Supplementary Material for: Metformin-Derived Growth Inhibition in Renal Cell Carcinoma Depends on miR-21-Mediated <b><i>PTEN</i></b> Expression

<b><i>Purpose:</i></b> Metformin (MF) acts as a tumour-suppressor in renal cell carcinoma (RCC) by inhibiting the <i>AKT</i>/<i>mTOR</i> pathway via <i>AMPK </i>activation. Here, we explore the influence of miR-21 and its target gene <i>PTEN</i> on MF effects in CAKI-1 and CAKI-2 cells. <b><i>Methods:</i></b> Proliferation assays (MTS) and qRT-PCR after transient transfection with pre- and anti-miR-21 and MF treatment were conducted. <i>AMPK</i>-dependency was assessed via transfection of siAMPK. The expression of <i>PTEN</i>, <i>AKT</i> and miR-21 after transient pre-miR-21 transfection and MF treatment was analysed. <b><i>Results:</i></b> We demonstrate that CAKI-1 cells, which were found to be less sensitive towards MF, showed a significant higher miR-21 and lower PTEN expression than CAKI-2. This was confirmed in a primary RCC collective (n = 28): miR-21 and <i>PTEN </i>expression correlated negatively. MF treatment lowered miR-21 <i>AMPK</i>-dependently and increased <i>PTEN</i> expression in the cell lines. Ectopic miR-21 regulation modulated MF sensitivity. Western blot analysis showed that pre-miR-21 transfection and MF treatment regulated <i>PTEN</i> expression with impact on <i>pAKT</i> levels in the cells. <b><i>Conclusions:</i></b> We show that differing MF sensitivity in RCC cells is associated with and mediated through the regulation of miR-21/<i>PTEN </i>expression with an impact on subsequent <i>AKT</i> signalling. This provides imaginable clinical implications regarding MF therapy of RCC patients for the future.