Supplementary Material for: Metformin Regulates the miR-205/VEGFA Axis in Renal Cell Carcinoma Cells – Exploring a Clinical Synergism with Tyrosine Kinase Inhibitors

Introduction: Metformin (MF) intake could be associated with a favorable outcome in Sunitinib (SUT)- and Axitinib (AX)-treated clear cell renal cell carcinoma (ccRCC) patients. Functionally, MF induces miR-205, a microRNA serving as a tumor suppressor in several cancers. Methods: qRT-PCR, viability assays and Western blotting analyzed MF and SUT/AX effects in RCC4 and 786-O cells. A tetracycline-inducible overexpression model was used to study the role of miR-205 and its known target gene VEGFA. We analyzed miR-205 and VEGFA within a public and an in-house ccRCC cohort. HUVEC (Human Umbilical Vein Endothelial Cell) sprouting assays examined miR-205 effects on angiogenesis initiation. To determine the influence of the von Hippel-Lindau tumor suppressor (VHL), we examined VHLwt re-expressing RCC4 and 786-O cells. Results: Viability assays confirmed a sensitizing effect of MF towards SUT/AX in RCC4 and 786-O cells. Overexpression of miR-205 diminished VEGFA expression – as did treatment with MF. Tumor tissue displayed a downregulation of miR-205 and an upregulation of VEGFA. Accordingly, miR-205 caused less and shorter vessel sprouts in HUVEC assays. Finally, VHLwt expressing RCC4, and 786-O cells displayed higher miR-205 and lower VEGFA levels. Conclusion: Our results support the protective role of MF in ccRCC and offer functional insights into the clinical synergism with tyrosine kinase inhibitors (TKI).