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DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B

Release time: Sept 29,2022 Reading volume: 153 Source: wfcms
Objective:To explore the molecular bases of Chinese medicine(CM) syndrome classification in chronic hepatitis B(CHB) patients in terms of DNA methylation,transcription and cytokines.Methods:Genome-wide DNA methylation and 48 serum cytokines were detected in CHB patients(DNA methylation:15 cases;serum cytokines:62 cases) with different CM syndromes,including dampness and heat of Gan(Liver) and gallbladder(CHB1,DNA methylation:5 cases,serum cytokines:15 cases),Gan stagnation and Pi(Spleen) deficiency(CHB2,DNA methylation:5 cases,serum cytokines:15 cases),Gan and Shen(Kidney) yin deficiency(CHB3,DNA methylation:5 cases,serum cytokines:16 cases),CHB with hidden symptoms(HS,serum cytokines:16 cases) and healthy controls(DNA methylation:6 cases).DNA methylation of a critical gene was further validated and its mRNA expression was detected on enlarged samples.Genome-wide DNA methylation was detected using Human Methylation 450 K Assay and further verified using pyrosequencing.Cytokines and mRNA expression of gene were evaluated using multiplex biometric enzyme-linked immunosorbent assay(ELISA)-based immunoassay and reverse transcription-quantitative polymerase chain reaction(RT-qPCR),respectively.Results:Totally 28,667 loci,covering 18,403 genes were differently methylated among CHB1,CHB2 and CHB3(P<0.05 and|△β value|> 0.17).Further validation showed that compared with HS,the hg19 CHR6:29691140 and its closely surrounded 2 CpG loci were demethylated and its mRNA expressions were significantly up-regulated in CHB1(P<0.05).However,they remained unaltered in CHB2(P>0.05).Levels of Interleukin(IL)-12 were higher in CHB3 and HS than that in CHB1 and CHB2 groups(P<0.05).Levels of macrophage inflammatory protein(MIP)-1αand MIP-1β were higher in CHB3 than other groups and leukemia inhibrtory factor level was higher in CHB1 and HS than CHB2 and CHB3 groups(P<0.05).IL-12,MIP-1α and MIP-1β concentrations were positively correlated with human leukocyte antigen F(HLA-F)mRNA expression(R~2=0.238,P<0.05;R~2=0.224,P<0.05;R~2=0.447,P<0.01;respectively).Furthermore,combination of HLA-F mRNA and differential cytokines greatly improved the differentiating accuracy among CHB1,CHB2 and HS.Conclusions:Demethylation of CpG loci in 5’ UTR of HLA-F may up-regulate its mRNA expression and HLA-F expression was associated with IL-12,MIP-1α and MIP-1β levels,indicating that HLA-F and the differential cytokines might jointly involve in the classification of CM syndromes in CHB.(Registration No.ChiCTR-RCS-13004001)

cytokine;chronic hepatitis B;DNA methylation;human leukocyte antigen F;Chinese medicine syndrome