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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

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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 450K Assay and furthered 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 |Δβ 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)-1α and MIP-1β were higher in CHB3 than other groups and leukemia inhibitory 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 (R2=0.238, P<0.05; R2=0.224, P<0.05; R=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.

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Authors and Affiliations

  1. Research Center for Traditional Chinese Medicine Complexity System, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China

    Xue-qing Hu, Yuan Zhou, Jian Chen, Yi-yu Lu, Qi-long Chen & Shi-bing Su

  2. Department of Medical Oncology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China

    Xue-qing Hu

  3. Academy of Integrative Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China

    Xue-qing Hu

  4. Department of Vascular Disease, Shanghai Traditional Chinese Medicine-Integrated Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China

    Jian Chen

  5. Department of Vascular Disease, Shanghai Traditional Chinese Medicine-Integrated Institute of Vascular Disease, Shanghai, 201203, China

    Jian Chen

  6. Institute of Liver Disease, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China

    Yi-yang Hu

Authors
  1. Xue-qing Hu
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  2. Yuan Zhou
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  3. Jian Chen
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  4. Yi-yu Lu
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  5. Qi-long Chen
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  7. Shi-bing Su
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Contributions

Su SB designed the study and revised the article. Hu YY collected the CHB samples in clinical. Hu XQ conducted the experiments, performed the analyses and wrote the manuscript. Chen J helped to draw the figures. Lu YY conducted the cytokines detection. Zhou Y and Chen QL offered suggestions for experiment performing and data analyses.

Corresponding author

Correspondence to Shi-bing Su.

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Conflict of Interest

The authors declare that they have no conflict of interests.

Supported by Key Program of National Natural Science Foundation of China (No. 81330084), National Science and Technology Major Special Project (No. 2012ZX10005001-004), National Key Research and Development: Special Project for Research on the Modernization of Traditional Chinese Medicine (No. 2018YFC1704204) and Science and Technology Commission of Shanghai Municipality (No.19YF1449900)

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Hu, Xq., Zhou, Y., Chen, J. et al. DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B. Chin. J. Integr. Med. 28, 501–508 (2022). https://doi.org/10.1007/s11655-021-3279-8

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  • DOI: https://doi.org/10.1007/s11655-021-3279-8

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