MicroRNA-505 is downregulated in human osteosarcoma and regulates cell proliferation, migration and invasion

Abstract

Recent studies have demonstrated that microRNAs (miRNAs/miRs) are involved in osteosarcoma tumorigenesis, progression, invasion and metastasis. For example, miR-505 plays important roles in human carcinogenesis; however, its exact function in osteosarcoma remains unclear. MicroRNA profiles of osteosarcoma and normal tissues were obtained by miRNA microarray assays, which were validated by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Then, high-mobility group box 1 (HMGB1) expression was evaluated by qRT-PCR and western blot analysis. The correlation between miR-505 and HMGB1 was analyzed by Pearson correlation. In vitro, the biological functions of miR-505 were examined by wound healing, MTT and Transwell assays and western blot analysis in MG63 cells transfected with miRNA mimics or empty vector. Luciferase assay was utilized to assess whether HMGB1 is a target of miR-505. miRNA microarrays revealed 26 aberrant miRNAs in osteosarcoma tissues; miR-505 showed the most pronounced decrease (P<0.01), which was significantly associated with TNM stage and metastasis status (P<0.05). In addition, HMGB1 was highly expressed in osteosarcoma tissues (P<0.01), with a significantly negative correlation with miR-505 (r=-0.6679, P<0.001). Furthermore, miR-505 inhibited proliferation, migration and invasion abilities of MG63 cells (P<0.01). Moreover, luciferase activity of the HMGB1-3'-UTR plasmid was suppressed following miR-505 binding (P<0.01). Finally, HMGB1 overexpression partly reversed the effects of miR-505 on MG63 cells. In conclusion, miR-505 levels are decreased in osteosarcoma tissues, and reduced miR-505 expression is significantly associated with poorer clinical prognosis in patients with osteosarcomas. miR-505 inhibits osteosarcoma cell proliferation, migration and invasion by regulating HMGB1.

Figure 1.

miRNA profile of osteosarcoma tissues. Five paired osteosarcoma tissue samples were randomly chosen for microRNA microarray assay (osteosarcoma tissues are indicated as O1-5; adjacent normal tissues are indicated as N1-5). The green bar indicates downregulated miRNAs, red part indicates upregulated miRNAs.

Figure 2.

Analysis of the expression levels of the 4 miRNAs in specimens using qRT-PCR. Compared with normal tissues, (C) miR-505 and (D) miR-16 showed higher expression in osteosarcoma tissues, whereas (A) miR-210 and (B) miR-135b showed lower expression. **P<0.01 vs. corresponding normal tissues.

Figure 3.

An inverse correlation between HMGB1 mRNA and miR-505 expression in osteosarcoma tissues. (A) The expression of HMGB1 was significantly increased in osteosarcoma tissues, compared with that in the adjacent normal tissues (**P<0.01). (B) Pearson correlation was used to analyze the relationship between miR-505 and HMGB1 mRNA in osteosarcoma tissues. (C and D) Western blot analysis shows the expression of HMGB1 protein in osteosarcoma tissues and adjacent normal tissues.

Figure 4.

miR-505 inhibits MG63 cell proliferation, migration and invasion in vitro. (A) miR-505 expression levels were assessed in different osteosarcoma cell lines and a normal osteoblastic cell line. (B) The expression of miR-505 was tested in cells transfected with or without miR-505 mimics. (C) MTT assay of MG63 cells transduced with empty or miR-505 vectors. (D and E) Wound healing assay was used to investigate the migration ability of the cells. (F and G) Transwell invasion assay was used to determine the invasion ability of the MG63 cells in the different groups. (H and I) Western blot analysis showed the differential expression of cell viability- and invasion-related proteins MMP2/9 and cyclin D1, among the three groups. *P<0.01 vs. miR-NC, **P<0.01 vs. normal osteoblastic cell line hFOB1.19.

Figure 5.

miR-505 directly targets HMGB1 in osteosarcoma cells. (A) Bioinformatic tools were used to predict the site that miR-505 binds the 3′-UTR of HMGB1. (B) miR-505 expression affects HMGB1 protein expression in U2-OS and MG63 cells. (C) HMGB1 mRNA was significantly suppressed in the miR-505 mimics group in U2-OS and MG63 cells, detected by RT-qPCR. (D) Luciferase activity after transfection with wild-type/mutant-type 3′-UTR constructs in HMGB1. *P<0.01 compared with miR-NC.

Figure 6.

Overexpression of HMGB1 partly abrogates miR-505-induced inhibitory effects on MG63 cells. (A) The expression of HMGB1 in different groups was detected by western blotting analyses. (B) Cell migration ability was detected by wound-healing assay in MG63 cells transfected with miR-505 mimics or miR-505 mimics/empty vector or miR-505 mimics/HMGB1. (C) MTT assay was performed in MG63 cells. (D) Transwell chamber was used for cell invasive ability detection. (E) Western blotting analyses were used to detect the expression of MMP-2/9 and cyclin D1 in MG63 cells. *P<0.01 compared with mimics/empty vector.