miR-133b induces chemoresistance of osteosarcoma cells to cisplatin treatment by promoting cell death, migration and invasion

Abstract

As an important chemotherapeutic agent for the treatment of osteosarcoma, the effectiveness of cisplatin is considered to be due to its unique properties, which allow it to penetrate the cell membrane and form various DNA-platinum adducts, resulting in genetic alterations or DNA damage. However, chemoresistance to cisplatin remains a major challenge for its use and chemotherapeutic effects. In the present study, an isogenic model of a cisplatin resistant osteosarcoma cell line, MG63-DDP, was generated from the original MG63 cell line. The expression level of microRNA (miR)-133b in the MG63-DDP cisplatin-resistant osteosarcoma cell line was analyzed by reverse transcription-quantitative polymerase chain reaction (PCR). Cisplatin-DNA adduct formation, cell death (carboxyfluorescein succinimidyl ester/propidium iodide staining) and clonogenic survival assays (crystal violet staining) were performed, comparing various cell types. The effect of miR-133b on migration (scratch wound assay) and invasion (Transwell assay) was also evaluated. Characterization studies have previously revealed an increased level of miR-133b in MG63-DDP cells compared with normal MG63 cells. Upregulation of miR-133b was associated with the accumulation of cisplatin-DNA adducts and an increase in cisplatin-induced cell death. Furthermore, increased miR-133b expression levels enhanced the migration and invasion of MG63 cells under cisplatin stress. Concordantly, in MG63-DDP cells the neutralization of miR-133b demonstrated opposite effects, as compared with the upregulation of miR-133b. To the best of our knowledge, the present study demonstrated for the first time that cisplatin-resistant MG63 cells exhibit an increased level of miR-133b expression. The endogenous expression level of miR-133b is sufficient for inducing cisplatin resistance, which suggests that miR-133b may be a biomarker for cisplatin resistance in osteosarcoma.

Keywords: MG63 cells; chemoresistance; cisplatin; microRNA-133b; osteosarcoma.

Figure 1.

Cisplatin-resistant MG63 cells demonstrate a higher clonogenic ability and proliferation, compared with non-resistant cells. (A) A clonogenic assay 14 days post-treatment and (B) an MTT proliferation assay were performed with MG63 and MG63-DDP cells. *P<0.05 and **P<0.01 vs. MG63. miR, microRNA; DDP, cisplatin.

Figure 2.

(A and B) miR-133b is significantly upregulated in MG63-DDP cells, compared with the original MG63 cells. *P<0.05. miR, microRNA; DDP, cisplatin.

Figure 3.

(A and B) miR-133b is sufficient and necessary for the induction of cisplatin resistance in MG63 cells. A clonogenic assay (14 days post-treatment) was performed to determine the clonogenic ability of MG63 cells transfected with scrambled miR-133b mimics (MG63/vector) or miR-133b mimics (MG63/miR-133b mimics), and MG63-DDP cells were transfected with scrambled anti-miR-133b mimics (MG63-DDP/vector) or anti-miR-133b mimics (MG63-DDP/anti-miR-133b mimics). *P<0.05, compared with MG63-DDP/vector; **P<0.01, compared with MG63-vector. miR, microRNA; DDP, cisplatin.

Figure 4.

miR-133b expression inhibits cisplatin-DNA adduct formation and cell death. (A) Cells were treated with cisplatin for 24 h, fixed and stained using the RC-18 antibody or (B and C) stained with carboxyfluorescein succinimidyl ester/propidium iodide for cytometry assay to determine the rate of cell death. *P<0.05 vs. (B) MG63 and MG63/vector, or (C) MG63-DDP and MG63-DDP/vector. miR, microRNA; DDP, cisplatin.

Figure 5.

miR-133b affects wound healing. A scratch was created using a 200 µl sterile pipette in 100% confluent cells. The effects of miR-133b on migration of (A) orignial MG63 cells or (B) MG63-DDP cells were detected separately. miR, microRNA; DDP, cisplatin.

Figure 6.

miR-133b promotes cell invasion in MG63 or MG63-DDP cells under cisplatin stress. The effects of miR-133b on invasion of (A) original MG63 cells or (B) DDP-MG63 cells were detected separately. miR, microRNA; DDP, cisplatin.