BLV-miRNA跨界調(diào)控人類靶基因預測及生物信息學分析

王雍，李思妍，何思銳，張迪，連帥，王建發(fā)，武瑞

BLV-miRNA跨界調(diào)控人類靶基因預測及生物信息學分析

王雍，李思妍，何思銳，張迪，連帥，王建發(fā)，武瑞

黑龍江八一農(nóng)墾大學動物科技學院/黑龍江省牛病防制重點實驗室，黑龍江大慶 163319

【】評估牛白血病病毒（BLV）來源的miRNAs跨界調(diào)控人源基因的風險。對BLV-miRNA可能帶來的食品安全問題及對人體健康可能造成何種影響進行前瞻性研究，為未來實際生產(chǎn)中地方流行性白血病防控措施執(zhí)行的必要性研究奠定基礎，對BLV與人類疾病間關(guān)聯(lián)性的研究提供理論指導。首先使用mirbase網(wǎng)站對BLV miRNA的成熟序列進行查詢，通過miRanda軟件對BLV編碼的10種miRNA（BLV-miR-B1-3P,5P、BLV-miR-B2-3P,5P、BLV-miR-B3-3P,5P、BLV-miR-B4-3P,5P、BLV-miR-B5-3P,5P）進行靶基因預測，并選取每個BLV-miRNA評分前10的候選靶基因（去除重復基因后共88個）進行功能分析，對受到多個BLV miRNA共同調(diào)控的候選靶基因使用RNAhybrid軟件進行二次預測驗證，并對其功能進行分析。BLV編碼的10種miRNA經(jīng)預測后分別獲得1 630—16 383個靶基因不等。對評分前十的共計88個候選靶基因進行功能分析后發(fā)現(xiàn)，其中18個基因無相關(guān)功能報道；36個候選靶基因與腫瘤性疾病的發(fā)生發(fā)展存在相關(guān)性。2個候選靶基因可以對細胞周期起調(diào)控作用；16個候選靶基因參與細胞信號轉(zhuǎn)導的調(diào)控；14個候選靶基因在細胞結(jié)構(gòu)/骨架蛋白的形成中發(fā)揮作用；細胞的增殖與凋亡的功能表現(xiàn)成拮抗關(guān)系，往往促進增殖的基因同時也可以抑制細胞凋亡，共有13個基因?qū)毎脑鲋澈偷蛲銎鹫{(diào)節(jié)作用，有趣的是，這13的候選靶基因?qū)毎鲋车蛲龉δ艿恼{(diào)節(jié)是雙向性的，但不能明確BLV miRNA對細胞的調(diào)節(jié)到底是更趨向于增殖還是凋亡，因此仍需要后續(xù)研究深入探討；2個候選靶基因?qū)毎只鹫{(diào)節(jié)作用；16個候選靶基因?qū)毎倪w移/侵襲功能起調(diào)節(jié)作用，再次提示BLV miRNA與腫瘤性疾病可能存在更重要的關(guān)聯(lián)性。7個候選靶基因可能在乳腺細胞的分化、遷移、侵襲過程中發(fā)揮重要作用，提示BLV與人乳腺癌相關(guān)性的研究中，可以從BLV miRNA的角度深入探討；BLV-B4-3P的2個候選靶基因Ⅰ型膠原α1鏈基因（COL1A1）、斷裂點簇集區(qū)（BCR）對人急性淋巴細胞白血?。ˋLL）具有調(diào)節(jié)作用。此外，可以被多個BLV miRNA共同靶向的候選靶基因均屬于黏蛋白家族（MUC5B、MUC12和 MUC16），且均可以在結(jié)腸中表達，對結(jié)腸黏膜的形成產(chǎn)生影響。外源性BLV miRNA可能跨界調(diào)控細胞周期、信號轉(zhuǎn)導、結(jié)構(gòu)/骨架、增殖、凋亡、分化、遷移/侵襲相關(guān)等細胞功能相關(guān)基因，破壞細胞結(jié)構(gòu)；BLV miRNA與人乳腺癌的相關(guān)性可能表現(xiàn)在人乳腺癌細胞的分化、遷移、和侵襲過程中；而BLV-miR-B4-3p本身與白血病相關(guān)miR 29a共享同一種子區(qū)域，可能對人急性淋巴細胞白血病的發(fā)生發(fā)展造成影響；外源性BLV miRNA具有靶向抑制黏蛋白基因（MUC5B、MUC12、MUC16）表達，通過破壞腸黏膜形成這一途徑，跨界調(diào)控人源基因的風險。

生物信息學；牛白血病病毒；跨界調(diào)控；人源基因；miRNA

0 引言

【研究意義】牛白血病病毒（BLV）是牛群中常見的傳染性病毒之一，在世界各地均有分布，陽性牛群會呈持續(xù)感染狀態(tài)，通常為亞臨床型，只有約5%的BLV陽性牛會表現(xiàn)出淋巴瘤的癥狀[1]。最新的調(diào)查結(jié)果顯示，BLV在我國牛群中的發(fā)病率約為49%[2]。牛群中出現(xiàn)BLV感染后，會出現(xiàn)產(chǎn)奶量下降[3]、平均壽命降低[4]、免疫失敗[5]等諸多問題?；贐LV臨床癥狀不明顯的特點，在目前的實際生產(chǎn)中并不能引起飼養(yǎng)者的重視，而BLV編碼的miRNA具有靶向調(diào)控人源基因的風險，可能會對乳制品食用者的健康產(chǎn)生威脅，因此對BLV miRNA的研究對乳制品安全風險的預防具有重要意義?！厩叭搜芯窟M展】乳汁中發(fā)現(xiàn)的BLV可能引發(fā)全球性的公共衛(wèi)生問題，1974年，McClure等用自然感染BLV奶牛乳汁（未巴氏消毒）飼喂6只初生黑猩猩，連續(xù)飼喂至第30周后全部黑猩猩出現(xiàn)了嗜睡、厭食、白細胞增多、貧血和進行性肺炎癥狀，第35周和第46周時有兩只黑猩猩分別死亡，病死黑猩猩被確診患有紅細胞白血病（erythroleukemia）[6]。1976年，Graves等發(fā)現(xiàn)BLV可以體外感染牛、羊、貓、蝙蝠、類人猿和人類細胞（人胚肺二倍體細胞）。2015年以來，美國、澳大利亞和阿根廷乳腺癌患者乳腺組織樣品中相繼被檢出BLV核酸片段[7]。2019年4月，Buehring等在美國加州當?shù)卣心剂?5名婦女志愿者，對其血液中BLV核酸和BLV IgA、IgM、IgG進行了檢測，發(fā)現(xiàn)BLV核酸陽性數(shù)為33，IgG和IgA抗體陽性數(shù)均為30，IgM抗體陽性數(shù)為55[8]。因此，雖然尚無充分證據(jù)表明BLV是導致人乳腺癌的元兇，但BLV引發(fā)的公共衛(wèi)生問題愈發(fā)受到關(guān)注。BLV作為逆轉(zhuǎn)錄病毒，但已經(jīng)有研究者通過高通量測序技術(shù)證實BLV可編碼miRNA，且在BLV陽性牛的肉和乳汁中發(fā)現(xiàn)了BLV miRNAs的存在[9-10]。機體內(nèi)BLV主要依靠miRNA調(diào)控牛淋巴細胞功能，當BLV感染后，其核酸序列整合到B淋巴細胞基因組中，導致B細胞在宿主體內(nèi)異常增殖[11]。但是由于B細胞淋巴瘤中缺乏5′LTR驅(qū)動的RNA聚合酶II（PolII），BLV在原代B細胞淋巴瘤中出現(xiàn)轉(zhuǎn)錄沉默，無法檢測到病毒mRNA和蛋白質(zhì)[12]?；诖?，研究者在對BLV致病機制的研究過程中發(fā)現(xiàn)，白血病牛B淋巴細胞中存在大量高度保守的由RNA聚合酶III（PolIII）轉(zhuǎn)錄的BLV編碼的miRNA[9]，也打破了RNA病毒不編碼miRNA以避免其基因組或mRNA的非生產(chǎn)性切割的傳統(tǒng)認知。miRNA相比mRNA具有極高的穩(wěn)定性，乳品中BLV-miRNA具有危害人類健康的潛在風險，BLV可編碼5類共計10種miRNA，其中miR-B4-3p、miR-B2-5p、miR-B5-5p、miR-B1-3p、miR-B5-3p與牛白血病的發(fā)生相關(guān)[13]，這些miRNA主要靶向顆粒酶A（GZMA）、棕櫚酰蛋白硫酯酶1（PPT1）、膜聯(lián)蛋白A1（ANXA1）、促分裂原活化蛋白激酶激酶1（MAP2K1）、磷酸肌醇3激酶（PIK3CG）、FBJ鼠科骨肉瘤病毒原癌基因（FOS）等癌相關(guān)基因，清除上述miRNA后BLV不能誘導白血病發(fā)生[14]。BLV與人類T淋巴細胞白血病病毒1型（HTLV-1）存在共同的致瘤機制和結(jié)構(gòu)高度相似的致瘤miRNA[15]。miRNA可以跨物種、跨代際穩(wěn)定存在，并在不同物種間、代際間跨界調(diào)控基因表達，且miRNA跨界調(diào)控的普適性規(guī)律已得到廣泛認可[16]。BLV修飾后的牛樹突狀細胞外泌體攜帶有致瘤miRNA，并且牛奶外泌體所攜miRNA可跨界調(diào)控人類免疫細胞功能[17]。【本研究切入點】前人對BLV可能對人類健康產(chǎn)生風險的研究主要集中于BLV與人乳腺癌之間的聯(lián)系，本文以miRNA跨界調(diào)控的普適性作為切入點， BLV作為一種可以編碼miRNA的逆轉(zhuǎn)錄病毒，其所編碼的miRNA可以在牛奶的外泌體中發(fā)現(xiàn)[10]，具有跨界調(diào)控人源基因的食品安全風險?！緮M解決的關(guān)鍵問題】通過miranda軟件，預測了BLV miRNAs可能調(diào)控的人源靶基因，并對BLV miRNAs的候選靶基因功能進行查詢并分析，對進一步認識BLV miRNAs跨界調(diào)控人源基因風險的研究具有重要的科學意義和實際價值。

1 材料與方法

1.1 信息采集

通過mirbase（www.mirbase.org）查詢BLV-miR- B1-3P，5P、BLV-miR-B2-3P，5P、BLV-miR-B3-3P，5P、BLV-miR-B4-3P，5P、BLV-miR-B5-3P，5P成熟序列（表1）。

表1 BLV miRNAs成熟序列

1.2 靶基因預測

miRanDa軟件主要通過miRNA和mRNA間的序列互補匹配程度和形成復合結(jié)構(gòu)的自由能兩個因素來判斷miRNA與候選靶基因的結(jié)合位點，根據(jù)序列匹配的打分值和對應的自由能預測結(jié)合位點的可能性，當分值大于150時，可以被認定為該miRNA 的候選靶基因，由于不依賴結(jié)合位點的保守性，可以更加廣泛的對miRNA的結(jié)合位點進行評估，因此我們在哈爾濱醫(yī)科大學李春權(quán)老師幫助下，于2019年7月在哈爾濱醫(yī)科大學大慶分校區(qū)應用miRanda（V1.9）軟件對BLV-miR-B1-3P，5P、BLV-miR-B2-3P，5P、BLV- miR-B3-3P，5P、BLV-miR-B4-3P，5P、BLV-miR-B5-3P，5P序列進行靶基因預測分析。

1.3 靶基因功能查詢

通過NCBI、Bing檢索工具檢索每個BLV-miRNA所預測的靶基因評分前十位的靶基因功能。通過RNAhybrid[18]對多個BLV-miRNA共同調(diào)控的靶基因進行二次預測驗證，當最小自由能（△G）≤-15 kcal·mol-1時結(jié)合的可能性較高。

2 結(jié)果

2.1 BLV miRNAs靶向人源基因預測

通過miRanda軟件對BLV miRNAs進行靶基因預測，BLV miRNAs分別獲得了1 630—16 383個靶基因不等（圖1）。

圖1 BLV miRNAs靶基因預測

2.2 BLV miRNAs靶向評分前十位基因功能分析

對BLV-miRNAs的靶基因進行了分別預測，并對每個BLV-miRNA評分排名前十的靶基因（去除重復基因后共88個）功能進行了整理分析，由于18個候選靶基因沒有相關(guān)功能報道，最終本研究對70個靶基因的功能進行了分析，其中有36個基因的功能與腫瘤的發(fā)生發(fā)展存在相關(guān)性。這些候選靶基因所表達的蛋白在細胞的周期、信號轉(zhuǎn)導、結(jié)構(gòu)/骨架、增殖、凋亡、分化、遷移/侵襲功能中發(fā)揮了重要作用（表2—7）。

2.3 BLV miRNAs靶向調(diào)控人源白血病的候選靶基因

BLV屬于反轉(zhuǎn)錄病毒科，丁型反轉(zhuǎn)錄病毒屬，在分類學上與人類T淋巴細胞白血病病毒I型最為接近。而BLV miRNAs進入人體后仍然存在調(diào)控人源白血病細胞的可能性，BLV-miR-B4-3p作為患病牛體內(nèi)表達量最高的BLV miRNA，在所有評分排名前十的候選靶基因中有兩個靶基因可以在人急性淋巴細胞白血?。ˋLL）的發(fā)生發(fā)展中發(fā)揮作用，且均為BLV-miR-B4-3p的候選靶基因（表8）。

2.4 BLV miRNAs靶向調(diào)控人源乳腺癌的候選靶基因

發(fā)現(xiàn)BLV miRNAs靶向調(diào)控的評分前十的候選靶基因中，有5個miRNA調(diào)控的7個候選靶基因在乳腺細胞的分化、遷移、侵襲生命活動中發(fā)揮了重要作用（表9）。

2.5 多個BLV miRNA共同靶向基因的最小自由能和二級結(jié)構(gòu)

在評分前10的候選靶基因中，可以同時被多個BLV miRNA共同靶向的候選靶基因聚類于MUC5B、MUC12、MUC16基因（同屬于黏蛋白家族）。我們通過RNAhybrid對這3個基因進行了二次預測驗證的結(jié)果顯示，這些BLV miRNA與靶基因結(jié)合的自由能均小于-15 kcal/mol（圖2—4）。

紅色核酸序列：候選靶基因序列；綠色核酸序列：miRNA序列；mfe：最小自由能

表2 參與調(diào)控細胞周期的候選靶基因

表3 參與細胞信號轉(zhuǎn)導的候選靶基因

表4 參與合成細胞結(jié)構(gòu)/骨架蛋白的候選靶基因

表5 參與細胞增殖/凋亡的候選靶基因

紅色核酸序列：候選靶基因序列；綠色核酸序列：miRNA序列；mfe：最小自由能

表6 參與細胞分化的候選靶基因

表7 參與細胞遷移/侵襲的候選靶基因

表8 參與調(diào)控人急性淋巴細胞白血病的候選靶基因

表9 參與調(diào)控人乳腺癌的候選靶基因

紅色核酸序列：候選靶基因序列；綠色核酸序列：miRNA序列；mfe：最小自由能

3 討論

20世紀90年代，URSIN等提出了一項前瞻性研究，研究了牛奶消費與癌癥之間存在可能聯(lián)系，評估了包括白血病、淋巴癌等癌癥類型，雖然此項調(diào)查沒有確定牛奶消費與癌癥總發(fā)病率之間的關(guān)系，但是牛奶消費量（每天兩杯）和淋巴器官癌癥，特別是淋巴癌之間存在密切聯(lián)系[53]，雖然它們之間是通過何種方式進行互作還需進一步研究，但這與我們所探究的BLV miRNAs通過食源途徑跨界調(diào)控人源基因的風險可以相互印證。

筆者查詢分析的70個候選靶基因中，36個候選靶基因的功能與腫瘤的發(fā)生發(fā)展存在相關(guān)性。這些候選靶基因所表達的蛋白在細胞的周期、信號轉(zhuǎn)導、結(jié)構(gòu)/骨架、增殖、凋亡、分化、遷移/侵襲功能中發(fā)揮重要作用。值得注意的是，調(diào)控細胞增殖與凋亡的候選靶基因均具有雙向性，即促進與抑制細胞增殖和凋亡功能的基因共同存在，但對凋亡和增殖功能起抑制作用的基因相對較多，被miRNA靶向后可能增強細胞的代謝速度。有16個候選靶基因?qū)毎倪w移/侵襲功能產(chǎn)生影響，遷移/侵襲作為癌細胞最主要的功能表達形式，同樣證實了BLV miRNAs與癌癥存在相關(guān)性。

BLV-miR-B4-3p作為BLV編碼的10種miRNA中，是患病牛體內(nèi)表達量最高的一個[10]，與白血病相關(guān)miR 29a共享同一種子區(qū)域[9]，人急性淋巴細胞白血病的融合轉(zhuǎn)錄本同樣作為BLV-miR-B4-3p的候選靶基因，提示BLV miRNA跨界進入人體后可能對人白血病的發(fā)生發(fā)展產(chǎn)生影響，雖然BLV與人類T淋巴細胞白血病病毒I型和Ⅱ（HTLV-1，2）同源性較高，但目前未見兩種疾病間相關(guān)性的研究，這也可能是未來BLV的研究方向之一。但一直以來，BLV最受大家關(guān)注的公共衛(wèi)生問題就是其與人乳腺癌之間的關(guān)系，雖然目前尚無證據(jù)表明BLV就是人源乳腺癌的重要致病因素之一，但近年來美國、澳大利亞、阿根廷等國家已經(jīng)相繼在乳腺癌患者的組織樣品中檢測到BLV基因片段[7]，BLV miRNAs作為BLV在機體內(nèi)致病功能的主要發(fā)動者[9, 54]，我們的研究顯示BLV miRNA在乳腺癌細胞的分化、遷移、侵襲過程中，均可以發(fā)揮重要作用，這一結(jié)論對BLV與人源乳腺癌之間相關(guān)性的研究有重要意義與實際價值。

BLV miRNAs的候選靶基因中，可以同時被多個BLV miRNA共同靶向的候選靶基因聚類于黏蛋白家族的MUC5B、MUC12、MUC16。其中MUC12可以編碼一種完整的膜糖蛋白，可以在結(jié)腸表面的凝膠狀分泌物中作為關(guān)鍵組成部分；雖然MUC5B是呼吸道黏蛋白的主要貢獻者，MUC16是一種眾所周知的卵巢癌標志物，但MUC5B在結(jié)腸中同樣存在大量表達，MUC16也可以作為結(jié)腸癌的獨立預后標志物[55]。鑒于miRNA所存在的跨界調(diào)控機制[16]，而結(jié)腸作為機體吸收水分的重要腸段，我們推測BLV miRNAs極有可能通過靶向調(diào)控結(jié)腸組織細胞，破壞黏蛋白功能，進而跨界進入人體，并通過這一途徑靶向調(diào)控人源基因。基于此，我們認為牛白血病病毒來源的miRNAs具有跨界調(diào)控人源基因的風險。

4 結(jié)論

外源性牛白血病病毒 miRNA可能跨界調(diào)控細胞周期、信號轉(zhuǎn)導、結(jié)構(gòu)/骨架、增殖、凋亡、分化、遷移/侵襲相關(guān)等細胞功能相關(guān)基因，破壞細胞結(jié)構(gòu)。

牛白血病病毒miRNA與人乳腺癌的相關(guān)性可能表現(xiàn)在人乳腺癌細胞的分化、遷移和侵襲過程中；而BLV-miR-B4-3p本身與白血病相關(guān)miR 29a共享同一種子區(qū)域，可能對人急性淋巴細胞白血病的發(fā)生發(fā)展造成影響。

外源性牛白血病病毒 miRNA具有靶向抑制黏蛋白基因（MUC5B、MUC12、MUC16）表達，通過破壞腸黏膜形成這一途徑，跨界調(diào)控人源基因的風險。

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Prediction and Bioinformatics Analysis of BLV-miRNA Transboundary Regulation of Human Target Genes

WANG Yong, LI SiYan, HE SiRui, ZHANG Di, LIAN Shuai, WANG JianFa, WU Rui

College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University/Heilongjiang Provincial Key Laboratory of Prevention and Control of Bovine Diseases, Daqing 163319, Heilongjiang

【】To assess risk of regulation of human-derived genes by miRNAs derived from bovine leukemia virus (BLV), the prospective research on the possible food safety problems and the possible impact on human health caused by BLV-miRNA were carried out, which would lay the foundation for the necessary research on the implementation of Enzootic Bovine Leukosis (EBL) prevention and control measures in actual production in the future, and provide theoretical guidance for the study of the relationship between BLV and human diseases.【】In this study, the mature sequence of BLV miRNAs was first queried using mirbase website, and the miRanda software was used to predict target genes. The predictive 10 miRNAs (BLV-miR-B1-3P,5P, BLV-miR-B2-3P, 5P, BLV-miR-B3-3P,5P, BLV-miR-B4-3P,5P, and BLV-miR-B5-3P,5P) were encoded by BLV. The top 10 candidate target genes of each BLV-miRNA score were selected for functional analysis, including a total of 88 duplicated genes. The candidate target genes co-regulated by multiple BLV miRNAs were verified by secondary prediction using RNAhybrid software, and their functions were analyzed. 【】The ten miRNAs encoded by BLV were predicted to obtain 1 630-16 383 target genes, respectively. After functional analysis of eighty-eight candidate target genes in the top ten, it was found that eighteen of them had no relevant functional reports. Thirty-six candidate target genes were related to the occurrence and development of neoplastic diseases. Two candidate target genes could regulate cell cycle. Sixteen candidate target genes were involved in the regulation of cell signal transduction. Fourteen candidate target genes played a role in the formation of structure/cytoskeleton proteins. The function of cell proliferation and apoptosis showed an antagonistic relationship and the genes that often promoting proliferation could also suppress apoptosis. A total of thirteen genes played a regulatory role in cell proliferation and apoptosis. Interestingly, the regulation of the thirteen candidate target genes on cell proliferation and apoptosis was bidirectional. However, it was not clear whether the regulation of BLV miRNA towards cells was more prone to proliferation or apoptosis, so further studies were still needed to discuss in depth. Two candidate target genes could regulate cell differentiation. The sixteen candidate target genes played a role in regulating cell migration/invasion function, again suggesting that BLV miRNA might have a more important correlation with neoplastic diseases. The seven candidate target genes might play an important role in the differentiation, migration and invasion of breast cells, suggesting that the study on the correlation between BLV and human breast cancer could be further discussed from the perspective of BLV miRNA. Two candidate target genotypes of BLV-B4-3P, Collagen 1 chain gene (COL1A1), had a regulatory effect on human acute lymphoblastic leukemia (ALL). In addition, candidate target genes that could be co-targeted by multiple BLV miRNAs belong to the mucin family (MUC5B, MUC12 and MUC16), and be expressed in the colon, influencing the formation of colon mucosa.【】Exogenous BLV miRNA might transboundary regulate cell cycle signal transduction structure/cytoskeleton proliferation apoptosis differentiation migration/invasion related cell function related genes and destroy cell structure. The correlation between BLV miRNA and human breast cancer might be shown in the process of differentiation, migration and invasion of human breast cancer cells. BLV-miR-B4-3p shared a seed sequence with miR 29a, which might affect the occurrence and development of human acute lymphoblastic leukemia. Exogenous BLV miRNA had the target of inhibiting the expression of mucin genes, such as MUC5B, MUC12, and MUC16, through the destruction of intestinal mucosa formation to achieve transboundary regulation of human gene risk.

bioinformatics; bovine leukemia virus; transboundary regulation; human-derived genes; miRNA

10.3864/j.issn.0578-1752.2021.03.019

2020-02-23；

2020-07-29

國家自然科學基金（2041340046）、黑龍江省自然科學基金（YQ2019C014）、研究生創(chuàng)新科研項目（YJSCX2019-Y39）

王雍，Tel：13251599676；E-mail：bywy0209@126.com。通信作者武瑞，Tel：13836961026；E-mail：fuhewu@126.com

（責任編輯 林鑒非）