miR-99b suppresses IGF-1R expression and contributes to inhibition of cell proliferation in human epidermal keratinocytes.

Condyloma acuminatum (CA) is a condition caused by the highly contagious human papillomavirus (HPV), characterized by warts that undergo abnormal cell proliferation. One of the important regulators of cell proliferation is microRNAs (miRNAs). In this study, we aimed to investigate the expression profile of miR-99b in HPV positive CA samples and normal skin. We found significantly lower miR-99b levels in CA samples than in normal skin. Therefore, we investigated the role of miR-99b in regulating the proliferation of primary cultured human epidermal keratinocytes, and found that forced expression of miR-99b inhibited proliferation and induced G1-phase arrest. Based on conserved sequences in 3'UTR for miR-99b binding, we identified the insulin-like growth factor-1 receptor (IGF-1R) gene as a direct target for miR-99b. Further, we confirmed the binding site for miR-99b in the IGF-1R 3'UTR by mutation using a luciferase reporter assay that showed decrease in luciferase activity in the presence of miR-99b in the construct with the wild-type 3'UTR, but not in the construct with the mutant 3'UTR. Moreover, miR-99b over-expression could down-regulate IGF-1R expression, and could repress the PI3K-AKT signaling pathway. Lastly, over-expression of IGF-1R reversed the inhibitory effect of miR-99b on keratinocyte proliferation. Taken together, our results suggest that IGF-1R levels may be modulated by miR-99b in CA: downregulation of miR-99b with concomitant upregulation of its target gene IGF-1R may over-induce the PI3K-AKT signaling pathway, leading to deregulated cell proliferation in CA.