0222). The p values

were calculated with Mann–Whitney T-test. Upstream region of SOX7 gene in lung cancer cell lines was highly methylated The mechanism underlying the down-regulation of SOX7 expression in lung cancer was explored. The upstream region of SOX7 gene has several dense CpG islands (Figure 4A). Primers for Dasatinib Bisulfite Sequencing and Methylation Specific PCR (MSP) assays were designed (Figure 4A). Bisulfite Sequencing analysis showed that the upstream CpG rich region (-687 to -440) was hypermethylated in all 7 of the examined NSCLC cell lines. The downstream region (-71 to +251) was VX-809 in vitro hypermethylated in two (H1975 and HCC2279) of 9 NSCLC cell lines (Figure 4B). MSP analysis confirmed the Bisulfite Sequencing technique, showing that the upstream region (-683 to -493) was highly methylated in eight (H23, H460, H820, H1299, H1975, HCC827, HCC2279, selleck products PC14) of the 9 NSCLC cell lines (Figure 4C and Table 3). As expected, we could not amplify either the upstream or downstream regions of the SOX7 gene in the HCC2935 cells consistent with

a homozygous deletion of the gene in these cells (data not shown). A perfect correlation between upstream methylation and SOX7 expression did not occur. HCC4006 had only modest positivity by MSP but did not express SOX7; and PC14 was methylated by MSP examination, but expressed SOX7. Also in contrast to the cell line data, the Bisulfite Sequencing analysis showed that the upstream region (-687 to -440) was hypermethylated in one of 5 lung tumor samples. We did not have RNA or protein available for these samples to examine SOX7 expression. The downstream region (-71 to +251) was neither methylated in NSCLC

nor matched normal samples (Figure 4D), which was consistent with the methylation pattern noted in the NSCLC cell lines. Figure 4 Methylation analysis of upstream regions of SOX7 gene . (A) Schematic illustration of CpG sites spanning 1,500 bp upstream and 350 bp downstream of the transcription start site of SOX7 transcription. Black arrow Fossariinae denotes the transcription start site (+1). Vertical pink bars denotes the CpG sites. Arrowed bars define the regions subjected to bisulfite sequencing (BS). Bars with circles at their end define the regions subjected to methylation specific PCR (MSP). (B) Bisulfite sequencing of SOX7 gene in NSCLC cell lines. Each circle in each horizontal row represent the analysis of a single clone of bisulfite-treated DNA encompassing either 20 or 35 CpG sites (-678 to -440, left panels; -71 to +251, right panels, respectively). Open and solid circles represent unmethylated and methylated CpG sites, respectively. (C) MPS analysis of upstream region of SOX7 gene in NSCLC cell lines. PCR products in lanes marked “U” and “M” are obtained with unmethylated-specific and methylated-specific primers, respectively.