乙型肝炎病毒包膜蛋白诱导肝癌细胞系内质网应激的研究

doi:10.3877/cma.j.issn.1674-1358.2016.03.027

目的

探讨乙型肝炎病毒前-S缺失和野生型外膜大蛋白（LHBs）诱导内质网应激反应。

方法

运用脂质体转染技术将pcDNA3.1-S、pcDNA3.1-L、pcDNA3.1-L△30和pcDNA3.1-L△182等真核表达质粒转染至Huh7细胞，并应用qRT-PCR和Western blot方法检测转染细胞前-S1、前-S2、HBsAg和GRP78的mRNA和蛋白表达水平。

结果

转染HBV包膜蛋白各组的HBsAg蛋白相对表达量（分别为0.92、0.83、0.91、1.75、2.53和2.06）高于空白对照（0.00）和pcDNA3.1（+）转染组（0.00）（F = 247.38、P = 0.000）；转染HBV包膜蛋白各组的GRP78的蛋白相对表达量（分别为1.4、1.47、1.55、1.75、1.8和1.9）高于空白对照（1.18）和pcDNA3.1(+)转染组（1.23）（F = 11.623、P = 0.000）；且GRP78蛋白相对表达量与前-S1、前-S2抗原和HBsAg表达量呈正相关（相关系数r分别为0.884、0.728和0.816）。

结论

前-S缺失型/野生型LHBs及HBsAg均能诱导Huh7细胞发生内质网应激反应，提示其可能参与肝癌发生。

关键词: 肝炎病毒, 乙型, 外膜大蛋白, 表面抗原, 葡萄糖调节蛋白78, 内质网应激

Objective

To explore endoplasmic reticulum stress in Huh7 cells induced by wild type and pre-S mutant envelope proteins of HBV.

Methods

The recombinant eukaryotic expression vectors of pcDNA3.1-S, pcDNA3.1-L, pcDNA3.1-L△30 and pcDNA3.1-L△182 were transfected into Huh7 cells through liposome transfection, then the protein exrpression of pre-S1, pre-S2, HBsAg and Grp78 were detected by Western blot, and Grp78 mRNA transcripts were detected by qRT-PCR.

Results

The relative expression of HBsAg protein in Huh7 cells transfected with recombinant eukaryotic expression vectors (0.92, 0.83, 0.91, 1.75, 2.53 and 2.06, respectively) were significantly higher than that in blank control cells (0.00) and that in cells transfected with pcDNA3.1(+) (0.00) (F = 247.38, P = 0.000). The relative expression of GRP78 protein in Huh7 cells transfected with recombinant eukaryotic expression vectors (1.4, 1.47, 1.55, 1.75, 1.8 and 1.9, respectively) were significantly higher than that in blank control cells (1.18) and that in cells transfected with pcDNA3.1(+) (1.23) (F = 11.623, P = 0.000). The relative expression of GRP78 protein had opositive correlation with that of pre-S1, pre-S2 and HBsAg (r = 0.884, 0.728 and 0.816, respectively).

Conclusions

Expression of envelope proteins of HBV could induce endoplasmic reticulum stress, indicating they might involve in hepatocarcinogenesis.

Key words: Hepatitis B virus, Hepatitis B virus large surface protein, Hepatitis B virus surface antigen, Glucose regulated protein 78, Endoplasmic reticulum stress

表1 RT-PCR所有引物序列、位置和长度

名称	序列（5′→3′）	位置	长度（bp）
18srRNA-F	CCTGGATACCGCAGCTAGGA	846～861	108
18srRNA-R	GCGGCGCAATACGAATGCCCC	953～932	108
GRP78-F	CCCTGTCTTCTCAGCATCAA	1 154～1 173	101
GRP78-R	TAGACCGGAACAGATCCATGT	1 273～1 253	101
HBsAg-F	TGCCCGTTTGTCCTCTACTT	466～485	101
HBsAg-R	CCGAAGGTTTTGTACAGCAA	584～565	101
LHBs-F	ATGGGAGGTTGGTCTTCC	2 848～2 865	116
LHBs-R	TTTGAGTTGGCTCCGAAC	2 963～2 946	116

表1 RT-PCR所有引物序列、位置和长度

名称	序列（5′→3′）	位置	长度（bp）
18srRNA-F	CCTGGATACCGCAGCTAGGA	846～861	108
18srRNA-R	GCGGCGCAATACGAATGCCCC	953～932	108
GRP78-F	CCCTGTCTTCTCAGCATCAA	1 154～1 173	101
GRP78-R	TAGACCGGAACAGATCCATGT	1 273～1 253	101
HBsAg-F	TGCCCGTTTGTCCTCTACTT	466～485	101
HBsAg-R	CCGAAGGTTTTGTACAGCAA	584～565	101
LHBs-F	ATGGGAGGTTGGTCTTCC	2 848～2 865	116
LHBs-R	TTTGAGTTGGCTCCGAAC	2 963～2 946	116

表2 GRP78、HBsAg和LHBs等mRNA相对表达量（±s）

分组	GRP78	HBsAg	LHBs
空白组	1.00 ± 0.12	1.00 ± 0.14	1.00 ± 0.17
pcDNA3.1	1.03 ± 0.18	2.40 ± 0.13	1.11 ± 0.18
pcDNA3.1-S	1.23 ± 0.28	16 561.36 ± 1 490.21 ^b	1.22 ± 0.21
pcDNA3.1-L	1.42 ± 0.22^a	22 847.78 ± 1 515.87^b	26.51 ± 1.68^b
pcDNA3.1-L△30	1.33 ± 0.21	11 877.42 ± 770.54^b	21.11 ± 2.37^b
pcDNA3.1-S + pcDNA3.1-L	1.37 ± 0.25	37 047.28 ± 1 680.17^b	23.24 ± 2.07^b
pcDNA3.1-S + pcDNA3.1-L△30	1.28 ± 0.27	31 560.34 ± 1 824.43^b	18.27 ± 1.17^b
pcDNA3.1-S + pcDNA3.1-L△183	1.43 ± 0.31	43 271.72 ± 2 863.84^{b *}	20.37 ± 1.85^b

表2 GRP78、HBsAg和LHBs等mRNA相对表达量（±s）

分组	GRP78	HBsAg	LHBs
空白组	1.00 ± 0.12	1.00 ± 0.14	1.00 ± 0.17
pcDNA3.1	1.03 ± 0.18	2.40 ± 0.13	1.11 ± 0.18
pcDNA3.1-S	1.23 ± 0.28	16 561.36 ± 1 490.21 ^b	1.22 ± 0.21
pcDNA3.1-L	1.42 ± 0.22^a	22 847.78 ± 1 515.87^b	26.51 ± 1.68^b
pcDNA3.1-L△30	1.33 ± 0.21	11 877.42 ± 770.54^b	21.11 ± 2.37^b
pcDNA3.1-S + pcDNA3.1-L	1.37 ± 0.25	37 047.28 ± 1 680.17^b	23.24 ± 2.07^b
pcDNA3.1-S + pcDNA3.1-L△30	1.28 ± 0.27	31 560.34 ± 1 824.43^b	18.27 ± 1.17^b
pcDNA3.1-S + pcDNA3.1-L△183	1.43 ± 0.31	43 271.72 ± 2 863.84^{b *}	20.37 ± 1.85^b

图1 HBsAg和LHBs mRNA相对表达量

图2 转染细胞Grp78、HBsAg、pre-S1和pre-S2等蛋白表达

图3 GRP78蛋白相对表达量与前-S1、前-S2抗原和HBsAg表达的相关性

1	El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis[J]. Gastroenterology,2007,132(7):2557-2576.
2	Chen BF. Clinical significance of the hepatitis B virus pre-S deletion[J]. Fu-Jen J Med,2010;8(10):85-95.
3	丁静娟，王梅，刘悦晖. 乙型肝炎病毒前-S基因变异与肝病进展的关系[J]. 中华传染病杂志,2007,25(6):332-337.
4	卓传尚，柳丽娟，吴秋芳, 等乙型肝炎病毒前S区缺失突变与乙型肝炎病毒相关肝细胞癌的关系[J]. 中华传染病杂志,2012,30(9): 553-554.
5	Liu SJ, Zhang HW, Gu CY, et al. Associations between hepatitis B virus mutations and the risk of hepatocellular carcinoma: a meta-analysis[J]. JNCI,2009,101(15):1066-1082.
6	Kao JH, Liu CJ, Jow GM, et al. Fine mapping of hepatitis B virus pre-S deletion and its association with hepatocellular carcinoma[J]. Liver Int,2012,32(9):1373-1381.
7	Sinn DH, Choi MS, Gwak GY, et al. Pre-S mutation is a significant risk factor for hepatocellular carcinoma development: a long-term retrospective cohort study[J]. Dig Dis Sci,2013,58:751-758.
8	Wang HC, Wu HC, Chen CF, et al. Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress[J]. AM J Pathol,2003,163(6):2441-2449.
9	Wang HC, Huang W, Lai MD, et al. Hepatitis B virus pre-S mutants, endoplasmic reticulum stress and hepatocarcinogenesis[J]. Cancer Sci,2006,97(8):683-688.
10	Li YW, Yang FC, Lu HQ, et al. Hepatocellular carcinoma and hepatitis B surface protein [J]. World J Gastroenterol,2016,22(6):1943-1952.
11	Montalbano R, Di Fazio P, Quint K, et al. Induction of endoplasmic reticulum-mediated stress pathways in liver cancer cell lines after overexpression of Hepatitis B virus envelope proteins[J]. J Gastroenterol,2012,50:5-39.
12	Lai E, Teodoro T, Volchuk A.Endoplasmic reticulum stress: signaling the unfolded protein response[J]. Physiology,2007,22:193-201.
13	Malhi H, Kaufman RJ. Endoplasmic reticulum stress in liver disease role of endoplasmic reticulum stress and unfolded protein responses in health and diseases[J]. J Hepatol,2011,54(4):795-809.
14	Mahdi AA, Rizvi SH, Parveen A. Role of endoplasmic reticulum stress and unfolded protein responses in health and diseases[J]. Indian J Clin Biochem,2016,31(2):127-137.
15	Dicks N, Gutierrez K, Michalak M, et al. Endoplasmic reticulum stress, genome damage, and cancer[J]. Front Oncol,2015,3(5):11.
16	Shuda M, Kondoh N, Imazeki N, et al. Activation of the ATF6, XBP1 and grp78 genes in human hepatocellular carcinoma: a possible involvement of the ER stress patheway in hepatocarcinogenesis[J]. J Hepatol,2003,38(5):605-614.
17	Chignard N, Shang S, Wang H, et al. Cleavage of endoplasmic reticulum proteins in hepatocellular carcinoma: detection of generated fragments in patient sera[J]. Gastroenterology,2006;130(7):2010-2022.
18	Wu X, Xin Z, Zhang W, et al. A missense polymorphism in ATF6 gene is associated with susceptibility to hepatocellular carcinoma probably by altering ATF6 level[J]. Int J Cancer,2014,135(1):61-68.
19	Rasheva VI, Domingos PM. Cellular responses to endoplasmic reticulum stress and apoptosis[J]. Apoptosis,2009,14(8):996-1007.
20	Ryoo HD. Long and short (timeframe) of endoplasmic reticulum stress-induced cell death[J]. FEBS J,2016. [Epub ahead of print].
21	王晓琳，邹桂舟，叶珺, 等乙肝表面抗原在慢性乙型肝炎病毒感染临床不同阶段的变化[J]. 实用医学杂志,2014,30(17):2765-2767.

[1]	卢天祺, 张巍, 周康, 毕士玉, 张羽, 杨秀华. 血流向量成像技术在不同Child-Pugh分级乙肝患者左心功能评价中的价值[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(04): 352-360.
[2]	刘莉莉, 贾建茹, 张晶, 周大琼, 刘江雨, 曹振环. 单纯慢性乙型肝炎患者与慢性乙型肝炎合并肝脂肪变患者的临床特征横断面研究[J/OL]. 中华实验和临床感染病杂志(电子版), 2024, 18(05): 270-277.
[3]	张雨, 杨松. 世界卫生组织《慢性乙型肝炎预防、诊断、关怀及治疗指南（2024年版）》解读[J/OL]. 中华实验和临床感染病杂志(电子版), 2024, 18(03): 129-134.
[4]	陈观梅, 左璇, 廖宝林. 慢性乙型肝炎新型免疫治疗研究进展[J/OL]. 中华实验和临床感染病杂志(电子版), 2024, 18(01): 7-10.
[5]	孙鸿坤, 艾虹, 陈正. 内质网应激介导的牙周炎骨改建失衡的研究进展[J/OL]. 中华口腔医学研究杂志(电子版), 2024, 18(04): 211-218.
[6]	刘敏思, 李荣, 李媚. 基于GGT与Plt比值的模型在HBV相关肝细胞癌诊断中的作用[J/OL]. 中华肝脏外科手术学电子杂志, 2024, 13(06): 831-835.
[7]	邓万玉, 陈富, 许磊波. 肝硬化与非肝硬化乙肝相关性肝癌患者术后无复发生存比较及其影响因素分析[J/OL]. 中华肝脏外科手术学电子杂志, 2024, 13(05): 670-674.
[8]	董佳, 王坤, 张莉. 预后营养指数结合免疫球蛋白、血糖及甲胎蛋白对HBV 相关慢加急性肝衰竭患者治疗后预后不良的预测价值[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(06): 555-559.
[9]	胡静, 杨秀锦, 侯志云. HBV感染患者外周血ISGs表达水平变化及其与干扰素治疗疗效的关系[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(04): 343-347.
[10]	江浩, 余宏圣, 杨碧兰, 阿布都克尤木·斯马依, 吴斌, 杨逸冬. 基于列线图模型对慢性乙型肝炎合并肝脏脂肪变性患者并发晚期肝纤维化的临床预测[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(02): 114-120.
[11]	王秀, 王义国. 益生菌联合恩替卡韦治疗乙型肝炎肝硬化临床疗效的meta分析[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(02): 164-171.
[12]	黄圣楷, 许斌, 苏健, 孙龙. 海南省2010~2020年乙型肝炎流行趋势的时间序列分析及预测[J/OL]. 中华临床医师杂志(电子版), 2024, 18(06): 555-561.
[13]	李玛, 闫凌, 席云, 李尚霞, 朱冬林. HBV RNA用于监测慢性乙型肝炎患者临床治愈的研究和应用[J/OL]. 中华临床实验室管理电子杂志, 2024, 12(02): 70-74.
[14]	刘春林, 刘畅, 赵东岩, 李端萍, 刘建梅, 罗秋林. 利用室内质控与能力验证数据评定HBV DNA和HCV RNA测量不确定度[J/OL]. 中华临床实验室管理电子杂志, 2024, 12(02): 91-96.
[15]	郭楠, 徐学俊. 富马酸替诺福韦二吡呋酯在慢性乙型肝炎患者中诱导的范科尼综合征并低磷软骨病一例[J/OL]. 中华诊断学电子杂志, 2024, 12(03): 173-177.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Endoplasmic reticulum stress in Huh7 cell induced by envelope proteins of hepatitis B virus

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Endoplasmic reticulum stress in Huh7 cell induced by envelope proteins of hepatitis B virus