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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.

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Upregulation of miR-329 inhibits the cell proliferation in glioma. A) MTT assays revealed that upregulation of miR-329 inhibited cell growth of glioma cell lines of T98G and LN18. B) Representative micrographs (left) and quantification (right) of crystal violet stained cell colonies. C) Upregulation of miR-329 inhibited glioma cell tumorigenicity as determined by the anchorage-independent growth assay. Representative micrographs (up) and quantification of colonies that were larger than 0.1 mm (down) were scored. D) Representative micrographs (left) and quantification of BrdU incorporating-cells after transfection with miR-329 or NC. Each bar represents the mean of three independent experiments. E) Flow cytometric analysis of the indicated glioma cells transfected with NC or miR-329. Each bar represents the mean of three independent experiments. * P < 0.05.
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Figure 2: Upregulation of miR-329 inhibits the cell proliferation in glioma. A) MTT assays revealed that upregulation of miR-329 inhibited cell growth of glioma cell lines of T98G and LN18. B) Representative micrographs (left) and quantification (right) of crystal violet stained cell colonies. C) Upregulation of miR-329 inhibited glioma cell tumorigenicity as determined by the anchorage-independent growth assay. Representative micrographs (up) and quantification of colonies that were larger than 0.1 mm (down) were scored. D) Representative micrographs (left) and quantification of BrdU incorporating-cells after transfection with miR-329 or NC. Each bar represents the mean of three independent experiments. E) Flow cytometric analysis of the indicated glioma cells transfected with NC or miR-329. Each bar represents the mean of three independent experiments. * P < 0.05.

Mentions: To explore the role of miR-329 downregulation in the development and progression of glioma, we examined its effect on cell proliferation. A MTT assay showed that miR-329 upregulation significantly inhibited the proliferation rate of LN18 and T98G glioma cells (Figure 2A), and this was further confirmed by a colony formation assay (Figure 2B). Strikingly, we found that enforced expression of miR-329 in LN18 and T98G glioma cells drastically inhibited their anchorage-independent growth ability (Figure 2C), as shown by decreased colony numbers and sizes, these results suggested that miR-329 upregulation inhibits glioma cell tumorigenicity in vitro. Using a BrdU incorporation assay, we found that the percentage of cells in S phase was dramatically decreased in miR-329-overexpressing LN18 (12.25%) and T98G (13.43%) cells compared with control cells (LN18 cells, 27.25%; T98G cells, 28.43%; Figure 2D). Similarly, the result of flow cytometry showed that miR-329 overexpression decreased the percentage of cells in S phase and significantly increased the percentage of cells in G1/G0 (Figure 2E). Collectively, our results suggest that miR-329 may induce the G1/S arrest and inhibit cell proliferation of glioma.


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

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

Upregulation of miR-329 inhibits the cell proliferation in glioma. A) MTT assays revealed that upregulation of miR-329 inhibited cell growth of glioma cell lines of T98G and LN18. B) Representative micrographs (left) and quantification (right) of crystal violet stained cell colonies. C) Upregulation of miR-329 inhibited glioma cell tumorigenicity as determined by the anchorage-independent growth assay. Representative micrographs (up) and quantification of colonies that were larger than 0.1 mm (down) were scored. D) Representative micrographs (left) and quantification of BrdU incorporating-cells after transfection with miR-329 or NC. Each bar represents the mean of three independent experiments. E) Flow cytometric analysis of the indicated glioma cells transfected with NC or miR-329. Each bar represents the mean of three independent experiments. * P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Upregulation of miR-329 inhibits the cell proliferation in glioma. A) MTT assays revealed that upregulation of miR-329 inhibited cell growth of glioma cell lines of T98G and LN18. B) Representative micrographs (left) and quantification (right) of crystal violet stained cell colonies. C) Upregulation of miR-329 inhibited glioma cell tumorigenicity as determined by the anchorage-independent growth assay. Representative micrographs (up) and quantification of colonies that were larger than 0.1 mm (down) were scored. D) Representative micrographs (left) and quantification of BrdU incorporating-cells after transfection with miR-329 or NC. Each bar represents the mean of three independent experiments. E) Flow cytometric analysis of the indicated glioma cells transfected with NC or miR-329. Each bar represents the mean of three independent experiments. * P < 0.05.
Mentions: To explore the role of miR-329 downregulation in the development and progression of glioma, we examined its effect on cell proliferation. A MTT assay showed that miR-329 upregulation significantly inhibited the proliferation rate of LN18 and T98G glioma cells (Figure 2A), and this was further confirmed by a colony formation assay (Figure 2B). Strikingly, we found that enforced expression of miR-329 in LN18 and T98G glioma cells drastically inhibited their anchorage-independent growth ability (Figure 2C), as shown by decreased colony numbers and sizes, these results suggested that miR-329 upregulation inhibits glioma cell tumorigenicity in vitro. Using a BrdU incorporation assay, we found that the percentage of cells in S phase was dramatically decreased in miR-329-overexpressing LN18 (12.25%) and T98G (13.43%) cells compared with control cells (LN18 cells, 27.25%; T98G cells, 28.43%; Figure 2D). Similarly, the result of flow cytometry showed that miR-329 overexpression decreased the percentage of cells in S phase and significantly increased the percentage of cells in G1/G0 (Figure 2E). Collectively, our results suggest that miR-329 may induce the G1/S arrest and inhibit cell proliferation of glioma.

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