下調(diào)TCF3抑制非小細(xì)胞肺癌細(xì)胞的增殖和遷移能力

王士猛

(山東省菏澤市立醫(yī)院胸外科，山東 菏澤 274000)

下調(diào)TCF3抑制非小細(xì)胞肺癌細(xì)胞的增殖和遷移能力

王士猛△

(山東省菏澤市立醫(yī)院胸外科，山東 菏澤 274000)

目的： 探索下調(diào)T細(xì)胞因子3(TCF3)抑制非小細(xì)胞肺癌細(xì)胞增殖和遷移的分子機(jī)制。方法: 運(yùn)用Lipofectamine 2000轉(zhuǎn)染法將siTCF3和陰性對(duì)照siRNA(NCsiRNA)轉(zhuǎn)染非小細(xì)胞肺癌A549和H1299細(xì)胞；運(yùn)用real-time PCR和Western blot分別測定TCF3的mRNA和蛋白水平；運(yùn)用螢光素酶報(bào)告基因?qū)嶒?yàn)測定TCF3的轉(zhuǎn)錄活性；MTT、克隆形成實(shí)驗(yàn)、Transwell實(shí)驗(yàn)和Annexin V-FITC/PI染色聯(lián)合流式細(xì)胞術(shù)分別測定細(xì)胞的活力、克隆形成能力、轉(zhuǎn)移能力及細(xì)胞凋亡率；Western blot檢測Wnt、c-Myc、基質(zhì)金屬蛋白酶(MMP)-9、MMP-13、金屬蛋白酶組織抑制物(TIMP)-1的蛋白表達(dá)水平。結(jié)果: 與NCsiRNA轉(zhuǎn)染組的細(xì)胞比較，siTCF3顯著抑制A549細(xì)胞和H1299細(xì)胞中TCF3的mRNA 和蛋白水平 (P<0.01)。TCF3轉(zhuǎn)錄活性和c-Myc蛋白表達(dá)水平明顯低于NCsiRNA細(xì)胞(P<0.05)。MTT實(shí)驗(yàn)結(jié)果顯示，培養(yǎng)24 h、48 h、72 h和96 h的A549-siTCF3和H1299-siTCF3細(xì)胞活力均顯著低于NCsiRNA細(xì)胞(P<0.05)。與NCsiRNA細(xì)胞相比，siTCF3顯著抑制A549細(xì)胞 和H1299細(xì)胞的克隆形成能力(P<0.01)。Transwell實(shí)驗(yàn)結(jié)果顯示A549-siTCF3和H1299-siTCF3細(xì)胞遷移數(shù)顯著低于A549-NCsiRNA和H1299-NCsiRNA組細(xì)胞(P<0.05)。流式細(xì)胞術(shù)分析結(jié)果顯示A549-siTCF3細(xì)胞和H1299-siTCF3細(xì)胞的凋亡率顯著高于A549-NCsiRNA和H1299-NCsiRNA細(xì)胞(P<0.01)。Western blot實(shí)驗(yàn)結(jié)果顯示，下調(diào)TCF3表達(dá)能抑制Wnt蛋白的表達(dá)，MMP-9和MMP-13的蛋白表達(dá)明顯降低，TIMP-1的蛋白表達(dá)增高。結(jié)論: siTCF3顯著抑制A549細(xì)胞和H1299細(xì)胞的增殖和遷移能力，并誘導(dǎo)細(xì)胞凋亡，其分子機(jī)制可能通過下調(diào)Wnt通路活性以及調(diào)控MMP家族關(guān)鍵成員的表達(dá)而實(shí)現(xiàn)。

非小細(xì)胞肺癌； T細(xì)胞因子3； 基質(zhì)金屬蛋白酶

肺癌已成為嚴(yán)重威脅人類健康與生命最常見的惡性腫瘤之一，其發(fā)病率和死亡率位居所有癌癥之首[1]。非小細(xì)胞肺癌(non-small cell lung cancer，NSCLC)是肺癌的主要類型，約占肺癌的80%～85%[2]。目前放療是NSCLC的主要治療方案，但NSCLC對(duì)放療產(chǎn)生耐受是導(dǎo)致預(yù)后不良和五年生存率低的主要原因[3]。因此，尋找和研發(fā)治療NSCLC的小分子靶向藥物是亟需解決的問題，已成為治療肺癌的重要課題。

T細(xì)胞因子3(T-cell factor 3，TCF3)是經(jīng)典Wnt信號(hào)通路的效應(yīng)分子之一。早期的研究發(fā)現(xiàn)TCF3基因缺失導(dǎo)致早期胚胎發(fā)育致死，表現(xiàn)為神經(jīng)和中胚層發(fā)育缺陷[4]。TCF3過表達(dá)能刺激人皮膚毛囊隆突表皮原始祖細(xì)胞分裂并抑制細(xì)胞分化。研究發(fā)現(xiàn)TCF3與Oct4、Sox2、Nanog基因啟動(dòng)子區(qū)域結(jié)合,拮抗這些基因的作用進(jìn)而抑制鼠胚胎干細(xì)胞的自我更新[5-6]。Wnt通路激活使TCF3從相應(yīng)的基因啟動(dòng)子上脫離，激活靶基因轉(zhuǎn)錄從而增強(qiáng)多能干細(xì)胞的分裂活性并延遲對(duì)分化刺激的應(yīng)答[7]。最近研究表明TCF3對(duì)腎癌、乳腺癌、胚胎癌等人類腫瘤的發(fā)生起著抑制或促進(jìn)作用[8]。目前TCF3的功能在肺癌中的研究甚少[9]，近新研究發(fā)現(xiàn)TCF3在肺癌病人手術(shù)切除的癌變組織及幾種NSCLC細(xì)胞系中均有表達(dá)，在正常16HBE細(xì)胞中亦有表達(dá)，而在癌旁組織中無表達(dá)。但TCF3對(duì)肺癌發(fā)生、發(fā)展是促進(jìn)還是抑制作用尚不清楚。本研運(yùn)用siRNA干擾技術(shù)下調(diào)人肺腺癌細(xì)胞系A(chǔ)549細(xì)胞和H1299細(xì)胞中TCF3的表達(dá)，體外研究下調(diào)TCF3表達(dá)對(duì)肺腺癌細(xì)胞增殖和遷移的影響及其可能的分子機(jī)制，為以TCF3為靶點(diǎn)研發(fā)治療肺癌的靶向藥物提供理論依據(jù)。

材 料 和 方 法

1 試劑和材料

非小細(xì)胞肺癌細(xì)胞系A(chǔ)549和H1299購自ATCC；MTT、DMSO和胰蛋白酶購自Sigma；DMEM培養(yǎng)基(Gibco)；胎牛血清和雙抗購自北京金博益生物技術(shù)有限公司；Annexin V-FITC/PI細(xì)胞凋亡檢測試劑盒購自南京凱基生物科技發(fā)展有限公司；TOP-Flash/FOP-Flash質(zhì)粒購自Upstate；Lipofectamine 2000轉(zhuǎn)染試劑購自Invitrogen；Dual-Luciferase Reporter Assay System購自Promega；Transwell板購自Corning；抗c-Myc和GAPDH抗體購自Abcam；抗CTF3、基質(zhì)金屬蛋白酶(matrix metalloproteinase，MMP)-9、MMP-13和金屬蛋白酶組織抑制物1(tissue inhibitor of metalloproteinase-1，TIMP-1)抗體購自Cell Signaling Technology；辣根過氧化物酶(HRP)標(biāo)記的IgG II 抗購自北京中杉金橋公司；Total RNA 提取試劑、反轉(zhuǎn)錄試劑盒(PrimeScript?RT reagent Kit with gDNA Eraser)和real-time PCR 試劑盒(SYBR?Premix Ex TaqTMⅡ)購自 TaKaRa；BCA蛋白濃度檢測試劑盒購自Thermo。

2 主要方法

2.1 細(xì)胞培養(yǎng) A549細(xì)胞和H1299細(xì)胞培養(yǎng)于DMEM培養(yǎng)基(含10% 胎牛血清、1%青霉素和1%鏈霉素)中，置于37 ℃、5% CO2及完全濕度培養(yǎng)箱內(nèi)，待細(xì)胞融合度達(dá)到80%～90%時(shí)進(jìn)行細(xì)胞傳代。

2.2 siRNA合成和細(xì)胞轉(zhuǎn)染 針對(duì)TCF3基因開放閱讀框序列，運(yùn)用Ambion的網(wǎng)上在線軟件(http://www.ambion.com/techlib/misc/siRNA_finder.html)設(shè)計(jì)siTCF3，同時(shí)設(shè)計(jì)陰性對(duì)照siRNA (negative control siRNA, NCsiRNA)，由上海生工合成。將生長良好的A549細(xì)胞和H1299細(xì)胞以細(xì)胞密度為每孔2×105個(gè)接種到6孔板中，培養(yǎng)至細(xì)胞融合度達(dá)到85%左右時(shí)按照Lipofectamine 2000說明書進(jìn)行轉(zhuǎn)染。先用無血清OptiMEM培養(yǎng)液將Lipofectamine 2000稀釋和配置siTCF3，然后將等體積Lipofectamine 2000與siTCF3輕輕混勻，室溫靜置10 min后加入細(xì)胞中使siTCF3終濃度為30 μmol/L，培養(yǎng)4 h后換成完全培養(yǎng)基，以轉(zhuǎn)染NCsiRNA為陰性對(duì)照，置于培養(yǎng)箱中培養(yǎng)用于后續(xù)實(shí)驗(yàn)。

2.3 Real-time PCR 轉(zhuǎn)染的細(xì)胞培養(yǎng)48 h后收集細(xì)胞，按照TRIzol試劑說明書提取總RNA，濃度測定并保存于-80 ℃。按照RT-PCR試劑盒說明書合成cDNA，反應(yīng)條件為42 ℃ 1 h、70 ℃ 5 min；4 ℃放置10 min。cDNA置于-80 ℃保存。取2 ng cDNA進(jìn)行real-time PCR反應(yīng)，擴(kuò)增引物見表1。反應(yīng)條件為95 ℃ 5 min；95 ℃ 30 s、58 ℃ 30 s、72 ℃ 40 s，35個(gè)循環(huán)；72 ℃ 10 min。運(yùn)用 SYBR Green Ⅱ 熒光染料法和IQ5TMReal-time PCR Detection System (Bio-Rad)進(jìn)行real-time PCR數(shù)據(jù)分析，結(jié)果經(jīng)內(nèi)參照GAPDH校正，mRNA相對(duì)表達(dá)量用 2-ΔΔCt表示。

表1 Real-time PCR引物序列

2.4 螢光素酶報(bào)告基因檢測 將轉(zhuǎn)染的A549和H1299細(xì)胞經(jīng)胰酶消化，計(jì)數(shù)后以每孔5×104個(gè)接種于24孔板中，待細(xì)胞融合度為70%左右時(shí)進(jìn)行細(xì)胞轉(zhuǎn)染。按照Lipofectamine 2000說明書將200 ng TOP-Flash報(bào)告質(zhì)粒和10 ng FOP-Flash海腎螢光素酶對(duì)照質(zhì)粒共轉(zhuǎn)染細(xì)胞，48 h后收集細(xì)胞。加入1×被動(dòng)裂解緩沖液重懸細(xì)胞，置于4 ℃裂解24 h，12 000 r/min，4 ℃離心10 min，收集上清并測定細(xì)胞上清中螢光素酶的活性。以海腎螢光素酶活性為內(nèi)參照計(jì)算樣品中螢火蟲螢光素酶的活性。

2.5 MTT實(shí)驗(yàn)測定細(xì)胞活力 將轉(zhuǎn)染的A549細(xì)胞和H1299細(xì)胞經(jīng)胰酶消化后，以每孔2.5×104個(gè)接種于96孔板中，于培養(yǎng)箱中分別在24 h、48 h、72 h、96 h時(shí)進(jìn)行MTT實(shí)驗(yàn)，每孔加入20 μL(5 g/L)MTT，37 ℃繼續(xù)培養(yǎng)4 h，棄掉培養(yǎng)液，每孔加入150 μL DMSO，置于搖床上室溫振蕩5 min，用酶標(biāo)儀測定492 nm處的吸光度(A)值，按下式計(jì)算細(xì)胞活力抑制率(%)=(對(duì)照組A均值-實(shí)驗(yàn)組A均值)/實(shí)驗(yàn)組A均值×100%。

2.6 克隆形成實(shí)驗(yàn) 轉(zhuǎn)染的A549細(xì)胞和H1299細(xì)胞經(jīng)胰酶消化后計(jì)數(shù)，細(xì)胞以每皿500個(gè)/接種于90 mm細(xì)胞培養(yǎng)皿中，置于37 ℃、5% CO2培養(yǎng)箱中培養(yǎng)，設(shè)3個(gè)平行皿，置于37 ℃、5% CO2孵箱中培養(yǎng)，直到通過出現(xiàn)肉眼能清晰觀察到可見的細(xì)胞克隆(約2周)；棄掉培養(yǎng)基，PBS洗滌，4%甲醛固定10 min，PBS洗滌，吉姆薩染色10 min，自來水清洗，晾干后計(jì)數(shù)，按下列公式計(jì)算細(xì)胞克隆形成率=(細(xì)胞克隆數(shù)平均值/鋪板細(xì)胞總數(shù))×100%。

2.7 細(xì)胞遷移實(shí)驗(yàn) 轉(zhuǎn)染的A549細(xì)胞和H1299細(xì)胞經(jīng)胰酶消化，然后在Transwell每個(gè)培養(yǎng)孔上室中加入2×104個(gè)細(xì)胞，下室為無血清培養(yǎng)基，設(shè)置空白對(duì)照組；37 ℃繼續(xù)培養(yǎng)6 h后，經(jīng)4%多聚甲醛固定、乙醇脫水、結(jié)晶紫染色、洗滌。用棉簽輕輕擦去上室的貼壁細(xì)胞，在顯微鏡下拍照并計(jì)數(shù)從Transwell上室遷移至微孔膜下層的細(xì)胞，每組設(shè)3個(gè)重復(fù)孔，通過每組Transwell的遷移細(xì)胞數(shù)比較細(xì)胞的遷移能力。

2.8 流式細(xì)胞術(shù)測定細(xì)胞凋亡 轉(zhuǎn)染的A549細(xì)胞和H1299細(xì)胞經(jīng)消化后以細(xì)胞密度為每孔3×104個(gè)接種接種到6孔板中，培養(yǎng)72 h后棄掉培養(yǎng)基，用PBS洗滌3次，無EDTA的胰酶消化，離心收集細(xì)胞，按照Annexin V/PI試劑盒說明書操作，先加入500 μL的binding buffer重懸細(xì)胞，再加入5 μL FITC標(biāo)記的Annexin V和5 μL PI混勻，室溫下避光孵育15 min，運(yùn)用流式細(xì)胞儀測定細(xì)胞凋亡。

2.9 Western blot實(shí)驗(yàn) 轉(zhuǎn)染的A549細(xì)胞和H1299細(xì)胞培養(yǎng)48 h。收集細(xì)胞，加入RIPA重懸細(xì)胞，超聲破碎、12 000 r/min，4 ℃離心10 min，按照BCA試劑盒說明書測定總蛋白濃度。每個(gè)樣本取30 μg進(jìn)行SDS-PAGE，將蛋白轉(zhuǎn)移到硝酸纖維素膜上，5%脫脂奶粉室溫封閉1 h，分別孵育 Ⅰ 抗(Wnt、c-Myc、CTF3、MMP-9、MMP-13和TIMP-1)，β-actin為內(nèi)參照，4 ℃過夜。TBST洗膜、孵育 II 抗，室溫1 h。ECL顯影后掃描，蛋白相對(duì)表達(dá)量經(jīng)內(nèi)參照歸一化后經(jīng)Quanity One軟件分析。

3 統(tǒng)計(jì)學(xué)處理

每個(gè)實(shí)驗(yàn)進(jìn)行3次獨(dú)立重復(fù)試驗(yàn)。應(yīng)用SPSS 17.0統(tǒng)計(jì)軟件進(jìn)行相關(guān)數(shù)據(jù)分析，結(jié)果用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，多組間的比較采用單因素方差分析(one-way ANOVA)，組間兩兩比較采用LSD法。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 A549和H1299細(xì)胞中TCF3的mRNA及蛋白表達(dá)情況

Real-time PCR結(jié)果顯示，siTCF3顯著抑制A549細(xì)胞和H1299細(xì)胞中TCF3 mRNA的表達(dá)顯著低于陰性對(duì)照組(P<0.01)。Western blot實(shí)驗(yàn)結(jié)果進(jìn)一步顯示，siTCF3明顯下調(diào)A549細(xì)胞和H1299細(xì)胞TCF3蛋白的表達(dá)，與陰性對(duì)照組比較差異具有統(tǒng)計(jì)學(xué)意義(P<0.01)，見圖1。

2 Wnt通路的轉(zhuǎn)錄活性及其靶基因c-Myc的蛋白表達(dá)水平

TOP-Flash/FOP-Flash雙螢光素酶報(bào)告基因結(jié)果顯示，與陰性對(duì)照組比較，siTCF3轉(zhuǎn)染組細(xì)胞的TOP-Flash/FOP-Flash比值明顯降低(P<0.01)，提示TCF3可能具有促進(jìn)轉(zhuǎn)錄調(diào)節(jié)的作用。Western blot實(shí)驗(yàn)結(jié)果顯示，與陰性對(duì)照組比較，siTCF3轉(zhuǎn)染組細(xì)胞的c-Myc蛋白表達(dá)量明顯降低(P<0.01)，見圖2、表2。

3 干擾TCF3表達(dá)對(duì)肺癌細(xì)胞增殖的影響

MTT實(shí)驗(yàn)結(jié)果顯示，轉(zhuǎn)染siTCF3的A549和H1299細(xì)胞培養(yǎng)24 h、48 h、72 h和96 h的細(xì)胞活力分別為(96.26±15.23)%、(78.93±11.27)%、(56.32±6.58)%、(43.54±5.52)%和(83.74±16.20)%、(65.31±9.52)%、(57.00±7.33)%、(36.90±4.38)%。除了24 h的A549細(xì)胞外，各時(shí)點(diǎn)的細(xì)胞活力顯著低于陰性對(duì)照組(P<0.05)，提示下調(diào)TCF3的表達(dá)能抑制A549細(xì)胞和H1299細(xì)胞的活力，見圖3。為了進(jìn)一步證明下調(diào)TCF3能抑制肺癌細(xì)胞增殖，我們運(yùn)用克隆形成實(shí)驗(yàn)發(fā)現(xiàn)下調(diào)TCF3表達(dá)能抑制A549和H1299細(xì)胞的克隆形成，與陰性對(duì)照組比較，siTCF3顯著抑制A549和H1299細(xì)胞的克隆形成能力，其克隆形成率分別為(0.58±0.03)和(0.24±0.01)，與對(duì)照組比較差異具有統(tǒng)計(jì)學(xué)意義(P<0.01)，見圖4。

Figure 1.The expression of TCF3 at mRNA and protein levels in the lung cancer cells with RNA interference. Mean±SD.n=3.**P<0.01vsNCsiRNA group.

圖1 RNA干擾肺癌細(xì)胞中TCF3 mRNA和蛋白表達(dá)

Figure 2.The effect of down-regulated TCF3 expression on the transcriptional activity of Wnt pathway and the expression of target gene c-Myc protein. A: the detection of transcriptional activity in Wnt pathway by TOP-Flash/FOP-Flash; B: the protein expression of c-Myc detected by Western blot. Mean±SD.n=3.**P<0.01vsNCsiRNA group.

圖2 下調(diào)TCF3表達(dá)對(duì)Wnt通路轉(zhuǎn)錄活性及靶基因c-Myc蛋白表達(dá)的影響

表2 Wnt通路的轉(zhuǎn)錄活性及Wnt靶基因c-Myc的蛋白表達(dá)水平

**P<0.01vsNCsiRNA group.

Figure 3.The effect of TCF3 down-regulation on the viability of lung cancer cells. Mean±SD.n=3.*P<0.05,**P<0.01vsNCsiRNA group.

圖3 下調(diào)TCF3的表達(dá)對(duì)肺癌細(xì)胞活力的影響

Figure 4.The effect of down-regulation of TCF3 expression on the colony formation ability of lung cancer A549 cells and H1299 cells. Mean±SD.n=3.**P<0.01vsNCsiRNA group.

圖4 下調(diào)TCF3的表達(dá)對(duì)肺癌細(xì)胞克隆形成能力的影響

4 干擾TCF3表達(dá)對(duì)肺癌細(xì)胞遷移能力的影響

Transwell實(shí)驗(yàn)結(jié)果顯示對(duì)照組細(xì)胞轉(zhuǎn)移通過肌質(zhì)膜的數(shù)量明顯增多，而siTCF3轉(zhuǎn)染的A549細(xì)胞和H1299細(xì)胞遷移至下室的細(xì)胞數(shù)顯著降低，與對(duì)照組比較差異具有統(tǒng)計(jì)學(xué)意義(P<0.05),見圖5。

Figure 5.The effect of TCF3 down-regulation on the migratory ability of lung cancer A549 cells and H1299 cells. Mean±SD.n=3.**P<0.01vsNCsiRNA group.

圖5 下調(diào)TCF3表達(dá)對(duì)A549和H1299遷移的影響

5 下調(diào)TCF3表達(dá)對(duì)肺癌細(xì)胞凋亡的影響

流式細(xì)胞術(shù)分析結(jié)果顯示在TCF3下調(diào)的A549細(xì)胞和H1299細(xì)胞中，其細(xì)胞凋亡率明顯增高，分別為(31.40±4.12)%和(39.52±3.58)%，與陰性對(duì)照組比較差異具有統(tǒng)計(jì)學(xué)意義，提示下調(diào)TCF3的表達(dá)能誘導(dǎo)A549和H1299細(xì)胞凋亡，見圖6。

Figure 6.The effect of TCF3 down-regulation on the apoptosis of lung cancer A549 cells and H1299 cells. Mean±SD.n=3.**P<0.01vsNCsiRNA group.

圖6 下調(diào)TCF3表達(dá)對(duì)A549和H1299細(xì)胞凋亡的影響

6 下調(diào)TCF3表達(dá)對(duì)肺癌細(xì)胞Wnt通路相關(guān)蛋白水平的影響

Western blot結(jié)果發(fā)現(xiàn)，下調(diào)TCF3的表達(dá)后，A549和H1299細(xì)胞中的Wnt、MMP-9和MMP-13的相對(duì)表達(dá)量顯著低于NCsiRNA細(xì)胞；TIMP-1蛋白的相對(duì)表達(dá)量顯著高于NCsiRNA細(xì)胞，見圖7。

Figure 7.The effect of TCF3 down-regulation on the expression of Wnt pathway-related proteins in lung cancer A549 cells and H1299 cells. Mean±SD.n=3.**P<0.01vsNCsiRNA group.

圖7 下調(diào)TCF3的表達(dá)對(duì)Wnt通路相關(guān)蛋白水平的影響

討 論

Wnt/β-catenin信號(hào)通路是哺乳動(dòng)物細(xì)胞內(nèi)高度保守的關(guān)鍵信號(hào)通路，其與肺癌的發(fā)生發(fā)展密切相關(guān)[10]。TCF3是TCF/LEF家族的重要成員之一，是經(jīng)典Wnt/β-catenin信號(hào)通路的關(guān)鍵成分。研究表明TCF3在一些分化程度較低的惡性腫瘤中表達(dá)[11]，但目前對(duì)TCF3在惡性腫瘤中的作用及機(jī)制研究較少。Lin等[11]證實(shí)TCF3可能通過下調(diào)Oct4抑制胚胎癌細(xì)胞的惡性表型。TCF3對(duì)乳腺癌組織的形成及維持癌細(xì)胞生長起著重要作用，但對(duì)乳腺癌細(xì)胞的侵襲及遷移能力無明顯影響[10]。然而，TCF3在肺癌中的作用及分子機(jī)制仍不清楚，相關(guān)的研究報(bào)道較少。

研究證實(shí)，在無Wnt信號(hào)刺激時(shí)TCF3與共阻遏因子Groucho、CtBP和HDAC等結(jié)合抑制Wnt基因轉(zhuǎn)錄[12-13]；異常的Wnt信號(hào)刺激下TCF3一方面作為β-catenin依賴的轉(zhuǎn)錄激活因子促進(jìn)Wnt靶基因的轉(zhuǎn)錄激活，同時(shí)也能作為轉(zhuǎn)錄抑制因子下調(diào)Wnt靶基因的轉(zhuǎn)錄[14]。本研究結(jié)果顯示，干擾肺癌細(xì)胞系A(chǔ)549細(xì)胞和H1299細(xì)胞中的TCF3表達(dá)后，Wnt通路轉(zhuǎn)錄活性明顯降低，其下游靶基因c-Myc的表達(dá)明顯下調(diào)，提示TCF3在肺癌細(xì)胞的Wnt通路中起著轉(zhuǎn)錄激活的功能?？赡艿臋C(jī)制為Wnt通路激活時(shí)，大量游離的β-catenin進(jìn)入細(xì)胞核，通過與Groucho/TLE競爭結(jié)合TCF3，將TCF3轉(zhuǎn)換為轉(zhuǎn)錄激活因子[15]。我們通過MTT、克隆形成實(shí)驗(yàn)證實(shí)TCF3對(duì)肺癌細(xì)胞的增殖活性的影響，結(jié)果發(fā)現(xiàn)降低TCF3的表達(dá)使A549細(xì)胞和H1299細(xì)胞的活力和克隆形成率顯著降低。為了進(jìn)一步探討TCF3影響A549和H1299細(xì)胞增殖的原因，我們運(yùn)用Annexin V-FITC/PI染色和流式細(xì)胞術(shù)進(jìn)行了細(xì)胞凋亡實(shí)驗(yàn)，結(jié)果顯示干擾TCF3表達(dá)72 h后細(xì)胞的凋亡率明顯升高。推測TCF3可能通過調(diào)控Wnt通路下游勘基因c-Myc[16]等細(xì)胞凋亡相關(guān)基因而影響肺癌細(xì)胞的生長。

基質(zhì)金屬蛋白酶基因家族在腫瘤細(xì)胞轉(zhuǎn)移過程中發(fā)揮關(guān)鍵作用[17]，MMP基因家族成員的表達(dá)與惡性腫瘤的高轉(zhuǎn)移活性密切相關(guān)[18]。研究證實(shí)MMP-1過表達(dá)能促進(jìn)前列腺癌的增殖和轉(zhuǎn)移[19]。本研究觀察到下調(diào)TCF3表達(dá)顯著抑制A549細(xì)胞和H1299細(xì)胞的遷移，由此我們推測TCF3可能通過調(diào)控MMP家族基因的表達(dá)影響肺癌細(xì)胞的轉(zhuǎn)移。我們?cè)贏549和H1299細(xì)胞中的研究發(fā)現(xiàn)，siTCF3顯著抑制MMP-9和MMP-13蛋白的表達(dá)，促進(jìn)基質(zhì)金屬蛋白酶抑制因子TIMP-1的蛋白表達(dá)，提示siTCF3可能通過抑制MMP-9和MMP-13的表達(dá)抑制A549和H1299細(xì)胞的遷移能力。Western blot實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)下調(diào)TCF3表達(dá)抑制Wnt蛋白的表達(dá)，但是本研究沒有進(jìn)一步證實(shí)MMP-9、MMP-13和TIMP-1表達(dá)與Wnt信號(hào)通路的關(guān)系，MMP家族成員中MMP-9、MMP-13和TIMP-1是否是Wnt通路靶基因？或者M(jìn)MP通路和Wnt通路形成一種交叉網(wǎng)絡(luò)共同調(diào)控肺癌細(xì)胞轉(zhuǎn)移，這方面工作值得進(jìn)一步研究。我們的研究發(fā)現(xiàn)，siTCF3顯著抑制A549和H1299細(xì)胞的增殖、遷移和誘導(dǎo)細(xì)胞凋亡，其分子機(jī)制可能通過下調(diào)Wnt通路活性以及調(diào)控MMP家族關(guān)鍵成員的表達(dá)而實(shí)現(xiàn)。提示TCF3有望成為治療肺癌的潛在靶向藥物，本研究為以TCF3為靶點(diǎn)研發(fā)治療肺癌的分子靶向藥物提供理論依據(jù)。

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(責(zé)任編輯： 陳妙玲， 羅 森)

Down-regulated TCF3 expression inhibits growth and migratory abilities of non-small cell lung cancer cells

WANG Shi-meng

(DepartmentofThoracicSurgery,HezeMunicipalHospital,Heze274000,China.E-mail: 48947925@qq.com)

AIM: To investigate the effects of T-cell factor 3 (TCF3) expression on the growth and migratory abilities of the non-small cell lung cancer cells (NSCLC) and the underlying mechanisms. METHODS: The non-small cell lung cancer cells A549 and H1299 were transfected with siTCF3 or negative control siRNA (NCsiRNA) using Lipofectamine 2000. The expression of TCF3 at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The activity of TCF3 transcription was measured using luciferase reporter gene assay. The cell viability, colony formation ability, migratory ability and apoptotic rate were monitored by MTT assay, colony formation assay, Transwell method and flow cytometry with Annexin V-FITC/PI double staining, respectively. The protein expression levels of Wnt, c-Myc, matrix metalloproteinase (MMP)-9, MMP-13 and tissue inhibitor of metalloproteinase-1 (TIMP-1) were detected by Western blot. RESULTS: Compared with A549-NCsiRNA and H1299-NCsiRNA cells, the mRNA and protein levels of TCF3 were significantly inhibited in A549-siTCF3 and H1299-siTCF3 cells (P<0.01). The activity of TCF3 transcription and levels of c-Myc protein expression remarkably decreased as compared with NCsiRNA cells (P<0.05). The viability of A549-siTCF3 cells and H1299-siTCF3 cells cultured for 24 h, 48 h, 72 h and 96 h were notably lower than that of NCsi-RNA cells (P<0.05). The colony formation ability was significantly inhibited by siTCF3 compared with NCsiRNA cells (P<0.01). The numbers of migratory A549-siTCF3 and H1299-siTCF3 cells were significantly lower than those of A549-NCsiRNA and H1299-NCsiRNA cells (P<0.05). The apoptotic rates of A549-siTCF3 cells and H1299-siTCF3 cells were notably higher than those of A549-NCsiRNA cells and H1299-NCsiRNA cells (P<0.01). Down-regulated TCF3 expression inhibited the protein expression of Wnt, decreased the protein levels of MMP-9 and MMP-13 and enhanced the protein level of TCF3. CONCLUSION: siTCF3 suppresses the abilities of growth and migration, and induces apoptosis in A549 cells and H1299 cells by down-regulating Wnt pathway and regulating expression of key MMP family genes.

Non-small cell lung cancer; T-cell factor 3; Matrix metalloproteinases

1000- 4718(2017)02- 0289- 08

2016- 08- 11

2016- 10- 09

R730.23

A

10.3969/j.issn.1000- 4718.2017.02.016

雜志網(wǎng)址： http://www.cjpp.net

△通訊作者 Tel: 0530-5613311; E-mail: 48947925 @qq.com