| MicroRNAs(miRNAs)are a class of single small non-coding RNA molecules which has the function on transcriptional regulation by complementary base pairing with its target mRNAs that led to specificity degradation or inhibit mRNAs translation.MiRNAs were widely existed in species,and participate in the regulation of biological growth,cell proliferation and differentiation,apoptosis and cancer,etc.In the field of insects,the miRNAs were found to be involved in regulating immune stress,and abnormal development.However,there were only 26 species has been in the annotation in the latest miRBase,there are still a large number of miRNAs undetected.In addition,as a result of miRNAs with temporal sexual expression characteristics,many miRNAs related to development still need to be further identified.Therefore,in order to explore miRNAs in insects abnormal development,wing development,the regulating function of sexual dimorphism development,embryonic development,this study in hemimetamorphosis insect Eupolyphaga sinensis Walker and holometabolism insect Tribolium castaneum using Solexa high-throughput sequencing,microfluidic chip experiments,RNA interference(RNA interference,RNAi)technology,miRNAs mimics and antagomirs treatmnets were carried on,and have get the following important results:1.Eupolyphaga sinensis Walker(Blattaria)is a sexually dimorphic insect,which is also an important source of material used in traditional chinese medicine.The male E.sinensis have shorter lifecycles and go through fewer instars than the female.Furthermore,the males have forewings,while the females are totally wingless.We used the Illumina/Solexa deep sequencing technology to sequence small RNA libraries prepared from the fourth-instar larvae of male and female E.sinensis.19,097,799 raw reads were yielded in total:7,817,445 reads from the female library and 11,280,354 from the male,respectively.As a result,we identified 168 known miRNAs and 204 novel miRNAs.Among them,100 miRNAs specific expressed in male,32 miRNAs specific expressed in female and 240 miRNAs were co-expressed.Moreover,45 miRNAs abundant in both female and the male samples were showed significantly different expression pattern,such as let-7-5p,miR-184,miR-8,miR-9a etc,and we validated 10 of them by Stem-loop qRT-PCR.Combined with previous researches in miRNAs function,our results indicated that these differentially expressed miRNAs are closely related to metamorphosis,wing formation,sexual dimorphism development and laid the foundation to miRNAs function investigation in insect development.2.To date,although some miRNAs have been discovered in Tribolium castaneum,while large numbers of miRNAs still require investigation as its stage-specific expression.For investigating miRNAs function in wing development and metamorphosis,we knocked down Dcr-1 and Ago-1 by RNAi in T.castaneum late larvar instar and found the deleption resulted in a series defects in T.castaneum development and metamorphosis.The expression level of metamorphosis associated miRNAs:let-7,miR-8,miR-100,and miR-184 were significantly declined,suggested miRNAs played important roles during this process.However,the Dcr-1 and Ago-1 deletion impaired all miRNAs expression levels,such serious defects could not influence by a single miRNA individual but multiple of clusters miRNAs that involved in the complex regulation network.To further investigate the directly regulation of miRNAs in metamorphosis,we firtly detected several key genes of juvenile hormone pathway and ecdysone pathway.After RNAi treatments,the transcription levels of Met and its downstream gene Kr-h1 were increased while Br-c and E74 were decreased,significantly.Then,11 miRNAs were found to target 22 sites of Met and 14 miRNAs were found to target 28 sites of Kr-h1.The results showed that miRNAs directly involved in the beetles metamorphosis.In order to discover miRNAs more widely in T.castaneum,high-throughput Illumina/Solexa sequencing was performed with a mixed sample that was collected from eight key developmental stages.In total,12,259,974 raw reads were obtained;7,116,806 mappable reads remained;and 2,175,311 high-quality miRNA reads were identified.At last,1,154 unique miRNAs were discovered containing 274 conserved miRNAs,108 known candidate miRNAs and 772 novel miRNAs.Genome locus analysis showed that miRNA clusters are abundant in T.castaneum and that the density of mirs loci in the X chromosome is higher than that of the euchromosome and is more abundant in the T.castaneum genome than other species.In addition,we found a certain number of miRNAs individuals have multiple isomers(iso-miRNAs)that provided evidence for miRNAs evolution mode of duplication.3.Currently studies showed that several individual miRNAs were key regulators in regulation of metamorphosis in insects,while the miRNA numbers and its function in the regulation network still unclear.Thus,we combined with T.castaneum miRNAs which identified by high-throughput Solexa sequencing in our previous study and other 2,308 arthropod miRNAs annoation in miRBase as microarray probes,constructed expression profiles of those miRNAs using a customized microarray experiment to further investigate.Finally,179 unique differential expressed miRNAs were screened out and classified into 9 clusters(cluster A-1),which displayed dynamic expressed among early embryo,late larva,early pupa,late pupa,and early adult developmental stages.Among them,5 miRNA clusters were stage-specific that includng 3 relative highly expression clusters of early embryo(cluster A),late larva(cluster B),early adult(cluster C)stage and 2 lowly expression clusters of early embryo(cluster G),early pupa(cluster F).The further ananlysis showed that cluster A and cluster F was caught our eye.In particular,cluster A with 53 embryo-specific highly expressed miRNAs targeted 747 candidate genes,but only significantly enrichment to 8 GO functional components,whereas cluster F with 5 early pupa-specific lowly expressed miRNAs targeted 234 candidate genes that significantly concentration to 65 GO terms and many of these targets were assocated with organ developmet and tissue development.The results suggested that miRNAs regulation networks may more specific during embryogenesis while more extensive in metamorphosis by its target genes.For further explored and validated miRNAs function in metamorphosis and reproduction process in vivo,specific antagomirs of 5 stage-specific miRNAs(miR-6-3p,miR-9a-3p,miR-9d-3p,miR-11-3p,and miR-13a-3p)which targeted Met and Kr-hl were injected into late larvar beetles.In brief,knockdown of these miRNAs significantly increased its candidate target genes Met and Kr-h1 expression levels but obvious reduced Vg expression,resulted in ecdysis arrest and reproduction defects,closely correlated the beetls development Our study screened out and identified differential expressed miRNAs during embryo and metamorphosis,further validation its function in vivo,which demonstrated that these miRNAs directly or indirectly involved in T.castaneum metamorphosis regulation networks and provided foundation in deeper studies of miRNAs functional analysis in insect development.4.The miR-8 was insect-specific microRNA,and have been found involved in flies development and immunity at present studies,whether it is involved in other biological functions is unclear and unexplored in T.castaneum.Here,miR-8-3p relativie highly expressed in late larva and early adult stage,enriched in fat body of larvar and head,epidermis of adult.Specific inhibition of miR-8-3p into T.castaneum late larvar led to body size reduced,approximately 50%displayed metamorphosis defects in development of wings,eyes,legs,and embryo in T.castaneum.Moreover,wingless,Eyg,Fpps,and Sema-1a were screened out as miR-8-3p targets by computational prediction and miRNA-mRNA interaction validation.Luciferase analysis further confirmed that Eyg is the new functional target of miR-8-3p.In brief,miR-8-3p is broadly involved in wings,eyes,and legs’ development through those target genes,played the extensive regulation role in T.castaneum. |
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