Differentially expressed microRNAs in diapausing versus HCl-treated <i>Bombyx</i> embryos

<div><p>Differentially expressed microRNAs were detected to explore the molecular mechanisms of diapause termination. The total small RNA of diapause-destined silkworm eggs and HCl-treated eggs was extracted and then sequenced using HiSeq high-throughput method. 44 novel miRNAs were discovered. Compared to those in the diapause-destined eggs, 61 miRNAs showed significant changes in the acid-treated eggs, with 23 being up-regulated and 38 being down-regulated. The potential target genes of differentially expressed miRNAs were predicted by miRanda. Gene Ontology and KEGG pathway enrichment analysis of these potential target genes revealed that they were mainly located within cells and organelles, involved in cellular and metabolic processes, and participated in protein production, processing and transportation. Two differentially expressed genes, <i>Bombyx mori SDH</i> and <i>Bmo-miR-2761-3p</i>, were further analyzed with qRT-PCR. <i>BmSDH</i> was significantly up-regulated in the HCl-treated eggs, while <i>Bmo-miR-2761-3p</i> was down-regulated. These results suggested that these two genes were well coordinated in silkworm eggs. Dual luciferase reporter assay demonstrated that <i>Bmo-miR-2761-3p</i> inhibited the expression of <i>BmSDH</i>.</p></div

Length distribution of sRNA.

<p>(A) Length distribution of sRNA in control eggs, (B) Length distribution of sRNA in HCl-treated eggs. Y asix represents the precentage of special sRNA in total, X axis represents length of sRNA.</p

Expressions pattern of <i>Bmo-miR-2761-3p</i> and <i>BmSDH</i> from 3 to 7 day old eggs of silkworm, <i>B</i>. <i>mori</i>.

<p>Each time point was replicated three times using independently collected samples. Error bar = 1 SD. (A) Relative <i>Bmo-miR-2761-3p</i> expression level; (B) Relative <i>BmSDH</i> expression level.</p

<i>Bmo-miR-2761-3p</i> down-regulated <i>BmSDH</i> in vitro.

<p>PC group represents cells transfected with positive control plasmid (<i>Bmo-miR-2761-3p</i> inhibitor inserted into <i>Luc2</i> 3’UTR), WT group represents cells transfected with plasmid containing <i>BmSDH</i> 3’UTR. “NC” represents cells transfected with the negative control mimics, “mimics” represents cells transfected with <i>Bmo-miR-2761-3p</i> mimics. Normalized firefly luciferase activity equals to firefly luciferase activity/Renilla luciferase activity. ** P < 0.01 indicates significant differences compared to the relevant control. Each group was replicated three times using independently collected samples. Error bar = 1 SD.</p

Distribution of ncRNA.

<p>(A) ncRNA distribution of total sRNA in control eggs, (B) ncRNA distribution of unique sRNA in control eggs, (C) ncRNA distribution of total sRNA in HCl-treated eggs, (D) ncRNA distribution of unique sRNA in HCl-treated eggs.</p

Primers used in this study.

<p>SL (Stem loop) primers were used for cDNA synthesis of miRNAs.</p

Gene ontology annotation of candidate target genes predicted by miRanda.

<p>Candidate target genes were classified into three categories: Biological Process, Cellular Component, and Molecular Function.</p

Venn chart for sRNA.

<p>(A) Venn chart for sRNA of total sRNA, (B) Venn chart for sRNA of unique sRNA.</p

KEGG pathways mapped based on miRNAs candidate target genes.

<p>Y axis represents KEGG pathway, X axis Rich factor represents the percentage of Candidate target genes enriched in pathway, the qvalue closer to 0 represents enriched more significant.</p

miRNAs with specific expression.

<p>miRNAs with specific expression.</p