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Overexpression of miR-506 suppresses proliferation and promotes apoptosis of osteosarcoma cells by targeting astrocyte elevated gene-1

View Article: PubMed Central - PubMed

ABSTRACT

There is increasing evidence that microRNAs (miRs) are implicated in tumor development and progression; however, their specific roles in osteosarcoma are not well understood. The aim of the present study was to investigate the role of miR-506 in the pathogenesis of osteosarcoma. The expression levels of miR-506 and astrocyte elevated gene-1 (AEG-1) mRNA were detected using quantitative polymerase chain reaction, and the protein levels of AEG-1, β-catenin, c-myc and cyclin D1 were determined using western blot analysis. The effects of miR-506 and AEG-1 on cell viability, colony forming ability and apoptosis were assessed using MTT assay, colony formation assay, and flow cytometry, respectively. Lucifer reporter assays were used to demonstrate whether AEG-1 is a direct target of miR-506. The present study identified that miR-506 was downregulated in osteosarcoma tissues and cells. Overexpression of miR-506 suppressed the proliferation and induced apoptosis in osteosarcoma cells in vitro and inhibited tumor formation in vivo. Overexpression of miR-506 significantly inhibited the luciferase activity of AEG-1 with a wild-type 3′-untranslated region, providing clear evidence that AEG-1 was a direct and functional downstream target of miR-506. Similar to the overexpression of miR-506, downregulation of AEG-1 lead to an inhibitory effect on osteosarcoma in vitro. Furthermore, overexpression of miR-506 or downregulation of AEG-1 inhibited the Wnt/β-catenin signaling pathway, and inhibition of this pathway by β-catenin small interfering RNA or CGP049090, a small molecule inhibitor, suppressed cell proliferation and induced apoptosis in vitro. Overall, the present data indicated that miR-506 functions as a tumor suppressor by targeting AEG-1 in osteosarcoma via the regulation of the Wnt/β-catenin signaling pathway.

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AEG-1 is a direct target of miR-506 in human osteosarcoma MG63 cells. (A) Specific binding locations of miR-506 and the 3′-UTR of AEG-1. (B) Luciferase reporter assay showed that the relative luciferase activity of AEG-1 3′-UTR was significantly decreased in MG63 cells co-transfected with wt or mut AEG-1 3′-UTR and miR-506 mimics. (C and D) Western blot analysis showed that the relative level of AEG-1 protein (64 kDa) was decreased in MG63 cells following transfection with miR-506 mimics, but was increased in human normal osteoblastic hFOB 1.19 cells following transfection with anti-miR-506 mimics. *P<0.05, **P<0.01 vs. miR-control; #P<0.05 vs. anti-miR-control. miR, microRNA; AEG-1, astrocyte elevated gene-1; UTR, untranslated region; wt, wild-type; mut, mutant.
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f4-ol-0-0-4827: AEG-1 is a direct target of miR-506 in human osteosarcoma MG63 cells. (A) Specific binding locations of miR-506 and the 3′-UTR of AEG-1. (B) Luciferase reporter assay showed that the relative luciferase activity of AEG-1 3′-UTR was significantly decreased in MG63 cells co-transfected with wt or mut AEG-1 3′-UTR and miR-506 mimics. (C and D) Western blot analysis showed that the relative level of AEG-1 protein (64 kDa) was decreased in MG63 cells following transfection with miR-506 mimics, but was increased in human normal osteoblastic hFOB 1.19 cells following transfection with anti-miR-506 mimics. *P<0.05, **P<0.01 vs. miR-control; #P<0.05 vs. anti-miR-control. miR, microRNA; AEG-1, astrocyte elevated gene-1; UTR, untranslated region; wt, wild-type; mut, mutant.

Mentions: Bioinformatics analysis using TargetScan suggested that miR-506 was a predicted to target AEG-1, and the ‘seed sequence’ of miR-506 matched the 3′-UTR of the AEG-1 mRNA (Fig. 4A). To confirm AEG-1 was directly targeted and regulated by miR-506 in MG63 cells, luciferase reporter genes were constructed using the AEG-1 3′-UTR and the mutant counterpart at the miR-506 binding regions, and these were co-transfected with miR-506 mimics or miR-control into MG63 cells. Overexpression of miR-506 significantly inhibited the luciferase activity of AEG-1 with the wild-type 3′-UTR (P=0.0018), but not with mutant 3′-UTR (Fig. 4B). To further determine whether miR-506 could functionally affect the expression of AEG-1, the present study determined if the expression level of AEG-1 was regulated by miR-506. The results demonstrated that overexpression of miR-506 suppressed the expression of AEG-1 in MG63 cells, and downregulation of miR-506 increased the level of AEG-1 in hFOB 1.19 cells (P=0.0168 and P=0.0401, respectively; Fig. 4C and D). Overall, these results suggest that the 3′-UTR of AEG-1 is a functional target site of miR-506 in osteosarcoma cells.


Overexpression of miR-506 suppresses proliferation and promotes apoptosis of osteosarcoma cells by targeting astrocyte elevated gene-1
AEG-1 is a direct target of miR-506 in human osteosarcoma MG63 cells. (A) Specific binding locations of miR-506 and the 3′-UTR of AEG-1. (B) Luciferase reporter assay showed that the relative luciferase activity of AEG-1 3′-UTR was significantly decreased in MG63 cells co-transfected with wt or mut AEG-1 3′-UTR and miR-506 mimics. (C and D) Western blot analysis showed that the relative level of AEG-1 protein (64 kDa) was decreased in MG63 cells following transfection with miR-506 mimics, but was increased in human normal osteoblastic hFOB 1.19 cells following transfection with anti-miR-506 mimics. *P<0.05, **P<0.01 vs. miR-control; #P<0.05 vs. anti-miR-control. miR, microRNA; AEG-1, astrocyte elevated gene-1; UTR, untranslated region; wt, wild-type; mut, mutant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4998420&req=5

f4-ol-0-0-4827: AEG-1 is a direct target of miR-506 in human osteosarcoma MG63 cells. (A) Specific binding locations of miR-506 and the 3′-UTR of AEG-1. (B) Luciferase reporter assay showed that the relative luciferase activity of AEG-1 3′-UTR was significantly decreased in MG63 cells co-transfected with wt or mut AEG-1 3′-UTR and miR-506 mimics. (C and D) Western blot analysis showed that the relative level of AEG-1 protein (64 kDa) was decreased in MG63 cells following transfection with miR-506 mimics, but was increased in human normal osteoblastic hFOB 1.19 cells following transfection with anti-miR-506 mimics. *P<0.05, **P<0.01 vs. miR-control; #P<0.05 vs. anti-miR-control. miR, microRNA; AEG-1, astrocyte elevated gene-1; UTR, untranslated region; wt, wild-type; mut, mutant.
Mentions: Bioinformatics analysis using TargetScan suggested that miR-506 was a predicted to target AEG-1, and the ‘seed sequence’ of miR-506 matched the 3′-UTR of the AEG-1 mRNA (Fig. 4A). To confirm AEG-1 was directly targeted and regulated by miR-506 in MG63 cells, luciferase reporter genes were constructed using the AEG-1 3′-UTR and the mutant counterpart at the miR-506 binding regions, and these were co-transfected with miR-506 mimics or miR-control into MG63 cells. Overexpression of miR-506 significantly inhibited the luciferase activity of AEG-1 with the wild-type 3′-UTR (P=0.0018), but not with mutant 3′-UTR (Fig. 4B). To further determine whether miR-506 could functionally affect the expression of AEG-1, the present study determined if the expression level of AEG-1 was regulated by miR-506. The results demonstrated that overexpression of miR-506 suppressed the expression of AEG-1 in MG63 cells, and downregulation of miR-506 increased the level of AEG-1 in hFOB 1.19 cells (P=0.0168 and P=0.0401, respectively; Fig. 4C and D). Overall, these results suggest that the 3′-UTR of AEG-1 is a functional target site of miR-506 in osteosarcoma cells.

View Article: PubMed Central - PubMed

ABSTRACT

There is increasing evidence that microRNAs (miRs) are implicated in tumor development and progression; however, their specific roles in osteosarcoma are not well understood. The aim of the present study was to investigate the role of miR-506 in the pathogenesis of osteosarcoma. The expression levels of miR-506 and astrocyte elevated gene-1 (AEG-1) mRNA were detected using quantitative polymerase chain reaction, and the protein levels of AEG-1, &beta;-catenin, c-myc and cyclin D1 were determined using western blot analysis. The effects of miR-506 and AEG-1 on cell viability, colony forming ability and apoptosis were assessed using MTT assay, colony formation assay, and flow cytometry, respectively. Lucifer reporter assays were used to demonstrate whether AEG-1 is a direct target of miR-506. The present study identified that miR-506 was downregulated in osteosarcoma tissues and cells. Overexpression of miR-506 suppressed the proliferation and induced apoptosis in osteosarcoma cells in vitro and inhibited tumor formation in vivo. Overexpression of miR-506 significantly inhibited the luciferase activity of AEG-1 with a wild-type 3&prime;-untranslated region, providing clear evidence that AEG-1 was a direct and functional downstream target of miR-506. Similar to the overexpression of miR-506, downregulation of AEG-1 lead to an inhibitory effect on osteosarcoma in vitro. Furthermore, overexpression of miR-506 or downregulation of AEG-1 inhibited the Wnt/&beta;-catenin signaling pathway, and inhibition of this pathway by &beta;-catenin small interfering RNA or CGP049090, a small molecule inhibitor, suppressed cell proliferation and induced apoptosis in vitro. Overall, the present data indicated that miR-506 functions as a tumor suppressor by targeting AEG-1 in osteosarcoma via the regulation of the Wnt/&beta;-catenin signaling pathway.

No MeSH data available.


Related in: MedlinePlus