Limits...
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 an epigenetic target gene of miR-101(A) LMO3 was predicted to be a target gene of miR-101 using the online software program TargetScan 5.1. (B) MiR-101 did not regulate the expression of the CPEB1 3′-UTR reporter constructs. The luciferase reporter assays were performed 48 h after transfection with the indicated pMIR-REPORT plasmid and a Renilla transfection control plasmid, which 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. An independent sample t-test was used. *p < 0.05. (C) MiR-101 inhibits the expression of LMO3 mRNA. A real-time PCR analysis was performed 48 h after transfection with miR-101 mimics and a scrambled control. An independent sample t-test was used. *p < 0.05. **p < 0.01. (D) MiR-101 knockdown enhances the expression of LMO3 mRNA. Real-time PCR analysis was performed 48 h after transfection with an inhibitor of miR-101 and a negative control. An independent sample t-test was used. **p < 0.01. (E) MiR-101 regulates the expression of the LMO3 protein. Western blot analysis was performed 72 h after transfection with miR-101 mimics, an inhibitor or a scrambled control. GAPDH was used as an internal control. (F) The methylation level of LMO3 was increased by miR-101 in U251 cells. The unmethylated and methylated CpG sites are indicated by open and closed circles, respectively. Each row indicates the sequencing result of one clone of the bisulfite-PCR product. The number of methylated CpGs was divided by the total number of true CpGs analyzed and is given as a percentage to the right of each BSP result. (G) An miR-101 inhibitor had no effect on the methylation status of LMO3, as determined via MSP in U251 cells. (H) An analysis of the promoter activity of the LMO3 core promoter constructs via luciferase reporter assays. The construct containing the sequence spanning the region from –431 to –281 was sufficient to mediate maximal promoter activity. The core promoter ranged from –431 to –281. PGL3-control is the positive control, and pGL3-enhancer is the negative control. An independent sample t-test was used. *p < 0.05. (I) The histone occupancy of the LMO3 promoter was affected by miR-101. A ChIP assay was used to detect the H3K4me2, H3K27me3, H3K9me3 and H4K20me3 occupancy of the LMO3 core promoter. U251 cells were transfected with miR-101 mimics or a scrambled control. An independent sample t-test was used. *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4480726&req=5

Figure 2: LMO3 is an epigenetic target gene of miR-101(A) LMO3 was predicted to be a target gene of miR-101 using the online software program TargetScan 5.1. (B) MiR-101 did not regulate the expression of the CPEB1 3′-UTR reporter constructs. The luciferase reporter assays were performed 48 h after transfection with the indicated pMIR-REPORT plasmid and a Renilla transfection control plasmid, which 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. An independent sample t-test was used. *p < 0.05. (C) MiR-101 inhibits the expression of LMO3 mRNA. A real-time PCR analysis was performed 48 h after transfection with miR-101 mimics and a scrambled control. An independent sample t-test was used. *p < 0.05. **p < 0.01. (D) MiR-101 knockdown enhances the expression of LMO3 mRNA. Real-time PCR analysis was performed 48 h after transfection with an inhibitor of miR-101 and a negative control. An independent sample t-test was used. **p < 0.01. (E) MiR-101 regulates the expression of the LMO3 protein. Western blot analysis was performed 72 h after transfection with miR-101 mimics, an inhibitor or a scrambled control. GAPDH was used as an internal control. (F) The methylation level of LMO3 was increased by miR-101 in U251 cells. The unmethylated and methylated CpG sites are indicated by open and closed circles, respectively. Each row indicates the sequencing result of one clone of the bisulfite-PCR product. The number of methylated CpGs was divided by the total number of true CpGs analyzed and is given as a percentage to the right of each BSP result. (G) An miR-101 inhibitor had no effect on the methylation status of LMO3, as determined via MSP in U251 cells. (H) An analysis of the promoter activity of the LMO3 core promoter constructs via luciferase reporter assays. The construct containing the sequence spanning the region from –431 to –281 was sufficient to mediate maximal promoter activity. The core promoter ranged from –431 to –281. PGL3-control is the positive control, and pGL3-enhancer is the negative control. An independent sample t-test was used. *p < 0.05. (I) The histone occupancy of the LMO3 promoter was affected by miR-101. A ChIP assay was used to detect the H3K4me2, H3K27me3, H3K9me3 and H4K20me3 occupancy of the LMO3 core promoter. U251 cells were transfected with miR-101 mimics or a scrambled control. An independent sample t-test was used. *p < 0.05.

Mentions: Having established the hypomethylation role for LMO3 in gliomas, we next aimed to clarify the regulatory mechanism of LMO3 expression. We used the online software TargetScan 5.1 (Cambridge, MA, USA) to predict potential miRNA binding sites in the 3′-UTR sequence of LMO3. LMO3 was predicted to be a target of miR-101 (Figure 2A), and the predicted binding sites were cloned downstream of the firefly luciferase gene in the pMIR-REPORT vector (Supplementary Figure S2). When the cells were cotransfected with miR-101 and pMIR-LMO3– 3′-UTR-WT, there was no significant reduction in luciferase activity compared with cells transfected with the negative control (Figure 2B). Our previous study demonstrated the suppressor role of miR-101 in gliomas [6]; this finding is consistent with the results of the present study, which showed that the overexpression of miR-101 (Supplementary Figure S3A) inhibited the expression of LMO3 (Figure 2C and 2E) and that the knockdown of miR-101 (Supplementary Figure S3B) could enhance the expression of LMO3 in glioma cell lines (Figure 2D and 2E). LMO3 can therefore be considered to be a new indirect target of miR-101 in gliomas. Our previous study confirmed that miR-101 suppresses its targets via histone modification [6]. To determine whether miR-101 inhibits the expression of LMO3 via histone modification, the methylation status of LMO3 was detected using BSP and MSP. The demethylation rate was decreased in the glioma cell lines following the transfection with the miR-101 mimics (Figure 2F). However, the effect of the miR-101 inhibitor on the methylation status of the LMO3 promoter was not significant (Figure 2G). Subsequently, we determined the core promoter region of LMO3. The luciferase reporter assay demonstrated that the core promoter ranged from –431 to –281 (Figure 2H). As shown in Figure 2I, we found that the occupancy of H3K4me2 and H3K27me3 was decreased, whereas the occupancy of H3K9me3 and H4K20me3 was increased at the LMO3 core promoter in the miR-101 mimic-treated cells compared to the control. These results indicate that miR-101 inhibits the expression of LMO3 epigenetically in glioma cells.


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 an epigenetic target gene of miR-101(A) LMO3 was predicted to be a target gene of miR-101 using the online software program TargetScan 5.1. (B) MiR-101 did not regulate the expression of the CPEB1 3′-UTR reporter constructs. The luciferase reporter assays were performed 48 h after transfection with the indicated pMIR-REPORT plasmid and a Renilla transfection control plasmid, which 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. An independent sample t-test was used. *p < 0.05. (C) MiR-101 inhibits the expression of LMO3 mRNA. A real-time PCR analysis was performed 48 h after transfection with miR-101 mimics and a scrambled control. An independent sample t-test was used. *p < 0.05. **p < 0.01. (D) MiR-101 knockdown enhances the expression of LMO3 mRNA. Real-time PCR analysis was performed 48 h after transfection with an inhibitor of miR-101 and a negative control. An independent sample t-test was used. **p < 0.01. (E) MiR-101 regulates the expression of the LMO3 protein. Western blot analysis was performed 72 h after transfection with miR-101 mimics, an inhibitor or a scrambled control. GAPDH was used as an internal control. (F) The methylation level of LMO3 was increased by miR-101 in U251 cells. The unmethylated and methylated CpG sites are indicated by open and closed circles, respectively. Each row indicates the sequencing result of one clone of the bisulfite-PCR product. The number of methylated CpGs was divided by the total number of true CpGs analyzed and is given as a percentage to the right of each BSP result. (G) An miR-101 inhibitor had no effect on the methylation status of LMO3, as determined via MSP in U251 cells. (H) An analysis of the promoter activity of the LMO3 core promoter constructs via luciferase reporter assays. The construct containing the sequence spanning the region from –431 to –281 was sufficient to mediate maximal promoter activity. The core promoter ranged from –431 to –281. PGL3-control is the positive control, and pGL3-enhancer is the negative control. An independent sample t-test was used. *p < 0.05. (I) The histone occupancy of the LMO3 promoter was affected by miR-101. A ChIP assay was used to detect the H3K4me2, H3K27me3, H3K9me3 and H4K20me3 occupancy of the LMO3 core promoter. U251 cells were transfected with miR-101 mimics or a scrambled control. An independent sample t-test was used. *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480726&req=5

Figure 2: LMO3 is an epigenetic target gene of miR-101(A) LMO3 was predicted to be a target gene of miR-101 using the online software program TargetScan 5.1. (B) MiR-101 did not regulate the expression of the CPEB1 3′-UTR reporter constructs. The luciferase reporter assays were performed 48 h after transfection with the indicated pMIR-REPORT plasmid and a Renilla transfection control plasmid, which 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. An independent sample t-test was used. *p < 0.05. (C) MiR-101 inhibits the expression of LMO3 mRNA. A real-time PCR analysis was performed 48 h after transfection with miR-101 mimics and a scrambled control. An independent sample t-test was used. *p < 0.05. **p < 0.01. (D) MiR-101 knockdown enhances the expression of LMO3 mRNA. Real-time PCR analysis was performed 48 h after transfection with an inhibitor of miR-101 and a negative control. An independent sample t-test was used. **p < 0.01. (E) MiR-101 regulates the expression of the LMO3 protein. Western blot analysis was performed 72 h after transfection with miR-101 mimics, an inhibitor or a scrambled control. GAPDH was used as an internal control. (F) The methylation level of LMO3 was increased by miR-101 in U251 cells. The unmethylated and methylated CpG sites are indicated by open and closed circles, respectively. Each row indicates the sequencing result of one clone of the bisulfite-PCR product. The number of methylated CpGs was divided by the total number of true CpGs analyzed and is given as a percentage to the right of each BSP result. (G) An miR-101 inhibitor had no effect on the methylation status of LMO3, as determined via MSP in U251 cells. (H) An analysis of the promoter activity of the LMO3 core promoter constructs via luciferase reporter assays. The construct containing the sequence spanning the region from –431 to –281 was sufficient to mediate maximal promoter activity. The core promoter ranged from –431 to –281. PGL3-control is the positive control, and pGL3-enhancer is the negative control. An independent sample t-test was used. *p < 0.05. (I) The histone occupancy of the LMO3 promoter was affected by miR-101. A ChIP assay was used to detect the H3K4me2, H3K27me3, H3K9me3 and H4K20me3 occupancy of the LMO3 core promoter. U251 cells were transfected with miR-101 mimics or a scrambled control. An independent sample t-test was used. *p < 0.05.
Mentions: Having established the hypomethylation role for LMO3 in gliomas, we next aimed to clarify the regulatory mechanism of LMO3 expression. We used the online software TargetScan 5.1 (Cambridge, MA, USA) to predict potential miRNA binding sites in the 3′-UTR sequence of LMO3. LMO3 was predicted to be a target of miR-101 (Figure 2A), and the predicted binding sites were cloned downstream of the firefly luciferase gene in the pMIR-REPORT vector (Supplementary Figure S2). When the cells were cotransfected with miR-101 and pMIR-LMO3– 3′-UTR-WT, there was no significant reduction in luciferase activity compared with cells transfected with the negative control (Figure 2B). Our previous study demonstrated the suppressor role of miR-101 in gliomas [6]; this finding is consistent with the results of the present study, which showed that the overexpression of miR-101 (Supplementary Figure S3A) inhibited the expression of LMO3 (Figure 2C and 2E) and that the knockdown of miR-101 (Supplementary Figure S3B) could enhance the expression of LMO3 in glioma cell lines (Figure 2D and 2E). LMO3 can therefore be considered to be a new indirect target of miR-101 in gliomas. Our previous study confirmed that miR-101 suppresses its targets via histone modification [6]. To determine whether miR-101 inhibits the expression of LMO3 via histone modification, the methylation status of LMO3 was detected using BSP and MSP. The demethylation rate was decreased in the glioma cell lines following the transfection with the miR-101 mimics (Figure 2F). However, the effect of the miR-101 inhibitor on the methylation status of the LMO3 promoter was not significant (Figure 2G). Subsequently, we determined the core promoter region of LMO3. The luciferase reporter assay demonstrated that the core promoter ranged from –431 to –281 (Figure 2H). As shown in Figure 2I, we found that the occupancy of H3K4me2 and H3K27me3 was decreased, whereas the occupancy of H3K9me3 and H4K20me3 was increased at the LMO3 core promoter in the miR-101 mimic-treated cells compared to the control. These results indicate that miR-101 inhibits the expression of LMO3 epigenetically in glioma cells.

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