Abstract 3780: Reciprocal regulation between DNA methyl transferase 3A and 3B and microRNAs 299-3p and -30e are the causal factors for down regulation of microRNAs targeting androgen receptors in prostate cancer
Authors: Ganapathy Kavya , Harrs Christian , Harris Samuel , Khatib Ayman , Park Jong , Chakrabarti Ratna 
Published: April 2023

Promoter hypermethylation is one of the events that downregulate microRNAs (miRNA), resulting in differential expression of genes and progression of cancer. Previously we showed down-regulation of two miRNAs, miR-299-3p and -30e that target androgen receptor (AR) in advanced prostate cancer (PCa), and their function as tumor suppressors. In this study, we examined the contributing events for the loss of expression of these miRNAs and if the expression varies in PCa tissues and cells from racially disparate group of patients. To test whether promoter hypermethylation is one of the reasons, we examined the effect of treatment of DNA methyl transferase (DNMT) inhibitor 5-Aza-2’-deoxycytidine (Aza-C) on expression of these miRNAs in PCa cells. We noted that the expression of miRs-299-3p, -30e and -34c, another AR-targeting miRNA can be restored in PCa cells upon Aza-C treatment, which suggests that promoter hypermethylation is possibly responsible for down regulation of these miRNAs. We noted that miR- miR-299-3p, -34c and -30e are differentially expressed in PCa tissues and cells from African American (AA) and European American (CA) patients. Through bisulfite sequencing, we showed differential promoter methylation of mir-34c and -299-3p genes in PCa cell lines from AA and CA origins, which can be reversed through treatment with Aza-C. Differential expression and activities of DNMT3A and DNMT3B were also noted in PCa cells. Analysis of miRNA target prediction databases suggested that both miRs-299-3p and -30e have one or more binding sites at the 3’UTRs of DNMT3A and DNMT3B, which was confirmed by luciferase reporter assays. Interaction of these DNMTs with miR-299-3p was further established by Argonaut (Ago)-2-based RNAIP using induced C4-2B PCa cells overexpressing miR-299-3p. Interaction of miR-30e with these DNMTs was also confirmed by transfection of biotinylated miRNA mimic and streptavidin-based RNA pulldown assays. Overexpression of miRs-299-3p and -30e in PCa cells downregulated expression of mRNAs and proteins, and the activity of DNMTs. Ectopic expression of miR-299-3p restored expression of miR-34c and -30e in PCa cells. Similarly, overexpression of miR-30e restored expression of miR-34c and -299-3p. These observations confirmed that the increased expression of miRs-30e or -299-3p overcomes the loss of expression of miRNAs including miRs-299-3p and -34c, which is potentially mediated by DNMT induced promoter hypermethylation. This study established a reciprocal regulatory loop of AR targeting miRNAs and DNMTs in PCa cells and provides a mechanistic insight into the aberrant expression of AR in advanced PCa as a result of down regulation of miRs-299-3p, -34c and -30e and stabilization of expression and activities of DNMTs.

Citation Format: Kavya Ganapathy, Christian Harrs, Samuel Harris, Ayman Khatib, Jong Park, Ratna Chakrabarti. Reciprocal regulation between DNA methyl transferase 3A and 3B and microRNAs 299-3p and -30e are the causal factors for down regulation of microRNAs targeting androgen receptors in prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3780.