In Vivo Analysis of miR-34a Regulated Glucose Metabolism Related Genes in Megalobrama amblycephala

Abstract

The Megalobrama amblycephala (M. amblycephala) is one of the most important economic freshwater fish in China. The molecular mechanism under the glucose intolerance responses which affects the growth performance and feed utilization is still confused. miR-34a was reported as a key regulator in the glucose metabolism, but how did the miR-34a exert its function in the metabolism of glucose/insulin in M. amblycephala was still unclear. In this study, we intraperitoneally injected the miR-34a inhibitor (80 nmol/100 g body weight) into M. amblycephala (fed with high starch diet, 45% starch) for 12 h, and then analyzed the gene expression profiling in livers by RNA-seq. The results showed that miR-34a expression in M. amblycephala livers was inhibited by injection of miR-34a inhibitor, and a total of 2212 differentially expressed genes (DEGs) were dysregulated (including 1183 up- and 1029 downregulated DEGs). Function enrichment analysis of DEGs showed that most of them were enriched in the peroxisome proliferator-activated receptor (PPAR), insulin, AMP-activated protein kinase (AMPK) and janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways, which were all associated with the glucose/lipid metabolic and biosynthetic processes. In addition, we examined and verified the differential expression levels of some genes involved in AMPK signaling pathway by qRT-PCR. These results demonstrated that the inhibition of miR-34a might regulate glucose metabolism in M. amblycephala through downstream target genes.

Keywords: Megalobrama amblycephala; RNA-seq; glucose metabolism; inhibition; miR-34a.

Figure 1

The relative expression level of miR-34a in livers of fishes treated with high starch diet. All fishes were treated with 80 nmol/100 g body weight miR-34a inhibitors (antagomiR-34a) for 12, 24, and 48 h, combined with high starch diet (45% wheat starch). Relative expression levels of miR-34a was detected using qRT-PCR. *** notes p < 0.001 vs. control.

Figure 2

Homologous species distribution of the annotated contigs.

Figure 3

Open reading frame (ORF) length distribution of the annotated contigs (A) and the predicted ORF length distribution of the unannotated contigs (B) in M. amblycephala.

Figure 4

Differentially expressed genes in M. amblycephala in response to miR-34a inhibitor treatment. (A) Blue represents upregulated genes and red represents the downregulated genes in antagomiR34a-12h group vs. control group. |log2FC(Fold change)| ≥ 1 and p-value ≤ 0.05; (B) MA plot of all DEGs, the y-axis represents the logarithm of fold change and the x-axis represents the logarithm of read counts. Red color represents DEGs and black color represents non-DEGs.

Figure 5

Gene expression patterns of RNA-Seq and qRT-PCR. β-actin was used as an internal control and used for the normalization of the expression level of each gene. Log-fold changes are expressed as the ratio of gene expression. Error bars represent standard error.

Figure 6

Gene ontology enrichments of all differentially expressed genes responded to miR-34a inhibitor treatment.

Figure 7

Glucose metabolic-related KEGG pathways for differentially expressed genes responded to miR-34a inhibitor treatment.

Figure 8

qRT-PCR analysis of some glucose metabolism-related genes involved in AMPK signaling pathway. * p < 0.05; ** p < 0.01. Data not included between the whiskers was plotted as an outlier with black dot.

Figure 9

The potential interaction and molecular mechanism of putative genes involved in glucose metabolism process regulated by miR-34a inhibition. Green indicates downregulation, while and red and yellow note upregulation in our study. Gray notes no detection of dysregulation. The blue, red and grey circles include related genes in JAK-STAT1 pathway, AMPK pathway and PPAR pathway, respectively. The green line with an arrow and the green line with a ball stand for positive and negative effect between genes, and the green dot line with an arrow and the green dot line with a ball stand for supposed positive and negative effect between genes.