Tumor Suppressor Function of miR-127-3p and miR-376a-3p in Osteosarcoma Cells

Abstract

Since the introduction of high-dose chemotherapy about 35 years ago, survival rates of osteosarcoma patients have not been significantly improved. New therapeutic strategies replacing or complementing conventional chemotherapy are therefore urgently required. MicroRNAs represent promising targets for such new therapies, as they are involved in the pathology of multiple types of cancer, and aberrant expression of several miRNAs has already been shown in osteosarcoma. In this study, we identified silencing of miR-127-3p and miR-376a-3p in osteosarcoma cell lines and tissues and investigated their role as potential tumor suppressors in vitro and in vivo. Transfection of osteosarcoma cells (n = 6) with miR-127-3p and miR-376a-3p mimics significantly inhibited proliferation and reduced the colony formation capacity of these cells. In contrast, we could not detect any influence of miRNA restoration on cell cycle and apoptosis induction. The effects of candidate miRNA restoration on tumor engraftment and growth in vivo were analyzed using a chicken chorioallantoic membrane (CAM) assay. Cells transfected with mir-127-3p and miR-376a-3p showed reduced tumor take rates and tumor volumes and a significant decrease of the cumulative tumor volumes to 41% and 54% compared to wildtype cells. The observed tumor suppressor function of both analyzed miRNAs indicates these miRNAs as potentially valuable targets for the development of new therapeutic strategies for the treatment of osteosarcoma.

Keywords: cancer therapy; chorioallanthoic membrane assay; miR-127-3p; miR-376a-3p; microRNA; osteosarcoma.

Figure 1

Silencing of miR-127-3p and miR-376a-3p in osteosarcoma cell lines and tissue. Quantitative real-time PCR analysis of (A) miR-127-3p and (B) miR-376a-3p expression in osteosarcoma cell lines (n = 7), osteosarcoma tissue (n = 8), and primary human osteoblasts (hOBs) (n = 5). Expression values were normalized to the expression of the small nuclear RNA U6 pseudogene (RNU6B). The white lines indicate the medians, the lower boundary of the box of the 25th percentile and the upper boundary of the box of the 75th percentile. The whiskers indicate the highest and lowest values. p-values were determined by the Mann–Whitney U test. (** p < 0.01). (C) Upregulation of miRNA expression by epigenetic modifiers. The osteosarcoma cell line 143B was treated for seven days with the indicated concentrations of 5′-Aza-2′-deoxycytidine (AZA) followed by a further three days of culture with or without the addition of phenylbutyric acid (PBA). After the incubation period, the expression of miR-127-3p and miR-376a-3p was quantified and normalized to the expression of RNU6B.

Figure 2

Restoration of endogenous levels of miR-127-3p and miR-376a-3p inhibits proliferation of osteosarcoma cells. Six osteosarcoma cell lines were transfected with miR-127-3p and miR-376a-3p mimics before cells were counted at the indicated time points. Non-transfected wildtype cells (WT) and cells transfected with a negative control miRNA (NC) served as controls. Experiments were done in triplicates. * p < 0.05, ** p < 0.01 compared to wildtype cells.

Figure 3

Mir-127-3p and miR-376a-3p reduce the colony forming capacity of osteosarcoma cells. (A) Cells transfected with miRNA mimics, a negative control miRNA (NC), or untreated wildtype cells (WT) were cultured for 10 days in soft agar, photographed, and colonies >500 µm² were counted using ImageJ software (* p < 0.05 compared to WT group). (B) Mean colony size of all five osteosarcoma cell lines normalized to the wildtype cells. (C) Representative photographs of colonies formed by MG-63 cells.

Figure 4

Cell cycle analysis of osteosarcoma cells with restored miRNA expression. The percentage of cells in the subG, G0/G1-, S-, and G2/M-phase was determined 96 h after transfection by flow cytometry. (A) Representative graphs of the cell line CAL-72. (B) Summary of the mean cell counts and standard deviations obtained from three independent experiments with six cell lines. (C) Percentage of apoptotic cells determined by NucView 488 staining 96 h after miRNA restoration. Positive control cells were treated with 2mM H₂O₂ for 4 h and further incubated for 24 h without the addition of H₂O₂.

Figure 5

Restoration of miR-127-3p and miR-376a-3p inhibits tumor growth in vivo. Osteosarcoma cells were transiently transfected with miRNA mimics, a negative control miRNA (NC), or left untreated (WT). Then, 72 h after transfection, cells were transplanted onto the chorioallantoic membrane (CAM) of fertilized chicken eggs (n = 15 per group). Tumors were resected on day 16 and the tumor volumes (A), ** p < 0.01, the tumor take rates (B), and the cumulative tumor volumes (C) were calculated (* p < 0.05). (D) Representative photographs of tumor xenografts from each treatment group.