Functional roles of antisense enhancer RNA for promoting prostate cancer progression

Rationale: Enhancer RNA (eRNA) bi-directionally expresses from enhancer region and sense eRNA regulates adjacent mRNA in cis and in trans. However, it has remained unclear whether antisense eRNAs in different direction are functional or merely a reflection of enhancer activation. Methods: Strand-specific, ribosome-minus RNA sequencing (RNA-seq) were performed in AR positive prostate cancer cells. RNA-seq, GRO-seq, ChIP-seq, 4C-seq and DNA-methylation-seq that published in our and other labs were re-analyzed to define bi-directional enhancer RNA and DNA methylation regions. Molecular mechanisms were demonstrated by 3C, ChIP, ChIRP, CLIP, RT-PCR and western blot assays. The biological functions of antisense-eRNA were assessed using mice xenograft model and RT-PCR analysis in human tissues. Results: In this study, we identified that antisense eRNA was regulated by androgen receptor (AR) activity in prostate cancer cells. Antisense eRNA negatively regulated antisense ncRNA in AR-related target genes' loci, through recruiting DNMT1 on the antisense enhancer in the gene-ending regions and elevating DNA methylation. Importantly, the chromatin exhibited a double looping manner that facilitated sense-eRNA to promoter and antisense-eRNA to gene-ending region in cis. Depletion of antisense eRNA impaired its neighbor mRNA expression, cancer growth and invasion. The expressions of antisense eRNA were correlated with biochemical recurrence and clinical marker PSA's levels in patients' tissues. Conclusions: The findings indicated that antisense eRNA was a functional RNA and may be a novel target that when suppressed improved prostate cancer therapy and diagnosis. New chromatin interaction among enhancer, promoter and gene-ending region might provide new insight into the spatiotemporal mechanism of the gene transcription and acting of bi-directional eRNAs.

Automated summary

In this study, it was found that antisense eRNA is regulated by androgen receptor activity in prostate cancer cells, negatively regulating antisense ncRNA in AR-related target genes' loci through DNA methylation. The chromatin interaction among enhancer, promoter, and gene-ending regions facilitated the expression of sense-eRNA and antisense-eRNA in cis, impacting cancer growth and patient outcomes. The suppression of antisense eRNA may serve as a potential novel target for improving prostate cancer therapy and diagnosis.