To investigate the role of miR-20a in tumorigenesis, we generated MDA-MB-231 cell lines stably expressing NC or miR-20a through the lentivirus expression system. Stable transfection of miR-20a markedly suppressed mRNA and protein expression of ATG16L1, SQSTM1 and BECN1 in MDA-MB-231 cells. miR-20a stable transfection also led to reduced LC3-II level and increased OPTN expression, indicating that miR-20a inhibits autophagic flux (Supplementary Figure S7). Nude mice injected with the miR-20a-expressing cells produced more tumors than the mice that were injected with the control cells (Figure 8b). Tumor volume was also significantly larger in mice that were injected with miR-20a-expressing cells (Figure 8c). These results suggest that miR-20a promotes tumor initiation and progression in vivo.