Study Of The Relationship Between DNA Methylation And Etiology Of Congenital Microtia

PurposeScreening for abnormal methylation in CpG islands and CpG sites through whole genome of congenital microtia to identify their associated genes. RNA expression levels of these genes are also evaluated to study the relationship between CpG islands methylation level and gene expression. Meanwhile, the changes of gene expression after the intervention of 5-Azacytidine which is one of demethylation agent are detected.The study was designed to discuss the relationship between abnormal methylation level of genes and the etiology of congenital microtia.MethodTo collect the residual ear cartilage of microtia as the research object,marked as experimental group; normal ear cartilage of patients without ear malformations as control, marked as control group.Choosing Nimblegen CpG promoter array based on methylated DNA immunoprecipitation to screen the 28226 CpG islands in the whole genome of both experimental group and control group,the signals on the microarray were scanned by high-resolution Chip scanner.The data were extracted and exported to excel by GenePix Pro6.0 software.According to the standard that one group is hypermethylation and the other group is hypomethylation,the genes with differential methylated CpG islands were selected. Choosing SpectroCHIP array based on matrix-assisted laser desorption/ionization-time of flight mass spectrography analysis to detect the methylation level of each CpG site in abnormal methyletion CpG islands of both experimental group and control group.The signals were analyzed by EpiTYPER software and the data were exported to excel.Using t-test to analyse data between groups,the CpG sites with differential methylation level were elected. Collecting both experimental group and control group to isolate and culture chondrocytes in vitro,the morphologic changes were observed under light microscope,the amount of chondrocytes was evaluated by cell counting kit-8 in order to generate cell growth curves,and the type II collagen was tested by immunohistochemisty staining.Choosing part of control group chondrocytes as control group 2 to interfere the growth by 5-Azacytidine in different concentration,the influence of cellular proliferation and toxicity were evaluated by cell growth curves and cellular viability.Using the real-time quantitative PCR to test the RNA expression level of abnormal methylated genes between each group in ear cartilage and chondrocytes,the Ct values were analyzed by Prizm 4 software to select the abnormal methylated genes with differential RNA expression.Result1. There were thirty-six CpG islands with differential methylated level in whole genome between experimental group and control group,in which twenty-nine CpG islands were connected with twenty-nine named genes.The profile of DNA methylation patterns along the entire genome was initially established.2. In the abnormal methylated CpG islands of four differential genes including COL18A1,MYH14,RBMY1A1 and ZIC3, six differentially methylated CpG sites were found with statistical significance.3. The biological character and specificity showed no differences between each chondrocyte generation of experimental group and control group.Cell proliferation and toxicity test proved that the 5-Azacytidine has a dose dependent suppressive effect on the growth of the normal chondrocyte.4. In the cartilage, RNA expression in COL18A1 of experimental group was lower than that in control group with very significant difference. RNA expression in MYH14 and RBMYA1 of experimental group were both higher than that in control group with significant difference. RNA expression in ZIC3 of experimental group was higher than that in control group without significant difference.In chondrocyte, RNA expression in COL18A1,MYH14 and RBMY1A1 of experimental group were all higher than that in control group without significant difference. RNA expression in COL18A1,MYH14 and RBMY1A1 of control group 2 were all higher than that in control group with significant difference.ConclusionThere were thirty-six CpG islands with differential methylated level in whole genome between congenital microtia ear cartilage and normal ear cartilage. Screening for differential methylated CpG sites in four abnormal methylated CpG islands in genes,it is found that six CpG sites had differential methylation level between experimental group and control group. It is considered that the demethylation effect of 5-Azacytidine could promote gene expression,and proved that gene expression was associated with DNA methylation.The RNA expression of MYH14 and RBMY1A1 in experimental group and control group matched the difference of CpG islands methylation level,so it is a conclusion that the regulation of gene expression is tightly connected with DNA methylation in microtia.Therefore,the etiology of congenital microtia may have a close relationship with DNA methylation.