The Expression of MCUR1 in OS Tissues
To explore the functional role of MCUR1 in the tumorigenesis of OS, we investigated the expression level of MCUR1 in OS tissues. The immunohistochemical (IHC) analysis revealed that MCUR1 appeared to be predominantly localized in the cytoplasm (Fig. 1A). Moreover, IHC score of 49 paired OS and adjacent nontumor tissues showed that the expression level of MCUR1 was significantly upregulated in OS tissues compared to nonmalignant tissues (6.286 ± 3.725 vs 1.939 ± 1.625, t = 8.514༌P < 0.001) (Fig. 1B). The expression level of MCUR1 was further examined in a series of OS cell lines (SAOS2、MG63、HOS、143B、U2OS) and normal human osteoblast cell line (hFOB). qRT-PCR and Western blot analysis indicated that MCUR1 was overexpressed in OS cells at both mRNA and protein levels, respectively, compared with normal human osteoblast cells (Fig. 1C and 1D).
MCUR1 Expression and Pathological Features of OS Patients
Patients with OS were categorized into two groups including MCUR1 high-expression group and MCUR1 low-expression according to the median level of MCUR1. By analyzing the pathological data, we found that the protein expression level of MCUR1 were associated with the tumor size and tumor stage (P < 0.05). However, no statistical differences exist in lymph node metastasis and distant metastasis between the MCUR1 low-expression group and MCUR1 high-expression group (P > 0.05) (Table. 1).
The Relationship Between MCUR1 Expression and Prognosis of OS Patients
The Kaplan-Meier analysis of 49 patients with OS showed that there was a higher overall 3-year survival rate in OS patients with low MCUR1 expression (87.5%) compared with those with high MCUR1 expression (35.4%) (log-rank P = 0.027). The median overall survival was 29 months (min 15.9- max 42.1 months) for OS patients with high MCUR1 expression. However, the overall survival was remarkably improved in OS patients with MCUR1 low-expression (Fig. 2).
Knockdown of MCUR1 Inhibits SAOS2 Cell Growth and Increases Cell Apoptosis
The R2 (Genomics Analysis and Visualization Platform) was employed to analyze the KEGG pathways which are closely related with MCUR1. The data implied that MCUR1 may possess important roles in apoptosis (Fig. 3A). To verify this hypothesis and to examine the effect of MCUR1 on OS cell growth, we have successfully established MCUR1 knocked-down SAOS2 cells by transfecting siRNA targeting MCUR1. As shown in Fig. 3B and 3C, both the qRT-PCR and Western blot data showed that the expression of MCUR1 was remarkably lower in siMCUR1 group compared to the corresponding controls. MTS assays were then carried out to measure the cell viability of SAOS2 cells. The result indicated that knock-down of MCUR1 suppressed the growth SAOS2 cells compared to the control cells (P < 0.01) (Fig. 3D). To investigate the effect of MCUR1 knockdown on the apoptosis of SAOS2 cells, the rate of SAOS2 cell apoptosis was measured by flow cytometry. Our data showed that the rate of SAOS2 cell apoptosis was higher in MCUR1-knocked down SAOS2 cells (30.867 ± 3.729)%、(32.633 ± 4.365)% compared with that of control cells (14.300 ± 2.066)% (Fig. 3E). These results suggested that knock-down of MCUR1 inhibited SAOS2 cell growth and increased the rate of SAOS2 cell apoptosis.
The Mechanism Underlying the MCUR1-Mediated SAOS2 Cell Apoptosis
Western blot experiments showed that the protein expression level of AKT was not obviously changed in MCUR1 knocked-down SAOS2 cells compared to the corresponding controls. However, the protein expression level of phospho-AKT (p-AKT) was decreased, whereas the protein levels of p53 were remarkably increased (Fig. 4A). As displayed in Fig. 4B, the mRNA level of p53 was not obviously changed in MCUR1 knocked-down SAOS2 cells compared with the control cells according to the qRT-PCR analysis. Furthermore, the IHC analysis revealed that the expression of p53 protein was inversely correlated with the protein level of MCUR1 in OS tissues (Fig. 4C and 4D). Collectively, these data indicated that MCUR1 promoted SAOS2 cell apoptosis may be through activating AKT/p53 pathway.
Regulation of MCUR1 in OS Tissues
MicroRNA usually plays significant roles in regulating the gene expression . Thus, the MicroRNA Data Integration Portal (mirDIP) was used to explore the possible microRNA that may be involved in mediating the expression of MCUR1 in OS. The data indicates that hsa-miR-506-3p is likely to harbor a key role in regulating MCUR1 (Fig. 5A). Thus, qRT-PCR assays were carried out to verify this hypothesis. The result showed that the upregulation of miR-506-3p resulted in a decrease of mRNA and protein levels of MCUR1 in SAOS2 cells compared with the corresponding controls (Fig. 5B and 5C). Moreover, Pearson correlation analysis revealed that the mRNA level of MCUR1 was inversely correlated with the level of miR-506-3p, suggesting that miR-506-3p may participate in regulating the expression of MCUR1 in SAOS2 cells (Fig. 5D).