Fig 1: miR-199a and miR-199b promote GC cell growth and migration via targeting Frizzled-6. (A) qRT-PCR of candidate target genes of miR-199a/b in SNU601-vec, SNU601-miR199a, and SNU601-miR199b cells. Relative values to those of SNU601-vec are presented. Expression levels were normalized to RPL32 mRNA levels. *P < 0.05, **P < 0.01; two-tailed Student’s t-test. (B) Luciferase reporter assay with 3′-UTRs of ABCC1, FZD6, and SLC9A8. 3′-UTRs containing putative miR-199a/b-binding sites (predicted using TargetScan) were cloned into a luciferase reporter vector (psiCHECK-2) and then co-transfected with the miR-199b mimic or control into 293T cells. Data are mean + SD (n = 3). P, two-tailed Student’s t-test. (C) Western blotting of Frizzled-6 in in SNU601-vec, SNU601-miR199a, and SNU601-miR199b cells. Actin was used as a loading control. Densitometric analysis of Western blot results (n = 3) is presented in the graph. (D) qRT-PCR of FZD6 in SNU601 transfected with non-targeting control (NTC) or FZD6 siRNAs (#1, #2, and #3). P, two-tailed Student’s t-test. (E) Transwell migration assay in SNU601 transfected with control (NTC) or FZD6 siRNAs. Cells (1 × 105/insert) were cultured in the upper wells for 24 h, and migrated cells were counted after staining with crystal violet. × 100 magnification. Data are mean + SD (n = 3). *P < 0.05, **P < 0.01; two-tailed Student’s t-test. (F) Cell growth analysis in SNU601 transfected with control (NTC) or FZD6 siRNA #1 (FZD6-KD). Cells were counted using an automated cell counter. Data are mean + SD (n = 3). P, two-way ANOVA. (G) qRT-PCR of FZD6 in SNU601-miR199a transfected with an empty vector (+ vec, pEGFP-N1) or murine Fzd6-expression vector (+ Fzd6). P, two-tailed Student’s t-test. (H) Cell growth analysis in SNU601-miR199a + vec and SNU601-miR199a + Fzd6. Cells were counted using an automated cell counter. Data are mean + SD (n = 3). P, two-way ANOVA. (I) Transwell migration assay in SNU601-miR199a + vec and SNU601-miR199a + Fzd6. Cells (1 × 105/insert) were cultured in the upper wells for 24 h, and migrated cells were counted after staining with crystal violet. × 100 magnification. Data are mean + SD (n = 3). P, two-tailed Student’s t-test. (J) Cell attachment assay with SNU601-miR199a + vec and SNU601-miR199a + Fzd6. Cells (3 × 105/well) were seeded on collagen-coated 24-well plates, and non-adherent cells were removed by washing with PBS after 30 min. Attached cells were measured by crystal violet staining. Data are mean + SD (n = 3). P, two-tailed Student’s t-test. (K, L) qRT-PCR of miR-199a, miR-199b (K), and FZD6 mRNA (L) in SNU601 cells transfected with miR-199a and miR-199b inhibitors (199a + b inhibitors) or NTC. Data are mean + SD (n = 3). P, two-tailed Student’s t-test. (M) Western blotting of Frizzled-6 in in SNU601 cells transfected with miR-199a + b inhibitors or NTC. Actin was used as a loading control. Densitometric analysis of Western blot results (n = 3) is presented in the graph. (N) Cell growth analysis in SNU601 cells transfected with miR-199a + b inhibitors or NTC. Cells were counted using an automated cell counter. Data are mean + SD (n = 3). P, two-way ANOVA. (O) Transwell migration assay in SNU601 cells transfected with miR-199a + b inhibitors or NTC. × 100 magnification. Data are mean + SD (n = 3). P, two-tailed Student’s t-test. (P) Cell attachment assay with SNU601 cells transfected with miR-199a + b inhibitors or NTC. Data are mean + SD (n = 3). P, two-tailed Student’s t-test.