Supplementary Material for: DNA Methylation-Mediated Lowly Expressed AOX1 Promotes Cell Migration and Invasion of Prostate Cancer

<b><i>Introduction:</i></b> DNA methylation regulates gene transcriptional functions in the pathogenesis of malignant diseases. In prostate cancer, several tumor suppressors are known to be tumor specifically methylated. <b><i>Methods:</i></b> In this study, 450K methylation data and mRNA expression data were accessed from The Cancer Genome Atlas-Prostate Adenocarcinoma database and analyzed bioinformatically. Methylation-specific PCR was used to examine the methylation condition in AOX1 promoter. qRT-PCR was applied to measure the mRNA expression of AOX1. Western blot was employed to detect the expressions of AOX1 and the EMT associated proteins. Transwell and scratch healing assays were used to examine the invasive and migratory abilities of the prostate cancer cells respectively. <b><i>Results:</i></b> AOX1 was lowly expressed and hypermethylated in the prostate cancer tissues and cells. Also, AOX1 was downregulated at protein level in prostate cancer cells. Knocking down AOX1 could promote cell migration and invasion in the prostate cancer cells. By using a DNA methylation inhibitor, 5-AzadC was found to promote the expression of AOX1 and reverse the promoting effects of short interfering RNA against AOX1 on cell migration and invasion. <b><i>Conclusion:</i></b> This study suggested that DNA methylation and low AOX1 level might be biomarkers for prostate cancer.

**引言**：DNA甲基化在恶性疾病的发病机制中调控基因的转录功能。在前列腺癌中，已有研究证实多种抑癌基因会发生肿瘤特异性甲基化。
**方法**：本研究从癌症基因组图谱-前列腺腺癌（The Cancer Genome Atlas-Prostate Adenocarcinoma）数据库获取450K甲基化芯片数据与mRNA表达谱数据，并进行生物信息学分析。采用甲基化特异性PCR（methylation-specific PCR）检测AOX1启动子区域的甲基化状态；通过实时定量逆转录PCR（quantitative real-time PCR, qRT-PCR）测定AOX1的mRNA表达水平；利用蛋白质印迹（Western blot）检测AOX1及上皮间质转化（Epithelial-Mesenchymal Transition, EMT）相关蛋白的表达情况；分别采用Transwell小室实验与划痕愈合实验检测前列腺癌细胞的侵袭与迁移能力。
**结果**：AOX1在前列腺癌组织与细胞中呈低表达且高甲基化状态，同时其蛋白水平在前列腺癌细胞中亦出现下调。敲低AOX1可增强前列腺癌细胞的迁移与侵袭能力。使用DNA甲基化抑制剂5-氮杂-2'-脱氧胞苷（5-AzadC）后，可上调AOX1的表达，并逆转小干扰RNA（short interfering RNA, siRNA）靶向AOX1所介导的细胞迁移与侵袭促进效应。
**结论**：本研究提示，DNA甲基化与AOX1低表达可作为前列腺癌的潜在生物标志物。