Figure 1.

Downregulation of CHL1 in NPC. (A) Quantification and characterization of mRNA levels in CHL1 of 95 primary NPC cases was obtained using qRT-PCR these results were contrasted with those obtained for normal tissue (N);. GAPDH was used as an internal control. (B) CHL1 Fold changes detected using qRT-PCR in 15 primary NPC tissues and their corresponding non-tumor tissues (N). CHL1 was normalized by internal control GAPDH (*P<0.01, independent Student t test). (C) qRT-PCR analysis of CHL1 expression in three NPC cell lines (C666, CNE2 and SUNE1) and NP69. The fold change expressions of CHLI detected were compared against those of the immortalized NP cell line, NP460. (D) The expression of CHL1 in the three NPC cell lines, NP 460 and NP69 were characterized by Western blot assay. (E) Mapping of the methylation status of CpG dinucleotides within the CHL1 promoter region was done with BGS in NPC cell lines (CNE2 and SUNE1) and normal cell line (NP460). A 400-bp region spanning through the CpG island with 18 CpG sites was analyzed. The methylation status at each CpG dinucleotide was represented in the pie charts. The chart shows the methylation activities at three levels indicated with these colours, black, white and grey circle representing completely methylated, completely unmethylated and partially methylated CpGs respectively. (F) Promoter methylation analysis of CHL1 in NPC cell lines was carried out by MSP, with Immortalized nasopharyngeal epithelial cell line NP460 as normal control. Expressed as M & U; M, methylated allele and U, unmethylated allele respectively. (G) Expression of CHLI by qPCR analysis after treatment with 5-aza-dC a demethylation agent in NPC cell lines (C666, CNE2 and SUNE1 cells). Again, GAPDH served as the internal control. Three representing NPC cell lines (C666, CNE2 and SUNE1) without treatment; Aza 10, cell lines treated with 10 µM 5-aza-2'-deoxycytidine; Aza 20, cell lines treated with 20 µM 5-aza-2'-deoxycytidine.