Bmo-miR-275 down-regulates expression of Bombyx mori sericin gene 2 in vitro.

Ping Qian,Xin Wang,Tao Jiang,Xingjia Shen,Fei Song,Chen Chen,Erjun Ling

doi:10.1371/journal.pone.0190464

Abstract

We hypothesized that bmo-miR-275 has a potential regulatory function regarding the expression of sericin gene 2 (BmSer-2). First, we examined the expression of bmo-miR-275 and its target gene BmSer-2 in seven different tissues from 5th instar day-3 silkworm larvae. The results showed that they were both specifically expressed in the middle silk gland, implying that spatio-temporal conditions are required for bmo-miR-275 to regulate the expression of BmSer-2. To test this hypothesis, we constructed a pri-bmo-miR-275 expressing plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-275-SV40] and BmSer-2-3´UTR recombinant reporter plasmids pGL3.0 [A3-luc-Ser-2-3′ UTR-SV40]. Finally, BmN cells were harvested and luciferase activity was detected. Results showed that luciferase activity was reduced significantly (P<0.05) in BmN cells co-transfected with pcDNA3.0 [ie1-egfp-pri-bmo-miR-275-SV40] and pGL3.0 [A3-luc-Ser-2-3’UTR-SV40], suggesting that bmo-miR-275 can down-regulate the expression of BmSer-2 in vitro. Our results improve the understanding of the regulatory function of Bombyx mori miRNA on the expression of genes regulating silk formation.

Highlights

IntroductionMicroRNAs (miRNAs) are a class of endogenous noncoding regulatory RNAs with a length of 19–22 nucleotides
MicroRNAs are a class of endogenous noncoding regulatory RNAs with a length of 19–22 nucleotides
We used bioinformatics analysis and found that BmSer-2 mRNA is a target of bmo-miR-275

Summary

Introduction

MicroRNAs (miRNAs) are a class of endogenous noncoding regulatory RNAs with a length of 19–22 nucleotides. Mature miRNAs are sheared from longer primary transcripts through a series of nuclease cleavage and modification processes and are assembled into RNAinduced silencing complexes. Mature miRNAs recognize target mRNAs through complimentary base-pairing and regulate silencing complexes according to the degree of complementarity with the target mRNA to degrade target mRNA or repress translation[1,2,3,4]. MiRNAs are mostly found in clusters in the genome[5], and most sequences are located in intergenic regions, indicating that the transcription of miRNA is independent of other gene transcription and that these species have their own mechanisms for transcriptional regulation. Studies have shown that animal and plant miRNAs are located in the 30 and 50 untranslated regions (UTRs) of the target mRNA[6,7,8].

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Conclusion