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miR‑542‑3p overexpression is associated with enhanced osteosarcoma cell proliferation and migration ability by targeting Van Gogh‑like 2.

Li H, Liu H, Pei J, Wang H, Lv H - Mol Med Rep (2014)

Bottom Line: It occurs predominantly in infants and adolescents, with an incidence of 4‑5 cases/100,000,000.Using a dual luciferase assay and western blot analysis, the present study confirmed that Van Gogh‑like 2, which is a non‑canonical Wnt pathway suppressor, was a target gene of miR‑542‑3p.Subsequently, the biological function of miR‑542‑3p in U2OS cells was examined, which revealed that overexpression of miR‑542‑3p can enhance the cell proliferation and migration ability of U2OS cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedics, Yidu Central Hospital, Weifang Medical College, Weifang, Shandong 262500, P.R. China.

ABSTRACT
Osteosarcoma is the most common histological form of primary bone cancer, which arises from osteoid tissue. It occurs predominantly in infants and adolescents, with an incidence of 4‑5 cases/100,000,000. The 5-year survival rate of patients with osteosarcoma has significantly improved over time; however, there remains a significant proportion of patients that respond poorly to chemotherapy. An improved understanding of the pathology of osteosarcoma is required to provide more effective treatment strategies, identify biomarkers and develop novel chemotherapeutic agents. Disturbance in microRNA (miRNA) expression has been identified in osteosarcoma tissues and cell lines; however, the roles of miRNA during osteosarcoma pathogenesis remain to be elucidated. In the present study, the expression levels of eight selected miRNAs were investigated in osteosarcoma tissues and the results revealed that the expression levels of miR‑542‑3p and miR‑542‑5p were significantly upregulated and the expression of miR‑199‑3p was significantly downregulated. Using a dual luciferase assay and western blot analysis, the present study confirmed that Van Gogh‑like 2, which is a non‑canonical Wnt pathway suppressor, was a target gene of miR‑542‑3p. Subsequently, the biological function of miR‑542‑3p in U2OS cells was examined, which revealed that overexpression of miR‑542‑3p can enhance the cell proliferation and migration ability of U2OS cells. This indicated that miR‑542‑3p may act as an oncogene in osteosarcoma pathogenesis. The findings of the present study may provide assistance in understanding the development of osteosarcoma and aid in the development of strategies for the diagnosis and treatment of osteosarcoma.

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Expression of VANGL2 is suppressed by miR-542-3p. (A) Schematic diagram for constructing the predicted miR-542-3p binding site into the pGL3 control vector. (B) Confirmation of the miR-542-3p target gene. U2OS cells were co-transfected with the miRNA control, miR-542-3p mimic, anti-miR control or miR-542-3p inhibitor and pGL3-VANGL2 for the dual-luciferase assay. PRL-TK, containing RL, was cotransfected with the 3′-UTR of VANGL2 for data normalization. (C) Mutation analysis of the miR-542-3p binding site. Following mutation of five nucleotides of the binding site of miR-542-3p in the 3′-UTR of VANGL2 (pGL3-VANGL2-Mu), the luciferase activity was significantly decreased in the U2OS cells cotransfected with the miR-542-3p mimics and pGL3-VANGL2 compared with those cotransfected with pGL3-VANGL2-Mu or pGL3. (D) VANGL2 protein levels in the miR-542-3p mimic and inhibitor-treated U2OS cells were detected by western blot analysis. miR, microRNA; VANGL2, Van Gogh-like 2; UTR, untranslated region; Mu, mutant; PRL-TK, thymidine kinase promoter-Renilla luciferase reporter plasmid.
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f2-mmr-11-02-0851: Expression of VANGL2 is suppressed by miR-542-3p. (A) Schematic diagram for constructing the predicted miR-542-3p binding site into the pGL3 control vector. (B) Confirmation of the miR-542-3p target gene. U2OS cells were co-transfected with the miRNA control, miR-542-3p mimic, anti-miR control or miR-542-3p inhibitor and pGL3-VANGL2 for the dual-luciferase assay. PRL-TK, containing RL, was cotransfected with the 3′-UTR of VANGL2 for data normalization. (C) Mutation analysis of the miR-542-3p binding site. Following mutation of five nucleotides of the binding site of miR-542-3p in the 3′-UTR of VANGL2 (pGL3-VANGL2-Mu), the luciferase activity was significantly decreased in the U2OS cells cotransfected with the miR-542-3p mimics and pGL3-VANGL2 compared with those cotransfected with pGL3-VANGL2-Mu or pGL3. (D) VANGL2 protein levels in the miR-542-3p mimic and inhibitor-treated U2OS cells were detected by western blot analysis. miR, microRNA; VANGL2, Van Gogh-like 2; UTR, untranslated region; Mu, mutant; PRL-TK, thymidine kinase promoter-Renilla luciferase reporter plasmid.

Mentions: To confirm whether VANGL2 was the target gene of miR-542-3p, a 516 bp segment of the VANGL2 3′-UTR, containing the interaction sites of miR-542-3p, was cloned into the pGL3 control vector (pGL3-VANGL2) downstream of the firefly luciferase reporter gene (Fig. 2A) to perform a dual luciferase assay. The U2OS cells were cotransfected with pGL3-VANGL2 and either the miR-542-3p mimic or inhibitor (Fig. 2B). Compared with the miRNA control, luciferase activity was significantly decreased by miR-542-3p by ~47.8% (P<0.05). Furthermore, luciferase activity was significantly increased by the miR-542-3p inhibitor compared with the anti-miR control by ~23.5% (P<0.05). These results indicated that miR-542-3p targets the 3′-UTR of VANGL2, leading to a change in firefly luciferase translation.


miR‑542‑3p overexpression is associated with enhanced osteosarcoma cell proliferation and migration ability by targeting Van Gogh‑like 2.

Li H, Liu H, Pei J, Wang H, Lv H - Mol Med Rep (2014)

Expression of VANGL2 is suppressed by miR-542-3p. (A) Schematic diagram for constructing the predicted miR-542-3p binding site into the pGL3 control vector. (B) Confirmation of the miR-542-3p target gene. U2OS cells were co-transfected with the miRNA control, miR-542-3p mimic, anti-miR control or miR-542-3p inhibitor and pGL3-VANGL2 for the dual-luciferase assay. PRL-TK, containing RL, was cotransfected with the 3′-UTR of VANGL2 for data normalization. (C) Mutation analysis of the miR-542-3p binding site. Following mutation of five nucleotides of the binding site of miR-542-3p in the 3′-UTR of VANGL2 (pGL3-VANGL2-Mu), the luciferase activity was significantly decreased in the U2OS cells cotransfected with the miR-542-3p mimics and pGL3-VANGL2 compared with those cotransfected with pGL3-VANGL2-Mu or pGL3. (D) VANGL2 protein levels in the miR-542-3p mimic and inhibitor-treated U2OS cells were detected by western blot analysis. miR, microRNA; VANGL2, Van Gogh-like 2; UTR, untranslated region; Mu, mutant; PRL-TK, thymidine kinase promoter-Renilla luciferase reporter plasmid.
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Related In: Results  -  Collection

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f2-mmr-11-02-0851: Expression of VANGL2 is suppressed by miR-542-3p. (A) Schematic diagram for constructing the predicted miR-542-3p binding site into the pGL3 control vector. (B) Confirmation of the miR-542-3p target gene. U2OS cells were co-transfected with the miRNA control, miR-542-3p mimic, anti-miR control or miR-542-3p inhibitor and pGL3-VANGL2 for the dual-luciferase assay. PRL-TK, containing RL, was cotransfected with the 3′-UTR of VANGL2 for data normalization. (C) Mutation analysis of the miR-542-3p binding site. Following mutation of five nucleotides of the binding site of miR-542-3p in the 3′-UTR of VANGL2 (pGL3-VANGL2-Mu), the luciferase activity was significantly decreased in the U2OS cells cotransfected with the miR-542-3p mimics and pGL3-VANGL2 compared with those cotransfected with pGL3-VANGL2-Mu or pGL3. (D) VANGL2 protein levels in the miR-542-3p mimic and inhibitor-treated U2OS cells were detected by western blot analysis. miR, microRNA; VANGL2, Van Gogh-like 2; UTR, untranslated region; Mu, mutant; PRL-TK, thymidine kinase promoter-Renilla luciferase reporter plasmid.
Mentions: To confirm whether VANGL2 was the target gene of miR-542-3p, a 516 bp segment of the VANGL2 3′-UTR, containing the interaction sites of miR-542-3p, was cloned into the pGL3 control vector (pGL3-VANGL2) downstream of the firefly luciferase reporter gene (Fig. 2A) to perform a dual luciferase assay. The U2OS cells were cotransfected with pGL3-VANGL2 and either the miR-542-3p mimic or inhibitor (Fig. 2B). Compared with the miRNA control, luciferase activity was significantly decreased by miR-542-3p by ~47.8% (P<0.05). Furthermore, luciferase activity was significantly increased by the miR-542-3p inhibitor compared with the anti-miR control by ~23.5% (P<0.05). These results indicated that miR-542-3p targets the 3′-UTR of VANGL2, leading to a change in firefly luciferase translation.

Bottom Line: It occurs predominantly in infants and adolescents, with an incidence of 4‑5 cases/100,000,000.Using a dual luciferase assay and western blot analysis, the present study confirmed that Van Gogh‑like 2, which is a non‑canonical Wnt pathway suppressor, was a target gene of miR‑542‑3p.Subsequently, the biological function of miR‑542‑3p in U2OS cells was examined, which revealed that overexpression of miR‑542‑3p can enhance the cell proliferation and migration ability of U2OS cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedics, Yidu Central Hospital, Weifang Medical College, Weifang, Shandong 262500, P.R. China.

ABSTRACT
Osteosarcoma is the most common histological form of primary bone cancer, which arises from osteoid tissue. It occurs predominantly in infants and adolescents, with an incidence of 4‑5 cases/100,000,000. The 5-year survival rate of patients with osteosarcoma has significantly improved over time; however, there remains a significant proportion of patients that respond poorly to chemotherapy. An improved understanding of the pathology of osteosarcoma is required to provide more effective treatment strategies, identify biomarkers and develop novel chemotherapeutic agents. Disturbance in microRNA (miRNA) expression has been identified in osteosarcoma tissues and cell lines; however, the roles of miRNA during osteosarcoma pathogenesis remain to be elucidated. In the present study, the expression levels of eight selected miRNAs were investigated in osteosarcoma tissues and the results revealed that the expression levels of miR‑542‑3p and miR‑542‑5p were significantly upregulated and the expression of miR‑199‑3p was significantly downregulated. Using a dual luciferase assay and western blot analysis, the present study confirmed that Van Gogh‑like 2, which is a non‑canonical Wnt pathway suppressor, was a target gene of miR‑542‑3p. Subsequently, the biological function of miR‑542‑3p in U2OS cells was examined, which revealed that overexpression of miR‑542‑3p can enhance the cell proliferation and migration ability of U2OS cells. This indicated that miR‑542‑3p may act as an oncogene in osteosarcoma pathogenesis. The findings of the present study may provide assistance in understanding the development of osteosarcoma and aid in the development of strategies for the diagnosis and treatment of osteosarcoma.

Show MeSH
Related in: MedlinePlus