陳夢(mèng)竹，王秀杰，夏立新

1) 深圳大學(xué)醫(yī)學(xué)部，呼吸疾病國家重點(diǎn)實(shí)驗(yàn)室深圳大學(xué)變態(tài)反應(yīng)分室，廣東深圳518060；2) 中國科學(xué)院遺傳與發(fā)育生物學(xué)研究所，北京100101

【生物工程/Bioengineering】

肺腺癌和肺鱗癌中mRNA可變剪接特征的比較

陳夢(mèng)竹1，王秀杰2，夏立新1

1) 深圳大學(xué)醫(yī)學(xué)部，呼吸疾病國家重點(diǎn)實(shí)驗(yàn)室深圳大學(xué)變態(tài)反應(yīng)分室，廣東深圳518060；2) 中國科學(xué)院遺傳與發(fā)育生物學(xué)研究所，北京100101

利用SUPPA(a super-fast pipeline for alternative splicing)軟件分析癌癥基因組數(shù)據(jù)庫癌基因組圖譜中肺腺癌和肺鱗癌樣本的RNA-Seq數(shù)據(jù)，發(fā)現(xiàn)在鑒定的7種主要可變剪接轉(zhuǎn)錄本中外顯子跳躍轉(zhuǎn)錄本發(fā)生頻率最高．通過癌組織與癌旁組織的比較，系統(tǒng)地鑒定了肺腺癌和肺鱗癌中差異表達(dá)的可變剪接轉(zhuǎn)錄本，發(fā)現(xiàn)約60%的差異可變剪接轉(zhuǎn)錄本是兩種肺癌亞型所共有的．功能富集分析表明，癌組織特異的可變剪接轉(zhuǎn)錄本主要富集在細(xì)胞周期調(diào)控、脫氧核糖核酸代謝和核糖核酸代謝等生物過程中，而癌旁組織特異的可變剪接轉(zhuǎn)錄本則主要富集在囊泡介導(dǎo)轉(zhuǎn)運(yùn)、多肽轉(zhuǎn)運(yùn)和脂質(zhì)轉(zhuǎn)運(yùn)等生物過程中．通過肺癌驅(qū)動(dòng)基因可變剪接轉(zhuǎn)錄本的比較分析，鑒定了兩種肺癌亞型共有或特有的可能具有癌驅(qū)動(dòng)功能的可變剪接轉(zhuǎn)錄本．

生物信息學(xué)；肺腺癌；肺鱗癌；可變剪接；外顯子跳躍；可變起始外顯子

肺癌不僅是全球死亡率最高的癌癥，且發(fā)病率在男性和女性中分別僅次于發(fā)病率最高的前列腺癌和乳腺癌[1-2]．臨床診斷的肺癌大多為惡性上皮腫瘤，根據(jù)腫瘤細(xì)胞的顯微形態(tài)分為小細(xì)胞肺癌(small cell lung cancer, SCLC)和非小細(xì)胞肺癌(non-small cell lung cancer, NSCLC)．非小細(xì)胞肺癌發(fā)病率約占肺癌總發(fā)病率的85%～90%，包括肺腺癌(lung adenocarcinoma, LUAD)和肺鱗癌(lung squamous carcinoma, LUSC)兩種主要亞型[3]．深入研究兩種肺癌亞型的基因組變異，有助于更好地理解肺腺癌和肺鱗癌在發(fā)病機(jī)理、臨床特征及診療方案等方面存在的差異[4-9]．

可變剪接是由一種信使核糖核酸(messenger ribonucleic acid, mRNA)前體通過選擇性剪接產(chǎn)生多種不同成熟mRNA的過程，約95%的人多外顯子基因發(fā)生可變剪接[10]．根據(jù)可變剪接發(fā)生的位置，mRNA的可變剪接類型轉(zhuǎn)錄本主要分為可變5′剪接位點(diǎn)(alternative 5′ splice-site, A5)、可變3′剪接位點(diǎn)(alternative 3′ splice-site, A3)、可變起始外顯子(alternative first exon, AF)、可變終止外顯子(alternative last exon, AL)、內(nèi)含子滯留(retained intron, RI)、外顯子跳躍(skipping exon, SE)和互斥外顯子(mutually exclusive exons, MX)[11-12]．全基因組分析表明特異發(fā)生在腫瘤細(xì)胞中的某些可變剪接對(duì)癌癥的發(fā)生具有重要作用[13]．例如，癌細(xì)胞中原癌基因和抑癌基因異常的剪接轉(zhuǎn)錄本可致原癌基因的持續(xù)激活和抑癌基因部分功能的喪失，從而誘發(fā)癌癥．癌癥相關(guān)基因的可變剪接與肺癌的進(jìn)展關(guān)系密切[14]．已有研究結(jié)果表明，細(xì)胞凋亡調(diào)節(jié)因子Bcl-x基因前體mRNA通過可變剪接形成Bcl-xL和Bcl-xS兩種成熟的mRNA，分別行使抑制凋亡和促進(jìn)凋亡的功能．肺癌細(xì)胞中產(chǎn)生Bcl-xL轉(zhuǎn)錄本的比例較高，打破Bcl-xL和Bcl-xS在控制細(xì)胞凋亡中的平衡，從而傾向于抑制細(xì)胞凋亡，促進(jìn)肺癌的發(fā)生[15]．

隨著高通量測(cè)序技術(shù)的廣泛應(yīng)用，全基因組水平對(duì)肺癌可變剪接變異特征的研究也逐步深入，但仍缺乏較為系統(tǒng)地對(duì)于肺腺癌和肺鱗癌兩種主要肺癌亞型的比較分析．本研究利用已有癌癥基因組數(shù)據(jù)庫癌基因組圖譜(the cancer genome atlas, TCGA)，對(duì)LUAD和LUSC癌組織與其對(duì)應(yīng)的癌旁組織在mRNA可變剪接方面的變異特征進(jìn)行了系統(tǒng)的比較分析，發(fā)現(xiàn)兩者共有和各自特有的可變剪接變異，探討了可變剪接變異在兩種癌癥進(jìn)展中的潛在作用機(jī)制的異同，為肺癌的靶向治療提供新思路．

1 研究方法

1.1 肺腺癌和肺鱗癌數(shù)據(jù)下載

從癌癥基因組數(shù)據(jù)庫(the cancer genome atlas，TCGA．https://tcga-data.nci.nih.gov/tcga/)下載571例LUAD和547例LUSC患者癌組織及相應(yīng)癌旁組織的轉(zhuǎn)錄本表達(dá)數(shù)據(jù)、基因組注釋和臨床信息，用于后續(xù)的分析．

1.2 可變剪接轉(zhuǎn)錄本的產(chǎn)生與PSI (percentage of spliced in per event)值計(jì)算

利用SUPPA(a super-fast pipeline for alternative splicing analysis)軟件[16]，并結(jié)合TCGA提供的基因組注釋信息(https://tcga-data.nci.nih.gov/tcgafiles/ftp_auth/distro_ftpusers/anonymous/other/GAF/GAF3.0/transcript.genome.v3_0.gaf.gz)，獲得LUAD和LUSC中存在的A5、 A3、 AF、 AL、 RI、 SE和MX可變剪接轉(zhuǎn)錄本， 并計(jì)算可變剪接轉(zhuǎn)錄本的PSI值．

1.3 差異可變剪接轉(zhuǎn)錄本的鑒定

根據(jù)PSI值，如果某個(gè)可變剪接轉(zhuǎn)錄本在癌組織中的PSI值(PSI癌組織)與正常組織中的PSI值(PSI癌旁組織)的差值滿足PSI癌組織-PSI癌旁組織> 0.1 或PSI癌組織-PSI癌旁組織< -0.1，該可變剪接轉(zhuǎn)錄本即為差異的可變剪接轉(zhuǎn)錄本．挑選在研究群體中出現(xiàn)頻率大于10%的可變剪接轉(zhuǎn)錄本做后續(xù)分析．若具有該差異可變剪接轉(zhuǎn)錄本的患者中PSI癌組織- PSI癌旁組織> 0.1的患者數(shù)T， 是PSI癌組織-PSI癌旁組織<-0.1的患者數(shù)N的2.0倍以上(T/N > 2.0)， 該差異的可變剪接轉(zhuǎn)錄本傾向于在癌組織中出現(xiàn)，則定義該差異的可變剪接轉(zhuǎn)錄本為癌特異的差異可變剪接轉(zhuǎn)錄本；若T/N< 0.5， 該差異的可變剪接轉(zhuǎn)錄本傾向于在癌旁組織中出現(xiàn)，則定義該差異的可變剪接轉(zhuǎn)錄本為癌旁特異的差異可變剪接轉(zhuǎn)錄本．

1.4 差異可變剪接轉(zhuǎn)錄本的比較

比較LUAD和LUSC中差異可變剪接轉(zhuǎn)錄本，獲得兩者共有和特有的差異可變剪接轉(zhuǎn)錄本，利用DAVID[17]和RVIGO(reduce visualize gene ontology)[17-18]軟件對(duì)其相應(yīng)基因進(jìn)行功能富集分析．利用Pfam數(shù)據(jù)庫[19]評(píng)估差異的可變剪接轉(zhuǎn)錄本對(duì)蛋白結(jié)構(gòu)域的影響，篩選與LUAD和(或)LUSC進(jìn)展相關(guān)的可變剪接轉(zhuǎn)錄本．

1.5 肺癌驅(qū)動(dòng)基因的篩選

在癌驅(qū)動(dòng)基因數(shù)據(jù)庫(a database for human cancer driver gene research, DriverDB；http://driverdb.tms.cmu.edu.tw/driverdbv2/index.php)網(wǎng)站上，選擇在15個(gè)已知的癌驅(qū)動(dòng)基因鑒定工具中(ActiveDriver、Dendrix、MDPFinder、Simon、Netbox、OncodriveFM、MutSigCV、MEMo、CoMDP、DawnRank、DriverNet、e-Driver、iPAC、MSEA和OncodriveCLUST)任意3個(gè)(至少3個(gè))都可以篩查到的驅(qū)動(dòng)基因作為最終癌癥驅(qū)動(dòng)基因的集合．

2 結(jié)果分析

2.1 LUAD和LUSC中差異可變剪接轉(zhuǎn)錄本的鑒定

通過TCGA數(shù)據(jù)庫下載LUAD和LUSC中轉(zhuǎn)錄本的表達(dá)數(shù)據(jù)．在571例LUAD患者中，有57例患者同時(shí)具有癌組織和對(duì)應(yīng)癌旁組織轉(zhuǎn)錄本表達(dá)數(shù)據(jù)，在547例LUSC患者中，有50例患者同時(shí)具有癌組織和對(duì)應(yīng)癌旁組織轉(zhuǎn)錄本表達(dá)數(shù)據(jù)，選取57例LUAD和50例LUSC患者的轉(zhuǎn)錄本表達(dá)數(shù)據(jù)進(jìn)行后續(xù)分析．

利用SUPPA軟件，基因組注釋信息以及轉(zhuǎn)錄本表達(dá)數(shù)據(jù)，獲得LUAD和LUSC中7種主要可變剪接類型(A5、A3、AF、AL、RI、SE和MX)的所有可變剪接轉(zhuǎn)錄本(圖1)，并計(jì)算每個(gè)可變剪接轉(zhuǎn)錄本在57對(duì)LUAD和50對(duì)LUSC樣本中相應(yīng)的PSI值，篩選差異的可變剪接轉(zhuǎn)錄本．將LUAD或LUSC群體中發(fā)生頻率在10%以上的差異可變剪接轉(zhuǎn)錄本，即在LUAD樣本中至少6人(57×10%)或在LUSC樣本中至少5人(50×10%)同時(shí)具有該差異可變剪接轉(zhuǎn)錄本，挑選出來進(jìn)行后續(xù)分析，并鑒定了LUAD和LUSC樣本中癌特異和癌旁特異的差異可變剪接轉(zhuǎn)錄本，用于后續(xù)分析(表1)．

圖1 七種主要可變剪接示意圖Fig.1 The schematic diagram of seven main alternative splicing (AS) types

對(duì)每種類型差異可變剪接轉(zhuǎn)錄本占總差異可變剪接轉(zhuǎn)錄本的百分比進(jìn)行統(tǒng)計(jì)發(fā)現(xiàn)，在LUAD和LUSC中，SE類型可變剪接轉(zhuǎn)錄本所占比例都最高，分別為43.62%和42.66%(圖2)．其中， MRE11A、 IRAK1、 FHL1、 TCF4、 NUMB和TERT基因的可變剪接轉(zhuǎn)錄本在肺癌可變剪接轉(zhuǎn)錄本的研究中已有報(bào)道[20-26]，部分證明了該分析結(jié)果的準(zhǔn)確性．

2.2 LUAD與LUSC樣本中差異可變剪接轉(zhuǎn)錄本的比較

通過比較LUAD和LUSC中差異可變剪接轉(zhuǎn)錄本，獲得了兩種肺癌亞型共有以及各自特有的差異可變剪接轉(zhuǎn)錄本，其中，共有的差異可變剪接轉(zhuǎn)錄本約為60%(圖3)．

表1 LUAD和LUSC差異可變剪接轉(zhuǎn)錄本中7種可變剪接類型統(tǒng)計(jì)結(jié)果

圖2 肺腺癌和肺鱗癌中7種類型可變剪接轉(zhuǎn)錄本的百分比統(tǒng)計(jì)Fig.2 Percentage of each AS type in LUAD or LUSC

圖3 肺腺癌和肺鱗癌中共有可變剪接轉(zhuǎn)錄本統(tǒng)計(jì)Fig.3 The percentage of the common differentially expressed AS transcripts of LUAD and LUSC out of the total differentially expressed AS transcripts

LUAD和LUSC樣本中共有和特有的差異可變剪接轉(zhuǎn)錄本及其對(duì)應(yīng)基因數(shù)目(括號(hào)內(nèi)標(biāo)注)的統(tǒng)計(jì)如圖4．其中，紅色數(shù)字表示肺腺癌和肺鱗癌癌組織共有的特異剪接轉(zhuǎn)錄本(基因)；藍(lán)色數(shù)字表示癌旁組織共有的特異剪接轉(zhuǎn)錄本(基因)．將癌組織和癌旁組織特異的可變剪接轉(zhuǎn)錄本所對(duì)應(yīng)的基因進(jìn)行后續(xù)的功能分析.

圖4 LUAD和LUSC樣本中共有和特有的差異可變剪接轉(zhuǎn)錄本(基因)的統(tǒng)計(jì)Fig.4 The number of total and common differentially expressed AS transcripts (genes) of each AS type in LUAD and LUSC

2.3 LUAD和LUSC樣本中共有差異可變剪接轉(zhuǎn)錄本對(duì)應(yīng)基因的功能分析

功能富集分析的結(jié)果表明，兩種肺癌亞型共有的癌組織特異的差異可變剪基因主要富集在細(xì)胞周期調(diào)控、脫氧核糖核酸(deoxyribonucleic acid，DNA)代謝和RNA代謝等生物過程中見圖5．其中，點(diǎn)的大小代表該GO term在GO注釋(gene ontology annotation, GOA)中的代表頻率；點(diǎn)的顏色代表每個(gè)GO term的lgP.

圖5 LUAD和LUSC樣本中共有的癌組織特異的差異可變剪接轉(zhuǎn)錄本對(duì)應(yīng)基因的功能富集分析(P< 0.05, Fisher精確檢驗(yàn))Fig.5 GO analyses of the common cancer-specific AS transcripts of LUAD and LUSC (P< 0.05, Fisher’s exact test)

而兩種肺癌亞型共有的癌旁組織特異的差異可變剪接轉(zhuǎn)錄本對(duì)應(yīng)基因主要富集在囊泡介導(dǎo)轉(zhuǎn)運(yùn)、多肽轉(zhuǎn)運(yùn)、脂質(zhì)轉(zhuǎn)運(yùn)和細(xì)胞凋亡等生物過程(圖6)，癌組織與癌組織特異的差異可變剪接基因的功能富集結(jié)果存在明顯差異．

圖6 LUAD和LUSC樣本中共有的癌旁組織特異的差異可變剪接轉(zhuǎn)錄本對(duì)應(yīng)基因的功能富集分析(P< 0.05, Fisher精確檢驗(yàn))Fig.6 GO analyses of the common normal-specific AS transcripts (P< 0.05, Fisher’s exact test)

2.4 癌驅(qū)動(dòng)基因的可變剪接分析

癌驅(qū)動(dòng)基因是致癌的關(guān)鍵基因．在LUAD和LUSC中分別篩選出359和254個(gè)癌驅(qū)動(dòng)基因．結(jié)果表明，LUAD和LUSC中共有的差異可變剪接轉(zhuǎn)錄本所對(duì)應(yīng)的基因?yàn)? 097個(gè)，分別包含了21個(gè)LUAD和14個(gè)LUSC的驅(qū)動(dòng)基因(表2和表3)．研究結(jié)果表明， MET基因產(chǎn)生的可變剪接轉(zhuǎn)錄本在第10個(gè)外顯子上存在跳躍后終止的現(xiàn)象，且可能影響其編碼蛋白的激酶結(jié)構(gòu)域，如圖7(a)．LUAD中VCAN基因產(chǎn)生的外顯子跳躍轉(zhuǎn)錄本可能破壞其編碼蛋白的EGF蛋白結(jié)構(gòu)域，如圖7(b)，LUSC中外顯子跳躍轉(zhuǎn)錄本可能使ITGA8缺失一個(gè)Integrin_alpha2結(jié)構(gòu)域，如圖7(c)．

表2 LUAD癌驅(qū)動(dòng)基因中共有的差異可變剪接轉(zhuǎn)錄本

(續(xù)表2)

表3 LUSC癌驅(qū)動(dòng)基因中共有的差異可變剪接轉(zhuǎn)錄本

(續(xù)表3)

圖7 LUAD和LUSC樣本中癌驅(qū)動(dòng)基因的共有差異可變剪接轉(zhuǎn)錄本舉例Fig.7 Examples of the common differentially expressed AS transcripts of cancer driven genes in LUAD and LUSC

結(jié)果表明，LUAD和LUSC中亞型特異的差異可變剪接轉(zhuǎn)錄本對(duì)應(yīng)的基因數(shù)目分別為632和941，其中，包括16個(gè)LUAD和17個(gè)LUSC的驅(qū)動(dòng)基因(表4和表5)．本研究發(fā)現(xiàn)ALDH1L1基因的LUAD亞型特異轉(zhuǎn)錄本包括兩個(gè)外顯子跳躍，如圖8(a)． PLCB1基因存在可變起始外顯子產(chǎn)生的可變剪接轉(zhuǎn)錄本，且該轉(zhuǎn)錄本特異地在LUSC中表達(dá)，如圖8(b)．

表4 驅(qū)動(dòng)基因中LUAD亞型特異的差異可變剪接轉(zhuǎn)錄本

表5 驅(qū)動(dòng)基因中LUSC亞型特異的差異可變剪接轉(zhuǎn)錄本Table 5 Subtype-specific differentially expressed AS transcripts of cancer driven genes in LUSC

(續(xù)表5)

圖8 癌驅(qū)動(dòng)基因中LUAD或LUSC亞型特異的差異可變剪接舉例Fig.8 Examples of the subtype-specific differentially expressed AS transcripts of cancer driven genes in LUAD or LUSC

3 討 論

TCGA癌癥基因組數(shù)據(jù)庫為研究者提供了豐富的癌癥數(shù)據(jù)資源，以便深入地挖掘數(shù)據(jù)的生物意義．本研究利用TCGA數(shù)據(jù)庫中肺腺癌和肺鱗癌樣本的轉(zhuǎn)錄本表達(dá)數(shù)據(jù)，獲得肺腺癌和肺鱗癌樣本中所有的可變剪接轉(zhuǎn)錄本及對(duì)應(yīng)的PSI值，通過比較癌組織和癌旁組織中PSI值，獲得肺腺癌和肺鱗癌樣本與相應(yīng)癌旁組織相比存在表達(dá)差異的可變剪接轉(zhuǎn)錄本，以及肺腺癌和肺鱗癌樣本中共有和特有的差異可變剪接轉(zhuǎn)錄本及對(duì)應(yīng)基因．并對(duì)兩種亞型共有的癌特異和癌旁特異的差異可變剪接轉(zhuǎn)錄本對(duì)應(yīng)的基因進(jìn)行了功能富集．系統(tǒng)地分析和比較兩種肺癌亞型驅(qū)動(dòng)基因的可變剪接差異，為理解肺腺癌和肺鱗癌發(fā)生機(jī)制的異同提供新線索．

兩種肺癌亞型共有的差異可變剪接基因功能富集的結(jié)果發(fā)現(xiàn)癌特異的差異可變剪接基因富集在細(xì)胞周期調(diào)控、DNA代謝和RNA代謝等生物過程中，而癌旁特異的差異可變剪接基因富集在囊泡介導(dǎo)轉(zhuǎn)運(yùn)、多肽轉(zhuǎn)運(yùn)、脂質(zhì)轉(zhuǎn)運(yùn)和細(xì)胞凋亡等生物過程中，表明癌特異和癌旁特異的差異可變剪接基因的功能富集存在明顯的差異．該結(jié)果一方面體現(xiàn)了這些基因在癌癥發(fā)生和發(fā)展過程中功能的不同，另一方面由于數(shù)據(jù)類型和SUPPA軟件對(duì)可變剪接鑒定方法的局限，不能排除轉(zhuǎn)錄本表達(dá)水平對(duì)癌特異和癌旁特異的差異可變剪接轉(zhuǎn)錄本鑒定結(jié)果的影響．

文獻(xiàn)[6]報(bào)道，肺腺癌樣本中MET基因產(chǎn)生第14個(gè)外顯子跳躍的可變剪接轉(zhuǎn)錄本，且與基因的功能和癌癥的進(jìn)展相關(guān)．通過鑒定，本研究發(fā)現(xiàn)在肺腺癌樣本中MET基因產(chǎn)生第10個(gè)外顯子跳躍導(dǎo)致翻譯提前終止的轉(zhuǎn)錄本，并且通過結(jié)構(gòu)域分析發(fā)現(xiàn)這一可變剪接可能會(huì)影響其編碼蛋白的激酶結(jié)構(gòu)域．在分析的57例肺腺癌患者和50例肺鱗癌患者中，分別有38.6%和16.0%的患者具有該可變剪接轉(zhuǎn)錄本．MET基因中該可變剪接轉(zhuǎn)錄本的功能則需進(jìn)一步研究．

癌驅(qū)動(dòng)基因是癌癥發(fā)生和發(fā)展的關(guān)鍵調(diào)控基因，本研究重點(diǎn)分析和比較了驅(qū)動(dòng)基因在肺腺癌和肺鱗癌中可變剪接轉(zhuǎn)錄本的異同．ALDH1L1基因產(chǎn)生存在兩個(gè)外顯子跳躍的可變剪接轉(zhuǎn)錄本，并且該轉(zhuǎn)錄本僅特異地在肺腺癌中表達(dá)．PLCB1在肺鱗癌中存在一個(gè)由可變外顯子起始產(chǎn)生的轉(zhuǎn)錄本，結(jié)構(gòu)域分析表明該肺鱗癌亞型特異的轉(zhuǎn)錄本可能影響其編碼蛋白的結(jié)構(gòu)域．該研究為肺腺癌和肺鱗癌的治療提供新的線索以上分析的結(jié)果是基于有限數(shù)據(jù)量進(jìn)行的比較，隨著未來數(shù)據(jù)量的不斷增長，肺腺癌和肺鱗癌樣本中可變剪接差異的比較會(huì)更加細(xì)致準(zhǔn)確．

結(jié) 語

肺癌是高發(fā)病率和高死亡率的癌癥．通過分析TCGA數(shù)據(jù)庫中兩種主要的非小細(xì)胞肺癌亞型(肺腺癌和肺鱗癌)樣本中癌組織和相應(yīng)癌旁組織轉(zhuǎn)錄本的表達(dá)數(shù)據(jù)，獲得了肺腺癌和肺鱗癌樣本在癌組織與其對(duì)應(yīng)癌旁組織中存在表達(dá)差異的7種主要類型的可變剪接轉(zhuǎn)錄本，并對(duì)其中癌驅(qū)動(dòng)基因的差異可變剪接模式進(jìn)行了細(xì)致的比較分析．該研究將有助于深入理解兩種肺癌亞型的發(fā)病機(jī)理，為肺腺癌和肺鱗癌的靶向治療提供新的參考．

/ References：

[1] Siegel R L, Miller K D, Jemal A. Cancer statistics, 2015[J]. CA: A Cancer Journal for Clinicians, 2015, 65(1): 5-29.

[2] Chen Wanqing, Zheng Rongshou, Baade P D, et al. Cancer statistics in China, 2015[J]. CA: A Cancer Journal for Clinicians, 2016, 66(2): 115-132.

[3] Chang J T H, Lee Y M, Huang R S. The impact of the Cancer Genome Atlas on lung cancer[J]. Translational Research, 2015, 166(6): 568-585.

[4] Subramanian J, Govindan R. Lung cancer in never smokers: a review[J]. Journal of Clinical Oncology, 2007, 25(5): 561-570.

[5] Hammerman P S, Lawrence M S, Voet D, et al. Comprehensive genomic characterization of squamous cell lung cancers[J]. Nature, 2012, 489(7417): 519-525.

[6] Collisson E A, Campbell J D, Brooks A N, et al. Comprehensive molecular profiling of lung adenocarcinoma[J]. Nature, 2014, 511(7511): 543-550.

[7] Paez J G, J?nne P A, Lee J C, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy[J]. Science, 2004, 304(5676): 1497-1500.

[8] Antonicelli A, Cafarotti S, Indini A, et al. EGFR-targeted therapy for non-small cell lung cancer: focus on EGFR oncogenic mutation[J]. International Journal of Medical Sciences, 2013, 10(3): 320-330.

[9] Rekhtman N, Paik P K, Arcila M E, et al. Clarifying the spectrum of driver oncogene mutations in biomarker-verified squamous carcinoma of lung: lack of EGFR/KRAS and presence of PIK3CA/AKT1 mutations[J]. Clinical Cancer Research, 2012, 18(4): 1167-1176.

[10] Pan Qun, Shai O, Lee L J, et al. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing[J]. Nature Genetics, 2008, 40(12): 1413-1415.

[11] Chen Mo, Manley J L. Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches[J]. Nature Reviews Molecular Cell Biology, 2009, 10(11): 741-754.

[12] Wahl M C, Will C L, Lührmann R. The spliceosome: design principles of a dynamic RNP machine[J]. Cell, 2009, 136(4): 701-718.

[13] Venables J P, Klinck R, Koh C, et al. Cancer-associated regulation of alternative splicing[J]. Nature Structural & Molecular Biology, 2009, 16(6): 670-676.

[14] Chen J, Weiss W A. Alternative splicing in cancer: implications for biology and therapy[J]. Oncogene, 2015, 34(1): 1-14.

[15] Takehara T, Liu Xiaolong, Fujimoto J, et al. Expression and role of Bcl-xL in human hepatocellular carcinomas[J]. Hepatology, 2001, 34(1): 55-61.

[16] Alamancos GP, Pagès A, Trincado JL, et al. Leveraging transcript quantification for fast computation of alternative splicing profiles[J]. RNA, 2015, 21(9): 1521-1531.

[17] Huang Dawei, Sherman B T, Lempicki R A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources[J]. Nature Protocols, 2009, 4(1): 44-57.

[18] Supek F, Bo?njak M,kunca N, et al. REVIGO summarizes and visualizes long lists of gene ontology terms[J]. PLoS One, 2011, 6(7): e21800.

[19] Finn R D, Coggill P, Eberhardt R Y, et al. The Pfam protein families database: towards a more sustainable future[J]. Nucleic Acids Research, 2016, 44(D1): D279-D285.

[20] Wamg Z N, Lo H S, Yang H, et al. Computational analysis and experimental validation of tumor-associated alternative RNA splicing in human cancer[J]. Cancer Research, 2003, 63(3): 655-657.

[21] Pio R, Blanco D, Pajares M J, et al. Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer[J]. BMC Genomics, 2010, 11: 352.

[22] Planque C, Choi Y H, Guyetant S, et al. Alternative splicing variant of kallikrein-related peptidase 8 as an independent predictor of unfavorable prognosis in lung cancer[J]. Clinical Chemistry, 2010, 56(6): 987-997.

[23] Fan Y C, Min L, Chen H, et al. Alternative splicing isoform of T cell factor 4 K suppresses the proliferation and metastasis of non-small cell lung cancer cells[J]. Genetics and Molecular Research : GMR, 2015, 14(4): 14009-14018.

[24] Misquitta-Ali CM, Cheng E, O’Hanlon D, et al. Global profiling and molecular characterization of alternative splicing events misregulated in lung cancer[J]. Molecular and Cellular Biology, 2011, 31(1): 138-150.

[25] Pelosi G, Schianchi E, Dell’orto P, et al. Detecting cell-free circulating hTERT mRNA in the plasma may identify a subset of nonsmall cell lung cancer patients[J]. Laboratory Investigation, 2006, 448(1): 7-15.

[26] Fujiwara M, Kamma H, Wu W, et al. Expression and alternative splicing pattern of human telomerase reverse transcriptase in human lung cancer cells[J]. International Journal of Oncology, 2004, 24(4): 925-930.

【中文責(zé)編：晨 兮；英文責(zé)編：艾 琳】

Comparison of alternative splicing in lung adenocarcinoma and lung squamous carcinoma

Chen Mengzhu1, Wang Xiujie2?, and Xia Lixin1?

1) State Key Laboratory of Respiratory Disease for Allergy at Shenzhen University, Health Science Center,Shenzhen University, Shenzhen 518060, Guangdong Province, P.R.China 2) Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, P.R.China

Lung cancer is a common disease with high morbidity and mortality. Studies have shown that alternative splicing (AS) is closely associated with lung carcinogenesis, but the difference of alternative splicing between lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) remains unclear. Here, we analyze the RNA-Seq data of LUAD and LUSC from TCGA using SUPPA software, and find that skipping exon (SE) is the most frequently occurred AS transcript among seven main AS transcripts identified. By comparing tumor tissues with normal ones, we systematically identify differentially expressed AS transcripts in both LUAD and LUSC, of which around 60% are identical between these two subtypes. Functional analysis reveals that tumor-specific AS transcripts are mainly enriched among genes associated with cell cycle regulation, deoxyribonucleic acid metabolism and RNA metabolism, whereas normal-specific AS transcripts are enriched among genes mainly involved in vesicle-mediated transport, peptide transport and lipid transport. By comparing AS transcripts of cancer-driven genes between LUAD and LUSC, we identify some common and subtype-specific AS transcripts with cancer-driven potentiality.

bioinformatics; lung adenocarcinoma (LUAD); lung squamous carcinoma (LUSC); alternative splicing (AS); skipping exon (SE); alternative first exon (AF)

Received：2016-06-18；Revised：2016-11-14；Accepted：2016-11-20

Foundation：National High-Tech Research and Development Program (2012AA020409); Shenzhen Science and Technology Basic Research Foundation(JCYJ20150525092941055)

? Corresponding author：Professor Wang Xiujie. E-mail: xjwang@genetics.ac.cn; Professor Xia Lixin. E-mail:xialixin@126.com

：Chen Mengzhu, Wang Xiujie, Xia Lixin. Comparison of alternative splicing in lung adenocarcinoma and lung squamous carcinoma[J]. Journal of Shenzhen University Science and Engineering, 2017, 34(1): 33-45.(in Chinese)

R 734.2；Q 811.4

A

10.3724/SP.J.1249.2017.01033

國家高技術(shù)研究發(fā)展計(jì)劃資助項(xiàng)目(2012AA020409)；深圳市知識(shí)創(chuàng)新計(jì)劃基礎(chǔ)研究資助項(xiàng)目(JCYJ20150525092941055)

陳夢(mèng)竹(1982—)，女，深圳大學(xué)博士后研究人員．研究方向：生物信息學(xué)．E-mail：mzchen@szu.edu.cn

引 文：陳夢(mèng)竹，王秀杰，夏立新．肺腺癌和肺鱗癌中mRNA可變剪接特征的比較[J]. 深圳大學(xué)學(xué)報(bào)理工版，2017，34(1)：33-45.