CRIP1生物學(xué)功能的定量蛋白質(zhì)組學(xué)分析

楊 超，霍 祎，鄧海騰

(清華大學(xué)生命科學(xué)學(xué)院教育部生物信息學(xué)重點(diǎn)實(shí)驗(yàn)室，北京 100084)

CRIP1生物學(xué)功能的定量蛋白質(zhì)組學(xué)分析

楊 超，霍 祎，鄧海騰

(清華大學(xué)生命科學(xué)學(xué)院教育部生物信息學(xué)重點(diǎn)實(shí)驗(yàn)室，北京 100084)

CRIP1(cysteine-rich intestinal protein 1)是含有雙鋅指結(jié)構(gòu)域的蛋白，在很多腫瘤細(xì)胞中高表達(dá)，但其在肺癌細(xì)胞中的生理學(xué)功能尚不明確。本研究應(yīng)用定量蛋白質(zhì)組學(xué)探究CRIP1過表達(dá)對(duì)肺癌順鉑耐藥細(xì)胞(A549/DDP)的影響及作用機(jī)制。運(yùn)用慢病毒載體系統(tǒng)構(gòu)建了CRIP1過表達(dá)的A549/DDP穩(wěn)轉(zhuǎn)細(xì)胞系，利用Western Blotting證實(shí)了單克隆細(xì)胞系中CRIP1的過表達(dá)。發(fā)現(xiàn)CRIP1過表達(dá)能夠加快細(xì)胞增殖，增加細(xì)胞的耐藥性。通過定量蛋白質(zhì)組學(xué)分析，鑒定了CRIP1過表達(dá)引起的蛋白質(zhì)組的變化，并且對(duì)上調(diào)和下調(diào)的蛋白進(jìn)行聚類分析。結(jié)果表明，CRIP1過表達(dá)上調(diào)了煙酰胺磷酸核糖轉(zhuǎn)移酶(NAMPT)和NAD依賴型氧化還原酶的表達(dá)，從而促進(jìn)細(xì)胞的增殖，并且提高了細(xì)胞的耐藥性。

CRIP1；過表達(dá)；生長速率；蛋白質(zhì)組學(xué)；煙酰胺磷酸核糖轉(zhuǎn)移酶(NAMPT)

CRIP1(cysteine-rich intestinal protein 1)屬于含有雙鋅指結(jié)構(gòu)域(即LIM結(jié)構(gòu)域)的蛋白質(zhì)[1]，其分子質(zhì)量為8.5 ku[2]，LIM結(jié)構(gòu)域與心衰和心臟的發(fā)育有關(guān)[3]。CRIP家族成員在胚胎發(fā)育過程中起著重要作用[4]，CRIP1會(huì)影響鋅離子的轉(zhuǎn)運(yùn)[5]。近期研究發(fā)現(xiàn)，CRIP1在許多腫瘤細(xì)胞中高表達(dá)，是一個(gè)潛在的腫瘤標(biāo)志物。在骨肉瘤中，CRIP1高表達(dá)是病癥減輕的標(biāo)志[6]。在乳腺癌的治療中，CRIP1是預(yù)后良好的標(biāo)志物，也可以作為乳腺癌分期的標(biāo)志物[7-8]。另外，CRIP1和Galectin-3在子宮內(nèi)膜癌中共表達(dá)，會(huì)影響患者預(yù)后和生存[9]；CRIP1和S100P是前列腺癌中的高表達(dá)基因，其表達(dá)水平受表觀遺傳調(diào)節(jié)[10]。CRIP1在肺癌細(xì)胞中的生物學(xué)功能尚不清楚。

近年來，蛋白質(zhì)組學(xué)已經(jīng)成為癌癥研究的重要工具[11-12]。串聯(lián)質(zhì)譜標(biāo)簽(TMT)是一種常用的蛋白定量技術(shù)，通過TMT試劑與多肽N端及賴氨酸殘基側(cè)鏈的胺基反應(yīng)，標(biāo)記酶解后的肽段。TMT試劑由反應(yīng)基團(tuán)、報(bào)告基團(tuán)和平衡基團(tuán)組成，不同樣本的同一肽段標(biāo)記后的質(zhì)荷比在一級(jí)譜圖中是相同的，而在二級(jí)質(zhì)譜中，報(bào)告基團(tuán)和平衡基團(tuán)之間的鍵斷裂，產(chǎn)生不同質(zhì)荷比的報(bào)告離子用于定量分析。

本研究擬通過建立CRIP1過表達(dá)穩(wěn)轉(zhuǎn)細(xì)胞系，闡明CRIP1對(duì)細(xì)胞生長和耐藥的影響，并且結(jié)合定量蛋白質(zhì)組學(xué)分析，揭示CRIP1調(diào)控腫瘤細(xì)胞生長和耐藥的生物學(xué)功能。

1 實(shí)驗(yàn)部分

1.1儀器

Dionex Ultimate 3000高效液相色譜儀、Orbitrap Fusion Tribrid液相色譜-質(zhì)譜儀、細(xì)胞CO2培養(yǎng)箱、酶標(biāo)儀：美國Thermo公司產(chǎn)品；臺(tái)式低溫高速離心機(jī)：德國Eppendorf公司產(chǎn)品；凝膠成像系統(tǒng)、核酸電泳儀、蛋白轉(zhuǎn)膜裝置、蛋白質(zhì)電泳裝置：美國Bio-Rad公司產(chǎn)品。

1.2材料

2×Taq PCR MasterMix、瓊脂糖凝膠回收試劑盒、質(zhì)粒小提試劑盒和DH5α：北京天根生化科技有限公司產(chǎn)品；AceQ qPCR SYBR Green Master Mix：南京諾唯贊生物科技有限公司產(chǎn)品； CRIP1抗體：英國Abcam公司產(chǎn)品；CCK8試劑盒：日本Dojindo公司產(chǎn)品；pcDNA3.1(+)、pLVX-IRES-Zsgreen1和A549/DDP：均為實(shí)驗(yàn)室保存；DMEM、RPMI 1640、PBS和雙抗：加拿大Wisent公司產(chǎn)品；DTT：德國Merck公司產(chǎn)品；IAM：美國Sigma公司產(chǎn)品；胰酶(質(zhì)譜級(jí))：美國Promega公司產(chǎn)品；TMT試劑和乙腈：美國Thermo公司產(chǎn)品。

1.3實(shí)驗(yàn)方法

1.3.1目的基因的獲取與載體構(gòu)建 首先從肺癌順鉑耐藥細(xì)胞(A549/DDP，下同)中提取總RNA，逆轉(zhuǎn)錄成cDNA，以cDNA為模板，所用上游引物為5’-CCCAAGCTTGGGATGGATTACAAGGACGATGACGATAAGCCC-AAGTGTCCCAAGTG-3’，下游引物為5’-CGCGGATCCGCGTTACTTGAAAGTGTG-GCTCTCGG-3’進(jìn)行聚合酶鏈?zhǔn)椒磻?yīng)(PCR)，產(chǎn)物回收。分別用HindIII和BamHI回收的目的片段和pcDNA3.1(+)載體進(jìn)行雙酶切。PCR目的片段與載體連接，進(jìn)行連接產(chǎn)物轉(zhuǎn)化。

構(gòu)建pLVX-CRIP1-IRES-Zsgreen1質(zhì)粒，以pcDNA3.1(+)-CRIP1質(zhì)粒為模板進(jìn)行亞克隆設(shè)計(jì)CRIP1引物，以5’-GGAATTCCATGGATTACAAGGACGATGACGATAAGCC-CAAGTGTCCCAAGTG-3’為上游引物，以5’-GCTCTAGAGCTTACTTGAAAGTGTGGCTCTCGG-3’為下游引物，進(jìn)行PCR實(shí)驗(yàn)和產(chǎn)物回收。用EcoRI和XbaI進(jìn)行雙酶切，依次進(jìn)行連接、轉(zhuǎn)化和測(cè)序。

1.3.2細(xì)胞培養(yǎng)與細(xì)胞轉(zhuǎn)染 用含10%胎牛血清的DMEM培養(yǎng)基培養(yǎng)293T細(xì)胞至70%左右用于慢病毒包裝。用3種輔助質(zhì)粒(pLP1∶pLP2∶VSVG=2∶3∶2)，pLVX-CRIP1-IRES-Zsgreen1作為主質(zhì)粒，對(duì)照組用pLVX-IRES-Zsgreen1作為主質(zhì)粒，分別在293T細(xì)胞中進(jìn)行慢病毒顆粒包裝，獲得包裝完成的慢病毒顆粒并進(jìn)行濃縮。

用含10%胎牛血清的RPMI 1640培養(yǎng)基培養(yǎng)A549/DDP細(xì)胞系用于過表達(dá)CRIP1，將A549/DDP鋪于24孔板，細(xì)胞密度為30%～40%，加入一定量濃縮后的慢病毒顆粒和終濃度7 mg/L的聚凝胺溶液，感染6 h，換成含有10%胎牛血清和1%雙抗的RPMI1640培養(yǎng)基繼續(xù)培養(yǎng)至48 h，可通過熒光顯微鏡觀察是否具有綠色熒光，長滿至100 mm平皿。

選取綠色熒光較強(qiáng)的平皿，作為選定的多克隆細(xì)胞，一部分用于實(shí)時(shí)熒光定量核算擴(kuò)增(qPCR)鑒定，一部分用于分選單克隆，用流式細(xì)胞儀分選GFP單細(xì)胞于96孔板，培養(yǎng)一段時(shí)間后挑選亮度較高的細(xì)胞系擴(kuò)大培養(yǎng)，進(jìn)一步鑒定。

1.3.3穩(wěn)轉(zhuǎn)細(xì)胞系的鑒定 提取上一步的多克隆細(xì)胞系，進(jìn)行CRIP1過表達(dá)qPCR鑒定。用Trizol試劑提取細(xì)胞總RNA，配置逆轉(zhuǎn)錄PCR反應(yīng)體系，對(duì)以cDNA為模板的CRIP1的qPCR引物進(jìn)行qPCR檢測(cè)，采用SYBR Green Master Mix配置反應(yīng)體系，每組3個(gè)平行實(shí)驗(yàn)，使用的儀器是Roche，結(jié)果用2-ΔΔCt法分析，內(nèi)參基因?yàn)锳ctin。

利用Western Blotting鑒定單克隆細(xì)胞系CRIP1過表達(dá)。選取2種熒光顯微鏡下較亮的過表達(dá)單克隆細(xì)胞系(CRIP1-OE1和CRIP1-OE2)和1種對(duì)照組細(xì)胞系(Control)提取全蛋白，用BCA(bicinchoninic acid)法測(cè)定并計(jì)算蛋白濃度，取等量的蛋白煮沸變性后，進(jìn)行SDS-PAGE電泳，轉(zhuǎn)PVDF膜，封閉1 h，CRIP1一抗于4 ℃孵育過夜并回收，洗膜，二抗于室溫孵育1 h，洗膜。加入ECL發(fā)光液反應(yīng)1 min，置于Bio-rad凝膠成像儀成像。

1.3.4細(xì)胞生長曲線的檢測(cè) 用胰酶將CRIP1-OE和Control細(xì)胞從100 mm培養(yǎng)皿消化下來，收取細(xì)胞；使用血球計(jì)數(shù)法將細(xì)胞濃度調(diào)整為30 000個(gè)/毫升，加入96孔板(每孔100 μL)，設(shè)置4組平行實(shí)驗(yàn)；分別在0、12、24、36、48、60、72、84、96 h以10∶1加入CCK8，2 h后測(cè)定450 nm吸光度值，并繪制細(xì)胞生長曲線，對(duì)比CRIP1-OE和Control組的細(xì)胞增殖速度。

1.3.5細(xì)胞順鉑耐藥性的檢測(cè) 用胰酶將CRIP1-OE和Control細(xì)胞從100 mm培養(yǎng)皿消化下來，以800 r/min離心5 min后收取細(xì)胞，用培養(yǎng)基重懸，使用血球計(jì)數(shù)法調(diào)整細(xì)胞濃度為20 000個(gè)/毫升，加入96孔板培養(yǎng)至貼壁牢固，設(shè)置3組平行實(shí)驗(yàn)；以0、40、80、120、160、200、240、320、400 μmol/L順鉑作用于細(xì)胞24 h，用CCK8計(jì)數(shù)，通過測(cè)量細(xì)胞的存活率研究CRIP1對(duì)細(xì)胞耐藥性的影響。

1.3.6定量蛋白質(zhì)組學(xué)分析 定量蛋白質(zhì)組學(xué)分析包括以下四個(gè)步驟：第一步，蛋白提取。利用胰酶分離100 mm培養(yǎng)皿中貼壁的CRIP1-OE細(xì)胞和Control組細(xì)胞，分別收集于1.5 mL的EP管，用PBS洗2～3次，然后，用適量的8 mol/L尿素充分裂解，高速離心20 min，取上清用BCA試劑盒測(cè)量蛋白濃度，取100 μg蛋白，稀釋至濃度1 g/L以下。

第二步，溶液內(nèi)酶解。在室溫下向蛋白溶液中加入適量的DTT，反應(yīng)1 h；避光并加入適量的IAM，反應(yīng)30 min；加入PBS稀釋尿素；按照與蛋白1∶50比例加入質(zhì)譜級(jí)胰酶，于37 ℃反應(yīng)14 h，用10% TFA終止反應(yīng)，調(diào)節(jié)pH為2左右。

第三步，樣品除鹽、TMT標(biāo)記和鑒定。用Sep-Pak C18除鹽柱，依次經(jīng)活化、平衡、上樣(3次過柱)、漂洗和洗脫5個(gè)步驟，使用的試劑依次是乙腈、0.1%TFA、50%ACN+0.1%TFA，除鹽后離心，用100 mmol/L TEAB復(fù)溶，調(diào)至pH 8；向CRIP1-OE組中加入TMT6-128試劑，Control組中加入TMT6-131試劑，于室溫反應(yīng)1 h，用羥胺終止反應(yīng)，合并兩種樣品，揮去TMT試劑中的乙腈，再次進(jìn)行除鹽、揮干，用0.1%FA復(fù)溶至100 μL，進(jìn)行HPLC分離肽段，后續(xù)在蛋白質(zhì)組學(xué)平臺(tái)上進(jìn)行二級(jí)質(zhì)譜鑒定。

第四步，蛋白質(zhì)組學(xué)數(shù)據(jù)分析。采用Protein Discoverer(Thermo)軟件搜索數(shù)據(jù)庫，找到鑒定蛋白。根據(jù)軟件評(píng)分，選取大于4分的蛋白質(zhì)進(jìn)行分析。上調(diào)蛋白的標(biāo)準(zhǔn)是TMT比值大于1.5，下調(diào)同理。用Panther軟件對(duì)上調(diào)和下調(diào)蛋白進(jìn)行聚類分析。

2 實(shí)驗(yàn)結(jié)果

2.1CRIP1過表達(dá)穩(wěn)轉(zhuǎn)細(xì)胞系構(gòu)建結(jié)果及鑒定

將構(gòu)建成功的真核表達(dá)載體pcDNA3.1(+)-CRIP1酶切后，經(jīng)瓊脂糖凝膠電泳鑒定的目的條帶符合預(yù)期大小，測(cè)序結(jié)果證實(shí)了目的條帶已與載體連接。亞克隆的測(cè)序結(jié)果證實(shí)了pLVX-CRIP1-IRES-Zsgreen1質(zhì)粒構(gòu)建成功。用pLVX-CRIP1-IRES-Zsgreen1質(zhì)粒包裝病毒顆粒侵染A549/DDP細(xì)胞后，選取較亮的多克隆細(xì)胞進(jìn)行qPCR分析，證明質(zhì)粒成功轉(zhuǎn)入細(xì)胞并表達(dá)CRIP1，示于圖1a。對(duì)篩選出的過表達(dá)細(xì)胞系CRIP1-OE1、CRIP1-OE2和轉(zhuǎn)入空載的Control細(xì)胞進(jìn)行Western Blotting分析，發(fā)現(xiàn)在CRIP1過表達(dá)細(xì)胞中CRIP1表達(dá)量明顯增加，證明過表達(dá)細(xì)胞系構(gòu)建成功，示于圖1b。

2.2CRIP1對(duì)細(xì)胞增殖和耐藥性的影響

用CCK8試劑盒測(cè)量細(xì)胞數(shù)目，根據(jù)繪制的細(xì)胞生長曲線，發(fā)現(xiàn)與對(duì)照組細(xì)胞相比，CRIP1-OE細(xì)胞系生長速度明顯加快，表明CRIP1能夠促進(jìn)細(xì)胞增殖。從第48 h開始，能夠明顯看到細(xì)胞數(shù)目的顯著差異，到96 h時(shí)，差異更加明顯，測(cè)量CRIP1-OE細(xì)胞吸光度為3.11，對(duì)照組為2.65，說明CRIP1-OE細(xì)胞的表型之一是增殖速度加快，示于圖2a。通過1.3.5節(jié)的方法檢測(cè)CRIP1-OE細(xì)胞的順鉑耐藥性，比較實(shí)驗(yàn)組和對(duì)照組細(xì)胞存活率，發(fā)現(xiàn)實(shí)驗(yàn)組的細(xì)胞存活率大于對(duì)照組。實(shí)驗(yàn)結(jié)果表明，CRIP1過表達(dá)增強(qiáng)了A549/DDP細(xì)胞的耐藥性，示于圖2b。

注：a.CRIP1過表達(dá)細(xì)胞和Control細(xì)胞中CRIP1 mRNA的水平；b.CRIP1過表達(dá)細(xì)胞和Control細(xì)胞中CRIP1蛋白的表達(dá)水平圖1 穩(wěn)轉(zhuǎn)細(xì)胞系中CRIP1過表達(dá)的驗(yàn)證Fig.1 Confirmation of CRIP1 overexpression in stably transfected cells

2.3CRIP1過表達(dá)穩(wěn)轉(zhuǎn)細(xì)胞系的蛋白質(zhì)組學(xué)分析

為了理解CRIP1在細(xì)胞中的生物學(xué)功能，對(duì)CRIP1過表達(dá)的A549/DDP細(xì)胞和對(duì)應(yīng)的Control細(xì)胞進(jìn)行基于TMT標(biāo)記的定量蛋白質(zhì)組學(xué)分析。共鑒定到4 525個(gè)蛋白，其中上調(diào)蛋白質(zhì)(比值>1.5)152個(gè)，下調(diào)蛋白質(zhì)(比值<0.67)142個(gè)。用Panther軟件對(duì)所有變化的蛋白質(zhì)進(jìn)行聚類分析，結(jié)果表明，CRIP1過表達(dá)能夠影響代謝過程、細(xì)胞組成、生物學(xué)調(diào)控、細(xì)胞粘附、細(xì)胞增殖等細(xì)胞過程，尤其對(duì)細(xì)胞初級(jí)代謝中含氮化合物的代謝有較大影響，示于圖3。

為了證實(shí)CRIP1的表達(dá)量變化，鑒定到了CRIP1蛋白胰酶降解后被TMT標(biāo)記的一個(gè)肽段：TLTSGGHAEHEGKPYCNHPCYAAMFGPK。該肽段的二級(jí)質(zhì)譜圖示于圖4a，其中碰撞碎裂后產(chǎn)生的b、y系列離子與肽段序列匹配，斷裂形成的TMT報(bào)告基團(tuán)質(zhì)量分別為128.13 Da和131.11 Da，其離子強(qiáng)度比值為2.4∶1，說明該肽段在CRIP1過表達(dá)細(xì)胞的含量是對(duì)照細(xì)胞的2.4倍。對(duì)CRIP1的全部二級(jí)譜圖的定量結(jié)果進(jìn)行平均，計(jì)算出CRIP1在過表達(dá)細(xì)胞系中升高了4倍左右。同理，鑒定了煙酰胺磷酸核糖轉(zhuǎn)移酶(NAMPT)降解后被TMT標(biāo)記的一個(gè)肽段VLEILGK，其二級(jí)圖譜顯示該肽段相較于對(duì)照細(xì)胞升高了1.7倍，將NAMPT的全部二級(jí)圖譜的定量結(jié)果進(jìn)行平均，計(jì)算出NAMPT在CRIP1過表達(dá)細(xì)胞系中升高了1.7倍，示于圖4b。同時(shí)，在定量蛋白質(zhì)組學(xué)數(shù)據(jù)中，發(fā)現(xiàn)NAD依賴型氧化還原酶表達(dá)量增加，例如AKR1C1和AKR1B10分別上調(diào)2.3倍和2.1倍，示于圖5。

注：a.CRIP1過表達(dá)細(xì)胞和Control細(xì)胞的生長曲線；b.順鉑刺激下CRIP1過表達(dá)細(xì)胞和Control細(xì)胞的存活率曲線圖2 CRIP1過表達(dá)對(duì)細(xì)胞生長和耐藥性的影響Fig.2 Effects of CRIP1 overexpression on cell growth and drug resistance

圖3 CRIP1過表達(dá)細(xì)胞與對(duì)照細(xì)胞差異表達(dá)蛋白的聚類分析Fig.3 GO analysis of differentially expressed proteins between CRIP1 overexpressing cells and the control cells

注：a.TMT標(biāo)記過的CRIP1酶解肽段TLTSGGHAEHEGKPYCNHPCYAAMFGPK的二級(jí)質(zhì)譜圖；b.TMT標(biāo)記過的NAMPT酶解肽段VLEILGK的二級(jí)質(zhì)譜圖圖4 CRIP1過表達(dá)細(xì)胞和Control細(xì)胞中CRIP1和NAMPT表達(dá)的定量蛋白質(zhì)組學(xué)分析Fig.4 Quantitative proteomics analysis of CRIP1 and NAMPT expression in CRIP1 overexpression and the control cells

圖5 蛋白質(zhì)組學(xué)中NAD依賴型氧化還原酶的表達(dá)Fig.5 Expression of oxidoreductase dependent on NAD in proteomics analysis

3 討論

本研究構(gòu)建了CRIP1過表達(dá)細(xì)胞系，發(fā)現(xiàn)CRIP1過表達(dá)可使細(xì)胞生長速度加快，耐藥性增強(qiáng)。通過蛋白質(zhì)組學(xué)分析，發(fā)現(xiàn)CRIP1過表達(dá)會(huì)影響細(xì)胞的代謝過程，尤其會(huì)導(dǎo)致NAMPT上調(diào)。NAMPT是細(xì)胞內(nèi)NAD合成的限速酶，可以將煙酰胺轉(zhuǎn)化為哺乳動(dòng)物的煙酰胺單核苷酸，進(jìn)而合成NAD[13]，主要影響補(bǔ)救途徑[14]。它在很多腫瘤中高表達(dá)，其中包括乳腺癌、卵巢癌、黑色素瘤等實(shí)體瘤[15]和血液瘤[16]。腫瘤細(xì)胞代謝與NAMPT的調(diào)節(jié)作用有關(guān)，可以通過影響NAD依賴的酶(如SIRT家族和PARP家族)[17-18]，維持細(xì)胞NAD水平。另外，NAMPT在清除細(xì)胞內(nèi)ROS方面，可以與GSH系統(tǒng)相互作用，從而減輕藥物處理引發(fā)的ROS對(duì)細(xì)胞的殺傷作用[19]。也有一些研究認(rèn)為，通過影響細(xì)胞內(nèi)NAD含量的變化，NAMPT保障了糖酵解和線粒體呼吸鏈代謝需求[20]，從而維持了線粒體穩(wěn)態(tài)并增加了細(xì)胞的耐藥性。NAMPT還與腫瘤預(yù)后有關(guān)[21-22]。其高表達(dá)可能導(dǎo)致細(xì)胞內(nèi)NAD的增加，從而引起NAD依賴的氧化還原酶表達(dá)上調(diào)，促進(jìn)肺癌細(xì)胞的生長。

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QuantitativeProteomicsAnalysisonBiologicalFunctionofCRIP1

YANG Chao, HUO Yi, DENG Hai-teng

(MOEKeyLaboratoryofBioinformatics,SchoolofLifeSciences,TsinghuaUniversity,Beijing100084,China)

The aim of the present study is to investigate the effects of cysteine-rich intestinal protein (CRIP1) over-expression on cellular processes using proteomics. CRIP1 is a protein that contains a double zinc finger domain, and is highly expressed in many tumor cells, but its roles in lung cancer cells remain elusive. In this study, the CRIP1 gene was cloned and used pLVX-IRES-Zsgreen1 lentiviral vector that encodes a GFP to transfect A549/DDP cells that was a cisplatin resistant cells as compared to A549 cells. The real-time quantitative PCR (qPCR) was used to confirm the transfection efficiency of CRIP1. Flow cytometry was used to isolate single cells, which was seeded into a 96-well plate and cultured to obtain monoclonal cell lines. The monoclonal cell lines with high brightness were screened by fluorescence microscopy and CRIP1 over-expression was confirmed by western blot analysis, indicating that a stable cell line was established successfully, in which CRIP1 was over-expressed. The growth curves of CRIP1 over-expression cells and the control cells were measured by the CCK8 assay. It showed that CRIP1 over-expression increased cell proliferation. CRIP1 over-expression cells and the control cells were also treated with cisplatin at different concentrations for 24 h and examined the viability of cisplatin-treated cells. Results showed that CRIP1 over-expression enhanced cells’ resistance to cisplatin.

To further explore the mechanism underlying CRIP1 over-expression mediated cellular processes, quantitative proteomics was applied to identify differentially expressed proteins between the control and CRIP1 over-expression cells. The quantitative proteomics was carried out using the TMTsixplexTMIsobaric Label Reagent, which modified the free amino group at the peptideN-terminus and lysine residues. Modified peptides generated reporter ions in MS/MS spectra to provide quantitative information about the proteins being analyzed. It was found that CRIP1 over-expression upregulated the expressions of nicotinamide phosphoribosyltransferase (NAMPT), which was the rate limiting enzyme in the scavenge pathway for production of the intracellular nicotinamide adenine dinucleotide (NAD). The CRIP1 over-expression mediated upregulation of NAMPT can increase the cellular NAD level. It was also revealed that CRIP1 over-expression upregulated expressions of NAD-dependent oxidoreductases, such as aldo-keto reductase family 1 member C1 (AKR1C1) and aldo-keto reductase family 1 member B10 (AKR1B10). Extensive studies have reported that NAD is essential for the cellular anti-oxidant systems and for maintaining mitochondrial integrity. The results suggest that the CRIP1 over-expression mediated NAMPT upregulation contributed to the enhanced cell growth and resistance to cisplatin.

cysteine-rich intestinal protein 1 (CRIP1); overexpression; growth rate; proteomics; nicotinamide phosphoribosyltransferase (NAMPT)

2017-04-17;

2017-05-25

科技部973項(xiàng)目(2014CBA02005)資助

楊 超(1991—)，女(滿族)，河北承德人，碩士研究生，生物學(xué)專業(yè)。E-mail: hiyangchao@163.com

鄧海騰(1964—)，男(漢族)，云南宣威人，博士生導(dǎo)師，從事蛋白質(zhì)組學(xué)研究。E-mail: dht@mail.tsinghua.edu.cn

O657.3

：A

：1004-2997(2017)04-0486-08

10.7538/zpxb.2017.0077