Analysis of microRNA profile of Anopheles sinensis by deep sequencing and bioinformatic approaches

Abstract

BackgroundmicroRNAs (miRNAs) are small non-coding RNAs widely identified in many mosquitoes. They are reported to play important roles in development, differentiation and innate immunity. However, miRNAs in Anopheles sinensis, one of the Chinese malaria mosquitoes, remain largely unknown.MethodsWe investigated the global miRNA expression profile of An. sinensis using Illumina Hiseq 2000 sequencing. Meanwhile, we applied a bioinformatic approach to identify potential miRNAs in An. sinensis. The identified miRNA profiles were compared and analyzed by two approaches. The selected miRNAs from the sequencing result and the bioinformatic approach were confirmed with qRT-PCR. Moreover, target prediction, GO annotation and pathway analysis were carried out to understand the role of miRNAs in An. sinensis.ResultsWe identified 49 conserved miRNAs and 12 novel miRNAs by next-generation high-throughput sequencing technology. In contrast, 43 miRNAs were predicted by the bioinformatic approach, of which two were assigned as novel. Comparative analysis of miRNA profiles by two approaches showed that 21 miRNAs were shared between them. Twelve novel miRNAs did not match any known miRNAs of any organism, indicating that they are possibly species-specific. Forty miRNAs were found in many mosquito species, indicating that these miRNAs are evolutionally conserved and may have critical roles in the process of life. Both the selected known and novel miRNAs (asi-miR-281, asi-miR-184, asi-miR-14, asi-miR-nov5, asi-miR-nov4, asi-miR-9383, and asi-miR-2a) could be detected by quantitative real-time PCR (qRT-PCR) in the sequenced sample, and the expression patterns of these miRNAs measured by qRT-PCR were in concordance with the original miRNA sequencing data. The predicted targets for the known and the novel miRNAs covered many important biological roles and pathways indicating the diversity of miRNA functions. We also found 21 conserved miRNAs and eight counterparts of target immune pathway genes in An. sinensis based on the analysis of An. gambiae.ConclusionsOur results provide the first lead to the elucidation of the miRNA profile in An. sinensis. Unveiling the roles of mosquito miRNAs will undoubtedly lead to a better understanding of mosquito biology and mosquito-pathogen interactions. This work lays the foundation for the further functional study of An. sinensis miRNAs and will facilitate their application in vector control.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2734-7) contains supplementary material, which is available to authorized users.

Too Many False Targets for MicroRNAs: Challenges and Pitfalls in Prediction of miRNA Targets and Their Gene Ontology in Model and Non‐model Organisms

“…The latter are more frequently used when genomic data from closely related species are unavailable, but not always. It seems that some researchers tend to overestimate the prediction accuracy of such software, to a degree that experimentally non‐validated miRNA targets are speculated to control various biological processes . This is probably due to the misconception that the parameters that these software programs take into account (e.g., computation of miRNA–mRNA favorable thermodynamics and secondary structure of mRNA) enable us to efficiently eliminate most false positives.…”

“…It seems that speculations about miRNA–target regulation, based on prediction alone are less frequent in highly studied model organisms as experimental support can be obtained relatively easily. However, when it comes to non‐model organisms, recent publications continue with bold speculations regarding the putative role of miRNAs that regulate targets that were chosen from a predicted list without any experimental support . A disturbing fact is that in non‐model organisms the search is often made by assuming miRNA–target recognition rules that might not reflect the biology of the studied organisms .…”

“…Notably, faulty target prediction is not restricted only to non‐bilaterian animals and lineages where the mechanism is unknown as some studies in bilaterian non‐model organisms are suffering from the same problems . These studies searched for seed‐matching targets, which is indeed the relevant mechanism for bilaterians; however, their conclusions about the importance of miRNA‐related targets are most likely wrong because of the reasons discussed in the previous chapter.…”

“…Studies of miRNAs often do not stop at lists of predicted targets. Many perform functional annotation on the predicted targets, followed by Gene Ontology (GO) enrichment analysis for finding biological pathways regulated by certain miRNAs . When significant enrichment for biological processes is detected, the level of confidence in the “newly discovered” miRNA‐regulated pathway is increased.…”

“…In summary, we discussed major problems related to computational predictions of miRNA targets and their regulatory pathways. The high level of noise in lists of predicted targets (Figures and ), the failure to consider the type of the relevant miRNA–target interaction, and the pitfalls of using GO enrichment analyses on predicted targets, altogether strongly suggest that miRNA‐regulated pathways being reported without experimental support are false.…”