MIR-99a and MIR-99b Modulate TGF-β Induced Epithelial to Mesenchymal Plasticity in Normal Murine Mammary Gland Cells

Epithelial to mesenchymal transition (EMT) is a key process during embryonic development and disease development and progression. During EMT, epithelial cells lose epithelial features and express mesenchymal cell markers, which correlate with increased cell migration and invasion. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that induces EMT in multiple cell types. The TGF-beta pathway is regulated by microRNAs (miRNAs), which are small non-coding RNAs regulating the translation of specific messenger RNAs. Herein, we identified mir-99a and mir-99b as two novel TGF-beta target miRNA genes, the expression of which increased during TGF-beta induced EMT of NMUMG cells. Mir-99a and mir-99b inhibition decreased TGF-beta activity by inhibiting SMAD3 phosphorylation, resulting in decreased migration and increased proliferation in response to TGF-beta. However, mir-99a and mir-99b inhibition was insufficient to block TGF-beta induced EMT of NMUMG cells. Mir-99a and mir-99b over-expression in epithelial NMUMG cells resulted in increased proliferation, migration and fibronectin expression, while E-cadherin and ZO-1 expression were negatively regulated. In conclusion, we identified mir-99a and mir-99b as two novel modulators of TGF-beta pathway that alter SMAD3 phosphorylation, in turn altering cell migration and adhesion of mesenchymal NMUMG cells. The effect of mir-99a and mir-99b over-expression on NMUMUG proliferation is dependent upon the epithelial or mesenchymal status of the cells. Our study suggests that mir-99a and mir-99b may function as modulators within a complex network of factors regulating TGF-beta induced breast epithelial to mesenchymal transition, as well as proliferation and migration of breast cancer cells, providing a possible target for future translationally oriented studies in this area.