PM2.5對(duì)人肺癌細(xì)胞A549遷移、侵襲能力的增強(qiáng)作用

楊丹，周偉強(qiáng)，楊彪，肖純凌,*

1. 沈陽(yáng)醫(yī)學(xué)院 藥理學(xué)教研室，沈陽(yáng) 110034 2. 沈陽(yáng)醫(yī)學(xué)院 遼寧省環(huán)境污染與微生態(tài)重點(diǎn)實(shí)驗(yàn)室，沈陽(yáng) 110034

PM2.5是指空氣動(dòng)力學(xué)直徑≤2.5 μm的顆粒物，是構(gòu)成可吸入顆粒物的主要部分[1]。眾多流行病學(xué)調(diào)查均顯示PM2.5與包括肺癌在內(nèi)的呼吸系統(tǒng)疾病關(guān)系密切[2]。長(zhǎng)期的PM2.5暴露能明顯增加肺癌患者的死亡率，同時(shí)可致確診后的肺癌患者的生存期縮短，說明PM2.5能夠促進(jìn)肺癌的進(jìn)展[3-4]。肺癌的進(jìn)展與其轉(zhuǎn)移密切相關(guān)，有研究表明：PM2.5可能通過促進(jìn)腫瘤新生血管形成、誘發(fā)炎癥反應(yīng)等促進(jìn)肺癌轉(zhuǎn)移，但其促進(jìn)肺癌轉(zhuǎn)移的具體機(jī)制仍不清楚[5-7]。以往對(duì)于PM2.5致呼吸系統(tǒng)損傷的分子機(jī)制的研究多集中于以較高濃度PM2.5所致的細(xì)胞損傷作用，而關(guān)于PM2.5對(duì)腫瘤細(xì)胞遷移及侵襲能力影響的實(shí)驗(yàn)研究仍較少，因此有必要進(jìn)行更多及更深入的研究[8-11]。

本研究采用細(xì)胞劃痕實(shí)驗(yàn)和transwell小室法檢測(cè)PM2.5對(duì)人肺癌細(xì)胞A549遷移、侵襲能力的影響，以期進(jìn)一步證實(shí)PM2.5具有促肺癌轉(zhuǎn)移的作用。同時(shí)本研究采用Western Blotting法檢測(cè)同細(xì)胞遷移、侵襲能力密切相關(guān)的Wnt/βcatenin通路活性及其下游cyclin D1、MMP-2、MMP-9和轉(zhuǎn)錄因子snail、slug蛋白表達(dá)水平的變化，分析該信號(hào)通路在PM2.5影響肺癌細(xì)胞A549遷移、侵襲能力中的作用，為今后深入研究PM2.5促肺癌轉(zhuǎn)移的機(jī)制提供實(shí)驗(yàn)依據(jù)。

1 材料與方法(Materials and methods)

1.1 細(xì)胞株和主要試劑

人肺癌細(xì)胞A549購(gòu)自中國(guó)科學(xué)院上海細(xì)胞生物學(xué)研究所細(xì)胞庫(kù)。胰酶及RPMI 1640培養(yǎng)基(Gibco公司)； Matrigel基質(zhì)膠(BD公司)；transwell小室(Costar公司)；snail和slug抗體(CST公司)；MMP-2、MMP-9、β-catenin及cyclin D1抗體(Santa Cruz公司)；細(xì)胞核蛋白抽提試劑盒(碧云天公司)。

1.2 PM2. 5 采集和處理

選擇遼寧省沈陽(yáng)市為采樣地區(qū)，采樣地點(diǎn)為城市中心。霧霾天氣時(shí)運(yùn)用高流量樣品顆粒采集器采集PM2.5樣品于濾膜上，采樣高度為距離地面約1.5 m。連續(xù)自動(dòng)采樣6個(gè)月，以1.13 m3·min-1流速每48 小時(shí)采集1份。采樣后的濾膜用鋁箔紙包裹后于-20 ℃冰箱中保存。制備PM2.5懸液時(shí)將采樣后的濾膜剪成約1 cm ×1 cm大小，浸泡在50 mL去離子水中。超聲震蕩收集PM2.5顆粒物，用多層紗布過濾震蕩液后制成PM2.5懸液。收集PM2.5懸液在13 000×g、4 ℃ 的條件下離心10 min后的底層顆粒物，高壓滅菌后真空冷凍干燥，-80 ℃保存。染毒前用無(wú)菌生理鹽水配制成所需的PM2.5混懸液并超聲處理[12-13]。

1.3 細(xì)胞培養(yǎng)及處理

人肺癌A549細(xì)胞株用RPMI 1640培養(yǎng)基(含10%新生牛血清、1% L-谷氨酰胺及1%青鏈霉素)培養(yǎng)于37 ℃、5 %CO2細(xì)胞培養(yǎng)箱中，細(xì)胞長(zhǎng)滿約90%時(shí)傳代。取對(duì)數(shù)生長(zhǎng)期的A549細(xì)胞，接種于培養(yǎng)皿或培養(yǎng)板內(nèi)，于37 ℃、5%CO2條件下培養(yǎng)24 h后更換不含血清及抗生素的RPMI 1640培養(yǎng)基。次日向培養(yǎng)基中加入不同濃度PM2.5或TGF-β1(10 ng·mL-1)后持續(xù)培養(yǎng)72 h。

1.4 MTT 比色法檢測(cè)細(xì)胞增殖

設(shè)立實(shí)驗(yàn)組(PM2.5終濃度為2.5、10、40和160 μg·mL-1)、對(duì)照組(只加細(xì)胞，不加PM2.5)和空白組(不加細(xì)胞，只加RPMI 1 640培養(yǎng)基及PM2.5)。體外培養(yǎng)72 h后，每孔加入20 μL MTT (終濃度5 g·L-1)，37 ℃繼續(xù)孵育4 h，棄上清，每孔加入150 μL DMSO 振蕩10 min，用酶標(biāo)儀在490 nm處測(cè)定吸光度(A)值，實(shí)驗(yàn)重復(fù)3次。按公式計(jì)算：

細(xì)胞生長(zhǎng)抑制率=(A處理組-A空白組)/(A對(duì)照組-A空白組)×100%

1.5 細(xì)胞劃痕實(shí)驗(yàn)

1.6 Transwell小室侵襲、遷移實(shí)驗(yàn)

細(xì)胞分組同劃痕實(shí)驗(yàn)。侵襲實(shí)驗(yàn)取200 μL細(xì)胞懸液種于鋪好Matrigel膠的Transwell上室中，下室加入600 μL含10%血清的培養(yǎng)基，于37 ℃、5% CO2細(xì)胞培養(yǎng)箱中孵育24 h，移出transwell小室，棉簽擦去上室的非遷移細(xì)胞，4%甲醛固定30 min后用0.1%結(jié)晶紫染色15 min，沖洗干凈后倒置風(fēng)干。在倒置顯微鏡下觀察并拍照，每個(gè)小室隨機(jī)取6個(gè)視野拍照，計(jì)數(shù)穿膜細(xì)胞個(gè)數(shù)。細(xì)胞遷移試驗(yàn)不需要鋪Matrigel膠，其余操作同侵襲實(shí)驗(yàn)。

1.7 胞核蛋白的抽提

按照細(xì)胞核蛋白與細(xì)胞漿蛋白抽提試劑盒說明進(jìn)行。用PBS洗一遍，刮下細(xì)胞，每20 μL細(xì)胞沉淀加入200 μL細(xì)胞漿蛋白抽提試劑A。最高速劇烈渦旋5 s，冰浴10～15 min。加入細(xì)胞漿蛋白抽提試劑B 10 μL，最高速劇烈渦旋5 s，冰浴1 min，4 ℃、13 000×g離心5 min。收集沉淀，加入50 μL細(xì)胞核蛋白抽提試劑。最高速劇烈渦旋30 s，把細(xì)胞沉淀完全懸浮并分散開。然后放回冰浴中，每隔1～2 min再高速劇烈渦旋30 s，共30 min。4 ℃、13 000×g離心10 min，立即吸取上清，即為抽提得到的細(xì)胞核蛋白。

1.8 Western Blotting

收集細(xì)胞總蛋白，SDS - PAGE電泳后轉(zhuǎn)印到PVDF膜上，用含5%脫脂奶粉的封閉液常溫封閉1 h，加入一抗，4 ℃反應(yīng)過夜，再用辣根過氧化物酶標(biāo)記的二抗室溫下反應(yīng)0.5 h，化學(xué)發(fā)光法顯影；利用Image J 軟件對(duì)目的條帶進(jìn)行灰度值檢測(cè)，以β-actin為內(nèi)參照分析蛋白相對(duì)表達(dá)量。

1.9 統(tǒng)計(jì)學(xué)處理

2 結(jié)果(Results)

2.1 PM2.5對(duì)A549細(xì)胞增殖抑制作用

PM2.5處理A549細(xì)胞72 h后采用MTT 法檢測(cè)各組吸光度，計(jì)算PM2.5對(duì)A549細(xì)胞的增殖抑制率。如圖1和表1所示，與對(duì)照組比較，2.5、10、40和160 μg·mL-1PM2.5組對(duì)A549細(xì)胞的增殖抑制率隨濃度升高而增加，分別為(3.08% ± 3.73%)、(10.37% ± 5.97%)、(34.00% ± 6.39%) 和(51.01% ± 8.37%)。 選取對(duì)A549細(xì)胞增殖抑制作用較弱的2個(gè)濃度(2.5 μg·mL-1和10 μg·mL-1)進(jìn)行后續(xù)實(shí)驗(yàn)。

圖1 PM2.5對(duì)A549細(xì)胞的生長(zhǎng)抑制作用Fig. 1 Inhibition of PM2.5 on growth of A549

表1 PM2.5對(duì)A549細(xì)胞的生長(zhǎng)抑制作用Table 1 Inhibition of PM2.5 on growth of A549

注：與對(duì)照組比較，*為P<0.05。

Note： compared with control group，*P<0.05.

圖2 PM2.5促進(jìn)A549細(xì)胞的遷移和侵襲注: A為細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)A549細(xì)胞遷移能力(×50)；2B為Transwell法檢測(cè)A549細(xì)胞遷移能力(a～d)和侵襲能力(e～h)(×200) 。Fig. 2 PM2.5 promotes migration and invasion of A549 cellsNote: A showed the migration ability of A549 cells detected by wound healing assay (×50); B showed the migration ability (a-d) and invasion ability (e-h) of A549 cells detected by transwell assay (×200).

圖3 Western Blotting 法檢測(cè)PM2.5 處理72 h后細(xì)胞核內(nèi)β-catenin蛋白表達(dá)的變化注：與對(duì)照組比較，*為 P<0.05。Fig. 3 The expression levels of β-catenin proteins after exposure to PM2.5 for 72 h were analyzed by Western BlottingNote: compared with control group, *P<0.05.

2.2 PM2.5可增強(qiáng)A549細(xì)胞的遷移、侵襲能力

劃痕實(shí)驗(yàn)結(jié)果如圖2A和表2所示：劃痕后24 h，對(duì)照組創(chuàng)面愈合率為(31.97%±1.80%)、2.5 μg·mL-1和10 μg·mL-1PM2.5組創(chuàng)面愈合率分別為(37.59%±3.08%)和(46.34%±5.19%)。與對(duì)照組相比，10 μg·mL-1PM2.5組A549細(xì)胞的創(chuàng)面愈合率明顯增加(P<0.05)。Transwell小室法遷移和侵襲實(shí)驗(yàn)結(jié)果如圖2B和表2 所示：對(duì)照組、2.5 μg·mL-1和10 μg·mL-1PM2.5組中穿過基膜的細(xì)胞數(shù)分別為(118.67 ± 4.5) 個(gè)和(32.33 ±2.73) 個(gè)，(122.50 ± 6.53) 個(gè)和(35.5 ± 5.17) 個(gè)，(165.67 ± 6.62) 個(gè)和(47.83 ± 2.04) 個(gè)。與對(duì)照組相比，10 μg·mL-1PM2.5組小室濾膜下表面的細(xì)胞個(gè)數(shù)明顯增多(P<0.05)。上述結(jié)果表明10 μg·mL-1PM2.5可使A549細(xì)胞的遷移、侵襲能力增高。

2.3 PM2.5增強(qiáng)A549細(xì)胞Wnt/β-catenin信號(hào)通路活性

表2 PM2.5促進(jìn)A549細(xì)胞的遷移和侵襲Table 2 PM2.5 promotes migration and invasion of A549 cells

注：與對(duì)照組比較，*為P<0.05。

Note： compared with control group,*P<0.05.

PM2.5處理A549細(xì)胞后，Western Blotting 法檢測(cè)細(xì)胞核內(nèi)β-catenin蛋白表達(dá)的變化。結(jié)果如圖3和表3所示：給予A549細(xì)胞2.5 μg·mL-1和10 μg·mL-1的PM2.5處理72 h后，與對(duì)照組相比，10 μg·mL-1PM2.5組細(xì)胞核內(nèi)β-catenin蛋白表達(dá)水平明顯增高(P<0.05)。

2.4 PM2.5上調(diào)A549細(xì)胞中cyclin D1、snail、slug、MMP-2 和MMP-9的蛋白表達(dá)

PM2.5處理A549細(xì)胞后，Western Blotting 法檢測(cè)細(xì)胞內(nèi)cyclin D1、snail、slug、MMP-2 和MMP-9蛋白表達(dá)的變化。結(jié)果如圖4和表4所示：給予A549細(xì)胞2.5 μg·mL-1和10 μg·mL-1的PM2.5處理72 h后，與對(duì)照組相比，10 μg·mL-1PM2.5組細(xì)胞內(nèi)cyclin D1、snail、slug、MMP-2 和MMP-9的蛋白表達(dá)明顯增高(P<0.05)。

3 討論(Discussion)

表3 PM2.5對(duì)肺癌A549細(xì)胞核內(nèi)β-catenin蛋白相對(duì)表達(dá)的影響Table 3 The effect of PM2.5 on the expression of β-catenin protein in A549 cells nucleus (±s，n=6)

注：與對(duì)照組比較，*為P<0.05。

Note： compared with control group,*P<0.05.

圖4 Western Blotting 法檢測(cè)加入PM2.5 72 h后細(xì)胞內(nèi)cyclin D1、snail、slug、MMP-2 和MMP-9蛋白表達(dá)的變化注：與對(duì)照組比較，*為 P<0.05。Fig. 4 The expression levels of cyclin D1, snail, slug, MMP-2 and MMP-9 proteins after treated with PM2.5 for 72 h were analyzed by Western BlottingNote: compared with control group, *P<0.05.

注：與對(duì)照組比較，*為P<0.05。

Note： compared with control group,*P<0.05.

腫瘤細(xì)胞的轉(zhuǎn)移是肺癌患者復(fù)發(fā)以及治療失敗的重要原因。以往關(guān)于PM2.5對(duì)肺癌影響的研究多集中其致癌細(xì)胞損傷或者死亡的作用方面[10, 14-16]，而關(guān)于PM2.5是否可促進(jìn)肺癌的早期轉(zhuǎn)移的研究尚少見報(bào)道。本研究通過transwell遷移、侵襲實(shí)驗(yàn)和細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)PM2.5對(duì)肺癌A549細(xì)胞遷移、侵襲能力的影響，結(jié)果顯示在無(wú)血清條件下較低濃度(10 μg·mL-1)的PM2.5作用72 h即可明顯增強(qiáng)肺癌細(xì)胞遷移、侵襲能力，說明PM2.5可能具有促進(jìn)肺癌的早期轉(zhuǎn)移的作用。

腫瘤細(xì)胞的轉(zhuǎn)移過程非常復(fù)雜，涉及多條信號(hào)通路、轉(zhuǎn)錄因子及酶的活化。Wnt/β-catenin信號(hào)傳導(dǎo)通路在腫瘤細(xì)胞轉(zhuǎn)移過程中起著重要作用，該通路激活時(shí)，過量的β-catenin從胞質(zhì)中轉(zhuǎn)移入細(xì)胞核，在核內(nèi)與轉(zhuǎn)錄因子T細(xì)胞因子/淋巴增強(qiáng)因子(T cell factor, TCF/lymphoid enhancer factor, LEF)相互作用，激活下游大量具有促進(jìn)腫瘤細(xì)胞轉(zhuǎn)移功能的基因轉(zhuǎn)錄，如基質(zhì)金屬蛋白酶、cyclin D1、slug、snail等，進(jìn)而增強(qiáng)腫瘤細(xì)胞轉(zhuǎn)移能力[17-20]。本研究通過檢測(cè)細(xì)胞核內(nèi)β-catenin的表達(dá)水平證明在無(wú)血清條件下較低濃度的PM2.5(10 μg·mL-1)作用72 h即可明顯激活肺癌A549細(xì)胞Wnt/β-catenin信號(hào)通路。

基質(zhì)金屬蛋白酶家族是降解細(xì)胞外基質(zhì)的重要酶類。腫瘤細(xì)胞周圍細(xì)胞外基質(zhì)的降解是腫瘤侵襲和轉(zhuǎn)移的必要條件，因此金屬蛋白酶家族在腫瘤細(xì)胞遷移和侵襲過程中發(fā)揮重要作用。多種腫瘤(包括非小細(xì)胞肺癌)中均可見金屬蛋白酶家族過表達(dá)?；|(zhì)金屬蛋白酶-2和基質(zhì)金屬蛋白酶-9是基質(zhì)金屬蛋白酶家族內(nèi)的重要成員，同屬于IV型明膠酶，主要降解細(xì)胞間基質(zhì)及基底膜主要成分IV型膠原，促進(jìn)腫瘤細(xì)胞侵襲和擴(kuò)散，因此其在腫瘤轉(zhuǎn)移過程中具有重要作用[20-21]。研究表明：Wnt/β-catenin信號(hào)通路的激活能夠使MMP-2和MMP-9的表達(dá)水平增高[18]。如Dong等[22]的研究表明：電離輻射可通過激活Wnt/β-catenin信號(hào)通路使其下游包括MMP-2和MMP-9在內(nèi)的多種基因表達(dá)上調(diào)，增強(qiáng)膠質(zhì)瘤細(xì)胞U87侵襲能力。本研究中Western Blotting實(shí)驗(yàn)結(jié)果表明：在無(wú)血清條件下較低濃度(10 μg·mL-1)的PM2.5作用72 h即可使MMP-2和MMP-9蛋白表達(dá)水平的上調(diào)，說明PM2.5增強(qiáng)肺癌A549細(xì)胞遷移、侵襲能力作用與其激活Wnt/β-catenin信號(hào)通路并通過上調(diào)其下游MMP-2和MMP-9蛋白表達(dá)相關(guān)。

cyclin D1是細(xì)胞周期蛋白家族的一個(gè)成員，在調(diào)控細(xì)胞周期進(jìn)展過程中發(fā)揮重要作用，此外cyclin D1還是一個(gè)癌基因，在多種腫瘤包括肺癌中呈高表達(dá)[23-24]。越來(lái)越多的研究表明，cyclin D1與腫瘤的轉(zhuǎn)移關(guān)系密切[25]。如Li等[26]的報(bào)道稱，cyclin D1通過抑制ROCK信號(hào)和轉(zhuǎn)移抑制因子TSP-1而增強(qiáng)細(xì)胞的運(yùn)動(dòng)能力。此外，細(xì)胞周期蛋白D1提高M(jìn)MPs的表達(dá)和活性，從而提高侵襲性。如Arato-Ohshima等[27]的研究表明cyclin D1蛋白的過度表達(dá)可增加MMP-2和MMP-9的活性并增強(qiáng)腦膠質(zhì)瘤細(xì)胞的侵襲性。

鋅指蛋白轉(zhuǎn)錄因子snail超家族包括snail(snail 1)和slug (snail 2)2個(gè)成員，二者作為轉(zhuǎn)錄因子，調(diào)控某些基因的表達(dá)，如下調(diào)細(xì)胞間的緊密連接成分如ZO-1及上調(diào)腫瘤細(xì)胞的金屬蛋白酶類的表達(dá)，這些均有助于增加細(xì)胞的遷徙轉(zhuǎn)移力，尤其常見于各種腫瘤的癌性細(xì)胞向遠(yuǎn)處轉(zhuǎn)移機(jī)制中[28-32]。如Wang等[33]的研究表明：snail促進(jìn)MMP-9表達(dá)，并且二者均與甲狀腺乳頭狀癌的淋巴結(jié)轉(zhuǎn)移有關(guān)。Li等[34]報(bào)道snail介導(dǎo)的MMP-2上調(diào)與細(xì)胞侵襲性增強(qiáng)有關(guān)。Qiao等[35]關(guān)于口腔鱗狀細(xì)胞癌的研究證明：snail通過上調(diào)MMP-2和MMP-9促進(jìn)細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化，轉(zhuǎn)移能力增強(qiáng)。Bolos等[36]和Chandler[37]研究發(fā)現(xiàn)，slug通過調(diào)控靶基因MMP-2影響細(xì)胞Ⅳ型膠原和明膠的降解，進(jìn)而參與腫瘤細(xì)胞的侵襲與轉(zhuǎn)移。Qiao等[35]和Yue等[38]報(bào)道slug能夠上調(diào)MMP-9的表達(dá)。Wnt/β-catenin信號(hào)轉(zhuǎn)導(dǎo)途徑可調(diào)控snail和slug表達(dá)水平，當(dāng)Wnt信號(hào)被激活，穩(wěn)定的β-catenin進(jìn)入細(xì)胞核內(nèi)與TCF/LEF相互作用，形成的復(fù)合物可使snail和slug轉(zhuǎn)錄，核內(nèi)水平增加[39]。

本研究中較低濃度的PM2.5(10 μg·mL-1)作用72 h即可明顯上調(diào)cyclin D1、snail和slug的蛋白表達(dá)水平，說明Wnt/β-catenin信號(hào)通路激活同時(shí)上調(diào)了某些其下游蛋白的表達(dá)，而這些蛋白的高表達(dá)又可進(jìn)一步上調(diào)MMP-2和MMP-9的表達(dá)，促進(jìn)腫瘤細(xì)胞的轉(zhuǎn)移和侵襲。

綜上所述，本研究發(fā)現(xiàn)將人肺癌A549細(xì)胞在無(wú)血清條件下暴露于較低濃度的PM2.5(10 μg·mL-1)72 h，能夠使細(xì)胞的侵襲、轉(zhuǎn)移能力增強(qiáng)，此作用與PM2.5活化Wnt/β-catenin信號(hào)傳導(dǎo)通路，上調(diào)細(xì)胞cyclin D1、MMP-2、MMP-9及轉(zhuǎn)錄因子snail和slug的蛋白表達(dá)水平有關(guān)。

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