MicroRNA‑34b promotes proliferation, migration and invasion of Ewing's sarcoma cells by downregulating Notch1

Abstract

Ewing's sarcoma is the second most frequent bone and soft tissue sarcoma, which is commonly driven by the Ewing's sarcoma breakpoint region 1‑friend leukemia integration 1 transcription factor (EWS‑FLI1) fusion gene. Since microRNAs (miRs) can act as either oncogenes or tumor suppressor genes in human cancer, and miR‑34b has been reported to act as a tumor suppressor, the role of miR‑34b in Ewing's sarcoma was investigated in the present study. The results demonstrated that miR‑34b expression levels were higher in tumor samples compared within normal tissue samples. Notably, miR‑34b expression levels were significantly higher in EWS‑FLI1‑positive samples compared within EWS‑FLI1‑negative samples. The effects of miR‑34b expression on cell proliferation, migration and invasion were also examined. miR‑34b expression was inhibited using small interfering (si)RNA targeting the fusion gene. Transfection of a miR‑34b precursor sequence into siRNA‑treated tumor cells resulted in a significant increase in cell growth, migration and invasion compared within the control group. In addition, the adhesive ability was increased in the Ewing's sarcoma cell line RD‑ES, but not A673, following miR‑34b upregulation. Conversely, downregulation of miR‑34b expression led to a significant decrease in cell growth, migration and invasion. Notch has previously been reported to serve either oncogenic or tumor suppressive roles in human cancer. The results indicated that Notch1 and its target genes, Hes family BHLH transcription factor 1 and Hes‑related family BHLH transcription factor with YRPW motif 1, were suppressed by miR‑34b directly In conclusion, EWS‑FLI1 may modulate miR‑34b expression directly or indirectly, and miR‑34b potentially has an oncogenic role in Ewing's sarcoma by downregulating Notch1.

Figure 1.

Association between the EWS-FLI1 fusion gene and miR-34b in tumor and NT samples. (A) EWS-FLI1 was not detected in three tumor samples and in NT. (incomplete data presented due to limited lanes). (B) EWS-ERG was detected in three tumor samples. Lanes 1–7, EWS-FLI1-positive samples; lanes 8–10, EWS-ERG-positive samples. (C) Expression levels of miR-34b were increased in tumor samples compared with in NT. In EWS-FLI1-positive samples, miR-34b relative expression was higher compared with in NT. **P<0.01 vs. NT, ^##P<0.01 vs. EWS-ERG positive samples. EWS, Ewing's sarcoma breakpoint region 1; FLI1, friend leukemia integration 1 transcription factor; miR, microRNA; NT, normal tissue.

Figure 2.

(A) Detection of EWS-FLI1 fusion gene expression in RD-ES and A673 cell lines using reverse transcription-polymerase chain reaction. (B) Expression levels of the fusion gene were downregulated in RD-ES and A-673 cells following siRNA transfection. (C) miR-34b expression in A673 cells was downregulated following siRNA transfection. (D) miR-34b expression in RD-ES cells was downregulated following siRNA transfection. (E) miR-34b expression was upregulated by the precursor of miR-34b in RD-ES and A-673 cells. (F) miR-34b expression was downregulated by the inhibitor of miR-34b in RD-ES and A-673 cells. BC, cell lines that were treated with lentiviral vector alone; NC(+), normal cell lines that were not transfected with siRNA; NS, cells transfected with non-targeting siRNA; siRNA, siRNA-transfected cells; NC(−), normal cell lines that were transfected with specific siRNA targeting the EWS-FLI1 fusion gene; Micro-up, NC(−) cell lines treated with precursor miR-34b sequences; Micro-down, NC(+) cell lines treated with complementary sequences of miR-34b. **P<0.01, ^##P<0.01. EWS, Ewing's sarcoma breakpoint region 1; FLI1, friend leukemia integration 1 transcription factor; miR, microRNA; siRNA, small interfering RNA.

Figure 3.

Proliferation and adhesion of RD-ES and A673 cells were detected using an MTT assay. (A) When miR-34b expression was upregulated, the siRNA-transfected RD-ES cells exhibited significantly increased proliferative capacity compared with cells in the control groups. (B) When miR-34b expression was upregulated, the siRNA-transfected A673 cells exhibited significantly increased proliferative capacity. (C) Proliferative ability of normal RD-ES cells was significantly inhibited when miR-34b expression was downregulated. (D) Proliferative of normal A673 cells was significantly inhibited when miR-34b expression was downregulated. (E) Overexpression of miR-34b significantly improved the adhesion of RD-ES cells, but had little effect on A673 cells. (F) Knockdown of miR-34b reduced the adhesive ability of RD-ES cells, but had little effect on A673 cells. BC, cell lines that were treated with lentiviral vector alone; NC(+), normal cell lines that were not transfected with siRNA; siRNA, siRNA-transfected cells; NC(−), normal cell lines that were transfected with specific siRNA; Micro-up, cell lines treated with precursor miR-34b sequences; Micro-down, cell lines treated with complementary sequences of miR-34b. *P<0.05 and **P<0.01. miR, microRNA; OD, optical density; siRNA, small interfering RNA.

Figure 4.

Effects of miR-34b on the migration and invasion of RD-ES cells in vitro. Magnification, ×200 (A) Migratory and (B) invasive ability of normal RD-ES cells was inhibited when miR-34b expression was downregulated (top panel). However, miR-34b overexpression improved the migratory and invasive ability of siRNA-transfected-RD-ES cells (bottom panel). Quantification of (C) EWS-FLI1 (−) and (D) EWS-FLI1 (+) cells. The number of cells that passed through the membrane was counted. BC, cell lines that were treated with lentiviral vector alone; NC(+), normal cell lines that were not transfected with siRNA; siRNA, siRNA-transfected cells; NC(−), normal cell lines that were transfected with specific siRNA; Micro-up, cell lines treated with precursor miR-34b sequences; Micro-down, cell lines treated with complementary sequences of miR-34b; EWS-FLI1 (+), cells that were not transfected with EWS-FLI1 fusion gene siRNA; EWS-FLI1 (−), cells that were transfected with EWS-FLI1 fusion gene siRNA against EWS-FLI1 gene. **P<0.01, ^##P<0.01. EWS, Ewing's sarcoma breakpoint region 1; FLI1, friend leukemia integration 1 transcription factor; miR, microRNA; siRNA, small interfering RNA.

Figure 5.

mRNA expression levels of Notch1, Hes-1 and Hey-1 demonstrated the negative association with that of EWS-FLI1 and miR-34b in RD-ES and A673 cells. (A) mRNA expression levels of Notch-1, Hes-1 and Hey-1 were increased when the expression of the EWS-FLI1 fusion gene was downregulated by siRNA in RD-ES and A673 cells. (B) Conversely, mRNA expression levels were inhibited after the siRNA-transfected cells were treated with miR-34b precursor sequences. (C) Notch-1, Hes-1 and Hey-1 mRNA expression was increased after miR-34b was downregulated in normal cells. BC, cell lines that were treated with lentiviral vector alone; NC(+), normal cell lines that were not transfected with siRNA; NS, cells transfected with non-targeting siRNA; siRNA, siRNA-transfected cells; NC(−), normal cell lines that were transfected with specific siRNA; MU, cell lines treated with precursor miR-34b sequences; MD, cell lines treated with complementary sequences of miR-34b. **P<0.01. EWS, Ewing's sarcoma breakpoint region 1; FLI1, friend leukemia integration 1 transcription factor; Hes1, Hes family BHLH transcription factor 1; Hey1, Hes-related family BHLH transcription factor with YRPW motif 1; miR, microRNA; siRNA, small interfering RNA.

Figure 6.

Protein expression levels of Notch1, Hes-1 and Hey-1 demonstrated the negative association with EWS-FLI1 and miR-34b in RD-ES and A673 cells. (A) The protein expression levels of Notch-1, Hes-1 and Hey-1 were increased following EWS-FLI1 repression in RD-ES cells. (B) The expression of Notch-1, Hes-1 and Hey-1 were decreased after miR-34b was upregulated in RD-ES cells in which EWS-FLI1 is repressed. (C) The expression of Notch-1, Hes-1 and Hey-1 were increased after miR-34b was downregulated in RD-ES cells. (D) The protein expression levels of Notch-1, Hes-1 and Hey-1 were increased following EWS-FLI1 repression in A673 cells. (E) The expression of Notch-1, Hes-1 and Hey-1 were decreased following miR-34b was upregulated in A673 cells in which EWS-FLI1 is repressed. (F) The expression of Notch-1, Hes-1 and Hey-1 were increased after miR-34b was downregulated in A673 cells. BC, cell lines that were treated with lentiviral vector alone; NC(+), normal cell lines that were not transfected with siRNA; NS, cells transfected with non-targeting siRNA; siRNA, siRNA-transfected cells; NC(−), normal cell lines that were transfected with specific siRNA; MU, cell lines treated with precursor miR-34b sequences; MD, cell lines treated with complementary sequences of miR-34b. EWS, Ewing's sarcoma breakpoint region 1; FLI1, friend leukemia integration 1 transcription factor; Hes1, Hes family BHLH transcription factor 1; Hey1, Hes-related family BHLH transcription factor with YRPW motif 1; miR, microRNA; siRNA, small interfering RNA.

Figure 7.

Dual-luciferase reporter assay. Relative luciferase activity was normalized to Renilla luciferase activity following co-transfection of RD-ES and A673 cells with the miR-34b inhibitor and pmiR-RB-REPORT construct containing WT or MUT NOTCH1 3′-UTR. Experiments were performed in triplicate. ^##P<0.01 and, **P<0.01 vs. NC+WT or Micro-down+MUT as illustrated by the lines. 3′-UTR, 3′-untranslated region; miR, microRNA; MUT, mutated; NC, negative control; WT, wild type.