miR-29 Family Inhibits Resistance to Methotrexate and Promotes Cell Apoptosis by Targeting COL3A1 and MCL1 in Osteosarcoma
Wei Xu, Zhikun Li, Xiaodong Zhu, Ruijun Xu, Youjia Xu
(Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China (mainland))
Med Sci Monit 2018; 24:8812-8821
MicroRNAs (miRNAs) play a crucial role in regulating diverse biological processes, including drug resistance. We investigated the potential roles of the miR-29 family in methotrexate (MTX) resistance in osteosarcoma.
MATERIAL AND METHODS: Two MTX-resistant osteosarcoma cell lines, MG-63/MTX and U2OS/MTX, were generated by continuous exposure to stepwise increasing concentrations of MTX. miR-29abc, COL3A1, and MCL1 mRNA expression levels were determined using quantitative real-time PCR (qRT-PCR). Protein expression levels of COL3A1 and MCL1 were detected by Western blot. Cell viability, IC50 value, and cell apoptosis were assessed by CCK-8 assay and flow cytometry, respectively. The target relationship between the miR-29 family and COL3A1 or MCL1 was confirmed by luciferase reporter assay.
RESULTS: miR-29a, miR-29b, and miR-29c were significantly downregulated in MG-63/MTX and U2OS/MTX cells and in chemotherapy poor-response osteosarcoma tissues. Overexpression of the miR-29 family sensitized MG-63/MTX and U2OS/MTX cells to MTX and obviously promoted cell apoptosis compared with negative control. COL3A1 and MCL1 were identified to be target genes of the miR-29 family, and transfection with miR-29abc mimics in MG-63/MTX and U2OS/MTX cells decreased COL3A1 and MCL1 mRNA and protein expression. Meanwhile, overexpression of COL3A1 and MCL1 partly neutralized the effects of the miR-29 family on MTX resistance and cell apoptosis.
CONCLUSIONS: Taken together, our findings suggested a tumor-suppressor role of the miR-29 family in control of MTX resistance and cell apoptosis through regulating COL3A1 or MCL1. Targeting the miR-29 family might provide new strategies to overcome the high-dosage MTX-induced cytotoxicity in osteosarcoma treatment.
Keywords: Methotrexate, MicroRNAs, Osteosarcoma