Methylation Level Of Genes And MiR-101-mediated Histone Methylation Modification Mechanism In Glioma

[The previous research of the project]Glioma is the most common malignant tumor with low survival rate in central nervous system. It's indicated that deregulation of epigenetic mechanism which contained abnormal miRNA expression, DNA methylation changes and histone modification played an important role in glioma carcinogenesis. DNA methylome in glioma was constructed by high throughput methylatioin DNA IP (MeDIP) combining with promoter and CpG islands chip in our previous research. By massarray and bisulfite sequencing PCR, it's verified that LRRC4, ANKDD1A, GAD1, HIST1H3E, PCDHA8, PCDHA13, PHOX2B, SIX3and SST are new hypermethylated genes in glioma. Our research also indicated that tumor suppressor microRNA miR-185inhibited the genome-wide methylation status by directly targeting DNMT1, and then regulated the methylation modification status and recovered the expression of the above9hypermethylated genes.[F10, POTEH, CPEB1, LMO3, ELFN2and PRDM16are new hypomethylated genes, and their methylation status and expression may be as prognosis mark for glioma patients]Based on the research above, Signalmap software was used to select12genes, including F10, POTEH, CPEB1, LM03, ELFN2, PRDM16, CD207, BAD, NRBP2, SLITRK5, SLC44A2and PGP from the74hypomethylated genes screened by methylation chip. These genes were tested in large scale of samples by BSP. It's revealed that there's no no difference between the methylation level of CD207, BAD, NRBP2, SLITRK5, SLC44A2and PGP in glioma tissues (n=6) and that in normal brain tissues (n=4), and F10(n=102), POTEH (n=102), CPEB1(n=56), LMO3(n=56), ELFN2(n=56), PRDM16(n=56) were hypomethylated in glioma tissues. In this study, we confirmed that they were new hypomethylated genes in glioma. The hypomethylation of these genes was associated to their high expression in glioma. The hypomethylation and high expression of these genes could be regarded as important prognosis marks of glioma patients, because the lower the methylation level was and the higher genes expressed, the poorer outcome patients haboring. While, hypomethylation of F10was correlated to patients' age; high expression of POTEH and hypomethylation of PRDM16was related to glioma pathology grade. Thus, high expression of POTEH and hypomethylation of PRDM16could be considered as important marks in the progression of glioma. The glioma tissues with POTEH high-expression or PRDM16hypomethylation level would be more malignant than those with POTEH low-expression or PRDM16hypermethylation.[miR-101, a significant mark of early stage, was loss or down expressed and related to the prognosis in glioma]It's proved that miR-101was down-regulated and acted as a tumor suppressor in multiple tumors. The expression of miR-101was detected in50glioma tissues specimens and10normal brain tissues samples. It's showed that the expression of miR-101was lower in glioma tissues than in normal brain tissues, and the expression in glioma grade Ⅰ was much higher than in other grades glioma tissues, but there's no difference among grade Ⅱ, Ⅲ and Ⅳ. It's considered that down-regulation of miR-101was concerned with initiation of glioma, but no relationship with the progression. There's also no association between miR-101expression and patients'age and gender. Low-expression of miR-101may be an important mark predicting initiation and prognosis of glioma patients. Patients with miR-101low-expression could harbor poor prognosis.[Hypomethylated genes CPEB1, PRDM16and ELFN2were target genes of miR-101, and their expression were inhibited directly by miR-101in glioma cells.]It's well-known that miRNAs play significant role by regulating gene expression in tumors. We assumed to analyze the upregulation mechanism of hypomethylation genes in the extent of gene regulation by miRNA. Subsequently, we predicted miRNAs which could regulate F10, POTEH, CPEB1, ELFN2, LM03and PRDM16. Interestedly, CPEB1, ELFN2, LM03and PRDM16were predicted to be targeted by the same miRNA——miR-101by online software Targetscan6.0. It's confirmed that miR-101could bind to the3'UTR of CPEB1, ELFN2and PRDM16and inhibit their expression. Although miR-101could not bind the3'UTR of LM03, it still down-regulated expression of LM03in glioma cells.[miR-101suppressed histone methylation of LMO3by targeting EZH2, EED and DNMT3A, and increased the methylation level of LMO3, decreased indirectly it's expression in glioma cells.]The previous study verified that histone methyltransferase EZH2was a direct target of miR-101. We predicted and demonstrated that histone methyltransferase EED and DNA methyltransferase DNMT3A were targets of miR-101. miR-101suppressed the expression of EZH2, EED and DNMT3A, inhibited activity of H3K4demethylase, H3K27methyltransferase and DNMT3A methyltransferase, and increase the activity of H3K9and DNMT methyltransferase. Because EZH2, EED and DNMT3A possesses methyltransferase activity, miR-101was much more likely to influence the histone methylation modification by regulating EZH2, EED and DNMT3A. The effect of miR-101, EZH2siRNA, EED siRNA and DNMT3A siRNA on histone methylation at LM03core promoter was detected. It's showed that miR-101decreased the occupancy of H3K27me3and H3K4me2and increased the occupancy of H3K9me3and H4K20me3at LM03core promoter, upregulated the methylation level of LM03promoter, indirectly inhibited the expression of LM03by targeting EZH2, EED and DNMT3A in glioma cells.[miR-101indirectly suppressed expression of CPEB1, ELFN2and PRDM16and affected their methylation levels by targeting EZH2, EED and DNMT3A and regulating histone methylation in glioma cells.]As miR-101regulated the methylation status and expression of LMO3through histone modification, it may regulate the methylation status of CPEB1, ELFN2and PRDM16in the same way. Hence, the effect of miR-101, EZH2siRNA, EED siRNA and DNMT3A siRNA on histone methylation and expression of CPEB1, ELFN2and PRDM16was detected. ChIP combining with qRT-PCR and BSP was used to verify that miR-101decreased the H3K4me2and H3K27me3occupancy at CPEB1, ELFN2and PRDM16core promoter and increased the H3K9me3and H4K20me3occupancy at CPEB1and PRDM16core promoter by targeting EZH2, EED and DNMT3A, then it recovered the methylation levels of CPEB1, ELFN2and PRDM16gene promoter, and indirectly down-regulating the expression of these hypomethylation genes[miR-101recovered the expression of hypermethylation gene LRRC4by down-regulating H3K27me3occupancy and hypomethylation level of LRRC4in glioma cells.]LRRC4is cloned in our lab and has been a glioma suppressor gene and its hypermethylation and down-expression is common in glioma. In order to clarify the mechanism of LRRC4regulation, miRNAs regulating LRRC4were predicted. miR-101was predicted to target LRRC4. Here, we indicated that miR-101could not bind to3'UTR of LRRC4, but it remain to upregulate the expression of LRRC4in glioma cells. miR-101decreased the occupancy of H3K27me3at LRRC4core promoter and induced hypomethylation of LRRC4by targeting EZH2, EED and DNMT3A.Token together, deregulation of gene methylation including hypermethylation and hypomethylation, plays an important role in carcinogenesis of glioma. Hypermethylation or hypomethylation of genes and their deregulated expression could be applied to predict the early diagnosis and prognosis of glioma. miRNAs are small noncoding RNA, around22-24nucleotides in size. They could not only directly regulate expression hyper/hypo-methylation genes by binding to3'-UTR of genes, but also regulate the methylation level and gene expression through histone and DNA methylation modification by targeting histone and DNA methyltransferases.