Genome-wide Analysis Of 5-hmC In The Peripheral Blood Of Systemic Lupus Erythematosus Patients Using HMeDIP-chip

Systemic lupus erythematosus is a typical systemic autoimmune disease, which involved diffuse connective tissues and characterized by immune inflammation. The clinical manifestations of SLE is complex and repeatedly, it may result to multiple damage in tissues and organs, of which the most involved is kidney. With its complex pathogenesis, involving genetic, immunologic and environmental factors and so on, it has a tendency of family inhabited and can be combined with other systemic autoimmune diseases. Studies have shown that non-coding RNA regulation, DNA methylation and histone modifications and other epigenetic regulation involved in the pathogenesis of SLE. The major forms of DNA methylation have 5-methylcytosine, N6-methyl-adenine and so on. Among this forms,5-hydroxymethylcytosine (5-hmC) is the hydroxylated forms of the well-known 5-methylcytosine (5-mC),5-hmC was found to be abundantly present in certain cell types, including a variety of mammalian cells, Plant tissue, embryonic stem cells. Compared with 5mC,5hmC levels was lower, but also have the same important biological significance in the regulation of gene expression, is an important epigenetic marks. As a stable DNA epigenetic modification of human genome which constitutes an important component of epigenetic,5-hmC is generally recognized and subjected of great concern, is a the new hotspot, but 5-hmC status changed in SLE environment remains unclear; Currently there are many studies about the pathogenesis of SLE, but still have not found SLE-specific biomarkers, so it is necessary to fully understand the existing research results, based on the scientific use of the results of previous studies, performed with innovative ways to further exploration and research, find new molecular markers of SLE disease activity. In summary, study about whether 5-hmC exception in the environment of SLE is very important, which may be able to provide new ideas for the treatment of SLE, and a deeper understanding of the pathogenesis of SLE.Aim:1. In the genome-wide, detected 5-hmC modification status of SLE patients and normal controls, gained 5-hmC genes which differentially expressed, discussed the correlation between 5-hmC and SLE and assessed the biological function of 5-hmC in SLE, in-depth analytical SLE pathogenesis; 2. Obtain the abundance and expression levels of SLE-specific 5-hmC candidate genes, it can be used as potential biomarkers and therapeutic targets, provide new evidence for the diagnosis and treatment of SLE.Method:Genome-wide analysis of 5-hmC in peripheral blood of 15 SLE patients and 15 normal controls by hMeDIP-Chip.1. Genomic DNA (gDNA) was extracted from the SLE patients and normal controls blood samples using a DNeasy Blood & Tissue Kit and genome was interrupted by ultrasound; 2. Immunoprecipitation; 3. DNA Labeling and Array Hybridization; 4. Image acquisition and data analysis, Comparative analysis the distribution of 5-hmC between SLE patients and normal controls.Screened the target gene which significant differences in the level of 5-hmC and further line of cluster analysis, gene ontology (gene ontology, GO) and pathway analysis; 5. The genes included TREX1, CDKN1A and CDKN1B that were analyzed and verified by Quantitative real-time PCR.Results:1. There were 1701 genes with significantly different 5-hmC levels at the promoter region in the SLE patients compared with the normal controls. Of these SLE-associated differentially hydroxymethylated genes,884 genes had higher 5-hmC levels, and 817 genes had lower 5-hmC levels in the SLE patients compared with the normal controls.3826 genes of the CpG islands showed significantly different 5-hmC levels in the SLE patients compared with the normal controls. Among these genes,2034 genes were hyper-hydroxymethylated and 1792 were hypo-hydroxymethylated; 2. Pathway analysis revealed a total of 175-hmC differences in signal transduction pathways, respectively, P less than 0.05, which histidine metabolic, P=0.000632576, Glycerolipid metabolism of triglycerides, P=0.005711304, calcium signaling pathway, P=0.005990646 highly correlated; 3. Out of the differentially hydroxymethylated genes, three were selected for validation, including three prime repair exonuclease 1 (TREX1), cyclin-dependent kinase inhibitor 1A (p21, Cipl; CDKN1A), and cyclin-dependent kinase inhibitor IB (p27, Kip1; CDKN1E). By GO analysis found that the purpose of these three genes associated with cellular process, DNA metabolic process, cellular response to stress; 4. The hydroxymethylation levels of these three genes were confirmed by quantitative RT-PCR.Conclusion:1. hMeDIP-chip is an accurate and reliable experimental technique can analysis the DNA 5-hmC state of genomic, with high sensitivity, specificity, good reproducibility, which makes use of magnetic beads to capture quite fast; 2.5-hmC modifications related with SLE, it may be able to reveal the pathogenesis of SLE, and provides a new way to identify the potential biomarkers; 3. The selected gene including TREX1, CDKN1A, CDKN1B, it is likely to be used as potential biomarkers and therapeutic targets, provide new evidence for the diagnosis and treatment of SLE.