Supplementary Material for: The Clinicopathological Significance and Correlative Signaling Pathways of an Autophagy-Related Gene, Ambra1, in Breast Cancer: a Study of 25 Microarray RNA-Seq Datasets and in-House Gene Silencing

<b><i>Background/Aims:</i></b> The activating molecule in Beclin1-regulated autophagy (Ambra1) has been observed to be over-expressed in several cancers, but the clinical contribution of Ambra1 in breast cancer (BC) remains unknown. Hence, in this study, we conducted a comprehensive investigation into the expression, biological role, and underlying functional mechanism of Ambra1 in BC. <b><i>Methods:</i></b> Microarray and RNA-seq datasets providing Ambra1 expression data were obtained from Gene Expression Omnibus (GEO), ArrayExpress, Oncomine, and The Cancer Genome Atlas (TCGA). Both standard mean deviation (SMD) and summary receiver operating characteristic methods were employed to assess Ambra1 expression in BC. We then silenced Ambra1 in MDA-MB-231 cells and performed <i>in vitro</i> experiments to explore the biological effects of Ambra1 on BC cells. Furthermore, differentially expressed genes (DEGs) after Ambra1 knock-down were profiled with a microarray and overlapped with the genes correlated with Ambra1 from Multi Experiment Matrix (MEM) and genes similar to Ambra1 from Gene Expression Profiling Interactive Analysis. These overlapping genes were collected for further bioinformatics analyses to investigate the underlying molecular mechanism of Ambra1 in BC. <b><i>Results:</i></b> A total of 25 microarray and RNA-seq datasets involving 2460 breast cancer samples were included. The pooled results demonstrated that Ambra1 was markedly up-regulated in BC tissues (SMD=0.39, 95% CI=0.15–0.63; P=0.002), and the Ambra1 level was also significantly related to the progression of BC, especially metastasis status (P=0.004). <i>In vitro</i> experiments suggested that the proliferation of MDA-MB-231 cells transfected with Ambra1 short hairpin RNA (sh-RNA 2450) showed a decreasing trend at 48 h compared with the control (CK) group. However, apoptosis was similar in cells transfected with Ambra1 sh-RNAs and in the CK cells. Furthermore, we performed a microarray-based comparison of genes after Ambra1 knock-down. The 828 DEGs from microarray analysis were intersected with 4266 Ambra1 co-expressed genes from MEM. Eventually, the overlapped 183 genes were found to be enriched in several well-known cancer-related pathways, including the MAPK signaling pathway, chronic myeloid leukemia pathway, and VEGF signaling pathway. <b><i>Conclusion:</i></b> These results indicate that the level of Ambra1 up-regulation is clearly related to tumorigenesis and progression of BC, probably via influencing several vital pathways. However, this hypothesis needs to be validated with more in-depth experiments in the future.