李智，王碩，侯科佐，臧丹，鄭春雷，車曉芳，曲秀娟，劉云鵬

(中國醫(yī)科大學(xué)附屬第一醫(yī)院，沈陽110001)

胃癌組織ATP1A2的表達及機制探討

李智，王碩，侯科佐，臧丹，鄭春雷，車曉芳，曲秀娟，劉云鵬

(中國醫(yī)科大學(xué)附屬第一醫(yī)院，沈陽110001)

目的 觀察胃癌組織ATP酶α2亞基(ATP1A2)表達情況，探討其與黏著斑通路、細胞黏附分子、鈣離子通路、細胞外基質(zhì)相關(guān)信號通路的關(guān)系。方法 從癌癥基因組圖譜(TCGA)數(shù)據(jù)庫下載并預(yù)處理胃癌RNASeqV2數(shù)據(jù)、從人類腫瘤相關(guān)基因表達匯編(GEO)數(shù)據(jù)庫下載胃癌樣本數(shù)據(jù)集GSE62254的series matrix數(shù)據(jù)，通過TCGA數(shù)據(jù)集納入胃癌患者293例，通過GEO數(shù)據(jù)集納入胃癌患者300例。根據(jù)表達譜數(shù)據(jù)，將胃癌組織ATP1A2表達由低到高排序，按33%、67%者將數(shù)據(jù)三等分，低于33%者為低表達，高于67%者為高表達，兩者之間為中表達。分析不同ATP1A2表達與胃癌患者臨床病理參數(shù)的關(guān)系，比較低、中、高表達者中位生存時間，利用基因集富集分析方法預(yù)測ATP1A2相關(guān)的基因通路。結(jié)果 TCGA數(shù)據(jù)集納入的293例胃癌患者中，ATP1A2低、中、高表達者分別為95、99、99例；不同ATP1A2表達與胃癌患者性別、年齡無關(guān)(P均>0.05)，與T分期、N分期、pTNM分期及組織分化程度有關(guān)(P均<0.05)；ATP1A2低、中、高表達者中位生存時間分別為70、36.9和26個月，三者間比較P<0.01。GEO數(shù)據(jù)集納入的300例胃癌患者中，ATP1A2低、中、高表達者分別為99、102、99例；不同ATP1A2表達與胃癌患者性別、年齡、T分期、N分期、pTNM分期及Lauren分型均有關(guān)(P均<0.05)；ATP1A2高表達者的中位生存時間為31個月，較低表達和中表達者中位生存時間明顯延長(P<0.01)。ATP1A2高表達富集了黏著斑通路、細胞黏附分子、鈣離子信號通路、細胞外基質(zhì)等相關(guān)的基因通路(P<0.05或<0.01)。結(jié)論 胃癌組織中ATP1A2高表達，其高表達預(yù)示患者預(yù)后不良；ATP1A2高表達導(dǎo)致黏著斑通路、細胞黏附分子、鈣離子信號通路、細胞外基質(zhì)等基因通路異?？赡苁瞧渥饔脵C制。

胃癌；癌癥基因組圖譜；人類腫瘤相關(guān)基因表達匯編；ATP酶α2亞基

胃癌是臨床常見的惡性腫瘤之一[1]，占所有惡性腫瘤相關(guān)死亡的第二位。多數(shù)胃癌患者就診時已屬晚期，失去了手術(shù)治療機會，其生存時間很難超過1年[2]。侵襲和轉(zhuǎn)移是胃癌患者短期內(nèi)死亡的主要原因。Na+-K+-ATP酶是廣泛表達于上皮細胞的一種跨膜蛋白，是進行主動運輸?shù)妮d體蛋白，由α、β、γ三種亞基組成[3,4]。近年研究發(fā)現(xiàn)，Na+-K+-ATP酶的α亞單位在包括非小細胞肺癌、腸癌和惡性腦膠質(zhì)瘤等多種惡性腫瘤中異常表達，并參與腫瘤細胞的侵襲、轉(zhuǎn)移等過程[5～8]。但目前的研究主要集中于α1和α3兩種亞單位，有關(guān)α2亞單位的研究較少。ATP酶α2亞單位(ATP1A2)位于染色體1q23.2，包含23個外顯子，其功能缺失會造成Na+-K+-ATP酶功能受到抑制，導(dǎo)致細胞外K+聚集、細胞內(nèi)Na+聚集，并通過Na+/Ca2+交換使細胞內(nèi)Ca2+增多，從而引發(fā)一系列疾病。近年研究認為，ATP1A2基因改變與腦膠質(zhì)瘤、多囊卵巢綜合征等關(guān)系密切，但其作用機制尚不清楚。2015年12月～2016年1月，本研究利用在線高通量數(shù)據(jù)，采用生存分析、基因集富集分析(GSEA)等方法探討ATP1A2在胃癌組織中的表達及意義。

1 資料與方法

1.1 資料收集 利用Bioconductor/TCGAbiolinks函數(shù)包從癌癥基因組圖譜(TCGA)數(shù)據(jù)庫(https://tcga-data.nci.nih.gov/tcga/)下載并預(yù)處理胃癌RNASeqV2數(shù)據(jù)(level 3)。從NCBI的人類腫瘤相關(guān)基因表達匯編(GEO)數(shù)據(jù)庫(http://www.ncbi.nlm.nih.gov/geo)下載胃癌樣本數(shù)據(jù)集GSE62254的series matrix數(shù)據(jù)。僅保留臨床資料和生存信息完整的病例，TCGA數(shù)據(jù)集共納入胃癌患者293例(男184例、女109例，中位年齡65歲；TNM分期：Ⅰ期41例、Ⅱ期109例、Ⅲ期125例、Ⅳ期18例)。GSE62254數(shù)據(jù)集共納入胃癌患者300例(男199例、女101例，中位年齡62歲；TNM分期：Ⅰ期30例、Ⅱ期96例、Ⅲ期95例、Ⅳ期77例，分期不詳2例。

1.2 胃癌組織ATP1A2表達情況及其與患者預(yù)后的關(guān)系 TCGA數(shù)據(jù)庫的293例胃癌患者采用RNA測序方法，GSE62254數(shù)據(jù)庫的300例胃癌患者采用微陣列方法，分別檢測胃癌組織中ATP1A2的表達。以探針信號強度反映ATP1A2的相對表達量，若同一基因信號強度不同，取其中位數(shù)。根據(jù)ATP1A2表達譜數(shù)據(jù)，將ATP1A2的相對表達量由低到高排序，按33%、67%將數(shù)據(jù)三等分，低于33%者為低表達，高于67%者為高表達，兩者之間為中表達。分析不同ATP1A2表達與胃癌患者臨床病理參數(shù)的關(guān)系，比較不同ATP1A2表達者的中位生存時間。

1.3 ATP1A2基因集GSEA 采用GSEA2.2.2版軟件，根據(jù)TCGA數(shù)據(jù)集ATP1A2表達的中位值(原始讀長:48.5)將患者分為低表達和高表達，選用GSEA網(wǎng)站MsigDB數(shù)據(jù)庫中的c2.cp.kegg.v5.1.symbols.gmt數(shù)據(jù)集作為參照基因集，分析不同ATP1A2表達對各參照基因集的影響。采用缺省加權(quán)富集統(tǒng)計的方法進行GSEA，設(shè)置隨機組合次數(shù)為1 000次。

1.4 統(tǒng)計學(xué)方法 采用SPSS17.0統(tǒng)計軟件。相關(guān)性分析采用Spearman秩相關(guān)檢驗。采用Kaplan Meier法繪制生存曲線，Log-rank檢驗進行比較。P<0.05為差異有統(tǒng)計學(xué)意義。在GSEA中按P<0.05且錯誤發(fā)現(xiàn)率(FDR)<0.25的基因集為顯著富集基因集。

2 結(jié)果

2.1 不同ATP1A2表達與胃癌患者臨床病理參數(shù)的關(guān)系 TCGA數(shù)據(jù)庫293例胃癌患者中，ATP1A2低、中、高表達分別為95、99、99例，不同ATP1A2表達與胃癌患者性別、年齡無關(guān)(P均>0.05)，與T分期、N分期、pTNM分期及組織分化程度顯著相關(guān)(P均<0.01)，具體見表1。

GEO數(shù)據(jù)庫300例胃癌患者中，ATP1A2低、中、高表達分別為99、102、99例，不同ATP1A2表達與患者性別、年齡、T分期、N分期、pTNM分期、Lauren分型等顯著相關(guān)(P均<0.05)，具體見表2。

2.2 不同ATP1A2表達者生存情況 分別利用TCGA及GSE62254數(shù)據(jù)集進行生存分析。結(jié)果顯示，在TCGA數(shù)據(jù)集中ATP1A2低、中、高表達者的中位生存時間分別為70、36.9和26個月，三者比較P<0.01(見插頁Ⅰ圖2A)。GSE62254數(shù)據(jù)集分析亦得出相似的結(jié)果，ATP1A2高表達者中位生存時間為31個月，中表達和低表達者中位生存時間未達到，均長于高表達組，組間比較P均<0.01(見插頁Ⅰ圖2B)。

表1 不同ATP1A2表達與TCGA數(shù)據(jù)庫293例胃癌患者臨床病理參數(shù)的關(guān)系

表2 不同ATP1A2表達與GEO數(shù)據(jù)庫300例胃癌患者臨床病理參數(shù)的關(guān)系

注：*2例患者的T分期及TNM分期不詳，**3例患者的Lauren分型不詳，未被列入分析。

2.3 ATP1A2的功能基因集GSEA情況 GSEA結(jié)果顯示，ATP1A2高表達樣本富集到黏著斑通路(P<0.01，F(xiàn)DR=0.000 4)、細胞黏附分子(P<0.01，F(xiàn)DR=0.000 4)、鈣離子信號通路(P<0.01，F(xiàn)DR=0.001 0)、細胞外基質(zhì)(P<0.05，F(xiàn)DR=0.022 0)等相關(guān)基因通路。見插頁Ⅰ圖3。

3 討論

Na+-K+-ATP酶作為信號轉(zhuǎn)導(dǎo)蛋白，參與機體生理功能的維持和病理狀態(tài)的發(fā)展[9]。有研究證實，Na+-K+-ATP酶是一種潛在的抗腫瘤作用靶標[10]。Na+-K+-ATP酶的不同亞基在不同腫瘤組織中異常表達。α1亞單位在非小細胞肺癌和腦膠質(zhì)瘤組織中表達升高[6，8]，而在結(jié)腸癌組織中表達降低[7]。α3亞單位在肝細胞癌和結(jié)直腸癌組織中表達升高[7，11]。異常表達的α1和α3亞單位參與腫瘤細胞的增殖、侵襲和轉(zhuǎn)移等過程。α2亞單位主要表達于骨骼肌、心肌和腦組織，目前的研究主要集中于α2亞單位與中樞神經(jīng)系統(tǒng)疾病的關(guān)系[12,13]。但α2亞單位是否參與胃癌的發(fā)生、發(fā)展尚并不清楚。本研究利用TCGA和GEO數(shù)據(jù)集，結(jié)合生物信息學(xué)分析方法，證實ATP1A2基因在胃癌組織中高表達，在TNM分期高和組織分化程度差的胃癌組織中表達更高。進一步評價ATP1A2基因?qū)υu估胃癌患者預(yù)后的作用，結(jié)果顯示ATP1A2高表達患者生存期更短，兩個數(shù)據(jù)集的結(jié)果基本一致。上述結(jié)果說明，胃癌組織中ATP1A2高表達者預(yù)后不良，故ATP1A2表達可作為評價胃癌患者預(yù)后的指標。

細胞外基質(zhì)通路、黏著斑通路、細胞黏附分子基因通路與癌細胞的侵襲和轉(zhuǎn)移密切相關(guān)[14]。癌細胞侵襲和轉(zhuǎn)移的過程包括細胞發(fā)生上皮間質(zhì)轉(zhuǎn)化(EMT)、緊密連接松散、integrin等細胞黏附分子表達和功能發(fā)生異常、基質(zhì)微環(huán)境改變、金屬骨架蛋白重排，進而導(dǎo)致癌細胞離開原發(fā)灶，穿透基底膜及細胞外基質(zhì)，伴隨循環(huán)系統(tǒng)種植于遠處[15]。有研究指出，癌細胞EMT的發(fā)生與細胞內(nèi)鈣離子通路密切相關(guān)[16,17]。在表皮生長因子刺激下，當癌細胞發(fā)生EMT時，細胞內(nèi)Ca2+水平快速恢復(fù)到基線水平。本研究GESA方法發(fā)現(xiàn)，ATP1A2基因高表達樣本富集到了鈣離子信號通路和細胞外基質(zhì)、黏著斑通路、細胞黏附分子等基因通路，提示ATP1A2基因可能通過改變Na+-K+-ATP酶活性干預(yù)細胞內(nèi)鈣離子通路，改變腫瘤微環(huán)境，進而參與胃癌的侵襲和轉(zhuǎn)移過程。下階段可以利用胃癌臨床標本，聯(lián)合檢測ATP1A2基因和相應(yīng)信號通路蛋白的表達，并評估其預(yù)后價值。另外，循環(huán)腫瘤細胞是近階段研究熱點[18]。其產(chǎn)生與癌細胞間的連接松散、黏附異常、細胞外基質(zhì)微環(huán)境改變等關(guān)系密切[19～21]。ATP1A2基因水平與循環(huán)腫瘤細胞檢出量的關(guān)系也值得進一步探討。外泌體(exosome)是細胞主動向胞外分泌的、大小均一的囊泡樣小體[22]。目前已知其具有影響細胞外基質(zhì)、改變細胞連接狀態(tài)等功能[23,24]。已有多個研究顯示，ATP酶改變與exosome的產(chǎn)生和功能有關(guān)[25,26]，但尚無ATP1A2基因與exosome的相關(guān)研究，這是我們今后研究的方向之一。

綜上所述，本研究利用TCGA及GEO數(shù)據(jù)庫推測出胃癌組織中ATP1A2高表達，其表達變化參與胃癌的發(fā)生、發(fā)展，并可作為預(yù)測患者預(yù)后的指標；ATP1A2高表達導(dǎo)致黏著斑通路、細胞黏附分子、鈣離子信號通路、細胞外基質(zhì)等基因通路異?？赡苁瞧渥饔脵C制。

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Expression of ATP1A2 in gastric cancer and its mechanism

LIZhi,WANGShuo,HOUKezuo,ZANGDan,ZHENGChunlei,CHEXiaofang,QUXiujuan,LIUYunpeng

(TheFirstHospitalofChinaMedicalUniversity,Shenyang110001,China)

Objective To observe the expression of ATP1A2 in gastric cancer tissues and to investigate the correlations of ATP1A2 expression with focal adhesion, cell adhesion molecules, calcium signaling and ECM receptor interaction pathways. Methods TCGA dataset which included 293 cases, and GEO dataset GSE62254 which included 300 cases, were collected. ATP1A2 expression was trisected into 3 levels: low, median, and high. The cutoff was set to 0.33 and 0.67. The relationships between ATP1A2 and clinicopathological characteristics as well as prognosis were assessed. GSEA was used to predict the functional gene sets modulated by ATP1A2. Results There were 95 cases of low expression, 99 of median expression and 99 of high expression in TCGA dataset. The expression of ATP1A2 was significantly associated with T stage, lymph node metastasis, TNM stage and differentiation (allP<0.01), but was not associated with gender and age (allP>0.05). The median survival durations were 70, 36.9, and 26 months in low, median, and high ATP1A2 expression subgroups, respectively. The P value was less than 0.05 in the log-ranked test. While in GSE 62254 dataset, there were 99 low expressed, 102 median expressed and 99 high expressed cases. Different expression of ATP1A2 was significantly associated with gender, age, T stage, N stage, pTNM stage and Lauren types (allP<0.05). The median survival duration in the high ATP1A2 subgroup was 31 months, while it was much longer in the median and low expression subgroups (P<0.01). In GSEA, focal adhesion, cell adhesion molecules, calcium signaling, and extracellular matrix receptor interaction pathways were enriched in samples with high ATP1A2 expression (P<0.05 orP<0.01). Conclusions ATP1A2 is highly expressed in gastric cancer tissues and the high expression indicates poor prognosis. The underlying mechanism might be that the high expression of ATP1A2 causes the abnormity of focal adhesion, cell adhesion molecules, calcium signaling and extracellular matrix receptor interaction pathways.

gastric carcinoma; cancer genome atlas; human cancer-related gene expression assembly; ATP1A2

國家自然科學(xué)基金資助項目(81302023)；“重大新藥創(chuàng)制”科技重大專項課題(2013ZX09303002)；遼寧省科學(xué)技術(shù)計劃項目(2014225013)。

李智(1978-)，男，副教授，研究方向為腫瘤生物信息學(xué)。E-mail： lizhi2018@hotmail.com

劉云鵬(1959-)，男，教授，研究方向為腫瘤分子生物學(xué)、生物信息學(xué)。E-mail： cmuliuyunpeng@hotmail.com

10.3969/j.issn.1002-266X.2016.24.002

R735.2

A

1002-266X(2016)24-0005-04

2016-01-12)