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MiR-136 modulates glioma cell sensitivity to temozolomide by targeting astrocyte elevated gene-1.

Wu H, Liu Q, Cai T, Chen YD, Liao F, Wang ZF - Diagn Pathol (2014)

Bottom Line: Recent studies have linked chemotherapy resistance to the altered expression of microRNAs (miRNAs).Downregulation of AEG-1 expression by siRNAs, U251 cells became more sensitive to the therapy of TMZ.In addition, the enhanced growth-inhibitory effect by the miR-136 mimics transfection was enhanced after the addition of AEG-1 siRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, The Third Xiangya Hospital, Central South University, 138 Tongzipo Road, Hexi Yuelu District, Changsha 400013, Hunan, China. dr_wangzhifei@126.com.

ABSTRACT

Background: Recent studies have linked chemotherapy resistance to the altered expression of microRNAs (miRNAs). Thus, miRNA-based approaches to modulating sensitivity to temozolomide (TMZ) may overcome chemoresistance. The aim of the present study was to investigate whether miR-136 could modulates glioma cell sensitivity to TMZ.

Methods: The proliferation of glioma U251 cell line was evaluated by MTT assay. The expression of astrocyte elevated gene-1 (AEG-1)was detected by real‑time polymerase chain reaction (RT-PCR)and Western blot. The luciferase reporter gene was used to test whether AEG-1 was the target of the miR-136.

Results: The MTT assay showed that U251 cells with miR-136 overexpression were significantly more sensitive to the therapy of TMZ than control cells. Luciferase assays revealed that miR-136 directly targeted the 3'UTR of AEG-1. qRT-PCR and western blotting analysis found that AEG-1 expression at the mRNA and protein levels decreased in the miR-136 mimic-treatment group relative to control group. Downregulation of AEG-1 expression by siRNAs, U251 cells became more sensitive to the therapy of TMZ. In addition, the enhanced growth-inhibitory effect by the miR-136 mimics transfection was enhanced after the addition of AEG-1 siRNA.

Conclusions: The present study provides the first evidence that miR-136 plays a key role in TMZ resistance by targeting the AEG-1 protein in glioma cell line, suggesting that miR-136 can be used to predict a patient's response to TMZ therapy as well as serve as a novel potential maker for glioma therapy.

Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_173.

No MeSH data available.


Related in: MedlinePlus

AEG-1 was a direct target of miR-136. A: the luciferase activity significantly decreased after co-transfection with psiCHECK-2/AEG-1 3’-UTR and miR-136 mimics in comparison with control cells. B: AEG-1 mRNA level was detected by qRT–PCR in U251 cells transfected with miR-136 mimic or the control. C: AEG-1 protein level was detected by Western blotting in U251 cells transfected with miR-136 mimic or the control.
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Fig2: AEG-1 was a direct target of miR-136. A: the luciferase activity significantly decreased after co-transfection with psiCHECK-2/AEG-1 3’-UTR and miR-136 mimics in comparison with control cells. B: AEG-1 mRNA level was detected by qRT–PCR in U251 cells transfected with miR-136 mimic or the control. C: AEG-1 protein level was detected by Western blotting in U251 cells transfected with miR-136 mimic or the control.

Mentions: Predicted miR-136 targets were identified using the algorithms of TargetScan and microRNA. Among hundreds of potential target genes, we specifically focused on AEG-1, as it has been consistently reported to be oncogene in various cancers. We identified a binding site for miR-136 in the 3’-UTR of AEG-1 mRNA. To confirm that miR-136 can bind to the predicted site, we performed a luciferase reporter assay in the 293 T cell line. Figure 2A showed that the luciferase activity significantly decreased after co-transfection with psiCHECK-2/ AEG-1 3’-UTR and miR-136 mimics in comparison with control cells. The results demonstrated that miR-136 specifically binded to the 3’-UTR of AEG-1 mRNA. The effect of miR-136 transfection on AEG-1 mRNA and protein expression was respectively assessed using qRT-PCR and Western blot in U251 cell line. As shown in Figure 2B and C, the qRT–PCR and western blotting analysis found that AEG-1 expression at the mRNA and protein levels decreased in the miR-136 mimic-treatment group relative to NC.Figure 2


MiR-136 modulates glioma cell sensitivity to temozolomide by targeting astrocyte elevated gene-1.

Wu H, Liu Q, Cai T, Chen YD, Liao F, Wang ZF - Diagn Pathol (2014)

AEG-1 was a direct target of miR-136. A: the luciferase activity significantly decreased after co-transfection with psiCHECK-2/AEG-1 3’-UTR and miR-136 mimics in comparison with control cells. B: AEG-1 mRNA level was detected by qRT–PCR in U251 cells transfected with miR-136 mimic or the control. C: AEG-1 protein level was detected by Western blotting in U251 cells transfected with miR-136 mimic or the control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4195982&req=5

Fig2: AEG-1 was a direct target of miR-136. A: the luciferase activity significantly decreased after co-transfection with psiCHECK-2/AEG-1 3’-UTR and miR-136 mimics in comparison with control cells. B: AEG-1 mRNA level was detected by qRT–PCR in U251 cells transfected with miR-136 mimic or the control. C: AEG-1 protein level was detected by Western blotting in U251 cells transfected with miR-136 mimic or the control.
Mentions: Predicted miR-136 targets were identified using the algorithms of TargetScan and microRNA. Among hundreds of potential target genes, we specifically focused on AEG-1, as it has been consistently reported to be oncogene in various cancers. We identified a binding site for miR-136 in the 3’-UTR of AEG-1 mRNA. To confirm that miR-136 can bind to the predicted site, we performed a luciferase reporter assay in the 293 T cell line. Figure 2A showed that the luciferase activity significantly decreased after co-transfection with psiCHECK-2/ AEG-1 3’-UTR and miR-136 mimics in comparison with control cells. The results demonstrated that miR-136 specifically binded to the 3’-UTR of AEG-1 mRNA. The effect of miR-136 transfection on AEG-1 mRNA and protein expression was respectively assessed using qRT-PCR and Western blot in U251 cell line. As shown in Figure 2B and C, the qRT–PCR and western blotting analysis found that AEG-1 expression at the mRNA and protein levels decreased in the miR-136 mimic-treatment group relative to NC.Figure 2

Bottom Line: Recent studies have linked chemotherapy resistance to the altered expression of microRNAs (miRNAs).Downregulation of AEG-1 expression by siRNAs, U251 cells became more sensitive to the therapy of TMZ.In addition, the enhanced growth-inhibitory effect by the miR-136 mimics transfection was enhanced after the addition of AEG-1 siRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, The Third Xiangya Hospital, Central South University, 138 Tongzipo Road, Hexi Yuelu District, Changsha 400013, Hunan, China. dr_wangzhifei@126.com.

ABSTRACT

Background: Recent studies have linked chemotherapy resistance to the altered expression of microRNAs (miRNAs). Thus, miRNA-based approaches to modulating sensitivity to temozolomide (TMZ) may overcome chemoresistance. The aim of the present study was to investigate whether miR-136 could modulates glioma cell sensitivity to TMZ.

Methods: The proliferation of glioma U251 cell line was evaluated by MTT assay. The expression of astrocyte elevated gene-1 (AEG-1)was detected by real‑time polymerase chain reaction (RT-PCR)and Western blot. The luciferase reporter gene was used to test whether AEG-1 was the target of the miR-136.

Results: The MTT assay showed that U251 cells with miR-136 overexpression were significantly more sensitive to the therapy of TMZ than control cells. Luciferase assays revealed that miR-136 directly targeted the 3'UTR of AEG-1. qRT-PCR and western blotting analysis found that AEG-1 expression at the mRNA and protein levels decreased in the miR-136 mimic-treatment group relative to control group. Downregulation of AEG-1 expression by siRNAs, U251 cells became more sensitive to the therapy of TMZ. In addition, the enhanced growth-inhibitory effect by the miR-136 mimics transfection was enhanced after the addition of AEG-1 siRNA.

Conclusions: The present study provides the first evidence that miR-136 plays a key role in TMZ resistance by targeting the AEG-1 protein in glioma cell line, suggesting that miR-136 can be used to predict a patient's response to TMZ therapy as well as serve as a novel potential maker for glioma therapy.

Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_173.

No MeSH data available.


Related in: MedlinePlus