DNA methylation profile and expression of surfactant protein A2 gene in lung cancer

Abstract

Knowledge of the methylation profile of genes allow for the identification of biomarkers that may guide diagnosis and effective treatment of disease. Human surfactant protein A (SP-A) plays an important role in lung homeostasis and immunity, and is encoded by two genes (SFTPA1 and SFTPA2). The goal of this study was to identify differentially methylated CpG sites in the promoter region of the SFTPA2 gene in lung cancer tissue, and to determine the correlation between the promoter's methylation profile and gene expression. For this, we collected 28 pairs of cancerous human lung tissue and adjacent noncancerous (NC) lung tissue: 17 adenocarcinoma (AC), 9 squamous cell carcinoma (SCC), and 2 AC with SCC features, and we evaluated DNA methylation of the SFTPA2 promoter region by bisulfite conversion. Our results identified a higher methylation ratio in one CpG site of the SFTPA2 gene in cancerous tissue versus NC tissue (0.36 versus 0.11, p = 0.001). When assessing AC samples, we also found cancerous tissues associated with a higher methylation ratio (0.43 versus 0.10, p = 0.02). In the SCC group, although cancerous tissue showed a higher methylation ratio (0.22 versus 0.11), this difference was not statistically significant (p = 0.35). Expression of SFTPA2 mRNA and total SP-A protein was significantly lower in cancer tissue when compared to adjacent NC tissue (p < 0.001), and correlated with the hypermethylated status of an SFTPA2 CpG site in AC samples. The findings of this pilot study may hold promise for future use of SFTPA2 as a biomarker for the diagnosis of lung cancer.

Keywords: SP-A; adenocarcinoma; methylation; squamous cell carcinoma.

Figure 1. SFTPA2 promoter CpG sites analyzed in this study

A schematic diagram of the region studied and the 14 CpG sites is shown (top panel). The sequence shown on the bottom panel (664 bp) corresponds to the region between nucleotides −1628 and −2292 upstream of the SFTPA2 transcription start site (NCBI reference sequence: NG_013046.1).

Figure 2. DNA Methylation ratios of CpG Site 2 in cancer and adjacent NC lung samples

Statistical differences were observed when all lung cancer tissues (AC+SCC) were compared to adjacent NC lung tissue (p=0.001, n=28), and the AC group to adjacent NC lung tissue (p=0.02, n=17). No statistically significant differences (p=0.35, n=9) were observed when the SCC and NC were compared. Results are expressed as mean ± SEM.

Figure 3. SFTPA2 Gene Expression in NC vs. Lung Cancer Samples

Expression of SFTPA2 mRNA in NC and lung cancer tissue was measured by Real Time PCR, and expressed as mean ± SEM. Both AC (n=12) and SCC (n=12) cancer samples expressed significantly lower levels of SFTPA2 (p<0.001) compared to NC adjacent tissues.

Figure 4. Total SP-A protein expression in NC vs. Lung Cancer Samples

Western Blot analysis of total SP-A and cyclophilin B (loading control) in NC and lung cancer tissue. A representative gel is shown for 4 NC/tumor pairs (2 AC and 2 SCC) of the total 8 pairs (4 AC and 4 SCC) analyzed.

Figure 5. DNMT gene expression in lung cancer and NC tissue

Expression of DNMT1 and DNMT3 mRNA in NC and lung cancer tissue was measured by Real Time PCR, and expressed as mean + SEM. Both AC (n=12) and SCC (n=12) tumor samples had significantly higher levels of DNMT1 and DNMT3 (p<0.05).

Figure 6. Predicted binding of transcription factors to CpG site 2

Binding of transcription factors was predicted in the SFTPA2 promoter sequence by the online software Patch (www.gene-regulation.com/cgi-bin/pub/programs/patch/bin/patch.cgi). The figure shows predictions for the region surrounding CpG site 2 (positions −2200/−2230 upstream of the SFTPA2 transcription start site). The box indicates the position of CpG site 2 (−2215).