Regulation Of MiR-2805 And MiR-0031-3p On The Expression Of Fibroin Light Chain Gene BmFib-L From Bombyx Mori

miRNAs are a class of endogenous functional small RNAs that are involved in cell differentiation,developmental morphologsis,nervous system development,muscle development,and maintenance of cell survival in animals,mainly by degrading target gene mRNAs or inhibiting translation at the post-transcriptional level.The expression of silk fibroin genes of Bombyx mori not only need the synergistic participation of transcription factors,but also closely relate to the regulation of miRNAs at the post-transcriptional level.Although there are many reports on regulation of silkworm protein expression,the mechanism of silk protein synthesis regulation is remained unclear.Study the regulation of miRNAs on silkworm protein expression is helpful to understand the function of miRNAs and provides new experimental data for elucidating the molecular regulation mechanism of silk protein synthesis.In this study,the silk fibroin light chain gene(BmFib-L)was used as the target gene,and two candidate miRNAs,namely miR-2805 and miR-0031-3p,were obtained by bioinformatics analysis.On the basis of semi-quantitative RT-PCR for expression level analysis,recombinant expression vectors for target gene and miRNAs were constructed respectively,and the Dual-Luciferase? Reporter Assay System and qRT-PCR method were used for analysis the regulation of miRNAs on the target gene at cell,tissue and individuall level.And the main results are as follows.1.Two candidate miRNAs were predicted which have poteneial regulation function on BmFib-L expressionB.mori miRNAs were downloaded from miRBase and 35 novel bmo-miRNAs that may be involved in silk protein regulation were obtained from previous high-throughput sequencing.Two candidates bmo-miR-2805 and bmo-miR-0031-3p were predicted with the RNAhybrid Software by using BmFib-L mRNA 3?-untranslated region(3?UTR)as the target based on pairing level of No.2 to 8 base of miRNA 5'-end to the target site and setting folding free energy <-20.0 kcal/mol.These candidates have potential regulatory effects on BmFib-L,and used for subsequent validation.2.A method was established for culture and gene transient expression analysis of silk glandThe healthy 5th instar larvae of silkworm were obtained,and the silk gland tissues were dissected and placed in a 12-well cell culture plate containing 800 ?L of TC-100 media,4silk glands per well,cultured at 27?.The constructed expression vector pcDNA3.0[ie1-egfp-SV40]containing an enhanced green fluorescent protein(eGFP)was transfected into silk gland tissues with EntransterTM-H4000 kit,and observed with a fluorescence microscope 48 h post transfection.The medium was clear and the silk gland tissues were intact and emitted.Bright greenfluorescence indicates that EGFP is expressed in the silk glands,indicating the method is feasible to perform transient expression of genes in vitro cultured silk gland tissue,which provides a new technical approach for study the molecular mechanism of silk protein gene expression regulation and a reference for culture and research of other silkworm tissues as well.3.miR-2805 up-regulates the expression of BmFib-L in tissue and individual levelThe sequences of pre-miR-2805 and BmFib-L 3'UTR were downloaded from NCBI,a miR-2805 expression plasmid pcDNA3.0[ie1-egfp-pre-miR-2805-SV40] and a BmFib-L 3'UTR luciferase report plasmid pGL3.0[A3-luc-Fib-L-3'UTR-SV40] were constructed,respectively.BmN cells(delivered from ovary of B.mori)were co-transfected with the above mentioned plasmids,and pRL-CMV(contains a renilla luciferase gene)was served as an intrinsic plasmid to validate the regulatory function of miR-2805 on BmFib-L by assay of dual luciferase activities,as well as artificially synthesized mimics and inhibitors of miR-2805.The results revealed that miR-2805 significantly down-regulated the expression of BmFib-L in BmN cells.To validate the regulatory function of miR-2805 in vivo,cultured silk glands isolated from day-2 5th instar larvae,more over day-2 5th instar larvae were injected with a transfection solution containing pcDNA3.0[ie1-egfp-SV40],pcDNA3.0[ie1-egfp-pre-miR-2805-SV40],mimic and inhibitor respectively.BmFib-L expression was analyzed by quantitative RT-PCR using total RNAs extracted from silk glands.The results showed that miR-2805 significantly up-regulated the expression of BmFib-L both in cultured silk glands and transfected individuals.4.miR-2805 up-regulates expression of BmFib-L by BmAwh and / or BmdimmTo find out the resean that miR-2805 differentially regulates BmFib-L expression in BmN cells and tissues or individuals,we designed primers of transcription factors(TFs)including BmSGF-1,BmSGF-3,BmMBF1,BmFMBP-1,BmAwh and Bmdimm,and analyzed the expression level of the TFs by using above RNAs as templates extracted from injected individuals,and A3 was an internal reference.The expression levels of BmAwh and Bmdimm were increased significantly in the larvae injected with miR-2805 expression vector pcDNA3.0[ie1-egfp-pre-miR-2805-SV40] and mimic,respectively.In contrast,inhibitor of miR-2805 suppressed the expression of BmAwh and Bmdimm.And bioinformatics analysis also showed miR-2805 could bind to the CDS region of BmAwh and the 5' UTR of Bmdimm.Thus we deduce that miR-2805 up-regulate the expression of BmFib-L by BmAwh and / or Bmdimm in vivo.5.miR-0031-3p down-regulates the expression of BmFib-LThe same method was used to construct the expression vector of miR-0031-3p and the mimic,mimc negative,inhibitor,inhibitor negative of miR-0031-3p were synthesized.Overexpression and inhibition of endogenous expression at the cellular level were designed andthe results showed that miR-0031-3p inhibited BmFib-L gene expression.At the same time,the inhibition function was verified at the tissue level,and the inhibitor and inhibitor negative control of miR-0031-3p were transfected into the cultured wells with silk gland tissue,and the expression level of target gene BmFib-L was detected by real-time PCR.The results showed that inhibitor inhibits the expression of miR-0031-3p and promotes the expression of BmFib-L,which means that miR-0031-3p can down-regulate the expression of BmFib-L.A method of verifying the injection of day-2 5th instar larvae was performed.The results again indicate that miR-0031-3p binds to BmFib-L 3'UTR to inhibit gene expression.The results are beneficial to understand the molecular mechanism of miRNAs and provide new experimental data for clarifying the regulatory mechanism of B.mori silk protein biosynthesis.