Identification of microRNAs in Silver Carp (Hypophthalmichthys molitrix) Response to Hypoxia Stress

Abstract

Hypoxia is one of the serious stresses in fish culture, which can lead to physical and morphological changes, and cause injury and even death to fish. Silver carp (Hypophthalmichthys molitrix) is an important economic fish and widely distributed in China. MicroRNA is a kind of endogenous non-coding single-stranded small RNA, which is involved in cell development, and immune response and gene expression regulation. In this study, silver carp were kept in the closed containers for hypoxia treatment by spontaneous oxygen consumption. The samples of heart, brain, liver and gill were collected, and the total RNAs extracted separately from the four tissues were mixed in equal amounts according to the concentration. Afterwards, the RNA pool was constructed for high-throughput sequencing, and based on the small RNA sequencing, the differentially expressed microRNAs were identified. Furthermore, their target gene prediction and enrichment analyses were carried out. The results showed that a total of 229 known miRNAs and 391 putative novel miRNAs were identified, which provided valuable resources for further study on the regulatory mechanism of miRNAs in silver carp under hypoxia stress. The authors verified 16 differentially expressed miRNAs by qRT-PCR, and the results were consistent with small RNA sequencing (sRNA-seq). The predicted target genes number of differentially expressed miRNAs was 25,146. GO and KEGG functional enrichment analysis showed that these target genes were mainly involved in the adaption of hypoxia stress in silver carp through biological regulation, catalytic activity and apoptosis. This study provides references for further study of interaction between miRNAs and target genes, and the basic data for the response mechanism under hypoxia stress in silver carp.

Keywords: Hypophthalmichthys molitrix; high-throughput sequencing; hypoxia stress; microRNA; silver carp.

Figure 1

Distribution statistics of small RNA fragment length in four samples. X-axis represents the length distribution of small RNA in T0, T1, T2 and T3 group. The Y axis represents the percentage of each length of small RNA. MiRNAs of 22 nt in length were the most common type.

Figure 2

Differently expressed miRNAs validated by qRT-PCR. Comparison was carried out between sRNA-Seq results and qRT-PCR validation results. The 16 selected miRNAs showed concordant expression patterns when the 2 different methods were used.

Figure 3

GO function classification of the differentially expressed genes comparison between the groups. (A) T0 vs. T1, (B) T0 vs. T2, (C) T0 vs. T3. The x-axis represents the number of genes and the y-axis represents different Gene Ontology (GO) term functional classification.

Figure 4

KEGG enrichment analysis of differentially expressed genes in different groups. (A) T0 vs. T1, (B) T0 vs. T2, (C) T0 vs. T3. The x-axis represents the number of genes and the y-axis represents different KEGG pathways.