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Theranostics 2021; 11(4):1780-1794. doi:10.7150/thno.51931 This issue Cite

Research Paper

Functional roles of antisense enhancer RNA for promoting prostate cancer progression

Chun-Wu Pan^1✉*, Simeng Wen^2*, Lei Chen¹, Yulei Wei³, Yuanjie Niu², Yu Zhao^4✉

1. Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China.
2. Department of Urology, The Second Hospital of Tianjin Medical University, Tianjin Medical University, Tianjin, 300211, China.
3. Department of Gynecology and Obstetrics, Tianjin First Central Hospital, Tianjin, 300192, China.
4. Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
*These authors contributed equally to this work.

✉ Corresponding authors: Yu Zhao, Ph.D. (E-mail: zhao.yuedu) and Chun-Wu Pan, M.D. (E-mail: panchunwucom).

Abstract

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.

Keywords: Antisense eRNA, Enhancer RNA, Antisense RNA, Looping, DNA methylation

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