A Primary Study On Effects And Mechanics Of Formaldehyde Induced Epigenetic Regulation

Epigenetic gene regulation by DNA methylation and associated factors is essential for mammalian development and also plays a major role in tumorigenesis. DNA methylation is the most common eukaryotic DNA modification and is one of the epigenetic (alteration in gene expression without a change in nucleotide sequence) phenomena. Genome stability, allele-specific expression of imprinted genes and masking of transposons and X chromosome inactivation are all clearly dependent of cytosine methylation. The effect of DNA methylation to oncogenesis appears to be important in current achievements.DNA methylation in eukaryotes involves addition of a methyl group to the carbon 5 position of the cytosine ring. This reaction is catalyzed by DNA methyltransferase in the context of the sequence 5' -CG-3' , which is also referred to as a CpG dinucleotide. In most vertebrates, 60-90% of the cytosines at CpG dinucleotides are methylated. During evolution, the dinucleotide CpG has been progressively eliminated from the genome of higher eukaryotes and is present at only 5% to 10% of its predicted frequency. Cytosine methylation appears to have played a major role in this process, because most CpG sites lost represent the conversion through deamination of methylcytosines to thymines. Approximately 70% to 80% of the remaining CpG sites contain methylated cytosines in most vertebrates, including humans. In contrast to the rest of the genome, smaller regions of DNA, called CpG islands, ranging from 0.5 to 5 kb and occurring on average every 100 kb, have distinctive properties. These regions are unmethylated, GC rich (60% to 70%), have a ratio of CpG to GpC of at least 0.6, and thus do not show any suppression of the frequency of the dinucleotide CpG. DNA methylation plays important roles in gene expression. It may suppress gene transcription by direct or (and) indirect inhibition of transcription factor binding. The density of DNA methylation, the intensity of promoter and the relative location of methylated DNA and promoter can all influence this inhibitory effect. Decreased levels of overall genomic methylation are common findings in tumorigenesis. This decrease in global methylation appears to begin early and before the development of frank tumor formation. Apart from the overall genomic hypomethylation, specific oncogenes have been observed to be hypomethylated in human tumors.An important event in tumor development appears to be down-regulation of tumor suppressor genes (TSGs) by hypermethylation of 5'CpGs. Hypermethylation takes place after DNA replication and is catalyzed by DNA methyltransferase using S-adenosylmethionine as the methyl donor. Three distinct families of DNA methyltransferase genes, DNMT1, DNMT2, and DNMT3, have been identified in mammalian cells. DNMT1 is known to maintain methylation, but the function of DNMT2 remains unknown. In addition, the methyl-CpG-binding protein MeCP2 or MBD2 can interact directly with DNMT1 and is associated with TSG repression in cell model .The DNMT3 family consists of two related genes, DNMT3a and DNMT3b. Both DNMT3a and DNMT3b function as de novo methyltransferases. These three types of DNA 5-cytosine-methyltransferases (DNMT1, 3a and 3b) have been identified and are associated with TSG hyper-methylation. Expression of DNMT1 mRNA, and protein in lung tumors was significantly higher than that in normal lung tissue. DNMT3a mRNA was expressed at either similar or higher levels in lung tumors whereas expression levels of DNMT3b mRNA were consistently higher. So overexpression and strong binding of various DNMTs may result in promoter hypermethylation of multiple TSGs and ultimately lead to lung tumorigenesis and poor prognosis.Methyl-CpG binding proteins (MeCPs) are a class of nucleic proteins which can attach to the methyl-CpG dinucleotide. MeCP2 is capable of binding to a single, symmetrically methylated CpG dinucleotide and binds to chromosomes at sites known to contain methylated DNA.In addition, the methyl-CpG-binding protein MeCP2 or MBD2 can interact directly with DNMT1 and is associated with TSG repression in cellular model. Knockdown mediation by RNA interference or by specific inhibition by antisense oligonucleotides of DNMT1 resulted in the reduction of promoter methylation in TSGs such as RASSF1A, p16 and CDH1.Formaldehyde is regarded as an important indoor air chemical pollutant for its extensive sources, high level, long-term and high toxicity. Many studies showed that indoor air formaldehyde induces various harmful effects on human being, including carcinogenicity. Some epidemiologic studies have linked occupational exposure to formaldehyde with cancers of the noses, nasopharynxes, and lungs. Nowadays, FA ranks in group AI (human carcinogen) in WHO document (2004). However, it is unclear whether these tumors arose through a genotoxic mechanism or as a consequence of cytotoxicity. In order to expole the relationship of formaldehyde and epigenetic gene regulation, We have conducted in vivo and in vitro studies to reveal transcription levels of mRNA for DNA methyltransferases (DNMTS) and methyl-CpG-binding proteins 2 (MeCP2) by formaldehyde exposure, and results were as followings:1 Effect of formaldehyde on expression of dnmts and mecp2 gene of Hela cellsThe results showed that formaldehyde could up-regulate the mRNA expression of mecp2 in a dose-dependent manner, resulting in elevated the mRNA expression of mecp2 with the increase of formaldehyde concentrations, and the increase was statistically significant when the concentration of formaldehyde was up to 1250μmol/L (compared with 0μmol/L, p<0.01). Meanwhile, 1250μmol/L formaldehyde could up-regulate the mRNA expression of dnmtl (p<0.05) and dnmt3b(p<0.01). This study suggested that FA may result in promoter hypermethylation of multiple tumor suppressor genes and lead to lung tumorigenesis and poor prognosis by dnmts and mecp2 pathway.2 Increased expression of dnmts and mecp2 gene in lung after gaseous FA expsoureIn this study, for in vivo clean air and 3.0 mg/m~3 gaseous formaldehyde exposures, two groups of mice were kept in two modified 8.4 L glass inhalation chambers respectively, with five rats together in each group for 72 h continuously, after that the lungs were removed for RNA extraction. The mRNA levels of dnmts and mecp2 gene were determined by semiquantitative RT-PCR.We observed that the mRNA expression of dnmtl, dnmt3b (compared with 0 mg/m~3, p<0.01) and mecp2 (p<0.05) was significantly increased in lungs of mice after exposure to 3.0mg/m~3 of formaldehyde.In conclusion, formaldehyde can up-regulate dnmtl and mecp2 mRNA expression in vivo. This study suggested a possible mechanism that FA may result in promoter hypermethylation of multiple tumor suppressor genes and lead to lung tumorigenesis and poor prognosis by dnmts and mecp2 pathway.