Abstract
Emerging evidence shows that ring finger protein 144A (RNF144A), a poorly characterized member of the Ring‐between‐Ring (RBR) family of E3 ubiquitin ligases, is a potential tumor suppressor gene. However, its regulatory mechanism in breast cancer remains undefined. Here, we report that RNF144A promoter contains a putative CpG island and the methylation levels of RNF144A promoter are higher in primary breast tumors than those in normal breast tissues. Consistently, RNF144A promoter methylation levels are associated with its transcriptional silencing in breast cancer cells, and treatment with DNA methylation inhibitor 5‐Aza‐2‐deoxycytidine (AZA) reactivates RNF144A expression in cells with RNF144A promoter hypermethylation. Furthermore, genetic knockdown or pharmacological inhibition of endogenous methyl‐CpG‐binding domain 4 (MBD4) results in increased RNF144A expression. These findings suggest that RNF144A is epigenetically silenced in breast cancer cells by promoter hypermethylation and MBD4.
Highlights
Ring finger protein 144A (RNF144A) belongs to the Ring- between-Ring (RBR) family of E3 ubiquitin ligases and is highly conserved in eukaryotes [1]
To directly analyze whether expression of RNF144A is regulated by promoter methylation, we examined the effects of DNA methylation inhibitor AZA on the reactivation of RNF144A in breast cancer cells by Quantitative real-time PCR (qPCR) and immunoblotting analyses. qPCR analysis results showed that treatment of MDA-M B-231 and BT20 cells, in which RNF144A promoter was hypermethylated, with AZA significantly reactivated RNF144A expression in a dose- dependent manner (Fig. 3C, left and middle panels, respectively)
We provide evidence for the first time that promoter hypermethylation is involved in transcriptional repression of RNF144A in breast cancer cells
Summary
Ring finger protein 144A (RNF144A) belongs to the Ring- between-Ring (RBR) family of E3 ubiquitin ligases and is highly conserved in eukaryotes [1]. The members of the RBR protein family contain three functional domains, including two separated RING finger domains and an in- between ring (IBR) domain [1]. Emerging evidence shows that RNF144A promotes apoptosis in response to DNA damage through targeting DNA-dependent protein kinase (DNA-PK) for ubiquitination and proteasomal degradation [5]. Our recent studies demonstrated that induced expression of RNF144A decreases the sensitivity of breast cancer cells to Epigenetic Silencing of RNF144A in Breast Cancer Cells poly(ADP-ribose) polymerase (PARP) inhibitor olaparib through targeting DNA repair protein PARP1 for ubiquitination and subsequent proteasomal degradation[6]. The regulatory mechanism of RNF144A in breast cancer remains unknown
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