51P - Functional characterization of the novel long intergenic non-coding RNA-RFC4, a transcript regulating chromosomal instability in prostate cancer

Background

Chromosomal instability (CIN) is hallmark of cancer associated to progression and aggressiveness in prostate cancer (PCa). In the search of the mechanisms controlling CIN, long non-coding RNAs (lncRNAs) have shown to play important roles in maintaining genome stability, nevertheless, characterization of these transcripts and the networks they control in CIN regulation is still poorly understood.

Methods

RNA-seq data from the prostate cell lines PrEC (stable) and LNCaP (unstable) was analyzed to find novel lncRNAs associated with CIN. Afterwards, experimental validation of the results in PrEC and LNCaP was performed with RT-qPCR and western blot. For the functional experiments, lncRNA knockdown (KD) was performed by transfecting cells with antisense oligonucleotides (ASOs) targeting the lncRNA of interest, followed by karyotyping to assess CIN in the transfected cells. Confirmation of the lncRNA KD was performed by qPCR.

Results

After analyzing and comparing RNA-Seq data from PrEC and LNCaP, a novel lncRNA was discovered in chromosome 3. This nuclear-retained transcript is adjacent to the protein coding gene RFC4, a gene associated to CIN that participates in DNA repair and replication. RT-qPCR analysis showed that while the lncRNA is upregulated in PrEC, RFC4 is downregulated, the opposite pattern was found in LNCaP, suggesting the lncRNA as a cis-repressor of RFC4. The functional experiments confirmed that the lncRNA is a transcriptional repressor of RFC4 but also a regulator of genome stability. After KD of the lncRNA in the stable cell line PrEC, an increase in RFC4 expression was observed as well as an increased number of tetraploid cells, suggesting CIN induction after lncRNA KD. To understand how the lncRNA regulates genome stability, RNA-Seq was performed in the KD experiments showing that lncRNA KD induced disruption of different pathways needed for proper DNA replication and chromosome segregation.

Conclusions

Together our results indicate that the novel lncRNA is a regulator of CIN through regulation of RFC4 and pathways important for maintaining genome stability. The lncRNA adjacent to the protein coding gene RFC4 could be a novel biomarker for PCa progression.

Editorial acknowledgement

Clinical trial identification

Legal entity responsible for the study

Unidad de Investigación Biomédica en Cáncer - Instituto Nacional de Cancerología - Instituto de Investigaciones Biomédicas - UNAM.

Funding

Unidad de Investigación Biomédica en Cáncer - Instituto Nacional de Cancerología - Instituto de Investigaciones Biomédicas - UNAM - CONACyT.

Disclosure

All authors have declared no conflicts of interest.