Forkhead box C1 is targeted by microRNA-133b and promotes cell proliferation and migration in osteosarcoma

Abstract

Forkhead box C1 (FOXC1) has been demonstrated to act as an oncogene in a number of malignant tumors, though its underlying mechanism of action in osteosarcoma (OS) remains unknown. The present study evaluated the expression and regulatory role of FOXC1 in OS. Reverse transcription-quantitative polymerase chain reaction and western blot data indicated that FOXC1 was significantly upregulated in OS tissues and cell lines when compared with adjacent non-tumor tissues (P<0.001) and normal human osteoblast cells (P<0.01), respectively. Moreover, levels of FOXC1 expression were significantly higher in OS at advanced clinical stage (III-IV) when compared with that at low clinical stage (I-II; P<0.001). Knockdown of FOXC1 expression caused a significant decrease in the proliferation and migration of OS U2OS cells (P<0.01), while overexpression of FOXC1 significantly promoted U2OS cell proliferation and migration (P<0.01), relative to control U2OS cells. Furthermore, FOXC1 was identified as a direct target of microRNA (miR)-133b, a reported tumor-suppressive miR in OS. The protein expression of FOXC1 was negatively regulated by miR-133b in U2OS cells (P<0.01), and miR-133b expression was inversely correlated with FOXC1 expression in OS. In conclusion, the present study demonstrated that FOXC1, targeted by miR-133b, may promote cell proliferation and migration in OS. Thus, FOXC1 may be a potential therapeutic target in the treatment of OS.

Keywords: forkhead box C1; microRNA; oncogene; osteosarcoma.

Figure 1.

Upregulation of FOXC1 in OS. (A) RT-qPCR was used to measure the levels of FOXC1 mRNA in OS tissues and adjacent non-tumor tissues. (B) RT-qPCR was used to assess the levels of FOXC1 mRNA in OS tissues at different clinical stages. (C) RT-qPCR and (D) western blot analysis were performed to measure the expression of FOXC1 at the mRNA and protein levels in OS cell lines and normal osteoblast hFoB1.19 cells. ^###P<0.001 vs. adjacent tissue; ^&&&P<0.001 vs. I–II stage OS. **P<0.01 vs. hFoB1.19 cells. FOXC1, forkhead box C1; OS, osteosarcoma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 2.

Effects of FOXC1 knockdown on OS cell proliferation and migration. The OS cell line U2OS was transfected with FOXC1 siRNA or with non-specific siRNA, as a control. (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blot analysis were performed to measure the expression of FOXC1 at the mRNA and protein levels. (C) MTT and (D) wound healing assays were conducted to assess the proliferation and migration of transfected cells. Magnification, ×40. **P<0.01 vs. control. FOXC1, forkhead box C1; OS, osteosarcoma; siRNA, small interfering RNA.

Figure 3.

Effects of FOXC1 overexpression on OS cell proliferation and migration. The OS cell line U2OS was transfected with a pcDNA3.1-FOXC1 expression plasmid or with a blank vector, as a control. (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blot analysis were performed to measure the expression of FOXC1 at the mRNA and protein levels. (C) MTT and (D) wound healing assays were conducted to assess the proliferation and migration of transfected cells. Magnification, ×40. **P<0.01 vs. control. FOXC1, forkhead box C1; OS, osteosarcoma.

Figure 4.

miR-133b targets FOXC1 in OS. (A) Targetscan software predicted that FOXC1 was a putative target gene of miR-133b, and that (B) their targeting relationship was evolutionally conserved. (C and D) WT- or MT-FOXC1 3′UTR was cloned into a pMIR-GLOTM Luciferase vector downstream of the F. luciferase coding region. (E) Luciferase activity was significantly decreased in U2OS cells co-transfected with miR-133b mimic and WT-FOXC1-3′UTR plasmid but not in cells co-transfected with miR-133b mimic and MT-FOXC1-3′UTR plasmid. **P<0.01 vs. miR-NC. FOXC1, forkhead box C1; miR, microRNA; OS, osteosarcoma; WT, wild type; MT, mutant type; 3′UTR, 3′ untranslated region; NC, negative control; SV40, simian vacuolating virus 40; R. luciferase, Renilla-glo luciferase; F. luciferase, firefly luciferase; HSV TK, herpes simplex virus thymidine kinase promoter; Sgf1, XhoI, PmeI and NotI, restriction sites.

Figure 5.

Targeting of FOXC1 by miR-133b in OS. U2OS cells were transfected with miR-133b or miR-NC. (A) RT-qPCR and (B) western blot analysis were performed to measure the expression of miR-133b and FOXC1 protein, respectively. U2OS cells were also transfected with miR-133b inhibitor or NC inhibitor. (C) RT-qPCR and (D) western blot analysis were performed to measure the expression of miR-133b and FOXC1 protein, respectively. **P<0.01 vs. NC. FOXC1, forkhead box C1; miR, microRNA; OS, osteosarcoma; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 6.

FOXC1 expression is inversely correlated with miR-133b expression in OS. (A) RT-qPCR was performed to assess the expression of miR-133b in OS tissues and adjacent non-tumor tissues. (B) RT-qPCR was performed to assess the expression of miR-133b in OS cell lines and normal osteoblast hFoB1.19 cells. (C) An inverse correlation was identified between the levels of miR-133b and FOXC1 mRNA in OS tissues. ^###P<0.001 vs. adjacent tissue; **P<0.01 vs. hFoB1.19 cells. FOXC1, forkhead box C1; miR, microRNA; OS, osteosarcoma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.