MicroRNA-199a-5p inhibits cisplatin-induced drug resistance via inhibition of autophagy in osteosarcoma cells

Abstract

Osteosarcoma (OS) is the most common cancer of the bone. Chemotherapy is commonly used for the clinical treatment of OS. However, chemoresistance to cisplatin [also known as diamminedichloridoplatinum (II) (DDP)] is a major obstacle for OS therapy, the underlying mechanism of which is not fully understood. The present study aimed to investigate the role of microRNA (miR)-199a-5p in the regulation of chemoresistance to DDP in OS cells. Reverse transcription-quantitative polymerase chain reaction demonstrated that the expression level of miR-199a-5p was significantly reduced in human OS MG63 cells. In addition, DDP treatment also upregulated the protein levels of light chain 3 (LC3)-II and Beclin1 as well as the ratio of LC3-II vs. LC3-I in MG63 cells, indicating that autophagy was activated. Restoration of miR-199a-5p expression promoted DDP-induced inhibition of MG63 cell proliferation and inhibited DDP-induced autophagy, as indicated by the reduced protein levels of LC3-II and Beclin1 and the ratio of LC3-II vs. LC3-I. Finally, luciferase reporter assay data revealed that miR-199a-5p directly targeted Beclin1 and negatively mediated Beclin1 expression at a post-transcriptional level in MG63 cells. In conclusion, our study suggests that miR-199a-5p promotes the cytotoxicity of DDP in OS cells via inhibition of autophagy. Therefore, miR-199a-5p/autophagy signaling is involved in chemoresistance and may become a potential target for the treatment of DDP-resistant OS.

Keywords: autophagy; chemotherapy resistance; cisplatin; microRNA-199a-5p; osteosarcoma.

Figure 1.

(A) Reverse transcription-quantitative polymerase chain reaction assay was conducted to determine the relative level of miR-199a-5p in MG63 cells treated with DDP for 6 h. (B) Western blot assay was conducted to examine the protein levels in each group. (C) The ratio of LC3-II vs. LC3-I in each group was determined. Non-treated MG63 cells were used as control. **P<0.01 vs. control. miR, microRNA; LC3, light chain 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DDP, diamminedichloridoplatinum (II).

Figure 2.

(A) Reverse transcription-quantitative polymerase chain reaction assay was conducted to determine the relative level of miR-199a-5p in MG63 cells transfected with miR-199a-5p mimic or miR-NC. Non-treated MG63 cells were used as control. **P<0.01 vs. control. (B) Western blot assay was conducted to examine the protein levels in DDP-treated MG63 cells transfected with miR-199a-5p mimic or miR-NC. **P<0.01 vs. DDP. (C) The ratio of LC3-II vs. LC3-I in each group was determined. **P<0.01 vs. DDP. miR, microRNA; LC3, light chain 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DDP, diamminedichloridoplatinum (II); NC, negative control.

Figure 3.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was conducted to determine cell proliferation in DDP-treated MG63 cells with or without transfection with miR-199a-5p or miR-NC. Non-transfected MG63 cells without DDP treatment were used as control. **P<0.01 vs. control. miR, microRNA; DDP, diamminedichloridoplatinum (II); NC, negative control.

Figure 4.

(A) TargetScan data indicated that Beclin1 was a target of miR-199a-5p. (B) The wild type and mutant type of Beclin1 3′-UTR containing the putative miR-199a-5p binding sequences are indicated. (C) MG63 cells were co-transfected with miR-199a-5p mimics or miR-NC and Luc-Beclin1 or Luc-mutant Beclin1 vector. Luciferase reporter assay was conducted to determine the luciferase activity in each group. MG63 cells transfected with only Luc-Beclin1 or Luc-mutant Beclin1 vector were used as control. **P<0.01 vs. control. miR/miRNA, microRNA; UTR, untranslated region; BECN1, Beclin1; Luc, luciferase; NC, negative control; hsa, Homo sapiens.

Figure 5.

(A) Reverse transcription-quantitative polymerase chain reaction assay was conducted to determine the relative level of miR-199a-5p in MG63 cells transfected with miR-199a-5p mimic or miR-199a-5p inhibitor. (B) Western blot assay was conducted to examine the protein level of Beclin1 in each group. Non-treated MG63 cells were used as control. **P<0.01 vs. control. miR, microRNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.