Limits...
MiRNA-329 targeting E2F1 inhibits cell proliferation in glioma cells.

Xiao B, Tan L, He B, Liu Z, Xu R - J Transl Med (2013)

Bottom Line: The E2F1 was identified as the target of miR-329.Overexpression of miR-329 blocked G1/S transition in LN18 and T98G cell lines, dramatically suppressed cell proliferation and the ability of colony formation.MiR-329 significantly decreased the phosphorylation levels of intracellular kinases Akt and expression of cyclin D1, but the expression of p21 was upregulated, cell growth was suppressed by inhibiting E2F1-mediated Akt pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Neurosurgery Department, General Hospital of Beijing Military Command of People's Liberation Army-PLA, Beijing 100700, P.R. China.

ABSTRACT

Background: MicroRNAs have recently emerged as key regulators of cancers, miR-329 located on 14q32.31 is one of down-regulated miRNAs in glioma, but the function and molecular mechanisms of miR-329 in determining the malignant phenotype of human glioma are elusive. This study therefore was conducted to investigate the role of miR-329 in biological behaviors of human glioma LN18 and T98G cell lines and its molecular mechanisms.

Methods: Nine patients with GBM were analyzed for the expression of miR-329 by quantitative RT-PCR. MiR-329 overexpression was established by transfecting miR-329 precursor into LN18 and T98G cells, and its effects on cell proliferation were studied using MTT assay, anchorage-independent growth ability assay, colony formation assays, Bromodeoxyuridine labeling and immunofluorescence.The effects of miR-329 on cell cycle were studied by flow cytometry. The target of miR-329 was determined by luciferase assays. The regulation of miR-329 on Akt pathway was determined by western blot.

Results: The E2F1 was identified as the target of miR-329. Overexpression of miR-329 blocked G1/S transition in LN18 and T98G cell lines, dramatically suppressed cell proliferation and the ability of colony formation. MiR-329 significantly decreased the phosphorylation levels of intracellular kinases Akt and expression of cyclin D1, but the expression of p21 was upregulated, cell growth was suppressed by inhibiting E2F1-mediated Akt pathway.

Conclusions: MiR-329 may inhibit cell proliferation in human glioma cells through regulating E2F1-mediated suppression of Akt pathway.

Show MeSH

Related in: MedlinePlus

MiR-329 suppresses E2F1 expression and Akt pathway. A) Predicted miR-329 target sequence (blue) in the 3′UTR of E2F1 (E2F1-3′UTR) and positions of the mutated nucleotides (green) in the miR-329 (miR-329 mut). B) Western blotting analysis of E2F1,pAkt, Akt, p21, cyclinD1 expression in cells transfected with miR-329 or the miR-329 inhibitor, β-actin served as the loading control. C) Western blotting analysis of GFP expression in the indicated cells, β-actin served as the loading control. D) Luciferase reporter assay of the indicated cells transfected with the pGL3-E2F1-3′UTR reporter and increasing amounts (10, 50 nM) of miR-329 mimic, miR-329 inhibitor or miR-329 mut oligonucleotides. Bars represent the mean ± SD of three independent experiments. * P <0.05. E) Luciferase reporter assay of the indicated cells transfected with pGL3-E2F1-3′UTR or pGL3-E2F1-3′UTR-mut reporter with increasing amounts (10, 50 nM) of miR-329 mimic. * P <0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750231&req=5

Figure 4: MiR-329 suppresses E2F1 expression and Akt pathway. A) Predicted miR-329 target sequence (blue) in the 3′UTR of E2F1 (E2F1-3′UTR) and positions of the mutated nucleotides (green) in the miR-329 (miR-329 mut). B) Western blotting analysis of E2F1,pAkt, Akt, p21, cyclinD1 expression in cells transfected with miR-329 or the miR-329 inhibitor, β-actin served as the loading control. C) Western blotting analysis of GFP expression in the indicated cells, β-actin served as the loading control. D) Luciferase reporter assay of the indicated cells transfected with the pGL3-E2F1-3′UTR reporter and increasing amounts (10, 50 nM) of miR-329 mimic, miR-329 inhibitor or miR-329 mut oligonucleotides. Bars represent the mean ± SD of three independent experiments. * P <0.05. E) Luciferase reporter assay of the indicated cells transfected with pGL3-E2F1-3′UTR or pGL3-E2F1-3′UTR-mut reporter with increasing amounts (10, 50 nM) of miR-329 mimic. * P <0.05.

Mentions: Analysis with the use of two publicly available algorithms (TargetScan and miRanda), we found that E2F1 mRNA is theoretically the target gene of miR-329 (Figure 4A). Importantly, western blotting analysis showed that ectopic expression of miR-329 dramatically decreased, but inhibition of miR-329 increased E2F1 protein expression in both LN18 and T98G glioma cells (Figure 4B). The pBABE-E2F1 overexpressing E2F1and pBABE-E2F1-3′UTR were respectively transfected into glioma cells with miR-329 mimic expressing using the Lipofectamine 2000 reagent. The result of colony formation assay showed overexpressing E2F1 significantly increased the proliferation rate of LN18 and T98G glioma cells compared with that cells expressing E2F1-3′UTR (Figure 5A), the rescuing experiment further confirmed that the inhibitory role of miR-329 in glioma cells may be mediated by E2F1. To examine whether miR-329 downregulation of E2F1 was mediated by the 3′-untranslated region (3′UTR) of E2F1, we subcloned the E2F1 3′UTR fragment, containing the miR-329 binding site, into pEGFP-C1 and pGL3 dual luciferase reporter vectors. As shown in Figure 4C, overexpressing miR-329 only decreased expression of a GFP vector containing the E2F1 3′UTR, but had no effect on GFP-γ-tubulin expression, the result suggested that miR-329 specifically affected the 3′UTR of E2F1. To validate that miR-329 can directly bind to and regulate the levels of E2F1 mRNA through the predicted binding sites, a mutant version of the reporter (pGL3–E2F1-3′UTR-mut plasmids) and altering bases in the putative miR-329 binding sites (miR-329 mut) were used in luciferase reporter assay. The consistent and dose-dependent reduction of luciferase activity was observed following miR-329 transfection in both glioma cells, the reporter assay revealed that the repressive effect of miR-329 on the luciferase activity of E2F1 3′UTR was abolished by miR-329 inhibitor but did not have the effect in the miR-329 mut group (Figure 4D). The overexpression of miR-329 also efficiently reduced the expression of the luciferase reporter in the pGL3–E2F1-3′UTR group but did not have the effect in the pGL3–E2F1-3′UTR-mut group (Figure 4E). Collectively, these results demonstrate that E2F1 is a bona fide target of miR-329.


MiRNA-329 targeting E2F1 inhibits cell proliferation in glioma cells.

Xiao B, Tan L, He B, Liu Z, Xu R - J Transl Med (2013)

MiR-329 suppresses E2F1 expression and Akt pathway. A) Predicted miR-329 target sequence (blue) in the 3′UTR of E2F1 (E2F1-3′UTR) and positions of the mutated nucleotides (green) in the miR-329 (miR-329 mut). B) Western blotting analysis of E2F1,pAkt, Akt, p21, cyclinD1 expression in cells transfected with miR-329 or the miR-329 inhibitor, β-actin served as the loading control. C) Western blotting analysis of GFP expression in the indicated cells, β-actin served as the loading control. D) Luciferase reporter assay of the indicated cells transfected with the pGL3-E2F1-3′UTR reporter and increasing amounts (10, 50 nM) of miR-329 mimic, miR-329 inhibitor or miR-329 mut oligonucleotides. Bars represent the mean ± SD of three independent experiments. * P <0.05. E) Luciferase reporter assay of the indicated cells transfected with pGL3-E2F1-3′UTR or pGL3-E2F1-3′UTR-mut reporter with increasing amounts (10, 50 nM) of miR-329 mimic. * P <0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750231&req=5

Figure 4: MiR-329 suppresses E2F1 expression and Akt pathway. A) Predicted miR-329 target sequence (blue) in the 3′UTR of E2F1 (E2F1-3′UTR) and positions of the mutated nucleotides (green) in the miR-329 (miR-329 mut). B) Western blotting analysis of E2F1,pAkt, Akt, p21, cyclinD1 expression in cells transfected with miR-329 or the miR-329 inhibitor, β-actin served as the loading control. C) Western blotting analysis of GFP expression in the indicated cells, β-actin served as the loading control. D) Luciferase reporter assay of the indicated cells transfected with the pGL3-E2F1-3′UTR reporter and increasing amounts (10, 50 nM) of miR-329 mimic, miR-329 inhibitor or miR-329 mut oligonucleotides. Bars represent the mean ± SD of three independent experiments. * P <0.05. E) Luciferase reporter assay of the indicated cells transfected with pGL3-E2F1-3′UTR or pGL3-E2F1-3′UTR-mut reporter with increasing amounts (10, 50 nM) of miR-329 mimic. * P <0.05.
Mentions: Analysis with the use of two publicly available algorithms (TargetScan and miRanda), we found that E2F1 mRNA is theoretically the target gene of miR-329 (Figure 4A). Importantly, western blotting analysis showed that ectopic expression of miR-329 dramatically decreased, but inhibition of miR-329 increased E2F1 protein expression in both LN18 and T98G glioma cells (Figure 4B). The pBABE-E2F1 overexpressing E2F1and pBABE-E2F1-3′UTR were respectively transfected into glioma cells with miR-329 mimic expressing using the Lipofectamine 2000 reagent. The result of colony formation assay showed overexpressing E2F1 significantly increased the proliferation rate of LN18 and T98G glioma cells compared with that cells expressing E2F1-3′UTR (Figure 5A), the rescuing experiment further confirmed that the inhibitory role of miR-329 in glioma cells may be mediated by E2F1. To examine whether miR-329 downregulation of E2F1 was mediated by the 3′-untranslated region (3′UTR) of E2F1, we subcloned the E2F1 3′UTR fragment, containing the miR-329 binding site, into pEGFP-C1 and pGL3 dual luciferase reporter vectors. As shown in Figure 4C, overexpressing miR-329 only decreased expression of a GFP vector containing the E2F1 3′UTR, but had no effect on GFP-γ-tubulin expression, the result suggested that miR-329 specifically affected the 3′UTR of E2F1. To validate that miR-329 can directly bind to and regulate the levels of E2F1 mRNA through the predicted binding sites, a mutant version of the reporter (pGL3–E2F1-3′UTR-mut plasmids) and altering bases in the putative miR-329 binding sites (miR-329 mut) were used in luciferase reporter assay. The consistent and dose-dependent reduction of luciferase activity was observed following miR-329 transfection in both glioma cells, the reporter assay revealed that the repressive effect of miR-329 on the luciferase activity of E2F1 3′UTR was abolished by miR-329 inhibitor but did not have the effect in the miR-329 mut group (Figure 4D). The overexpression of miR-329 also efficiently reduced the expression of the luciferase reporter in the pGL3–E2F1-3′UTR group but did not have the effect in the pGL3–E2F1-3′UTR-mut group (Figure 4E). Collectively, these results demonstrate that E2F1 is a bona fide target of miR-329.

Bottom Line: The E2F1 was identified as the target of miR-329.Overexpression of miR-329 blocked G1/S transition in LN18 and T98G cell lines, dramatically suppressed cell proliferation and the ability of colony formation.MiR-329 significantly decreased the phosphorylation levels of intracellular kinases Akt and expression of cyclin D1, but the expression of p21 was upregulated, cell growth was suppressed by inhibiting E2F1-mediated Akt pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Neurosurgery Department, General Hospital of Beijing Military Command of People's Liberation Army-PLA, Beijing 100700, P.R. China.

ABSTRACT

Background: MicroRNAs have recently emerged as key regulators of cancers, miR-329 located on 14q32.31 is one of down-regulated miRNAs in glioma, but the function and molecular mechanisms of miR-329 in determining the malignant phenotype of human glioma are elusive. This study therefore was conducted to investigate the role of miR-329 in biological behaviors of human glioma LN18 and T98G cell lines and its molecular mechanisms.

Methods: Nine patients with GBM were analyzed for the expression of miR-329 by quantitative RT-PCR. MiR-329 overexpression was established by transfecting miR-329 precursor into LN18 and T98G cells, and its effects on cell proliferation were studied using MTT assay, anchorage-independent growth ability assay, colony formation assays, Bromodeoxyuridine labeling and immunofluorescence.The effects of miR-329 on cell cycle were studied by flow cytometry. The target of miR-329 was determined by luciferase assays. The regulation of miR-329 on Akt pathway was determined by western blot.

Results: The E2F1 was identified as the target of miR-329. Overexpression of miR-329 blocked G1/S transition in LN18 and T98G cell lines, dramatically suppressed cell proliferation and the ability of colony formation. MiR-329 significantly decreased the phosphorylation levels of intracellular kinases Akt and expression of cyclin D1, but the expression of p21 was upregulated, cell growth was suppressed by inhibiting E2F1-mediated Akt pathway.

Conclusions: MiR-329 may inhibit cell proliferation in human glioma cells through regulating E2F1-mediated suppression of Akt pathway.

Show MeSH
Related in: MedlinePlus