Supplementary Material for: MicroRNA-224 Promotes Pancreatic Cancer Cell Proliferation and Migration by Targeting the TXNIP-Mediated HIF1α Pathway G.Zhu L.Zhou H.Liu Y.Shan X.Zhang 2018 <b><i>Background/Aims:</i></b> MicroRNAs (miRNAs) have been shown to participate in the development of pancreatic ductal adenocarcinoma (PDAC) by modulating multiple cellular processes. Increased miR-224 expression enhances proliferation and metastasis in human cancers. This study aimed to investigate the role of miR-224 and its underlying mechanism of action in PDAC. <b><i>Methods:</i></b> BrdU, MTT, and cell migration assays were performed to determine cell proliferation, viability, and migration, respectively. The binding sites of miR-224 were identified using a luciferase reporter system, whereas protein expression of target genes was determined by immunoblotting and immunofluorescence analyses. A BALB/c nude mouse xenograft model was used to evaluate the role of miR-224 <i>in vivo</i>. <b><i>Results:</i></b> We demonstrated that miR-224 expression was enhanced in PDAC cells and tissues, and was related to migration and proliferation. Noticeably, miR-224 overexpression promoted the proliferation, migration, and metastasis of Panc1 cells, while miR-224 inhibition had the reverse effect on PDAC cells. Moreover, we found that thioredoxin-interacting protein (TXNIP) is a target of miR-224. The results also indicated that miR-224 inversely regulated TXNIP by binding directly to its 3′-untranslated region, which resulted in the activation of hypoxia-inducible factor 1α (HIF1α). Further, either TXNIP re-expression or HIF1α depletion abolished the effects of miR-224 on the proliferation and migration of PDAC cells <i>in vitro</i> and <i>in vivo</i>. Regarding the relationship of TXNIP and HIF1α, we found that TXNIP mediated the nuclear export of HIF1α and its degradation by forming a complex with HIF1α. <b><i>Conclusion:</i></b> The miR-224-TXNIP-HIF1α axis may be useful in developing novel therapies for PDAC.