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

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

Abstract

Abstract:

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

Chuanshang Zhuo, Lijuan Liu, Haihua Xie, Shengcong Li. Endoplasmic reticulum stress in Huh7 cell induced by envelope proteins of hepatitis B virus[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2016, 10(03): 375-379.

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Figures/Tables 5

References 21

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.

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

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

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

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

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