The Study On Regulation By Bmo-miR-79 To Its Target Gene BmEm4

MicroRNAs(miRNA) are a kind of small non-coding RNAs, which play a very important role at post-transcriptional level in organism. MicroRNAs always complementary combine with the target squences in the 3’UTR of the mRNAs via the RISC compound, which can lead to the mRNA degradation or inhibit mRNA translating into the related proteins. One kind of miRNA always can regulate target genes with the same target sites; At the same time, one target gene may be regulated by different kinds of miRNAs. E(spl)m4 was a miRNA-targeted gene in Drosophila and existd in the end of Notch signaling pathway. MiRNA could participate in the pathway to regulate neural development of Drosophila by regulating the E(spl)m4. We have cloned a homologue of E(spl)m4 gene from Bombyx mori and named it BmEm4 and have got the polyclonal antibody of BmEm4 protein with high titer. According to the bioinformatics analysis, we have found that there were multiple potential binding sequences in the 3’UTR of the BmEm4, which could combine with mi RNAs(3 Brd box, 1 GY box, 2 K box and so on). In this work, we studied the regulation function of bmo-miR-79 to BmEm4, which could contribute to the study on influence of miRNAs upon Notch signaling pathway in Bombyx mori,.Based on achievement before, we futher carried out the dual-luciferase expriment to verify the target genes of bmo-miR-79. At first, we cloned the 3’UTR of BmEm4 with normal and Brd box-, K box-mutational sequences into the plasmid pIEx-1-Rluc-Luc(a dual luciferase plasmid bulit in the laboratory) by PCR. We co-tranfected these two recombinant vectors and the bmo-miR-79, Negative control which were synthesized in vitro to BmN cells. The dual luciferase reporter gene detection result showed that the fluorescent signal values of firefly luciferase catalytic substrates were reduced by co-transfection with recombinant vector of normal BmEm4 3’UTR sequence and bmo-miR-79 compared with negative control in the BmN cells. At the same time, the result of co-transfection with mutational recombinant vector and bmo-miR-79 had no significant difference compared with negative control. The result showed that BmEm4 was bmo-mi R-79-targeted gene and its sites were in the Brd box and K box of the 3’UTR.In order to futher verify the regulation function of bmo-miR-79 to BmEm4, we used different concentrations of bmo-miR-79 mimics to transfect BmN cells to let its content be up-regulated, and used the Western Blotting method to detect the BmEm4 expression spectrum at different times after transfection. The expression levels of BmEm4 were all down-regulated after transfection with different concentrations of bmo-miR-79 and the inhibition effect of high concentration of bmo-miR-79 was remarkable that the protein expression was nearly 50 % down-regulated after 72 h. In order to verify the regulation function of bmo-miR-79 to BmEm4 in different stages of Bombyx mori, we researched the expression spectrum of bmo-miR-79 and BmEm4. The result showed that the difference of expression level of BmEm4 and bmo-miR-79 was obvious in different stages of Bombyx mori. The translational level of BmEm4 was inhibit in different degree when compared with the transcriptional level and the order of inhibition degree from high to low was pupa, egg and moth. The result futher proved that bmo-miR-79 could regulate the expression of BmEm4 on post-transcriptional level in different stages of Bombyx mori, by which it could go deep into regulating silkworm Notch signaling pathway.Previous study has found that BmEm4 enriched in RNAs combined with BmAGO2, as a potential miRNA target gene. The EMSA experiment futher verified that bmo-miR-79 could combine with BmAGO2 which was bioactive. The result indicated that bmo-miR-79 might regulate BmEm4 on post-transcriptional level by combining BmAGO2 to form mi RISC. The result conformed the previous study that miRNA preformed the function of gene regulation based on the miRISC which constituted by AGO protein.BmEm4 is one of members of the family of Notch signaling pathways, and there is little research on Notch signaling pathways of Bombyx mori. This study is based on the lepidoptera insect model organism silkworm which is important agricultural economy insects in our country. It is of great significance for further research on many gene’s regulation mechanism by miRNAs in the process of growth and development of Bombyx mori.