內(nèi)質(zhì)網(wǎng)應(yīng)激前后肝癌細(xì)胞中C/EBP同源蛋白及X-盒-結(jié)合蛋白-1的表達(dá)*

李利波， 潘　婭， 陳騰祥

(貴州醫(yī)科大學(xué)， 貴州 貴陽　550004)

?

內(nèi)質(zhì)網(wǎng)應(yīng)激前后肝癌細(xì)胞中C/EBP同源蛋白及X-盒-結(jié)合蛋白-1的表達(dá)*

李利波， 潘婭**， 陳騰祥**

(貴州醫(yī)科大學(xué)， 貴州 貴陽550004)

目的： 觀察內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)標(biāo)志蛋白X-盒-結(jié)合蛋白-1(XBP-1)和C/EBP同源蛋白(CHOP)在正常肝細(xì)胞及不同分化程度肝癌細(xì)胞中的表達(dá)。方法： 將培養(yǎng)獲得的正常肝細(xì)胞LO2、高分化肝癌細(xì)胞株HepG2及低分化肝癌細(xì)胞株SMMC-7721細(xì)胞分為無水乙醇未處理組(ERS前組)和無水乙醇處理組(ERS組)，ERS組的3種細(xì)胞分別給予無水乙醇處理獲得ERS細(xì)胞模型； 采用Western Blot方法檢測3種細(xì)胞ERS前后XBP-1、CHOP表達(dá)。結(jié)果： 在ERS前組中，XBP-1表達(dá)從高到低依次為LO2、HepG2、SMMC-7721；ERS組中，XBP-1在3種細(xì)胞中的表達(dá)都上調(diào)，與ERS前組比較差異有統(tǒng)計學(xué)意義(P<0.05)，增加量從高到低依次為SMMC-7721、LO2及HepG2，3種細(xì)胞間兩兩比較差異無統(tǒng)計學(xué)意義(P>0.05)；ERS前后，HepG2細(xì)胞中均無CHOP表達(dá)；ERS前組中，CHOP表達(dá)高低依次為SMMC-7721、LO2；ERS組中，CHOP在LO2細(xì)胞及SMMC-7721細(xì)胞表達(dá)均上調(diào)，與ERS前組比較差異有統(tǒng)計學(xué)意義(P<0.05)，而在SMMC-7721細(xì)胞中表達(dá)上調(diào)更明顯(與LO2細(xì)胞比較，P<0.05)。結(jié)論： 發(fā)生ERS后，LO2、HepG2及SMMC-7721細(xì)胞中XBP-1的表達(dá)水平都升高，分化程度低的SMMC-7721細(xì)胞中的XBP-1及CHOP的表達(dá)升高更明顯。

肝腫瘤； 肝細(xì)胞； 內(nèi)質(zhì)網(wǎng)應(yīng)激; X-盒-結(jié)合蛋白-1; C/EBP同源蛋白

內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress，ERS)是指在多種病理生理因素的干擾下，內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)被打破，錯誤折疊和未折疊蛋白質(zhì)在內(nèi)質(zhì)網(wǎng)腔內(nèi)聚集，導(dǎo)致內(nèi)質(zhì)網(wǎng)功能和Ca2+平衡紊亂[1]。ERS主要包括未折疊蛋白反應(yīng)(UPR)、內(nèi)質(zhì)網(wǎng)超負(fù)荷反應(yīng)(EOR)和固醇調(diào)節(jié)級聯(lián)反應(yīng)(SREBP)，研究認(rèn)為癌癥相關(guān)的病理機(jī)制與內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)失衡誘導(dǎo)ERS和UPR途徑有關(guān)[2-4]；而ERS反應(yīng)過強(qiáng)或持續(xù)時間過久，這些反應(yīng)不足以恢復(fù)內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)，則最終引起細(xì)胞凋亡[5-6]。誘導(dǎo)內(nèi)質(zhì)網(wǎng)中應(yīng)激蛋白表達(dá)是UPR 的主要效應(yīng)之一, 這些蛋白包括內(nèi)質(zhì)網(wǎng)分子伴侶, 例如CHOP、XBP-1等，因此這些蛋白質(zhì)水平的變化可提示ERS的發(fā)生[7-9]。本研究通過觀察XBP-1、CHOP在正常肝細(xì)胞、肝癌HepG2及SMMC-7721細(xì)胞在ERS前后表達(dá)水平的變化，探討不同分化程度肝癌細(xì)胞與ERS的關(guān)系。

1　材料與方法

1.1細(xì)胞及試劑

正常肝細(xì)胞LO2、高分化肝癌細(xì)胞株HepG2和低分化肝癌細(xì)胞株SMMC-7721購買于上海生命科學(xué)研究所，XBP-1和CHOP抗體為ABCAM公司產(chǎn)品，BCA Protein Assay KitP0010S、RIPA裂解液(強(qiáng))P0013B購買于上海碧云天公司。Prestained protein marker 00161543、ECL-PLUS/KitM3121/1859022購買于thermo公司，醫(yī)用X光片038401501購買于carestream公司，SDS-PAGE 蛋白電泳儀 VE-180，蛋白轉(zhuǎn)膜儀VE-186為上海天能公司產(chǎn)品。

1.2方法

1.2.1細(xì)胞培養(yǎng)及處理人正常肝細(xì)胞LO2及高分化人肝癌細(xì)胞系HepG2細(xì)胞用DMEM加10%胎牛血清、37 ℃、5% CO2細(xì)胞培養(yǎng)箱中培養(yǎng)，培養(yǎng)1周后傳代至第3代，細(xì)胞貼壁生長。低分化人肝癌細(xì)胞系SMMC-7721細(xì)胞用RMPI1640加10%胎牛血清、37 ℃、5% CO2細(xì)胞培養(yǎng)箱中培養(yǎng)，首次傳代23 d，2周后傳至第3代。將收集的3種細(xì)胞分成兩組，一組進(jìn)行ERS處理(ERS組)，一組不予ERS處理(ERS前組)。誘導(dǎo)ERS的方法為用150 mmol/L無水乙醇作用于HepG2、SMMC-7721及L-02細(xì)胞48 h，棄去無水乙醇，PBS 洗滌2 次，獲得ERS后的3種細(xì)胞。

1.2.2CHOP及XBP-1蛋白的表達(dá)用Western Blot方法檢測，分別提取培養(yǎng)獲得的ERS前后的3種細(xì)胞樣品，PBS洗滌兩次后刮下細(xì)胞轉(zhuǎn)移入EP管中，用預(yù)冷的2×Lysis Buffer裂解，冰上裂解10～15 min后超聲破碎細(xì)胞，離心15 min，取上清液提取蛋白樣品。根據(jù)CHOP及XBP-1的蛋白分子量制膠，樣品上樣，恒壓80 V，電泳2 h，在4 ℃及300 mA恒流條件下電轉(zhuǎn)150 min，將蛋白轉(zhuǎn)移到PVDF膜上，用封閉液(含5%脫脂牛奶的TBST溶液)室溫封閉PVDF膜1 h、4 ℃過夜；TBST洗膜3次、10 min/次，然后顯色。將膠片進(jìn)行掃描、拍照，用凝膠圖像處理系統(tǒng)分析目標(biāo)條帶的分子量和凈光密度值，檢測3種細(xì)胞在發(fā)生ERS前后細(xì)胞裂解液中的CHOP及XBP-1蛋白的表達(dá)。

1.3統(tǒng)計學(xué)處理

采用SPSS 21軟件進(jìn)行統(tǒng)計分析，采用one-way ANOVA法、重復(fù)測量方差分析法分析3種細(xì)胞中這CHOP及XBP-1蛋白在ERS前后水平的差異。P<0.05為差異有統(tǒng)計學(xué)意義。

2　結(jié)果

2.1XBP-1表達(dá)

ERS前組：3種細(xì)胞中，XBP-1在LO2細(xì)胞中表達(dá)最高，其次為HepG2細(xì)胞，在SMMC-7721細(xì)胞中表達(dá)最低。ERS組：3種細(xì)胞中，XBP-1表達(dá)均上調(diào)，與ERS前組比較差異有統(tǒng)計學(xué)意義(P<0.05)，增加量從高到低依次為SMMC-7721、LO2及hepG2細(xì)胞，但增加量在3種細(xì)胞間兩兩比較，差異無統(tǒng)計學(xué)意義(P>0.05)。見圖1、表1。

圖1　ERS前后XBP-1在SMMC-7721、LO2及hepG2細(xì)胞中的表達(dá)(Western Blot)Fig.1　The expression of XBP-1 in three kinds of cells before and after ethanol treatment

細(xì)胞光密度值ERS前組ERS組變化量LO21.16±0.221.29±0.25(1)0.13±0.02HepG20.96±0.571.07±0.64(1)0.12±0.005SMMC-77210.62±0.530.90±0.77(1)0.28±0.17

(1)與ERS前組比較，P<0.05

2.2CHOP表達(dá)

ERS前后，CHOP在HepG2細(xì)胞中均無表達(dá)；ERS前組中CHOP在LO2細(xì)胞中表達(dá)較低，而在SMMC-7721細(xì)胞中表達(dá)較高；ERS組中，CHOP在LO2細(xì)胞及SMMC-7721細(xì)胞表達(dá)均上調(diào)，與ERS前組比較差異有統(tǒng)計學(xué)意義(P<0.05)；CHOP在ERS前后表達(dá)水平的增加量在SMMC-7721細(xì)胞中變化較大，在LO2細(xì)胞中變化較小，差異有統(tǒng)計學(xué)意義(P<0.05)。見圖2、表2。

圖2　ERS前后LO2及SMMC-7721細(xì)胞中CHOP的表達(dá)(Western Blot)Fig.2　The altered expression of CHOP in two kinds of cells before and after ethanol treatment

細(xì)胞光密度值ERS前組ERS組變化量LO20.97±0.420.99±0.40(1)0.019±0.0005SMMC-77210.89±0.160.95±0.17(1)0.066±0.008(2)

(1)與ERS前組比較，P<0.05；(2)與LO2比較，P<0.05

3　討論

當(dāng)ER內(nèi)通路被阻斷時，ER所具有的調(diào)節(jié)蛋白質(zhì)折疊、轉(zhuǎn)錄后修飾、脂質(zhì)和類固醇合成、基因表達(dá)、調(diào)節(jié)細(xì)胞代謝和鈣信號等多種功能將不能發(fā)揮，ER腔內(nèi)錯誤折疊蛋白質(zhì)的累積將最終導(dǎo)致 ERS[10]。ERS可通過促凋亡反應(yīng)誘導(dǎo)腫瘤細(xì)胞的凋亡，亦可通過抗凋亡反應(yīng)導(dǎo)致腫瘤細(xì)胞的無限增殖，這體現(xiàn)了ERS對腫瘤細(xì)胞作用的矛盾方面，提示ERS的反應(yīng)程度或反應(yīng)類型對腫瘤的作用不同。近年來，ERS誘導(dǎo)腫瘤細(xì)胞凋亡的研究已經(jīng)成為腫瘤治療研究的熱點(diǎn)。誘導(dǎo)內(nèi)質(zhì)網(wǎng)的應(yīng)激蛋白表達(dá)是UPR 的主要效應(yīng)之一, 這些蛋白包括內(nèi)質(zhì)網(wǎng)分子伴侶, 例如GRP78、CHOP、XBP-1、PDI等。所以ERS一般用參與UPR的標(biāo)志性分子來提示ERS的發(fā)生。研究發(fā)現(xiàn)，ERS時CHOP的表達(dá)量在轉(zhuǎn)錄水平上大大增加，認(rèn)為CHOP參與調(diào)節(jié)下游凋亡相關(guān)基因的表達(dá)，CHOP高表達(dá)時ER對蛋白質(zhì)折疊修飾功能受到影響，引起細(xì)胞分裂周期停滯及DNA損傷，促進(jìn)凋亡發(fā)生，因此往往將CHOP作為ERS凋亡途徑被激活的主要標(biāo)志之一[11-13]。XBP-1是ER中蛋白質(zhì)折疊能力的主要調(diào)控者之一，它通過調(diào)控蛋白陪襯分子和ER中的相關(guān)降解蛋白表達(dá)來發(fā)揮其增加ER對蛋白質(zhì)折疊能力的作用[14-17]。當(dāng)ERS發(fā)生時，ER網(wǎng)腔中未折疊或錯誤折疊的蛋白質(zhì)堆積，刺激定位于ER膜的肌醇酶1(inositolrequiring1,IRE1)二聚化而具有蛋白激酶和核糖核酸酶的活性，將XBP1-U剪接成具有高度轉(zhuǎn)錄活性的XBP1-S。因此，XBP-1的表達(dá)上調(diào)也常常作為ERS標(biāo)志。

本研究發(fā)現(xiàn)，在無水乙醇未處理時，XBP-1在正常肝細(xì)胞中表達(dá)最高，其次為HepG2細(xì)胞，在SMMC-7721細(xì)胞中表達(dá)最低，經(jīng)無水乙醇處理后表達(dá)均上調(diào)在SMMC-7721細(xì)胞中XBP-1表達(dá)水平變化最大，其次為LO2細(xì)胞，變化最小的為hepG2細(xì)胞。提示XBP-1 ERS后XBP-1表達(dá)變化與細(xì)胞分化程度不相關(guān)。正常肝細(xì)胞中CHOP表達(dá)較SMMC-7721細(xì)胞中高，HepG2細(xì)胞未見CHOP表達(dá)，ERS后兩種細(xì)胞中CHOP上調(diào)，在SMMC-7721細(xì)胞中CHOP表達(dá)水平變化較大，而LO2細(xì)胞變化較小。提示CHOP在ERS前后的表達(dá)水平變化與細(xì)胞分化程度相關(guān)，細(xì)胞分化程度越低，CHOP 表達(dá)水平變化越大。

本研究證實了無水乙醇處理誘導(dǎo)發(fā)生ERS后，低分化肝癌SMMC-7721細(xì)胞中XBP-1及CHOP表達(dá)水平升高。ERS后，CHOP、XBP-1等ERS標(biāo)志物的表達(dá)水平變化是與惡性肝癌細(xì)胞的分化程度相關(guān)的，分化程度越低，CHOP、XBP-1等ERS標(biāo)志物表達(dá)水平變化越大。

[1] kaufman RJ.Orchestrating the unfolded protein response inhealth and disease[J].J Clin Invest, 2002(10):1389-1398.

[2] Xu Y, Chiu L F, He QY, et al. Tubeimoside-1 exerts cytotoxicity in Hela cells through mitochondrial dysfunction and endoplasmic reticulum stress pathways [J]. J Proteome Res, 2009(3): 1585-1593.

[3] Mujcic H, Rzymski T, Rouschop KM, et al. Hypoxic activation of the unfolded protein response (UPR) induces expression of the metastasis-associated gene LAMP3[J]. Radiother Oncol, 2009(3): 450-459.

[4] Sun SY, Liu XG, Zou W, et al. The farnesyl transferase inhibitor lonafarnib induces CCAAT/enhancer-binding protein homologous protein-dependent expression of death receptor 5, leading to induction of apoptosis in human cancer cells[J]. Biol Chem, 2007(26): 18800-18809.

[5] Volmer R，van der Ploeg K，Ron D． Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains[J]． Proc Natl Acad Sci USA， 2013(12):4628-4633．

[6] Hetz C，Chevet E，Harding HP． Targeting the unfolded protein response in disease[J]． Nat Rev Drug Discov， 2013( 9) : 703-719．

[7] Jauhiainen A, Thomsen C, Str?mbom L, et al. Distinct cytoplasmic and nuclear functions of the stress induced protein DDIT3/CHOP/GADD153[J]. PLoS One, 2012(4):e33208.

[8] Irie Y, Saeki M, Tanaka H, et al. Methamphetamine induces endoplasmic reticulum stress related gene CHOP/Gadd153/ddit3 in dopaminergic cells[J]. Cell Tissue Res, 2011(2):231-241.

[9] Benham AM. The protein disulfide isomerase family: key players in health and disease[J]. Antioxid Redox Signal, 2012(16):781-789.

[10]Zhou M, Jacob A, Ho N, et al. Downregulation of protein disulfide isomerase in sepsis and its role in tumor necrosis factor-alpha release[J]. Crit Care, 2008(12):100.

[11]Robinson A,Hines V,Eiden K,et al．Protein disfide isomerase over expression increase secretion of foreign protein in sac-charomyces cerevisiae [J].Biotechnology, 1994(12):381-384.

[12]Goplen D, Wang J, Enger PO, et al. Protein disulfide isomerase expression is related to the invasive properties of malignant glioma[J]. Cancer Res， 2006(66):9895-9902.

[13]Henders hot LM.The ER function BiP is a master regulat or of ER funct ion[J].Mt Sinai J Med, 2004(5):289-297.

[14]Hsu TA,Watson S, Eiden JJ,et al．Rescue of immunoglobulins from insolubility is facilitated by PDI in the baculovirus expression system[J].Protein Expr Purif, 1996(7):281-288.

[15]Tabata Y, Takano K, Ito T, et al. Vaticanol B, a resveratrol tetramer, regulates endoplasmic reticulum stress and inflammation[J]. Am J Physiol Cell Physiol， 2007(293):C411-C418.

[16]Hsu TA,Watson S, Eiden JJ,et al．Rescue of immunoglobulins from insolubility is facilitated by PDI in the baculovirus expression system[J].Protein Expr Purif, 1996(7):281-288.

[17]Xu S, Sankar S, Neamati N. Protein disulfide isomerase: a promising target for cancer therapy[J]. Drug Discov Today, 2014(19): 222-240.

(2016-05-21收稿，2016-08-31修回)

中文編輯： 文箐潁； 英文編輯： 趙毅

The Expression of XBP-1 and CHOP Alteration in the Normal Liver Cells and Liver Cancer Cells before and after RES

LI Libo, PAN Ya, CHEN Tengxiang

(GuizhouMedicalUniversity,Guiyang550004,Guizhou)

Objective: To observe the alteration of ERS marker protein X-box-binding protein 1 (XBP -1) and C/EBP homologous protein (CHOP) in normal liver cells LO2, expression of high differentiation of primary liver cancer cell line HepG2 and low expression in the differentiation of primary liver cancer cell line SMMC-7721. Methods: Cultivation of LO2 cells and HepG2, SMMC-7721 cells was divided into anhydrous ethanol untreated group before (ERS) and anhydrous ethanol treatment group (ERS), ERS group of three kinds of cells were given anhydrous ethanol processing to gain ERS model. Western blot method was adopted to detect the expression level of CHOP and XBP-1 before and after anhydrous ethanol intervention. Results: Before and after ERS, XBP-1 was expressed in three kinds of cells which had not been given anhydrous ethanol processing. Expression level from high to low in turn for LO2 cells, HepG2 cells, SMMC - 7721 cells. After treated with anhydrous ethanol, expression of XBP-1 in three kinds of cells rose; compared with before ERS, of which there was statistically significant difference in the expression of XBP-1 in three kinds of cells between before ERS and after ERS (P<0.05). The increase was not statistically significant compared between among the three (P>0.05). Before and after ERS, CHOP in HepG2 cells had no expression. Its expression in LO2 cells is higher, SMMC-7721 cells expression is lower. Compared with before ERS, the expression of CHOP in LO2 and SMMC-7721 cell lysate significantly up-regulated after ERS (P<0.05). The alteration of Expression levels varied considerably in SMMC-7721 cells which is more than LO2 cells,it was statistically significant (P<0.05). Conclusion: After ERS, CHOP, XBP-1 is associated with malignant cancer of the liver cell differentiation degree, the lower of the degree of differentiation, the greater the expression level changes.

liver carcinoma; hepatocellular; endoplasmic reticulum stress; X box-binding protein-1; C/EBP homologous protein

貴州省科技廳科技項目[黔科合LG(2001)008]； 貴州省教育廳自然科學(xué)研究項目[黔教合(2008)022]

E-mail:710232517@qq.com; 371251826@qq.com

R735.7；R363

A

1000-2707(2016)09-1033-04

10.19367/j.cnki.1000-2707.2016.09.010

**

網(wǎng)絡(luò)出版時間：2016-09-13網(wǎng)絡(luò)出版地址：http://www.cnki.net/kcms/detail/52.5012.R.20160913.2240.050.html