MicroRNA-129-5p regulates proliferation and apoptosis of osteosarcoma cells by down-regulating PAK5 expression / 肿瘤

ABSTRACT

Objective: To investigate the effects of microRNA-129-5p (miR-129-5p) on the proliferation and apoptosis of osteosarcoma cells, and to explore the possible molecular mechanism. Methods: The expression of miR-129-5p in osteosarcoma MG63 cells and osteoblasts hFOB1.19 cells was determined by real-time fluorescent quantitative PCR (RFQ-PCR). After miR-129-5p mimics were transfected into osteosarcoma MG63 cells, the expression of miR-129-5p was detected by RFQ-PCR to verify the transfection efficiency. CCK-8 assay and plate clony formation experiment were used to detect the cell proliferative ability, while FCM was performed to analyze the cell apoptosis. Double luciferase reporter gene system was used to verify the interaction between miR-129-5p and the target gene p21-activated kinase 5 (PAK 5). The expressions of PAK5 mRNA and protein in MG63 cells with miR-129-5p overexpression were tested by RFQ-PCR and Western blotting, while the expression levels of phospho-glycogen synthase kinase-3β (p-GSK3β) and β-catenin were detected by Western blotting. After siRNA-PAK5, PAK5 overexpressed plasmids and miR-129-5p mimics＋ PAK5 overexpressed plasmids were separately transfected into osteosarcoma MG63 cells, the above methods were used to further verify the molecular mechanism of miR-129-5p regulating the proliferation and apoptosis of osteosarcoma cells. Results: The level of miR-129-5p in MG63 cells was lower than that in hFOB1.19 cells (P < 0.05). After the transfection with miR-129-5p mimics, the proliferation and clony formation abilities of MG63 cells were decreased (P < 0.001, P < 0.01), while the apoptosis rate was increased (P < 0.05). miR-129-5p regulated negatively the expression of PAK 5 gene (P < 0.05). After the transfection with miR-129-5p mimics or siRNA-PCK5, the expression levels of PAK5 mRNA and protein were decreased (all P < 0.01), while the expressions of p-GSK-3β and β-catenin proteins were also decreased (both P < 0.05, both P < 0.01). The overexpression of PAK5 could restore the effects of miR-129-5p on proliferation and apoptosis of osteosarcoma cells (both P < 0.05). Conclusion: miR-129-5p can inhibit the proliferation ability of osteosarcoma cells, and promote apoptosis by down-regulating the expression of PAK 5 gene. The mechanism may be associated with the expression regulation of p-GSK-3β and β-catenin proteins.