Supplementary Material for: Role of Aquaporin-5 in Gallbladder Carcinoma

<b><i>Background/Purpose:</i></b> Aquaporins (AQPs) are important in controlling bile formation. However, the exact role in human gallbladder carcinogenesis has not yet been defined. <b><i>Methods:</i></b> AQP-5-expressing gallbladder carcinoma (GBC) cell lines (NOZ) were transfected with anti-AQP-5 small interfering RNA (siRNA). Growth, migration, invasion assay, and drug susceptibility tests were performed. Next, microRNA (miRNA) expression was analyzed by miRNA oligo chip (3D-Gene®). AQP-5 and AQP-5-related miRNA target gene expressions were also analyzed using tissue microarray (TMA) in 44 GBC samples. <b><i>Results:</i></b> Treatment with AQP-5 siRNA decreased cell proliferation, migration, and invasion. On the other hand, those cells increased IC<sub>50</sub> of gemcitabine. By performing miRNA assays, miR-29b, -200a, and -21 were shown to be highly overexpressed in cells treated with AQP-5 siRNA NOZ. When focusing on miR-21, phosphatase and tensin homolog (PTEN) was found to be a target of miR-21. In the TMA, AQP-5/PTEN coexpression was significantly associated with the depth of invasion and MIB-1 index (p = 0.003, 0.010). Survival of patients with a high AQP-5/PTEN coexpression was longer than that of patients with a low coexpression (p = 0.003). <b><i>Conclusions:</i></b> Our result suggested that miR-21 and PTEN may contribute to the role of AQP-5 in GBC. AQP-5 and PTEN cascades are favorable biomarkers of GBC.