Department of Obstetrics and Gynecology, the First Affiliated Hospital of Air Force Military Medical University, Xi'an, China.
Environ Toxicol. 2022 Aug;37(8):1957-1967. doi: 10.1002/tox.23542. Epub 2022 Apr 20.
Recently, it was covered that cellular retinoic acid-binding protein 2 (CRABP2) is upregulated in ovarian cancer and participates in tumor progression, however, the specific mechanism remains to be explored. The pcDNA-CRABP2 or si-CRABP2 was transfected into SKOV3 and OVCAR3 ovarian cancer cells, respectively, and we observed that overexpression of CRABP2 inhibited cell apoptosis, promoted cell invasion and expression of epithelial mesenchymal transition (EMT) marker proteins, and transfection of si-CRABP2 had the opposite effect. Furthermore, we predicted that EZH2 interacted with CRABP2, and overexpression of CRABP2 promoted EZH2 expression, knockdown of CRABP2 inhibited EZH2 expression, and co-immunoprecipitation assay confirmed their binding relationship. The SKOV3 and OVCAR3 cells were then incubated with pcDNA-CRABP2 alone together with si-EZH2, and we found that si-EZH2 reversed the effect of pcDNA-CRABP2 on promotion of EZH2 expression, cell invasion and EMT maker protein levels. Next, we found that EZH2 could bind to DNMT1, and overexpression of EZH2 inhibited TRIM16 expression and knockdown of EZH2 promoted TRIM16 expression. Moreover, the promoter of TRIM16 contains the CpG island, and ChIP assay observed enriched DNMT1 on the promoter of TRIM16, and overexpression of EZH2 increased the promoter methylation level of TRIM16 and knockdown of EZH2 suppressed the methylation. The SKOV3 cells were incubated with si-EZH2 alone or combined with si-TRIM16, and we found that si-TRIM16 reversed the effect of si-EZH2. In vivo studies showed that knockdown of CRABP2 inhibited tumor volume and weight, suppressed the expression of EZH2 and EMT related proteins vimentin and snail, and increased the expression of TRIM16 and E-cadherin.
最近有研究表明，细胞视黄酸结合蛋白 2（CRABP2）在卵巢癌中上调并参与肿瘤进展，但具体机制仍需探讨。分别将 pcDNA-CRABP2 或 si-CRABP2 转染到 SKOV3 和 OVCAR3 卵巢癌细胞中，结果发现 CRABP2 的过表达抑制细胞凋亡，促进上皮间质转化（EMT）标志物蛋白的表达，而 si-CRABP2 的转染则产生相反的效果。此外，我们预测 EZH2 与 CRABP2 相互作用，CRABP2 的过表达促进 EZH2 的表达，CRABP2 的敲低抑制 EZH2 的表达，并且免疫共沉淀实验证实了它们的结合关系。然后将 SKOV3 和 OVCAR3 细胞与 pcDNA-CRABP2 共孵育，同时加入 si-EZH2，发现 si-EZH2 逆转了 pcDNA-CRABP2 对 EZH2 表达、细胞侵袭和 EMT 标志物蛋白水平的促进作用。接下来，我们发现 EZH2 可以与 DNMT1 结合，EZH2 的过表达抑制 TRIM16 的表达，而 EZH2 的敲低则促进 TRIM16 的表达。此外，TRIM16 的启动子含有 CpG 岛，ChIP 实验观察到 DNMT1 在 TRIM16 启动子上富集，EZH2 的过表达增加了 TRIM16 的启动子甲基化水平，而 EZH2 的敲低抑制了甲基化。单独孵育 si-EZH2 或与 si-TRIM16 共孵育 SKOV3 细胞，发现 si-TRIM16 逆转了 si-EZH2 的作用。体内研究表明，CRABP2 的敲低抑制了肿瘤体积和重量，抑制了 EZH2 和 EMT 相关蛋白 vimentin 和 snail 的表达，增加了 TRIM16 和 E-cadherin 的表达。
