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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

MiR-101 suppresses the binding of USF and MZF1 to the LMO3 promoter(A) USF and MZF1 bound to the LMO3 promoter. A ChIP assay was performed to detect the binding of USF and MZF1 to the LMO3 promoter in U251 cells. Normal mouse IgG was used as a negative control. (B) The expression of LMO3 was regulated by USF and MZF1. A real-time PCR analysis was performed 48 h after transfection with USF siRNA, MZF1 siRNA or a scrambled control. An independent sample t-test was used. **p < 0.01. (C) MiR-101 regulated the expression of USF and MZF1. Western blotting was performed 72 h after transfection with miR-101 mimics, miR-101 inhibitor or a negative control. GAPDH was used as an internal control. (D) The transcription factor occupancy of the LMO3 promoter was affected by miR-101. A ChIP assay was performed to evaluate the USF and MZF1 occupancy of the LMO3 core promoter. Left: U251 cells transfected with miR-101 mimics and a negative control. Right: U251 cells transfected with a miR-101 inhibitor and a negative control. An independent sample t-test was used. *p < 0.05. **p < 0.01.
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Figure 4: MiR-101 suppresses the binding of USF and MZF1 to the LMO3 promoter(A) USF and MZF1 bound to the LMO3 promoter. A ChIP assay was performed to detect the binding of USF and MZF1 to the LMO3 promoter in U251 cells. Normal mouse IgG was used as a negative control. (B) The expression of LMO3 was regulated by USF and MZF1. A real-time PCR analysis was performed 48 h after transfection with USF siRNA, MZF1 siRNA or a scrambled control. An independent sample t-test was used. **p < 0.01. (C) MiR-101 regulated the expression of USF and MZF1. Western blotting was performed 72 h after transfection with miR-101 mimics, miR-101 inhibitor or a negative control. GAPDH was used as an internal control. (D) The transcription factor occupancy of the LMO3 promoter was affected by miR-101. A ChIP assay was performed to evaluate the USF and MZF1 occupancy of the LMO3 core promoter. Left: U251 cells transfected with miR-101 mimics and a negative control. Right: U251 cells transfected with a miR-101 inhibitor and a negative control. An independent sample t-test was used. *p < 0.05. **p < 0.01.

Mentions: To identify the mechanism of the miR-101 regulation of LMO3, we predicted the potential transcription factors on the LMO3 promoter using TFSEARCH ver. 1.3. The results indicated that USF and MZF1 bind to the LMO3 promoter, which was verified via the ChIP assay (Figure 4A). As shown in Figure 4B, the expression of LMO3 was decreased by the inhibition of USF and MZF1 (Supplementary Figure S4, lower line). Furthermore, overexpression of miR-101 inhibited the expression of USF and MZF1, and the inhibition of miR-101 enhanced the expression of USF and MZF1 (Figure 4C). The effects of miR-101 on the binding of USF and MZF1 to the LMO3 promoter was detected via the ChIP assay. The data showed that the overexpression of miR-101 disrupted the binding of USF and MZF1 to the LMO3 promoter (Figure 4D). These results illustrate that miR-101 inhibits the binding of USF and MZF1 to the LMO3 promoter and inhibits the expression of LMO3.


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)

MiR-101 suppresses the binding of USF and MZF1 to the LMO3 promoter(A) USF and MZF1 bound to the LMO3 promoter. A ChIP assay was performed to detect the binding of USF and MZF1 to the LMO3 promoter in U251 cells. Normal mouse IgG was used as a negative control. (B) The expression of LMO3 was regulated by USF and MZF1. A real-time PCR analysis was performed 48 h after transfection with USF siRNA, MZF1 siRNA or a scrambled control. An independent sample t-test was used. **p < 0.01. (C) MiR-101 regulated the expression of USF and MZF1. Western blotting was performed 72 h after transfection with miR-101 mimics, miR-101 inhibitor or a negative control. GAPDH was used as an internal control. (D) The transcription factor occupancy of the LMO3 promoter was affected by miR-101. A ChIP assay was performed to evaluate the USF and MZF1 occupancy of the LMO3 core promoter. Left: U251 cells transfected with miR-101 mimics and a negative control. Right: U251 cells transfected with a miR-101 inhibitor and a negative control. An independent sample t-test was used. *p < 0.05. **p < 0.01.
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Figure 4: MiR-101 suppresses the binding of USF and MZF1 to the LMO3 promoter(A) USF and MZF1 bound to the LMO3 promoter. A ChIP assay was performed to detect the binding of USF and MZF1 to the LMO3 promoter in U251 cells. Normal mouse IgG was used as a negative control. (B) The expression of LMO3 was regulated by USF and MZF1. A real-time PCR analysis was performed 48 h after transfection with USF siRNA, MZF1 siRNA or a scrambled control. An independent sample t-test was used. **p < 0.01. (C) MiR-101 regulated the expression of USF and MZF1. Western blotting was performed 72 h after transfection with miR-101 mimics, miR-101 inhibitor or a negative control. GAPDH was used as an internal control. (D) The transcription factor occupancy of the LMO3 promoter was affected by miR-101. A ChIP assay was performed to evaluate the USF and MZF1 occupancy of the LMO3 core promoter. Left: U251 cells transfected with miR-101 mimics and a negative control. Right: U251 cells transfected with a miR-101 inhibitor and a negative control. An independent sample t-test was used. *p < 0.05. **p < 0.01.
Mentions: To identify the mechanism of the miR-101 regulation of LMO3, we predicted the potential transcription factors on the LMO3 promoter using TFSEARCH ver. 1.3. The results indicated that USF and MZF1 bind to the LMO3 promoter, which was verified via the ChIP assay (Figure 4A). As shown in Figure 4B, the expression of LMO3 was decreased by the inhibition of USF and MZF1 (Supplementary Figure S4, lower line). Furthermore, overexpression of miR-101 inhibited the expression of USF and MZF1, and the inhibition of miR-101 enhanced the expression of USF and MZF1 (Figure 4C). The effects of miR-101 on the binding of USF and MZF1 to the LMO3 promoter was detected via the ChIP assay. The data showed that the overexpression of miR-101 disrupted the binding of USF and MZF1 to the LMO3 promoter (Figure 4D). These results illustrate that miR-101 inhibits the binding of USF and MZF1 to the LMO3 promoter and inhibits the expression of LMO3.

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