王星，姜立，張沄，陳肖燕

(1.西南醫(yī)科大學(xué)附屬醫(yī)院炎癥與變態(tài)反應(yīng)實(shí)驗(yàn)室，四川瀘州646000；2.廣東省人民醫(yī)院醫(yī)學(xué)研究中心，廣東廣州510080)

OAZ1基因穩(wěn)定表達(dá)SCC15細(xì)胞株的構(gòu)建及其對(duì)舌鱗狀細(xì)胞癌上皮間質(zhì)化的影響

王星1，姜立2，張沄1，陳肖燕2

(1.西南醫(yī)科大學(xué)附屬醫(yī)院炎癥與變態(tài)反應(yīng)實(shí)驗(yàn)室，四川瀘州646000；2.廣東省人民醫(yī)院醫(yī)學(xué)研究中心，廣東廣州510080)

目的探索鳥(niǎo)氨酸脫羧酶抗酶(OAZ1)對(duì)舌鱗癌細(xì)胞株SCC15上皮間質(zhì)化的影響及相關(guān)機(jī)制。方法構(gòu)建框移位點(diǎn)突變的OAZ1過(guò)表達(dá)慢病毒表達(dá)載體pLVX-Neo-OAZ1-IRES-ZsGreen，包裝病毒并感染SCC15細(xì)胞，采用有限稀釋法挑取熒光強(qiáng)度較高的單克隆細(xì)胞株；利用RT-PCR和Western blot檢測(cè)OAZ1以及上皮間質(zhì)化相關(guān)基因(E-cadherin、Vimentin、N-cadherin、Zeb1、Snail、Tw ist1、TGF-β1、Smad1)的mRNA水平和蛋白水平。結(jié)果成功構(gòu)建了OAZ1基因穩(wěn)定表達(dá)的SCC15細(xì)胞株，且OAZ1的過(guò)表達(dá)可以抑制Vimentin、N-cadherin、TGF-β1的mRNA和蛋白水平表達(dá)；此外，OAZ1可以促進(jìn)Smad1的蛋白水平降低。結(jié)論OAZ1可以抑制舌鱗癌細(xì)胞的上皮間質(zhì)化，其機(jī)制可能涉及TGF-β1信號(hào)通路和Smad1信號(hào)通路的抑制。

鳥(niǎo)氨酸脫羧酶抗酶；舌磷狀細(xì)胞癌；慢病毒；上皮間質(zhì)化

鳥(niǎo)氨酸脫羧酶抗酶(OAZ1)最早發(fā)現(xiàn)其可與鳥(niǎo)氨酸脫羧酶(ODC)結(jié)合，并靶向使之被26S蛋白酶體降解，從而減少細(xì)胞內(nèi)多胺的水平，影響基因的復(fù)制、轉(zhuǎn)錄以及細(xì)胞的增殖[1]。更多研究表明，作為抑癌基因，OAZ1的功能具有多樣性，其不僅可通過(guò)誘導(dǎo)CyclinD1、Mps1的非泛素化降解影響細(xì)胞增殖以及細(xì)胞的遺傳穩(wěn)定性[2-3]；此外，OAZ1還具有誘導(dǎo)細(xì)胞分化與凋亡的作用[4-5]。

舌鱗狀細(xì)胞癌(TSCC)是口腔癌中最常見(jiàn)的惡性腫瘤，一般惡性程度高，生長(zhǎng)快，浸潤(rùn)性強(qiáng)，轉(zhuǎn)移早，預(yù)后差，復(fù)發(fā)率高[6]。近年的統(tǒng)計(jì)資料顯示，舌鱗狀細(xì)胞癌的發(fā)病率呈現(xiàn)明顯上升的趨勢(shì)，并且有發(fā)病低齡化的傾向[7]。上皮間質(zhì)化(EMT)是指上皮細(xì)胞經(jīng)轉(zhuǎn)分化(即由高分化狀態(tài)向低分化狀態(tài)轉(zhuǎn)化)變?yōu)榫哂虚g質(zhì)細(xì)胞表型的生物學(xué)現(xiàn)象，其不僅可使腫瘤細(xì)胞獲得遷移和侵襲特征，還可使腫瘤細(xì)胞獲得干細(xì)胞特性[8]。EMT是上皮來(lái)源的舌鱗狀細(xì)胞癌發(fā)生轉(zhuǎn)移的關(guān)鍵啟動(dòng)步驟，參與舌鱗狀細(xì)胞癌侵襲轉(zhuǎn)移[9]。大量研究表明，高分化舌鱗狀細(xì)胞癌的預(yù)后較低分化鱗狀細(xì)胞癌更好[10]，且在多種舌鱗癌細(xì)胞系中，OAZ1的表達(dá)水平降低[11]。進(jìn)一步研究表明，在舌鱗癌細(xì)胞中異位表達(dá)外源OAZ1可誘導(dǎo)細(xì)胞分化[12]。然而，OAZ1是否可以通過(guò)誘導(dǎo)舌鱗癌細(xì)胞分化抑制EMT尚不明確。本研究應(yīng)用慢病毒將OAZ1框移位點(diǎn)突變基因?qū)隨CC15細(xì)胞，獲得OAZ1基因穩(wěn)定表達(dá)的SCC15細(xì)胞株，并進(jìn)一步探索OAZ1對(duì)EMT的影響。

1 材料與方法

1.1 材料DMEM/F12培養(yǎng)基；胎牛血清(Gibco,USA)；Lentivirus質(zhì)粒系統(tǒng)由深圳百恩微生物公司提供；限制性內(nèi)切酶Eco RⅠ、XbaⅠ，T4DNA Ligase購(gòu)自美國(guó)NEB公司；OAZ1,Vimentin,N-cadherin抗體購(gòu)至abcam公司；TGF-β1，Smad1抗體購(gòu)至Santa Crus公司；G418為Merck公司產(chǎn)品；PrimeScript RT試劑盒、SYBR Premix E×Taq、RNAisoPlus、RNA free dd H2O購(gòu)自TaKaRa公司。

1.2 方法

1.2.1 慢病毒表達(dá)載體的構(gòu)建本實(shí)驗(yàn)選擇含有OAZ1基因框移位點(diǎn)缺失突變的質(zhì)粒pET-32a-OAZ1 (前期已經(jīng)構(gòu)建完成)作為模板，PCR擴(kuò)增OAZ1框移位點(diǎn)缺失突變基因(687 bp，205位的T缺失)，并且在片段兩端分別引入Eco RⅠ和XbaⅠ酶切位點(diǎn)。pLVX-Neo-IRES-ZsGreen載體與擴(kuò)增的缺失突變基因產(chǎn)物經(jīng)Eco RⅠ和XbaⅠ雙酶切并連接，連接產(chǎn)物pLVX-Neo-OAZ1-IRES-ZsGreen轉(zhuǎn)化JM 109感受態(tài)細(xì)胞，擴(kuò)大培養(yǎng)后抽提質(zhì)粒，酶切測(cè)序鑒定。

1.2.2 慢病毒包裝利用磷酸鈣沉淀法將四質(zhì)粒系統(tǒng)共轉(zhuǎn)染293T細(xì)胞，24 h后更換新鮮培養(yǎng)基，當(dāng)細(xì)胞熒光強(qiáng)度達(dá)最強(qiáng)時(shí)，收集含病毒的培養(yǎng)基上清，0.22 μm針頭濾器過(guò)濾獲得OAZ1慢病毒，分裝后置于-80℃冰箱保存?zhèn)溆谩?/p>

1.2.3 細(xì)胞培養(yǎng)與穩(wěn)轉(zhuǎn)株的篩選SCC15細(xì)胞用含10%胎牛血清的DMEM/F12(100 μg/m L鏈霉素、100 IU/m L青霉素)，于37℃、5%CO2培養(yǎng)箱中培養(yǎng)。細(xì)胞按1×105/孔接種于六孔板，用OAZ1慢病毒和作為對(duì)照的GFP慢病毒感染SCC15細(xì)胞，共培養(yǎng)24 h后換液繼續(xù)培養(yǎng)。細(xì)胞出現(xiàn)綠色熒光后，用G418 (1 000 μg/m L)進(jìn)行篩選，擴(kuò)大培養(yǎng)后使用有限稀釋法挑取帶綠色熒光的單克隆細(xì)胞。

1.2.4 定量PCR檢測(cè)OAZ1、E-cadherin、Vimentin、N-cadherin、Zeb1、Snail、Tw ist1、TGF-β1的mRNA水平按RNAiso Plus試劑盒操作說(shuō)明提取細(xì)胞總RNA。使用PrimeScript RT試劑盒將RNA逆轉(zhuǎn)錄成cDNA，Real-time RT-PCR體系10 μL，采用兩步法PCR擴(kuò)增程序：95℃預(yù)變性30 s；95℃15 s，58℃45 s，40個(gè)PCR循環(huán)；55℃～95℃，0.5℃/s變溫速度讀取熔解曲線。引物序列見(jiàn)表1。

基因名稱OAZ1(NM_004152)引物-F -R -F -R -F -R -F -R -F -R -F -R -F -R -F -R -F -R引物序列(5'-3') TCTCCCTCCACTGCTGTAGTAACC GTTGAGAATCCTCGTCTTGTCGTT CTACAATGCCGCCATCGCTTA CACTGATGACTCCTGTGTTCCTGTT TATGAAGGAGGAAATGGCT AGTTTGGAAGAGGCAGAGA GGTAATCCTCCCAAATCAA GGTAATCCTCCCAAATCAA AAGTGGCGGTAGATGGTAA TTGTTGTATGGGTGAAGCA CGAGTGGTTCTTCTGCGCTA CTGCTGGAAGGTAAACTCTGGA GCCGGAGACCTAGATGTCATT CCCACGCCCTGTTTCTTTGA GACAGCAGGGATAACACACT ATGAGAAGCAGGAAAGGC CGCTGAGTACGTCGTGGAGTC GCTGATGATCTTGAGGCTGTTGTC引物長(zhǎng)度(bp) 198 E-cadherin(NM_001317184.1)98 Vimentin(NM_003380.3)140 N-cadherin(NM_001308176.1)175 Zeb1(NM_001128128.2)104 Snail(NM_005985.3)157 Tw ist1(NM_000474.3)151 TGF-β1(NM_000660.5)107 GAPDH(NM_002046)172

1.2.5 Western blot使用預(yù)冷磷酸鹽緩沖液(PBS)洗細(xì)胞三次，加入裂解液(含PMSF)提取的細(xì)胞總蛋白。用12%的分離膠進(jìn)行SDS-PAGE凝膠電泳，電轉(zhuǎn)至PVDF膜上，5%脫脂奶粉于室溫封閉2 h，一抗4℃孵育過(guò)夜。二抗4℃孵育2 h，ECL法顯影，應(yīng)用FluorChem 8900軟件進(jìn)行掃描灰度分析。

1.3 統(tǒng)計(jì)學(xué)方法應(yīng)用SPSS16.0軟件進(jìn)行數(shù)據(jù)分析，計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差()表示，多組間比較采用單因素方差分析，以P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 構(gòu)建慢病毒表達(dá)載體pLVX-Neo-OAZ1-IRES -ZsGreen以pET-32a-OAZ1質(zhì)粒為模板，PCR擴(kuò)增OAZ1框移位點(diǎn)缺失突變基因(687 bp,205位的T缺失)，并且在片段兩端分別引入Eco RⅠ和XbaⅠ酶切位點(diǎn)，測(cè)序鑒定顯示擴(kuò)增片段準(zhǔn)確無(wú)誤，205位的T已經(jīng)缺失。測(cè)序結(jié)果如圖1。將擴(kuò)增得到的OAZ1目的片段連接到pLVX-Neo-IRES-ZsGreen慢病毒表達(dá)載體中，連接產(chǎn)物pLVX-Neo-OAZ1-IRES-ZsGreen轉(zhuǎn)化JM 109感受態(tài)細(xì)胞，擴(kuò)大培養(yǎng)后抽提質(zhì)粒，經(jīng)酶切測(cè)序鑒定pLVX-Neo-OAZ1-IRES-ZsGreen重組載體構(gòu)建成功(圖2)。

圖1 OAZ1突變基因的測(cè)序結(jié)果

圖2 pLVX-Neo-OAZ1-IRES-ZsGreen雙酶切驗(yàn)證

2.2 病毒包裝及OAZ1基因穩(wěn)定表達(dá)SCC15細(xì)胞株的構(gòu)建四質(zhì)粒系統(tǒng)共轉(zhuǎn)染293T細(xì)胞，更換新鮮培養(yǎng)基后24 h熒光強(qiáng)度達(dá)到最高(圖3)，故在此時(shí)離心收集含病毒的上清液，過(guò)濾獲得OAZ1慢病毒。使用獲得的慢病毒感染HEK293細(xì)胞，更換新鮮培養(yǎng)基后24 h于熒光顯微鏡下計(jì)數(shù)熒光細(xì)胞數(shù)目，生物學(xué)滴度測(cè)定結(jié)果顯示，OAZ1病毒為2×107TU/m L(圖4)。使用OAZ1慢病毒及GFP慢病毒感染SCC15細(xì)胞，然而G418篩選后，SCC15/OAZ1細(xì)胞株的熒光率未達(dá)到75%以上(圖5)。故使用有限稀釋法挑取9株熒光強(qiáng)度較高的單克隆細(xì)胞進(jìn)行培養(yǎng)(圖6A)。Western blot和定量PCR驗(yàn)證OAZ1的表達(dá)情況。結(jié)果顯示，相對(duì)于對(duì)照組SCC15細(xì)胞和轉(zhuǎn)染空載體的SCC15/GFP細(xì)胞，實(shí)驗(yàn)組SCC15/OAZ1-1，SCC15/OAZ1-7，SCC15/ OAZ1-8，SCC15/OAZ1-9等4株單克隆細(xì)胞中OAZ1的mRNA水平均顯著上調(diào)(圖6B)。進(jìn)一步Western blot結(jié)果顯示，SCC15/OAZ1-8細(xì)胞中OAZ1蛋白水平上調(diào)最為顯著(圖6C)。故選擇SCC15/OAZ1-8細(xì)胞用于后續(xù)的研究。

圖3 四質(zhì)粒系統(tǒng)轉(zhuǎn)染293T細(xì)胞包裝病毒(×10)

圖4 倍比稀釋法測(cè)定慢病毒滴度(×10)

圖5 OAZ1慢病毒和GFP慢病毒穩(wěn)定轉(zhuǎn)染SCC15細(xì)胞株(×20)

圖6 OAZ1穩(wěn)定轉(zhuǎn)染的單克隆細(xì)胞株篩選(×20)

2.3 OAZ1抑制舌鱗癌細(xì)胞SCC15的上皮間質(zhì)化檢測(cè)EMT相關(guān)基因(E-cadherin、Vimentin、N-cadherin、Zeb1、Snail、Twist1、TGF-β1)的mRNA水平，結(jié)果顯示，在SCC15/OAZ1細(xì)胞中，Vimentin，N-cadherin和TGF-β1的mRNA水平顯著降低，而E-cadherin、Zeb1、Snail、Twist1的mRNA水平?jīng)]有明顯變化(圖7A)。Western blot結(jié)果顯示，OAZ1的過(guò)表達(dá)可抑制Vimentin、N-cadherin和TGF-β1的蛋白水平(圖7B)，表明OAZ1可以抑制舌鱗癌細(xì)胞SCC15的上皮間質(zhì)化，其機(jī)制可能涉及TGF-β1的轉(zhuǎn)錄抑制。進(jìn)一步研究結(jié)果顯示，相對(duì)于對(duì)照組SCC15細(xì)胞和轉(zhuǎn)染空載體的SCC15/GFP細(xì)胞，實(shí)驗(yàn)組SCC15/OAZ1中Smad1的蛋白水平顯著降低，然而其mRNA水平?jīng)]有明顯變化(圖7A，B)。提示，OAZ1可能通過(guò)促進(jìn)Smad1的降解抑制SCC15細(xì)胞的上皮間質(zhì)化。

圖7 OAZ1抑制舌鱗癌細(xì)胞SCC15的上皮間質(zhì)化

3 討論

OAZ1在細(xì)胞增殖分化以及凋亡中起重要調(diào)控作用，其因在多胺代謝中的重要調(diào)控地位以及誘導(dǎo)部分細(xì)胞內(nèi)重要大分子的非泛素依賴降解作用而備受關(guān)注[5,13]?，F(xiàn)已證實(shí)OAZ1作為一個(gè)抑癌基因具有抗腫瘤特性[14]。研究顯示，在舌鱗癌癌細(xì)胞中，OAZ1可以誘導(dǎo)細(xì)胞向終末分化，且抑制細(xì)胞的增殖[12]。然而，OAZ1是否參與調(diào)控舌鱗癌細(xì)胞上皮間質(zhì)化過(guò)程尚不明確，故對(duì)其進(jìn)行深入研究，將有助于OAZ1在舌鱗狀細(xì)胞癌治療中的研究應(yīng)用。

為了研究OAZ1對(duì)舌鱗癌細(xì)胞上皮間質(zhì)化的影響，本研究利用攜帶OAZ1框移位點(diǎn)缺失突變的慢病毒構(gòu)建OAZ1穩(wěn)定表達(dá)的舌鱗癌細(xì)胞株SCC15/OAZ1。然而經(jīng)G418篩選后，SCC15/OAZ1細(xì)胞株的熒光率未達(dá)到75%以上。為了進(jìn)一步提高陽(yáng)性細(xì)胞的百分率，我們通過(guò)有限稀釋法挑取帶綠色熒光的單克隆細(xì)胞進(jìn)行培養(yǎng)。經(jīng)定量PCR和Western blot檢測(cè)發(fā)現(xiàn)，SCC15/OAZ1-8細(xì)胞中OAZ1的mRNA水平和蛋白水平均顯著高于對(duì)照組，表明我們已經(jīng)成功構(gòu)建OAZ1穩(wěn)定表達(dá)細(xì)胞株。

上皮間質(zhì)化在舌鱗癌轉(zhuǎn)移中發(fā)揮關(guān)鍵作用。E-cadherin是鈣粘蛋白超家族成員，可介導(dǎo)細(xì)胞間連接，其作為上皮標(biāo)志物在鱗狀細(xì)胞癌中表達(dá)水平顯著下調(diào)[15]。然而，定量結(jié)果顯示在SCC15/OAZ1細(xì)胞中E-cadherin的mRNA表達(dá)水平?jīng)]有顯著變化。研究表明，在上皮間質(zhì)化過(guò)程中，間質(zhì)細(xì)胞標(biāo)志物Vimentin、N-cadherin以及EMT相關(guān)的轉(zhuǎn)錄因子Zeb1、Snail和Tw ist1的表達(dá)水平上調(diào)[16-17]。我們的研究結(jié)果顯示，在SCC15/OAZ1細(xì)胞中間質(zhì)細(xì)胞標(biāo)志物Vimentin、N-cadherin的mRNA水平顯著降低，然而EMT相關(guān)的轉(zhuǎn)錄因子Zeb1、Snail和Tw ist1的mRNA水平?jīng)]有明顯變化。進(jìn)一步的實(shí)驗(yàn)表明，在SCC15/OAZ1細(xì)胞中Vimentin、N-cadherin蛋白水平顯著降低。這些結(jié)果表明，在舌鱗癌細(xì)胞中OAZ1的過(guò)表達(dá)可以抑制上皮間質(zhì)化，然而其機(jī)制可能并不涉及調(diào)控Zeb1、Snail和Tw ist1等轉(zhuǎn)錄因子的表達(dá)。

TGF-β1信號(hào)通路在促進(jìn)腫瘤細(xì)胞上皮間質(zhì)化中扮演重要作用，TGF-β1可以通過(guò)激活Smad2/3信號(hào)通路促進(jìn)上皮間質(zhì)化[18]。故我們檢測(cè)了OAZ1對(duì)TGF-β1的影響，結(jié)果顯示，OAZ1可以抑制TGF-β1的mRNA水平以及蛋白水平。此外，有研究顯示，BMPs/Smad1信號(hào)通路的激活同樣可以促進(jìn)上皮間質(zhì)化[19-23]。而OAZ1可與Smad1以及HsN3形成Smad1-HsN3-OAZ1三元復(fù)合物，并且介導(dǎo)Smad1的非泛素依賴性降解[24-25]。故我們檢測(cè)了Smad1的蛋白水平，結(jié)果顯示OAZ1可以促進(jìn)Smad1的降解。因此推測(cè)，OAZ1可能通過(guò)抑制TGF-β1信號(hào)通路和Smad1信號(hào)通路抑制舌鱗癌細(xì)胞的上皮間質(zhì)化。然而OAZ1抑制舌鱗癌細(xì)胞上皮間質(zhì)化的確切分子機(jī)制還需進(jìn)一步研究。

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Establishment of SCC15 cell line stably expressing human OAZ1 gene and its influence on the epithelialmesenchymal transition of tongue squamous cell carcinoma.

WANG Xing1,JIANG Li2,ZHANG Yun1,CHEN Xiao-yan2.1.Laboratory of Inflammation and Allergy,the Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan,CHINA;2.Medical Research Center,Guangdong General Hospital,Guangzhou 510080,Guangdong, CHINA

Objective To investigate the role of OAZ1 in epithelial mesenchymal transition(EMT)in tongue squamous cell carcinoma cell lines SCC15 and explore its related mechanism.Methods The lentivirus vector pLVX-Neo-OAZ1-IRES-ZsGreen in which the target gene OAZ1 was modified at the frameshift site was constructed, packaged lentivirus and infected SCC15 cells,and the monoclonal cell lines w ith high fluorescence intensity were obtained by lim ited diluted method.Real-time PCR and Western blot was used to detect the expression of OAZ1 and the genes(E-cadherin,Vimentin,N-cadherin,Zeb1,Snail,Tw ist1,TGF-β1,Smad1)related w ith EMT.Results The SCC15 cell line w ith stable expression of OAZ1 gene was successfully constructed.The overexpression of OAZ1 inhibited the protein level and mRNA level of the Vimentin,N-cadherin,TGF-β1.Furthermore,the OAZ1 would promote the degradation of Smad1.Conclusion OAZ1 can inhibit the EMT of tongue squamous cell carcinoma cell lines through the possible mechanism related w ith the inhibition of TGF-β1and Smad1 pathway.

Ornithine decarboxylase antizyme 1(OAZ1);Tongue squamous cell carcinoma(TSCC);Lentivirus;Epithelial mesenchymal transition(EMT)

R739.86

A

1003—6350（2017）13—2065—07

10.3969/j.issn.1003-6350.2017.13.001

2017-04-14)

國(guó)家自然科學(xué)基金(編號(hào)：30901757)

姜立。E-mail：lijiang3434@yeah.net