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MeCP2 suppresses LIN28A expression via binding to its methylated-CpG islands in pancreatic cancer cells.

ABSTRACT: LIN28A aberrant expression contributes to the development of human malignancies. However, the LIN28A expression profile remains to be clarified. Herein, we report that LIN28A expression is directly associated with the methylation status of its two CpG island sites in pancreatic cancer cells. First, Bisulfite sequencing reveals that PANC1 cells possess the higher methylation rate at LIN28A CpG islands compared with SW1990 and PaTu8988 cells. Subsequently, LIN28A expression is increased at both mRNA and protein levels in pancreatic cancer cells treated with 5-Aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor. Further Chromatin immunoprecipitation (ChIP) assays indicate that methyl-CpG-binding protein 2 (MeCP2) binds preferentially to the two hypermethylated CpG islands sites at LIN28A promoter compare to MBD3. Expectedly, MeCP2 knockdown transcriptionally activates LIN28A expression in above cells, rather than MBD3 knockdown. Moreover, LIN28A overexpression remarkably improves OCT4, NANOG and SOX2 expression, and the ability of sphere and colony formation, and enhances the capacities of invasion in PaTu8988 and SW1990 cells, whereas LIN28A knockdown significantly inhibits the above malignant behaviors in PANC1 cells. These findings suggest that LIN28A is epigenetically regulated via MeCP2 binding to methylated-CpG islands, and may play a crucial role in pancreatic cancer progression.

SUBMITTER: Xu M

PROVIDER: S-EPMC4924729 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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MeCP2 suppresses LIN28A expression via binding to its methylated-CpG islands in pancreatic cancer cells.

Xu Min M Bian Shihui S Li Jie J He Junbo J Chen Hui H Ge Lu L Jiao Zhijun Z Zhang Youli Y Peng Wanxin W Du Fengyi F Mo Yinyuan Y Gong Aihua A

Oncotarget 20160301 12

LIN28A aberrant expression contributes to the development of human malignancies. However, the LIN28A expression profile remains to be clarified. Herein, we report that LIN28A expression is directly associated with the methylation status of its two CpG island sites in pancreatic cancer cells. First, Bisulfite sequencing reveals that PANC1 cells possess the higher methylation rate at LIN28A CpG islands compared with SW1990 and PaTu8988 cells. Subsequently, LIN28A expression is increased at both mR ...[more]

PMID: 26910839

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Project description:BackgroundMammalian CpG islands (CGIs) normally escape DNA methylation in all adult tissues and developmental stages. However, in our previous study we unexpectedly identified many methylated CGIs in human peripheral blood leukocytes. Methylated CpG dinucleotides convert to TpG dinucleotides through deaminization of their cytosine bases more frequently than hypomethylated CpG dinucleotides. Therefore, we wondered how methylated CGIs in germline or non-germline cells maintain their CpG-rich sequences. It is known that events such as germline hypomethylation, CpG selection, biased gene conversion (BGC), and frequent CpG fixation can contribute to the maintenance of CpG-rich sequences in methylated CGIs in germline or non-germline cells. However, it has not been investigated which of the processes maintain CpG-rich sequences of methylated CGIs in each genomic position.ResultsIn this study, we comprehensively examined the contribution of the processes described above to the maintenance of CpG-rich sequences in methylated CGIs in germline and non-germline cells which were classified by genomic positions. Approximately 60-80% of CGIs with high methylation in H1 cell line (H1-HM) in all the genomic positions showed a low average CpG→TpG/CpA substitution rate. In contrast, fewer than half the numbers of CGIs with H1-HM in all the genomic positions showed a low average CpG→TpG/CpA substitution rate and low levels of methylation in sperm cells (SPM-LM). Furthermore, a small fraction of CGIs with a low average CpG→TpG/CpA substitution rate and high levels of methylation in sperm cells (SPM-HM) showed CpG selection. On the other hand, independent of the positions in genes, most CGIs with SPM-HM showed a slightly higher average TpG/CpA→CpG substitution rate compared with those with SPM-LM.ConclusionsRelatively high numbers (approximately 60-80%) of CGIs with H1-HM in all the genomic positions preserve their CpG-rich sequences by a low CpG→TpG/CpA substitution rate caused mainly by their SPM-LM, and for those with SPM-HM partly by CpG selection and TpG/CpA→CpG fixation. BGC has little contribution to the maintenance of CpG-rich sequences of CGIs with SPM-HM which were classified by genomic positions.

Project description:Hypermethylation of promoter CpG islands is strongly correlated to transcriptional gene silencing and epigenetic maintenance of the silenced state. As well as its role in tumor development, CpG island methylation contributes to the acquisition of resistance to chemotherapy. Differential Methylation Hybridisation (DMH) is one technique used for genome-wide DNA methylation analysis. The study of such microarray data sets should ideally account for the specific biological features of DNA methylation and the non-symmetrical distribution of the ratios of unmethylated and methylated sequences hybridised on the array. We have therefore developed a novel algorithm tailored to this type of data, Methylation Linear Discriminant Analysis (MLDA).MLDA was programmed in R (version 2.7.0) and the package is available at CRAN 1. This approach utilizes linear regression models of non-normalised hybridisation data to define methylation status. Log-transformed signal intensities of unmethylated controls on the microarray are used as a reference. The signal intensities of DNA samples digested with methylation sensitive restriction enzymes and mock digested are then transformed to the likelihood of a locus being methylated using this reference. We tested the ability of MLDA to identify loci differentially methylated as analysed by DMH between cisplatin sensitive and resistant ovarian cancer cell lines. MLDA identified 115 differentially methylated loci and 23 out of 26 of these loci have been independently validated by Methylation Specific PCR and/or bisulphite pyrosequencing.MLDA has advantages for analyzing methylation data from CpG island microarrays, since there is a clear rational for the definition of methylation status, it uses DMH data without between-group normalisation and is less influenced by cross-hybridisation of loci. The MLDA algorithm successfully identified differentially methylated loci between two classes of samples analysed by DMH using CpG island microarrays.

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MeCP2 suppresses LIN28A expression via binding to its methylated-CpG islands in pancreatic cancer cells.

Publications

MeCP2 suppresses LIN28A expression via binding to its methylated-CpG islands in pancreatic cancer cells.

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