Regulation Of MiR-2845 And MiR-3385-3p On The Expression Of Fibroin Light Chain Gene From Bombyx Mori

The silkworm Bombyx mori is an important economic insect with important economic value,and a lepidoptera model organism.Its silk glands have the strong ability to synthesize silk proteins efficiently and specifically.Micro RNAs(miRNAs)are a class of endogenous non-coding small RNAs in a length of 19-22 nts and play important regulatory roles in organism.Researches have shown that B.mori miRNAs(bmo-miRNAs)involve in regulation of expression of silk protein genes.Although there are many reports about regulation of bmo-miRNAs on silk protein synthesis,its regulatory mechanism needs further study.It is of great improtance to study the fine regulation effect of miRNAs on the expression of silk fibroin light chain gene(BmFib-L)in the silkworm.The BmFib-L was used as the target gene in this study,two candidate miRNAs,bmo-miR-2845 and bmo-miR-3385-3p,were screened out through bioinformatics prediction analysis.Fluorescence quantitative PCR(q RT-PCR)analysis showed that the two candidate miRNAs and BmFib-L were expressed at high levels in the posterior silk glands(PSG)of the 5th instar larvae,suggesting the possibility of regulation between them.To verify the regulation effect of the two miRNAs on BmFib-L,recombinant expression vectors of two miRNAs and target gene-reporter gene were constructed respectively.Mimics and inhibitors of two candidate miRNAs were synthesized artificially,and the regulatory effects of two candidate miRNAs on target gene BmFib-L were verified in Bm N cells,in vitro cultured silk gland tissues of 5th instar larval and in individuals respectively.The main results achieved were as follows.1.Two candidate miRNAs with potential regulation on BmFib-L were screened out by bioinformaticsThe 3? UTR sequence of BmFib-L was obtained from Silkworm Genome Database in NCBI.The mature sequence of miRNAs were obtained from previous sequencing results of small RNA in our laboratory and miRBase database.Based on the pairing level of the 2nd to 8th bases of the 5' end seed sequence and the folding free energy <-20.0 kcal/mol,two candidate bmo-miRNAs,bmo-miR-2845 and bmo-miR-3385-3p with potential regulatory effect on BmFib?L were screened out by the RNAhybrid Software online.The q RT-PCR method was employed to analyze the expression levels of miRNAs and BmFib-L in PSG of 5th instar larvae from day-1 to day-7 and the bmo-miRNAs in different tissues of the 5th instar day-3 larvae of B.mori.Results showed that miR-2845 and miR-3385-3p were higher expresed in the PSG,indicating that in the PSG,there is the spatio-temporal condition for these two candidate miRNAs to regulate BmFib-L.2.Successfully constructed the recombinant expression vectors of bmo-miRNAs and its target gene-reporter genePre-miR-2845 and pre-miR-3385-3p were cloned and connected to p MD19-T vector,cloned into pc DNA3.0 [ie1-egfp-SV40] vector,respectively after double digestion of Bam HI and Hind? to construct pc DNA3.0[ie1-egfp-pre-miR-2845-SV40] and pc DNA3.0[ie1-egfp-pre-miR-3385-3p-SV40]expression vectors.Similarly,BmFib-L 3? UTR was cloned into p GL3.0[A3-luc-SV40]vector after Xba? and Fse?digestion,and the recombinant expression vector p GL3.0[A3-luc-BmFib-L-3? UTR-SV40] was constructed.3.miR-2845 and miR-3385-3p significantly inhibit the expression of BmFib-L gene in Bm N cellsThe recombinant expression vector ware co-transfected into Bm N cells with p RL-CMV as the internal,and the regulation function of miRNAs on BmFib-L ware detected by double luciferase assay.Then mimic and inhibitor of miRNAs were artificially synthesized and their regulation functions on BmFib-L were analyzed by over expression and inhibitory expression.The results showed that miR-2845 and miR-3385-3p repress the expression of BmFib-L in Bm N cells.Then the target sites of miRNAs on the target gene BmFib-L were mutated,and the corresponding mutant vectors were constructed and co-transfected into Bm N cells as described above.The results showed that miRNAs had no inhibitory effect on BmFib-L after the mutation of the target sites,demonstrating the target sites predicted by bioinformatics were all correct.4.miR-2845 and miR-3385-3p significantly inhibit the expression of BmFib-L in vivoTo verify the regulatory function of miRNAs on BmFib-L in vivo,the silkworm larvae were dissected on the day-2 of the 5th instar to obtain the complete silk gland tissues.According to the same process as co-transfection in Bm N cells,the experimental group pc DNA3.0 [ie1-egfp-pre-miRNA-SV40],mimic,inhibitor and their corresponding control group pc DNA3.0 [ie1-egfp-SV40],mimic NC,inhibitor NC were co-transfected into the cultured silk gland tissue in vitro.In the same way,corresponding expression vectors incubated with transfection reagents were injected into the coelom of the larvae on day-2 of the 5th instar.Silk gland tissues were collected after 48 h,total RNA of different treatment groups was extracted,and the expression of BmFib-L after different treatment groups was detected by q RT-PCR,to measure the regulation of miRNAs and mimic,inhibitor on BmFib-L at individual level.The results showed that miR-2845 and miR-3385-3p significantly down regulate the expression of BmFib-L in cultured silk glands and in larva.The results of this study are beneficial for understanding the regulation function of miRNA,and provide new experimental data for elucidating the molecular mechanism of silk protein expression regulation and improving the molecular regulatory network.