Department of Gastroenterology, Pingxiang People's Hospital of Southern Medical University, Pingxiang, Jiangxi 337000, P.R. China.
Department of Gastroenterology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, P.R. China.
Oncol Rep. 2021 Dec;46(6). doi: 10.3892/or.2021.8201. Epub 2021 Oct 7.
The secreted frizzled related proteins (SFRPs) are extracellular inhibitors of WNT pathway signaling. Methyl‑CpG binding domain protein 2 (MBD2) and enhancer of zeste homolog 2 (EZH2) are core members of the methylated DNA binding domain (MBD) and polycomb group (PcG) protein families for epigenetic regulation, respectively. This study aimed to ascertain the potential role of MBD2 and EZH2 proteins in colorectal cancer (CRC) and its effects on the expression of SFRP. Bioinformatics, real‑time quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect the expression of MBD2, EZH2, and SFRP in CRC cell lines and tissues. The functions of MBD2 and EZH2 in regards to cell proliferation, cell cycle distribution, apoptosis and invasion were examined in CRC cell lines. Methylation‑specific PCR (MSP) was used to detect the methylation status of the SFRP promoter. The results revealed that the mRNA expression levels of SFRP were significantly decreased in CRC tissues and cell lines compared to these levels in the adjacent tissues and NCM460, respectively. However, the mRNA levels of EZH2 and MBD2 genes were highly expressed in CRC cell lines. We found that reducing MBD2 and EZH2 expression together remarkably inhibited and decreased the proliferation, migration and invasion abilities of the CRC cell lines compared to reducing one of each. Flow cytometric analysis showed that knockdown of MBD2 and EZH2 together in CRC affected cell apoptosis and the cell cycle progression more effectively than knockdown of one of each. The mRNA expression of SFRP1 was reactivated by silencing of MBD2 but not EZH2 in SW480 and HCT116 cells. SFRP4 and SFRP5 mRNA expression was reactivated by silencing of EZH2 but not MBD2 only in SW480 cells. However, depletion of both MBD2 and EZH2 restored SFRP1, SFRP2, SFRP4, and SFRP5 mRNA expression more effectively in CRC cells. Interestingly, there was no significant change in the methylation status of SFRP1, SFRP2, SFRP4, and SFRP5 gene promoter between before and after interference with MBD2, EZH2, and both. In conclusion, our results suggest that silencing of MBD2 and EZH2 simultaneously was able to rescue the expression of SFRP and inhibit the proliferation of CRC cells more effectively. However, the underlying regulatory mechanism system of MBD2 and EZH2 for SFRP in CRC requires further research.
分泌卷曲相关蛋白（SFRPs）是 WNT 信号通路的细胞外抑制剂。甲基化CpG 结合域蛋白 2（MBD2）和增强子的锌指蛋白 2（EZH2）分别是 DNA 甲基化结合域（MBD）和多梳抑制复合物（PcG）蛋白家族中表观遗传调控的核心成员。本研究旨在确定 MBD2 和 EZH2 蛋白在结直肠癌（CRC）中的潜在作用及其对 SFRP 表达的影响。生物信息学、实时定量聚合酶链反应（qPCR）和 Western blot 分析用于检测 CRC 细胞系和组织中 MBD2、EZH2 和 SFRP 的表达。在 CRC 细胞系中检测 MBD2 和 EZH2 对细胞增殖、细胞周期分布、凋亡和侵袭的功能。甲基化特异性 PCR（MSP）用于检测 SFRP 启动子的甲基化状态。结果显示，与相邻组织和 NCM460 相比，CRC 组织和细胞系中 SFRP 的 mRNA 表达水平显著降低。然而，CRC 细胞系中 EZH2 和 MBD2 基因的 mRNA 水平高度表达。我们发现，与单独降低其中一种基因相比，同时降低 MBD2 和 EZH2 的表达可显著抑制和降低 CRC 细胞系的增殖、迁移和侵袭能力。流式细胞术分析表明，与单独降低一种基因相比，CRC 中同时敲低 MBD2 和 EZH2 可更有效地影响细胞凋亡和细胞周期进程。沉默 SW480 和 HCT116 细胞中的 MBD2 可重新激活 SFRP1 的 mRNA 表达，但不能重新激活 EZH2。沉默 EZH2 仅可在 SW480 细胞中重新激活 SFRP4 和 SFRP5 的 mRNA 表达，但不能重新激活 MBD2。然而，在 CRC 细胞中，同时耗尽 MBD2 和 EZH2 可更有效地恢复 SFRP1、SFRP2、SFRP4 和 SFRP5 mRNA 的表达。有趣的是，在干扰 MBD2、EZH2 和两者之后，SFRP1、SFRP2、SFRP4 和 SFRP5 基因启动子的甲基化状态在前后之间没有明显变化。总之，我们的结果表明，同时沉默 MBD2 和 EZH2 能够更有效地恢复 SFRP 的表达并抑制 CRC 细胞的增殖。然而，MBD2 和 EZH2 对 CRC 中 SFRP 的调控机制需要进一步研究。
