胥丰恺　古　杰　王　琳　卢春来　葛　棣　袁云锋　蒋家好

(复旦大学附属中山医院胸外科　上海　200032)

CAPN4与食管鳞形细胞癌增殖和迁移关系的体外研究

胥丰恺古杰王琳卢春来△葛棣袁云锋蒋家好

(复旦大学附属中山医院胸外科上海200032)

【摘要】目的探讨钙蛋白酶小亚基1(calpain small subunit 1,CAPN4)在食管鳞形细胞癌细胞系中的表达水平,分析其与食管鳞形细胞癌细胞增殖和迁移能力的相关性。 方法挑选人正常食管上皮细胞系HEEC及食管鳞形细胞癌细胞系EC109与KYSE510,应用Western blot检测细胞系中CAPN4蛋白质水平,应用RNA干扰下调EC109与KYSE510中CAPN4的表达水平,通过CCK-8与Transwell实验分别检测细胞增殖与迁移能力的变化。同时用Western blot检测细胞系中增殖和迁移相关蛋白质水平。结果CAPN4在EC109与KYSE510中的蛋白质水平显著高于HEEC,下调EC109与KYSE510中CAPN4蛋白质水平后,细胞增殖与迁移能力均显著降低(P<0.05),MMP-2蛋白质水平下调。 结论CAPN4在食管鳞形细胞癌中高表达,促进食管癌增殖和迁移,并与下游分子MMP-2的表达水平密切相关。

【关键词】食管鳞形细胞癌;钙蛋白酶小亚基1;增殖;迁移

食管癌是一种常见的消化道恶性肿瘤。在中国,食管癌发病率位列第五,死亡率位列第四,病理类型以鳞形细胞癌为主,发病率显著高于腺癌[1]。由于食管癌易于迁移和转移的特性,尽管数十年来食管癌的诊治水平在不断提高,但其五年生存率并未得到显著改善[2]。因此,深入研究影响食管鳞形细胞癌迁移和转移的相关机制,具有重要的临床意义。

钙蛋白酶(calpain,CAPN)为一组保守的半胱氨酸蛋白酶,在生物体内广泛表达。该分子具有钙依赖活性,并与细胞骨架重塑、细胞存活以及细胞迁移密切相关[3-5]。有研究表明,在一些肿瘤细胞中CAPN出现异常表达,例如:Moretti等[6-7]发现CAPN3在黑色素瘤中表达下调,而上调CAPN3水平可显著降低黑色素瘤细胞的增殖能力;Lee等[8-9]发现CAPN6在子宫颈鳞状上皮逐步癌变进展过程中表达水平呈阶梯式上升。钙蛋白酶小亚基(calpain small subunit 1,CAPN4)为μ-calpain的调控亚基,影响胚胎早期的细胞死亡水平[10-12]。目前已有报道在肝细胞癌、肝内胆管细胞癌、肾透明细胞癌、神经胶质瘤、鼻咽癌、非小细胞肺癌等肿瘤中,CAPN4的表达上调,并影响肿瘤细胞的迁移或增殖能力[13-20]。然而,目前国内外尚无相关文献报道CAPN4在食管鳞形细胞癌中的相关研究。

本研究拟检测CAPN4在食管鳞形细胞癌细胞系中的表达水平,以探究其与食管癌迁移和增殖的相关性。

材 料 和 方 法

细胞培养与转染人正常食管上皮细胞系HEEC,人食管鳞形细胞癌细胞系EC109与KYSE510培养均使用DMEM+10%胎牛血清,于37 ℃、5%CO2孵箱中培养。EC109与KYSE510细胞系培养达到50%～60%密度时,按照Lipo2000说明书予以转染,siRNA-CAPN4正义链:5′-CGAUCAGGGACCAUUUGCAdTdT-3′,反义链:5′-UGCAAAUGGUCCCUGAUCGTdTd-3′ (上海拓然生物科技有限公司)。对照组siRNA-NC为非特异性非相关的RNA片段。转染对应siRNA片段后24 h进行CCK-8与Transwell实验，48 h收集细胞总蛋白。

Transwell与CCK-8实验通过Transwell实验来检测细胞迁移能力。不同细胞(5 × 104个)以200 μL无血清培养液重悬后,接种于Transwell上室(Costar,6.5 mm 小室,8 μm 聚碳酸酯膜),下室内加入600 μL含20%血清的DMEM培养液,培养24 h后取出上室,无菌棉签擦去上室培养液与未穿透的细胞,结晶紫染色,于倒置显微镜下随机选取5个视野进行计数;通过CCK-8实验来检测细胞增殖能力。细胞接种于96孔板,转染对应siRNA片段后24 h、48 h、72 h、96 h分别于对应孔中加入CCK-8试剂10 μL,混匀孵育1～2 h,采用酶标仪测定其在450 nm的吸光度(D)。

细胞总蛋白提取与Western blot检测以RIPA裂解细胞,4 ℃下14 000× g离心10 min后取上清液。取5～10 μL样品于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离,转至聚偏二氟乙烯膜,用含5%脱脂奶粉的TBST(Tris-Hcl,NaCl,Tween-20)溶液封闭2 h,加入对应一抗于4 ℃孵育过夜,以TBST洗涤4次后,加入二抗于室温孵育1.5 h,再以TBST洗涤4次后,采用ECL试剂曝光显色。应用一抗如下:β-actin (HRP-60008,proteintech);CAPN4 (ab28237,Abcam),E-cadherin (ab1416,Abcam),Vimentin (C0414,Santa Cruz Biotechnology),MMP-2 (BA0569,BOSTER)。

统计学方法采用SPSS 13.0统计软件对结果进行分析。每项实验重复3次,采用t检验比较各实验组之间结果的差异。P<0.05为差异具有统计学意义。

结果

食管鳞形细胞癌细胞系中CAPN4蛋白质水平上调Western blot结果显示,CAPN4在EC109与KYSE510细胞系中的蛋白表达水平比HEEC细胞系中的高出约0.5倍,差异具有统计学意义(P<0.01,图1A)。转染siRNA-CAPN4后,EC109与KYSE510细胞系中CAPN4的蛋白表达水平显著下调约4倍,差异均具有统计学意义(P<0.001,图1B)。

A:Western blot assay was performed to detect the CAPN4 expression in HEEC,EC109 and KYSE510.B:The effect of the knockdown was validated through Western blot analyses.β-actin was used as an internal reference.(1)P<0.01,(2)P<0.001.

图1CAPN4在HEEC、EC109与KYSE510细胞系中的

蛋白表达水平及RNA干扰效果

Fig 1The expression of CAPN4 in HEEC,EC109 and

KYSE510 cell lines,and the effect of RNA interference

下调CAPN4表达水平抑制食管鳞形细胞癌细胞的迁移与增殖能力通过Transwell实验检测肿瘤细胞的迁移能力,结果显示:下调CAPN4的表达后,EC109与KYSE510细胞系的迁移能力较对照组显著下降(P<0.01,图2A);通过CCK-8实验检测肿瘤细胞的增殖能力,结果显示:EC109细胞系于干扰CAPN4后48 h起增殖能力显著低于对照组;KYSE510细胞系于干扰CAPN4后72 h起增殖能力显著低于对照组(P<0.001,图2B)。

CAPN4与MMP-2表达水平密切相关Western blot结果显示,下调EC109与KYSE510细胞系中CAPN4蛋白质水平后,MMP-2蛋白质水平显著下调,E-cadherin与Vimentin蛋白质水平未见明显变化(图2C)。

A:The migration ability was assessed with Transwell(original magnification,×100).B:The proliferation ability was assessed with CCK-8 assay at 24,48,72,and 96 hours after transfection.C:Western blot was performed to analyze the shift of related protein expression level after knockdown of CAPN4.β-actin was used as an internal reference.ns:no significance,(1)P<0.01,(2)P<0.001.

图2下调CAPN4后,EC109与KYSE510细胞系迁移与增

殖能力下降,相关蛋白MMP-2表达水平下调

Fig 2After downregulating CAPN4 in EC109 and KYSE510,

the proliferation and migration ability descended,and the

related protein MMP-2 was downregulated

讨论

恶性肿瘤增殖与迁移是一个多步骤的复杂过程,也是导致患者死亡的主要原因[21-22]。CAPN4为一种钙蛋白酶小亚基,在调节CAPN的稳定性和活性过程中起到非常重要的作用[11]。在成纤维细胞中,CAPN4缺失可造成细胞迁移率降低,并造成细胞黏附的异常[10,23]。故有研究者推测CAPN4可能影响肿瘤细胞的迁移。Bai等[14]最早在肝细胞癌肝移植患者中研究CAPN4的作用,并发现CAPN4的过表达可促进肝细胞癌患者肝移植后的肿瘤迁移与远处转移。此外,在肝内胆管细胞癌、肾透明细胞癌、神经胶质瘤、鼻咽癌、非小细胞肺癌等肿瘤中,CAPN4均被证明表达上调,并影响肿瘤细胞的迁移或增殖能力,对于恶性肿瘤进展具有非常重要的意义[15-20]。

本研究采用Western blot法来检测人正常食管上皮细胞系HEEC以及食管鳞形细胞癌细胞系EC109与KYSE510中CAPN4的蛋白质水平,结果发现EC109与KYSE510中CAPN4的蛋白质显著高于HEEC。这与既往报道CAPN4在多种其他恶性肿瘤中的研究结果一致[13-20],提示CAPN4在食管鳞形细胞癌细胞中高表达。

我们在体外实验研究中,利用CCK-8和Transwell检测食管鳞形细胞癌增殖和迁移能力。结果显示:RNA干扰下调CAPN4表达后,肿瘤细胞的增殖与迁移能力均显著降低。既往文献报道中,在不同的恶性肿瘤中,一致发现CAPN4能促进肿瘤细胞迁移,但对于细胞增殖能力的影响尚存在争议。Dai等[13]发现在肝细胞癌中,CAPN4能同时显著促进肿瘤细胞增殖与转移,而Zhang等[15]研究肝内胆管细胞癌中CAPN4的作用,发现CAPN4虽然能显著促进肿瘤细胞迁移与转移能力,但与肿瘤细胞的增殖能力无显著相关性。不同肿瘤类型的差异可能造成CAPN4作用的差异,本研究结果与Dai等[13]的结果一致,并提示CAPN4蛋白质水平与食管鳞形细胞癌的增殖与迁移能力呈正相关,该分子可能在食管鳞形细胞癌进展过程中起到重要作用。

影响恶性肿瘤进展与转移的的机制包括:上皮-间质转化(epithelial-mesenchymal transition,EMT)、肿瘤细胞黏附、蛋白水解酶降解组织屏障等[23]。本课题组前期研究结果证明:CAPN4能够通过调节MMP-2以调节非小细胞肺癌的迁移能力[20],Zheng等[19]在鼻咽癌中发现一致的结果。MMP家族是一类锌依赖的肽链内切酶,参与细胞外基质降解和新生血管的形成等过程,已被证实与恶性肿瘤细胞进展与转移密切相关。在本研究中,我们下调CAPN4表达水平后,发现食管鳞形细胞癌细胞系内MMP-2蛋白质水平显著下调,而EMT的标志物E-cadherin与Vimentin蛋白质水平未见明显变化,推测CAPN4有可能通过调节MMP-2的表达从而促进食管鳞形细胞癌增殖与迁移[20],今后会进一步探究。

目前,食管癌的非手术治疗以放、化疗为主,大量研究致力于探寻食管癌靶向治疗的靶点,目前已有报道EGFR、HER2+、VEGF、c-MET、PI3K/Akt/mTOR 等通路的靶向药物已完成Ⅱ、Ⅲ期临床研究,但是结果并不令人满意。目前应用于临床的靶向药物如尼妥珠单抗、西妥昔单抗等,均为针对EGFR的靶向药物,但临床疗效尚无确切依据[25]。深入研究CAPN4影响食管癌侵袭和转移的信号通路和相关机制,将助于有望研发针对性的靶向药物,为食管癌治疗提供新的方向。

综上所述,本研究结果证明CAPN4在食管鳞形细胞癌细胞中高表达且促进食管鳞形细胞癌细胞增殖与迁移,并与下游分子MMP-2的表达水平密切相关。因此我们将深入研究CAPN4与食管癌迁移和增殖的相关分子信号通路,寻找相关的节点分子,这将具有重要的临床意义。

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In vitro study of the correlation between CAPN4 and the proliferation and migration in esophageal squamous cell carcinoma

XU Feng-kai, GU Jie, WANG Lin, LU Chun-lai△, GE Di, YUAN Yun-feng, JIANG Jia-hao

(DepartmentofThoracicSurgery,ZhongshanHospital,FudanUniversity,Shanghai200032,China)

【Abstract】Objective To evaluate the protein level of Calpain small subunit 1 (CAPN4) in the cell lines of esophageal squamous cell carcinoma (ESCC),and the correlation between CAPN4 and ESCC cells＇s proliferation and migration.MethodsThe human normal esophageal epithelial cell line HEEC and human ESCC cell lines EC109 and KYSE510 were picked up for experiments.Western blot was performed to detect the CAPN4 protein level in each cell line.Knockdown of CAPN4 expression was performed through the RNA interference.CCK-8 and Transwell were applied to compare the ability of proliferation and migration change after knockdown of CAPN4.Western blot was performed to detect the related protein level for proliferation and migration in each ESCC cell line.ResultsCAPN4 protein level was upregulated in ESCC cell lines EC109 and KYSE510. Knockdown of the CAPN4 expression could inhibit cell proliferation and migration in vitro (P<0.05),and also downregulate the MMP-2 protein level.ConclusionsCAPN4 is upregulated in ESCC and promotes cancer cells proliferation and migration.CAPN4 is also significantly correlated with the expression of downstream MMP-2.

【Key words】esophageal squamous cell carcinoma;calpain small subunit 1;proliferation;migration

【中图分类号】R655.4

【文献标识码】A

doi:10.3969/j.issn.1672-8467.2016.03.006

(收稿日期:2016-01-27;编辑:王蔚)

国家自然科学基金(81302099,81372313,81401876);复旦大学附属中山医院人才培养计划;复旦大学卓学计划

△Corresponding authorE-mail:lu.chunlai@zs-hospital.sh.cn

*This work was supported by the National Natural Science Foundation of China(81302099,81372313,81401876),Personnel Training Plan of Zhongshan Hospital,Fudan University and Zhouxue Program of Fudan University.