慢性HBV感染者外周血单个核细胞中miR-194-5p和MagT1表达水平检测及其意义

doi:10.13481/j.1671-587x.20200328

吉林大学学报(医学版) ›› 2020, Vol. 46 ›› Issue (03): 607-613.doi: 10.13481/j.1671-587x.20200328

• 临床研究 • 上一篇

慢性HBV感染者外周血单个核细胞中miR-194-5p和MagT1表达水平检测及其意义

林晓娥¹, 吴景华², 黎洁¹, 刘洋³, 谌晓楠³, 祝朝前³, 李广平¹

1. 河北省唐山市工人医院检验科, 河北唐山 063000;
2. 华北理工大学附属医院检验科, 河北唐山 063000;
3. 河北省唐山市工人医院肝胆外科, 河北唐山 063000

收稿日期:2019-07-12 发布日期:2020-06-11
通讯作者: 林晓娥,副主任技师(Tel:0315-3722303,E-mail:xiaoe721@163.com) E-mail:xiaoe721@163.com
作者简介:林晓娥(1975-),女,河北省唐山市人,副主任技师,医学硕士,主要从事检验医学方面的研究。
基金资助:
河北省自然科学基金资助课题（H2016209007）；河北省唐山市科技局科技支撑计划项目资助课题（12130296b）

Detection of expression levels of miR-194-5p and MagT1 in peripheral blood mononuclear cells of patients with chronic HBV infection and their significances

LIN Xiaoe¹, WU Jinghua², LI Jie¹, LIU Yang³, SHEN Xiaonan³, ZHU Chaoqian³, LI Guangping¹

1. Department of Clinical Laboratory, Tangshan Worker's Hospital, Tangshan 063000, China;
2. Department of Clinical Laboratory, Affiliated Hospital, North China University of Science and Technology, Tangshan 063000, China;
3. Department of Hepatobiliary Surgery, Tangshan Worker's Hospital, Tangshan 063000, China

Received:2019-07-12 Published:2020-06-11

摘要/Abstract

摘要： 目的：检测慢性乙型肝炎病毒（HBV）感染者外周血单个核细胞（PBMCs）中miR-194-5p和镁离子（Mg²⁺）转运蛋白1（MagT1）表达水平，阐明其可能的临床意义。方法：收集40例健康体检者（健康对照组）、40例HBV携带者（HBV携带组）、40例轻中度慢性乙型肝炎（轻中度CHB组）和40例重度慢性乙型肝炎（重度CHB组）患者的临床资料。采集各组研究对象外周血并利用Ficoll-Hypaque密度梯度离心法分离PBMCs，采用RT-PCR法检测各组研究对象PBMCs中miR-194-5p及MagT1 mRNA表达水平，Western blotting法检测各组研究对象PBMCs中MagT1蛋白表达水平，检测各组研究对象外周血和PBMCs中Mg⁺水平，流式细胞术检测各组研究对象PBMCs中CD8+T淋巴细胞表面分子程序性死亡受体1（PD-1）和自然杀伤细胞受体2群D分子（NKG2D）表达水平。在293T细胞中分别转染miR-194-5p mimic质粒（miR-194-5p mimic组）和miR-NC质粒（miR-NC组），应用双荧光素酶报告基因实验检测2组293T细胞中荧光素酶活性。结果：与健康对照组比较，HBV携带组、轻中度CHB组和重度CHB组患者PBMCs中MagT1 mRNA和蛋白表达水平、Mg²⁺水平及CD8+T淋巴细胞表面分子NKG2D表达水平均明显降低（P<0.05），miR-194-5p mRNA及CD8+T淋巴细胞表面分子PD-1表达水平升高（P<0.05）。与HBV携带组和轻中度CHB组比较，重度CHB组患者PBMCs中MagT1 mRNA和蛋白表达水平、Mg²⁺水平及CD8+T淋巴细胞表面分子NKG2D表达水平均降低（P<0.05），miR-194-5p mRNA及CD8+T细胞表面分子PD-1表达水平降低（P<0.05）。与HBV携带组比较，轻中度CHB组患者PBMCs中MagT1 mRNA和蛋白表达水平、Mg²⁺水平及CD8+T淋巴细胞表面分子NKG2D表达水平降低（P<0.05），miR-194-5p mRNA及CD8+T淋巴细胞表面分子PD-1表达水平升高（P<0.05）。双荧光素酶报告基因实验，MagT1是miR-194-5p靶基因。结论：miR-194-5p可能通过靶向下调MagT1表达，引起MagT1介导的Mg²⁺内流障碍，造成CD8+T淋巴细胞免疫活性降低，导致乙型肝炎慢性发展。

关键词: 乙型肝炎病毒, 外周血单个核细胞, miR-194-5p, 镁离子转运蛋白1

Abstract: Objective: To detect the expression levels of miR-194-5p and magnesium transporter protein1(MagT1) in peripheral blood mononuclear cells (PBMCs) of the patients with chronic hepatitis B virus(HBV) infection, and to investigate their possible clinical significances. Methods: The clinical materials of 40 healthy subjects (healthy control group), 40 cases of asymptomatic hepatitis B carriers(HBV carrier group), 40 patients with mild-moderate chronic hepatitis B (mild-moderate CHB group) and 40 patients with severe chronic hepatitis B (severe CHB group) were collected. The peripheral blood of the subjects in various groups was collected and the PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation. The expression levels of miR-194-5p and MagT1 mRNA in PBMCs of the subjects in various groups were detected by RT-PCR assay, the expression levels of MagT1 protein in PBMCs of the subjects in various groups were detected by Western blotting method,the levels of Mg²⁺ in peripheral blood and PBMCs of the subjects in various groups were detected, and the expression levels of CD8+ T lymphocyte surface molecules programmed death receptor 1(PD-1) and natural killer cell receptor 2 group D molecule (NKG2D) in PBMCs of the subjects in various groups were analyzed by flow cytometry. The miR-194-5p mimic (miR-194-5p mimic group)and miR-NC(miR-NC group) were transfected into the 293T cells, and then the dual luciferase reporter gene assay was used to detect the luciferase activities in 293T cells in two groups. Results: Compared with healthy control group, the expression levels of MagT1 mRNA and protein, the expression levels of CD8+ T lymphocyte surface molecule NKG2D and the levels of Mg²⁺ in PBMCs of the patients in HBV carrier group, mild-moderate CHB group and severe CHB group were decreased(P<0.05),the expression levels of miR-194-5p mRNA in PBMCs and CD8+ T lymphocyte surface molecule PD-1 of the patients were increased(P<0.05).Compared with HBV carrier group and mild-moderate CHB group,the expression levels of MagT1 mRNA and protein, the expression level of CD8+ T lymphocyte surface molecule NKG2D and the level of Mg²⁺ in PBMCs of the patients in severe CHB group were decreased (P<0.05), and the expression levels of miR-194-5p mRNA in PBMCs and CD8+ T lymphocyte surface molecule PD-1 of the patients were increased (P<0.05).Compared with HBV carrier group, the expression levels of MagT1 mRNA and protein, the expression level of CD8+ T lymphocyte surface molecule NKG2D and the level of Mg²⁺ in PBMCs of the patients in mild-moderate CHB group were decreased(P<0.05),and the expression levels of miR-194-5p mRNA in PBMCs and CD8+ T lymphocyte surface molecule PD-1 of the patients were increased (P<0.05).The dual luciferase reporter gene assay results confirmed that MagT1 was a miR-194-5p target gene. Conclusion: MiR-194-5p may lead to chronic development of hepatitis B by down-regulating the expression of MagT1, causing MagT1-mediated Mg²⁺ influx disorder and resulting in the decrease of the immune activity of CD8+ T lymphocyte.

Key words: hepatitis B virus, peripheral blood mononuclear cell, miR-194-5p, magnesium transporter protein 1

中图分类号:

R512.62

林晓娥, 吴景华, 黎洁, 刘洋, 谌晓楠, 祝朝前, 李广平. 慢性HBV感染者外周血单个核细胞中miR-194-5p和MagT1表达水平检测及其意义[J]. 吉林大学学报(医学版), 2020, 46(03): 607-613.

LIN Xiaoe, WU Jinghua, LI Jie, LIU Yang, SHEN Xiaonan, ZHU Chaoqian, LI Guangping. Detection of expression levels of miR-194-5p and MagT1 in peripheral blood mononuclear cells of patients with chronic HBV infection and their significances[J]. Journal of Jilin University(Medicine Edition), 2020, 46(03): 607-613.

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慢性HBV感染者外周血单个核细胞中miR-194-5p和MagT1表达水平检测及其意义

Detection of expression levels of miR-194-5p and MagT1 in peripheral blood mononuclear cells of patients with chronic HBV infection and their significances

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