The tumor suppressor role of miR-124 in osteosarcoma

Abstract

MicroRNAs have crucial roles in development and progression of human cancers, including osteosarcoma. Recent studies have shown that miR-124 was down-regulated in many cancers; however, the role of miR-124 in osteosarcoma development is unknown. In this study, we demonstrate that expression of miR-124 is significantly downregulated in osteosarcoma tissues and cell lines, compared to the adjacent tissues. The expression of miR-124 in the metastases osteosarcoma tissues was lower than that in non- metastases tissues. We identified and confirmed Rac1 as a novel, direct target of miR-124 using prediction algorithms and luciferase reporter gene assays. Overexpression of miR-124 suppressed Rac1 protein expression and attenuated cell proliferation, migration, and invasion and induced apoptosis in MG-63 and U2OS in vitro. Moreover, overexpression of Rac1 in miR-124-transfected osteosarcoma cells effectively rescued the inhibition of cell invasion caused by miR-124. Therefore, our results demonstrate that miR-124 is a tumor suppressor miRNA and suggest that this miRNA could be a potential target for the treatment of osteosarcoma in future.

Figure 1. The expression of miR-124 in human osteosarcoma cell lines and tissues.

(A) The patient who were diagnosed as in osteosarcoma in H&E staining (original magnification, ×100). (B) The expression of miR-124 in four human osteosarcoma cell lines (MG-63, U2OS, SOSP-9607, and SAOS-2) and four primary tissues (C) and adjacent non-neoplastic tissues (N) using real-time PCR. (C) miR-124 was detected in 70 osteosarcoma patients by real-time PCR. Data is presented as log 2 of fold change of GC tissues relative to non-tumor adjacent tissues. (D) The expression of miR-124 in the osteosarcoma tissues was lower than that in non-tumor adjacent tissues. P<0.01. (E) The expression of miR-124 in the metastases osteosarcoma tissues was lower than that in non- metastases tissues. Experiments were performed three times. All data uses t test and is shown as mean±SD.

Figure 2. Overexpression of miR-124 inhibits osteosarcoma cell growth and affects cell cycle.

(A) Expression levels of miR-124 were examined by real-time PCR after transfection of 50 nmol/L of miR-124 mimics or sramble or no transfection or miR-124 inhibitor. (B) Growth of MG-63 and U2OS cells were shown after transfection with 50 nmol/L of miR-10b mimics or sramble or no transfection or miR-124 inhibitor. The growth index as assessed at 0, 24, 48, 72, and 96 hours. (C)The effect of miR-124 on cell cycle progression of MG-63 and U2OS cells after 48-h incubation detected by flow cytometry analysis. Experiments were performed three times. Bars are mean±SD, *p<0.05, **p<0.01 and ***p<0.001.

Figure 3. Overexpression of miR-124 inhibits osteosarcoma cell migration and invasion.

(A) MG-63 and U2OS cells were not transfected or transfected with 50 nmol/L of miR-10b mimics or sramble or inhibitor for 24 hours, and wounds were made. The relative ratio of wound closure per field is shown. (B) (C) MG-63 and U2OS cells were not transfected or transfected with 50 nmol/L of miR-10b mimics or sramble or inhibitor for 24 hours, and migration (B) or transwell invasion (C) assay was performed. The relative ratio of invasive cells per field is shown. Magnification for identification of migration is ×400 and invasion ×40. All data is shown as mean±SD. *p<0.05, **p<0.01 and ***p<0.001.

Figure 4. Rac1 is a direct target of miR-124.

(A) Predicted duplex formation between human Rac1 3′-UTR and miR-124, Rac1 3′-UTR is highly conserved in different species. Upper panel, sequence alignment of miR-124 with binding site on the Rac1 3′-UTR. Lower panel, sequence of the miR-124 binding site within the Rac1 3′-UTR of four species. (B) MiR-124 cannot alter mRNA level of Rac1 by real-time PCR. (C) Luciferase activity of wild-type (WT-UTR) or mutant (MUT-UTR). (D) Rac1 protein expression in MG-63 cells were transfected with 50 nmol/L of miR-124 mimics , sramble or not transfected.

Figure 5. Overexpression of Rac1 inpairs miR-124-induced inhibition of invasion in MG-63 cells.

(A) Rac1 is over-expression in osteosarcoma tissues. Western blot analysis of Rac1 protein expression in six patients whose miR-124 expression was down-regulated in osteosarcoma tissues. (B) Overexpression of Rac1 incresed Rac1 protein expression. (C) MG-63 cells were transfected with miR-124 mimics and/or Rac expression vector, and transwell invasion assay was performed. Overexpression of Rac1 inpairs miR-124-induced inhibition of invasion. All data is shown as mean±SD. *p<0.05, **p<0.01 and ***p<0.001.