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

Background/Aims: 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. Methods: 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 in vitro 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. Results: 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). In vitro 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. Conclusion: 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.

背景与研究目的：已有研究发现，Beclin1调控自噬中的激活分子（Ambra1）在多种癌症中呈过表达状态，但Ambra1在乳腺癌（BC）中的临床作用仍不明确。因此，本研究全面探讨了Ambra1在乳腺癌中的表达情况、生物学功能及潜在作用机制。

方法：从基因表达综合数据库（Gene Expression Omnibus, GEO）、阵列表达数据库（ArrayExpress）、Oncomine数据库及癌症基因组图谱（The Cancer Genome Atlas, TCGA）中获取包含Ambra1表达数据的基因芯片与RNA测序数据集。采用标准化均数差（standard mean deviation, SMD）与综合受试者工作特征曲线两种方法评估Ambra1在乳腺癌中的表达情况。随后在MDA-MB-231细胞中沉默Ambra1基因，并开展体外实验以探究Ambra1对乳腺癌细胞的生物学作用。此外，通过基因芯片分析了Ambra1基因敲低后的差异表达基因（differentially expressed genes, DEGs），并将其与多实验矩阵数据库（Multi Experiment Matrix, MEM）中与Ambra1相关的基因、基因表达谱交互分析数据库中与Ambra1功能相似的基因取交集。对获得的交集基因进行进一步生物信息学分析，以阐明Ambra1在乳腺癌中的潜在分子机制。

结果：共纳入25项基因芯片与RNA测序数据集，涉及2460例乳腺癌样本。合并分析结果显示，Ambra1在乳腺癌组织中显著上调（SMD=0.39，95%置信区间CI=0.15~0.63；P=0.002），且Ambra1表达水平与乳腺癌进展密切相关，尤其是与转移状态显著相关（P=0.004）。体外实验结果表明，与对照组（CK）相比，转染Ambra1短发夹RNA（short hairpin RNA, sh-RNA 2450）的MDA-MB-231细胞在48h时的增殖能力呈下降趋势，但Ambra1 sh-RNA转染组细胞与对照组细胞的凋亡水平无明显差异。此外，本研究通过基因芯片分析了Ambra1敲低后的基因表达差异，将基因芯片分析得到的828个DEGs与MEM数据库中4266个Ambra1共表达基因取交集，最终获得183个重叠基因。富集分析显示，这些基因显著富集于多个经典癌症相关通路，包括MAPK信号通路、慢性髓系白血病通路及VEGF信号通路。

结论：上述结果表明，Ambra1表达上调与乳腺癌的发生及进展密切相关，其潜在机制可能与调控多条关键信号通路有关。但该假说仍需未来开展更深入的实验加以验证。