miR-454 is down-regulated in osteosarcomas and suppresses cell proliferation and invasion by directly targeting c-Met

Abstract

Objectives: Osteosarcoma is the most common primary bone malignancy of children and young adults. Increasing evidence has shown that microRNAs (miRNAs) are associated with cancer development, but, little is known concerning the role of miR-454 in osteosarcoma.

Materials and methods: qRT-PCR was performed to detect expression of miR-454 in osteosarcoma cell lines and tissues. To understand its role in osteosarcoma, we reintroduced expression of miR-454 in the MG-63 cell line by transfection with miR-454 mimics or inhibitors. CCK-8 assay and an invasion assay were used to detect the functional role of miR-454. Luciferase assay and western blot analysis were performed to detect the target gene of miR-454.

Results: miR-454 was found to be down-regulated in osteosarcoma tissues and cell lines. Its over-expression inhibited tumour growth and invasion and its down-regulation promoted cell proliferation and invasion. Subsequent investigation revealed that c-Met was a direct and functional target of miR-454 in osteosarcoma. Overexpression of miR-454 impaired c-Met-induced cell proliferation and invasion. Finally, miR-454 was found to be inversely correlated to c-Met expression in human osteosarcoma tissues.

Conclusions: Reduced-expression of miR-454 in osteosarcoma cells promoted tumour growth by targeting c-Met, thus miR-454 may be a potential therapy target for this tumour.

Figure 1

miR‐454 is frequently down‐regulated in both osteosarcoma clinical samples and cell lines. (a) qRT‐PCR analysis of miR‐454 expression in human osteosarcoma cell lines (MG‐63, U2OS, SOSP‐9607 and SAOS‐2) and one human osteoblast cell line (hFOB). Expression of miR‐454 was normalized to U6 snRNA. (b) Relative miR‐454 expression levels in osteosarcoma tissues and their corresponding adjacent normal tissues. Expression of miR‐454 was normalized to U6 snRNA.(c) qRT‐PCR analysis of miR‐454 expression in 30 pairs of osteosarcomas and their corresponding adjacent normal tissues. Data are presented as log 2‐fold change of osteosarcomas relative to non‐tumour adjacent tissues.(c) Relative miR‐454 expression levels in osteosarcomas and their corresponding adjacent normal tissues. (d) Loss of miR‐454 levels in patients with osteosarcoma was associated with considerably shortened disease‐free survival. ***P < 0.001.

Figure 2

Cell proliferation and invasion was inhibited by miR‐454 in human osteosarcomas. (a) qRT‐PCR analysis of miR‐454 expression after transfection of miR‐454 mimics or scramble or control or inhibitor. (b) CCK8 assay used to evaluate proliferation of MG‐63 cells after transfection with miR‐454 mimics or scramble or control or inhibitor. (c) Invasion analysis of MG‐63 cells after treatment with miRNA mimics, inhibitors or scramble or control; relative ratio of invasive cells per field is shown on the right, *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3

miR‐454 directly targeted c‐Met 3′‐ UTR. (a) Sequences of miR‐454 binding sites within the human c‐Met 3′UTRs and schematic reporter constructs; in this panel, c‐Met‐WT represents reporter constructs containing the entire 3′UTR sequences of c‐Met, and c‐Met‐MUT represents reporter constructs containing mutated nucleotides. (b) Analysis of relative luciferase activities of c‐Met‐WT and c‐Met‐MUT. Error bars are derived from triplicate experiments. (c) qRT‐PCR analysis of c‐Met mRNA expression in MG‐63 cells after treatment with miRNA mimics, inhibitors, scramble or control. Expression of c‐Met was normalized to GAPDH. (d) Western blot analysis of c‐Met expression in MG‐63 cells transfected with miR‐454 mimics, inhibitors, scramble or control. GAPDH was also detected as loading control. ***P < 0.001.

Figure 4

c‐Met was involved in miR‐454‐induced repression of osteosarcoma cell proliferation and invasion. (a) Western blot analysis of c‐Met in MG‐63 cells transfected with either pCDNA–c‐Met or pCDNA empty vector. GAPDH was also detected as loading control. (b) Cell growth curves of MG‐63 cells transfected with different combinations, using CCK‐8. (c) Invasion analysis of MG‐63 cells treated with different combinations. Relative ratio of invasive cells per field is shown right. *P < 0.05,**P < 0.01, ***P < 0.001.

Figure 5

miR‐454 expression was inversely correlated to c‐Met expression in osteosarcomas. (a) qRT‐PCR analysis of c‐Met expression in human osteosarcoma cell lines (MG‐63, U2OS, SOSP‐9607 and SAOS‐2) and one human osteoblast cell line (hFOB). Expression of c‐Met was normalized to GAPDH. (b) qRT‐PCR analysis of c‐Met expression in 30 pairs of osteosarcomas and their corresponding adjacent normal tissues. Data presented as log 2‐fold change of osteosarcomas relative to non‐tumour adjacent tissues. (c) Relative c‐Met expression levels in osteosarcomas and their corresponding adjacent normal tissues. Expression of c‐Met was normalized to GAPDH. (d) Analysis of correlation of miR‐454 and c‐Met expression in osteosarcomas. (Two‐tailed Pearson's correlation analysis, r ² = −0.688; P < 0.001, n = 30). Data presented as log 2‐fold change of osteosarcomas relative to non‐tumour adjacent tissues. ***P < 0.001