内质网应激相关蛋白1对衣霉素诱导HepG2细胞内质网应激的影响

doi:10.3877/cma.j.issn.2095-655X.2014.01.006

中华诊断学电子杂志 ›› 2014, Vol. 02 ›› Issue (01) : 32 -37. doi: 10.3877/cma.j.issn.2095-655X.2014.01.006

所属专题：总编推荐；文献；

基础研究

内质网应激相关蛋白1对衣霉素诱导HepG2细胞内质网应激的影响

肖元元¹, 韩峻峰¹, 毛月芹¹, 王倩倩¹, 魏美林¹, 殷峻¹, 黄金伟², 魏丽¹^,^†(

)

^1. 200233　上海交通大学医学院附属第六人民医院内分泌代谢科　上海市糖尿病研究所
^2. 上海交通大学无锡研究院

收稿日期:2013-10-27 出版日期:2014-02-26
通信作者: 魏丽
基金资助:
国家自然科学基金(81370956); 上海交通大学无锡研究院交大专项资金(2011JDZX021)

Effects of stress-associated endoplasmic reticulum protein 1 on endoplasmic stress induced by the tunicamycin in HepG2 cells

Yuanyuan Xiao¹, Yueqin Mao¹, Qianqian Wang¹, Meilin Wei¹, Junfeng Han¹, jun Yin¹, Jinwei Huang², Li Wei¹^,^†()

^1. Department of Endocrinology and Metabolism, Shanghai Jiaotong University Affiliated Sixth People′s Hospital, Shanghai 200233, China

Received:2013-10-27 Published:2014-02-26
Corresponding author: Li Wei
About author:
Corresponding author: WEI Li, Email: weili63@hotmail.com

全文 ( ) 下载PDF ( ) 导出引用

引用本文：

肖元元, 韩峻峰, 毛月芹, 王倩倩, 魏美林, 殷峻, 黄金伟, 魏丽. 内质网应激相关蛋白1对衣霉素诱导HepG2细胞内质网应激的影响[J]. 中华诊断学电子杂志, 2014, 02(01): 32-37.

Yuanyuan Xiao, Yueqin Mao, Qianqian Wang, Meilin Wei, Junfeng Han, jun Yin, Jinwei Huang, Li Wei. Effects of stress-associated endoplasmic reticulum protein 1 on endoplasmic stress induced by the tunicamycin in HepG2 cells[J]. Chinese Journal of Diagnostics(Electronic Edition), 2014, 02(01): 32-37.

目的

研究过表达内质网应激相关蛋白1(SERP1)对衣霉素诱导肝癌HepG2细胞内质网应激的影响。

方法

以衣霉素诱导HepG2细胞发生内质网应激，将细胞分为以下5组：正常对照组、衣霉素组、衣霉素＋0.25μg SERP1转染组、衣霉素＋0. 5μg SERP1转染组和衣霉素＋1.0μg SERP1转染组，每组实验重复3次；采用MTT法检测不同浓度与作用时间的衣霉素对HepG2细胞存活率的影响，以吸光度(A)值表示。Western blot法检测各组细胞内内质网应激标志蛋白葡萄糖调节蛋白(GRP78)、C/EBP同源蛋白(CHOP)以及钙联蛋白的表达水平。采用SPSS 15.0统计软件进行统计学分析，比较蛋白表达水平。

结果

与对照组相比，衣霉素处理组HepG2细胞中内质网应激标志性蛋白GRP78、CHOP及Calnexin蛋白表达量显著升高，分别为对照处理组的3.8倍(t＝11.5，P<0.05)、1.3倍(t＝3.498，P<0.05)和1.4倍(t＝4.1，P<0.05)，差异均有统计学意义；随着SERP1过表达量的逐渐升高，变化呈现剂量依赖性。随着SERP1转染剂量的增加，各组GRP78蛋白的表达较单独衣霉素处理组分别下降了12%[(1.83±0.29)A值，(1.61±0.13)A值，t＝2.36，P>0.05]、24%和30%[(1.83±0.29)A值，(1.40±0.11)A值，(1.27±0.21)A值；F＝50.56，P<0.05]，CHOP蛋白的表达水平分别下降了23%, 29%和34%[(1.0±0.15)A值，(0.79±0.07)A值，(0.72±0.55)A值，(0.67±0.14)A值；F＝9.532，P<0.05]，Calnexin蛋白的表达水平分别下降了5%[(1.20±0.18)A值，(1.15±0.13)A值；P>0.05]、24%和28%[(1.20±0.18)A值，(0.92±0.07)A值，(0.87±0.18)A值；F＝8.116，P<0.05]。

结论

外源性过表达SERP1蛋白通过下调内质网应激蛋白的表达，降低HepG2细胞内质网应激水平，缓解内质网应激介导的细胞损伤。

关键词: 内质网应激, 内质网应激相关蛋白1, 葡萄糖调节蛋白78, C/EBP同源蛋白, 钙联蛋白

Backgroud

Endoplasmic reticulum stress was induced by the accumulation and aggregation of unfolded proteins due to stresses that disturbed the cellular energy levels, the redox state, or Ca²⁺ concentration, and leading to the unfolded protein response (UPR) pathway. The hepatic UPR was activated in several forms of liver disease. Recent data showed that the role of the UPR in hepatic cells have identified molecular mechanisms that may underlie the association between UPR activation and liver disease. SERP1 was known as ribosome-associated membrane protein 4 (RAMP4), was homologous to yeast suppressor of SecY 6 protein (YSY6p) which suggested a role in pathways controlling membrane protein biogenesis at the ER level. Expression of SERP1 was enhanced during cellular stress, causing accumulation of unfolded proteins in the ER.By interaction with the molecular chaperone calnexin, SERP1/RAMP4 could control biogenesis of membrane proteins and take participate in the endoplasmic reticulum stress.Objective To study the effects of stress-associated endoplasmic reticulum protein 1(SERP1) on the endoplasmic reticulum stress induced by the tunicamycin in HepG2 cells.

Methods

The tunicamycin was used to induce endoplasmic reticulum stress in the HepG2 cells.We divided the cells into 5 groups: normal control group, tunicamycin treated group, tunicamycin + 0.25μg/μl SERP1 transfected group, tunicamycin + 0.25μg/μl SERP1 transfected group, tunicamycin + 0.5μg/μl SERP1 transfected group, tunicamycin + 1μg/μl SERP1 transfected group. Each experiment was repeated three times.MTT was used to detect the effect on the survival rate of the HepG2 cells and selected the optimal concentration and time of tunicamycin treatment.Western blot was used to detect the standard of expression of endoplasmic reticulum stress spcific mark proteins, glucose-regulated protein 78(GRP78), C/EBP homologous protein (CHOP) and calnexin.

Results

Compared with the control group, the expression levels of GRP78, CHOP and calnexin were significantly increased in the tunicaymicin treated group, which were 3.8 times, 1.3 times and 1.4 times respectively. With the increasing amount of transfection, SERP1 over expression was found to relieve the expression of GRP78 12%(1.838±0.29, 1.6±0.132, P>0.05), 24% and 30%(1.838±0.29, 1.40±0.11, 1.27±0.21, F=50.56, P<0.01)compared with the tunicamycin group, the expression of CHOP were decreased by 23%, 29% and 34%(1.0±0.15, 0.79±0.07, 0.72±0.55, 0.67±0.14, F=9.532, P<0.01)respectively and calnexin were decreased by 5%(1.20±0.18, 1.15±0.13, P>0.05)、24%和28%(1.20±0.18, 0.92±0.07, 0.87±0.18, F=8.116, P<0.01)respectively, which were induced by tunicamycin treatment.

Conclusion

SERP1 overexpression could attenuate the ER stress induced by tunicamycin, and may reduce the cell damage mediated by the ER stress.

Key words: Endopasmic reticulum stress, Stress-associated Endoplasmic Reticulum Protein 1, Glucose-regulated protein 78, C/EBP homologous protein, Calnexin

图1 不同浓度衣霉素干预HepG2细胞12，24，36，48h对细胞存活率的影响

图2 不同浓度组SERP1质粒转染HepG2细胞后的荧光染色图像(×200)

图3 免疫印迹法测定各组HePG2细胞中内质网应激标志性蛋白表达情况

表1 转染的HePG2细胞过表达SERP1对GRP78、CHOP和Calnexin蛋白表达的影响(每组n＝5，A值，±s)

组别	GRP78	CHOP	Calnexin
对照组	0.48±0.04	0.78±0.07	0.88±0.08
衣霉素组	1.83±0.29^a	1.01±0.15^a	1.20±0.18^a
衣霉素＋0.25mg/L SERP1组	1.61±0.13^a	0.79±0.07^c	1.15±0.13^b
衣霉素＋0. 5mg/L SERP1组	1.40±0.11^ac	0.72±0.06^c	0.92±0.07^c
衣霉素＋1mg/L SERP1组	1.27±0.21^ac	0.67±0.14^c	0.88±0.18^c
F值	50.56	9.53	8.12
P值	<0.05	<0.05	<0.05

表1 转染的HePG2细胞过表达SERP1对GRP78、CHOP和Calnexin蛋白表达的影响(每组n＝5，A值，±s)

组别	GRP78	CHOP	Calnexin
对照组	0.48±0.04	0.78±0.07	0.88±0.08
衣霉素组	1.83±0.29^a	1.01±0.15^a	1.20±0.18^a
衣霉素＋0.25mg/L SERP1组	1.61±0.13^a	0.79±0.07^c	1.15±0.13^b
衣霉素＋0. 5mg/L SERP1组	1.40±0.11^ac	0.72±0.06^c	0.92±0.07^c
衣霉素＋1mg/L SERP1组	1.27±0.21^ac	0.67±0.14^c	0.88±0.18^c
F值	50.56	9.53	8.12
P值	<0.05	<0.05	<0.05

[1]	Bravo R,Parra V,Gatica D, et al. Endoplasmic reticulum and the unfolded protein response: dynamics and metabolic integration[J]. Int Rev Cell Mol Biol, 2013, 301(2): 215-290.
[2]	Henkel A,Green RM. The unfolded protein response in Fatty liver disease[J]. Semin Liver Dis, 2013, 33(4): 321-329.
[3]	邢燕,叶山东,洪海鸥, 等. 健康体检人群体重指数与血脂水平及非酒精性脂肪肝关系的研究[J]. 中华诊断学电子杂志, 2013, 1(1): 41-44.
[4]	Malhi H,Kaufman RJ.Endoplasmic reticulum stress in liver disease[J]. J Hepatol, 2011, 54(4): 795-809.
[5]	Walter P,Ron D. The unfolded protein response: from stress pathway to homeostatic regulation[J]. Science. 2011, 334(6059): 1081-1086.
[6]	Ozcan U,Cao Q,Yilmaz E, et al. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes[J]. Science, 2004, 306(5695): 457-461.
[7]	Puri P,Mirshahi F,Cheung O, et al.Activation and dysregulation of the unfolded protein response in nonalcoholic fatty liver disease[J]. Gastroenterology, 2008, 134(2): 568-576.
[8]	Ji C,Mehrian-Shai R,Chan C, et al. Role of CHOP in hepatic apoptosis in the murine model of intragastric ethanol feeding[J]. Alcohol Clin Exp Res, 2005, 29(8): 1496-1503.
[9]	Zeng T,Xie KQ.Ethanol and liver：recent advances in the mechanisms of ethanol-induced hepatosteatosis[J]. Arch Toxicol, 2009, 83(12): 1075-1081.
[10]	Duvigneau JC,Kozlov AV,Zifko C, et al.Reperfusion does not induce oxidative stress but sustained endoplasmic reticulum stress in livers of rats subjected to traumatic-hemorrhagic shock[J]. Shock, 2010, 33(3): 289-298.
[11]	Mencin A,Seki E,Osawa Y, et al.Alpha-1 antitrypsin Z protein (PiZ) increases hepatic fibrosis in a murine model of cholestasis[J]. Hepatology, 2007, 46(5): 1443-1452.
[12]	Kaplowitz N,Than TA,Shinohara M, et al.Endoplasmic reticulum stress and liver injury[J]. Semin Liver Dis, 2007, 27(4): 367-377.
[13]	赵明,王为东,李玉军. 肝细胞肝癌患者CD147 P53的表达及临床意义[J]. 济宁医学院学报, 2012, 35(5): 347-349.
[14]	Barba-Espín G,Dedvisitsakul P,Hägglund P, et al.Gibberellic Acid-induced aleurone layers responding to heat shock or tunicamycin provide insight into the N-glycoproteome, protein secretion, and endoplasmic reticulum stress[J]. Plant Physiol, 2014, 164(2): 951-965.
[15]	Tiffany-Castiglioni E,Qian Y. ER chaperone-metal interactions：links to protein folding disorders[J]. Neurotoxicology, 2012, 33(3): 545-557.
[16]	Liu D,Zhang M,Yin H. Signaling pathways involved in endoplasmic reticulum stress-induced neuronal apoptosis[J]. Int J Neurosci, 2013, 123(3): 155-162.
[17]	Yamaguchi A,Hori O,Stern DM, et al.Stress-associated endoplasmic reticulum protein 1 (SERP1)/Ribosome-associated membrane protein 4 (RAMP4) stabilizes membrane proteins during stress and facilitates subsequent glycosylation[J]. J Cell Biol, 1999, 147(6): 1195-1204.
[18]	Schroder K,Martoglio B,Hofmann M, et al.Control of glycosylation of MHC class II-associated invariant chain by translocon-associated RAMP4[J]. EMBO J, 1999, 18(17): 4804-4815.
[19]	Malhotra JD,Kaufman RJ.The endoplasmic reticulum and the unfolded protein response[J]. Semin Cell Dev Biol, 2007, 18(6): 716-731.
[20]	Hebert DN,Molinari M. In and out of the ER：protein folding, quality control, degradation, and related human diseases[J]. Physiol Rev, 2007, 87(4): 1377-1408.
[21]	Groenendyk J,Michalak M. Acta Biochim Pol.Endoplasmic reticulum quality control and apoptosis[J]. Acta Biochim Pol, 2005, 52(2): 381-395.

[1]	徐婷婷, 王晓东. 内质网应激及Wolframin蛋白质与妊娠相关疾病研究现状[J]. 中华妇幼临床医学杂志(电子版), 2020, 16(04): 373-380.
[2]	杨浩, 李毅, 梁琰. 硫化氢调控微小核糖核酸对内质网应激影响的研究进展[J]. 中华损伤与修复杂志(电子版), 2019, 14(04): 307-310.
[3]	邹明, 李毅. 硫化氢对组织损伤后内质网应激影响的研究进展[J]. 中华损伤与修复杂志(电子版), 2017, 12(05): 378-381.
[4]	杨思园, 李辉, 李鑫, 蒋荣猛, 马成杰, 魏红山, 李兴旺. TOLL样受体信号转导通路在内质网应激致炎症反应中的作用[J]. 中华实验和临床感染病杂志(电子版), 2017, 11(03): 222-227.
[5]	林锐钿, 李子涵, 古丽莎. 内质网应激在骨代谢及其稳态中的作用[J]. 中华口腔医学研究杂志(电子版), 2021, 15(05): 309-313.
[6]	张翠荣. 脂联素通过PERK/eIF2a介导的内质网应激通路调节子宫内膜癌细胞增殖、凋亡和胰岛素敏感性[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(04): 240-245.
[7]	雷艳, 詹世淮, 陈俊秋, 董会月, 林榕, 陈津, 王水良, 黄梁浒. CHOP双重调控衣霉素诱导的DU-145细胞凋亡及自噬的研究[J]. 中华细胞与干细胞杂志(电子版), 2018, 08(05): 257-263.
[8]	郭兆安, 孙丽娜. 内质网应激及药物干预对糖尿病肾病足细胞损伤的作用[J]. 中华肾病研究电子杂志, 2021, 10(02): 96-99.
[9]	苏晓乐, 闫冰娟, 乔晞, 王艳红, 王利华. 阿托伐他汀调控PERK/eIF2α/CHOP通路减轻造影剂诱导的大鼠肾小管上皮细胞凋亡[J]. 中华肾病研究电子杂志, 2019, 08(03): 102-108.
[10]	刘天喜, 宋琼, 许国双, 刘永鑫. X-盒结合蛋白1条件性基因敲除小鼠模型的建立[J]. 中华肾病研究电子杂志, 2019, 08(01): 13-18.
[11]	龚军, 孙峰, 金聪慧, 葛建娟, 宋佳烨, 杨磊. FOXM1和GRP78在进展期胃癌组织中的表达及其临床意义[J]. 中华临床医师杂志(电子版), 2019, 13(08): 566-571.
[12]	赵玉青, 柴颖儒, 江莉, 安国霞, 柴晏. 高糖环境对心血管系统的影响及尼可地尔在心血管疾病中的应用[J]. 中华临床医师杂志(电子版), 2017, 11(21): 2373-2376.
[13]	郭如烨, 孟黎明, 陈楠, 宋玉莹, 尹海燕, 郭岩. Apelin/APJ系统对帕金森病模型的神经保护作用及机制研究进展[J]. 中华诊断学电子杂志, 2023, 11(04): 276-282.
[14]	赵倩倩, 姜颖哲, 班博, 邵倩. 内质网应激相关基因调控骨生长发育的研究进展[J]. 中华诊断学电子杂志, 2018, 06(04): 286-288.
[15]	李祖寅, 周志杰, 晏滨, 王晓亮. 内质网应激在非酒精性脂肪肝病中的作用[J]. 中华肥胖与代谢病电子杂志, 2020, 06(02): 122-126.

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

Effects of stress-associated endoplasmic reticulum protein 1 on endoplasmic stress induced by the tunicamycin in HepG2 cells

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

Effects of stress-associated endoplasmic reticulum protein 1 on endoplasmic stress induced by the tunicamycin in HepG2 cells