Relationship between methylation of SOX11 gene and cervical cancer / 西安交通大学学报(医学版)

Objective: To detect the methylation status of SOX11 gene in normal cervix, cervical cancer tissues and cervical cancer cell lines so as to explore the relationship between methylation status and expression of SOX11 gene. Methods: DNA methylation status of SOX11 gene in normal cervical, cervical cancer tissues and cervical cancer cell lines was analyzed by bisulfite sequencing and TA cloning assay. The expression of SOX11 mRNA in cervical cancer tissues, cell lines and normal cervical was detected by RT-PCR. Results: The average methylation rate of SOX11 in cervical cancer tissues (81.07%) was significantly higher than that in normal cervical tissues (12.86%) (P<0.001). The methylation status of SOX11 in HeLa, SiHa, C33-A and CaSki cells was all hypermethylated with the methylation of 90.71%, 97.14%, 77.14% and 99.64%, respectively. The expression of SOX11 mRNA in cervical cancer tissues was significantly lower than that in normal cervixes (P<0.001). The SOX11 gene expression was significantly negatively correlated with its promoter hypermethylation (P<0.001, r=-0.808). After demethylation agent 5-azacytidine treatment, SOX11 mRNA expression in cervical cancer cell lines was significantly increased. Further analysis showed that HPV infection in cervical cancer might increase SOX11 promotor methylation. Conclusion: SOX11 gene in cervical cancer is silenced by the hypermethylation of DNA in the promoter region.

Research on methylation of FHIT,hMLH1,p16,RAR-beta,Reprimo and TIMP3 genes in gastric cancer patients / 实用肿瘤学杂志

Objective The aim of this study was to investigate the methylation status of fragile histidine triad( FHIT) gene, human mutl homolog 1(hMLH1)gene,p16 gene,retinoic acid receptor beta( RAR-beta) gene,Reprimo gene and tissue inhibitor of metalloproteinase 3 ( Timp3) gene in gastric cancer and corresponding paracancerous tissues. Methods The methylation levels of FHIT,hMLH1,p16,RAR-beta,Reprimo and TIMP3 genes in 42 clinically resected gastric cancer specimens and 42 corresponding paracancerous tissues were detected by sodium bisulfite sequencing. Results The average methylation rates of the genes in gastric cancer and corresponding paracancerous tissues were:FHIT(1. 50% ,1. 36% ),hMLH1(4. 77% ,0. 48% ),p16(9. 63% ,10. 36% ), RAR-beta(4. 75% ,4. 17% ),Reprimo(9. 71% ,3. 76% )and TIMP3 genes(18. 34% ,14. 06% ). Compared with the paracancerous control group,the average methylation rate of Reprimo gene was only statistically different in gastric cancer patients(P=0. 00787). The difference in methylation rate of Reprimo gene promoter in gastric cancer patients with the degree of tissue differentiation was sta-tistically significant(P<0. 05). Conclusion There has methylation in the cytosine guanidine dinucleotide island of the Reprimo gene promoter region in gastric cancer. The high methylation rate of the Reprimo gene can be used as a potential biomarker for gastric cancer to detect the early stage of gastric cancer.

HeteroMeth: A Database of Cell-to-cell Heterogeneity in DNA Methylation / 基因组蛋白质组与生物信息学报·英文版

DNA methylation is an important epigenetic mark that plays a vital role in gene expression and cell differentiation. The average DNA methylation level among a group of cells has been extensively documented. However, the cell-to-cell heterogeneity in DNA methylation, which reflects the differentiation of epigenetic status among cells, remains less investigated. Here we established a gold standard of the cell-to-cell heterogeneity in DNA methylation based on single-cell bisulfite sequencing (BS-seq) data. With that, we optimized a computational pipeline for estimating the heterogeneity in DNA methylation from bulk BS-seq data. We further built HeteroMeth, a database for searching, browsing, visualizing, and downloading the data for heterogeneity in DNA methylation for a total of 141 samples in humans, mice, Arabidopsis, and rice. Three genes are used as examples to illustrate the power of HeteroMeth in the identification of unique features in DNA methylation. The optimization of the computational strategy and the construction of the database in this study complement the recent experimental attempts on single-cell DNA methylomes and will facilitate the understanding of epigenetic mechanisms underlying cell differentiation and embryonic development. HeteroMeth is publicly available at http://qianlab.genetics.ac.cn/HeteroMeth.

A influência de polimorfismos de base única na metilação de DNA em genes de receptores olfatórios / Single nucleotide polymorphisms lead to differential DNA methylation in odorant receptor genes

Os genes de receptores olfatórios (OR) pertencem a uma família de proteínas de membrana formada por cerca de 1000 genes no genoma de camundongo. Os genes OR são expressos de forma monogênica e monoalélica nos neurônios olfatórios (OSNs). No entanto, ainda não está claro o mecanismo que permite essa forma de expressão peculiar, sobretudo, qual o papel da metilação de DNA nesse processo. Nosso estudo determinou o padrão de metilação de DNA da região promotora e codificadora do gene Olfr17. Em células de epitélio olfatório (MOE) de camundongos adultos, observamos na região codificadora (CDS) do gene uma frequência de metilação em dinucleotídeos CpG 58%, enquanto que na sua região promotora ela foi bem mais baixa. Os níveis de metilação do Olfr17 em MOE de embrião (E15.5) e fígado foram similares aos observados em MOE de animais adultos. Em seguida, analisamos se a metilação de DNA pode regular a expressão gênica do Olfr17. Utilizando animais transgênicos onde os neurônios olfatórios que expressam Olfr17 também expressam GFP, pudemos selecionar neurônios olfatórios GFP+ e analisar a metilação do gene Olfr17, que está ativo nestas células. Verificamos que o padrão geral de metilação do Olfr17, tanto na região CDS como na região promotora, não se altera quando este gene está ativo. Este resultado indica que alterações na metilação do gene Olfr17 não são necessárias para que este receptor seja expresso. Finalmente, verificamos que a região promotora do gene Olfr17, de duas linhagens de camundongos diferentes, a C57BL/6 e a 129, possuem dois polimorfismos de base única (SNPs) que alteram o conteúdo CpG. Devido a estes SNPs, a linhagem 129 apresenta dois sítios CpG adicionais, inexistentes na linhagem C57BL/6. Nossas análises mostraram que estes CpGs são frequentemente metilados, o que torna o promotor do Olfr17 de 129 significativamente mais metilado que o promotor de C57BL/6. Em seguida, nós analisamos o nível de expressão no MOE dos dois alelos de Olfr17, o 129 e o C57BL/6, utilizando ensaios de RT-qPCR. Estes experimentos demonstraram que o nível de expressão do alelo 129, que possui 3 CpGs metiladas em seu promotor, é menor que o do alelo C57BL/6, que apresenta apenas uma CpG que é pouco metilada em seu promotor. Nossos resultados sugerem que as alterações na região promotora influenciam a probabilidade com que o gene OR é escolhido para ser expresso no MOE

Olfactory receptor (OR) genes belong to a large family of membrane proteins composed of 1000 genes in the mouse genome. The OR genes are expressed in the olfactory sensory neurons (OSNs) in a monogenic and monoallelic fashion. However, the mechanisms that govern OR gene expression are unclear. Here we asked whether DNA methylation plays a role in the regulation of OR gene expression. We first determined the DNA methylation pattern in the coding (CDS) and promoter regions of the odorant receptor gene Olfr17. In olfactory epithelium (MOE) cells, the CpG methylation level in the CDS is 58% but is much lower in the promoter region of the gene. In embryonic MOE (E15.5) and liver, the levels of Olfr17 DNA methylation are similar to the ones shown in adult MOE. We next analyzed whether DNA methylation is involved in Olfr17 regulation. We isolated GFP+ neurons from transgenic mice that coexpress GFP with Olfr17, and analyzed the DNA methylation pattern of the Olfr17, which is active in these cells. We found that the general methylation pattern, both, in the coding and promoter regions is not altered in the active gene. These results indicate that changes in DNA methylation are not required for the activation of Olfr17. Finally, we found that the Olfr17 promoter region from two different mouse strains, C57BL/6 and 129, has two single-nucleotide polymorphisms (SNPs) that alter the CpG content. The SNPs lead to the existence of two additional CpGs in the 129 allele, which are absent in the C57BL/6 allele. These CpGs are frequently methylated, making the 129 Olfr17 promoter significantly more methylated than the Olfr17 promoter from C57BL/6. We next performed RT-qPCR experiments to analyze the expression levels of the 129 and C57BL/6 Olfr17 alleles in the MOE. These experiments showed that the expression level of the 129 Olfr17 allele, which contains three methylated CpGs in its promoter region, is lower than the one from C57BL/6, which contains only one, undermethylated CpG, in its promoter. Our results suggest that these promoter modifications regulate the probability of the OR gene choice

Application strategy of DNA methylation and bisultite sequencing in medicinal plants / 中草药

DNA methylation is one of the most widely epigenetics phenomena, methylated DNA can regulate the phenotype of plants without changing the nucleotide sequence. In higher plants, there are three methylated sites, CG, CHG, and CHH (H stands for A, C, or T). They often occur in symmetrical sequences CG and repetitive sequence, and methylation degrees and patterns are different among different species. The methylation of functional gene promoters can suppress the gene expression in medicinal plants, and then affect the accumulation of secondary metabolites, resulting in quality variations. Among many methods for the DNA methylation detection, bisulfite sequencing can identify the site and extent of DNA methylation at single nucleotide level, which is the gold standard for DNA methylation analysis. Through optimizing primers and improving technology, it can effectively reveal the DNA methylation status for medicinal plants and provide technical support and a new research direction for clarifying some problems such as quality variations in medicinal plants, which are not completely explained by classical genetics.

5'-flanking regulatory sequence methylation of the Boule gene in the testis tissue of infertile men with Sertoli cell-only syndrome / 中华男科学杂志

<p><b>Objective</b>To investigate the 5'-flanking regulatory sequence methylation status of the Boule gene in the testis tissue of infertile men with Sertoli cell-only syndrome (SCOS).</p><p><b>METHODS</b>We collected biopsy samples of the testis tissue from 12 men with obstructive azoospermia (the control group) and 15 cases of SCOS, all without varicocele, cryptorchidism, or infectious disease. We extracted genomic DNA from the testis tissue of the SCOS patients, analyzed the characteristics of the 5'-flanking regulatory sequence of the Boule gene using the bioinformatics method, and detected the methylation status of the Boule gene by sodium bisulfite sequencing.</p><p><b>RESULTS</b>A CpG island was observed in the 5'-flanking regulation region of the Boule gene. The methylation level of the Boule gene was remarkably higher in the SCOS group than in the obstructive azoospermia controls (61.4% vs 21.7%, P<0.01), with significant differences in the methylation levels of 14 CpG sites, namely, －58 bp, －50 bp, －48 bp, －38 bp, －28 bp, －24 bp, －20 bp, －15 bp, －1 bp, +5 bp, +8 bp, +15 bp, +29 bp, and +58 bp.</p><p><b>CONCLUSIONS</b>The methylation level of the Boule gene is significantly higher in the SCOS patients than in the obstructive azoospermia males, which suggests that the changes in Boule methylation may be associated with spermatogenic dysfunction.</p>

Expression and promoter methylation of Kras gene in thymic lymphomas induced by ionizing radiation / 吉林大学学报(医学版)

Objective To study the changes of mRNA and protein expressions of Kras gene in thymic lymphomas induced by ionizing radiation,and to detect the methylation of CpG islands in promoter region of Kras gene,then to investigate the mechanisms for the occurrence of radiation carcinogenesis.Methods The thymic lymphoma models of BALB/c mice were made by X-ray irradiation,then the total RNA was extracted,cDNA was synthesized and the total protein was extracted from both thymic lymphoma tissue and normal thymus tissue;the mRNA and protein expressions of Kras gene in thymic lymphoma tissue and normal thymus tissue were detected by RT-PCR and Western blotting method, and the methylation of CpG islands in promoter region of Kras gene was detected by bisulfite sequencing PCR. Results The mRNA expression level of Kras gene in thymic lymphoma tissue was significantly higher than that in normal thymus tissue(P<0.01).The protein expression level in thymic lymphoma tissue was about 1.41 times higher than that in normal thymus tissue;4 CpG sites were methylated detected by bisulfite sequencing PCR in normal thymus tissue, however, 1 CpG site was methylated in thymic lymphoma tissue,the CpG islands in promoter region of Kras gene were demethylation state in thymic lymphoma. Conclusion Ionizing radiation can cause the changes of mRNA and protein expression levels of Kras gene in thymic lymphoma tissue by demethylation state of Kras gene,eventually lead to the occurrence of tumor;it might be one of the mechanisms for the occurrence of radiation carcinogenesis.

A Visualization Tool for Computational Analysis of DNA Methylation Level Using Bisulfite Sequencing Data

Methylation of cytosine is a post-synthesis modification that does not affect the primary DNA sequence but greatly influences gene expression level and phenotypes of an organism. As high-throughput sequencing of bisulfite-treated DNA is the most efficient method to identify methylated sites, several tools to map sequencing reads on a reference are available. But tools to visualize and to interpret the methylation level of methylation sites are currently insufficient. Herein, we present a novel tool to visualize the methylation level of CpG sites.