Yonsei Med J.  2018 Jan;59(1):141-147. 10.3349/ymj.2018.59.1.141.

microRNA-183 is Essential for Hair Cell Regeneration after Neomycin Injury in Zebrafish

• Kim CW
• Han JH
• Wu L
• Choi JY

• KMID: 2418861
• DOI: http://doi.org/10.3349/ymj.2018.59.1.141

Abstract

PURPOSE
microRNAs (miRNAs) are non-coding RNAs composed of 20 to 22 nucleotides that regulate development and differentiation in various organs by silencing specific RNAs and regulating gene expression. In the present study, we show that the microRNA (miR)-183 cluster is upregulated during hair cell regeneration and that its inhibition reduces hair cell regeneration following neomycin-induced ototoxicity in zebrafish.
MATERIALS AND METHODS
miRNA expression patterns after neomycin exposure were analyzed using microarray chips. Quantitative polymerase chain reaction was performed to validate miR-183 cluster expression patterns following neomycin exposure (500 µM for 2 h). After injection of an antisense morpholino (MO) to miR-183 (MO-183) immediately after fertilization, hair cell regeneration after neomycin exposure in neuromast cells was evaluated by fluorescent staining (YO-PRO1). The MO-183 effect also was assessed in transgenic zebrafish larvae expressing green fluorescent protein (GFP) in inner ear hair cells.
RESULTS
Microarray analysis clearly showed that the miR-183 cluster (miR-96, miR-182, and miR-183) was upregulated after neomycin treatment. We also confirmed upregulated expression of the miR-183 cluster during hair cell regeneration after neomycin-induced ototoxicity. miR-183 inhibition using MO-183 reduced hair cell regeneration in both wild-type and GFP transgenic zebrafish larvae.
CONCLUSION
Our work demonstrates that the miR-183 cluster is essential for the regeneration of hair cells following ototoxic injury in zebrafish larvae. Therefore, regulation of the miR-183 cluster can be a novel target for stimulation of hair cell regeneration.

Keyword

microRNA; ototoxicity; regeneration; zebrafish

MeSH Terms

Animals
Animals, Genetically Modified
Cell Count
Gene Expression Profiling
Gene Expression Regulation/drug effects
Gene Knockdown Techniques
Green Fluorescent Proteins/metabolism
Hair Cells, Auditory/drug effects/*physiology
Larva/drug effects/genetics
MicroRNAs/genetics/*metabolism
Morpholinos/pharmacology
Neomycin/toxicity
Regeneration/drug effects/*genetics
Zebrafish/*genetics
MicroRNAs
Morpholinos
Neomycin
Green Fluorescent Proteins

Figure

• 

  Fig. 1 Experimental design for the effect of microRNA in hair cell regeneration. MO-183 injection into zebrafish larvae immediately after fertilization. Larvae were treated with neomycin (500 µM) for 2 h at 72 hpf. Total RNA sampling and hair cell counting was performed at 4, 12, 24, and 48 h after neomycin treatment. hpf, hours post fertilization; MO, morpholino.

• 

  Fig. 2 miRNA expression microarray data after neomycin (500 µM) exposure for 2 h in zebrafish larvae. The data are expressed as log2 transformed fold change in miRNA expression at 4, 12, 24, and 48 h after neomycin treatment, compared to non-treated larvae. Only those miRNAs which showed 2- or more fold change at any time point, compared to control, are shown. Arrows indicated miRNAs up-regulated in three time points. *miR-183 clusters. miRNA, microRNA.

• 

  Fig. 3 Expression of microRNAs after neomycin exposure. miR-96 (A), miR-182 (B), and miR-183 (C) expression in zebrafish larvae after neomycin treatment was compared with that from non-treated samples (n=3 at each time point). *p<0.05. miR, miRNA.

• 

  Fig. 4 In situ hybridization of miR-183 after MO injection. (A) The larvae injected with standard MO shows clear miR-183 expression in neuromast cells at 122 hours post fertilization (arrows). (B) In contrast, miR-183 expression disappeared in larvae injected with MO-183. Scale bar, 50 µm. miR, miRNA; MO, morpholino.

• 

  Fig. 5 The effect of miR-183 knockdown on hair cell regeneration after neomycin-induced ototoxicity. (A) Hair cells labeled with Y-PRO1 are regenerating 24 h after neomycin treatment in zebrafish larvae injected with control MO. (B) Hair cells are partially regenerating in larvae injected with MO-183 (1 mM). (C) Summarized data from six independent experiments. Scale bar, 100 µm. *p<0.05. miR, miRNA; MO, morpholino.

• 

  Fig. 6 The effect of miRNA-183 knockdown on hair cell regeneration in the inner ear of pou4f3::GFP zebrafish larvae. (A) Hair cells and otoliths are mostly regenerated in larvae injected with control MO. (B) Hair cells and otoliths are partially regenerated in larvae injected with MO-183. MO, morpholino; GFP, green fluorescent protein.

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