MicroRNA-33b suppresses migration and invasion by targeting c-Myc in osteosarcoma cells

Abstract

MicroRNAs have emerged as fundamental regulators in gene expression through silencing gene expression at the post-transcriptional and translational levels. Osteosarcoma is the most common type of primary malignant bone tumor and is characterized by complex genetic changes and resistance to conventional treatments. In our study, the role of miR-33b in the progression and metastasis of osteosarcoma was investigated. Our results showed that miR-33b was significantly downregulated in osteosarcoma tissue and cell lines. Overexpression of miR-33b significantly inhibited cell proliferation, migration, and invasion in the MG-63 osteosarcoma cell line. Moreover, we also showed that c-Myc was negatively regulated by miR-33b at the posttranscriptional level, via a specific target site within the 3'UTR. Overexpression of c-Myc impaired miR-33b-induced inhibition of proliferation and invasion in osteosarcoma cells. The expression of c-Myc was frequently downregulated in osteosarcoma tumors and cell lines and was inversely correlated with miR-33b expression. Thus, our findings suggest that miR-33b inhibits osteosarcoma cells migration and invasion by targeting the c-Myc gene, acting as tumor suppressor. The findings of this study contribute to current understanding of the functions of miR-33b in osteosarcoma.

Figure 1. miR-33b is downregulated in osteosarcoma.

(A) The expression relative expression levels were determined by qRT-PCR in human osteoblast cell line (hFOB) and osteosarcoma cells (SOSP-9607, U2OS, MG-63, SAOS-2). (B) qRT-PCR analysis of miR-33b expression in 60 pairs osteosarcoma tissues and their corresponding adjacent normal bone tissues. The expression of miR-33b was normalized to U6 snRNA. (C) Relative miR-33b expressionlevels in osteosarcoma tissues and their corresponding adjacent normal bone tissues. (D) Association between miRNA level between pM stage (No metastasis and Metastasis). Student’s t test was used to analyze the significant differences between the tumor and normal tissues. Correlation of miR-33b expression with patients’clinicopathological variables was evaluated by one-way analysis of variance test. ***p<0.001.

Figure 2. Overexpression of miR-33b inhibits proliferation, migration and invasion of osteosarcoma cells.

(A) qRT-PCR analysis of miR-33b expression after the transfection of miR-33b mimics, inhibitors or scramble or untreated. (B) The CCK8 assay used to evalute the proliferation of MG-63 cells after transfection with the miR-33b mimics, inhibitors or scramble or untreated. (C) Migration assays of MG-63 cells after treatment with miRNA mimics, inhibitors or scramble or no transfection; the relative ratio of migrative cells per field is shown. (D) Invasion analysis of MG-63 cells after treatment with miRNA mimics, inhibitors or scramble or no untreated; the relative ratio of invasive cells per field is shown below, least significant difference (LSD) test was used to analyze the significant differences. *p<0.05, ** p<0.01, and ***p<0.001.

Figure 3. miR-33b targets c-Myc in osteosarcoma cells.

(A) The sequences of miR-33b binding sites within the human c-Myc 3′UTRs and schematic reporter constructs, in this panel, c-Myc-WT represent the reporter constructs containing the entire 3′UTR sequences of c-Myc. C-Myc-MUT represent the reporter constructs containing mutated nucleotides. (B) The analysis of the relative luciferase activities of c-Myc-WT, c-Myc-MUT in 293T cells. The error bars are derived from triplicate expriments. (C) qRT-PCR analysis of c-Myc mRNA expression in MG-63 cells after treatment with miRNA mimics or scramble or untreated. The expression of c-Myc was normalized to GAPDH. (D)Western blot analysis of c-Myc expression in MG-63 cells transfected with miR-33b mimics or scramble or untreated. GAPDH was also detected as a loading control. (E) The ratio signal of c-Myc to GAPDH in each lane was determined. Student’s t test was used to analyze the significant differences. ***p<0.001.

Figure 4. Overexpression of c-Myc impairs miR-33b-induced inhibition of proliferation and invasion in osteosarcoma cells.

(A) Western blot analysis of c-Myc protein expression in MG-63 cells co-transfected with either a miR-33b mimics or scramble and pCDNA-c-Myc or pCDNA empty vector; GAPDH was also detected as a loading control. (B) The CCK8 assay used to evalute the proliferation of MG-63 cells transfected with different combinations. (C) Invasion assay of MG-63 cells treated with different combinations. The ratio of invasive cells per field is shown right. LSD test was used to analyze the significant differences. *p<0.05,** p<0.01,***p<0.001.

Figure 5. miR-33b negatively regulates c-Myc gene expression.

(A) The relative mRNA of c-Myc expression levels were determined by qRT-PCR in human osteoblast cell line (hFOB) and osteosarcoma cells (SOSP-9607, U2OS, MG-63, SAOS-2). (B) Western blot analysis of c-Myc expression in human osteoblast cell line (hFOB) and osteosarcoma cells (SOSP-9607, U2OS, MG-63, SAOS-2). (C) qRT-PCR analysis of c-Myc expression in 40 pairs osteosarcoma tissues and their corresponding adjacent normal bone tissues. The expression of c-Myc was normalized to GAPDH. The expression of c-Myc in osteosarcoma tissues was significant higher than in the corresponding adjacent normal bone tissues. (D) Analysis of correlation of miR-33b and c-Myc expression in osteosarcoma tissues. Student’s t test was used to analyze the significant differences between the osteosarcoma cells and osteoblast cell line. ***p<0.001.