Study On The Epigenetic Mechanism Of Bilirubin-induced Neuronal Damage Based On DNA Methylation Sequencing

Objectives To explore the role of DNA methylation modification in bilirubin-induced neuronal damage based on DNA methylation sequencing,so as to reveal the pathogenesis of hyperbilirubinemia,and provid a new"target"for the diagnosis and treatment of hyperbilirubinemia.Methods?1?Establishment of primary culture system of rat cerebral cortical neurons and treatment of bilirubin:The neonatal SD rats born within 24h after birth were selected and cerebral cortical neurons were isolated and cultured in serum-free neuronal Neurobasal medium at 37?in a 5%CO₂ incubator for 72h.The culture medium was replaced,and bilirubin was added with final concentrations of 0,25,50,and 100?mol/L in culture medium.The neurons were harvested after culturing for 24h under the same conditions.?2?DNA methylation sequencing:Whole genome DNA extracted by CTAB method from the harvested cells of control group and high bilirubin group cells was sequenced using RRBS technology,followed by bioinformatics analysis.Observing the differences in DNA methylation base C sites between control group and high bilirubin group to screen out candidate different methylation genes,which then were annotated with GO and KEGG databases.?3?Two differentially methylated genes including Plagg1 and Rps6ka3 which are involved in apoptosis were screened out to verify the expression of the two genes at mRNA and protein levels by using RT-PCR and Western Blot in order to verify DNA methylation sequencing results..Results?1?Sequencing results showed that each sample yielded 6.20G Clean Data,and the total Clean Reads bases was 37.23Gbp,and Q30 reached 85.09%.The average comparison efficiency between samples and reference genome was about 49.46%,and the conversion rate of sulfite was about 99.40%.The CG type methylation sites of the sample accounted for about85.10%of all methylation sites.?2?Through analysising on the different methylation region between samples,830 different methylation genes of CG methylation type were screened out,of which 409 were hypermethylated and 421 were hypomethylated.There were 61 genes with different methylation in their promoter region.The different methylation genes were annotated and classified into 62 functional terms in the GO database,such as apoptosis,cell differentiation,transcriptional expression,synapses,and nucleus.236 pathways annotated in the KEGG database including calcium signaling pathways,synaptic transmission,and synaptic plasticity.?3?RT-PCR results showed,with the increase of bilirubin concentration,the mRNA level of Plagg1 was gradually increased with a significant dose-effect relationship.Each group was significantly different compared with control group?F=10.31,P<0.05?.However,the mRNA level of Rps6ka3showed a downward trend with the increase of bilirubin concentration.There was a significant dose-effect relationship and all groups were significantly different compared with control group?F=5.675,P<0.05?.?4?Western Blot results showed that compared with control group,Plagl1protein expression significantly increased in three groups?F=6.289,p<0.05?.Compared with control group,The expression of Rps6ka3 protein significantly decreased in all groups(?F=13.48,p<0.01?Conclusions?1?Bilirubin exposure can lead to change of DNA methylation level of genes in rat neurons in vitro.DNA methylation modification may play an important role in bilirubin-induced neuronal damage.?2?Bilirubin Exposure activates the expression of Plagl1 gene both at mRNA and protein levels in cultured cerebral cortical neurons in vitro,which is consistent with the hypomethylation level in the promoter region of this gene.?3?Bilirubin Exposure can inhibit the expression of Rps6ka3 gene both at mRNA and protein levels in cultured cerebral cortical neurons in vitro,which is inconsistent with the hypermethylation level in the promoter region of this gene.It deserves further research whether there are any other more complex mechanisms in the expression and regulation of Rps6ka3 gene.?4?Both Plagl1 and Rps6ka3 are involved in the regulation of cell apoptosis.We discovered that bilirubin exposure can change the expression of these two genes.The results suggest that there may be more extensive mechanisms in neuronal apoptosis induced by bilirubin.