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NEAT1 polyA-modulating antisense oligonucleotides reveal opposing functions for both long non-coding RNA isoforms in neuroblastoma

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Abstract

Many long non-coding RNAs (lncRNA) are highly dysregulated in cancer and are emerging as therapeutic targets. One example is NEAT1, which consists of two overlapping lncRNA isoforms, NEAT1_1 (3.7 kb) and NEAT1_2 (23 kb), that are functionally distinct. The longer NEAT1_2 is responsible for scaffolding gene-regulatory nuclear bodies termed paraspeckles, whereas NEAT1_1 is involved in paraspeckle-independent function. The NEAT1 isoform ratio is dependent on the efficient cleavage and polyadenylation of NEAT1_1 at the expense of NEAT1_2. Here, we developed a targeted antisense oligonucleotide (ASO) approach to sterically block NEAT1_1 polyadenylation processing, achieving upregulation of NEAT1_2 and abundant paraspeckles. We have applied these ASOs to cells of the heterogeneous infant cancer, neuroblastoma, as we found higher NEAT1_1:NEAT1_2 ratio and lack of paraspeckles in high-risk neuroblastoma cells. These ASOs decrease NEAT1_1 levels, increase NEAT1_2/paraspeckles and concomitantly reduce cell viability in high-risk neuroblastoma specifically. In contrast, overexpression of NEAT1_1 has the opposite effect, increasing cell proliferation. Transcriptomic analyses of high-risk neuroblastoma cells with altered NEAT1 ratios and increased paraspeckle abundance after ASO treatment showed an upregulation of differentiation pathways, as opposed to the usual aggressive neuroblastic phenotype. Thus, we have developed potential anti-cancer ASO drugs that can transiently increase growth-inhibiting NEAT1_2 RNA at the expense of growth-promoting NEAT1_1 RNA. These ASOs, unlike others that degrade lncRNAs, provide insights into the importance of altering lncRNA polyadenylation events to suppress tumorigenesis as a strategy to combat cancer.

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Availability of data and materials

R2: Genomics Analysis and Visualization Platform is a web based genomics analysis and visualization application, available at (https://r2.amc.nl). RNA-sequencing data (Fig. 6) can be found on GEO with number GSE133562 and reviewer token: wdqduskcfpmtzed.

Abbreviations

NEAT1:

Nuclear Paraspeckle Assembly Transcript 1

NONO:

Non-POU domain-containing octamer-binding protein

MYCN-V:

Myc myelocytomatosis viral-related oncogene, neuroblastoma derived

BoostPS:

Boost Paraspeckle

PMO:

Morpholino

2’OMe:

2’O-methyl

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Acknowledgements

We acknowledge the Centre for Microscopy, Characterisation and Analysis at The University of Western Australia for SIM microscopy. We thank other members of the Fox lab for helpful discussions about the manuscript.

Funding

This work was supported by a research grant from the Cancer Council of Western Australia to AHF and RL [APP1126667] and AHF [APP1106644], a Cancer Council of Western Australia Honours Scholarship (2017) to JALC and a grant from the National Health and Medical Research Council of Australia [APP1147496] to AHF, CSB and SF.

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AN conducted all wet-lab experiments associated with the neuroblastoma cell lines, designed the experiments, analysed the data and co-wrote the paper. JALC conducted the bioinformatics analyses, analysed the data and co-wrote the Methods section. RL designed the experiments, conducted work done with the U2OS, A549 and HCT116 cell lines, and contributed intellectually to the paper. AH acquired the SIM images. JC conducted MYCN overexpression followed by Western blotting and RT-qPCRs in SK-N-AS cells. TL provided the neuroblastoma cell lines, and provided intellectual input. SW and SF designed and provided reagents for the 2’O methyl ASO work and provided intellectual input. AHF designed the experiments, analysed the data and co-wrote the paper.

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Correspondence to Archa H. Fox.

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SF and SDW are named on patents licensed to Sarepta Therapeutics and act as consultants to the company.

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Naveed, A., Cooper, J.A., Li, R. et al. NEAT1 polyA-modulating antisense oligonucleotides reveal opposing functions for both long non-coding RNA isoforms in neuroblastoma. Cell. Mol. Life Sci. 78, 2213–2230 (2021). https://doi.org/10.1007/s00018-020-03632-6

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  • DOI: https://doi.org/10.1007/s00018-020-03632-6

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