Lin Lecheng, Chen Lingli, Jing Yajie, Chen Zhihong
School of Basic Medicine, Youjiang Medical University for Nationalities, Baise, 533000, China.
Suzhou Engineering Research Center of Natural Medicine and Functional Food, School of Biological and Food Engineering, Suzhou University, Suzhou, 234000, China.
BMC Biotechnol. 2025 Aug 27;25(1):90. doi: 10.1186/s12896-025-01031-y.
The discovery of novel DNA methyltransferase (DNMT) inhibitors as anticancer agents represents a significant milestone in pharmaceutical research. However, the absence of robust high-throughput screening methods for these compounds has substantially hindered their development.
In this study, we found that the epithelial splicing regulatory protein 1 (ESRP1) was underexpressed in renal cell carcinoma (RCC) cells. ESRP1 overexpression induced G1-phase arrest and inhibited the proliferation of RCC cells by downregulating cyclin A2 expression. Furthermore, the ESRP1 promoter was hypermethylated in RCC cells, and treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNMT inhibitor, effectively demethylated the CpG sites within the promoter region of ESRP1, thereby upregulating the transcriptional activity of the ESRP1 promoter and gene expression both in vitro and in vivo. Additionally, we constructed a bioluminescent reporter gene (designated ESRP1-P-Luc2) by fusing the promoter sequence of the ESRP1 gene with the luciferase gene using molecular cloning techniques. Bioluminescence imaging revealed that 5-Aza-CdR treatment could upregulate the expression of the reporter gene both in vitro and in vivo.
Our results demonstrate that in RCC cells, ESRP1 promoter hypermethylation is accompanied by downregulation of its expression level; restoring ESRP1 expression can induce cell cycle G1-arrest and inhibit RCC cell proliferation by downregulating cyclin A2 expression; ESRP1-P-Luc2 may serve as a useful tool for monitoring the effects of DNMT inhibitor anticancer drugs at both the cellular level and in living animals, thereby providing a potential tool for high-throughput screening (HTS) of such drugs.
新型DNA甲基转移酶（DNMT）抑制剂作为抗癌药物的发现是药物研究中的一个重要里程碑。然而，缺乏针对这些化合物的强大高通量筛选方法严重阻碍了它们的开发。
在本研究中，我们发现上皮剪接调节蛋白1（ESRP1）在肾细胞癌（RCC）细胞中表达下调。ESRP1过表达诱导G1期阻滞，并通过下调细胞周期蛋白A2的表达抑制RCC细胞的增殖。此外，ESRP1启动子在RCC细胞中发生高甲基化，用DNMT抑制剂5-氮杂-2'-脱氧胞苷（5-Aza-CdR）处理可有效使ESRP1启动子区域内的CpG位点去甲基化，从而在体外和体内上调ESRP1启动子的转录活性和基因表达。此外，我们使用分子克隆技术将ESRP1基因的启动子序列与荧光素酶基因融合，构建了一个生物发光报告基因（命名为ESRP1-P-Luc2）。生物发光成像显示，5-Aza-CdR处理在体外和体内均可上调报告基因的表达。
我们的结果表明，在RCC细胞中，ESRP1启动子高甲基化伴随着其表达水平的下调；恢复ESRP1表达可诱导细胞周期G1期阻滞，并通过下调细胞周期蛋白A2的表达抑制RCC细胞增殖；ESRP1-P-Luc2可作为在细胞水平和活体动物中监测DNMT抑制剂抗癌药物效果的有用工具，从而为这类药物的高通量筛选（HTS）提供潜在工具。
