MicroRNAs show diverse and dynamic expression patterns in multiple tissues of Bombyx mori

Shiping Liu 0 Song Gao 0 Danyu Zhang 0 Jiyun Yin 0 Zhonghuai Xiang 0 Qingyou Xia 0 1 0 The Key Sericultural Laboratory of Agricultural Ministry, College of Biotechnology, Southwest University , Chongqing 400715 , PR China 1 Institute of Agricultural and Life Sciences, Chongqing University , Chongqing, 400030 , PR China Background: MicroRNAs (miRNAs) repress target genes at the post-transcriptional level, and function in the development and cell-lineage pathways of host species. Tissue-specific expression of miRNAs is highly relevant to their physiological roles in the corresponding tissues. However, to date, few miRNAs have been spatially identified in the silkworm. Results: We establish for the first time the spatial expression patterns of nearly 100 miRNAs in multiple normal tissues (organs) of Bombyx mori females and males using microarray and Northern-blotting analyses. In all, only 10 miRNAs were universally distributed (including bmo-let-7 and bmo-bantam), while the majority were expressed exclusively or preferentially in specific tissue types (e.g., bmo-miR-275 and bmo-miR-1). Additionally, we examined the developmental patterns of miRNA expression during metamorphosis of the body wall, silk glands, midgut and fat body. In total, 63 miRNAs displayed significant alterations in abundance in at least 1 tissue during the developmental transition from larvae to pupae (e.g., bmo-miR-263b and bmo-miR-124). Expression patterns of five miRNAs were significantly increased during metamorphosis in all four tissues (e.g., bmo-miR-275 and bmo-miR305), and two miRNA pairs, bmo-miR-10b-3p/5p and bmo-miR-281-3p/5p, showed coordinate expression. Conclusions: In this study, we conducted preliminary spatial measurements of several miRNAs in the silkworm. Periods of rapid morphological change were associated with alterations in miRNA expression patterns in the body wall, silk glands, midgut and fat body during metamorphosis. Accordingly, we propose that corresponding ubiquitous or tissue-specific expression of miRNAs supports their critical roles in tissue specification. These results should facilitate future functional analyses. - Background MicroRNAs (miRNAs) are an extensive class of small (~22 nucleotides) regulatory RNAs found in a wide range of eukaryotic organisms and viruses [1,2]. Increasing evidence shows that miRNAs function in several biological processes, including development, cellular differentiation, proliferation, metabolism and apoptosis [1,3-5]. Single miRNAs may regulate hundreds of different target genes at the post-transcriptional level, and extensively control more than 30% of animal genes [3,6]. Plant miRNAs regulate the expression of other genes by cleaving perfect or nearly perfect complementary sites within the transcribed regions [7], whereas animal miRNAs repress the translational expression of genes by partially binding to the complementary target sites at 3untranslated regions (3UTRs) [8,9]. The first members of the miRNA family discovered in invertebrates, lin-4 and let-7, are expressed in Caenorhabditis elegans at distinct stages of development and regulate the timing of larval transition through cell-fate decisions [8,10]. In insects, bantam miRNA regulates cell proliferation and death by targeting the apoptosis gene hid (wrinkled) [11]. Drosophila miR-14 is implicated in fat metabolism, stress resistance and cell death [12]. Interestingly, most of these well-characterized miRNAs are highly conserved between invertebrates and vertebrates, resulting in maintenance of their regulatory functions across species [13]. The tissue-specific expression patterns of miRNAs observed in many species [14-17] are considered a prerequisite for specifying and maintaining tissue identity [18]. Moreover, tissue- and cell-specific expression patterns are directly associated with the physiological functions of miRNAs. For instance, miR-1 is specifically expressed in cardiac and skeletal muscle precursor cells, and regulates cardiomyocyte proliferation in vertebrates [19]. In Drosophila, miRNA-1 is strongly expressed throughout the mesoderm of early embryos and subsequently in somatic, visceral, and pharyngeal muscles and the dorsal vessel, and functions in the post-mitotic growth of larval muscle [20]. miR-122 is an abundant liver-specific miRNA [21], which facilitates replication of the hepatitis C viral RNA in humans [22] and regulates cholesterol and fatty-acid metabolism [23,24]. Establishment of the tissue-specific distribution of miRNAs in these species offers critical insights into the roles of corresponding miRNAs in tissue specification and cell lineage decisions. B. mori, a characteristic representative of Lepidoptera, has long been used as a model for lepidopteran biology [25]. A number of silkworm miRNAs have been experimentally identified and temporally characterized [26-30]. However, only the spatial expression patterns of let-7 have been extensively characterized in the silkworm to date [29]. In this study, we established the distribution of silkworm miRNAs in fifth-instar day 3 larvae and their spatiotemporal expression patterns in the body wall, silk glands, midgut and fat body during metamorphosis. Results and Discussion Identification of miRNAs in multiple tissues of fifth-instar day 3 larvae To determine the global spatial expression patterns of miRNAs in silkworm, we designed a DNA oligonucleotide-based microarray examining 92 unique miRNAs with 106 antisense probes [30]. To verify results, all probes for miRNAs and controls were printed in triplicate on two parallel blocks. The microarray raw data and processed files have been deposited in the NCBI Gene Expression Omnibus [31] and are accessible through GEO Series accession number GSE18039 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE18039. To make the inter-slide signals comparable, all net signals of fifth-instar day 5 larvae were normalized on the basis of U6 and 5 S rRNA signals. All internal and external controls were deleted after normalization. In all, only 10 miRNAs (No.37-46), including bmo-let-7, bmo-miR-34b and bmo-bantam, were ubiquitously expressed in all tissues examined, whereas individual tissues were enriched for distinct sets of miRNAs (Table 1). For example, strong expression signals of miR-124 (No.1) and miR-263b (No.4) were specifically detected in the head. Similarly, miR-124 is restricted to the central nervous system of the fruit fly [17] and only expressed in the brain of mouse [32]. The Drosophila orthologue of bmo-miR-7 has been identified in the nervous system, and may be related to segmentation, sensory organ development, and Notch signal transduction [17]. In the silkworm, miR-7 (No.10) was expressed in the head and gonads in both sexes, as well as in the body wall in males. Three miRNAs, miR-288 (No.14), miR-278 (No.16) and miR-13a (No.17) were solely detected in the head, body wall and malpighian tubules. A previous study shows that miR-274 is exp (...truncated)