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miR-93 promotes cell proliferation in gliomas through activation of PI3K/Akt signaling pathway.

Jiang L, Wang C, Lei F, Zhang L, Zhang X, Liu A, Wu G, Zhu J, Song L - Oncotarget (2015)

Bottom Line: Multiple regulators, such as phosphatase and tensin homolog (PTEN) and PH domain leucine rich repeat protein phosphatases (PHLPP), have also found to be involved in suppression of the PI3K/Akt signaling pathway.Furthermore, we found that overexpressing miR-93 promoted, but inhibition of miR-93 reduced, glioma cell proliferation and cell-cycle progression.Therefore, our results suggest that miR-93 might play an important role in glioma progression and uncover a novel mechanism for constitutive PI3K/Akt activation in gliomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Guangzhou Medical University, Guangzhou 510182, China.

ABSTRACT
The PI3K/Akt signaling pathway is frequently activated in various human cancer types and plays essential roles in development and progression of cancers. Multiple regulators, such as phosphatase and tensin homolog (PTEN) and PH domain leucine rich repeat protein phosphatases (PHLPP), have also found to be involved in suppression of the PI3K/Akt signaling pathway. However, how suppressive effects mediated by these regulators are concomitantly disrupted in cancers, which display constitutively activated PI3K/Akt signaling, remains puzzling. In the present study, we reported that the expression of miR-93 was markedly upregulated in glioma cell lines and clinical glioma tissues. Statistical analysis revealed that miR-93 levels significantly correlated with clinicopathologic grade and overall survival in gliomas. Furthermore, we found that overexpressing miR-93 promoted, but inhibition of miR-93 reduced, glioma cell proliferation and cell-cycle progression. We demonstrated that miR-93 activated PI3K/Akt signaling through directly suppressing PTEN, PHLPP2 and FOXO3 expression via targeting their 3'UTRs. Therefore, our results suggest that miR-93 might play an important role in glioma progression and uncover a novel mechanism for constitutive PI3K/Akt activation in gliomas.

No MeSH data available.


Related in: MedlinePlus

miR-93 activates PI3K/Akt signaling(A) Relative Akt activity in the indicated cells, determined by K-LISA Akt Activity assay. (B) Western blotting analysis of phosphorylated Akt (p-Akt) (Ser 473), total Akt, Cyclin D1 and p27Kip1 protein levels in indicated cells. α-Tubulin was used as a loading control. (C) The mRNA expression level of Cyclin D1 and p27Kip1, determined by real-time PCR analysis. (D) Quantification of colonies formed determined by colony formation assay in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). (E) Quantification of colonies determined by anchorage-independent growth ability assay in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). (F) Quantification of BrdUrd incorporating-cells in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). (G) The mRNA expression level of Cyclin D1 and p27Kip1, determined by real-time PCR analysis, in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). Experiments were repeated at least 3 times with similar results, and error bars represent ± SD. *P < 0.05.
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Figure 6: miR-93 activates PI3K/Akt signaling(A) Relative Akt activity in the indicated cells, determined by K-LISA Akt Activity assay. (B) Western blotting analysis of phosphorylated Akt (p-Akt) (Ser 473), total Akt, Cyclin D1 and p27Kip1 protein levels in indicated cells. α-Tubulin was used as a loading control. (C) The mRNA expression level of Cyclin D1 and p27Kip1, determined by real-time PCR analysis. (D) Quantification of colonies formed determined by colony formation assay in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). (E) Quantification of colonies determined by anchorage-independent growth ability assay in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). (F) Quantification of BrdUrd incorporating-cells in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). (G) The mRNA expression level of Cyclin D1 and p27Kip1, determined by real-time PCR analysis, in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). Experiments were repeated at least 3 times with similar results, and error bars represent ± SD. *P < 0.05.

Mentions: It has been demonstrated that PTEN and PHLPP2 are critical negative regulators of PI3K/Akt signaling, which prompted us to further examine whether dysregulation miR-93 alters the activity of PI3K/Akt signaling in glioma cells. As shown in Figure 6A and 6B, overexpressing miR-93 significantly increased, but silencing miR-93 decreased, the Akt activity and the expression of phosphorylated Akt (Ser 473) in glioma cells. Consistently, the transcriptional and translational levels of Cyclin D1 and p27kip1, two downstream effectors of PI3K/Akt signaling, were also significantly alliterated in the miR-93-deregulated glioma cells (Figure 6B and 6C). These results indicate that miR-93 activates PI3K/Akt signaling. Furthermore, we examined whether activation of PI3K/Akt signaling contributed to miR-93-mediated gliomas cell proliferation. As shown in Figure 6D and 6E, inactivation of PI3K/Akt signaling using Akt inhibitor significantly decreased the growth rates of miR-93-transduced glioma cells, analyzed by colony formation and anchorage-independent growth assays. Moreover, the percentage of BrdUrd incorporating-cells and Cyclin D1 expression significantly decreased, but the expression of p27kip1 increased, in the miR-93-transduced cells treated with Akt inhibitor (Figure 6F and 6G). Taken together, our results demonstrate that PI3K/Akt signaling plays essential function during miR-93-induced glioma cells proliferation.


miR-93 promotes cell proliferation in gliomas through activation of PI3K/Akt signaling pathway.

Jiang L, Wang C, Lei F, Zhang L, Zhang X, Liu A, Wu G, Zhu J, Song L - Oncotarget (2015)

miR-93 activates PI3K/Akt signaling(A) Relative Akt activity in the indicated cells, determined by K-LISA Akt Activity assay. (B) Western blotting analysis of phosphorylated Akt (p-Akt) (Ser 473), total Akt, Cyclin D1 and p27Kip1 protein levels in indicated cells. α-Tubulin was used as a loading control. (C) The mRNA expression level of Cyclin D1 and p27Kip1, determined by real-time PCR analysis. (D) Quantification of colonies formed determined by colony formation assay in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). (E) Quantification of colonies determined by anchorage-independent growth ability assay in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). (F) Quantification of BrdUrd incorporating-cells in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). (G) The mRNA expression level of Cyclin D1 and p27Kip1, determined by real-time PCR analysis, in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). Experiments were repeated at least 3 times with similar results, and error bars represent ± SD. *P < 0.05.
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Figure 6: miR-93 activates PI3K/Akt signaling(A) Relative Akt activity in the indicated cells, determined by K-LISA Akt Activity assay. (B) Western blotting analysis of phosphorylated Akt (p-Akt) (Ser 473), total Akt, Cyclin D1 and p27Kip1 protein levels in indicated cells. α-Tubulin was used as a loading control. (C) The mRNA expression level of Cyclin D1 and p27Kip1, determined by real-time PCR analysis. (D) Quantification of colonies formed determined by colony formation assay in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). (E) Quantification of colonies determined by anchorage-independent growth ability assay in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). (F) Quantification of BrdUrd incorporating-cells in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). (G) The mRNA expression level of Cyclin D1 and p27Kip1, determined by real-time PCR analysis, in indicated glioma cell lines, treated with Akt inhibitor (0.5 μM). Experiments were repeated at least 3 times with similar results, and error bars represent ± SD. *P < 0.05.
Mentions: It has been demonstrated that PTEN and PHLPP2 are critical negative regulators of PI3K/Akt signaling, which prompted us to further examine whether dysregulation miR-93 alters the activity of PI3K/Akt signaling in glioma cells. As shown in Figure 6A and 6B, overexpressing miR-93 significantly increased, but silencing miR-93 decreased, the Akt activity and the expression of phosphorylated Akt (Ser 473) in glioma cells. Consistently, the transcriptional and translational levels of Cyclin D1 and p27kip1, two downstream effectors of PI3K/Akt signaling, were also significantly alliterated in the miR-93-deregulated glioma cells (Figure 6B and 6C). These results indicate that miR-93 activates PI3K/Akt signaling. Furthermore, we examined whether activation of PI3K/Akt signaling contributed to miR-93-mediated gliomas cell proliferation. As shown in Figure 6D and 6E, inactivation of PI3K/Akt signaling using Akt inhibitor significantly decreased the growth rates of miR-93-transduced glioma cells, analyzed by colony formation and anchorage-independent growth assays. Moreover, the percentage of BrdUrd incorporating-cells and Cyclin D1 expression significantly decreased, but the expression of p27kip1 increased, in the miR-93-transduced cells treated with Akt inhibitor (Figure 6F and 6G). Taken together, our results demonstrate that PI3K/Akt signaling plays essential function during miR-93-induced glioma cells proliferation.

Bottom Line: Multiple regulators, such as phosphatase and tensin homolog (PTEN) and PH domain leucine rich repeat protein phosphatases (PHLPP), have also found to be involved in suppression of the PI3K/Akt signaling pathway.Furthermore, we found that overexpressing miR-93 promoted, but inhibition of miR-93 reduced, glioma cell proliferation and cell-cycle progression.Therefore, our results suggest that miR-93 might play an important role in glioma progression and uncover a novel mechanism for constitutive PI3K/Akt activation in gliomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Guangzhou Medical University, Guangzhou 510182, China.

ABSTRACT
The PI3K/Akt signaling pathway is frequently activated in various human cancer types and plays essential roles in development and progression of cancers. Multiple regulators, such as phosphatase and tensin homolog (PTEN) and PH domain leucine rich repeat protein phosphatases (PHLPP), have also found to be involved in suppression of the PI3K/Akt signaling pathway. However, how suppressive effects mediated by these regulators are concomitantly disrupted in cancers, which display constitutively activated PI3K/Akt signaling, remains puzzling. In the present study, we reported that the expression of miR-93 was markedly upregulated in glioma cell lines and clinical glioma tissues. Statistical analysis revealed that miR-93 levels significantly correlated with clinicopathologic grade and overall survival in gliomas. Furthermore, we found that overexpressing miR-93 promoted, but inhibition of miR-93 reduced, glioma cell proliferation and cell-cycle progression. We demonstrated that miR-93 activated PI3K/Akt signaling through directly suppressing PTEN, PHLPP2 and FOXO3 expression via targeting their 3'UTRs. Therefore, our results suggest that miR-93 might play an important role in glioma progression and uncover a novel mechanism for constitutive PI3K/Akt activation in gliomas.

No MeSH data available.


Related in: MedlinePlus