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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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Inhibition of miR-329 promotes cell proliferation in glioma. A) MTT assays revealed that inhibition of miR-329 increased cell growth of glioma cell lines of T98G and LN18. B) Representative micrographs (left) and quantification (right) of crystal violet stained cell colonies. C) Inhibition of miR-329 increased the anchorage-independent growth of glioma cells. 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 inhibitor or NC. E) Flow cytometric analysis of the indicated glioma cells transfected with NC or miR-329 inhibitor. Each bar represents the mean of three independent experiments. * P < 0.05.
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Figure 3: Inhibition of miR-329 promotes cell proliferation in glioma. A) MTT assays revealed that inhibition of miR-329 increased cell growth of glioma cell lines of T98G and LN18. B) Representative micrographs (left) and quantification (right) of crystal violet stained cell colonies. C) Inhibition of miR-329 increased the anchorage-independent growth of glioma cells. 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 inhibitor or NC. E) Flow cytometric analysis of the indicated glioma cells transfected with NC or miR-329 inhibitor. Each bar represents the mean of three independent experiments. * P < 0.05.

Mentions: We further examined the effect of miR-329 inhibition on cell proliferation in glioma. Consistent with above mentioned results, MTT and colony formation assays showed that miR-329 suppression dramatically increased the growth rate of both LN18 and T98G glioma cells as compared with that of control cells transfected with negative control (NC) (Figures 3A and 3B). In addition, the anchorage-independent growth ability of LN18 and T98G glioma cells was significantly increased in response to miR-329 inhibitor (Figure 3C). Furthermore, we found that transfection of the miR-329 inhibitor drastically increased the percentage of cells in the S peak but decreased the percentage of cells in the G0/G1 peak (Figures 3D and 3E).These results suggested that the proliferative effect of inhibiting miR-329 in glioma cells may occur through regulation of G1/S transition.


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

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

Inhibition of miR-329 promotes cell proliferation in glioma. A) MTT assays revealed that inhibition of miR-329 increased cell growth of glioma cell lines of T98G and LN18. B) Representative micrographs (left) and quantification (right) of crystal violet stained cell colonies. C) Inhibition of miR-329 increased the anchorage-independent growth of glioma cells. 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 inhibitor or NC. E) Flow cytometric analysis of the indicated glioma cells transfected with NC or miR-329 inhibitor. Each bar represents the mean of three independent experiments. * P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Inhibition of miR-329 promotes cell proliferation in glioma. A) MTT assays revealed that inhibition of miR-329 increased cell growth of glioma cell lines of T98G and LN18. B) Representative micrographs (left) and quantification (right) of crystal violet stained cell colonies. C) Inhibition of miR-329 increased the anchorage-independent growth of glioma cells. 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 inhibitor or NC. E) Flow cytometric analysis of the indicated glioma cells transfected with NC or miR-329 inhibitor. Each bar represents the mean of three independent experiments. * P < 0.05.
Mentions: We further examined the effect of miR-329 inhibition on cell proliferation in glioma. Consistent with above mentioned results, MTT and colony formation assays showed that miR-329 suppression dramatically increased the growth rate of both LN18 and T98G glioma cells as compared with that of control cells transfected with negative control (NC) (Figures 3A and 3B). In addition, the anchorage-independent growth ability of LN18 and T98G glioma cells was significantly increased in response to miR-329 inhibitor (Figure 3C). Furthermore, we found that transfection of the miR-329 inhibitor drastically increased the percentage of cells in the S peak but decreased the percentage of cells in the G0/G1 peak (Figures 3D and 3E).These results suggested that the proliferative effect of inhibiting miR-329 in glioma cells may occur through regulation of G1/S transition.

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