Targeting miR-29 induces apoptosis of osteosarcoma MG-63 cells via regulation of TGF-β1/PUMA signal

OBJECTIVE: Recent studies have shown that high miR-29 expression was associated with poor prognosis in patients with osteosarcoma (OS). However, the exact role and mechanisms of miR-29 in human OS remains speculative. Here, we identify a connection between miR-29 and TGF-beta 1/PUMA signaling in this context. PATIENTS AND METHODS: MG-63 cells were treated with anti-miR-29 for 48 h. Cell growth and apoptosis in vitro were detected by MTT, colony formation and flow cytometry assay. The effect of the miR-29 inhibitor on the growth of MG-63 cells was also evaluated in a MG-63 mouse model. Human recombinant TGF-beta 1 (rh TGF-beta 1) and PUMA siRNA transfection were used to assess the signal pathway. miR-29, TGF-beta 1, PUMA, and caspase-3 protein expression were detected by Western blotting assay and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays. RESULTS: Knockdown of miR-29 resulted in 80% decrease of miR-29 compared to the negative control. Knockdown of miR-29 significantly downregulated TGF-beta 1 and upregulated PUMA expression, and decreased MG-63 cell growth by 70%, impaired colony formation by approximately 80%, and increased MG-63 cell apoptosis by 40%. Knockdown of PUMA reversed miR29 silencing-induced proliferation and apoptosis of MG-63 cells. Restoration of TGF-beta 1 decreased PUMA expression. In murine engraftment models of MG-63, we showed that knockdown of miR-29 was able to reduce tumor growth. This was accompanied by decreased levels of TGF-beta 1 and increased levels of PUMA in these tumors. CONCLUSIONS: Targeting miR-29 exhibits significant in vivo and in vitro anti-tumor activities in OS through a novel mechanism resulting in inhibition of TGF-beta 1 expression and inducing PUMA expression.