Effects of SMYD3 and MLL5 on histone methylation of Foxp3 gene in children with Kawasaki disease / 中华风湿病学杂志

ABSTRACT

Objective To investigate the effects of SMYD3 and MLL5 on histone methylation of Transcription factor forkhead box protein 3 (Foxp3) gene and its roles in the immunological pathogenesis of Kawasaki disease (KD). Methods Forty-two children with KD and 26 age-matched healthy children were consented to participate in this study. Co-Immunoprec-ipitation and real-time polymerase chain reaction (PCR) was performed to determine Foxp3-associated histone methylation levels of H3K4me3 and H3K27me3, and binding levels of SMYD3 and MLL5 with Foxp3 gene in CD4+T cells. The proportion of CD4+CD25high Foxp3+cells (Treg) and protein levels of Foxp3, cytotoxic T lymphocyte associated antigen-4 (CTLA4), TGF-βRⅡand pSmad3 were analyzed by flow cytometry. Quantitative real-time PCR was used to evaluate levels of Foxp3, interleukin (IL)-10, GITR, TGF-βRⅠand RARαmRNA in CD4+T cells. Plasma concentrations of TGF-βand retinol acid (RA) were measured by enzyme-linked Immunosorbent assay. Independent-samples t-test was used as the statistical method in this study. Results ① The proportion of Treg, expression levels of Foxp3 and molecules associated with suppressive function of Treg cells(IL-10, GITR and CTLA4), and histone methylation levels of H3K4me3 associating with promoter, conserved non-coding DNA sequence (CNS) 1 and CNS2 of Foxp3 gene decreased remarkably during acute KD [Promoter(5.4±1.8)%vs (9.1±2.2)%;CNS1(2.6±0.9)% vs (3.8±1.1)%; CNS2 (2.4±0.8)% vs (4.2±1.0)%; t=5.50, 6.02, 9.56, 7.92, 7.97, 4.76, 7.73, 5.01, 8.66; P0.05). ② Binding levels of SMYD3 and MLL5 with Foxp3 gene in CD4+T cells were down-regulated significantly during acute KD (t=6.63, 6.15; P<0.05), and restored to some extent after IVIG treatment (t=5.36, 4.56; P<0.05). Positive correlations between binding levels of SMYD3 and MLL5 and expression level of Foxp3 mRNA were detected in patients with acute KD (r=0.62、0.45, P<0.05). Furthermore, Binding levels of SMYD3 and MLL5 with Foxp3 gene in KD-CAL+group were lower than those in KD-CAL- group (t=4.11, 4.31; P<0.05). ③ Compared with healthy controls, plasma concentration of TGF-β and RA, and expressions of TGF-βRⅡ, TGF-βRⅠ, pSmad3 and RARα were down-regulated during acute KD (t=11.54, 12.81, 7.43, 16.10, 8.25, 12.06; P<0.05), and elevated remarkably after IVIG treatment (t=8.40, 6.24, 5.94, 11.78, 6.27, 8.30; P<0.05). Simultaneously, all the items aforementioned in KD-CAL+ group were found to be lower than those in KD-CAL-group (t=3.58, 3.30, 3.82, 5.27, 4.71, 3.78; P<0.05). Conclusion Hypomethylation of H3K4me3 associated with Foxp3 gene caused by insufficient binding levels of SMYD3/MLL5 may be involved with immune dysfunction in Kawasaki disease.