Downstream effects of dre-miR-210-5p in zebrafish primary ovarian cell culture

Abstract

MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in post-transcriptional gene regulation in many cellular functions and are highly conserved throughout evolution. In teleost fish species, miRNAs are believed to play a role in the reproductive system, but more research is needed to better understand the functions of miRNAs in fish gonads. Furthermore, miR-210 has previously been described to be involved in many processes, such as hypoxia response, angiogenesis, cell proliferation and male infertility. The aim of this study was, first, to develop an in vitro model in fish to study the functions of miRNAs and second, to identify target genes of dre-miR-210-5p by establishing a primary ovarian cell culture in zebrafish (Danio rerio). The cell culture was performed by isolating ovaries from adult female fish (n=4) which were incubated with dre-miR-210-5p mimic or scramble miRNA mimic. Cell survival was studied by flow cytometry analysis and fluorescent microscopy. Following, the effect of dre-miR-210-5p on ovarian cells was uncovered by RNA-sequencing, identifying [~]6,000 targeted-genes differentially expressed (DEGs), of which [~]2,600 downregulated and [~]3,400 upregulated. GO term and KEGG pathway analyses showed downregulated genes involved in cell cycle processes and reproduction-related pathways while, in contrast, immune-related pathways were upregulated after miR-210 mimic treatment in the ovarian cells. To provide the molecular mechanisms triggered by miRNA-210, seed regions of targeted genes were identified and in silico analysis classified DEGs as potential biomarkers in reproduction or immune cell types. These results support the crosstalk between the reproductive and immune system in which dre-miR-210-5p plays a key role in the transcriptomic alteration in the fish ovaries. Take home messagemiR-210 promotes the immune response and suppresses oocyte meiosis in the ovarian cells in zebrafish