It should be noted that the level of SOX9 protein in the NSCLC cell lines and clinical lung cancer tissues was paralleled with the mRNA expression level, indicating the possibility that the increase of SOX9 in NSCLC might be largely attributable to regulation at the mRNA level. Figure 2 Differential expression of SOX9 in human NSCLC tissues (T) and their paired normal lung tissue (N), each pair obtained from the same patient. A, Western Gefitinib order blotting analysis of SOX9 protein expression in eight paired tumor and normal tissue samples, average tumor/normal ratios of SOX9 protein expression quantified using the LabWorks software. Expression levels were normalized with β-actin. B,
average tumor/normal ratios of SOX9 expression were quantified by real-time RT-PCR. Expression levels were normalized for GAPDH. Bars, SD from three independent experiments Correlation between increased expression of SOX9 and malignancy of NSCLC To determine whether the expression level of SOX9 protein
is associated with the histological characteristics of NSCLC, 142 paraffin-embedded, archived NSCLC clinical specimens, which selleck chemicals llc included 32 cases of stage I, 25 cases of stage II, 58 cases of stage III, and 27 cases of stage IV lung cancers, were examined by immunohistochemical staining with an antibody against human SOX9. As shown in Figure 3A, SOX9 was found to be upregulated in NSCLC (c and d, stage I NSCLC; e and f, stage II NSCLC; g and h, stage III NSCLC; and i and j, stage IV NSCLC) compared with that in the normal lung tissue (Figure 3). As summarized in Table 1, SOX9 protein was detected in 135 of 142 (95.1%) cases. High levels of SOX9 were present in areas containing tumor cells next in primary NSCLC tissues (Figure 3A, c-j). In contrast, SOX9 was barely detectable in normal lung tissue (Figure 3A and 3B). Quantitative analysis indicated that the average mean absorbance of SOX9 staining in stage I-IV tumors was statistically significantly higher than in normal lung tissue (P < 0.01; Figure 3B). Figure 3 Immunohistochemical
analysis of SOX9 expression in normal lung tissue and primary NSCLC. A, thin sections of paraffin-embedded specimens of a total of 2 normal lung tissues and 142 primary NSCLC specimens including AJCC grade I to IV NSCLC samples were stained by immunohistochemistry using an anti-SOX9 antibody. a and b, normal lung tissue; c and d, AJCC grade I NSCLC; e and f, AJCC grade II NSCLC; g and h, AJCC grade III NSCLC; i and j, AJCC grade IV. Amplification, 200 (a, c, e, g, and i) and 400 (b, d, f, h and j). Immunohistochemical analyses were done two independent times on each of the samples with similar results. B, statistical quantification of the average mean absorbance of SOX9 staining between normal lung tissues (4 cases) and NSCLC specimens of different AJCC grades (30 random cases per grade).