Study On Aberrant Epigenetic Modifications In Peripheral Blood Mononuclear Cells And Skin Lesions From Patients With Psoriasis Vulgaris

Background Psoriasis vulgaris, a common, chronic and relapsing inflammatory skin disease, is the commonest subtype of psoriasis clinically associated with epidermal hyperplasia and vascular proliferation. The etiology and pathogenesis of psoriasis are still unclear. Patients with psoriasis usually have a recurring manifestation, characterized by aggravation in winter and remission in summer. Factors promoting or aggravating the disease include infection, mental tension and stress events, trauma, surgery, pregnancy, smoking and certain drugs. More and more evidence indicates that environmental factors play significant roles in the occurrence and development of psoriasis. Psoriasis vulgaris is a T-cell-mediated autoimmune skin disease associated with epidermal hyperplasia and vascular proliferation, although putative autoantigen remains unknown. Previous studies demonstrated that epigenetic modification plays an important role in the occurrence and development of autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). However, the role and mechanism of epigenetic regulation in the pathogenesis of psoriasis remains unclear.Epigenetics, referring to stable and heritable changes in gene expression independent of alterations in DNA coding sequence, comprises of DNA methylation, histone modifications and microRNAs.DNA methylation, the most characterized epigenetic modification, takes place almost exclusively at the 5'-carbon position of cytosine residues within CpG pairs, which is catalyzed by DNA methyltransferases (DNMTs) with S-adenosyl-methionine (SAM) as the methyl donor. DNA methylation exists in many kinds of organisms such as bacterial, plants and mammalians, which is a modification style in nature. In general, DNA methyaltion is associated with gene siliencing, while DNA hypomethylation is responsible for gene activation. Up to now, DNA hypermethylation in promoter regions have been found to be associated with gene silencing of a great number of tumor suppressor genes, while DNA hypomethylation will lead to over-expression of genes which are suppressed in normal conditions.Histone modifications are another important epigenetic mechanism. Histones are the alkaline proteins binding to eukaryotic DNA, consisting of H1, H2A, H2B, H3 and H4. An octamer is constructed by H2A, H2B, H3 and H4, surrounded by 146 bp of DNA. The post-translational modifications on each histone subtype include acetylation, methylation, phosphorylation, ubiquitination, ADP ribosylation and so on, which can interact each other or with other factors to provide binding sites for transcriptional regulation proteins activiating or inhibiting gene expression. The study on histone modifications focuses on the acetylation and methylation of lysine residues of N-terminal tails. Histone acetylation can neutralize the positive charge in histones and therefore lossens their interaction with DNA backbone with the negatively charge, resulting in chromatin structure more open and accessible for transcriptional factors. In contrast, methylation in different sites of histones may either activates or repress gene expression.In recent years, microRNAs, a class of newly-discovered small RNA, are believed to be involved in post-transcriptional regulation of gene expression in eukaryotic cells. MicroRNAs are genome-encoded 21-23nt RNAs targeting the 3'untranslated region (3'-UTR) of specific messenger RNAs (mRNA) for degradation or translational repression. It has been proved that microRNAs play important roles in growth, development, cell proliferation, apoptosis, lipid metabolism and tumor formation as well as many other physiological and pathological processes. microRNAs exist in the majority of multi-cell organisms and account for 2% among the genome. Microarray is the best choice to detect the expression of microRNA.To investigate the question whether there are any changes in epigenetic modifications in psoriasis, we detected DNA methylation status, histone acetylation/methylation levels and microRNAs expression in patients with psoriasis vulgaris. Firstly, we detected the global genomic DNA methylaiton status in peripheral blood monouclear cells (PBMCs) and skin lesions as well as mRNA expression levels of DNA methyltransferase (DNMTs) and Methylated CpG binding proteins (MBDs) in PBMCs; secondly, we detected mRNA level and promoter methylation status of p14ARF in skin lesions. We also measured the global histone H3/H4 acetylation and H3-K4/H3-K27 methylation levels in PBMCs and mRNA levels of histone acetyltransferases (HATs) and histone deacetylases (HDACs); finally, miRNA expression profiling and real-time PCR were carried out to identify the aberrant microRNA expression in PBMCs from psoriatic patients. Our study will contribute to uncover whether epigenetic modifications are involved in the occurence and development of psoriasis and establish foundations for comprehensive and deep study on the pathogenesis of psoriasis, thereby providing the novel clues for the diagnosis and treatment for psoriasis. Objective To investigate global DNA methylation and the expression of genes that regulate methylation in patients with psoriasis vulgaris.Methods Global DNA methylation level in PBMCs from 30 psoriatic patients and 20 healthy controls was quantified by an ELISA-like reaction using 5-methylcytosine antibody. Skin samples from 30 psoriatic patients and 15 healthy controls were labeled immunohistochemically with mouse monoclonal anti-5-methylcytosine antibody. The expression of enzymes involved in DNA methylation was measured using real-time PCR. The mRNA level and methylation status of the tumor suppressor gene p14ARF was assessed by real-time PCR and methylation-specific PCR, respectively.Results Compared with healthy controls, PBMCs genomic DNA was significantly hypermethylated in patients with psoriasis vulgaris (p=0.011), and DNMT1 mRNA expression was upregulated (p=0.011). Expression levels of MBD2 and MeCP2 mRNA were significantly lowered in PBMCs from psoriatic patients than healthy controls (p=0.043, p=0.000). Anti-5-methylcytosine labeling was increased in psoriatic skin lesions compared to healthy controls (p=0.007), and there was a significantly positive correlation between 5-methylcytosine labeling and Psoriasis Area and Severity Index (PASI) scores (r=0.868, p=0.000). The tumor suppressor gene p14ARF exhibited decreased mRNA expression (p=0.006) and increased promoter methylation in psoriatic skin lesions compared to healthy skin tissues (p=0.036).Conclusion Aberrant DNA methylation may play an important role in the pathogenesis of psoriasis vulgaris. Objective To investigate global histone H3/H4 acetylation, H3K4/H3K27 methylation levels and the mRNA expression of enzymes involved in histone modifications in patients with psoriasis vulgaris.Methods Global histone H3/H4 acetylation and H3K4/H3K27 methylation in PBMCs from 30 psoriatic patients and 20 normal controls were assayed using the EpiQuikTM global histone H3/H4 acetylation, and H3K4/H3K27 methylation assay kit, respectively. The mRNA levels of enzymes involved in histone acetylation/methylation were measured by real-time PCR.Results Global histone H4 hypoacetylation was observed in PBMCs from patients with psoriasis vulgaris compared with normal controls (p=0.005). There were no significant differences in H3 acetylation and H3K4/H3K27 methylation levels between psoriatic patients and normal controls (p>0.05). The expression of HDAC1 mRNA was upregulated (p=0.005), while mRNA levels of P300, CBP, SIRT1 were decreased in PBMCs from patients with psoriasis vulgaris compared to healthy controls (p=0.024, p=0.006, p=0.002, respectively).Conclusion Abnormal histone acetylation may contribute to development of psoriasis. Objective To investigate the roles of microRNAs in the pathogenesis of psoriasis vulgaris.Methods PBMCs from 9 patients with psoriasis vulgaris and 9 normal controls were extracted by Ficoll-Hypaque density gradient centrifugation. Total RNA including microRNAs was extracted using TRIzol reagent. MicroRNAs with differential expression were detected by Affymetrix microRNAs chips and the results were confirmed in 30 patients with psoriasis vulgaris and 20 healthy controls by real-time PCR.Results The results from Affymetrix microRNAs chips showed that miR-210, miR-584, miR193b and miR-501-3p were overexpressed in PBMCs from patients with psoriasis compared to normal controls (p<0.01), all of which were confirmed in 30 patients and 20 healthy controls by real-time PCR. In addition, the increased expression of miR-203 was also identified in PBMCs from patients with psorisis vulgaris (p<0.01).Conclusion Aberrant microRNAs expression may play a role in the pathogenesis of psoriasis vulgaris.