ABSTRACT
Microprocessor, the complex of Drosha and DGCR8, promotes the processing of primary microRNA to precursor microRNA, which is a crucial step for microRNA maturation. So far, no convenient assay systems have been developed for observing this step in vivo. Here we report the establishment of highly sensitive cellular systems where we can visually monitor the function of Microprocessor. During a series of screening of transfectants with fusion genes of the EGFP cDNA and primary microRNA genes, we have obtained certain cell lines where introduction of siRNA against DGCR8 or Drosha strikingly augments GFP signals. In contrast, these cells have not responded to Dicer siRNA; thus they have a unique character that GFP signals should be negatively and specifically correlated to the action of Microprocessor among biogenesis of microRNA. These cell lines can be useful tools for real-time analysis of Microprocessor action in vivo and identifying its novel modulators.
Subject(s)
MicroRNAs/metabolism , NIH 3T3 Cells , Proteins/metabolism , Ribonuclease III/metabolism , Animals , Base Sequence , DEAD-box RNA Helicases/genetics , Endoribonucleases/genetics , Gene Fusion , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Humans , Mice , MicroRNAs/genetics , Molecular Sequence Data , Proteins/genetics , RNA-Binding Proteins , Ribonuclease III/genetics , TransfectionABSTRACT
To search for the intracellular signaling pathway critical for the pulmonary metastasis of osteosarcoma, we examined two osteosarcoma cell lines with different metastatic capability, Dunn and LM8. While parental Dunn is poorly metastatic to lung, LM8, derived from Dunn by in vivo selection through pulmonary metastasis, displays clear capability of pulmonary metastasis. We found that LM8 had higher levels of Akt phosphorylation and Ezrin expression than Dunn. In contrast, no clear difference was observed between Dunn and LM8 in other signaling mediators, including MAPK, SAPK and Stat3. In contrast to Dunn, LM8 secreted higher levels of matrix metalloproteinase-2 and -9, showed higher levels of invasiveness and cell motility, and displayed strong pulmonary metastasis. Inhibition of PI3K-Akt signaling in LM8 by a PI3K inhibitor, LY294002, or by a dominant negative form of Akt, resulted in suppression of MMP secretion, in vitro invasiveness, cell locomotion and in vivo pulmonary metastasis. In contrast, expression of an active form of Akt in Dunn substantially activated its MMP secretion, in vitro invasiveness, cell locomotion and in vivo pulmonary metastasis. Taken together, our results demonstrate, for the first time, an important role of Akt signaling in pulmonary metastasis of osteosarcoma.
