Methylation of the SPARC gene promoter and its clinical implication in pancreatic cancer

Abstract

Background: The secreted protein acidic and rich in cysteine (SPARC) plays a pivotal role in regulating cell-matrix interactions and tumor angiogenesis, proliferation, and migration. Detection of SPARC gene methylation may be useful as a tumorigenesis marker for early detection of pancreatic cancer.

Methods: Methylation of the SPARC gene transcriptional regulation region (TRR) was detected using bisulfite-specific (BSP) PCR-based sequencing analysis in 40 cases of pancreatic cancer and the adjacent normal tissues, 6 chronic pancreatitis tissues, and 6 normal pancreatic tissues. BSP cloning-based sequencing analysis was also performed in selected cases. Clinicopathological data from the cancer patients were collected and analyzed.

Results: Analysis of SPARC gene TRR methylation showed two hypermethylation wave peak regions: CpG Region 1 (CpG site 1-7) and CpG Region 2 (CpG site 8-12). Pancreatic tissues have shown methylation in both regions with gradual increases from normal, chronic pancreatitis, and adjacent normal tissues to cancerous tissues. However, Methylation of CpG Region 2 was more sensitive than CpG Region 1 in pancreatic tumorigenesis. Furthermore, the methylation level of CpG Region 2 was associated with increased tumor size and exposure to the risk factors (tobacco smoke and alcohol consumption) for developing pancreatic cancer.

Conclusion: Methylation of the SPARC gene, specifically CpG Region 2, may be an early event during pancreatic tumorigenesis and should be further evaluated as a tumorigenesis marker for early detection of pancreatic cancer.

Figure 1

Detection of SPARC gene TRR methylation. (A) Illustration of the SPARC gene TRR and topology of the BSP primer. The black bar indicates the analyzed region. The bold "G" indicates the transcriptional start site. The bold italic "CG" indicates the location of 12 CpG island sites. The underlined sequence indicates the primers for BSP. Blue and red rectangles indicate the Sp1 and AP1 binding consensus sequences, respectively. The red triangles indicate the region whose representative sequence analyses were showed in Figure 1B. (B) Representative sequencing data of the SPARC gene TRR in four different groups of pancreatic tissues obtained using BSP PCR-based sequencing analysis. CpG dinucleotides "C" in the objective sequence are shown in red. The red, yellow, green, light blue, and deep blue dots under the analyzed sequence represent different methylation ratios, respectively.

Figure 2

Methylation pattern of the SPARC gene TRR in pancreatic tissue samples. The pattern consists of two hypermethylation wave peak regions including CpG region 1 (CpG site 1--7) and CpG region 2 (CpG site 8--12). The curve was fitted to the mean methylation ratios of pancreatic cancer tissues using the MACD (moving average convergence/divergence) method.

Figure 3

Methylation level of CpG region 1 (A) and CpG region 2 (B) in the SPARC gene TRR in pancreatic tissues. All data are reported as means ± 95% CI. #, the pancreatic cancer tissues are compared to the corresponding tumor adjacent normal tissues, chronic pancreatitis tissues, or normal pancreatic tissues, p < 0.05. &, the corresponding tumor adjacent normal tissues are compared to the real normal pancreatic tissues, p < 0.05.

Figure 4

Methylation status of 12 CpG island sites and the methylation level of CpG Region 1 and CpG Region 2 in the samples determined using BSP cloning-based sequencing analysis. BSP cloning-based sequencing analysis was performed on real normal pancreatic tissues (N4 and N7), white blood cells (W6 and W8) of two healthy volunteers, pancreatic cancer cell lines (PANC1 and Patu8988), pancreatic cancer tissues (PC09, PC179, and PC186), and the corresponding adjacent normal tissues (PN09, PN179, and PN186). Black dot, methylated; white dot, unmethylated.