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: 854
Abstract：
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）
Keywords：
cytokine;chronic hepatitis B;DNA methylation;human leukocyte antigen F;Chinese medicine syndrome
[ FULL TEXT ]
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）
Keywords：
cytokine;chronic hepatitis B;DNA methylation;human leukocyte antigen F;Chinese medicine syndrome
[ FULL TEXT ]
