Supplementary Material for: MicroRNAs Associated with Epithelial-Mesenchymal Transition Can Be Targeted to Inhibit Peritoneal Dissemination of Human Scirrhous Gastric Cancers

Objectives: Scirrhous gastric cancers grow rapidly, and frequently invade the peritoneum. Such peritoneal dissemination properties markedly reduce patient survival. Thus, an effective means for inhibiting peritoneal dissemination is urgently required. Methods: We previously established a cell line, HSC-58, from a scirrhous gastric cancer patient, and further successfully isolated a metastatic line, 58As9, in nude mice upon orthotopic inoculation. Using the lines, we examined the mechanism underlying peritoneal dissemination from the viewpoint of microRNA (miRNA) expression. Results: miRNA array and qRT-PCR analysis showed that the expressions of epithelial-mesenchymal transition (EMT)-associated miRNAs such as miR-200c and miR-141 were significantly low in 58As9. Using 58As9 with stably overexpressing miR-200c, miR-141, or both, together with a luciferase reporter assay, we found that miR-200c targeted zinc finger E-box-binding homeobox 1 (ZEB1) and miR-141 targeted ZEB2. The overexpressed lines reversed the EMT status from mesenchymal to epithelial in 58As9, and significantly reduced the invasion activity and peritoneal dissemination for a significant prolongation of survival in the orthotopic tumor models in nude mice. Conclusions: EMT-associated miRNAs such as miR-200c and miR-141 and their target genes ZEB1/ZEB2 have good potential for antiperitoneal dissemination therapy in patients with scirrhous gastric cancers.