胥豐愷　古　杰　王　琳　盧春來　葛　棣　袁云鋒　蔣家好

(復旦大學附屬中山醫(yī)院胸外科　上?！?00032)

CAPN4與食管鱗形細胞癌增殖和遷移關系的體外研究

胥豐愷古杰王琳盧春來△葛棣袁云鋒蔣家好

(復旦大學附屬中山醫(yī)院胸外科上海200032)

【摘要】目的探討鈣蛋白酶小亞基1(calpain small subunit 1,CAPN4)在食管鱗形細胞癌細胞系中的表達水平,分析其與食管鱗形細胞癌細胞增殖和遷移能力的相關性。 方法挑選人正常食管上皮細胞系HEEC及食管鱗形細胞癌細胞系EC109與KYSE510,應用Western blot檢測細胞系中CAPN4蛋白質(zhì)水平,應用RNA干擾下調(diào)EC109與KYSE510中CAPN4的表達水平,通過CCK-8與Transwell實驗分別檢測細胞增殖與遷移能力的變化。同時用Western blot檢測細胞系中增殖和遷移相關蛋白質(zhì)水平。結(jié)果CAPN4在EC109與KYSE510中的蛋白質(zhì)水平顯著高于HEEC,下調(diào)EC109與KYSE510中CAPN4蛋白質(zhì)水平后,細胞增殖與遷移能力均顯著降低(P<0.05),MMP-2蛋白質(zhì)水平下調(diào)。 結(jié)論CAPN4在食管鱗形細胞癌中高表達,促進食管癌增殖和遷移,并與下游分子MMP-2的表達水平密切相關。

【關鍵詞】食管鱗形細胞癌;鈣蛋白酶小亞基1;增殖;遷移

食管癌是一種常見的消化道惡性腫瘤。在中國,食管癌發(fā)病率位列第五,死亡率位列第四,病理類型以鱗形細胞癌為主,發(fā)病率顯著高于腺癌[1]。由于食管癌易于遷移和轉(zhuǎn)移的特性,盡管數(shù)十年來食管癌的診治水平在不斷提高,但其五年生存率并未得到顯著改善[2]。因此,深入研究影響食管鱗形細胞癌遷移和轉(zhuǎn)移的相關機制,具有重要的臨床意義。

鈣蛋白酶(calpain,CAPN)為一組保守的半胱氨酸蛋白酶,在生物體內(nèi)廣泛表達。該分子具有鈣依賴活性,并與細胞骨架重塑、細胞存活以及細胞遷移密切相關[3-5]。有研究表明,在一些腫瘤細胞中CAPN出現(xiàn)異常表達,例如:Moretti等[6-7]發(fā)現(xiàn)CAPN3在黑色素瘤中表達下調(diào),而上調(diào)CAPN3水平可顯著降低黑色素瘤細胞的增殖能力;Lee等[8-9]發(fā)現(xiàn)CAPN6在子宮頸鱗狀上皮逐步癌變進展過程中表達水平呈階梯式上升。鈣蛋白酶小亞基(calpain small subunit 1,CAPN4)為μ-calpain的調(diào)控亞基,影響胚胎早期的細胞死亡水平[10-12]。目前已有報道在肝細胞癌、肝內(nèi)膽管細胞癌、腎透明細胞癌、神經(jīng)膠質(zhì)瘤、鼻咽癌、非小細胞肺癌等腫瘤中,CAPN4的表達上調(diào),并影響腫瘤細胞的遷移或增殖能力[13-20]。然而,目前國內(nèi)外尚無相關文獻報道CAPN4在食管鱗形細胞癌中的相關研究。

本研究擬檢測CAPN4在食管鱗形細胞癌細胞系中的表達水平,以探究其與食管癌遷移和增殖的相關性。

材 料 和 方 法

細胞培養(yǎng)與轉(zhuǎn)染人正常食管上皮細胞系HEEC,人食管鱗形細胞癌細胞系EC109與KYSE510培養(yǎng)均使用DMEM+10%胎牛血清,于37 ℃、5%CO2孵箱中培養(yǎng)。EC109與KYSE510細胞系培養(yǎng)達到50%～60%密度時,按照Lipo2000說明書予以轉(zhuǎn)染,siRNA-CAPN4正義鏈:5′-CGAUCAGGGACCAUUUGCAdTdT-3′,反義鏈:5′-UGCAAAUGGUCCCUGAUCGTdTd-3′ (上海拓然生物科技有限公司)。對照組siRNA-NC為非特異性非相關的RNA片段。轉(zhuǎn)染對應siRNA片段后24 h進行CCK-8與Transwell實驗，48 h收集細胞總蛋白。

Transwell與CCK-8實驗通過Transwell實驗來檢測細胞遷移能力。不同細胞(5 × 104個)以200 μL無血清培養(yǎng)液重懸后,接種于Transwell上室(Costar,6.5 mm 小室,8 μm 聚碳酸酯膜),下室內(nèi)加入600 μL含20%血清的DMEM培養(yǎng)液,培養(yǎng)24 h后取出上室,無菌棉簽擦去上室培養(yǎng)液與未穿透的細胞,結(jié)晶紫染色,于倒置顯微鏡下隨機選取5個視野進行計數(shù);通過CCK-8實驗來檢測細胞增殖能力。細胞接種于96孔板,轉(zhuǎn)染對應siRNA片段后24 h、48 h、72 h、96 h分別于對應孔中加入CCK-8試劑10 μL,混勻孵育1～2 h,采用酶標儀測定其在450 nm的吸光度(D)。

細胞總蛋白提取與Western blot檢測以RIPA裂解細胞,4 ℃下14 000× g離心10 min后取上清液。取5～10 μL樣品于十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳分離,轉(zhuǎn)至聚偏二氟乙烯膜,用含5%脫脂奶粉的TBST(Tris-Hcl,NaCl,Tween-20)溶液封閉2 h,加入對應一抗于4 ℃孵育過夜,以TBST洗滌4次后,加入二抗于室溫孵育1.5 h,再以TBST洗滌4次后,采用ECL試劑曝光顯色。應用一抗如下:β-actin (HRP-60008,proteintech);CAPN4 (ab28237,Abcam),E-cadherin (ab1416,Abcam),Vimentin (C0414,Santa Cruz Biotechnology),MMP-2 (BA0569,BOSTER)。

統(tǒng)計學方法采用SPSS 13.0統(tǒng)計軟件對結(jié)果進行分析。每項實驗重復3次,采用t檢驗比較各實驗組之間結(jié)果的差異。P<0.05為差異具有統(tǒng)計學意義。

結(jié)果

食管鱗形細胞癌細胞系中CAPN4蛋白質(zhì)水平上調(diào)Western blot結(jié)果顯示,CAPN4在EC109與KYSE510細胞系中的蛋白表達水平比HEEC細胞系中的高出約0.5倍,差異具有統(tǒng)計學意義(P<0.01,圖1A)。轉(zhuǎn)染siRNA-CAPN4后,EC109與KYSE510細胞系中CAPN4的蛋白表達水平顯著下調(diào)約4倍,差異均具有統(tǒng)計學意義(P<0.001,圖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.

圖1CAPN4在HEEC、EC109與KYSE510細胞系中的

蛋白表達水平及RNA干擾效果

Fig 1The expression of CAPN4 in HEEC,EC109 and

KYSE510 cell lines,and the effect of RNA interference

下調(diào)CAPN4表達水平抑制食管鱗形細胞癌細胞的遷移與增殖能力通過Transwell實驗檢測腫瘤細胞的遷移能力,結(jié)果顯示:下調(diào)CAPN4的表達后,EC109與KYSE510細胞系的遷移能力較對照組顯著下降(P<0.01,圖2A);通過CCK-8實驗檢測腫瘤細胞的增殖能力,結(jié)果顯示:EC109細胞系于干擾CAPN4后48 h起增殖能力顯著低于對照組;KYSE510細胞系于干擾CAPN4后72 h起增殖能力顯著低于對照組(P<0.001,圖2B)。

CAPN4與MMP-2表達水平密切相關Western blot結(jié)果顯示,下調(diào)EC109與KYSE510細胞系中CAPN4蛋白質(zhì)水平后,MMP-2蛋白質(zhì)水平顯著下調(diào),E-cadherin與Vimentin蛋白質(zhì)水平未見明顯變化(圖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.

圖2下調(diào)CAPN4后,EC109與KYSE510細胞系遷移與增

殖能力下降,相關蛋白MMP-2表達水平下調(diào)

Fig 2After downregulating CAPN4 in EC109 and KYSE510,

the proliferation and migration ability descended,and the

related protein MMP-2 was downregulated

討論

惡性腫瘤增殖與遷移是一個多步驟的復雜過程,也是導致患者死亡的主要原因[21-22]。CAPN4為一種鈣蛋白酶小亞基,在調(diào)節(jié)CAPN的穩(wěn)定性和活性過程中起到非常重要的作用[11]。在成纖維細胞中,CAPN4缺失可造成細胞遷移率降低,并造成細胞黏附的異常[10,23]。故有研究者推測CAPN4可能影響腫瘤細胞的遷移。Bai等[14]最早在肝細胞癌肝移植患者中研究CAPN4的作用,并發(fā)現(xiàn)CAPN4的過表達可促進肝細胞癌患者肝移植后的腫瘤遷移與遠處轉(zhuǎn)移。此外,在肝內(nèi)膽管細胞癌、腎透明細胞癌、神經(jīng)膠質(zhì)瘤、鼻咽癌、非小細胞肺癌等腫瘤中,CAPN4均被證明表達上調(diào),并影響腫瘤細胞的遷移或增殖能力,對于惡性腫瘤進展具有非常重要的意義[15-20]。

本研究采用Western blot法來檢測人正常食管上皮細胞系HEEC以及食管鱗形細胞癌細胞系EC109與KYSE510中CAPN4的蛋白質(zhì)水平,結(jié)果發(fā)現(xiàn)EC109與KYSE510中CAPN4的蛋白質(zhì)顯著高于HEEC。這與既往報道CAPN4在多種其他惡性腫瘤中的研究結(jié)果一致[13-20],提示CAPN4在食管鱗形細胞癌細胞中高表達。

我們在體外實驗研究中,利用CCK-8和Transwell檢測食管鱗形細胞癌增殖和遷移能力。結(jié)果顯示:RNA干擾下調(diào)CAPN4表達后,腫瘤細胞的增殖與遷移能力均顯著降低。既往文獻報道中,在不同的惡性腫瘤中,一致發(fā)現(xiàn)CAPN4能促進腫瘤細胞遷移,但對于細胞增殖能力的影響尚存在爭議。Dai等[13]發(fā)現(xiàn)在肝細胞癌中,CAPN4能同時顯著促進腫瘤細胞增殖與轉(zhuǎn)移,而Zhang等[15]研究肝內(nèi)膽管細胞癌中CAPN4的作用,發(fā)現(xiàn)CAPN4雖然能顯著促進腫瘤細胞遷移與轉(zhuǎn)移能力,但與腫瘤細胞的增殖能力無顯著相關性。不同腫瘤類型的差異可能造成CAPN4作用的差異,本研究結(jié)果與Dai等[13]的結(jié)果一致,并提示CAPN4蛋白質(zhì)水平與食管鱗形細胞癌的增殖與遷移能力呈正相關,該分子可能在食管鱗形細胞癌進展過程中起到重要作用。

影響惡性腫瘤進展與轉(zhuǎn)移的的機制包括:上皮-間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)、腫瘤細胞黏附、蛋白水解酶降解組織屏障等[23]。本課題組前期研究結(jié)果證明:CAPN4能夠通過調(diào)節(jié)MMP-2以調(diào)節(jié)非小細胞肺癌的遷移能力[20],Zheng等[19]在鼻咽癌中發(fā)現(xiàn)一致的結(jié)果。MMP家族是一類鋅依賴的肽鏈內(nèi)切酶,參與細胞外基質(zhì)降解和新生血管的形成等過程,已被證實與惡性腫瘤細胞進展與轉(zhuǎn)移密切相關。在本研究中,我們下調(diào)CAPN4表達水平后,發(fā)現(xiàn)食管鱗形細胞癌細胞系內(nèi)MMP-2蛋白質(zhì)水平顯著下調(diào),而EMT的標志物E-cadherin與Vimentin蛋白質(zhì)水平未見明顯變化,推測CAPN4有可能通過調(diào)節(jié)MMP-2的表達從而促進食管鱗形細胞癌增殖與遷移[20],今后會進一步探究。

目前,食管癌的非手術治療以放、化療為主,大量研究致力于探尋食管癌靶向治療的靶點,目前已有報道EGFR、HER2+、VEGF、c-MET、PI3K/Akt/mTOR 等通路的靶向藥物已完成Ⅱ、Ⅲ期臨床研究,但是結(jié)果并不令人滿意。目前應用于臨床的靶向藥物如尼妥珠單抗、西妥昔單抗等,均為針對EGFR的靶向藥物,但臨床療效尚無確切依據(jù)[25]。深入研究CAPN4影響食管癌侵襲和轉(zhuǎn)移的信號通路和相關機制,將助于有望研發(fā)針對性的靶向藥物,為食管癌治療提供新的方向。

綜上所述,本研究結(jié)果證明CAPN4在食管鱗形細胞癌細胞中高表達且促進食管鱗形細胞癌細胞增殖與遷移,并與下游分子MMP-2的表達水平密切相關。因此我們將深入研究CAPN4與食管癌遷移和增殖的相關分子信號通路,尋找相關的節(jié)點分子,這將具有重要的臨床意義。

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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

【中圖分類號】R655.4

【文獻標識碼】A

doi:10.3969/j.issn.1672-8467.2016.03.006

(收稿日期:2016-01-27;編輯:王蔚)

國家自然科學基金(81302099,81372313,81401876);復旦大學附屬中山醫(yī)院人才培養(yǎng)計劃;復旦大學卓學計劃

△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.