In vivo miRNA delivery in whitefish: Synthetic MiR92b-3p uptake and the efficacy of gene expression silencing. Issue 1 (January 2019)

• Record Type: Journal Article
• Authors: Brzuzan, Paweł; Woźny, Maciej; Lewczuk, Bogdan; Florczyk, Maciej; Gomułka, Piotr; Budzińska, Paulina; Wesołowski, Michał; Dobosz, Stefan
• Is Part Of: Experimental biology and medicine. Volume 244:Issue 1(2019)
• Page Start: 52
• Page End: 63
• Publication Date: 2019-01
• Subjects: cdo1 -- in vivo screening -- oligonucleotides mimicking miRNAs -- RNA-Seq -- transfection -- imaging; Physiology -- Periodicals; Biology, Experimental -- Periodicals; Medicine, Experimental -- Periodicals; 610.72
• DOI: 10.1177/1535370218824573
• Languages: English
• ISSNs: 1535-3702

Abstract

Here, we provide the first report of an efficient method of in vivo delivery of MiRNA92b-3p mimic by intraperitoneal injection to whitefish, a Teleost fish. Juvenile whitefish were exposed to synthetic MiR92b-3p suspended in Invivofectamine 3.0 transfection reagent. After 24 and 48 h of the treatment, the blood, liver, spleen, brain, and heart of the fish were sampled to track the uptake of the synthetic miRNA and to assess its specific and off-target effects. RT-qPCR indicated that, within the first 24 h of treatment, MiR92b-3p levels were higher in the blood plasma, liver, and spleen of the injected fish than in the control fish that were administered only solvent vehicle, which was further confirmed by screening the organs with a fluorescently labeled MiR92b-3p probe. We then found that, 48 h after the injection, the mimic decreased the mRNA levels of its putative downstream targets (p53, cdkn1a, and pcna) by approximately 50%, which may indicate that MiR92b-3p was functional. Further profiling of mRNA expression in the liver by RNA sequencing showed perturbations that did not prompt global, specific silencing but instead produced significant differences in expression of a number of genes 48 h after transfection with the mimic. Neither histological nor ultrastructural analyses showed any pathological changes in the liver structure of the exposed fish, and biochemical measurements of the fishes' blood did not differ between the experimental groups. Together, these results indicate that the MiR92b-3p mimic was effectively delivered to the liver of the injected fish, and that the short-term treatment did not cause any apparent toxic effects. This methodology of miRNA mimic delivery has utility not only for the study of miRNA-dependent silencing mechanisms in fish, but also gives reasons to anticipate significant progress in the development of both miRNA diagnostic markers and therapeutic targets in liver injury. Impact statement: The delivery of short snippets of RNA, such as synthetic miRNA agents, is an essential step for achieving RNA-mediated knockdown, which has not been studied in sufficient detail in fish. Our results indicate that a MiR92b-3p mimic may be effectively delivered via intraperitoneal injection to the spleen and the liver of whitefish, and that it likely achieves functionality without causing any apparent toxic effects in the challenged animals. We report the novel finding that the MiR92b-3p mimic reduced the in vivo liver mRNA expression levels of its putative pro-apoptotic targets (p53, cdkn1a, and pcna), and important metabolic genes, e.g. cdo1 . This shows that this methodology of MiR92b-3p mimic transfection in vivo may be a useful tool for studies that investigate the molecular pathways that confer pro-proliferative and anti-apoptotic phenotypes or those that regulate intracellular metabolism in fish and other vertebrates.