Evidence from the above-mentioned in vitro experiments indicates that miR-20a suppresses autophagy pathway, induces ROS and DNA damage response. To further define the clinical relevance of our findings, we examined miR-20a expression in normal mammary tissues (n=30) and breast cancer tissues (n=30) by in situ hybridization. Consistent with the TCGA miRNA-seq data set analysis, miR-20a expression was markedly higher in tumor tissues (Figures 6a and b). Immunohistochemical staining of consecutive sections of triple-negative breast cancer specimens revealed that tumor tissues expressed lower levels of BECN1, ATG16L1 and SQSTM1 than normal tissues (Figure 6c). We also performed immunoblotting to explore the expression of BECN1, ATG16L1 and SQSTM1 in tissues from triple-negative subtype. Compared with the normal mammary tissues, breast cancer tissues showed an obvious reduction in BECN1, ATG16L1 and SQSTM1 levels, whereas OPTN expression was higher in cancer tissues (Figure 6d).