Direct Sequencing, Identification And Analysis Of The Expression Profile Of MiRNAs In Aedes Albopictus

Background:MicroRNAs (miRNAs) are non-coding 21-23 nt RNAs that play important gene-regulatory roles in animals and plants.Through specific base-pairing with mRNAs, these tiny RNAs induce mRNA degradation or translational repression, or both. Many miRNA genes are transcribed by RNA polymeraseâ…¡, yielding primary miRNAs (pri-miRNAs) of hundreds to thousands of bases in length. A given pri-miRNA can be either monocistronic, containing a sequence for one mature miRNA, or polycistronic, containing a sequence for multiple mature miRNA products. In Drosophila, the primary miRNA is processed by a Drosha-Pasha complex to yield small stem-loop structures that are approximately 70 nucleotides in length called pre-miRNAs. These pre-miRNA are then exported to the cytoplasm and processed by Dicer-1 to form an miRNA:miRNA* duplex, which is further separated by a helicase and normally the miRNA* strand is rapidly degraded and the other strand is chosen as the mature miRNA. Mature miRNAs associate with an Argonaute protein and bind their mRNA targets, which are often in the 3 untranslated region(UTR), resulting in inhibition of translation or possibly target mRNA degradation according to the complementation degree.Usually mRNA degradation is the main way in plants and translational repression in animals.miRNAs are widely distributed in various species,like animals,plants,viruses etc. Many miRNAs are highly conserved across divergent species and that have temporal and spatial specific expression patterns. miRNAs have been found to play important roles in apoptosis, cancer, development,differentiation, neurogenesis,inflammation, longevity, and viral infection.As the earlier pattern of the insect miRNA research, It is estimated that approximately 110 different miRNAs are expressed across the different life stages of D. melanogaster. There are reports describing Anopheles gambiae miRNAs on the basis of computational prediction or similarity to known miRNAs from other species. Direct experimental evidence for mosquito miRNAs has been reported in anopheline mosquitoes using small-scale sequencing. Using high throughput sequencing and bioinformatics approaches, the first systematic analysis of miRNAs in Ae. Aegypti has been recently reported. As a whole, there are hundreds of conserved as well as mosquito-specific miRNAs have been identitied in mosquitoes. Expression profile analysis of several miRNAs from early embryo to adult stages revealed that they may play important roles during embryonic development and during blood feeding. Understanding the functions of mosquito miRNAs will undoubtedly contribute to a better understanding of mosquito biology including longevity, reproduction, and mosquito-pathogen interactions, which are important to disease transmission.Aedes albopictus, as the main vector of Dengue virus, is widely distributed in southern China and Southeast Asia. Recently, the research on the miRNA of Aedes albopictus C7/10 cell has been reported in vitro,but no research in vivo.Using high throughput sequencing and bioinformatics approaches, we performed the first systematic analysis of miRNAs in Aedes albopictus.In the current study, we substantially expanded the list of miRNAs for all mosquitoes and uncovered a number of novel and mosquito-specific miRNAs. Northern and small RNA sequencing revealed several miRNAs that may play important roles during embryonic development, metamorphosis and blood feeding.The intensive study of the target prediction and function of mosquito miRNAs will undoubtedly contribute to a better understanding of mosquito biology as well as the prevention and control work of mosquitoes in the future. Objective:1. To complete the deep sequencing of the whole small RNA in Aedes albopictus using high throughput sequencing and bioinformatics approaches, and identify the known miRNAs and predict the novel ones.2. Using northern blot to confirm the small RNA sequencing results and analyzethe expression profiles of six mosquito-specific miRNAs in different developmental stages. We also compare the expression level of certain miRNAs in female before and after blood feeding.Methods:1. The small RNA sequencing of Aedes albopictus1.1 Sample collection of six different development stages of Aedes albopictusSix samples were prepared for solexa sequencing, such as embryo, larvae, pupa, male mosquito, female mosquito.1.2 Extraction and identification of total RNAAll samples were first frozen in liquid nitrogen and total RNA were isolated according to the instruction of Trizol. Perform the concentration and purity test and then identify the quality of the total RNA use 15% denaturing polyacrylamide gel.1.3 Solexa sequencingAfter detecting purity with Agilent Bioanalyzer, the 18-30nt small RNAs are isolated from total RNA, the 3'and 5'termini of RNA molecules are joined with special adapters respectively and are reversely transcribed into cDNA library for single-end sequencing.1.4 Basic biological information analysisAfter removing the low quality tags, adaptor and contamination formed by adaptor-adaptor ligation, we perform the advance analysis for the "clean" sequences. 1.5 Advanced biological information analysisAll sequences were annotated through the alignment with rRNAetc(non-coding RNA such as rRNA,tRNA,snRNA,snoRNA),known miRNA,repeat and degradation fragments of exon or intron, predict the novel miRNA use the small RNA which match none of these sequences. 2. Expression profile of miRNAs in Aedes albopictus and differential expression of some miRNAs in female before and after blood feeding.2.1 Sample collection of six different development stages of Aedes albopictusSix samples were prepared for northern, such as embryo,â… andâ…¡instars Larvae,â…¢andâ…£instars Larvae,pupa, male mosquito, female mosquito.2.2 Extraction and identification of total RNAAll samples were frozen on liquid nitrogen immediately following collection. Total RNAs from six developmental stages of Aedes albopictus were extracted with a mirVanaTM miRNA Isolation Kit(Ambion). Perform the concentration and purity test and then identify the quality of the total RNA use 15% denaturing polyacrylamide gel.2.3 Expression profiles of miRNAs across different development stages in Aedes albopictus.Equal amount of total RNA were loaded onto 15% denaturing polyacrylamide gel,after electrophoresis then go on with semi-dry transfer, crosslinking, hybridization, wash membranes,exposure and detection.2.4 Differential expression of some miRNAs in female before and after blood feeding.Equal amount of total RNA were loaded onto 15% denaturing polyacrylamide gel,after electrophoresis then go on with semi-dry transfer, crosslinking, hybridization,wash membranes,exposure and detection.Results:1. We obtained approximately 1801570 small RNA sequences including 12663936 reads by lllumina solexa sequencing. In general,92 different known miRNAs were uncovered by comparing with the miRBase14.0 miRNA registry and 46 novel miRNAs were predicted using mireap.2. All miRNAs consist of six conserved and six lineage-specific miRNAs, most of which were temporal expressed across different development stages in Aedes albopictus, were experimental verified by northern blot.3. Three miRNAs (miR-184,miR-998,miR-989) showed an high expression in blood-fed female Aedes albopictus compared with sugar-fed ones. Surprisingly, miR-Nl,which is not detected in sugar-fed mosquito, starts to express after blood feeding.Conclusion:1. Using solexa high throughput sequencing and bioinformatics approaches, we performed the first systematic analysis of miRNAs in Aedes albopictus.Totally, we identitied 92 known miRNAs and predicted 46 novel miRNAs.2. Some miRNAs consist of conserved and lineage-specific miRNAs were experimental verified by northern blot. And most of them were temporal expressed across different development stages in Aedes albopictus, which revealed that they may play important roles in the growing development of mosquito.3. Four miRNAs showed an high expression after blood feeding which suggested that they may play important regulation roles during embryonic development and reproduction.