CPEB1, a histone-modified hypomethylated gene, is regulated by miR-101 and involved in cell senescence in glioma

Abstract

Epigenetic mechanisms have important roles in carcinogenesis. We certified that the mRNA translation-related gene cytoplasmic polyadenylation element-binding protein 1 (CPEB1) is hypomethylated and overexpressed in glioma cells and tissues. The knockdown of CPEB1 reduced cell senescence by regulating the expression or distribution of p53 in glioma cells. CPEB1 is also regulated directly by the tumor suppressor miR-101, a potential marker of glioma. It is known that the histone methyltransferase enhancer of zeste homolog 2 (EZH2) and embryonic ectoderm development (EED) are direct targets of miR-101. We demonstrated that miR-101 downregulated the expression of CPEB1 through reversing the methylation status of the CPEB1 promoter by regulating the presence on the promoter of the methylation-related histones H3K4me2, H3K27me3, H3K9me3 and H4K20me3. The epigenetic regulation of H3K27me3 on CPEB1 promoter is mediated by EZH2 and EED. EZH2 has a role in the regulation of H3K4me2. Furthermore, the downregulation of CPEB1 induced senescence in a p53-dependent manner.

Figure 1

CPEB1 is overexpressed due to promoter hypomethylation and correlates with a poor outcome in glioma. (a) Schematic diagram of CpG dinucleotides within the CPEB1 promoter. The nucleotide number is relative to the transcription start site of CPEB1. The red line indicates the region tested with BSP; the blue line indicates the region detected with MSP. (b) The methylation status of CPEB1 in glioma cell lines was detected with MSP. U, unmethylated primer, M, methylated primer. (c) BSP of the upstream regulatory region of CPEB1 was performed for representative tissues (N, normal brain tissue, T, glioma sample). For each sample, at least five separate clones were sequenced, and the results are shown here. Unmethylated CpG sites are shown as open circles, whereas methylated CpG sites are indicated by closed circles. For each row of circles, the sequence results for an individual clone of the bisulfite-PCR product are given. The number of methylated CpGs divided by the total number of true CpGs analyzed is given as a percentage on the right of each BSP result. *P<0.05. (d) Real-time PCR was used to detect the expression of CPEB1. The expression level of CPEB1 in the normal brain tissue was much lower than that in the four glioma cell lines. *P<0.05. (e) The expression of CPEB1 in normal brain tissues and glioma tissues was tested by ISH. (f) The correlation between CPEB1 protein expression in the tumor and the OS of the glioma patients. The patients with a high level of CPEB1 expression had a poor outcome. (g) The correlation between CPEB1 methylation in the tumor tissue and the OS of the glioma patients. The patients with hypomethylation of CPEB1 had a shorter OS than those with normal levels of CPEB1 methylation

Figure 2

CPEB1 reduces senescence by regulating p53 distribution in glioma cells. (a) A cell growth assay was used to analyze the function of CPEB1 siRNA in glioma cell proliferation. *P<0.05. (b) The effect of CPEB1 siRNA on senescence in glioma cell lines as determined with SA-β-gal staining. Arrows indicate the green senescent cells. *P<0.05. (c) Transmission electron micrographs of the morphology of glioma cells transfected with CPEB1 siRNA. (d) The regulation of p53 and p21 by CPEB1 siRNA. (e) The changes in the cellular distribution of p53 induced by CPEB1 siRNA in the U87 cell line

Figure 3

CPEB1 is a direct and epigenetic target of miR-101. (a) miR-101 binds to the 3′-UTR of CPEB1 as shown by the predicted binding between miR-101 and the 7mer seed matches in the CPEB1 3′-UTR. miR-101 regulates the expression of the CPEB1 3′-UTR reporter constructs. The luciferase reporter assays were performed 48 h after transfection with the indicated pMIR-REPORT plasmids and a renilla transfection control plasmid that were cotransfected with miR-101 or a relevant scrambled control. The data shown are the mean±S.D. of six replicates and are representative of three-independent experiments. *P<0.05. (b) miR-101 regulates CPEB1 expression. A real-time PCR analysis was performed 48 h after transfection with miR-101 mimics, an inhibitor of miR-101 or a scrambled control. A western blot analysis was performed 72 h after transfection with miR-101 mimics, an inhibitor or a scrambled control. The gain of miR-101 function assay was performed in U251 and U87 cells. The loss of miR-101 function assay was performed in U251 cells. *P<0.05. (c) The expression of CPEB1 is regulated by EZH2 siRNA and EED siRNA. A real-time PCR analysis was performed 48 h after transfection with EZH2 siRNA, EED siRNA or a scrambled control. A western blot analysis was performed 72 h after transfection with EZH2 siRNA, EED siRNA or a scrambled control. *P<0.05. (d) A luciferase reporter assay defines the position of the CPEB1 core promoter. 0 is the TSS. The core promoter ranged from −307 to −207. pGL3-control is the positive control, pGL3-enhancer is the negative control. The relative activity as compared with the pRL-TK plasmid is shown. Where the relative luciferase activity is higher than the pGL3-enhancer is considered to embody the core promoter region. *P<0.05. (e) A ChIP assay was performed to detect the H3K4me2, H3K27me3, H3K9me3 and H4K20me3 occupancy of the CPEB1 core promoter. U251 and U87 cells transfected with miR-101 mimics, EZH2 siRNA and EED siRNA were analyzed. *P<0.05. (f) The methylation status of CPEB1 is affected by miR-101. The unmethylated CpG sites are shown as open circles, whereas the methylated CpG sites are indicated by closed circles. For each row of circles, the sequence results for an individual clone of the bisulfite-PCR product are given. The number of methylated CpGs divided by the total number of true CpGs analyzed is given as a percentage on the right of each BSP result. The methylation level of CPEB1 is increased by miR-101 in the U251 and U87 cells as determined with BSP. MiR-101 mimics reverse the methylation status of CPEB1 as determined with MSP in U251 cells

Figure 3

CPEB1 is a direct and epigenetic target of miR-101. (a) miR-101 binds to the 3′-UTR of CPEB1 as shown by the predicted binding between miR-101 and the 7mer seed matches in the CPEB1 3′-UTR. miR-101 regulates the expression of the CPEB1 3′-UTR reporter constructs. The luciferase reporter assays were performed 48 h after transfection with the indicated pMIR-REPORT plasmids and a renilla transfection control plasmid that were cotransfected with miR-101 or a relevant scrambled control. The data shown are the mean±S.D. of six replicates and are representative of three-independent experiments. *P<0.05. (b) miR-101 regulates CPEB1 expression. A real-time PCR analysis was performed 48 h after transfection with miR-101 mimics, an inhibitor of miR-101 or a scrambled control. A western blot analysis was performed 72 h after transfection with miR-101 mimics, an inhibitor or a scrambled control. The gain of miR-101 function assay was performed in U251 and U87 cells. The loss of miR-101 function assay was performed in U251 cells. *P<0.05. (c) The expression of CPEB1 is regulated by EZH2 siRNA and EED siRNA. A real-time PCR analysis was performed 48 h after transfection with EZH2 siRNA, EED siRNA or a scrambled control. A western blot analysis was performed 72 h after transfection with EZH2 siRNA, EED siRNA or a scrambled control. *P<0.05. (d) A luciferase reporter assay defines the position of the CPEB1 core promoter. 0 is the TSS. The core promoter ranged from −307 to −207. pGL3-control is the positive control, pGL3-enhancer is the negative control. The relative activity as compared with the pRL-TK plasmid is shown. Where the relative luciferase activity is higher than the pGL3-enhancer is considered to embody the core promoter region. *P<0.05. (e) A ChIP assay was performed to detect the H3K4me2, H3K27me3, H3K9me3 and H4K20me3 occupancy of the CPEB1 core promoter. U251 and U87 cells transfected with miR-101 mimics, EZH2 siRNA and EED siRNA were analyzed. *P<0.05. (f) The methylation status of CPEB1 is affected by miR-101. The unmethylated CpG sites are shown as open circles, whereas the methylated CpG sites are indicated by closed circles. For each row of circles, the sequence results for an individual clone of the bisulfite-PCR product are given. The number of methylated CpGs divided by the total number of true CpGs analyzed is given as a percentage on the right of each BSP result. The methylation level of CPEB1 is increased by miR-101 in the U251 and U87 cells as determined with BSP. MiR-101 mimics reverse the methylation status of CPEB1 as determined with MSP in U251 cells

Figure 4

miR-101 is frequently downregulated in human glioma cell lines and tissues and is associated with a poor prognosis. (a) The expression of miR-101 in normal brain tissue and glioma cell lines. The real-time PCR data indicate that miR-101 is downregulated in glioma cell lines. *P<0.05. (b) The expression of miR-101 in glioma tissues and normal brain tissues. The ISH assay indicates that miR-101 is expressed at high levels in normal brain tissue, whereas the expression is lost in glioma tissues. (c) The correlation between miR-101 expression in the tumor and the OS of glioma patients

Figure 5

miR-101 acts as a tumor suppressor and induces senescence in glioma cell lines. (a) The inhibition of proliferation by miR-101 in glioma cells. (b) A wound-healing assay analyzes the effect of miR-101 on glioma cells. *P<0.05. (c) The effect of miR-101 on senescence in glioma cells was measured with SA-β-gal staining. Arrows point to the green senescent cells. *P<0.05. (d) Transmission electron micrographs of the morphology of the glioma cells transfected with miR-101 mimics. The U251 cells exhibited cytoplasmic and nuclear concentration, chromatin aggregation at the periphery of the nucleus and mitochondrial vacuolar degeneration after transfection with miR-101. Two different lesions were observed in the treated U87 cells: one was cellular edema, including in the cytoplasm, nucleus and mitochondria; the other was cellular condensation, including cytoplasmic, nuclear and mitochondrial vacuolar degeneration

Figure 6

miR-101 induces glioma cell senescence in a p53-dependent manner. (a) Western blot analysis of the expression of p53 and p21. (b) Western blot analyzes the distribution changes in U87 cells