Figure 6

Negative regulation of miR-99a expression by the IGF1R signaling pathway. (A) Downregulation of miR-99a in response to IGF1 treatment. After serum starvation, cells were treated with vehicle, 10% FBS or 10 nM IGF1. (B) Immunoblot analysis revealed increased levels of IGF1R protein following IGF1-induced downregulation of miR-99a. After serum starvation, cells were treated with vehicle, 10% FBS or 10 nM IGF1. The protein levels were normalized against an internal control α-tubulin. Ratios were determined by dividing the normalized protein levels in OEC-M1 cells with different conditions to that in complete medium (CM). The means in the lower panel were measured by averaging the ratios from independent blots. (C) The PI3K inhibitor LY294002 and (D) MAPK kinase inhibitor PD98059 suppressed the IGF1-induced downregulation of miR-99a. After serum starvation, cells were treated with vehicle, 10 nM IGF1, or combination of LY294002/PD98059 and IGF1. Expression of miR-99a was normalized against an endogenous control U6. The relative expression of miR-99a was determined by normalizing the expression of miR-99a in OEC-M1 cells in different conditions to that in cells in complete medium (CM). Bar, SE; *p < 0.05; **p < 0.01; ***p < 0.001. (E) Proposed model of the relationship between the activation of the IGF1R signaling pathway and miR-99a expression. The activation of the IGF1R signaling pathway initiated the inhibitory signal leading to decreased miR-99a expression. Following the suppression of the IGF1R signaling pathway by inhibitors, or its silencing by the removal of stimulation, we observed increased miR-99a expression because of the removal of the inhibitory signal. This reciprocal regulation of miR-99a and IGF1R signaling augmented the activation of the IGF1R signaling pathway in response to IGF1 stimulation and accelerated its inactivation following the removal of stimulation.