| BackgroundMicroRNAs (miRNAs) are～22-nucloetide RNAs that modulate gene expression by targeting cognate mRNAs for cleavage or translational repression. They are endogenous and widely distributed in metazoans and plants. In the latest MirBase vision16.0, there are 15172 miRNAs in 142 species and most miRNAs are highly conserved across divergent species. Estimates of the extent of miRNA gene regulation vary from 4% of transcripts in the Drosophila ovary to a third of human genes. At present miRNA is the most important regulator and they have been shown to be involved in the regulation of many biological processes including apoptosis, cancer, embryonic development, immunity and infection.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 in animals. Each miRNA may have multiple target genes and several miRNAs can regulate the same gene simultaneously. Functional studies of miRNAs in C. elegans, Drosophila, mouse, human and other species suggested that the amount of miRNAs varied significantly in different tissues or at different developmental stages, similar to many other molecules involved in gene expression regulation. The specific temporal and spatial expression of miRNAs suggested that miRNA might be involved in gene regulation. Because the miRNA plays an important role in gene regulation, it becomes a new hotspot after small interfering RNA in studies of gene function, biological evolution and disease control. For mosquitos, in 2005, Wang et al, for the first time, identified 38 miRNAs in Anopheles gambiae by computational prediction of similarity to known miRNAs from other species. In 2007, Winter et al. experimentally identified 18 miRNAs from Anopheles gambiae including three miRNAs unique to mosquito. They used a combination of shot-gun cloning and bioinformatic analysis method. In addition, they found that Dicer1 and Agol mRNAs knocked out A. gambiae was more sensitive to the infection of Plasmodium, suggesting that miRNAs might be involved in the defence reaction of Anopheles. Mead and Li also experimentally identified miRNAs in An. stephensi and Ae. Aegypti, as well as finding that miRNA played a regulatory role in development and disease transmission. In 2010. Scalsky infected Culex mosquitoes with WNV and two miRNAs, miR-92 and miR-989, and resulted in significant changes of miRNA expression levels following WNV infection, suggesting that miRNAs might be linked to virus infection in mosquitos.Aedes albopictus, as the main vector of Dengue virus, is widely distributed in southern China and Southeast Asia. Rapid increase of Dengue fever (DF) incidence in recent years has made it a serious public health threat to nearly half of the world's population. In recent years, Guangdong province has had the highest number of DF epidemics, with cases reported every year since 1997. The prevalence of dengue fever in Guangdong, not only poses a serious health hazard, but also often translate into sudden public health events, which brought society instability and serious impact on economic development. As no DF vaccine is currently available, the only effective means for DF prevention is mosquito vector including Aedes aegypti and Aedes albopictus, but basic research such as mosquito susceptibility to dengue virus and spread as well as Modern biological technology in mosquito vectors and infectious disease control application, is still lacking. Therefore, it's very important for control and prevention of dengue fever to discover Aedes albopictus the susceptibility.Before we carryed out the present study, there are no answeres for the following questions:(1) Do miRNAs exist in A.albopictus? (2) What are the sequences of miRNA in A.albopictus and what's their expression pattern? (3) If miRNA play any role in the infection of dengue virus and is there any possiblity to block dengue transmission by targeting on miRNA?The present study employes the highthroughput sequencing capacity provided by Solexa GA to study the OMIC of miRNA in A.albopictus, and explores detailed expression pattersn of miRNAs at different developmental stages. Solexa Genome Analyzer is one of next -generation sequencing technologies produced by Illumina, it has priorities of high accuracy, high throughput, high sensitivity and cost-effective. The method has been widely used in genome sequence, transcriptome sequence and small RNA sequencing studies. Determining miRNAs using Solexa hasa high potential to identify novel miRNA as well as determining the experssion profiles of known and novel miRNAs. The Solexa sequencing technique does not need to clone a library and the sequencing read length is compatible with the length of mature miRNAs. These characters made the Solexa sequencing particularly useful for studying miRNA. The experimental process is very simple and cost-effective, and the method dramatically increases the throughput and has a deeper coverage. To date, small RNA in chicken somites, Bombyx mori, Apis mellifora, Locust and culex were identified by Solexa sequencing which expanded the list of miRNAs and accelerated of the study of small RNAs. In the present study, we performed the first systhematic analysis of miRNAs in Aedes albopictus using a combination of Solexa high throughput sequencing and bioinformatics analysis, and compared with miRNAs in other species according to miRBase virsion14.0. Our result substantially expanded the list of miRNAs for mosquitoes and uncovered a number of novel and mosquito-specific miRNAs. Northern blot and Solexa sequencing found that some miRNAs changed during mosquito development and blood feeding. Using Solexa high throughput sequencing and bioinformatics approaches, we analyzed the expression difference of miRNA in Ae.albopictus infected with dengue virus and predicted targets of these miRNAs in dengue virus's genes, providing potential clues for regulation mechanism of dengue virus in mosquitoes.Objective 1. Analyze miRNAs in Aedes albopictus using Solexa high throughput sequencing and bioinformatics approaches, enrich the list of mosquito miRNAs, and find novel and mosquito -specific miRNAs.2. Analyze expression patterns of miRNAs in different developmental stages in mosquito using Solexa sequencing and northern blot technologies, and find miRNAs that play potential roles during development and blood feeding activies.3. Analyze expression difference of miRNA in Ae.albopictus between infected and uninfected by dengue virus and predicted target genes of miRNAs in dengue virus using Solexa high throughput sequencing and bioinformatics approaches.Methods1 Developing methods for determining miRNAs in Aedes albopictus by Solexa sequencing and bioinformatics analysis:1.1 Sample collection:Six samples were prepared for solexa sequencing, including embryo, larvae, pupa, male mosquito, female mosquito.1.2 Extraction of total RNA and quality control:All samples were firstly frozen in liquid nitrogen and total RNA were isolated according to the user instruction of Trizol. The concentration of RNA was determined using nucleic acid quantitator. The purity was analyzed by 15% denaturing polyacrylamide gel and Agilent Bioanalyzer.1.3 Identification of small RNA by the traditional clone library and sequencing method:Using TA clone to construct library and sequenced by the traditional Sanger capillary sequencing method1.4 Solexa sequencing:After detecting purity with Agilent Bioanalyzer, the 18-30nt small RNAs were 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.1.5 Raw data analysis:The low quality tags, adaptors, and contaminations formed by adaptor-adaptor ligation were removed and the residual reads were analyzed. 1.6 SmallRNAs maping:All sequences were annotated through the alignment with rRNA, tRNA, snRNA, snoRNA, repeat and degradation fragments of exon or intron. The residual sequences were labled as unannotated and were used to predict the novel miRNA.1.7 Identification of known miRNA and prediction of novel miRNA:Clean reads were aligned against the Ae. Aegypti genome (Ae. Albopictus genome has not sequenced so far and Ae. Aegypti is the most closely related species) using SOAP. Sequences with perfect match or one mismatch were retained for further analysis. To further analyze the RNA secondary structures,100 nucleotides of genomic sequence flanking each side of matched sequences (potential miRNAs) were extracted, and the secondary structures were predicted using RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) and analyzed by MIREAP (http://sourceforge.net/projicts/mireap)using default settings.1.8 Identification of miRNAs by northern blot.2 Systhematic analysis of miRNAs in Aedes albopictus:2.1 Analysis of miRNAs in egg, lava, pupa, adult, and blood female in Aedes albopictus by Solexa sequencing and bioinformatics analysis2.2 Comparison with Culex, Drosophila, Apis mellifera, Bombyx mori, Gallus gallus, Danio rerio, Mus musculus, Humus sapient in miRBase.2.3 Clustering of miRNA genes:Find miRNA cluster based on the cutoff in 1000nt in one chromosomeaccording according to method of Altuvia.3 Expression profiles of miRNAs in Aedes albopictus analyzed by Solexa sequencing and northern blot:3.1 Expression difference of miRNAs:The total RNAs from embryo, larvae, pupae, male adult, female adult fed sugar and blood were used for solexa sequencing respectively, and the protocol described as above. One hundred twenty one miRNAs from each sample were analyzed by Solexa reads. All expression data are normalized as following:Expression of normalization=Expression of miRNA /expression of total miNRA from sample xnormalization coefficient. With reference to female, compare miRNAs expression with other samples. 3.2 Expression profiles at different developmental stages were analyzed with MeV by hierarchical clustering.3.3 Identification of expression profiles in different stages in mosquito by northern blot.4 Analysis of miRNAs in Ae. Albopictus infected by dengue virus by Solexa:4.1 Comparison of miRNAs infected and uninfected mosquito and exploration of dengue-specific miRNAs.4.2 Prediction of target genes of miRNAs on dengue virus genome by RNAhybrid.Results1 After filtering linker sequences and ambiguous reads, a total of 12663936 clean small RNA reads were obtained for the mixed sample of multiple developmental stages. We aligned sequencing reads to rRNAs, repeat, exon, intron and known miRNA used by NCBI GenBank dabase (http:www.ncbi.nlm.nih.gov/), and there are 5094481,986,51020,307750.2368768 reads espectively.2 The 79 known miRNAs and 6 novel miRNAs were identified by SOAP and MIREAP and all of them have hairpins with length of 70bp.3 Northern blot determined miR-184 in each developmental stage, and miR-2941 only in egg stage, supporting the Solexa high-throughputing result.4 91 known miRNAs and 37 novel miRNAs in 6 developmental stages were uncovered.5 Comparison of known miRNAs in Ae.albopictus with other species showed that, although the known miRNAs are highly conserved throughout widely divergent animal taxa, the pattern of Ae.albopictus miRNA is more similar to insect than vertebrates.6 Comparison of novel miRNAs in Culex and Anopheles gambiae genomes showed that miR-B-m0097, miR-F-m0156 andmiR-M-m0019 was found in Anopheles gambiae, and 10 novel miRNAs such as miR-2941, miR-2943, and miR-M-m0036 were in Culex, while the rest are Aedes-specific.7 There are 19 out of 128 total miRNAs that were highly similar, such as miR-137-1 and miR-137-2, miR-263a and miR-263b, miR-2941-1 and miR-2941-2, etc.8 There were 8 clusters of miRNAs that showing more than one miRNA hairpin sequences within 1 kb. A miRNA gene cluster within an intron of a gene coding for a serinethreonine kinase group protein. The miRNA gene cluster contained miR-306b, miR-79 and miR-9b.9 The heatmap showed a systematic overview of expression patterns of 129 miRNAs. Clustering of the 6 samples showed that the adult samples (male and sugar-fed and blood-fed females) formed a group, while the other 3 developmental stages formed the other one. We observed obvious stage specific expression of miRNAs in different developmental stages. Hierarchical clustering showed many miRNAs were highly expressed in either the embryo (cluster 1) or male (cluster 4) stages.31 miRNAs were highly expressed in the embryo, among them the mosquito specific miRNAs including miR-2941, miR-2943, miR-2944, miR-2945, and miR-E-m0165. There were 54 miRNAs highly expressed in the male adult, besides some of them were also expressed in female adults. The northern blot of let-7, miR-210, miR-1890, miR-1891 also proved that those miRNA were mainly expressed in the lateral developmental stages, but not being expressed before the pupae stage.10 We further determined 6 conserved miRNAs and 6 mosquito specific miRNAs using northern blot to confirm the illumina sequencing results. All 12 miRNAs showed positive expression, at least at one of the 5 developmental stages, with signals at～22 nt by northern blot. Grossly, the presence/ absence patterns in 5 developmental stages are consistent to the illumina results for all determined miRNAs.11 The expression of a large proportion of miRNAs (70) was increased after blood-feeding, and some (29) were dysregulated. The increasing expression of miR-2941, miR-184, miR-998, and miR-989 has also being proved by northern blot data.12 The expression of a large proportion of miRNAs has changed after infected by dengue virus. There are 38 miRNAs decreased and 21 miRNAs increased after the infection of dengue virus.13 According to the target prediction and expression profile,23 target genes were identified on dengue genes, aiming at non-structural proteins (1,2,4,5) and envelope proteins.Conclusions1 Using Solexa high throughput sequencing and bioinformatics approaches, we performed the first systhematic analysis of miRNAs in Ae. Albopictus, substantially expanded the list of miRNAs for all mosquitoes, and uncovered a number of novel mosquito-specific miRNAs.2 Expression profiles of miRNAs in Ae. Albopictus suggested that several miRNAs may play important roles in the development. High similar result between Solexa and northern bolt demonstrated that Solexa sequencing have a high accuracy.3 Expression levels of several miRNAs have changed after blood feeding, indicatingthat they might be related with embryonic development and reproduction. The result could be helpful for understanding disease transmission in mosquitos.4 Expression of miRNAs was altered after infectionof dengue virus, suggesting that miRNAs may play important regulation roles inviral infection in the mosquito host.
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