Increased CXCR4 expression in AsPC1 pancreatic carcinoma cells with RNA interference-mediated knockdown of DNMT1 and DNMT3B.
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.
Przybylski
M
M
Department of Biochemistry and Molecular Biology, Poznań University of Medical Sciences, 6, Swiecickiego Street, 60-781 Poznań, Poland.
Kozłowska
A
A
Pietkiewicz
P P
PP
Lutkowska
A
A
Lianeri
M
M
Jagodzinski
P P
PP
D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics; D000077209:Decitabine; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D010190:Pancreatic Neoplasms / Q000235:genetics*; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D019718:Receptors, CXCR4 / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D000097584:DNA Methyltransferase 3B
D001374:Azacitidine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D004273:DNA, Neoplasm; D000077209:Decitabine; D019718:Receptors, CXCR4; D000097584:DNA Methyltransferase 3B; C527276:CXCR4 protein, human; C000619380:DNMT1 protein, human