Methylation Regulation of NANOGP8 Expression in Lung Cancer Cell Line

The nearly identified retrogene NANOGP8 is from a stem cell transcription factor NANOG gene, which expressed in multiple cancers,but generally not in normal tissues and its function is related to cancer development and progression. To investigate the relationship between NANOGP8 expression level and methylation sites at the gene promoters. Bisulphate Sequencing in lung cancer cell line A549 as well as the normal fibroblast cells were performed. Genomic DNA was extracted from these cells and treated with bisulphate,then PCR was performed in the NANOG promoter,encompassing a total of 10 CpGs within nucleotides-1449 to-1295 and-1168 to-952 relative to the transcription start site( TSS) reported by Park. Two regions including 9 CpGs in the potential NANOGP8 promoter,which located within nucleotides-1529 to-1401 and-690 to-587 relative to the TSS of NANOGP8 predicted by Methprimer program were also examined. PCR products were then ligated to pGEM-T vector. The recombinant plasmid was transformed into E. coli DH5α and eight clones for each region were sequenced and blasted. There are no significant changes of the percentage of clones with methylation in NANOG promoter in A549 cell line compared to in the fibroblast cells( 65% vs. 67. 5%,P = 0. 62). However, the percentage at the two regions of NANOGP8 shows a significant increase from A549 cell line to the fibroblast cells( 12. 5% vs. 59. 7%,P 0. 001). NANOGP8 transcriptional level was detected by RT-PCR in both cell lines and found NANOGP8 is highly expressed in A549 cell line but not in the fibroblast cells. The methylation status of 5'-flanking regions of human NANOGP8 is associated with the NANOGP8 expression in lung cell line and may relate to lung cancer development and progression.