Methylation of secreted frizzled-related protein 1 (SFRP1) promoter downregulates Wnt/β-catenin activity in keloids
Methylation of secreted frizzled-related protein 1 (SFRP1) promoter downregulates Wnt/β-catenin activity in keloids
复制标题
分泌型卷曲相关蛋白 1 (SFRP1) 启动子的甲基化下调瘢痕疙瘩中 Wnt/β-连环蛋白的活性
DOI：
10.1007/s10735-018-9758-3
复制
发表时间：
2018-04-01
影响因子：
3.2
通讯作者：
Hu, Dahai
中科院分区：
文献类型：
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作者：
Liu, Jiaqi;Zhu, Huayu;Hu, Dahai
瘢痕疙瘩是一种良性皮肤病，在遗传易感个体的伤口愈合过程中形成。为了更好地控制瘢痕疙瘩并了解其分子机制，本研究筛选GEO数据库瘢痕疙瘩基因芯片数据，并鉴定分泌型卷曲相关蛋白1（SFRP 1）启动子的高甲基化。随后，在57例瘢痕疙瘩与正常皮肤组织中评估了超甲基化以及mRNA和蛋白质水平。从组织中分离成纤维细胞用于体外基因调控的评估。所使用的方法是生物信息学分析、携带SFRP 1 cDNA的慢病毒感染、qRT-PCR、western blot、免疫组织化学、荧光素酶报告基因测定、甲基化特异性PCR和甲基化DNA免疫沉淀-qPCR、ELISA和/或5-Aza-2 '-脱氧胞苷处理。结果显示，瘢痕疙瘩组织中SFRP 1启动子的甲基化程度高于正常皮肤组织。瘢痕疙瘩组织和瘢痕疙瘩成纤维细胞中SFRP 1基因启动子甲基化导致SFRP 1 mRNA和蛋白表达下调。5-Aza处理显著上调瘢痕疙瘩成纤维细胞中SFRP 1的mRNA和蛋白水平。此外，DNMT 1表达的敲低，而不是DNMT 3a或DMNT 3b的表达，是导致瘢痕疙瘩成纤维细胞中SFRP 1启动子的高甲基化和SFRP 1 mRNA和蛋白的上调的原因。此外，携带SFRP 1 cDNA的慢病毒感染可显著抑制瘢痕疙瘩成纤维细胞中Wnt/beta-catenin信号通路的活性以及beta-catenin和alpha-SMA的mRNA和蛋白表达。总之，由于SFRP 1启动子的超甲基化而导致的SFRP 1表达诱导的Wnt/β-连环蛋白信号传导的丢失可能与瘢痕疙瘩的发展相关，这表明SFRP 1可能是瘢痕疙瘩治疗的治疗靶点。
Keloid, a benign skin disorder, forms during wound healing in genetically susceptible individuals. To better control keloid and understand the molecular mechanisms, this study screened gene hypermethylations of GEO database microarray data on keloids and identified the hypermethylation of the secreted frizzled related protein-1 (SFRP1) promoter. Subsequently, hypermethylation and mRNA and protein levels were assessed in 57 cases of keloid vs. normal skin tissues. Fibroblasts from tissues were isolated for the assessment of gene regulation in vitro. The methods used were bioinformatic analysis, lentiviral infection carrying SFRP1 cDNA, qRT-PCR, western blot, immunohistochemistry, luciferase reporter assay, methylation-specific PCR and methylated DNA immunoprecipitation-qPCR, ELISA, and/or 5-Aza-2'-deoxycytidine treatment. The data revealed that the SFRP1 promoter was hypermethylated in keloid tissues, compared with that in normal skin tissues. The SFRP1 promoter methylation contributed to the downregulation of SFRP1 mRNA and protein in keloid tissues and keloid fibroblasts. The 5-Aza treatment significantly upregulated SFRP1 mRNA and protein level in keloid fibroblasts. Furthermore, the knockdown of DNMT1 expression, and not the expression of DNMT3a or DMNT3b, was responsible for the hypermethylation of the SFRP1 promoter and upregulation of SFRP1 mRNA and protein in keloid fibroblasts. In addition, the infection of lentivirus carrying SFRP1 cDNA significantly inhibited the signaling activity of Wnt/beta-catenin and the mRNA and protein expression of beta-catenin and alpha-SMA in keloid fibroblasts. In summary, the lost SFRP1 expression-induced Wnt/beta-catenin signaling due to the hypermethylation of the SFRP1 promoter could associate with keloid development, suggesting that SFRP1 might be a therapeutic target for keloid treatment.