VIRMA-Dependent N6-Methyladenosine Modifications Regulate the Expression of Long Non-Coding RNAs CCAT1 and CCAT2 in Prostate Cancer.

Daniela Barros-Silva,Elena S Martens-Uzunova,Jorge Oliveira,Rui Henrique,João Lobo,Carmen Jerónimo,Catarina Guimarães-Teixeira,Isa Carneiro

doi:10.3390/cancers12040771

Daniela Barros-Silva, Elena S Martens-Uzunova + Show 6 more

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https://doi.org/10.3390/cancers12040771

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Abstract

RNA methylation at position N6 in adenosine (m6A) and its associated methyltransferase complex (MTC) are involved in tumorigenesis. We aimed to explore m6A biological function for long non-coding RNAs (lncRNAs) in prostate cancer (PCa) and its clinical significance. m6A and MTC levels in PCa cells were characterized by ELISA and western blot. Putative m6A-regulated lncRNAs were identified and validated by lncRNA profiler qPCR array and bioinformatics analysis, followed by m6A/RNA co-immunoprecipitation. Impact of m6A depletion on RNA stability was assessed by Actinomycin D assay. The association of m6A-levels with PCa prognosis was examined in clinical samples. Higher m6A-levels and VIRMA overexpression were detected in metastatic castration-resistant PCa (mCRPC) cells (p < 0.05). VIRMA knockdown in PC-3 cells significantly decreased m6A-levels (p = 0.0317), attenuated malignant phenotype and suppressed the expression of oncogenic lncRNAs CCAT1 and CCAT2 (p < 0.00001). VIRMA depletion and m6A reduction decreased the stability and abundance of CCAT1/2 transcripts. Higher expression of VIRMA, CCAT1, and CCAT2 as a group variable was an independent predictor of poor prognosis (HR = 9.083, CI95% 1.911–43.183, p = 0.006). VIRMA is a critical factor sustaining m6A-levels in PCa cells. VIRMA downregulation attenuates the aggressive phenotype of PCa by overall reduction of m6A-levels decreasing stability and abundance of oncogenic lncRNAs.

Highlights

Prostate cancer (PCa) is a highly prevalent malignancy, being the second most common cancer and the fifth leading cause of cancer-related death in men [1]
M6 A is installed by the methyltransferase complex (MTC), a multi-component protein complex including a heterodimer of two methyltransferase-like proteins, METTL3 and METTL14, the Wilms’ tumor 1-associating protein (WTAP), and the Vir-like methylation at position N6 in adenosine (m6 A) methyltransferase associated protein (VIRMA)
Inspection of the genomic regions encoding each one of the MTC subunits revealed that VIRMA is the most commonly altered MTC protein in prostate cancer (PCa) (8% of the samples), whereas the other MTC subunits disclose lower frequencies of alterations (1% for METTL3, 3% for METTL14, and 1.8% for WTAP) (Figure 1A)

Summary

Introduction

Prostate cancer (PCa) is a highly prevalent malignancy, being the second most common cancer and the fifth leading cause of cancer-related death in men [1]. A proportion of patients develops resistance (Castration-Resistant Prostate Cancer, CRPC) and progresses to lethal metastatic disease (mCRPC). RNA activity in cells is tightly regulated by different chemical modifications that can be dynamic and adaptable [2]. RNA methylation at position N6 in adenosine (m6 A) is a highly prevalent epitranscriptomic modification in messenger and non-coding RNAs (ncRNAs) that affects splicing, translation, and stability [3]. METTL14 is important for substrate recognition, activity and specificity, while WTAP and VIRMA function as regulatory components. WTAP stabilizes the heterodimer formed by METTL3/14, plays a critical role for the nuclear localization of MTC, and can affect RNA alternative splicing. VIRMA is required for the full methylation program in mammals and its silencing causes a substantial reduction in m6 A levels during embryonic development [8]

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