Figure 2.

Inhibition of miR-491 Stimulates OS Metastasis and Chemoresistance In Vitro and In Vivo

(A) Inhibition of miR-491 stimulated OS cell invasion in both U2OS and Saos-2 cells. Cells were transfected with negative control oligonucleotides (NC) or antisense oligonucleotides of miR-491 (ASO miR-491). After 24 hr of transfection, cells were subjected to an invasion assay. (B) Inhibition of miR-491 reduced E-cadherin expression and increased MMP-9 expression in OS cells. Indicated cells were transfected with NC or ASO miR-491. After 72 hr of transfection, cells were subjected to western blot analysis. (C) Inhibition of miR-491 stimulates OS lung metastasis in vivo. 143B cells were transfected with miR-491-antisense oligonucleotides expressing plasmid or empty vector. After 48 hr of transfection, cells were injected into the intramedullary cavity of the tibia of 6-week-old nude mice (n = 5 per group). The mice were sacrificed 4 weeks after cell injection, and the lung surface nodules were counted under microscopy. (D) Inhibition of miR-491 suppressed CDDP-induced cell growth inhibition in both U2OS and Saos-2 cells. After 24 hr of transfection with the indicated oligonucleotides, cells were plated into a 96-well plate. 12 hr after seeding, cells were incubated with or without CDDP (10 μM) for 48 hr and then subjected to an MTT assay. (E) Inhibition of miR-491 attenuated CDDP-induced apoptosis in U2OS and Saos-2 cells. 24 after transfection with the indicated oligonucleotides, cells were plated into a six-well plate. After 12 hr of seeding, cells were incubated with or without CDDP (10 μM) for 24 hr and then subjected to a flow cytometry assay. (F) Inhibition of miR-491 promotes tumor growth and induces resistance to CDDP in MG63 xenograft models. Stably expressing miR-491-antisense cells were injected subcutaneously into nude mice (n = 8 per group). After the tumor size reached approximately 100 mm³, the mice were started on a treatment of either PBS or CDDP (10 mg/kg body weight). The mice were sacrificed after 3 weeks of CDDP treatment, and the tumor weight was measured. (G) Apoptotic cells were detected using a TUNEL assay in the indicated xenograft tumor samples. (H) Inhibition of miR-491 promotes cancer cell proliferation in MG63 xenograft models. Tumor tissues from the MG63 xenograft model were stained with Ki-67. *p < 0.05; **p < 0.01; ***p < 0.001.