HLA-F DNA甲基化、转录水平及血清细胞因子与慢性乙型肝炎患者中医证候分型相关

Xue-qing HU; Yuan ZHOU; Jian CHEN; Yi-yu LU; Qi-long CHEN; Yi-yang HU; Shi-bing SU

doi:10.1007/s11655-021-3279-8

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HLA-F DNA甲基化、转录水平及血清细胞因子与慢性乙型肝炎患者中医证候分型相关

Original Article | Updated：2022-06-27

- HLA-F DNA甲基化、转录水平及血清细胞因子与慢性乙型肝炎患者中医证候分型相关
- DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B
- Chinese Journal of Integrative Medicine 2022年28卷第6期页码：501-508
- 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
  2.Department of Medical Oncology,Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai (201203), China
  3.Academy of Integrative Medicine, Shanghai University of Traditional Chinese Medicine,Shanghai (201203), China
  4.Department of Vascular Disease,Shanghai Traditional Chinese Medicine-Integrated Hospital,Shanghai University of Traditional Chinese Medicine, Shanghai(201203), China
  5.Department of Vascular Disease, Shanghai Traditional Chinese Medicine-Integrated Institute of Vascular Disease, Shanghai (201203), China
  6.Institute of Liver Disease, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai (201203), China
- Author bio：
  
  Prof. SU Shi-bing, E-mail: shibingsu07@163.com
- Funds：
  
  Key Program of National Natural Science Foundation of China(81330084);National Science and Technology Major Special Project(2012ZX10005001-004);National Key Research and Development: Special Project for Research on the Modernization of Traditional Chinese Medicine(2018YFC1704204);Science and Technology Commission of Shanghai Municipality(19YF1449900)
- DOI：10.1007/s11655-021-3279-8
  中图分类号：
- 纸质出版日期：2022-06-01，
  
  网络出版日期：2021-01-09，
  
  录用日期：2019-11-25
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Xue-qing HU, Yuan ZHOU, Jian CHEN, 等. HLA-F DNA甲基化、转录水平及血清细胞因子与慢性乙型肝炎患者中医证候分型相关[J]. Chinese Journal of Integrative Medicine, 2022,28(6):501-508.

Xue-qing HU, Yuan ZHOU, Jian CHEN, et al. DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B[J]. Chinese Journal of Integrative Medicine, 2022,28(6):501-508.
Xue-qing HU, Yuan ZHOU, Jian CHEN, 等. HLA-F DNA甲基化、转录水平及血清细胞因子与慢性乙型肝炎患者中医证候分型相关[J]. Chinese Journal of Integrative Medicine, 2022,28(6):501-508. DOI： 10.1007/s11655-021-3279-8.

Xue-qing HU, Yuan ZHOU, Jian CHEN, et al. DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B[J]. Chinese Journal of Integrative Medicine, 2022,28(6):501-508. DOI： 10.1007/s11655-021-3279-8.

摘要

目的:

从DNA甲基化、转录及细胞因子角度

探讨慢性乙型肝炎 (慢乙肝) 患者中医证候分型的生物学基础.

方法:

检测15例慢乙肝患者 (5例肝胆湿热证、5例肝郁脾虚证、5例肝肾阴虚证)及6名健康者的全基因组DNA甲基化水平; 并检测62例慢乙肝患者 (包括15例肝胆湿热证、15例肝郁脾虚证、16例肝肾阴虚证及16例隐证) 的48种血清细胞因子水平. 经全基因组DNA甲基化水平分析

找出关键甲基化基因人白细胞抗原F (HLA-F)

采用焦磷酸测序扩大样本进一步验证其DNA甲基化水平

并通过酶联免疫吸附测定 (ELISA) 检测血清细胞因子

采用逆转录定量聚合酶链反应 (RT-qPCR) 检测HLA-F转录水平.

结果:

667个DNA甲基化位点

覆盖18

403个DNA. 肝胆湿热证、肝郁脾虚证和肝肾阴虚证患者甲基化水平之间有明显差异(

0.05 和 |Δβ value|

0.17). 与健康者比较

肝胆湿热证患者 hg19 HLA-F CHR6: 29691140 及相邻的两个位点均发生去DNA甲基化

且HLA-F转录水平上调 (

0.05)

但肝郁脾虚证中均未改变(

0.05). 在肝肾阴虚证和隐证患者中

IL-2水平高于肝胆湿热证和肝郁脾虚证. MIP-1α和MIP-1β水平在肝肾阴虚证中较高 (

0.05)

而白血病抑制因子(LIF)水平在肝胆湿热证和隐证中较高(

0.05). IL-12

MIP-1α 和MIP-1β 水平与 HLA-F mRNA 表达量呈正相关 (依次为R2=0.238

0.05; R2 = 0.224

0.05; R=0.447

0.01). 结合HLA-F转录水平及差异细胞因子提高了慢乙肝证候分型的准确性.

结论:

HLA-F 位于5' UTR区域的CpG位点去甲基化可促进HLA-F转录. HLA-F 表达与血清IL-12

MIP-1α 和 MIP-1β 水平相关

二者可能共同参与慢乙肝患者证候分型.

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 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 (

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 (

0.05). However

they remained unaltered in CHB2 (

0.05). Levels of Interleukin (IL)-12 were higher in CHB3 and HS than that in CHB1 and CHB2 groups (

0.05). Levels of macrophage inflammatory protein (MIP)-1α and 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 (

0.05). IL-12

MIP-1α and MIP-1β concentrations were positively correlated with human leukocyte antigen F (HLA-F) mRNA expression (

=0.238

0.05;

=0.224

0.05;

=0.447

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)

关键词

Keywords

cytokinechronic hepatitis BDNA methylationhuman leukocyte antigen FChinese medicine syndrome

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