The ESRP1 promoter reporter can function as an in vivo sensor of DNA methyltransferase inhibition.

Background: 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.

Results: 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.

Conclusions: 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.