農(nóng)順強(qiáng) 陳曉昊 許桂丹 韋武均 彭彬 周律 鄧益斌
【摘要】?目的?對(duì)公共基因表達(dá)數(shù)據(jù)庫(kù)(GEO)中的HBV相關(guān)肝癌數(shù)據(jù)進(jìn)行生物信息學(xué)分析,探討長(zhǎng)鏈非編碼RNA(lncRNA)與mRNA在HBV相關(guān)肝癌中的作用。方法?從GEO數(shù)據(jù)庫(kù)下載HBV相關(guān)肝癌數(shù)據(jù)集,利用相應(yīng)R包對(duì)數(shù)據(jù)集的差異表達(dá)基因進(jìn)行計(jì)算與比較,獲取差異基因并構(gòu)建lncRNA-mRNA共表達(dá)網(wǎng)絡(luò)、蛋白質(zhì)相互作用(PPI)網(wǎng)絡(luò)和模塊分析;繼而對(duì)共表達(dá)網(wǎng)絡(luò)中的mRNA進(jìn)行基因本體論(GO)功能注釋和京都基因與基因組百科全書(KEGG)路徑分析;并分析與關(guān)鍵lncRNA高度相關(guān)的mRNA與患者生存曲線的相關(guān)性。結(jié)果?通過(guò)基因分析獲得HBV相關(guān)肝癌高度相關(guān)的差異lncRNA 30個(gè),mRNA 676個(gè)。共表達(dá)的mRNA主要參與的GO通路有細(xì)胞黏附、細(xì)胞運(yùn)動(dòng)的負(fù)調(diào)控、生物黏附、酶聯(lián)受體蛋白信號(hào)通路、細(xì)胞趨化性;KEGG通路主要有類固醇激素生物合成、視黃醇代謝、PI3K-Akt信號(hào)通路、ECM-受體相互作用、化學(xué)致癌作用、黏著斑、Rap1信號(hào)通路等。與關(guān)鍵lncRNA(DNM3OS、HAND2-AS1、HELLPAR、AC126118.1、FAM230E、LINC01139、LINC01943、AL022344.1和PCAT7)高度相關(guān)的mRNA(GGT5、CEP55、DPT、TEK、TRIP13、STAB2、ESM1和MS4A1)與患者生存曲線有顯著相關(guān)性(P<0.01)。結(jié)論?lncRNA (DNM3OS、HAND2-AS1、HELLPAR、AC126118.1、FAM230E、LINC01139、LINC01943、AL022344.1和PCAT7)對(duì)HBV相關(guān)肝癌的發(fā)生發(fā)展及預(yù)后具有重要作用,為HBV相關(guān)HCC發(fā)生發(fā)展的機(jī)制研究、預(yù)后指標(biāo)、藥物治療靶點(diǎn)的選擇等提供參考。
【關(guān)鍵詞】?乙肝病毒;肝細(xì)胞癌;lncRNA-mRNA共表達(dá)網(wǎng)絡(luò);生物標(biāo)志物
中圖分類號(hào):R735.7?文獻(xiàn)標(biāo)志碼:A?DOI:10.3969/j.issn.1003-1383.2021.01.004
【Abstract】?Objective?To perform bioinformatics analysis on HBV related liver cancer data in public gene expression omnibus (GEO), so as to investigate the role of long non-coding RNA (lncRNA) and mRNA in HBV related hepatocellular carcinoma(HCC).Methods?Data set of HBV related HCC were downloaded from GEO, and differentially expressed genes in the data set were calculated and compared by corresponding R packets to obtain differentially expressed genes and construct lncRNA-mRNA co-expression network, protein-protein interaction (PPI) network and module analysis. And then, gene ontology (GO) functional annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were performed on mRNA in co-expression networks, and correlation between mRNA highly related to key lncRNA and patient survival curve was analyzed.Results?By genetic analysis, 30 highly correlated differential lncRNAs and 676 mRNAs were obtained for HBV related HCC. Main GO pathways involved in co-expressed mRNA were cell adhesion, negative regulation of cell movement, bioadhesion, enzyme-linked receptor protein signaling pathway and chemotaxis. KEGG pathways included steroid hormone biosynthesis, retinol metabolism, PI3K-Akt signaling pathway, ECM-receptor interaction, chemical carcinogenesis, focal adhesion and Rap1 signaling pathway, etc. mRNAs (GGT5, CEP55, DPT, TEK, TRIP13, STAB2, ESM1 and MS4A1) that highly co-expressed with the key lncRNAs (DNM3OS, HAND2-AS1, HELLPAR, AC126118.1, FAM230E, LINC01139, LINC01943, AL022344.1 and PCAT7) were significantly correlated with the survival curve of HBV related HCC patient(P < 0.01).Conclusion?lncRNA (DNM3OS, HAND2-AS1, HELLPAR, AC126118.1, FAM230E, LINC01139, LINC01943, AL022344.1 and PCAT7) plays a crucial role in the occurrence, development and prognosis of HBV-related HCC, and provide reference for the study of mechanism, prognostic index, and selection of drug therapeutic targets, etc.
【Key words】?HBV; HCC; lncRNA-mRNA co-expression network; biomarker
肝細(xì)胞癌(hepatocellular carcinoma,HCC)是常見的惡性腫瘤之一,且惡性程度較高,其發(fā)病率居全球第六位、病死率居第四位[1]。慢性乙型肝炎病毒(HBV)感染是大多數(shù)高危HCC地區(qū)的主要危險(xiǎn)因素。HBV參與肝細(xì)胞的癌變、侵襲和轉(zhuǎn)移,在肝癌的發(fā)生發(fā)展中起著至關(guān)重要的作用[2~4]。由于缺乏特征性臨床表現(xiàn),HCC的大部分診斷多處于晚期,預(yù)后較差。盡管部分文獻(xiàn)報(bào)道了一些用于早期診斷的血清生物標(biāo)志物,但結(jié)果并不十分令人滿意[5~7]。因此,探求新的早期發(fā)現(xiàn)與早期干預(yù)標(biāo)志物,對(duì)于改善肝癌患者預(yù)后和提高其長(zhǎng)期生存率尤為重要。
長(zhǎng)鏈非編碼RNA(lncRNA)一般是指長(zhǎng)度大于200個(gè)核苷酸(nucleotide,nt),缺乏或者僅有微弱蛋白編碼能力的RNA[8]。lncRNA以多種方式調(diào)節(jié)表觀遺傳、轉(zhuǎn)錄及轉(zhuǎn)錄后、翻譯和翻譯后水平的基因表達(dá)[9~10],積極參與腫瘤生長(zhǎng)、轉(zhuǎn)移和復(fù)發(fā)等多方面的調(diào)節(jié)[11~12]。lncRNA已經(jīng)被證實(shí)與肝癌細(xì)胞的增殖、凋亡、侵襲和轉(zhuǎn)移、血管生成及預(yù)后密切相關(guān)[13~14],參與肝癌進(jìn)展過(guò)程中的各種生物學(xué)過(guò)程。例如,肝癌中表達(dá)上調(diào)的ZEB1-AS1通過(guò)調(diào)節(jié)上皮-間質(zhì)轉(zhuǎn)化誘導(dǎo)標(biāo)志物的表達(dá)水平,參與HCC中的腫瘤生長(zhǎng)和轉(zhuǎn)移,且高表達(dá)ZEB1-AS1的患者與高轉(zhuǎn)移復(fù)發(fā)和預(yù)后不良相關(guān)[15]。HULC在HCC中上調(diào),與HBV感染及HCC患者腫瘤生長(zhǎng)相關(guān),并與其腫瘤分級(jí)、轉(zhuǎn)移和耐藥性相關(guān)[11]。雖然與HCC的lncRNA相關(guān)研究在逐日增多,但lncRNA在HBV相關(guān)肝癌發(fā)生發(fā)展進(jìn)程中的作用及機(jī)制研究仍舊十分局限。
芯片技術(shù)、二代測(cè)序和其他基因檢測(cè)技術(shù)及生物信息學(xué)的發(fā)展,為疾病的診斷和治療靶點(diǎn)的發(fā)掘提供了新的技術(shù)手段[16]。本研究利用生物信息學(xué)方法,從GEO數(shù)據(jù)庫(kù)中下載獲得HBV相關(guān)肝癌基因數(shù)據(jù),構(gòu)建lncRNA-mRNA共表達(dá)網(wǎng)絡(luò),獲取關(guān)鍵lncRNA,通過(guò)基因本體論(gene ontology,GO)和京都基因與基因組百科全書(kyoto encyclopedia of genes and genomes,KEGG)分析HBV相關(guān)肝癌共表達(dá)mRNA所富集的信號(hào)通路,分析與關(guān)鍵lncRNA共表達(dá)的mRNA及HBV相關(guān)肝癌患者生存時(shí)間的相關(guān)性,探究關(guān)鍵lncRNA在肝癌發(fā)生、發(fā)展進(jìn)程中的作用機(jī)制,從而為lncRNA在作為HBV相關(guān)肝癌的診斷、預(yù)后和治療靶點(diǎn)的潛在生物標(biāo)志物提供新的見解及有價(jià)值的信息。
1?資料與方法
1.1?數(shù)據(jù)材料
從美國(guó)國(guó)立生物技術(shù)信息中心(national center for biotechnology information,NCBI)的GEO數(shù)據(jù)庫(kù)中,檢索HBV相關(guān)肝癌芯片表達(dá)數(shù)據(jù)集。選取GSE55092、GSE19665芯片表達(dá)數(shù)據(jù)集行后續(xù)分析。所選數(shù)據(jù)集均基于Affmetrix公司[HG-U133_Plus_2]平臺(tái),其中GSE55092包括49例肝癌和91例癌旁正常樣本,GSE19665包括肝癌和癌旁正常樣本各5例。以GSE14520的218例HBV相關(guān)肝癌數(shù)據(jù)做共表達(dá)mRNA的生存分析。
1.2?篩選差異表達(dá)基因
首先,我們從Affymetrix官方網(wǎng)站(www.affymetrix.com)下載芯片探針序列FASTA格式文件。然后,使用SeqMap工具將HG-U133_Plus_2芯片的探針序列與GENCODE的人類基因組(GRCh38)(https://www.gencodegenes.org/)(release 30)和lncRNA基因序列進(jìn)行比對(duì),獲取非編碼RNA(non-coding RNA)和信使RNA(mRNA)探針信息。使用R語(yǔ)言中的limma[17]包標(biāo)準(zhǔn)化數(shù)據(jù),將HBV肝癌組與HBV正常組比較,以差異倍數(shù)2倍(|logFC|>1),P<0.05為標(biāo)準(zhǔn),獲取差異基因;使用R語(yǔ)言中Robust Rank Aggreg包[18],根據(jù)log Fold Change值對(duì)基因進(jìn)行排序,并選出兩個(gè)數(shù)據(jù)集中都存在差異表達(dá)的基因。
1.3?基因共表達(dá)分析
選取在2個(gè)數(shù)據(jù)集中,HBV肝癌和HBV正?;颊咧芯町惐磉_(dá)的lncRNA和mRNA,納入共表達(dá)分析網(wǎng)絡(luò)。根據(jù)基因表達(dá)值,計(jì)算所有篩選出的差異表達(dá)lncRNA與mRNA兩兩間的皮爾森相關(guān)系數(shù),選取相關(guān)系數(shù)絕對(duì)值|r|>0.75,且校正后P<0.05的lncRNA和mRNA對(duì),導(dǎo)入Cytoscape 3.72構(gòu)建共表達(dá)網(wǎng)絡(luò)。
1.4?差異表達(dá)基因功能和通路富集分析
利用DAVID(https://david.ncifcrf.gov/)對(duì)共表達(dá)的差異基因進(jìn)行GO富集分析及KEGG分析,以P<0.05為富集標(biāo)準(zhǔn)。
1.5?蛋白質(zhì)相互作用網(wǎng)絡(luò)構(gòu)建
將共表達(dá)的mRNA,用在線數(shù)據(jù)String(https://string-db.org/)進(jìn)行蛋白質(zhì)互作(PPI)分析及MCODE模塊識(shí)別,將結(jié)果網(wǎng)絡(luò)引入Cytoscape進(jìn)行可視化,選取基因模塊,利用DAVID網(wǎng)站進(jìn)行信號(hào)通路分析。
1.6?生存分析
使用R語(yǔ)言的“Survival”包行Kaplan-Meier生存分析,分析與lncRNA存在共表達(dá)關(guān)系的差異mRNA和HBV肝癌患者生存時(shí)間相關(guān)性,檢驗(yàn)方法為L(zhǎng)og-rankχ2,以α=0.05為檢驗(yàn)水準(zhǔn)。
2?結(jié)?果
2.1?差異基因篩選
根據(jù)篩選條件,在數(shù)據(jù)集GSE55092中得到差異mRNA 1182個(gè),上調(diào)基因412個(gè),下調(diào)基因770個(gè);差異lncRNA 103個(gè),上調(diào)基因59個(gè),下調(diào)基因44個(gè)。在數(shù)據(jù)集GSE19665中得到差異mRNA 2147個(gè),上調(diào)基因833個(gè),下調(diào)基因1314個(gè);差異lncRNA 333個(gè),上調(diào)基因195個(gè),下調(diào)基因138個(gè)(圖1)。根據(jù)log Fold Change值,對(duì)2個(gè)數(shù)據(jù)集差異基因進(jìn)行排序,然后對(duì)2個(gè)數(shù)據(jù)集進(jìn)行Robust Rank Aggreg分析,得到HBV肝癌相關(guān)可信度高的差異lncRNA 30個(gè)、mRNA 676個(gè)。
2.2?LncRNA-mRNA共表達(dá)網(wǎng)絡(luò)分析
通過(guò)共表達(dá)網(wǎng)絡(luò)構(gòu)建(共表達(dá)相關(guān)系數(shù)絕對(duì)值|r|>0.75,且校正后P<0.05),發(fā)現(xiàn)有17個(gè)lncRNA及82個(gè)mRNA具有高度共表達(dá)關(guān)系。其中DNM3OS、HAND2-AS1、HELLPAR居于共表達(dá)網(wǎng)絡(luò)核心位置,與眾多mRNA均具有共表達(dá)關(guān)系(圖2)。
2.3?差異基因功能富集及通路富集分析
對(duì)共表達(dá)的mRNA,通過(guò)DAVID進(jìn)行GO分析發(fā)現(xiàn),差異基因主要參與多細(xì)胞生物過(guò)程的負(fù)向調(diào)控、細(xì)胞黏附、細(xì)胞運(yùn)動(dòng)的負(fù)調(diào)控、生物黏附、酶聯(lián)受體蛋白信號(hào)通路、細(xì)胞趨化性、單加氧酶活性跨膜受體蛋白酪氨酸激酶活性等。KEGG分析發(fā)現(xiàn),共表達(dá)差異基因主要參與類固醇激素生物合成、補(bǔ)體和凝血級(jí)聯(lián)、視黃醇代謝信號(hào)通路、ECM-受體相互作用、化學(xué)致癌作用、黏著斑、Rap1信號(hào)通路等(圖3)。
2.4?共表達(dá)差異mRNA的蛋白質(zhì)互作網(wǎng)絡(luò)分析
將PPI網(wǎng)絡(luò)引入Cytoscape進(jìn)行可視化,使用MCODE插件進(jìn)行模塊識(shí)別,模塊1包含28個(gè)基因及357連線,模塊2包含10個(gè)基因及29連線(圖4)。對(duì)模塊進(jìn)行KEGG通路富集,結(jié)果顯示模塊1主要與卵母細(xì)胞減數(shù)分裂、細(xì)胞周期相關(guān);模塊2主要與視黃醇代謝、化學(xué)致癌作用、藥物代謝-細(xì)胞色素P450、代謝途徑、類固醇激素生物合成、細(xì)胞色素P450對(duì)異生素的代謝、亞油酸代謝、藥物代謝-其他酶等信號(hào)通路相關(guān)(表1)。
2.5?與關(guān)鍵lncRNA共表達(dá)的mRNA與HCC患者生存曲線相關(guān)性
為了進(jìn)一步探討上述關(guān)鍵lncRNA在HBV相關(guān)肝癌發(fā)生、發(fā)展中的作用,使用GSE14520數(shù)據(jù)分析與關(guān)鍵lncRNA存在高相關(guān)性的mRNA與HBV相關(guān)肝癌患者生存預(yù)后關(guān)系。K-M曲線結(jié)果顯示,與9個(gè)關(guān)鍵lncRNA(DNM3OS、HAND2-AS1、HELLPAR、AC126118.1、FAM230E、LINC01139、LINC01943、AL022344.1和PCAT7)共表達(dá)的16個(gè)mRNA與HBV相關(guān)肝癌患者存在顯著相關(guān)性(P<0.05),其中8個(gè)mRNA存在高度相關(guān)性(P<0.01)。見表2、圖5。
3?討?論
肝癌是一種發(fā)病率及病死率高的、世界范圍的、常見的消化系統(tǒng)腫瘤。雖然有多種治療方法,但其臨床預(yù)后仍很差;鑒于早期診斷對(duì)生存的重要性,急需更準(zhǔn)確和更特異的診斷及預(yù)后標(biāo)志物。越來(lái)越多的研究報(bào)道lncRNA在HBV相關(guān)肝細(xì)胞癌發(fā)生中起著至關(guān)重要的作用[19],然而,關(guān)于lncRNAs作為預(yù)測(cè)性生物標(biāo)志物和治療靶點(diǎn)的研究仍然非常有限。
LncRNA通過(guò)表觀遺傳修飾、轉(zhuǎn)錄調(diào)控等多種方式影響其靶基因mRNA的表達(dá)[12]。LncRNA-mRNA共表達(dá)分析是鑒定lncRNA潛在靶基因和進(jìn)一步研究lncRNA在疾病中生物學(xué)功能的常用方法。本文運(yùn)用生物信息學(xué)技術(shù),對(duì)NCBI-GEO數(shù)據(jù)庫(kù)中HBV相關(guān)肝癌的數(shù)據(jù)進(jìn)行綜合分析,篩選出HBV相關(guān)肝癌可信度高的差異lncRNA及mRNA表達(dá)譜,并對(duì)差異表達(dá)基因行l(wèi)ncRNA-mRNA共表達(dá)分析。GO功能富集分析發(fā)現(xiàn)共表達(dá)網(wǎng)絡(luò)中的mRNA主要影響多細(xì)胞生物過(guò)程的負(fù)向調(diào)控、細(xì)胞黏附、細(xì)胞運(yùn)動(dòng)的負(fù)調(diào)控、生物黏附、細(xì)胞周期進(jìn)程、酶聯(lián)受體蛋白信號(hào)通路、細(xì)胞趨化性、單加氧酶活性、跨膜受體蛋白酪氨酸激酶活性等。以上結(jié)果提示,共表達(dá)差異mRNA在HBV相關(guān)肝癌的細(xì)胞增殖和轉(zhuǎn)移中起重要作用。KEGG分析發(fā)現(xiàn),共表達(dá)差異基因主要參與類固醇激素生物合成、補(bǔ)體和凝血級(jí)聯(lián)、視黃醇代謝、PI3K-Akt信號(hào)通路、ECM-受體相互作用、化學(xué)致癌作用、黏著斑、Rap1信號(hào)通路等。以上顯著富集的與代謝相關(guān)的信號(hào)通路,包括類固醇激素生物合成、視黃醇代謝、化學(xué)致癌作用等信號(hào)通路主要為涉及細(xì)胞色素P450酶系統(tǒng)(CYP)的蛋白編碼RNA;CYP在藥物代謝中起著重要作用,參與內(nèi)源性和外源性物質(zhì)的代謝,包括藥物和環(huán)境化合物[20],其通過(guò)化學(xué)致癌物的生物活性催化、癌癥治療藥物的激活、作為癌癥治療的靶點(diǎn)和代謝酶等多種機(jī)制參與多種癌癥[21]。黏著斑是一種膜相關(guān)的高分子集合體,將肌動(dòng)蛋白、細(xì)胞骨架和整合素與細(xì)胞外基質(zhì)連接起來(lái),其在維持細(xì)胞張力和細(xì)胞生存的信號(hào)轉(zhuǎn)導(dǎo)中起著重要作用,參與調(diào)節(jié)腫瘤細(xì)胞的EMT過(guò)程,調(diào)控腫瘤的侵襲和轉(zhuǎn)移[22~23]。PI3K-Akt通路調(diào)節(jié)腫瘤細(xì)胞的增殖和存活,其活性異常不僅能導(dǎo)致細(xì)胞惡性轉(zhuǎn)化,而且與腫瘤細(xì)胞的遷移、黏附、腫瘤血管生成以及細(xì)胞外基質(zhì)的降解等相關(guān)[24~25]。有研究表明,HBV感染通過(guò)促進(jìn)NF-κB與IAP家族成員細(xì)胞凋亡蛋白2(cIAP2)啟動(dòng)子的結(jié)合激活PI3K/AKT/NF-κB信號(hào)通路,在肝臟中誘導(dǎo)cIAP2表達(dá)而致肝癌[26]。細(xì)胞外基質(zhì)(ECM)由基底膜細(xì)胞間質(zhì)組成,可影響細(xì)胞分化、增殖、黏附、形態(tài)發(fā)生和表型表達(dá)等,在腫瘤的轉(zhuǎn)移中起重要的組織屏障作用。腫瘤細(xì)胞通過(guò)與ECM中組分黏附后激活或分泌降解酶來(lái)消耗基質(zhì),形成局部溶解的腫瘤細(xì)胞轉(zhuǎn)移通道。膠原在ECM影響腫瘤細(xì)胞生物學(xué)行為的功能密切相關(guān)[27],膠原降解是腫瘤細(xì)胞侵襲和轉(zhuǎn)移的關(guān)鍵環(huán)節(jié)之一,ECM中膠原蛋白及其他蛋白主要被基質(zhì)金屬蛋白酶(MMPs)降解,如肝細(xì)胞癌組織MMP-2高于癌旁組織,可用于判斷肝癌的侵襲轉(zhuǎn)移。Ras蛋白是原癌基因ras的表達(dá)產(chǎn)物,Ras相關(guān)蛋白1(Rap1)為一種小的GTPase,通過(guò)調(diào)節(jié)整合素和其他黏附分子在不同細(xì)胞類型中的功能,在細(xì)胞-細(xì)胞和細(xì)胞-基質(zhì)相互作用中起主導(dǎo)作用,它控制著細(xì)胞增殖、黏附、細(xì)胞間連接形成和細(xì)胞極性等多種過(guò)程;Rap1信號(hào)通路的異常激活,可促進(jìn)肝癌細(xì)胞的增殖和侵襲轉(zhuǎn)移[28]。這進(jìn)一步說(shuō)明本研究中發(fā)現(xiàn)的lncRNA 及其共表達(dá)差異mRNA在乙肝相關(guān)肝癌的發(fā)生、發(fā)展中起重要作用,研究這些通路將有助于闡明HBV相關(guān)肝癌增殖和侵襲的機(jī)制,并有助于預(yù)測(cè)腫瘤的進(jìn)展和病人的預(yù)后。
本研究中發(fā)現(xiàn)的關(guān)鍵lncRNA在肝癌或其他腫瘤中有不同程度的報(bào)道。如DNM3OS在口腔癌、胃癌及其細(xì)胞系中表達(dá)上調(diào),與細(xì)胞活力及侵襲轉(zhuǎn)移相關(guān)[29~30],本研究發(fā)現(xiàn)DNM3OS在HBV相關(guān)肝癌中表達(dá)下調(diào),其機(jī)理還有待進(jìn)一步研究。HAND2-AS1在肝癌組織及細(xì)胞系中均呈低表達(dá),其過(guò)表達(dá)降低了HCC細(xì)胞的活力和增殖[31]。有研究發(fā)現(xiàn),HELLP綜合征相關(guān)長(zhǎng)鏈非編碼RNA(HELLPAR/LINC-HELLP)可激活大量與細(xì)胞周期有關(guān)的基因,通過(guò)改變?cè)鲋乘俾驶蛲顺黾?xì)胞周期而對(duì)滋養(yǎng)層細(xì)胞分化產(chǎn)生負(fù)面影響,減少增殖和侵襲,減少孕早期絨毛外滋養(yǎng)層細(xì)胞從增生型到浸潤(rùn)型的分化,且阻斷HELLP突變位點(diǎn)可降低絨毛外滋養(yǎng)層細(xì)胞的侵襲能力[32~33];與本研究中預(yù)測(cè)HBV相關(guān)肝癌中表達(dá)下調(diào)的HELLPAR可能通過(guò)細(xì)胞周期的調(diào)控影響HCC進(jìn)程一致。LINC01139在HCC組織和細(xì)胞系中過(guò)表達(dá),并且與晚期TNM分期、淋巴結(jié)轉(zhuǎn)移和HCC患者臨床預(yù)后不良有關(guān),敲低LINC01139可抑制HCC細(xì)胞的增殖、轉(zhuǎn)移;相關(guān)機(jī)制研究顯示,LINC01139可能充當(dāng)miR-30的海綿來(lái)上調(diào)MYBL2表達(dá)而表現(xiàn)出HCC的致癌作用[34]。PCAT7在前列腺癌、非小細(xì)胞肺癌和鼻咽癌等惡性腫瘤中高表達(dá),與腫瘤的侵襲轉(zhuǎn)移相關(guān)[35~37],本研究發(fā)現(xiàn)PCAT7在HBV相關(guān)肝癌中表達(dá)下調(diào),其機(jī)理還有待進(jìn)一步研究。
本研究發(fā)現(xiàn)另4個(gè)lncRNA可能在HBV相關(guān)肝癌中有重要作用,并預(yù)測(cè)其功能。反義lncRNA可通過(guò)多種機(jī)制在轉(zhuǎn)錄水平或轉(zhuǎn)錄后水平調(diào)控相應(yīng)正義的mRNA,從而發(fā)揮生物學(xué)功能。AC126118.1是LRRFIP2的反義鏈lncRNA,LRRFIP2基因編碼的蛋白質(zhì)與MYD88一起結(jié)合胞質(zhì)尾部toll樣受體4(TLR4),其導(dǎo)致NF-κB信號(hào)傳導(dǎo)的激活;NF-κB在細(xì)胞增殖和凋亡的相關(guān)基因調(diào)控中起關(guān)鍵作用,與腫瘤的發(fā)生發(fā)展密切相關(guān)[38]。FAM230E是基因間長(zhǎng)鏈非編碼RNA基因家族成員,其序列相似性為230,該家族成員基因通過(guò)相似基因序列復(fù)制形成,特別是在人類染色體低拷貝重復(fù)序列(LCR)或節(jié)段性復(fù)制中,其功能尚未見相關(guān)報(bào)道[39~40]。另外本研究發(fā)現(xiàn)的長(zhǎng)鏈非編碼RNA LINC01943和AL022344.1也未見有相關(guān)文獻(xiàn)的報(bào)道,其在HBV相關(guān)肝癌中的功能及機(jī)制有待進(jìn)一步研究。
生存分析發(fā)現(xiàn),共表達(dá)網(wǎng)絡(luò)中16個(gè)mRNA與HBV相關(guān)肝癌患者的生存率顯著相關(guān),其中8個(gè)mRNA具有高度相關(guān)性。如細(xì)胞分裂蛋白調(diào)節(jié)因子1(PRC1)編碼微管相關(guān)蛋白,在有絲分裂和細(xì)胞周期調(diào)控中起重要作用,敲低PRC1與微管相關(guān)因子可抑制胞質(zhì)分裂,并顯著減輕HCC的發(fā)展和肝損傷,高表達(dá)PRC1增加了HCC細(xì)胞化療耐藥性,并與不良預(yù)后相關(guān)[41~42]。CEP55在細(xì)胞質(zhì)分裂中有重要作用,其可通過(guò)調(diào)節(jié)JAK2/STAT3/MMPs信號(hào)傳導(dǎo)來(lái)促進(jìn)HCC細(xì)胞遷移和侵襲[43]。DPT編碼一種細(xì)胞外基質(zhì)蛋白,該蛋白在細(xì)胞-基質(zhì)相互作用和基質(zhì)組裝中發(fā)揮作用;DPT在肝癌中低表達(dá)且其表達(dá)水平與癌癥轉(zhuǎn)移和患者預(yù)后密切相關(guān),其過(guò)表達(dá)顯著抑制了體外HCC細(xì)胞遷移和體內(nèi)肝內(nèi)轉(zhuǎn)移[44];DPT可能通過(guò)與TGF-β1相互作用或其他的潛在機(jī)制影響HCC的發(fā)生和進(jìn)展[45]。TEK編碼受體蛋白屬蛋白酪氨酸激酶Tie2家族,為內(nèi)皮細(xì)胞特異性受體,在調(diào)節(jié)血管生成和重塑中起重要作用;有研究顯示,TEK低表達(dá)的透明細(xì)胞腎細(xì)胞癌患者預(yù)后較差并具有良好的診斷區(qū)分性,有望成為新的腫瘤標(biāo)志物[46],這與我們?cè)贖BV相關(guān)HCC中的發(fā)現(xiàn)基本一致。STAB2基因編碼跨膜受體蛋白,其可作用于血管生成、細(xì)胞黏附或受體清除等生物過(guò)程;在腫瘤細(xì)胞和淋巴管內(nèi)皮細(xì)胞共培養(yǎng)的mRNA譜分析中,透明質(zhì)酸受體STAB2、血管生成因子apelin受體(APLNR)和糖基化酶MAN1A1呈低表達(dá),其參與腫瘤進(jìn)程機(jī)制可能為促進(jìn)腫瘤的淋巴轉(zhuǎn)移[47]。研究表明,內(nèi)皮細(xì)胞特異性分子-1(ESM1)在肝癌中過(guò)表達(dá),可作為預(yù)測(cè)HCC生長(zhǎng)和死亡的獨(dú)立危險(xiǎn)因素[48]。以上結(jié)果進(jìn)一步證實(shí)了本研究發(fā)現(xiàn)的lncRNA在HBV相關(guān)肝癌的發(fā)生、發(fā)展中有著重要作用。
生物信息學(xué)作為一門新興學(xué)科,目前已成為尋找癌癥診斷及治療靶點(diǎn)的一種分析方法。本研究通過(guò)生物信息學(xué)方法,綜合分析了lncRNA及其共表達(dá)mRNA在HBV相關(guān)肝癌發(fā)生發(fā)展中的作用,為HBV相關(guān)HCC發(fā)生發(fā)展的機(jī)制研究、預(yù)后指標(biāo)、藥物治療靶點(diǎn)的選擇等提供參考。
參?考?文?獻(xiàn)
[1]?BRAY F,F(xiàn)ERLAY J,SOERJOMATARAM I,et al.Global cancer statistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J].CA:A Cancer J Clin,2018,68(6):394-424.
[2]?LIU Y C,LU L F,LI C J,et al.Hepatitis B virus X protein induces RHAMM-dependent motility in hepatocellular carcinoma cells via PI3K-Akt-oct-1 signaling[J].Mol Cancer Res,2020,18(3):375-389.
[3]?ZHENG S,WU H,WANG F,et al.The oncoprotein HBXIP facilitates metastasis of hepatocellular carcinoma cells by activation of MMP15 expression[J].Cancer Manag Res,2019,11:4529-4540.
[4]?ZHA Y, YAO Q, LIU J S, et al. Hepatitis B virus X protein promotes epithelial-mesenchymal transition and metastasis in hepatocellular carcinoma cell line HCCLM3 by targeting HMGA2[J]. Oncol Lett, 2018,16(5):5709-5714.
[5]?ALI O M, EL AMIN H A, SHARKAWY Y L, et al. Golgi Protein 73 versus Alpha-Fetoprotein as a New Biomarker in Early Diagnosis of Hepatocellular Carcinoma[J]. Int J Gen Med, 2020,13:193-200.
[6]?CHO H J,BAEK G O,SEO C W,et al.Exosomal microRNA-4661-5p-based serum panel as a potential diagnostic biomarker for early-stage hepatocellular carcinoma[J].Cancer Med,2020,9(15):5459-5472.
[7]?MA X L, TANG W G, YANG M J, et al. Serum STIP1, a Novel Indicator for Microvascular Invasion, Predicts Outcomes and Treatment Response in Hepatocellular Carcinoma[J]. Front Oncol, 2020,10:511.
[8]?IYER M K, NIKNAFS Y S, MALIK R, et al. The landscape of long noncoding RNAs in the human transcriptome[J]. Nat Genet, 2015,47(3):199-208.
[9]?ZHANG X P, WANG W, ZHU W D, et al. Mechanisms and Functions of Long Non-Coding RNAs at Multiple Regulatory Levels[J]. Int J Mol Sci, 2019,20(22):5573.
[10]?PANDEY R R, KANDURI C. Transcriptional and Posttranscriptional Programming by Long Noncoding RNAs[J]. Prog Mol Subcell Biol, 2011,51:1-27.
[11]?KLEC C, GUTSCHNER T, PANZITT K, et al. Involvement of long non-coding RNA HULC (highly up-regulated in liver cancer) in pathogenesis and implications for therapeutic intervention[J]. Expert Opin Ther Targets, 2019,23(3):177-186.
[12]?HUANG Z, ZHOU J K, PENG Y, et al. The role of long noncoding RNAs in hepatocellular carcinoma[J]. Mol Cancer, 2020,19(1):77.
[13]?HUANG J F, GUO Y J, ZHAO C X, et al. Hepatitis B virus X protein (HBx)-related long noncoding RNA (lncRNA) down-regulated expression by HBx (Dreh) inhibits hepatocellular carcinoma metastasis by targeting the intermediate filament protein vimentin[J]. Hepatology, 2013,57(5):1882-1892.
[14]?XU D, YANG F, YUAN J H, et al. Long noncoding RNAs associated with liver regeneration 1 accelerates hepatocyte proliferation during liver regeneration by activating Wnt/β-catenin signaling[J]. Hepatology, 2013,58(2):739-751.
[15]?LAN T, CHANG L, WU L, et al. Downregulation of ZEB2-AS1 decreased tumor growth and metastasis in hepatocellular carcinoma[J]. Mol Med Rep, 2016,14(5):4606-4612.
[16]?魏超, 張曉, 高杰. 長(zhǎng)鏈非編碼RNA與mRNA在胰腺癌中的差異表達(dá)及其預(yù)后價(jià)值[J]. 癌變·畸變·突變, 2019,31(2):119-126,132.
[17]?RITCHIE M E, PHIPSON B, WU D, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies[J]. Nucleic Acids Res, 2015,43(7):e47.
[18]?BOLSTAD B M, IRIZARRY R A, STRAND M, et al. A comparison of normalization methods for high densityoligonucleotide array data based on variance and bias[J]. Bioinformatics, 2003, 19(2): 185-193.
[19]?CHAUHAN R, LAHIRI N. Tissue-?and Serum-Associated Biomarkers of Hepatocellular Carcinoma[J]. Biomark Cancer, 2016,8(Suppl 1):37-55.
[20]?TORNIO A, BACKMAN J T. Cytochrome P450 in Pharmacogenetics: An Update[J]. Adv Pharmacol, 2018,83:3-32.
[21]?ALZAHRANI A M, RAJENDRAN P. The Multifarious Link between Cytochrome P450s and Cancer[J]. Oxid Med Cell Longev, 2020,2020:3028387.
[22]?LIU C Y,LIN H H,TANG M J,et al.Vimentin contributes to epithelial-mesenchymal transition cancer cell mechanics by mediating cytoskeletal organization and focal adhesion maturation[J].Oncotarget,2015,6(18):15966-15983.
[23]?SHAH P P,F(xiàn)ONG M Y,KAKAR S S.PTTG induces EMT through integrin αVβ3-focal adhesion kinase signaling in lung cancer cells[J].Oncogene,2012,31(26):3124-3135.
[24]?MAYER I A,ARTEAGA C L.The PI3K/AKT pathway as a target for cancer treatment[J].Annu Rev Med,2016,67:11-28.
[25]?RUIZ-SAENZ A, DREYER C, CAMPBELL M R, et al. HER2 Amplification in Tumors Activates PI3K/Akt Signaling Independent of HER3[J]. Cancer Res, 2018,78(13):3645-3658.
[26]?LIAN J P,ZOU Y H,HUANG L,et al.Hepatitis B virus upregulates cellular inhibitor of apoptosis protein 2 expression via the PI3K/AKT/NF-κB signaling pathway in liver cancer[J].Oncol Lett,2020,19(3):2043-2052.
[27]?JABOńSKA-TRYPU A, MATEJCZYK M, ROSOCHACKI S. Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM) enzymes in collagen degradation, as a target for anticancer drugs[J]. J Enzyme Inhib Med Chem, 2016,31(sup1):177-183.
[28]?MO S J, HOU X, HAO X Y, et al. EYA4 inhibits hepatocellular carcinoma growth and invasion by suppressing NF-κB-dependent RAP1 transactivation[J]. Cancer Commun (Lond), 2018,38(1):9.
[29]?FANG X,TANG Z,ZHANG H,et al.Long non-coding RNA DNM3OS/miR-204-5p/HIP1 axis modulates oral cancer cell viability and migration[J].J Oral Pathol Med,2020,49(9):865-875.
[30]?WANG S, NI B, ZHANG Z, et al. Long non-coding RNA DNM3OS promotes tumor progression and EMT in gastric cancer by associating with Snail[J]. Biochem Biophys Res Commun, 2019,511(1):57-62.
[31]?BI H Q,LI Z H,ZHANG H.Long noncoding RNA HAND2-AS1 reduced the viability of hepatocellular carcinoma via targeting microRNA-300/SOCS5 axis[J].Hepatobiliary Pancreat Dis Int,2020,19(6):567-574.
[32]?VAN DIJK M,VISSER A,BUABENG K M,et al.Mutations within the LINC-HELLP non-coding RNA differentially bind ribosomal and RNA splicing complexes and negatively affect trophoblast differentiation[J].Hum Mol Genet,2015,24(19):5475-5485.
[33]?VAN DIJK M,THULLURU H K,MULDERS J,et al.HELLP babies link a novel lincRNA to the trophoblast cell cycle[J].J Clin Invest,2012,122(11):4003-4011.
[34]?LI Z B, CHU H T, JIA M, et al. Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family[J]. Biochem Biophys Res Commun, 2020,525(3):581-588.
[35]?LANG C, DAI Y, WU Z, et al. SMAD3/SP1 complex-mediated constitutive active loop between lncRNA PCAT7 and TGF-β signaling promotes prostate cancer bone metastasis[J]. Mol Oncol, 2020,14(4):808-828.
[36]?LIU Q, WU Y, XIAO J, et al. Long Non-Coding RNA Prostate Cancer-Associated Transcript 7 (PCAT7) Induces Poor Prognosis and Promotes Tumorigenesis by Inhibiting mir-134-5p in Non-Small-Cell Lung (NSCLC)[J]. Med Sci Monit, 2017,23:6089-6098.
[37]?LIU Y, TAO Z, QU J, et al. Long non-coding RNA PCAT7 regulates ELF2 signaling through inhibition of miR-134-5p in nasopharyngeal carcinoma[J]. Biochem Biophys Res Commun, 2017,491(2):374-381.
[38]?VERZELLA D, PESCATORE A, CAPECE D, et al. Life, death, and autophagy in cancer: NF-κB turns up everywhere[J]. Cell Death Dis, 2020,11(3):210.
[39]?DELIHAS N.Formation of a family of long intergenic noncoding RNA genes with an embedded translocation breakpoint motif in human chromosomal low copy repeats of 22q11.2—some surprises and questions[J].NcRNA,2018,4(3):16.
[40]?DELIHAS N. A family of long intergenic non-coding RNA genes in human chromosomal region 22q11.2 carry a DNA translocation breakpoint/AT-rich sequence[J]. PLoS One, 2018,13(4):e0195702.
[41]?WANG Y, SHI F, XING G H, et al. Protein Regulator of Cytokinesis PRC1 Confers Chemoresistance and Predicts an Unfavorable Postoperative Survival of Hepatocellular Carcinoma Patients[J]. J Cancer, 2017,8(5):801-808.
[42]?LIU X, LI Y, MENG L, et al. Reducing protein regulator of cytokinesis 1 as a prospective therapy for hepatocellular carcinoma[J]. Cell Death Dis, 2018,9(5):534.
[43]?LI M,GAO J,LI D,et al.CEP55 promotes cell motility via JAK2dSTAT3dMMPs cascade in hepatocellular carcinoma[J].Cells,2018,7(8):99.
[44]?FU Y,F(xiàn)ENG M X,YU J,et al.DNA methylation-mediated silencing of matricellular protein dermatopontin promotes hepatocellular carcinoma metastasis by α3β1 integrin-Rho GTPase signaling[J].Oncotarget,2014,5(16):6701-6715.
[45]?LI X,F(xiàn)ENG P,OU J,et al.Dermatopontin is expressed in human liver and is downregulated in hepatocellular carcinoma[J].Biochemistry:Mosc,2009,74(9):979-985.
[46]?HA M, SON Y R, KIM J, et al. TEK is a novel prognostic marker for clear cell renal cell carcinoma[J]. Eur Rev Med Pharmacol Sci, 2019,23(4):1451-1458.
[47]?OLIVEIRA-FERRER L,MILDE-LANGOSCH K,EYLMANN K,et al.Mechanisms of tumor-lymphatic interactions in invasive breast and prostate carcinoma[J].Int J Mol Sci,2020,21(2):602.
[48]?VILLA E,CRITELLI R,LEI B,et al.Neoangiogenesis-related genes are hallmarks of fast-growing hepatocellular carcinomas and worst survival.Results from a prospective study[J].Gut,2016,65(5):861-869.
(收稿日期:2020-10-26?修回日期:2020-12-04)
(編輯:潘明志)