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MiR-101 reverses the hypomethylation of the LMO3 promoter in glioma cells.

Liu X, Lei Q, Yu Z, Xu G, Tang H, Wang W, Wang Z, Li G, Wu M - Oncotarget (2015)

Bottom Line: MiR-101 decreased the expression of LMO3 by reversing the methylation status of the LMO3 promoter and by inhibiting the presence of the methylation-related histones H3K4me2 and H3K27me3 and increasing the presence of H3K9me3 and H4K20me3 on the promoter.It was determined that miR-101 decreases the occupancy of H3K27me3 by inhibiting EZH2, DNMT3A and EED and decreases the H3K9me3 occupancy on the LMO3 promoter via SUV39H1, SUV39H2, G9a and PHF8.Furthermore, miR-101 suppresses the expression of LMO3 by decreasing USF and MZF1.

View Article: PubMed Central - PubMed

Affiliation: Hunan Provincial Tumor Hospital and the Affiliated Tumor Hospital of Xiangya Medical School, Central South University, Changsha 410013, Hunan, China.

ABSTRACT
LIM-only protein 3 (LMO3), a member of the LIM-only protein group, is a new DNA methylation gene that was identified in gliomas via the MeDIP-Chip in our previous study. In this study, we found that LIM-only protein 3 (LMO3) is hypomethylated and overexpressed in glioma cells and tissues. The overexpression of LMO3 was correlated with a poor prognosis in glioma patients, and LMO3 was indirectly inhibited by the tumor suppressor miR-101, which is a potential prognosis marker of gliomas. MiR-101 decreased the expression of LMO3 by reversing the methylation status of the LMO3 promoter and by inhibiting the presence of the methylation-related histones H3K4me2 and H3K27me3 and increasing the presence of H3K9me3 and H4K20me3 on the promoter. It was determined that miR-101 decreases the occupancy of H3K27me3 by inhibiting EZH2, DNMT3A and EED and decreases the H3K9me3 occupancy on the LMO3 promoter via SUV39H1, SUV39H2, G9a and PHF8. Furthermore, miR-101 suppresses the expression of LMO3 by decreasing USF and MZF1.

No MeSH data available.


Related in: MedlinePlus

LMO3 is overexpressed due to promoter hypomethylation and is correlated with a poor outcome in gliomas(A) A schematic diagram of the CpG dinucleotides within the LMO3 promoter. The nucleotide number is relative to the transcription start site of LMO3. The red line indicates the region that was tested with BSP; the blue line indicates the region that was detected with MSP. (B) A BSP of the upstream regulatory region of LMO3 was performed for the 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. The unmethylated CpG sites are shown as open circles, whereas the methylated CpG sites are indicated with 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 to the right of each BSP result. (C) The methylation status of LMO3 was detected using BSP in the glioma samples (n = 50) and normal brain tissues (n = 10). This was verified using an independent sample t-test. *p < 0.01. (D) The methylation status of LMO3 in the glioma cell lines was detected using MSP. U, unmethylated primer; M, methylated primer. (E) Real-time PCR was used to detect the expression of LMO3. The expression level of LMO3 in the normal brain tissue was much lower than in the four glioma cell lines. This was verified using an independent sample t-test. *p < 0.01. (F) The expression of LMO3 in normal brain tissues and glioma tissues was tested using ISH. (G) The correlation between the LMO3 methylation in the tumor tissues and the OS of the glioma patients. The patients with hypomethylation of LMO3 had a shorter OS than those with normal levels of LMO3 methylation. The Kaplan–Meier method was used. (H) The correlation between the expression of the LMO3 protein in the tumor and the OS of the glioma patients. The patients with a high level of LMO3 expression had a poor outcome. The Kaplan–Meier method was used.
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Figure 1: LMO3 is overexpressed due to promoter hypomethylation and is correlated with a poor outcome in gliomas(A) A schematic diagram of the CpG dinucleotides within the LMO3 promoter. The nucleotide number is relative to the transcription start site of LMO3. The red line indicates the region that was tested with BSP; the blue line indicates the region that was detected with MSP. (B) A BSP of the upstream regulatory region of LMO3 was performed for the 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. The unmethylated CpG sites are shown as open circles, whereas the methylated CpG sites are indicated with 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 to the right of each BSP result. (C) The methylation status of LMO3 was detected using BSP in the glioma samples (n = 50) and normal brain tissues (n = 10). This was verified using an independent sample t-test. *p < 0.01. (D) The methylation status of LMO3 in the glioma cell lines was detected using MSP. U, unmethylated primer; M, methylated primer. (E) Real-time PCR was used to detect the expression of LMO3. The expression level of LMO3 in the normal brain tissue was much lower than in the four glioma cell lines. This was verified using an independent sample t-test. *p < 0.01. (F) The expression of LMO3 in normal brain tissues and glioma tissues was tested using ISH. (G) The correlation between the LMO3 methylation in the tumor tissues and the OS of the glioma patients. The patients with hypomethylation of LMO3 had a shorter OS than those with normal levels of LMO3 methylation. The Kaplan–Meier method was used. (H) The correlation between the expression of the LMO3 protein in the tumor and the OS of the glioma patients. The patients with a high level of LMO3 expression had a poor outcome. The Kaplan–Meier method was used.

Mentions: We previously demonstrated that the LMO3 gene, located at 12p12.3, is hypomethylated in glioma tissues [3]. To further confirm the hypomethylation of LMO3 in gliomas, we designed and validated the BSP and MSP methods (Figure 1A). The CpG dinucleotides were heavily methylated in the normal brain samples of Cohort 1 (n = 4, 88.4 ± 9.9%) and Cohort 2 (n = 10, 76.9% ± 12.6%), whereas methylation was decreased in the glioma samples (Cohort 2, n = 50, 10.7% ± 13.6%; Cohort 1, n = 6, 40.1 ± 21.9%) (p < 0.05 or 0.001) (Figure 1B and Supplementary Figure S1). Forty-three of the 50 (86.0%) samples were hypomethylated (p < 0.001) (Figure 1C). A decrease in the methylation was observed in the glioma cell lines (Figure 1D). There were no statistically significant correlations between the sex, age or histological grade and LMO3 hypomethylation (Table 1). The expression of LMO3 in normal brain tissues was lower compared to the glioma cell lines (Figure 1E), and increased expression of the LMO3 protein was found in the glioma cell lines and in 37 of the 50 glioma tissues (Figure 1F). There were no statistical correlations between the sex, age or histological grade and the expression of LMO3 (Table 2). A comparison of the methylation status with the protein expression revealed that 34 of the 37 tumors that had a high level of LMO3 expression were hypomethylated (Table 1). There was a significant relationship between the hypomethylation of the LMO3 promoter and the overexpression of the LMO3 protein (χ2-test, p < 0.05, Table 1). The correlation between the LMO3 expression, methylation status and overall survival (OS) was statistically significant (Figure 1G and 1H). These results suggest that LMO3 overexpression and hypomethylation may be involved in glioma carcinogenesis, and LMO3 plays a potential role in glioma prognosis.


MiR-101 reverses the hypomethylation of the LMO3 promoter in glioma cells.

Liu X, Lei Q, Yu Z, Xu G, Tang H, Wang W, Wang Z, Li G, Wu M - Oncotarget (2015)

LMO3 is overexpressed due to promoter hypomethylation and is correlated with a poor outcome in gliomas(A) A schematic diagram of the CpG dinucleotides within the LMO3 promoter. The nucleotide number is relative to the transcription start site of LMO3. The red line indicates the region that was tested with BSP; the blue line indicates the region that was detected with MSP. (B) A BSP of the upstream regulatory region of LMO3 was performed for the 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. The unmethylated CpG sites are shown as open circles, whereas the methylated CpG sites are indicated with 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 to the right of each BSP result. (C) The methylation status of LMO3 was detected using BSP in the glioma samples (n = 50) and normal brain tissues (n = 10). This was verified using an independent sample t-test. *p < 0.01. (D) The methylation status of LMO3 in the glioma cell lines was detected using MSP. U, unmethylated primer; M, methylated primer. (E) Real-time PCR was used to detect the expression of LMO3. The expression level of LMO3 in the normal brain tissue was much lower than in the four glioma cell lines. This was verified using an independent sample t-test. *p < 0.01. (F) The expression of LMO3 in normal brain tissues and glioma tissues was tested using ISH. (G) The correlation between the LMO3 methylation in the tumor tissues and the OS of the glioma patients. The patients with hypomethylation of LMO3 had a shorter OS than those with normal levels of LMO3 methylation. The Kaplan–Meier method was used. (H) The correlation between the expression of the LMO3 protein in the tumor and the OS of the glioma patients. The patients with a high level of LMO3 expression had a poor outcome. The Kaplan–Meier method was used.
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Related In: Results  -  Collection

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Figure 1: LMO3 is overexpressed due to promoter hypomethylation and is correlated with a poor outcome in gliomas(A) A schematic diagram of the CpG dinucleotides within the LMO3 promoter. The nucleotide number is relative to the transcription start site of LMO3. The red line indicates the region that was tested with BSP; the blue line indicates the region that was detected with MSP. (B) A BSP of the upstream regulatory region of LMO3 was performed for the 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. The unmethylated CpG sites are shown as open circles, whereas the methylated CpG sites are indicated with 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 to the right of each BSP result. (C) The methylation status of LMO3 was detected using BSP in the glioma samples (n = 50) and normal brain tissues (n = 10). This was verified using an independent sample t-test. *p < 0.01. (D) The methylation status of LMO3 in the glioma cell lines was detected using MSP. U, unmethylated primer; M, methylated primer. (E) Real-time PCR was used to detect the expression of LMO3. The expression level of LMO3 in the normal brain tissue was much lower than in the four glioma cell lines. This was verified using an independent sample t-test. *p < 0.01. (F) The expression of LMO3 in normal brain tissues and glioma tissues was tested using ISH. (G) The correlation between the LMO3 methylation in the tumor tissues and the OS of the glioma patients. The patients with hypomethylation of LMO3 had a shorter OS than those with normal levels of LMO3 methylation. The Kaplan–Meier method was used. (H) The correlation between the expression of the LMO3 protein in the tumor and the OS of the glioma patients. The patients with a high level of LMO3 expression had a poor outcome. The Kaplan–Meier method was used.
Mentions: We previously demonstrated that the LMO3 gene, located at 12p12.3, is hypomethylated in glioma tissues [3]. To further confirm the hypomethylation of LMO3 in gliomas, we designed and validated the BSP and MSP methods (Figure 1A). The CpG dinucleotides were heavily methylated in the normal brain samples of Cohort 1 (n = 4, 88.4 ± 9.9%) and Cohort 2 (n = 10, 76.9% ± 12.6%), whereas methylation was decreased in the glioma samples (Cohort 2, n = 50, 10.7% ± 13.6%; Cohort 1, n = 6, 40.1 ± 21.9%) (p < 0.05 or 0.001) (Figure 1B and Supplementary Figure S1). Forty-three of the 50 (86.0%) samples were hypomethylated (p < 0.001) (Figure 1C). A decrease in the methylation was observed in the glioma cell lines (Figure 1D). There were no statistically significant correlations between the sex, age or histological grade and LMO3 hypomethylation (Table 1). The expression of LMO3 in normal brain tissues was lower compared to the glioma cell lines (Figure 1E), and increased expression of the LMO3 protein was found in the glioma cell lines and in 37 of the 50 glioma tissues (Figure 1F). There were no statistical correlations between the sex, age or histological grade and the expression of LMO3 (Table 2). A comparison of the methylation status with the protein expression revealed that 34 of the 37 tumors that had a high level of LMO3 expression were hypomethylated (Table 1). There was a significant relationship between the hypomethylation of the LMO3 promoter and the overexpression of the LMO3 protein (χ2-test, p < 0.05, Table 1). The correlation between the LMO3 expression, methylation status and overall survival (OS) was statistically significant (Figure 1G and 1H). These results suggest that LMO3 overexpression and hypomethylation may be involved in glioma carcinogenesis, and LMO3 plays a potential role in glioma prognosis.

Bottom Line: MiR-101 decreased the expression of LMO3 by reversing the methylation status of the LMO3 promoter and by inhibiting the presence of the methylation-related histones H3K4me2 and H3K27me3 and increasing the presence of H3K9me3 and H4K20me3 on the promoter.It was determined that miR-101 decreases the occupancy of H3K27me3 by inhibiting EZH2, DNMT3A and EED and decreases the H3K9me3 occupancy on the LMO3 promoter via SUV39H1, SUV39H2, G9a and PHF8.Furthermore, miR-101 suppresses the expression of LMO3 by decreasing USF and MZF1.

View Article: PubMed Central - PubMed

Affiliation: Hunan Provincial Tumor Hospital and the Affiliated Tumor Hospital of Xiangya Medical School, Central South University, Changsha 410013, Hunan, China.

ABSTRACT
LIM-only protein 3 (LMO3), a member of the LIM-only protein group, is a new DNA methylation gene that was identified in gliomas via the MeDIP-Chip in our previous study. In this study, we found that LIM-only protein 3 (LMO3) is hypomethylated and overexpressed in glioma cells and tissues. The overexpression of LMO3 was correlated with a poor prognosis in glioma patients, and LMO3 was indirectly inhibited by the tumor suppressor miR-101, which is a potential prognosis marker of gliomas. MiR-101 decreased the expression of LMO3 by reversing the methylation status of the LMO3 promoter and by inhibiting the presence of the methylation-related histones H3K4me2 and H3K27me3 and increasing the presence of H3K9me3 and H4K20me3 on the promoter. It was determined that miR-101 decreases the occupancy of H3K27me3 by inhibiting EZH2, DNMT3A and EED and decreases the H3K9me3 occupancy on the LMO3 promoter via SUV39H1, SUV39H2, G9a and PHF8. Furthermore, miR-101 suppresses the expression of LMO3 by decreasing USF and MZF1.

No MeSH data available.


Related in: MedlinePlus