNMT3B enhanced promoter demethylation and CXCR4 expression.
- The demethylating agent 5-Aza-2'-deoxycytidine showed the most significant increase in CXCR4 expression.
Conclusions:
- DNMT1 plays a primary role in maintaining CXCR4 promoter methylation in AsPC1 cells.
- DNMT3B may act as an accessory enzyme modulating CXCR4 expression.
- Targeting DNMT1 could be a therapeutic strategy for pancreatic cancer by influencing CXCR4 signaling.
