Bom-miR-2805 upregulates the expression of Bombyx mori fibroin light chain gene in vivo.

ABSTRACT

MicroRNAs (miRs) are inner regulatory RNAs mainly by regulating expression of genes at the posttranscriptional level. To investigate the regulatory function of Bombyx mori (B. mori) fibroin protein genes, the mRNA 3'-untranslated region (3'-UTR) of fibroin light chain gene (BmFib-L) was used as the target and one miRNA, miR-2805 was predicted by using the Software. miR-2805 expression plasmid pcDNA3.0[ie1-egfp-pre-miR-2805-SV40] and BmFib-L 3'-UTR plasmid pGL3.0[A3-luc-Fib-L-3'-UTR-SV40] were constructed, respectively. The mentioned plasmids were cotransfected in BmN cells, and the regulatory function of miR-2805 on BmFib-L was detected by assay of dual luciferase activities, as well as synthesized mimic and inhibitor of miR-2805. The results revealed that miR-2805 significantly downregulated the expression of BmFib-L in BmN cells. To validate the function of miR-2805 in vivo, cultured silk glands or larvae were injected with solution containing pcDNA3.0[ie1-egfp-SV40], pcDNA3.0[ie1-egfp-pre-miR-2805-SV40], mimic, inhibitor respectively. BmFib-L expression was analyzed by quantitative reverse transcription polymerase chain reaction using total RNAs extracted from silk glands. The results showed that miR-2805 significantly upregulated the expression of BmFib-L in both cultured tissues and individuals. To find out how miR-2805 differentially regulates BmFib-L expression in cells and tissues or individuals, we analyzed the expression level of transcription factors (TFs) involved in expression of silk protein genes. The results showed that miR-2805 upregulated the expression of TFs BmAwh and Bmdimm. These results suggest that miR-2805 may up-regulate the expression of BmFib-L interaction with BmAwh and/or Bmdimm in vivo. These findings are beneficial to clarify the molecular mechanism of miRNAs in regulating B. mori silk protein biosynthesis.