PMC

Effects of miR-21KD on miRNA expression and IFN-induced gene expression in B16 cells in vitro. B16 cells transduced with anti-miR-21 lentivirus (miR-21KD) or with empty vector (EV) were treated with IFN (1,000 units/ml), and total RNA assayed for miRNA expression (A) or IFN-induced gene expression by qPCR (n = 3) (B). Error bars, S.D. **, p < 0.01; ***, p < 0.001.

Characterization of IFN-induced miR-21 expression in B16 melanoma cells in vitro. B16 cells were treated with IFN at 1,000 units/ml for the indicated times (A) or at the indicated doses for 6 h (B), and miR-21 was quantified by qPCR. IFN-induced ISG15 induction is shown for comparison (n = 3). Error bars, S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Role of STAT3 in IFN-induced miRNA expression in B16 melanoma cells in vitro. B16 cells transduced with EV or F705-STAT3 were treated with IFN (1,000 units/ml) and cell lysates were prepared and immunoblotted as indicated (A), or after 6 h of IFN treatment the indicated miRNAs were quantified by qPCR (n = 3) (B). Error bars, S.D. **, p < 0.01. NS, not significant.

Decreased metastatic potential of miR-21KD B16 melanoma cells in vivo. Mice were injected into the tail vein with 10⁶ EV or miR-21KD B16 cells. A, bioluminescent imaging of mice injected with luciferin performed at day 16 (n = 8). B, Kaplan-Meier analysis of survival data (n = 8). C, mean body weight of mice at day 18 (n = 8). Error bars, S.D. *, p < 0.05; **, p < 0.01.

Effects of miR-21KD on B16 melanoma cell proliferation, apoptosis, migration, and protein expression in vitro. A–C, EV and miR-21KD B16 cells were treated with IFN (1,000 units/ml), and at daily intervals cell numbers were determined in a Coulter Counter (n = 3) (A), or after 48 h apoptosis was determined by cell death ELISA or by Annexin V-staining (n = 3) (B), or after 24 h cell migration was determined by transwell migration assays (n = 3) (C). D, EV and miR-21KD B16 cells were lysed, and the expression of key target proteins was determined by immunoblotting with anti-PTEN, PDCD4, BTG2, and SPRY2. Error bars, S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.