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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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Related in: MedlinePlus

Expression analysis of miR-329 in glioma cell lines and glioma tissues. A) Real-time PCR analysis of miR-329 expression in three nonneoplastic brain specimens and primary glioma tissues of nine individual patients. B) Real-time PCR analysis of miR-329 expression in primary normal human astrocytes (NHA) and glioma cell lines (including A172, LN340, U118MG, LN464, SNB19, LN18, T98G, and U251MG). The average miR-329 expression was normalized by U6 expression. Each bar represents the mean of three independent experiments. * P < 0.05. C) Western blotting analysis of E2F1 in primary normal human astrocytes (NHA) and glioma cell lines (including A172, LN340, U118MG, LN464, SNB19, LN18, T98G, and U251MG), β-actin served as the loading control.
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Figure 1: Expression analysis of miR-329 in glioma cell lines and glioma tissues. A) Real-time PCR analysis of miR-329 expression in three nonneoplastic brain specimens and primary glioma tissues of nine individual patients. B) Real-time PCR analysis of miR-329 expression in primary normal human astrocytes (NHA) and glioma cell lines (including A172, LN340, U118MG, LN464, SNB19, LN18, T98G, and U251MG). The average miR-329 expression was normalized by U6 expression. Each bar represents the mean of three independent experiments. * P < 0.05. C) Western blotting analysis of E2F1 in primary normal human astrocytes (NHA) and glioma cell lines (including A172, LN340, U118MG, LN464, SNB19, LN18, T98G, and U251MG), β-actin served as the loading control.

Mentions: First, we examined miR-329 expression in GBM cell lines. Real-time RT-PCR was performed on a panel of 8 human GBM cell lines and primary normal human astrocytes. MiR-329 expression of each cell line was compared to the average expression level of primary normal human astrocytes (NHA). As shown in Figure 1B, miR-329 expression levels of all cell lines were lower than that of NHA, while expression levels of E2F1 in the cell lines were higher (Figure 1C). Downregulation of miR-329 was also found in clinical samples compared with nonneoplastic brain specimens (Figure 1A).


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

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

Expression analysis of miR-329 in glioma cell lines and glioma tissues. A) Real-time PCR analysis of miR-329 expression in three nonneoplastic brain specimens and primary glioma tissues of nine individual patients. B) Real-time PCR analysis of miR-329 expression in primary normal human astrocytes (NHA) and glioma cell lines (including A172, LN340, U118MG, LN464, SNB19, LN18, T98G, and U251MG). The average miR-329 expression was normalized by U6 expression. Each bar represents the mean of three independent experiments. * P < 0.05. C) Western blotting analysis of E2F1 in primary normal human astrocytes (NHA) and glioma cell lines (including A172, LN340, U118MG, LN464, SNB19, LN18, T98G, and U251MG), β-actin served as the loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Expression analysis of miR-329 in glioma cell lines and glioma tissues. A) Real-time PCR analysis of miR-329 expression in three nonneoplastic brain specimens and primary glioma tissues of nine individual patients. B) Real-time PCR analysis of miR-329 expression in primary normal human astrocytes (NHA) and glioma cell lines (including A172, LN340, U118MG, LN464, SNB19, LN18, T98G, and U251MG). The average miR-329 expression was normalized by U6 expression. Each bar represents the mean of three independent experiments. * P < 0.05. C) Western blotting analysis of E2F1 in primary normal human astrocytes (NHA) and glioma cell lines (including A172, LN340, U118MG, LN464, SNB19, LN18, T98G, and U251MG), β-actin served as the loading control.
Mentions: First, we examined miR-329 expression in GBM cell lines. Real-time RT-PCR was performed on a panel of 8 human GBM cell lines and primary normal human astrocytes. MiR-329 expression of each cell line was compared to the average expression level of primary normal human astrocytes (NHA). As shown in Figure 1B, miR-329 expression levels of all cell lines were lower than that of NHA, while expression levels of E2F1 in the cell lines were higher (Figure 1C). Downregulation of miR-329 was also found in clinical samples compared with nonneoplastic brain specimens (Figure 1A).

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