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Role of microRNA-26b in glioma development and its mediated regulation on EphA2.

Wu N, Zhao X, Liu M, Liu H, Yao W, Zhang Y, Cao S, Lin X - PLoS ONE (2011)

Bottom Line: A binding site for miR-26b was identified in the 3'UTR of EphA2.Vasculogenic mimicry (VM) experiments were performed to further confirm the effects of miR-26b on the regulation of EphA2, and the results showed that miR-26b inhibited the VM processes which regulated by EphA2.EphA2 is a direct target of miR-26b, and the down-regulation of EphA2 mediated by miR-26b is dependent on the binding of miR-26b to a specific response element of microRNA in the 3'UTR region of EphA2 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.

ABSTRACT

Background: MicroRNAs (miRNAs) are short, non-coding RNAs that regulate the expression of multiple target genes. Deregulation of miRNAs is common in human tumorigenesis. Low level expression of miR-26b has been found in glioma cells. However, its underlying mechanism of action has not been determined.

Methodology/principal findings: Real-time PCR was employed to measure the expression level of miR-26b in glioma patients and cells. The level of miR-26b was inversely correlated with the grade of glioma. Ectopic expression of miR-26b inhibited the proliferation, migration and invasion of human glioma cells. A binding site for miR-26b was identified in the 3'UTR of EphA2. Over-expression of miR-26b in glioma cells repressed the endogenous level of EphA2 protein. Vasculogenic mimicry (VM) experiments were performed to further confirm the effects of miR-26b on the regulation of EphA2, and the results showed that miR-26b inhibited the VM processes which regulated by EphA2.

Significance: This study demonstrated that miR-26b may act as a tumor suppressor in glioma and it directly regulates EphA2 expression. EphA2 is a direct target of miR-26b, and the down-regulation of EphA2 mediated by miR-26b is dependent on the binding of miR-26b to a specific response element of microRNA in the 3'UTR region of EphA2 mRNA.

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Ectopic expression of miR-26b inhibits glioma cell proliferation in vitro.(A) The proliferation of glioma cell lines, U251 and C6. Cells were first transfected with miR-26b duplex, negative control RNA duplex, co-transfected with 26b-DP and miR-26b specific antisense oligonucleotides (26b-AS) or negative antisense oligonucleotides (NC-AS). After incubation for 48 h, cell proliferation rates were analyzed by MTT assay. The growth rates of cells transfected with NC-DP were defined as 1.0. **P<0.01, as compared to NC-DP group. (B) The cell proliferation inhibition rates of glioma cell lines of U251 and C6 after miR-26b transfection at certain time points. 26b-DP was transfected into U251 or C6 cells, and the cell proliferation inhibition rates were evaluated by MTT assay at 12, 24, 36, 48 and 72 h. The inhibitory rates of cells were the percentage of the ratio of cells proliferation transfected with miR-26b to that transfected with NC-DP.
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pone-0016264-g002: Ectopic expression of miR-26b inhibits glioma cell proliferation in vitro.(A) The proliferation of glioma cell lines, U251 and C6. Cells were first transfected with miR-26b duplex, negative control RNA duplex, co-transfected with 26b-DP and miR-26b specific antisense oligonucleotides (26b-AS) or negative antisense oligonucleotides (NC-AS). After incubation for 48 h, cell proliferation rates were analyzed by MTT assay. The growth rates of cells transfected with NC-DP were defined as 1.0. **P<0.01, as compared to NC-DP group. (B) The cell proliferation inhibition rates of glioma cell lines of U251 and C6 after miR-26b transfection at certain time points. 26b-DP was transfected into U251 or C6 cells, and the cell proliferation inhibition rates were evaluated by MTT assay at 12, 24, 36, 48 and 72 h. The inhibitory rates of cells were the percentage of the ratio of cells proliferation transfected with miR-26b to that transfected with NC-DP.

Mentions: We next determined the effect of miR-26b on the proliferation of glioma cells. The growth ability of glioma cells was determined by MTT assay. As shown in Fig. 2A, over-expression of miR-26b resulted in the growth inhibition of both U251 human glioma cells and C6 rat glioma cells. However, the growth inhibition induced by miR-26b was abolished when an antisense of miR-26b (26b-AS) was introduced into the cells (Fig. 2A). In contrast, substituting the 26b-AS with a negative control single strand RNA (NC-AS), similar inhibition effect on cell proliferation was found as the transfected 26b-DP alone (Fig. 2A). We examined the inhibitory effect of miR-26b on glioma cells at different time points and found that the maximum inhibition was at the 48 h (Fig. 2B). The inhibition rates were 34.28% and 29.547% in U251 and C6 cells transfected with 26b-DP, respectively (Fig. 2B). These results suggest that miR-26b plays a key role in the proliferation of some glioma cells, and might function as a tumor suppressor in glioma cell lines.


Role of microRNA-26b in glioma development and its mediated regulation on EphA2.

Wu N, Zhao X, Liu M, Liu H, Yao W, Zhang Y, Cao S, Lin X - PLoS ONE (2011)

Ectopic expression of miR-26b inhibits glioma cell proliferation in vitro.(A) The proliferation of glioma cell lines, U251 and C6. Cells were first transfected with miR-26b duplex, negative control RNA duplex, co-transfected with 26b-DP and miR-26b specific antisense oligonucleotides (26b-AS) or negative antisense oligonucleotides (NC-AS). After incubation for 48 h, cell proliferation rates were analyzed by MTT assay. The growth rates of cells transfected with NC-DP were defined as 1.0. **P<0.01, as compared to NC-DP group. (B) The cell proliferation inhibition rates of glioma cell lines of U251 and C6 after miR-26b transfection at certain time points. 26b-DP was transfected into U251 or C6 cells, and the cell proliferation inhibition rates were evaluated by MTT assay at 12, 24, 36, 48 and 72 h. The inhibitory rates of cells were the percentage of the ratio of cells proliferation transfected with miR-26b to that transfected with NC-DP.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3021542&req=5

pone-0016264-g002: Ectopic expression of miR-26b inhibits glioma cell proliferation in vitro.(A) The proliferation of glioma cell lines, U251 and C6. Cells were first transfected with miR-26b duplex, negative control RNA duplex, co-transfected with 26b-DP and miR-26b specific antisense oligonucleotides (26b-AS) or negative antisense oligonucleotides (NC-AS). After incubation for 48 h, cell proliferation rates were analyzed by MTT assay. The growth rates of cells transfected with NC-DP were defined as 1.0. **P<0.01, as compared to NC-DP group. (B) The cell proliferation inhibition rates of glioma cell lines of U251 and C6 after miR-26b transfection at certain time points. 26b-DP was transfected into U251 or C6 cells, and the cell proliferation inhibition rates were evaluated by MTT assay at 12, 24, 36, 48 and 72 h. The inhibitory rates of cells were the percentage of the ratio of cells proliferation transfected with miR-26b to that transfected with NC-DP.
Mentions: We next determined the effect of miR-26b on the proliferation of glioma cells. The growth ability of glioma cells was determined by MTT assay. As shown in Fig. 2A, over-expression of miR-26b resulted in the growth inhibition of both U251 human glioma cells and C6 rat glioma cells. However, the growth inhibition induced by miR-26b was abolished when an antisense of miR-26b (26b-AS) was introduced into the cells (Fig. 2A). In contrast, substituting the 26b-AS with a negative control single strand RNA (NC-AS), similar inhibition effect on cell proliferation was found as the transfected 26b-DP alone (Fig. 2A). We examined the inhibitory effect of miR-26b on glioma cells at different time points and found that the maximum inhibition was at the 48 h (Fig. 2B). The inhibition rates were 34.28% and 29.547% in U251 and C6 cells transfected with 26b-DP, respectively (Fig. 2B). These results suggest that miR-26b plays a key role in the proliferation of some glioma cells, and might function as a tumor suppressor in glioma cell lines.

Bottom Line: A binding site for miR-26b was identified in the 3'UTR of EphA2.Vasculogenic mimicry (VM) experiments were performed to further confirm the effects of miR-26b on the regulation of EphA2, and the results showed that miR-26b inhibited the VM processes which regulated by EphA2.EphA2 is a direct target of miR-26b, and the down-regulation of EphA2 mediated by miR-26b is dependent on the binding of miR-26b to a specific response element of microRNA in the 3'UTR region of EphA2 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.

ABSTRACT

Background: MicroRNAs (miRNAs) are short, non-coding RNAs that regulate the expression of multiple target genes. Deregulation of miRNAs is common in human tumorigenesis. Low level expression of miR-26b has been found in glioma cells. However, its underlying mechanism of action has not been determined.

Methodology/principal findings: Real-time PCR was employed to measure the expression level of miR-26b in glioma patients and cells. The level of miR-26b was inversely correlated with the grade of glioma. Ectopic expression of miR-26b inhibited the proliferation, migration and invasion of human glioma cells. A binding site for miR-26b was identified in the 3'UTR of EphA2. Over-expression of miR-26b in glioma cells repressed the endogenous level of EphA2 protein. Vasculogenic mimicry (VM) experiments were performed to further confirm the effects of miR-26b on the regulation of EphA2, and the results showed that miR-26b inhibited the VM processes which regulated by EphA2.

Significance: This study demonstrated that miR-26b may act as a tumor suppressor in glioma and it directly regulates EphA2 expression. EphA2 is a direct target of miR-26b, and the down-regulation of EphA2 mediated by miR-26b is dependent on the binding of miR-26b to a specific response element of microRNA in the 3'UTR region of EphA2 mRNA.

Show MeSH
Related in: MedlinePlus