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

Purpose: Metformin (MF) acts as a tumour-suppressor in renal cell carcinoma (RCC) by inhibiting the AKT/mTOR pathway via AMPK activation. Here, we explore the influence of miR-21 and its target gene PTEN on MF effects in CAKI-1 and CAKI-2 cells. Methods: Proliferation assays (MTS) and qRT-PCR after transient transfection with pre- and anti-miR-21 and MF treatment were conducted. AMPK-dependency was assessed via transfection of siAMPK. The expression of PTEN, AKT and miR-21 after transient pre-miR-21 transfection and MF treatment was analysed. Results: 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 PTEN expression correlated negatively. MF treatment lowered miR-21 AMPK-dependently and increased PTEN 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 PTEN expression with impact on pAKT levels in the cells. Conclusions: We show that differing MF sensitivity in RCC cells is associated with and mediated through the regulation of miR-21/PTEN expression with an impact on subsequent AKT signalling. This provides imaginable clinical implications regarding MF therapy of RCC patients for the future.