Exploring miR-9 Involvement in Ciona intestinalis Neural Development Using Peptide Nucleic Acids

Abstract

The microRNAs are small RNAs that regulate gene expression at the post-transcriptional level and can be involved in the onset of neurodegenerative diseases and cancer. They are emerging as possible targets for antisense-based therapy, even though the in vivo stability of miRNA analogues is still questioned. We tested the ability of peptide nucleic acids, a novel class of nucleic acid mimics, to downregulate miR-9 in vivo in an invertebrate model organism, the ascidian Ciona intestinalis, by microinjection of antisense molecules in the eggs. It is known that miR-9 is a well-conserved microRNA in bilaterians and we found that it is expressed in epidermal sensory neurons of the tail in the larva of C. intestinalis. Larvae developed from injected eggs showed a reduced differentiation of tail neurons, confirming the possibility to use peptide nucleic acid PNA to downregulate miRNA in a whole organism. By identifying putative targets of miR-9, we discuss the role of this miRNA in the development of the peripheral nervous system of ascidians.

Bisphenol A affects the development and the onset of photosymbiosis in the acoelSymsagittifera roscoffensis

“…The sequence of the LNA probe (5'-TTGGCATTCACCGCGTGCCTTA -3') was designed complementary to the S. roscoffensis miR-124 sequence previously reported (Sempere et al, 2007). A protocol of whole mount in situ hybridization with LNA probes was specifically optimized modifying those used for other marine invertebrates (Mercurio et al, 2020(Mercurio et al, , 2019a(Mercurio et al, , 2019b. Briefly, samples fixed in MOPS fixative were rinsed several times in PBT and hybridized with LNA probe at 55°C in 50% formamide, 5x SSC, 100 μg/ml yeast RNA; 50 μg/ml Heparin; 0.1% Tween-20 for 5 days.…”