Methylation of secreted frizzled-related protein 1 (SFRP1) promoter downregulates Wnt/β-catenin activity in keloids

Abstract

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/β-catenin and the mRNA and protein expression of β-catenin and α-SMA in keloid fibroblasts. In summary, the lost SFRP1 expression-induced Wnt/β-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.

Epigenetic modification mechanisms involved in keloid: current status and prospect

“…The treatment of 5-Aza-dc, as an inhibitor of DNA methyltransferase, appeared to conspicuously escalated SFRP1 level and coordinated Wnt/β-catenin activity in KFs. Furthermore, a functional verification experiment ulteriorly authenticated that downgrade DNMT1 instead of DNMT3a or DMNT3b was responsible for the hypermethylation of the SFRP1 promoter and upregulation of SFRP1 expression in KFs [52]. These two findings indicated that DNA methylation and histone acetylation might cooperatively participate in regulating SFRP1 expression, and the mutual relationship seems to be bidirectional with each able to affect the other.…”

“…Subsequently, the appliance of Trichostatin A (TSA) and 5-Aza-2′-deoxycytidine (5-aza-dc) determined that SFRP1 expression in KFs was increased almost 15-fold by TSA but not by 5-aza-dc, speculating that silencing of SFRP1 was not due to hypermethylation but histone modification. However, recent studies by Liu et al presented that the lost SFRP1 expression due to the hypermethylation of the SFRP1 promoter likewise might play a vital role in the pathological progress of keloid [52]. The treatment of 5-Aza-dc, as an inhibitor of DNA methyltransferase, appeared to conspicuously escalated SFRP1 level and coordinated Wnt/β-catenin activity in KFs.…”

Bioinformatics analysis and verification of gene targets for benign tracheal stenosis

“…Zhang found the genes related to the pathogenesis of oralsquarous cell carcinoma (OSCC) utilizing bioinformatics analysis, and further verified these molecules might lead to OSCC through the PI3K‐AKT pathway, suggesting the relevant molecules may be a molecular target for specific diagnosis and therapy (Zhang, Feng, et al, 2018; Zhang, Zhang, et al, 2018). In addition, Liu found abnormally expressed molecules in multiple scar tissues by bioinformatics analysis and further verified that SFRP1(OMIM:604156) might take part in the occurrence of scar by regulating Wnt/β‐catenin (Liu et al, 2018). However, there are few reports on the use of bioinformatics analysis to explore the mechanisms of acute tracheal injury and TS.…”

Downregulation of Epac Reduces Fibrosis and Induces Apoptosis Through Akt Signaling in Human Keloid Fibroblasts

“…Fibroblasts were grown to 95%-100% confluency and then scratched in each well using a sterile 200 mL tip. 18 Without ESI-09 application, fibroblasts migrated from the edge of the scratch and completely covered the area cleared by the scratch 24 h after wounding. ESI-09 application increased the amount of time required for HKFs and HDFs to cover the same blank area, thus cell migration was significantly decreased by ESI-09 in a dose-dependent manner (Fig.…”