Methylation status of p16 gene and expressions of related genes in keloid tissue and cultured keloid fibroblasts

Abstract

Objective To explore the role of p16 gene methylation in fibroblasts in the occurrence and development of keloid. Methods Skin tissue specimens were resected from the lesions of patients with keloid and normal skin of healthy human controls. Fibroblasts were isolated from these tissue specimens and subjected a primary culture. An immunohistochemical analysis was performed to measure the expression of p16 protein in tissue specimens, real-time fluorescence-based quantitative PCR to determine the mRNA expression level（expressed as 2-△ △ Ct）of p16 and DNA methyltransferases（DNMTs）in fibroblasts, and bisulfite sequencing PCR（BSP）to estimate the methylation status of p16 gene in the tissue specimens and primary fibroblasts. Results The keloid fibroblasts（KFbs）showed significantly lower mRNA expression of p16 gene（0.64 ± 0.18 vs. 1.92 ± 0.23, t = 10.54, P < 0.05）, but significantly higher mRNA expressions of 3 DNMTs（DNMT1: 2.58 ± 0.23 vs. 1.13 ± 0.21, t = 11.22, P < 0.05; DNMT3A: 4.87 ± 0.46 vs. 2.38 ± 0.32, t = 10.81, P < 0.05; DNMT3B: 1.57 ± 0.12 vs. 0.57 ± 0.16, t = 12.45, P < 0.05）compared with the normal fibroblasts（NFbs）. The DNA methylation rate in the p16 gene promoter region was significantly increased in keloid tissue（1.81% ± 0.46%）and KFbs（3.15% ± 0.94%）compared with normal skin tissue（0.90% ± 0.35%, F = 14.23, P < 0.01）and NFbs（0.17% ± 0.29%, F = 37.62, P < 0.01）. Conclusions The methylation and low expression of p16 gene in KFbs may be associated with the uncontrolled growth of keloid, and DNMTs may play a role in the pathogenesis of keloid. Key words: Keloid; Fibroblasts; Genes, p16; Methylation; Methyltransferases