RNA interference-mediated knockdown of DNMT1 and DNMT3B induces CXCL12 expression in MCF-7 breast cancer and AsPC1 pancreatic carcinoma cell lines.
It has been recently demonstrated that in colonic carcinoma, CXCL12 expression undergoes epigenetic regulation by methylation of cytosine in cytosine-guanosine (CpG) dinucleotides of the promoter sequence. Using lentiviral vectors, we generated stable RNA interference-mediated knockdown of DNMT1 and DNMT3B in MCF-7 breast cancer and AsPC1 pancreatic carcinoma cell lines. Employing reverse transcription real-time quantitative PCR and immunofluorescence analysis, we determined re-expression levels of CXCL12 transcript and protein in these cells. Bisulfite sequencing revealed that the level of promoter demethylation appeared more effective in cells expressing DNMT1 siRNA than in those expressing DNMT3B siRNA, and this correlated with higher expression of CXCL12. Moreover, the combined expression of DNMT1 and DNMT3B siRNAs enhanced promoter demethylation that was associated only with a slight increase of CXCL12 expression. However, the demethylating agent 5-Aza-2'-deoxycytidine exhibited the strongest effect on promoter demethylation, which correlated with the highest expression level of CXCL12 transcript and protein in MCF-7 and AsPC1 cells. Our findings suggest that DNMT1 plays a key role in maintenance of methylation, and DNMT3B may act as an accessory DNA methyltransferase to epigenetically silence CXCL12 expression in MCF-7 and AsPC1 cells.
Sowińska
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Department of Biochemistry and Molecular Biology, Poznań University of Medical Sciences, 6 Swiecickiego St, 60-781, Poznań, Poland.
Jagodzinski
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D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000096:biosynthesis*; D018899:CpG Islands; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000096:biosynthesis*; D019175:DNA Methylation; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008954:Models, Biological; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000097584:DNA Methyltransferase 3B
D054377:Chemokine CXCL12; D019743:Chemokines, CXC; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D012333:RNA, Messenger; D000097584:DNA Methyltransferase 3B; C515255:CXCL12 protein, human; C000619380:DNMT1 protein, human