miR-200c增加H2228細(xì)胞對(duì)克唑替尼和紫杉醇及順鉑敏感性的研究

高海祥，曹 磊

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·論著·

miR-200c增加H2228細(xì)胞對(duì)克唑替尼和紫杉醇及順鉑敏感性的研究

高海祥，曹 磊

背景 miR-200c可以增加腫瘤細(xì)胞對(duì)紫杉醇、順鉑的敏感性及表皮生長(zhǎng)因子受體(EGFR)陽(yáng)性肺癌細(xì)胞對(duì)分子靶向藥物厄洛替尼、吉非替尼及阿法替尼的敏感性，但其是否可以增加棘皮動(dòng)物微管相關(guān)樣蛋白4(EML4)-間變性淋巴瘤激酶(ALK)融合基因陽(yáng)性肺腺癌細(xì)胞(ALK陽(yáng)性肺癌細(xì)胞)對(duì)克唑替尼、紫杉醇和順鉑的敏感性尚無相關(guān)研究。目的 觀察miR-200c是否可以增加H2228細(xì)胞對(duì)克唑替尼、紫杉醇及順鉑的敏感性，并探討其機(jī)制。方法 2014年2月—2015年5月，培養(yǎng)H2228細(xì)胞并進(jìn)行轉(zhuǎn)染，根據(jù)轉(zhuǎn)染物的不同，將細(xì)胞分為增強(qiáng)轉(zhuǎn)染組(轉(zhuǎn)染miR-200c mimics)、增強(qiáng)對(duì)照組(轉(zhuǎn)染miR-200c mimics陰性對(duì)照)、抑制轉(zhuǎn)染組(轉(zhuǎn)染miR-200c inhibitor)、抑制對(duì)照組(轉(zhuǎn)染miR-200c inhibitor陰性對(duì)照)。采用實(shí)時(shí)熒光定量PCR法(Real-time PCR法)檢測(cè)H2228細(xì)胞中miR-200c基因表達(dá)水平，MTT法檢測(cè)不同濃度抗腫瘤藥物〔克唑替尼(50、100、200 nmol/L)、紫杉醇(6.25、12.50、25.00 nmol/L)、順鉑(25、50、100 nmol/L)〕作用下H2228細(xì)胞增殖水平，Transwell法檢測(cè)H2228細(xì)胞遷移率，Real-time PCR法檢測(cè)增強(qiáng)轉(zhuǎn)染組、增強(qiáng)對(duì)照組H2228細(xì)胞中E-鈣黏蛋白、N-鈣黏蛋白、波狀蛋白、CD24mRNA表達(dá)水平，Western blotting法檢測(cè)增強(qiáng)轉(zhuǎn)染組、增強(qiáng)對(duì)照組H2228細(xì)胞中E-鈣黏蛋白、N-鈣黏蛋白、波狀蛋白、CD24表達(dá)水平。結(jié)果 增強(qiáng)轉(zhuǎn)染組H2228細(xì)胞中miR-200c基因表達(dá)水平高于增強(qiáng)對(duì)照組(P<0.05)。抑制轉(zhuǎn)染組H2228細(xì)胞中miR-200c基因表達(dá)水平低于抑制對(duì)照組(P<0.05)。增強(qiáng)轉(zhuǎn)染組各濃度抗腫瘤藥物作用下H2228細(xì)胞增殖水平均低于增強(qiáng)對(duì)照組(P<0.05)。抑制轉(zhuǎn)染組各濃度抗腫瘤藥物作用下H2228細(xì)胞增殖水平均高于抑制對(duì)照組(P<0.05)。增強(qiáng)轉(zhuǎn)染組H2228細(xì)胞遷移率小于增強(qiáng)對(duì)照組(P<0.05)。抑制轉(zhuǎn)染組H2228細(xì)胞遷移率大于抑制對(duì)照組(P<0.05)。增強(qiáng)轉(zhuǎn)染組H2228細(xì)胞中E-鈣黏蛋白及其mRNA表達(dá)水平高于增強(qiáng)對(duì)照組，N-鈣黏蛋白、波狀蛋白、CD24及其mRNA表達(dá)水平低于增強(qiáng)對(duì)照組(P<0.05)。結(jié)論 miR-200c可以通過使肺癌細(xì)胞發(fā)生間質(zhì)-上皮轉(zhuǎn)變(MET)而增加H2228細(xì)胞對(duì)克唑替尼、紫杉醇及順鉑的敏感性。

抗藥性,腫瘤；H2228；miR-200c；克唑替尼；紫杉醇；順鉑

高海祥，曹磊.miR-200c增加H2228細(xì)胞對(duì)克唑替尼和紫杉醇及順鉑敏感性的研究[J].中國(guó)全科醫(yī)學(xué)，2016，19(36)：4478-4483.[www.chinagp.net]

GAO H X,CAO L.Effect of increased H2228 cells through miR-200c on sensitivity of crizotinib,paclitaxel and cisplatin[J].Chinese General Practice，2016，19(36)：4478-4483.

2016年CHEN等[1]研究顯示，肺癌是男性最常見的惡性腫瘤，是女性第二位高發(fā)的惡性腫瘤，而在病死率中，無論是男性還是女性，肺癌均為癌癥相關(guān)病死率的第一位。在肺癌的治療上，第三代含鉑聯(lián)合化療(PBC)具有良好的治療效果，可以使70%～80%接受一線化療的患者臨床獲益，但晚期肺癌患者總生存期(OS)較差，為8～10個(gè)月；與PBC相比，分子靶向藥物治療極大地提高了患者治療有效率和OS[2]。研究顯示，應(yīng)用厄洛替尼前后患者的OS存在明顯差異(17.6個(gè)月與27.2個(gè)月)[3]，但是，無論是化療藥物還是分子靶向藥物，耐藥的發(fā)生不可避免[4]，因此，尋找一種可以提高化療、分子靶向藥物治療敏感性的方法，延緩耐藥的發(fā)生也是解決腫瘤耐藥的新途徑。多項(xiàng)研究表明，miR-200c可以增加紫杉醇、順鉑及分子靶向藥物厄洛替尼、吉非替尼及阿法替尼對(duì)表皮生長(zhǎng)因子受體(EGFR)陽(yáng)性肺癌細(xì)胞的抑制作用，甚至可以增加EGFR野生型肺癌細(xì)胞對(duì)厄洛替尼的敏感性[5-8]。但是，miR-200c是否可以增加克唑替尼對(duì)棘皮動(dòng)物微管相關(guān)樣蛋白4(EML4)-間變性淋巴瘤激酶(ALK)融合基因陽(yáng)性肺腺癌細(xì)胞(ALK陽(yáng)性肺癌細(xì)胞)的抑制作用，目前尚無相關(guān)報(bào)道?；诖耍狙芯繉iR-200c轉(zhuǎn)染ALK陽(yáng)性肺癌細(xì)胞H2228細(xì)胞，觀察轉(zhuǎn)染前后細(xì)胞對(duì)紫杉醇、順鉑及克唑替尼敏感性的變化。

1 材料與方法

1.1 實(shí)驗(yàn)材料 H2228細(xì)胞株為ALK陽(yáng)性肺癌細(xì)胞，購(gòu)于ATCC(American Type Culture Collection)公司。

1.2 主要試劑及儀器 ALK抑制劑克唑替尼購(gòu)自美國(guó)Sigma-Aldrich公司；miR-200c mimics、miR-200c inhibitor及相應(yīng)陰性對(duì)照購(gòu)自美國(guó)invitrogen公司；RPMI 1640培養(yǎng)基、胎牛血清、胰蛋白酶以及MTT溶液購(gòu)自美國(guó)Gibco公司；鼠抗人E-鈣黏蛋白、N-鈣黏蛋白、波狀蛋白、CD24單克隆抗體購(gòu)自美國(guó)Abcam公司；辣根過氧化物酶(HRP)標(biāo)記抗鼠和兔的二抗購(gòu)自美國(guó)Fementas公司。

1.3 實(shí)驗(yàn)方法

1.3.1 細(xì)胞培養(yǎng)與轉(zhuǎn)染 2014年2月—2015年5月，將肺腺癌H2228細(xì)胞株從液氮中取出，迅速置于37 ℃水浴箱中解凍后移至離心管中，添加RPMI 1640培養(yǎng)基，輕輕吹打細(xì)胞，800 r/min離心5 min(離心半徑3 cm)，去上清液，完全培養(yǎng)液重懸細(xì)胞，調(diào)整至合適的細(xì)胞密度并接種于培養(yǎng)瓶中，放入37 ℃、5% CO2細(xì)胞培養(yǎng)箱中培養(yǎng)，次日更換1次培養(yǎng)液，磷酸鹽緩沖液(PBS)清洗，0.25%胰蛋白酶消化傳代。取對(duì)數(shù)生長(zhǎng)期H2228細(xì)胞接種在6孔板中，每孔中加入不含抗生素的培養(yǎng)液，調(diào)整細(xì)胞密度并于37 ℃培養(yǎng)箱中培養(yǎng)，使細(xì)胞貼壁生長(zhǎng)，密度達(dá)80%左右進(jìn)行轉(zhuǎn)染。將細(xì)胞接種于96孔板中，根據(jù)轉(zhuǎn)染物的不同，將細(xì)胞分為增強(qiáng)轉(zhuǎn)染組(轉(zhuǎn)染miR-200c mimics)、增強(qiáng)對(duì)照組(轉(zhuǎn)染miR-200c mimics陰性對(duì)照)、抑制轉(zhuǎn)染組(轉(zhuǎn)染miR-200c inhibitor)、抑制對(duì)照組(轉(zhuǎn)染miR-200c inhibitor陰性對(duì)照)。轉(zhuǎn)染方法：將A、B液混合形成mimics-lipofectamine 2000復(fù)合物，加入細(xì)胞培養(yǎng)基中，來回輕柔搖晃細(xì)胞培養(yǎng)基，然后放入培養(yǎng)箱中，放置4～6 h換液，換成含有血清和雙抗的培養(yǎng)基，繼續(xù)培養(yǎng)或進(jìn)行傳代。

1.3.2 實(shí)時(shí)熒光定量PCR法(Real-time PCR法)檢測(cè)H2228細(xì)胞中miR-200c基因表達(dá)水平 取各組對(duì)數(shù)生長(zhǎng)期H2228細(xì)胞，TRIZOL法提取H2228細(xì)胞的總RNA，將miRNA反轉(zhuǎn)錄為cDNA，參照Real-time PCR說明書檢測(cè)miR-200c基因表達(dá)水平。PCR擴(kuò)增反應(yīng)體系：SYBR Premix Ex Taq(2×)5 μl、PCR 正向引物(10 μmol/L)0.2 μl、PCR 反向引物(10 μmol/L)0.2 μl、DNA模板1 μl、雙蒸水(ddH2O)3.6 μl；PCR反應(yīng)條件：95 ℃預(yù)變性2 min，95 ℃變性15 s，60 ℃退火60 s，共40個(gè)循環(huán)。miR-200c上游引物：5′-CAGTGCGTGTCGTGGAGT-3′，下游引物：5′-GGCCTAATACTGCCGGGTAAT-3′；U6上游引物：5′-CGCTTCACGAATTTGCGTGTCAT-3′，下游引物：5′-GCTTCGGCAGCACATATACTAAAAT-3′。擴(kuò)增完畢后，以U6為內(nèi)參照基因，得到miR-200c基因表達(dá)水平。實(shí)驗(yàn)重復(fù)3次。

1.3.3 MTT法檢測(cè)不同濃度抗腫瘤藥物(克唑替尼、紫杉醇、順鉑)作用下H2228細(xì)胞增殖水平 取各組對(duì)數(shù)生長(zhǎng)期的H2228細(xì)胞，胰蛋白酶消化，培養(yǎng)液稀釋細(xì)胞懸液，并接種到96孔板，200 μl/孔，每組細(xì)胞均設(shè)置空白調(diào)零孔(不含細(xì)胞的完全培養(yǎng)基)。于37 ℃ 5% CO2培養(yǎng)箱中培養(yǎng)24 h。取出96孔板，吸去舊培養(yǎng)基，PBS洗滌2次，將細(xì)胞分別加入不同濃度的待測(cè)藥物中(克唑替尼：50、100、200 nmol/L；紫杉醇：6.25、12.50、25.00 nmol/L；順鉑：25、50、100 nmol/L)，于37 ℃ 5% CO2培養(yǎng)箱中培養(yǎng)44 h。取出96孔板，每孔加MTT溶液(5 mg/ml)20 μl，繼續(xù)孵育4 h，終止培養(yǎng)，每孔加150 μl二甲基亞砜(DMSO)，脫色搖床低速震蕩10 min，溶解結(jié)晶物。采用酶聯(lián)免疫檢測(cè)儀于490 nm波長(zhǎng)處檢測(cè)各孔吸光度值(OD值)，即H2228細(xì)胞增殖水平。實(shí)驗(yàn)重復(fù)3次。

1.3.4 Transwell法檢測(cè)H2228細(xì)胞遷移率 將Transwell小室置入無菌的24孔板中。各組取對(duì)數(shù)生長(zhǎng)期H2228細(xì)胞，用含0.1%胎牛血清的McCoy′s 5A培養(yǎng)基懸浮，計(jì)數(shù)，稀釋至密度為1×106/ml，每個(gè)上室中分別加入200 μl細(xì)胞懸液。24孔板下室中均加入600 μl含有30%胎牛血清的McCoy′s 5A培養(yǎng)基。37 ℃ 5% CO2培養(yǎng)箱中繼續(xù)培養(yǎng)48 h后棄去孔中培養(yǎng)液，PBS洗滌2次，將4 ℃ 4%甲醛溶液500 μl/孔加入下室，固定30 min，用棉簽輕輕擦掉上層未遷移細(xì)胞，再用0.1%龍膽紫500 μl染色20 min，清水洗滌3遍以上。顯微鏡下隨機(jī)選取5個(gè)視野(×400)觀察細(xì)胞，計(jì)數(shù)并采集圖像，計(jì)算H2228細(xì)胞遷移率。實(shí)驗(yàn)重復(fù)3次。

1.3.5 Real-time PCR法檢測(cè)增強(qiáng)轉(zhuǎn)染組、增強(qiáng)對(duì)照組H2228細(xì)胞中E-鈣黏蛋白、N-鈣黏蛋白、波狀蛋白、CD24mRNA表達(dá)水平 操作方法同Real-time PCR法檢測(cè)miR-200c基因表達(dá)水平。E-鈣黏蛋白上游引物：5′-TGGACAGGGAGGATTTTGAG-3′，下游引物：5′-ACCTGAGGCTTTGGATTCCT-3′；N-鈣黏蛋白上游引物：5′-CCACAGCTCCACCATATGACT-3′，下游引物：5′-CCCCAGTCGTTCAGGTAATC-3′；波狀蛋白上游引物：5′-AGTGCCTGGAACGTCAGATG-3′，下游引物：5′-CAGCAGCTTCCTGTAGGTGG-3′；CD24上游引物：5′-AGAGATAACCCTGCCCGAGG-3′，下游引物：5′-GTCTAGCAGGATGCTGGGTG-3′；內(nèi)參GAPDH上游引物：5′-CGGATTTGGTCGTATTGGG-3′，下游引物：5′-TGCTGGAAGATGGTGATGGGATT-3′。實(shí)驗(yàn)重復(fù)3次。

1.3.6 Western blotting法檢測(cè)增強(qiáng)轉(zhuǎn)染組、增強(qiáng)對(duì)照組H2228細(xì)胞中E-鈣黏蛋白、N-鈣黏蛋白、波狀蛋白、CD24表達(dá)水平 取各組對(duì)數(shù)生長(zhǎng)期H2228細(xì)胞，提取總蛋白，采用含有蛋白酶抑制劑的蛋白裂解液(RIPA)裂解細(xì)胞，4 ℃ 12 000×g離心30 min，取上清液總蛋白；采用BCA法測(cè)定蛋白質(zhì)濃度；蛋白樣品100 ℃水浴變性，按次序上樣至電泳道，十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)并轉(zhuǎn)印到聚偏二氟乙烯(PVDF)膜上，將PVDF膜放入含有一抗(鼠抗人E-鈣黏蛋白、N-鈣黏蛋白、波狀蛋白、CD24單克隆抗體)的封閉稀釋液中，4 ℃靜置孵育過夜。TBST洗膜3次，加入HRP標(biāo)記抗鼠和兔的二抗稀釋液，室溫下反應(yīng)1～2 h或4 ℃反應(yīng)過夜，再次TBST洗膜，增強(qiáng)化學(xué)發(fā)光法使蛋白條帶發(fā)光并在X線片上曝光顯影，計(jì)算E-鈣黏蛋白、N-鈣黏蛋白、波狀蛋白、CD24表達(dá)水平。實(shí)驗(yàn)重復(fù)3次。

2 結(jié)果

2.1miR-200c基因表達(dá)水平 增強(qiáng)轉(zhuǎn)染組H2228細(xì)胞中miR-200c基因表達(dá)水平(310.7±19.8)高于增強(qiáng)對(duì)照組(1.0±0.1)，差異有統(tǒng)計(jì)學(xué)意義(t=-27.225，P=0.001)。抑制轉(zhuǎn)染組H2228細(xì)胞中miR-200c基因表達(dá)水平(0.3±0.0)低于抑制對(duì)照組(1.0±0.2)，差異有統(tǒng)計(jì)學(xué)意義(t=5.393，P=0.033)。

2.2 不同濃度抗腫瘤藥物(克唑替尼、紫杉醇、順鉑)作用下H2228細(xì)胞增殖水平 增強(qiáng)轉(zhuǎn)染組各濃度抗腫瘤藥物(克唑替尼、紫杉醇、順鉑)作用下H2228細(xì)胞增殖水平均低于增強(qiáng)對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見表1)。抑制轉(zhuǎn)染組各濃度抗腫瘤藥物(克唑替尼、紫杉醇、順鉑)作用下H2228細(xì)胞增殖水平均高于抑制對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見表2)。

2.3H2228細(xì)胞遷移率 增強(qiáng)轉(zhuǎn)染組H2228細(xì)胞遷移率(31.0±2.6)小于增強(qiáng)對(duì)照組(54.7±3.5)，差異有統(tǒng)計(jì)學(xué)意義(t=16.289，P=0.004)。抑制轉(zhuǎn)染組H2228細(xì)胞遷移率(79.7±6.7)大于抑制對(duì)照組(53.3±6.0)，差異有統(tǒng)計(jì)學(xué)意義(t=-8.021，P=0.015，見圖1，本文圖1彩圖見本刊官網(wǎng)www.chinagp.net電子期刊相應(yīng)文章附件)。

2.4 增強(qiáng)轉(zhuǎn)染組、增強(qiáng)對(duì)照組H2228細(xì)胞中E-鈣黏蛋白、N-鈣黏蛋白、波狀蛋白、CD24mRNA表達(dá)水平 增強(qiáng)轉(zhuǎn)染組H2228細(xì)胞中E-鈣黏蛋白mRNA表達(dá)水平高于增強(qiáng)對(duì)照組，N-鈣黏蛋白、波狀蛋白、CD24mRNA表達(dá)水平低于增強(qiáng)對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見表3)。

Table 3 Comparison of the mRNA expression levels of E-cadherin,N-cadherin,corrugated protein,and CD24in H2228 cells in enhanced transfection group and enhanced control group

組別E-鈣黏蛋白mRNAN-鈣黏蛋白mRNA波狀蛋白mRNACD24mRNA增強(qiáng)對(duì)照組10±0210±0110±0110±03增強(qiáng)轉(zhuǎn)染組24±0102±0004±0102±01t值-146981597176165772P值0005000400170029

Table1ComparisonofcellproliferationlevelsofH2228undertheeffectofantineoplasticdrugs(crizotinib,paclitaxelandcisplatin)inenhancedtransfectiongroupandenhancedcontrolgroup

組別克唑替尼(nmol/L)50 100 200紫杉醇(nmol/L)625 1250 2500順鉑(nmol/L)25 50 100增強(qiáng)對(duì)照組608±30471±39324±12777±27586±55371±26752±18558±26341±03增強(qiáng)轉(zhuǎn)染組443±19368±17168±17627±17459±17246±25659±17379±33194±49t值59165752185037175576943897087173825026P值002700290003001900290048001900030037

Table2ComparisonofcellproliferationlevelsofH2228undertheeffectofantineoplasticdrugs(crizotinib,paclitaxelandcisplatin)ininhibitorytransfectiongroupandinhibitorycontrolgroup

組別克唑替尼(nmol/L)50 100 200紫杉醇(nmol/L)625 1250 2500順鉑(nmol/L)25 50 100抑制對(duì)照組592±45451±39312±05780±45613±30352±29754±45552±27347±12抑制轉(zhuǎn)染組736±17551±53385±24882±23753±31605±61917±23671±37494±49t值-8300-5399-5593-4629-4925-5347-4544-6962-5157P值001400330031004400390033004500200036

圖1 Transwell法檢測(cè)H2228細(xì)胞遷移率

2.5 增強(qiáng)轉(zhuǎn)染組、增強(qiáng)對(duì)照組H2228細(xì)胞中E-鈣黏蛋白、N-鈣黏蛋白、波狀蛋白、CD24表達(dá)水平 增強(qiáng)轉(zhuǎn)染組H2228細(xì)胞中E-鈣黏蛋白表達(dá)水平高于增強(qiáng)對(duì)照組，N-鈣黏蛋白、波狀蛋白、CD24表達(dá)水平低于增強(qiáng)對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見表4、圖2)。

Table 4 Comparison of the expression levels of E-cadherin,N-cadherin,corrugated protein,and CD24in H2228 cells in enhanced transfection group and enhanced control group

組別E-鈣黏蛋白N-鈣黏蛋白波狀蛋白CD24增強(qiáng)對(duì)照組10±0210±0110±0110±03增強(qiáng)轉(zhuǎn)染組21±0002±0004±0002±00t值-749311276100695778P值0017000800100029

圖2 Western blotting法檢測(cè)增強(qiáng)轉(zhuǎn)染組、增強(qiáng)對(duì)照組H2228細(xì)胞中E-鈣黏蛋白、N-鈣黏蛋白、波狀蛋白、CD24表達(dá)水平的SDS-PAGE圖

Figure 2 SDS-PAGE of the expression levels of E-cadherin,N-cadherin,corrugated protein,and CD24in H2228 cells in enhanced transfection group and enhanced control group by Western blotting method

3 討論

miRNA-200c是目前研究較多的微小RNA(microRNAs，簡(jiǎn)稱miRNAs)之一，miRNAs是一類進(jìn)化上高度保守的內(nèi)源性非編碼單鏈小RNA，其可以調(diào)節(jié)機(jī)體細(xì)胞的發(fā)育、分化、增殖、代謝及凋亡等關(guān)鍵環(huán)節(jié)[9]。多項(xiàng)研究表明，miR-200c與多種腫瘤相關(guān)，被認(rèn)為是具有治療潛力的腫瘤標(biāo)志物[10]，其不僅與肺癌的發(fā)生發(fā)展密切相關(guān)[11-12]，還可以增強(qiáng)腫瘤細(xì)胞對(duì)放療的敏感性[13-14]，同時(shí)增強(qiáng)多種化療藥物對(duì)腫瘤細(xì)胞的抑制作用，甚至增加分子靶向藥物EGFR-酪氨酸激酶抑制劑(TKI)對(duì)EGFR突變型肺癌的抗腫瘤活性[5-8]。本研究旨在檢測(cè)miR-200c 是否可以增加ALK-TKI藥物克唑替尼及化療藥物紫杉醇、順鉑對(duì)ALK陽(yáng)性肺癌細(xì)胞的抗腫瘤活性并進(jìn)一步探討其機(jī)制，為臨床應(yīng)用提供理論基礎(chǔ)。

本研究結(jié)果顯示，增強(qiáng)轉(zhuǎn)染組H2228細(xì)胞中miR-200c基因表達(dá)水平高于增強(qiáng)對(duì)照組，抑制轉(zhuǎn)染組H2228細(xì)胞中miR-200c基因表達(dá)水平低于抑制對(duì)照組，增強(qiáng)轉(zhuǎn)染組各濃度抗腫瘤藥物(克唑替尼、紫杉醇、順鉑)作用下H2228細(xì)胞增殖水平均低于增強(qiáng)對(duì)照組，抑制轉(zhuǎn)染組各濃度抗腫瘤藥物(克唑替尼、紫杉醇、順鉑)作用下H2228細(xì)胞增殖水平均高于抑制對(duì)照組，說明克唑替尼、紫杉醇及順鉑對(duì)高表達(dá)miR-200c細(xì)胞的抑制作用明顯增強(qiáng)，而對(duì)低表達(dá)miR-200c細(xì)胞的抑制作用則明顯減弱，表明miR-200c可以增加H2228細(xì)胞對(duì)分子靶向藥物克唑替尼及常規(guī)化療藥物紫杉醇和順鉑的敏感性，這與既往研究結(jié)果相似[5-7]。

有文獻(xiàn)報(bào)道，E-鈣黏蛋白是上皮細(xì)胞的特征性分子學(xué)標(biāo)志物，而N-鈣黏蛋白、波狀蛋白是間質(zhì)細(xì)胞的特征性分子學(xué)標(biāo)志物，通過調(diào)節(jié)這些分子學(xué)標(biāo)志物可以誘發(fā)細(xì)胞發(fā)生上皮及間質(zhì)之間的相互轉(zhuǎn)化[15]。CD24為腫瘤細(xì)胞上皮-間質(zhì)轉(zhuǎn)變(EMT)表型及干細(xì)胞表型的特征性分子標(biāo)志物[16]。本研究結(jié)果顯示，增強(qiáng)轉(zhuǎn)染組H2228細(xì)胞遷移率小于增強(qiáng)對(duì)照組，抑制轉(zhuǎn)染組H2228細(xì)胞遷移率大于抑制對(duì)照組，說明miR-200c抑制了細(xì)胞的遷移活性，表明過表達(dá)miR-200c后，細(xì)胞有間質(zhì)-上皮轉(zhuǎn)變(MET)可能，為進(jìn)一步證實(shí)，本研究組檢測(cè)了增強(qiáng)轉(zhuǎn)染組、增強(qiáng)對(duì)照組H2228細(xì)胞中E-鈣黏蛋白、N-鈣黏蛋白、波狀蛋白、CD24及其mRNA表達(dá)水平，結(jié)果顯示，增強(qiáng)轉(zhuǎn)染組H2228細(xì)胞中E-鈣黏蛋白及其mRNA表達(dá)水平高于增強(qiáng)對(duì)照組，N-鈣黏蛋白、波狀蛋白、CD24及其mRNA表達(dá)水平低于增強(qiáng)對(duì)照組。

多項(xiàng)研究顯示，腫瘤細(xì)胞對(duì)抗腫瘤藥物敏感性降低的過程有趨向間質(zhì)化的表現(xiàn)，通過對(duì)耐藥細(xì)胞的研究發(fā)現(xiàn)，多種耐藥細(xì)胞發(fā)生了EMT表現(xiàn)：阿霉素體外誘導(dǎo)乳腺癌細(xì)胞發(fā)生EMT，而且該細(xì)胞表現(xiàn)出侵襲轉(zhuǎn)移能力增強(qiáng)和多藥耐藥現(xiàn)象[17]，耐5-氟尿嘧啶的乳腺癌細(xì)胞株也觀察到了耐藥細(xì)胞的EMT現(xiàn)象[18]，此外，耐厄洛替尼、吉非替尼及克唑替尼的肺癌細(xì)胞也發(fā)生了不同性質(zhì)的EMT改變[19-21]。因此，逆轉(zhuǎn)癌細(xì)胞EMT的變化，可以增加癌細(xì)胞對(duì)化療藥物及分子靶向藥物的敏感性[5-6,21]。miR-200c最主要功能之一是調(diào)節(jié)細(xì)胞上皮及間質(zhì)轉(zhuǎn)變的病理生理過程即EMT和MET。EMT和MET是兩個(gè)可逆的、相反的過程，miR-200c通過直接調(diào)節(jié)E盒結(jié)合鋅指蛋白-1(ZEB1)及其下游基因E-鈣黏蛋白來調(diào)節(jié)EMT及MET之間的轉(zhuǎn)變，從而進(jìn)一步調(diào)節(jié)腫瘤多方面的生物學(xué)特征[22]。

本研究尚有不足之處，目前人工調(diào)節(jié)miRNA變化的體內(nèi)試驗(yàn)還鮮有報(bào)道[13]。miR-200c可以調(diào)節(jié)多種細(xì)胞因子而增加肺癌治療效果，但只是停留在細(xì)胞或動(dòng)物實(shí)驗(yàn)水平上，臨床上肺癌患者并無明顯受益，如何將miR-200c應(yīng)用于臨床或通過調(diào)節(jié)miR-200c上下通路而改變其表達(dá)尚待進(jìn)一步研究及論證，隨著miR-200c研究的進(jìn)一步深入，相信miR-200c將在腫瘤的早期診斷、治療及預(yù)后的判斷方面有更加廣泛的前景。

綜上所述，miR-200c通過使細(xì)胞發(fā)生MET改變而增加H2228細(xì)胞對(duì)克唑替尼、紫杉醇及順鉑的敏感性。

作者貢獻(xiàn)：高海祥進(jìn)行實(shí)驗(yàn)設(shè)計(jì)與實(shí)施、資料收集整理、撰寫論文并對(duì)文章負(fù)責(zé)；曹磊進(jìn)行實(shí)驗(yàn)實(shí)施、評(píng)估、質(zhì)量控制及審校。

本文無利益沖突。

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(本文編輯：崔麗紅)

Effect of Increased H2228 Cells through miR-200c on Sensitivity of Crizotinib,Paclitaxel and Cisplatin

GAO Hai-xiang,CAO Lei.

The First Department of Pulmonology Disease,Hebei General Hospital,Shijiazhuang 050051,China

GAO Hai-xiang,the First Department of Pulmonology Disease,Hebei General Hospital,Shijiazhuang 050051,China;E-mail:gaohaixiang0801@163.com

Background The miR-200c can increase the sensitivity of tumor cells on paclitaxel and cisplatin,and the sensitivity of positive lung carcinoma cells in epidermal growth factor receptor(EGFR) on molecular targeted drug such as erlotinib hydrochloride tablets,gefitinib and afatinib.However,there are no related studies on whether it can increase the sensitivity of echinodermata microtubule-associated protein-like 4(EML4)-anaplastic lymphoma kinase(ALK) fusion gene positive lung adenocarcinoma cells on crizotinib,paclitaxel and cisplatin.Objective To observe whether miR-200c can increase the sensitivity of H2228 cells on crizotinib,paclitaxel and cisplatin,and to explore its mechanism.Methods From February 2014 to May 2015,H2228 cells were cultured and transfected.According to the different transfectants,the cells were divided into enhanced transfection group(transfected with miR-200c mimics),enhanced control group(transfected miR-200c mimics negative control),inhibitory transfection group(transfected with miR-200c inhibitor) and inhibitory control group(transfected with miR-200c inhibitor negative control).The real-time fluorescence quantification PCR method was used to detect the expression level of miR-200c gene in H2228 cells,MTT assay was performed to measure the proliferation level of H2228 cells under antineoplastic drugs〔ketazidine(50,100,200 mmol/L),paclitaxel(6.25,12.50,25.00 mmol/L) and cisplatin(25,50,100 nmol/L)〕 of different concentrations,Transwell assay was used to measure the migration rate of H2228 cells,Real-time PCR method was applied to detect the expression level of E-cadherin,N-cadherin,corrugated protein and CD24mRNA in H2228 cells in transfection group and enhanced control group,Western blotting was used to measure the expression levels of E-cadherin,N-cadherin,corrugated protein and CD24in H2228 cells in enhanced transfection group and enhanced control group.Results The gene expression level of miR-200c in H2228 cells in enhanced transfection group was significantly higher than that in the enhanced control group(P<0.05).The gene expression level of miR-200c in H2228 cells in inhibitory transfection group was significantly lower than that in inhibitory control group(P<0.05).The cell proliferation level of H2228 under the effect of antineoplastic drugs of different concentrations in enhanced transfection group was lower than that in enhanced control group(P<0.05).The proliferation level of H2228 cells under the effect of the effect of antineoplastic drugs of different concentrations in inhibitory transfection group was higher than that in inhibitory control group(P<0.05).The migration rate of H2228 cells in enhanced transfection group was greater than that in enhanced control group(P<0.05).The migration rate of H2228 cells in inhibitory transfection group was greater than that in the inhibitory control group(P<0.05).The expression level of E-cadherin and its mRNA in H2228 cells in enhanced transfection group was higher than that in enhancedcontrol group,and the expression level of N-cadherin,corrugated protein,CD24and their mRNA was lower than that in enhanced control group(P<0.05).Conclusion Through the mesenchymal epithelial transiton in lung carcinoma cells,miR-200c can enhance the sensitivity of H2228 cells on crizotinib,paclitaxel and cisplatin.

Drug resistance,neoplasm;H2228;miR-200c;Crizotinib;Paclitaxel;Cisplatin

河北省2016年度醫(yī)學(xué)科學(xué)研究重點(diǎn)課題計(jì)劃項(xiàng)目(20160065)

050051 河北省石家莊市，河北省人民醫(yī)院呼吸一科(高海祥)，呼吸二科(曹磊)

高海祥，050051 河北省石家莊市，河北省人民醫(yī)院呼吸一科；E-mail：gaohaixiang0801@163.com

R 915 R 979.19

A

10.3969/j.issn.1007-9572.2016.36.015

2016-07-05；

2016-10-13)