李伯和，　袁　磊

(漯河醫(yī)學(xué)高等?？茖W(xué)校，河南 漯河 462002)

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地高辛對(duì)缺氧誘導(dǎo)的乳腺癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化和侵襲的影響*

李伯和，袁磊△

(漯河醫(yī)學(xué)高等專科學(xué)校，河南 漯河 462002)

[摘要]目的： 本研究旨在探究地高辛對(duì)缺氧誘導(dǎo)乳腺癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化、遷移和侵襲能力的影響，并探討其分子機(jī)制。方法：選取人乳腺癌MCF-7細(xì)胞作為研究對(duì)象，采用CoCl2模擬化學(xué)缺氧條件，采用細(xì)胞劃痕實(shí)驗(yàn)測(cè)量細(xì)胞遷移率，采用Transwell侵襲實(shí)驗(yàn)檢測(cè)細(xì)胞侵襲力，采用Western blot方法檢測(cè)人乳腺癌MCF-7細(xì)胞缺氧誘導(dǎo)因子-1α(HIF-1α)、Snail、E-cadherin和vimentin蛋白表達(dá)的變化。結(jié)果：缺氧使MCF-7細(xì)胞從多角形上皮形態(tài)轉(zhuǎn)變成梭形間質(zhì)細(xì)胞形態(tài)，細(xì)胞間隙增大，而在地高辛作用下缺氧的MCF-7細(xì)胞未發(fā)生明顯的上皮間質(zhì)轉(zhuǎn)化。細(xì)胞劃痕實(shí)驗(yàn)和Transwell侵襲實(shí)驗(yàn)結(jié)果顯示，經(jīng)CoCl2處理的MCF-7細(xì)胞的遷移和侵襲能力均顯著增強(qiáng)(P<0.01)，地高辛可抑制CoCl2誘導(dǎo)的細(xì)胞遷移和侵襲(P<0.01)。與control組相比，CoCl2組細(xì)胞的HIF-1α、Snail和vimentin蛋白表達(dá)水平顯著升高(P<0.01)，E-cadherin的蛋白表達(dá)水平顯著降低(P<0.01)；CoCl2+digoxin組細(xì)胞的HIF-1α、E-cadherin和vimentin蛋白表達(dá)水平與control組相比差異均無(wú)統(tǒng)計(jì)學(xué)顯著性，Snail蛋白表達(dá)水平雖略高于control組(P<0.05)，但與CoCl2組細(xì)胞相比顯著降低(P<0.01)。結(jié)論：地高辛可通過(guò)下調(diào)HIF-1α和Snail蛋白表達(dá)抑制缺氧誘導(dǎo)的MCF-7細(xì)胞上皮間質(zhì)轉(zhuǎn)化和侵襲。

[關(guān)鍵詞]地高辛； 上皮間質(zhì)轉(zhuǎn)化； 缺氧誘導(dǎo)因子-1α； 細(xì)胞侵襲； MCF-7細(xì)胞

在實(shí)體腫瘤的發(fā)生發(fā)展過(guò)程中，由于腫瘤細(xì)胞的快速增殖以及腫瘤組織自身血供不足，導(dǎo)致實(shí)體腫瘤組織內(nèi)部普遍處于缺氧狀態(tài)[1]。長(zhǎng)期處于缺氧環(huán)境會(huì)促使腫瘤細(xì)胞外基質(zhì)的結(jié)構(gòu)發(fā)生改變、細(xì)胞間黏附作用下降、細(xì)胞運(yùn)動(dòng)性和侵襲性增強(qiáng)[2]。缺氧誘導(dǎo)因子-1α(hypoxia inducible factor-1α，HIF-1α)是廣泛表達(dá)于脊椎動(dòng)物細(xì)胞內(nèi)的一種調(diào)控缺氧應(yīng)答的轉(zhuǎn)錄因子[3]。在缺氧狀態(tài)下，腫瘤細(xì)胞高表達(dá)HIF-1α，參與腫瘤細(xì)胞的存活、增殖、侵襲和轉(zhuǎn)移、放化療耐藥等過(guò)程[4-5]。地高辛(digoxin)是一種從毛花洋地黃中提純制得的強(qiáng)心苷，可選擇性與心肌細(xì)胞膜Na+/K+-ATP酶結(jié)合而抑制其活性，用于治療充血性心力衰竭和某些室上性心律失常。近年來(lái)越來(lái)越多的證據(jù)顯示，地高辛對(duì)多種腫瘤具有抑制作用[6-7]。本研究以人乳腺癌MCF-7細(xì)胞為研究對(duì)象，探究地高辛對(duì)CoCl2誘導(dǎo)的MCF-7遷移和侵襲的影響及其分子生物學(xué)機(jī)制。

材料和方法

1材料

人乳腺癌MCF-7細(xì)胞由本校分子醫(yī)學(xué)實(shí)驗(yàn)中心提供。胎牛血清(fetal bovine serum，F(xiàn)BS)和RPMI—1640培養(yǎng)基購(gòu)自HyClone；地高辛(digoxin)、氯化鈷(CoCl2)和4’,6-二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole，DAPI)購(gòu)自Sigma；E-cadherin、vimentin、HIF-1α、Snail和β-actin抗體購(gòu)自Santa Cruz；Transwell小室購(gòu)自Corning。

2方法

2.1細(xì)胞培養(yǎng)與實(shí)驗(yàn)分組人乳腺癌MCF-7細(xì)胞用含10% FBS的RPMI-1640培養(yǎng)基，置于37 ℃、5% CO2培養(yǎng)箱中培養(yǎng)。待細(xì)胞匯合度達(dá)到80%后，將細(xì)胞隨機(jī)分為3組：(1)對(duì)照(control)組：將細(xì)胞置于無(wú)血清RPMI-1640培養(yǎng)基中培養(yǎng)24 h；(2)CoCl2組：將細(xì)胞置于含有200 μmol/L CoCl2的無(wú)血清RPMI-1640培養(yǎng)基中培養(yǎng)24 h；(3)CoCl2+digoxin組：將細(xì)胞置于含有200 μmol/L CoCl2和100 nmol/L地高辛的無(wú)血清RPMI-1640培養(yǎng)基中培養(yǎng)24 h。倒置相差顯微鏡下觀察細(xì)胞形態(tài)變化。

2.2細(xì)胞劃痕實(shí)驗(yàn)取對(duì)數(shù)生長(zhǎng)期細(xì)胞以每孔1×106個(gè)細(xì)胞接種于6孔板，置于37 ℃、5% CO2細(xì)胞培養(yǎng)箱中培養(yǎng)，待細(xì)胞長(zhǎng)到融合成單層狀態(tài)時(shí)，將細(xì)胞置于無(wú)血清培養(yǎng)基中培養(yǎng)24 h，用無(wú)菌的100 μL移液器槍頭在底部劃線，吸去細(xì)胞培養(yǎng)液，用PBS沖洗細(xì)胞3次，按上述實(shí)驗(yàn)分組分別加入相應(yīng)的培養(yǎng)基，置于37 ℃、5% CO2細(xì)胞培養(yǎng)箱中培養(yǎng)，在0和24 h用倒置顯微鏡進(jìn)行拍照，用ImageJ軟件測(cè)量劃痕面積，計(jì)算平均遷移距離(S)，每孔測(cè)量5處，取平均值，每組設(shè)3復(fù)孔。遷移率(migration rate，MR；%)=S實(shí)驗(yàn)組/S對(duì)照組×100%。

2.3Transwell侵襲實(shí)驗(yàn)將Matrigel基質(zhì)膠與RPMI-1640培養(yǎng)基按1∶6稀釋，取50 μL均勻鋪到Transwell小室上室內(nèi)，將小室放入24孔板中，37 ℃恒溫孵育3 h使其成凝膠狀。取對(duì)數(shù)生長(zhǎng)期細(xì)胞，按上述實(shí)驗(yàn)分組，用配置好的各實(shí)驗(yàn)組培養(yǎng)基制備2.5×108/L的細(xì)胞懸液，取200 μL加入上室內(nèi)，在24孔板中加入600 μL含10% FBS的RPMI-1640培養(yǎng)基，置于37 ℃、5% CO2細(xì)胞培養(yǎng)箱中培養(yǎng)24 h。取出小室，用棉簽擦去上室內(nèi)的細(xì)胞，經(jīng)4%多聚甲醛固定和DAPI染色后，于熒光顯微鏡下取5個(gè)視野拍照計(jì)數(shù)，取平均值，每組設(shè)3復(fù)孔。侵襲率(invasion rate，IR；%)=侵襲細(xì)胞數(shù)實(shí)驗(yàn)組/侵襲細(xì)胞數(shù)對(duì)照組×100%。

2.4Western blot檢測(cè)蛋白水平裂解各組細(xì)胞提取總蛋白，用BCA法定量后，進(jìn)行SDS-PAGE電泳并轉(zhuǎn)移至PVDF膜。封閉液(5% BSA/TBST)封閉1 h，加入Ⅰ抗(1∶1 000稀釋)，4 ℃孵育過(guò)夜，TBST洗膜3次，加入Ⅱ抗(1∶1 000稀釋)室溫下孵育1 h，TBST洗膜3次，加入ECL進(jìn)行發(fā)光反應(yīng)，暗室X膠片顯影，膠片用凝膠成像系統(tǒng)攜帶的白色光源拍照，用ImageJ軟件測(cè)定各蛋白條帶灰度值。

3統(tǒng)計(jì)學(xué)處理

用SPSS 16.0統(tǒng)計(jì)軟件進(jìn)行分析。數(shù)據(jù)均采用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，多組間比較采用單因素方差分析(one-way ANOVA),并用Bonferroni校正的t檢驗(yàn)進(jìn)行組間兩兩比較。以P<0.05為差異有統(tǒng)計(jì)學(xué)顯著性。

結(jié)果

1地高辛抑制CoCl2誘導(dǎo)的MCF-7細(xì)胞上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transiton,EMT)

在倒置相差顯微鏡下可見，對(duì)照組細(xì)胞呈多角形上皮形態(tài), 細(xì)胞之間緊密相接。經(jīng)200 μmol/L CoCl2作用24 h后，細(xì)胞形態(tài)轉(zhuǎn)變?yōu)殚g質(zhì)細(xì)胞形態(tài)，表現(xiàn)為細(xì)胞形態(tài)拉長(zhǎng)呈梭形，細(xì)胞間隙增大。CoCl2+digoxin組細(xì)胞依然呈上皮形態(tài)，未發(fā)生明顯的EMT,見圖1。

2地高辛抑制CoCl2誘導(dǎo)的MCF-7細(xì)胞遷移

細(xì)胞劃痕實(shí)驗(yàn)結(jié)果顯示，CoCl2組細(xì)胞遷移率為155.97%±10.43%，顯著高于control組(P<0.01)；CoCl2+digoxin組細(xì)胞遷移率為113.96%±9.06%，與control組相比無(wú)統(tǒng)計(jì)學(xué)差異，這表明地高辛可抑制CoCl2誘導(dǎo)的細(xì)胞遷移，見圖2。

3地高辛抑制CoCl2誘導(dǎo)的MCF-7細(xì)胞侵襲

Transwell實(shí)驗(yàn)結(jié)果顯示，CoCl2組細(xì)胞侵襲率為233.80%±15.49%，顯著高于control組(P<0.01)；CoCl2+digoxin組細(xì)胞侵襲率為112.68%±11.27%，與control組相比差異無(wú)統(tǒng)計(jì)學(xué)顯著性，這表明地高辛可抑制CoCl2誘導(dǎo)的細(xì)胞侵襲，見圖3。

4CoCl2對(duì)HIF-1α、Snail、E-cadherin和vimentin蛋白表達(dá)的影響

Western blot結(jié)果顯示，與control組相比，CoCl2組細(xì)胞HIF-1α、Snail和vimentin蛋白表達(dá)水平顯著升高(P<0.01)，E-cadherin蛋白表達(dá)水平顯著降低(P<0.01)；CoCl2+digoxin組細(xì)胞HIF-1α、E-cad-herin和vimentin蛋白表達(dá)水平均與control組相比差異無(wú)統(tǒng)計(jì)學(xué)顯著性， Snail蛋白表達(dá)水平雖略高于control組(P<0.05)，但與CoCl2組細(xì)胞相比顯著降低(P<0.01)，見圖4、5。

Figure 1.Digoxin inhibited CoCl2-induced epithelial-mesenchymal transition in MCF-7 cells.

圖1地高辛抑制CoCl2誘導(dǎo)的MCF-7細(xì)胞上皮間質(zhì)轉(zhuǎn)化

Figure 2.Digoxin inhibited CoCl2-induced migration of MCF-7 cells. Mean±SD.n=3.**P<0.01vscontrol group.

圖2地高辛抑制CoCl2誘導(dǎo)的MCF-7細(xì)胞遷移

討論

缺氧在腫瘤的遷移侵襲過(guò)程中發(fā)揮著重要作用，其中的一個(gè)重要機(jī)制就是腫瘤細(xì)胞發(fā)生EMT[8]。EMT是指細(xì)胞通過(guò)去分化由多邊形上皮樣形態(tài)轉(zhuǎn)變?yōu)樗笮伍g葉性細(xì)胞形態(tài)，獲得更具運(yùn)動(dòng)能力表型的過(guò)程。本研究采用CoCl2處理MCF-7細(xì)胞模擬缺氧，導(dǎo)致MCF-7細(xì)胞發(fā)生EMT，而地高辛可顯著抑制CoCl2誘導(dǎo)的EMT，如圖1所示。缺氧也使得MCF-7細(xì)胞的遷移和侵襲能力均顯著增強(qiáng)，該作用也同樣被地高辛所阻斷。以上結(jié)果表明，地高辛對(duì)缺氧所導(dǎo)致的MCF-7細(xì)胞侵襲能力的增強(qiáng)具有顯著的抑制作用。

EMT的一個(gè)主要特征為E-cadherin表達(dá)減少。E-cadherin是一種鈣依賴性細(xì)胞黏附分子，在維持正常上皮細(xì)胞形態(tài)和細(xì)胞極性中發(fā)揮重要作用[9]。

Figure 3.Digoxin inhibited CoCl2-induced invasion of MCF-7 cells. Mean±SD.n=3.**P<0.01vscontrol group.

圖3地高辛抑制CoCl2誘導(dǎo)的MCF-7細(xì)胞侵襲

Figure 4.The effect of digoxin on the protein levels of E-cadhe-rin and vimentin in the MCF-7 cells. Mean±SD.n=3.**P<0.01vscontrol group.

圖4地高辛對(duì)MCF-7細(xì)胞E-cadherin和vimentin蛋白水平的影響

EMT的另一個(gè)主要特征為以細(xì)胞角蛋白為主細(xì)胞骨架轉(zhuǎn)化為以vimentin為主的細(xì)胞骨架[10]。vimentin是中間絲蛋白的一種，與微管和肌動(dòng)蛋白微細(xì)絲一起組成細(xì)胞骨架。本研究結(jié)果顯示，缺氧可使MCF-7細(xì)胞E-cadherin蛋白表達(dá)水平顯著降低，vimentin蛋白表達(dá)水平顯著升高，而地高辛可阻斷缺氧對(duì)MCF-7細(xì)胞E-cadherin蛋白和vimentin蛋白表達(dá)的影響。

Figure 5.The effect of digoxin on the protein levels of HIF-1α and Snail in the MCF-7 cells. Mean±SD.n=3.**P<0.01vscontrol group;##P<0.01vsCoCl2group.

圖5地高辛對(duì)MCF-7細(xì)胞HIF-1α和Snail蛋白水平的影響

為探究地高辛抑制缺氧誘導(dǎo)EMT的機(jī)制，我們觀察了地高辛對(duì)缺氧條件下MCF-7細(xì)胞Snail蛋白表達(dá)水平的影響。Snail屬轉(zhuǎn)錄因子Snail家族成員，該家族包括Snail1(Snail)、Snail2(Slug)和Snail3(Smuc)，其羧基末端高度保守，含有4～6個(gè)鋅指結(jié)構(gòu)，其中Snail羧基末端含有4個(gè)鋅指結(jié)構(gòu)。Snail可特異性地與E-cadherin啟動(dòng)子中的E-box作用元件相結(jié)合而抑制E-cadherin蛋白的表達(dá)[11]，同時(shí)Snail也可上調(diào)vimentin蛋白表達(dá)[12]，在調(diào)控EMT過(guò)程中發(fā)揮著關(guān)鍵性作用[13]。本研究發(fā)現(xiàn)缺氧可使MCF-7細(xì)胞Snail蛋白表達(dá)水平顯著升高，該作用可被地高辛抑制。

HIF-1也是缺氧誘導(dǎo)腫瘤細(xì)胞發(fā)生EMT的重要因子[8, 14]。HIF-1是由HIF-1α和HIF-1β兩個(gè)亞單位組成的異源二聚體。HIF-1α是HIF-1的功能亞基，由826個(gè)氨基酸殘基構(gòu)成，其C端有一個(gè)富含脯氨酸-絲氨酸-蘇氨酸(Pro/Ser/Thr)的氧依賴降解結(jié)構(gòu)域(oxygen-dependent degradation domain，ODD)。在常氧條件下，HIF-1α的ODD中Pro402和Pro564被脯氨酸羥化酶結(jié)構(gòu)域蛋白(prolyl hydroxylase domain protein，PHD)羥化，被羥化的HIF-1α與腫瘤抑制蛋白(von Hippel-Lindau syndrome protein，pVHL)結(jié)合后經(jīng)泛素-蛋白酶體途徑降解。缺氧條件下PHD失活，HIF-1α因不能被羥基化而降解受阻，HIF-1α與HIF-1β在核內(nèi)形成二聚體HIF-1，HIF-1與靶基因啟動(dòng)子或增強(qiáng)子的缺氧反應(yīng)元件(hypoxia response element，HRE)結(jié)合從而調(diào)節(jié)其轉(zhuǎn)錄表達(dá)。HIF-1可直接上調(diào)Snail基因表達(dá)[15]。Wong等[16]在人三陰乳腺癌細(xì)胞MDA-MB-231中發(fā)現(xiàn)地高辛可在轉(zhuǎn)錄水平顯著抑制缺氧誘導(dǎo)的HIF-1α表達(dá)。與此相一致，本研究結(jié)果顯示，地高辛可顯著下調(diào)缺氧誘導(dǎo)的HIF-1α蛋白表達(dá)。

綜上所述，缺氧可通過(guò)富集HIF-1α上調(diào)Snail蛋白表達(dá)，導(dǎo)致MCF-7細(xì)胞發(fā)生EMT，使其遷移和侵襲能力顯著增強(qiáng)。地高辛則可通過(guò)下調(diào)HIF-1α和Snail蛋白表達(dá)抑制缺氧誘導(dǎo)的MCF-7細(xì)胞上皮間質(zhì)轉(zhuǎn)化和侵襲。

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(責(zé)任編輯： 林白霜， 羅森)

Effect of digoxin on hypoxia-induced epithelial-mesenchymal transition and invasion in human breast carcinoma MCF-7 cells

LI Bai-he, YUAN Lei

(LuoheMedicalCollege,Luohe462002,China.E-mail:fzyx_yl@163.com)

[ABSTRACT]AIM: To investigate the effect of digoxin on hypoxia-induced epithelial-mesenchymal transition (EMT), migration and invasion in human breast carcinoma MCF-7 cells. METHODS: MCF-7 cells were treated in vitro with a chemical hypoxia inducer cobalt chloride (CoCl2) to imitate hypoxia. Cell migration was observed by wound healing assay, and cell invasion was measured by Transwell invasion assay. The protein levels of hypoxia-inducible factor-1α (HIF-1α), Snail, E-cadherin and vimentin in MCF-7 cells were detected by Western blot. RESULTS: Digoxin inhibited CoCl2-induced EMT and reversed the mesenchymal phenotype. CoCl2 enhanced the abilities of migration and invasion (P<0.01), significantly decreased the expression of E-cadherin and increased the expression of HIF-1α, Snail and vimentin (P<0.01), but these effects were blocked by digoxin. CONCLUSION: Digoxin inhibits CoCl2-induced EMT and invasion most likely via HIF1-α-Snail signaling pathway.

[KEY WORDS]Digoxin; Epithelial-mesenchymal transition; Hypoxia-inducible factor-1α; Cell invasion; MCF-7 cells

[文章編號(hào)]1000- 4718(2016)05- 0852- 05

[收稿日期]2015- 11- 13[修回日期] 2015- 12- 28

*[基金項(xiàng)目]河南省科技廳科技發(fā)展計(jì)劃項(xiàng)目(No. 142102310466)；漯河醫(yī)學(xué)高等?？茖W(xué)校自然科學(xué)研究計(jì)劃項(xiàng)目(No. 2014-S-LMC09)

通訊作者△Tel： 0395-2969424； E-mail： fzyx_yl@163.com

[中圖分類號(hào)]R737.9; R730.23

[文獻(xiàn)標(biāo)志碼]A

doi:10.3969/j.issn.1000- 4718.2016.05.014

雜志網(wǎng)址： http://www.cjpp.net