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Communication Dans Un Congrès Année : 2023

Antisense oligonucleotides targeting an oncogenic mitochondrial long non-coding mtRNA to inhibit equine skin tumor cells proliferation

Résumé

Introduction Cancer treatment is highly constrained by chemotherapy side-effects. ASncmtRNA family are mitochondrial long-non-coding RNAs, previously identified in mice and humans, containing both sense and antisense transcripts transcribed from 16S rRNA mDNA region (1), showing differential expression between tumor and normal cells. Knockdown of the ASncmtRNAs induces proliferative arrest and apoptosis in several tumor cell lines and mouse models without affecting normal cells (2-5). The aim of our project is to translate this technology into an equine skin tumor model by the design and screening of ASO candidates directed to the Equus caballus ASncmtRNAs ortholog (eca-ASncmtRNA) in two equine tumor cell culture models. Melanoma is a high prevalent in gray horses (6) and sarcoid tumors is a firosarcoma caused by BPV viral infection (7). Material and methods Equine eca-ASncmtRNAs were characterized using successive RT-PCR and amplicons Sanger sequencing. We designed in silico targeting the eca-ASncmtRNA. Melanoma cell lines (6) and sarcoid biopsy cultures were transfected with ASO candidates and controls and selected by differential anti-proliferative and cell death induction in tumor cells versus normal proliferative myoblasts. ATP luminescent cell viability assays and cell counts were used to perform screenings followed by flow cytometric cell death dye-based assays. Full phosphorotioate (PS) and locked nucleic acids (LNA) oligos were evaluated. Results Cell proliferation screenings showed three interesting ASO candidates inducing a significant reduction in cell viability and death in the melanoma cell line at 500nM at 72h post-transfection (65% reduction versus ASO-C, IC50 253nM). Flow cytometric cell death analyses showed ASO-08 induced cell death and increased mid-apoptotic cells significantly compared to ASO-C control (scrambled ASO). ASO-08 cell death induction was similar to doxorubicin treatment. ASO-08 repeated transfection increases the anti-proliferative effect (65 to 89%) at 500nM but not at 250nM compared to ASO-C. ASO-08 with full PS modifications induces higher anti-proliferative and cell death effects compared to the same sequence with LNA modifications. In sarcoid cell cultures, ASO-08 induced a higher anti-proliferative effect and cell death at 250nM for 72h (66% reduction versus ASO-C). Doxorubicin treatment did not significantly induce any anti-proliferative effect in these cells. Conclusion ASOs directed to the equine eca-ASncmtRNA induce selective antiproliferative effects in the two tumor cell models. Oligonucleotide antisense therapy will be evaluated in horse skin cancer cases by ASO local injections. References: 1-Villegas et al (2007) PMCID: PMC2175360 2-Burzio et al (2009) PMCID: PMC2695051 3-Vidaurre et al (2014) PMCID: PMC4175353 4-Fitzpatrick et al (2019) PMCID: PMC6541642 5-Lobos-Gonzalez et al (2016) PMCID: PMC5295434 6-Rosengren et al (2008) PMID: 18641652 7-Ogluszka et al (2012) PMID: 33461443
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hal-04510279 , version 1 (05-04-2024)

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Christopher Fitzpatrick, Sophie Dhorne-Pollet, Fany Blanc, Giorgia Egidy, Eric Barrey. Antisense oligonucleotides targeting an oncogenic mitochondrial long non-coding mtRNA to inhibit equine skin tumor cells proliferation. OTS OLIGO MEETING - 19th Annual Meeting, 19th Annual Meeting Oligonucleotide Therapeutics Society, Oct 2023, BARCELONA, Spain. ⟨hal-04510279⟩
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