MicroRNA-14 regulates larval development time in Bombyx mori

https://doi.org/10.1016/j.ibmb.2017.12.009Get rights and content

Highlights

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    The conserved invertebrate miR-14 plays a role in ecdysteroid regulated development in Bombyx mori.

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    Ubiquitous transgenic overexpression of miR-14 using the GAL4/UAS system delayed larval development.

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    miR-14 disruption using the transgenic CRISPR/Cas9 system led to a precocious wandering stage.

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    E75 and EcR-B are putative target genes of miR-14 in B. mori.

Abstract

MicroRNAs (miRNA) regulate multiple physiological processes including development and metamorphosis in insects. In the current study, we demonstrate that a conserved invertebrate miRNA-14 (miR-14) plays an important role in ecdysteroid regulated development in the silkworm Bombyx mori, a lepidopteran model insect. Ubiquitous transgenic overexpression of miR-14 using the GAL4/UAS system resulted in delayed silkworm larval development and smaller body size of larva and pupa with decrease in ecdysteriod titers. On the contrary, miR-14 disruption using the transgenic CRISPR/Cas9 system led to a precocious wandering stage with increase in ecdysteriod titers. We identified that the hormone receptor E75 (E75) and the ecdysone receptor isoform B (ECR-B), which both serve as essential mediators in the ecdysone signaling pathway, as putative target genes of miR-14 by in silico target prediction. Dual-luciferase reporter assays confirmed the binding of miR-14 to the 3′UTRs of E75 and ECR-B in a mammalian HEK293T cell line. Furthermore, transcription levels of E75 and ECR-B were significantly affected in both miR-14 overexpression and knockout transgenic animals. Taken together, our data suggested that the canonical invertebrate miR-14 is a general regulator in maintaining ecdysone homeostasis for normal development and metamorphosis in B. mori.

Introduction

MicroRNAs (miRNAs) comprise a group of short, non-coding RNAs which function by binding to protein elongation factors to suppress the process of mRNA translation and facilitate mRNA degradation via the RNA-induced silencing complex (RISC) complex (Carthew and Sontheimer, 2009, Djuranovic et al., 2011, Djuranovic et al., 2012). miRNAs participate in many physiological processes in insects and their roles in regulating insect development and metamorphosis has been investigated over the course of the last decade. For example, RNAi analysis of the Dicer-1 gene, a key component in miRNA processing, revealed that miRNAs are involved in the regulation of insect metamorphosis in Blattella germanica (Gomez-Orte and Belles, 2009). In Drosophila melanogaster, Let-7 and miR-125 mutants exhibited defects in neuromusculature which finally affected eclosion and adult behavior (Caygill and Johnston, 2008, Sokol et al., 2008). Loss of miR-8 function reduced eclosion at high temperatures in Drosophila (Kennell et al., 2012). In the silkworm, Bombyx mori, down-regulation of Let-7 led to development arrest during the larval-larval and larval-pupal transition (Ling et al., 2014). In addition, an invertebrate-specific miRNA, miR-2, has been demonstrated to play a critical role in wing morphogenesis in B. mori (Ling et al., 2015). Another conserved invertebrate miR-14, the seed sequence of which can only be found in insects, suppresses apoptosis-driven genes reaper, grim, hid, and dronc (Xu et al., 2003) and inhibit apoptosis through actinomycin-D in Drosophila (Kumarswamy and Chandna, 2010). Furthermore, miR-14 is expressed in the insulin-producing neurosecretory cells in Drosophila adult brain to control metabolism through its target gene, a zinc-finger protein sugarbabe (Varghese et al., 2010). The synthesis of insulin-like peptides (ilp) is also regulated by miR-14 and the Drosophila miR-14 mutants show increased lipid storage and a decrease in ilp transcripts (Varghese et al., 2010).

Ecdysone is a steroid hormone which is essential for insect development, since it triggers each of the major developmental transitions in the life cycle. The actions of ecdysone are mediated by a heterodimeric receptor consisting of the ecdysone receptor (ECR) and its partner molecule, the retinoid X receptor homolog ultraspiracle (USP) (Thomas et al., 1993, Yao et al., 1993). This hormone/receptor complex activates a small set of early-response genes encoding several transcription factors that activate a large set of downstream late-response genes. Pulses of 20E signals initiate major developmental transitions in insects, including egg hatching, larval-larval molting, and larval-pupal-adult metamorphosis (Riddiford et al., 2000). In Drosophila, miR-14 suppresses the translation of ECR mRNA, resulting in very low or no expression of ECR-encoded genes (Varghese and Cohen, 2007). High 20E titers drive increased expression of ECR and reduce the expression of miR-14, leading to up-regulation of 20E-regulated genes. Hence, miRNAs represent an additional tier of gene regulation during post-embryonic development in Drosophila.

Numerous miRNAs have been extensively investigated in model insects such as the fruit fly and mosquito, but still limited in other insect species, largely due to lack of genetic tools. Recent emergence of the genome-editing tool CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/RNA-guided Cas9 nucleases) provides a robust toolkit for loss-of-function analysis in non-drosophilid insects including B.mori (Li et al., 2015, Xu et al., 2017, Zhang et al., 2017). This technique has been successfully established in several insect species, and it also has been used to disrupt miRNA expression and dissected the regulation of miRNA target genes (Zhang et al., 2016). In the present study, we disrupted miR-14 using the transgenic CRISPR/Cas9 system, resulting in precocious wandering behavior in silkworm larvae. We also used the GAL4/UAS system to overexpress miR-14 and silkworm larval development time was delayed with reduced body size in transgenic silkworms. Furthermore, through in silico prediction and a dual-luciferase reporter (DLR) assay, we identified E75 and ECR-B as target genes of miR-14 and expression levels of these two genes were significantly altered in either miR-14 overexpression or disruption animals. Our data thus reveals that miR-14 regulates ecdysone signaling by targeting E75 and ECR-B to modulate larval development time in B. mori.

Section snippets

Silkworm strains and rearing

The multivoltine, nondiapausing silkworm strain Nistari was used for germ line transformation and subsequent experiments. Larvae were reared on fresh mulberry leaves or artificial diets at 25 °C.

Plasmid construction and transgenic transformation

To construct the transgenic UAS-miR-14 overexpression line, pre-bmo-miR-14 (pre-miR-14) and 5′-and-3′ end flanking sequences of genomic DNA were amplified by PCR using the primers OV-miR-14-F/OV-miR-14-R which are listed in Table 1. The amplified 708 bp fragments were sub-cloned into the piggyBac

Expression of miR-14 was enhanced by 20-Hydroxyecdysone (20E)

The previous study reported that miR-14 was constantly expressed during entire developmental stages in B. mori (He et al., 2008). Here we further investigated spatial expression of miR-14 by using qPCR and the result showed that miR-14 was ubiquitously expressed in all investigated tissues, with relative higher expression in the Malpighian tubule, epidermis, middle silk gland and midgut (Fig. 1A). We also investigated the temporal expression of miR-14 from day-one of the fifth instar larval

Discussion

The major contribution of the current study is the application of GAL4/UAS and CRISPR/Cas9 systems to characterize the biological functions of miR-14 in B. mori. As a conserved miRNA in insects, miR-14 is an important regulator of various biological processes (Xu et al., 2003). In miR-14-OV silkworms, the relative expression of mature miR-14 and pre-miR-14 were significantly up-regulated, resulting in decreased ecdysteriod titers and larval development delay with smaller body sizes of larvae

Conflicts of interest

The authors have no conflicts of interest to declare.

Acknowledgements

This work was supported by grants from National Science Foundation of China (31530072 and 31420103918) and Chinese Academy of Sciences (XDB11010600 and KJZD-EW-L12-02).

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