MBD2 and EZH2 regulate the expression of SFRP1 without affecting its methylation status in a colorectal cancer cell line

Abstract

Secreted frizzled-related protein 1 (SFRP1), which is an extracellular inhibitor involved in Wnt signalling, is downregulated by promoter hypermethylation in the early stages of colorectal tumorigenesis. Polycomb (PCG) and methyl-CpG-binding domain (MBD) proteins that serve a role in epigenetic gene regulation. The aim of the present study was to determine the role of PCG and MBD proteins in the regulation of SFRP1 gene expression in colorectal cancer (CRC), specifically in CRC cell lines and the human embryo intestinal mucosa cell line CCC-HIE-2. The methylation status of the SFRP1 gene promoter were analysed using methylation-specific PCR (MSP), whereas SFRP1 mRNA expression was analysed using reverse transcription-quantitative PCR. The association between PCG and MBD proteins and the SFRP1 gene was assessed, where associated proteins were screened by chromatin immunoprecipitation and their expression were subsequently knocked down using RNA interference to determine their role in the regulation of SFRP1 gene expression. The SFRP1 promoter was demonstrated to be hypermethylated in CRC cell lines and partially methylated in the non-cancerous cell line CCC-HIE-2. SFRP1 mRNA expression was significantly lower in CRC cell lines compared with that of CCC-HIE-2 cells. The expression of PCGs enhancer of zeste homolog 2 (EZH2) and BMI1, along with MBD2, was indicated to be upregulated with SFRP1 methylation in HCT116 and SW480 cells. The SFRP1 promoter region was enriched with EZH2 in CCC-HIE-2 cells and enriched with EZH2 and MBD2 in SW480 cells, whereas none of the proteins examined were indicated on the SFRP1 promoter in HCT116 cells. The expression of SFRP1 was reactivated by MBD2 small interfering (si)RNA but not by EZH2 siRNA in SW480 cells, but combined MBD2 and EZH2 knockdown effectively restored SFRP1 gene expression without affecting the methylation status of the SFRP1 promoter. In conclusion, data from the present study revealed that MBD2 and EZH2 regulated SFRP1 expression without affecting the hypermethylation of SFRP1 in CRC cell lines. Instead, the regulation of SFRP1 expression may be through a distinct mechanism, which warrants further investigation.

The long and short non-coding RNAs modulating EZH2 signaling in cancer

“…For instance, moderate and high expression levels of EZH2 are associated with unfavorable prognosis of patients with ovarian cancer [ 52 ]. Overall, EZH2 is a critical player in cancer [ 53 – 56 ] and can affect proliferation, metastasis, and therapy response of cancer cells. However, there are studies showing that EZH2 can also function as a tumor-suppressor.…”

Identifying missing pieces in color vision defects: a genome-wide association study in Silk Road populations

“…MBD2 encodes for the methyl-CpG binding domain protein 2, a protein that binds specific methylated sequences, involved in transcription regulation, both repressing and activating the transcription of methylated sites. Moreover, MBD2 encoded protein is associated with many types of cancer, such as breast (Liu et al, 2021), renal (Li et al, 2020), colorectal (Yu et al, 2020) and prostate (Patra et al, 2003). Furthermore, MBD2 is reported to be involved in the T-cells development (Cheng et al, 2021).…”

Expression of Tumor Suppressor SFRP1 Predicts Biological Behaviors and Prognosis: A Potential Target for Oral Squamous Cell Carcinoma

“…Since previous studies have confirmed SFRP1 to be an important regulator of cancer cell motility in skin tumor and colorectal cancer cell lines [ 26 , 27 ], few studies have reported the cell characteristics in OSCC. To achieve effective inhibition and overexpression in our study, we took advantage of adenovirus vectors to knock down and overexpress the target gene.…”