Yu Jun, Xie Yang, Liu Yuting, Wang Feng, Li Mengying, Qi Jian
Department of Gastroenterology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, P.R. China.
Hubei Clinical Center and Key Laboratory of Intestinal and Colorectal Diseases, Wuhan, Hubei 430071, P.R. China.
Exp Ther Med. 2020 Dec;20(6):242. doi: 10.3892/etm.2020.9372. Epub 2020 Oct 22.
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.
分泌型卷曲相关蛋白1（SFRP1）是一种参与Wnt信号传导的细胞外抑制剂，在结直肠癌发生的早期阶段，其因启动子高甲基化而下调。多梳（PCG）蛋白和甲基化CpG结合域（MBD）蛋白在表观遗传基因调控中发挥作用。本研究的目的是确定PCG和MBD蛋白在结直肠癌（CRC）中SFRP1基因表达调控中的作用，特别是在CRC细胞系和人胚胎肠黏膜细胞系CCC-HIE-2中。使用甲基化特异性PCR（MSP）分析SFRP1基因启动子的甲基化状态，而使用逆转录定量PCR分析SFRP1 mRNA表达。评估PCG和MBD蛋白与SFRP1基因之间的关联，通过染色质免疫沉淀筛选相关蛋白，随后使用RNA干扰敲低它们的表达，以确定它们在SFRP1基因表达调控中的作用。结果表明，SFRP1启动子在CRC细胞系中发生高甲基化，在非癌细胞系CCC-HIE-2中发生部分甲基化。与CCC-HIE-2细胞相比，CRC细胞系中SFRP1 mRNA表达显著降低。在HCT116和SW480细胞中，随着SFRP1甲基化，PCG蛋白zeste同源物2（EZH2）和BMI1以及MBD2的表达上调。在CCC-HIE-2细胞中，SFRP1启动子区域富含EZH2，在SW480细胞中富含EZH2和MBD2，而在HCT116细胞的SFRP1启动子上未检测到所研究的任何蛋白。在SW480细胞中，MBD2小干扰（si）RNA可使SFRP1表达重新激活，而EZH2 siRNA则不能，但联合敲低MBD2和EZH2可有效恢复SFRP1基因表达，且不影响SFRP1启动子的甲基化状态。总之，本研究数据表明，在CRC细胞系中，MBD2和EZH2调节SFRP1表达，但不影响SFRP1的高甲基化。相反，SFRP1表达的调节可能通过一种独特的机制，这值得进一步研究。
