趙學(xué)亮，王文龍，呼和巴特爾

·動(dòng)物及獸醫(yī)生物技術(shù)·

捻轉(zhuǎn)血矛線蟲丙硫咪唑耐藥相關(guān)lncRNA的轉(zhuǎn)錄組學(xué)測序與分析

趙學(xué)亮，王文龍，呼和巴特爾

內(nèi)蒙古農(nóng)業(yè)大學(xué) 獸醫(yī)學(xué)院 農(nóng)業(yè)農(nóng)村部動(dòng)物疾病臨床診療技術(shù)重點(diǎn)實(shí)驗(yàn)室，內(nèi)蒙古 呼和浩特 010018

分析捻轉(zhuǎn)血矛線蟲敏感株和耐藥株中長鏈非編碼RNA (Long non-coding RNA，lncRNA)的表達(dá)譜，探討lncRNA與捻轉(zhuǎn)血矛線蟲丙硫咪唑耐藥機(jī)制的關(guān)聯(lián)性，為捻轉(zhuǎn)血矛線蟲耐藥機(jī)理提供依據(jù)。文中對(duì)捻轉(zhuǎn)血矛線蟲敏感株和耐藥株進(jìn)行cDNA測序文庫構(gòu)建，使用Illumina HiSeq 4000平臺(tái)進(jìn)行雙端測序，篩選出差異的lncRNA，基于順式調(diào)控(cis) 和反式調(diào)控(trans) 對(duì)差異顯著的lncRNA進(jìn)行靶基因預(yù)測，并對(duì)靶基因進(jìn)行過Gene Ontology (GO)功能注釋和KEGG Pathway富集分析，利用FPKM法估計(jì)lncRNA和mRNA的表達(dá)水平。結(jié)果表明，敏感株和耐藥株候選lncRNA分別為6 377和6 356個(gè)，兩個(gè)文庫中篩選出168個(gè)差異顯著的lncRNA，其中在敏感株中表達(dá)上調(diào)有92個(gè)，表達(dá)下調(diào)76個(gè)。篩選得到的差異顯著lncRNA候選靶基因416個(gè)，這些基因共注釋到641條GO terms和92條信號(hào)通路；其中富集到耐藥性相關(guān)的通路有藥物代謝-其他酶、藥物代謝-細(xì)胞色素P450、細(xì)胞色素P450對(duì)異生素的代謝等。綜上表明，部分lncRNA介導(dǎo)的靶基因與捻轉(zhuǎn)血矛線蟲丙硫咪唑耐藥性相關(guān)，lncRNA在捻轉(zhuǎn)血矛線蟲耐藥性中具有潛在的重要的調(diào)節(jié)作用。探究了對(duì)于敏感蟲株和耐藥蟲株中l(wèi)ncRNA的表達(dá)譜，發(fā)現(xiàn)了敏感蟲株和耐藥蟲株中差異表達(dá)的lncRNA，有助于找出捻轉(zhuǎn)血矛線蟲如何抵抗丙硫咪唑的發(fā)生機(jī)制，為探討捻轉(zhuǎn)血矛線蟲丙硫咪唑耐藥機(jī)制提供科學(xué)的依據(jù)。

捻轉(zhuǎn)血矛線蟲，丙硫咪唑，耐藥性，轉(zhuǎn)錄組，長鏈非編碼RNA

捻轉(zhuǎn)血矛線蟲是反芻動(dòng)物最常見的消化道線蟲之一，由于缺乏有效的疫苗，長期以來對(duì)捻轉(zhuǎn)血矛線蟲病的防治幾乎主要依靠丙硫咪唑、伊維菌素等化學(xué)藥物。然而，隨著該藥物幾十年以來的反復(fù)頻繁使用，導(dǎo)致捻轉(zhuǎn)血矛線蟲耐藥性的逐漸產(chǎn)生，給畜牧業(yè)造成嚴(yán)重的經(jīng)濟(jì)損失[1]。檢測影響捻轉(zhuǎn)血矛線蟲丙硫咪唑耐藥相關(guān)基因表達(dá)情況是了解耐藥機(jī)制的基礎(chǔ)，同時(shí)可以篩選與丙硫咪唑耐藥相關(guān)的候選基因。1986年，在南非首次報(bào)道了寄生蟲對(duì)丙硫咪唑的耐藥性，由于驅(qū)蟲藥物頻繁使用，使寄生蟲多次與藥物接觸后，對(duì)藥物敏感性下降甚至消失，導(dǎo)致藥物對(duì)寄生蟲無 效[2]。國內(nèi)外許多學(xué)者在捻轉(zhuǎn)血矛線蟲耐藥性流行病學(xué)調(diào)查、耐藥性檢測方法和耐藥分子機(jī)制的研究中取得了大量的研究成果。但有關(guān)捻轉(zhuǎn)血矛線蟲耐藥性組學(xué)的研究較少[3]，尤其是關(guān)于長鏈非編碼RNA (Long non-coding RNA，lncRNA) 的研究仍屬未知。

lncRNA是長度大于200 bp的非編碼RNA，調(diào)控方式主要分為兩類，一類為順式調(diào)控，另一類為反式調(diào)控。研究表明，lncRNA在細(xì)胞周期、表觀遺傳和天然免疫等眾多生命活動(dòng)中發(fā)揮重要作用[4]。近年來，在人、綿羊、野豬上均鑒定到lncRNA，部分lncRNA與人類癌癥耐藥、腫瘤耐藥有著密切的聯(lián)系。Li等通過對(duì)lncRNA測序研究，揭示了lncARSR在肝臟癌癥細(xì)胞中對(duì)阿霉素耐藥性中的關(guān)鍵作用，表明lncARSR可作為肝癌預(yù)后生物標(biāo)志物和藥物治療靶點(diǎn)[5]。相比于人類疾病耐藥lncRNA，寄生蟲耐藥相關(guān)的lncRNA功能研究較滯后。本研究利用RNA-seq，對(duì)捻轉(zhuǎn)血矛線蟲丙硫咪唑敏感株和耐藥株中l(wèi)ncRNA與mRNA的結(jié)構(gòu)特征(長度分布、ORF長度、外顯子數(shù)目) 以及表達(dá)量進(jìn)行比較，對(duì)差異表達(dá)lncRNA進(jìn)行篩選，并基于反式調(diào)控機(jī)制進(jìn)行靶基因預(yù)測以及功能注釋分析，篩選出關(guān)鍵lncRNA。從lncRNA水平深入探索綿羊捻轉(zhuǎn)血矛線蟲對(duì)丙硫咪唑耐藥機(jī)制，同時(shí)有望確定綿羊捻轉(zhuǎn)血矛線蟲丙硫咪唑耐藥功能基因，為進(jìn)一步從轉(zhuǎn)錄組水平闡釋捻轉(zhuǎn)血矛線蟲丙硫咪唑耐藥機(jī)制提供重要理論依據(jù)。

1 材料與方法

1.1 材料

本研究捻轉(zhuǎn)血矛線蟲丙硫咪唑敏感株和耐藥株均采自內(nèi)蒙古自治區(qū)烏蘭察布市察右后旗(北緯41°3′–41°59′，東經(jīng)112°42′–113°30′，平均海拔1 500 m左右)，具體方法見參考文獻(xiàn)[6]。

1.2 RNA提取及質(zhì)控

選取敏感株雌蟲、敏感株雄蟲、耐藥株雌蟲和耐藥株雄蟲各30條，參照Qiagen+TRK1002 (LC Science，Houston，TX) 說明書分別對(duì)4個(gè)樣本進(jìn)行總RNA提取，用Bioanalyzer 2100和RNA 6000 Nano Lab Chip Kit進(jìn)行總RNA質(zhì)量和濃度檢測，以RIN值>7.0為合格的標(biāo)準(zhǔn)。

1.3 去rRNA鏈特異性文庫構(gòu)建

四個(gè)樣本總RNA經(jīng)過質(zhì)檢合格后，將敏感株雌蟲及雄蟲核苷酸等量混合，將耐藥株的雌蟲及雄蟲核苷酸等量混合，然后進(jìn)行敏感株和耐藥株文庫構(gòu)建。利用Illumina公司的Ribo-Zero? rRNA Removal Kit試劑盒分別對(duì)rRNA進(jìn)行捕獲去除，剩余的RNA在高溫條件下利用二價(jià)陽離子進(jìn)行片段化。以此為模板，用六堿基隨機(jī)引物合成第一鏈cDNA，隨后加入大腸桿菌DNA polymeraseⅠ與RNase H進(jìn)行第二鏈cDNA合成，將這些DNA與RNA的復(fù)合雙鏈轉(zhuǎn)化成DNA雙鏈，同時(shí)在二鏈中摻入dUTP，將雙鏈DNA的末端補(bǔ)齊為平末端。再在其兩端各加上一個(gè)A堿基，使其能夠與末端帶有T堿基的接頭進(jìn)行連接，利用磁珠對(duì)其片段大小進(jìn)行篩選和純化。以UDG酶消化二鏈，再通過PCR，使其形成片段大小為300 bp (±50 bp) 的文庫。最后使用Illumina HiSeq 4000 (LC Bio，China)測序平臺(tái)按照標(biāo)準(zhǔn)操作對(duì)其進(jìn)行雙端測序，讀長2×150 bp。

1.4 轉(zhuǎn)錄組數(shù)據(jù)分析

首先使用Cutadapt[7]去除測序過程中測序接頭及低質(zhì)量序列，然后利用Bowtie2[8]和Tophat2[9]軟件將測序得到的序列比對(duì)到捻轉(zhuǎn)血矛線蟲參考基因組上，再利用StringTie[10]對(duì)轉(zhuǎn)錄本進(jìn)行組裝。

識(shí)別lncRNA的關(guān)鍵是如何區(qū)分編碼轉(zhuǎn)錄本和非編碼轉(zhuǎn)錄本，本研究首先去除已知mRNA和具有編碼潛能的未知mRNA，然后去除小于200 bp和reads覆蓋度小于3的轉(zhuǎn)錄本，再利用CPC_Score (Score≤?1)[11]和CNCI_Score(Length≥200且exon≥1且score≤0)[12]軟件綜合預(yù)測lncRNA，能較好地從預(yù)測得到的新轉(zhuǎn)錄本中排除具蛋白編碼能力的轉(zhuǎn)錄本，通過轉(zhuǎn)錄本含外顯子數(shù)目≥1、已知lncRNA信息確定、與捻轉(zhuǎn)血矛線蟲非lncRNA序列比對(duì)等一系列過濾條件后，利用類代碼(Class code)o (與參考轉(zhuǎn)錄本的外顯子重疊)、I (內(nèi)含子區(qū)域的轉(zhuǎn)錄本)、u (基因間轉(zhuǎn)錄本)、j (已知轉(zhuǎn)錄本的潛在亞型) 及x (與參考轉(zhuǎn)錄本反義鏈的外顯子重疊) 作為新lncRNA轉(zhuǎn)錄本的候選。

對(duì)敏感株和耐藥株lncRNA的表達(dá)量(FPMK值)進(jìn)行差異分析，以|log2(差異倍數(shù))|>1且<0.05作為顯著差異表達(dá)基因篩選的閾值。將順式調(diào)控定義為染色體上下游100 kb范圍內(nèi)存在差異表達(dá)的lncRNA與差異表達(dá)的mRNA，反式調(diào)控定義為lncRNA跨染色體調(diào)控基因的表達(dá)，主要基于lncRNA與mRNA序列之間形成二級(jí)結(jié)構(gòu)所需要自由能的大小[13]。并然后利用GO和KEGG Pathway數(shù)據(jù)庫對(duì)lncRNA靶向的差異mRNA進(jìn)行功能注釋和代謝通路分析。

2 結(jié)果與分析

2.1 RNA-Seq測序質(zhì)量評(píng)估

對(duì)捻轉(zhuǎn)血矛線蟲敏感株和耐藥株分別構(gòu)建cDNA文庫并進(jìn)行轉(zhuǎn)錄組測序，將得到的數(shù)據(jù)進(jìn)行質(zhì)控和組裝，去除低質(zhì)量的數(shù)據(jù)，敏感株和耐藥株分別獲得86 162 364和89 454 054個(gè)讀長，Q30質(zhì)量值百分率分別達(dá)到了97.53%和97.38%，表明測序質(zhì)量較高，符合后續(xù)分析要求。將有效序列比對(duì)到捻轉(zhuǎn)血矛線蟲參考基因組，結(jié)果顯示，敏感蟲株與參考基因組的比對(duì)率為85.97%，耐藥蟲株與參考基因組的比對(duì)率為94.56%。

2.2 lncRNA的鑒定和特征比較

使用帶有“u”、“i”、“x”、“j”和“o”的5類代碼作為新lncRNA轉(zhuǎn)錄本的候選，共鑒定出6 505個(gè)新的lncRNA，其中敏感株6 377個(gè)，耐藥株6 356個(gè)(表1)，同時(shí)鑒定出16 209個(gè)mRNA。對(duì)lncRNA與mRNA的結(jié)構(gòu)特征(長度分布、和外顯子數(shù)目) 進(jìn)行統(tǒng)計(jì)，結(jié)果表明，長度小于500 bp的lncRNA在總lncRNA中的分布密度較高，占58.4%，而mRNA的長度分布主要集中在大于1 000 bp，占總mRNA的44.0% (圖1)。外顯子統(tǒng)計(jì)結(jié)果表明，鑒定出的lncRNA中有很大一部分僅含有1–2個(gè)外顯子，占lncRNA總數(shù)的81.6%，mRNA中的外顯子個(gè)數(shù)分布則較均勻(圖2)。利用FPKM值歸一化轉(zhuǎn)錄本表達(dá)量，根據(jù)中位數(shù)可以看出mRNA表達(dá)水平高于lncRNA (圖3)。

2.3 lncRNA差異表達(dá)分析

利用FPKM值對(duì)兩樣本進(jìn)行歸一化處理，以<0.05及l(fā)og2 (fold change)>1或log2 (fold change)

表1 lncRNA的鑒定

圖1 敏感株與耐藥株lncRNA和mRNA轉(zhuǎn)錄本長度分布

圖2 敏感株與耐藥株lncRNA和mRNA外顯子數(shù)目分布

圖3 lncRNA和mRNA轉(zhuǎn)錄本表達(dá)水平及數(shù)量

圖4 樣本間的差異lncRNA散點(diǎn)圖

2.4 lncRNA靶基因預(yù)測

通過皮爾森相關(guān)系數(shù)和自由能值對(duì)lncRNA進(jìn)行靶基因預(yù)測，結(jié)果顯示，共得到562個(gè)潛在靶基因，其中潛在順式調(diào)控(cis)靶基因146個(gè)，潛在反式調(diào)控(trans)靶基因416個(gè)。以<0.05及l(fā)og2(fold change)>1或log2(fold change)

2.5 差異lncRNA靶基因GO和KEGG富集分析

通過對(duì)鑒定出的所有l(wèi)ncRNA和mRNA的表達(dá)量情況進(jìn)行相關(guān)性分析，而后從中篩選出與lncRNA的表達(dá)模式極顯著相關(guān)的mRNA，從而對(duì)該lncRNA靶基因進(jìn)行功能的預(yù)測和KEGG富集分析。

GO分析結(jié)果顯示，差異顯著lncRNA的靶基因共注釋到641條GO terms，其中生物學(xué)過程317條，細(xì)胞組分113條，分子功能211條。主要注釋到代謝過程、蛋白水解、線蟲發(fā)育、蟲卵孵化死亡；細(xì)胞膜、細(xì)胞器、細(xì)胞質(zhì)；轉(zhuǎn)移酶 活性、核苷酸結(jié)合、ATP結(jié)合、蛋白結(jié)合等 (圖5)。

KEGG富集分析結(jié)果顯示，捻轉(zhuǎn)血矛線蟲敏感株和耐藥株差異表達(dá)lncRNA的靶基因共富集到92條通路，富集程度較高的前10條通路如表3所示，其中與耐藥性相關(guān)的通路有藥物代謝-其他酶(Drug metabolism-other enzymes)、藥物代謝-細(xì)胞色素P450 (Drug metabolism- cytochrome P450)、細(xì)胞色素P450對(duì)異生素的代謝(Metabolism of xenobiotics by cytochrome P450) 等。

3 討論

細(xì)菌的耐藥機(jī)制較為簡單，研究較成熟，而多細(xì)胞真核生物的耐藥性顯然要復(fù)雜得多，涉及真核生物和真核細(xì)胞雙重的藥物代謝、藥效機(jī)制。隨著高通量測序的發(fā)展，越來越多的lncRNA受到廣大學(xué)者的關(guān)注，目前已經(jīng)鑒定的lncRNA主要與癌癥、腫瘤耐藥相關(guān)[14-15]。迄今為止，關(guān)于寄生蟲耐藥性的lncRNA，特別是有關(guān)捻轉(zhuǎn)血矛線蟲的研究未見報(bào)道。在本研究中以捻轉(zhuǎn)血矛線蟲耐藥性為出發(fā)點(diǎn)，對(duì)敏感株和耐藥株進(jìn)行比較轉(zhuǎn)錄組學(xué)測序分析，鑒定出lncRNA并篩選出差異候選lncRNA的靶基因，在轉(zhuǎn)錄組層面為探討捻轉(zhuǎn)血矛線蟲耐藥性與lncRNA的關(guān)聯(lián)提供有價(jià)值的數(shù)據(jù)。并且mRNA表達(dá)水平高于lncRNA，與組織中的表達(dá)趨勢一致[15]。

在敏感株和耐藥株中分別鑒定出6 377個(gè)和 6 356個(gè)lncRNA，以log2 (差異倍數(shù))>1或log2 (差異倍數(shù))

圖5 GO分析差異lncRNA靶基因

表2 差異表達(dá)基因最大的10個(gè)lncRNA及其上調(diào)和下調(diào)靶基因

表3 DElncRNA差異靶基因KEGG富集前10通路

KEGG Pathway富集結(jié)果顯示前10條通路中，包括藥物代謝-其他酶(Drug metabolism-other enzymes)、藥物代謝-細(xì)胞色素(P450 Drug metabolism-cytochrome P450)、細(xì)胞色素P450對(duì)異生素的代謝(Metabolism of xenobiotics by cytochrome P450)、谷胱甘肽代謝(Glutathione metabolism) 等，這些信號(hào)通路均與耐藥性有關(guān)，可能是DElncRNA共表達(dá)基因的重要KEGG途徑，相關(guān)的lncRNA可能潛在地參與捻轉(zhuǎn)血矛線蟲丙硫咪唑耐藥性的過程。該通路下的lncRNA可能在丙硫咪唑藥物選擇壓力下，通過基因表達(dá)量的變化來發(fā)揮耐藥性的作用。

在差異表達(dá)基因最大的10個(gè)lncRNA中，其靶基因細(xì)胞色素P450在敏感株中的表達(dá)水平顯著低于耐藥株。細(xì)胞色素P450是寄生蟲體內(nèi)藥物代謝過程重要的系統(tǒng)，主要起著解毒等作用，目前在昆蟲、按蚊對(duì)殺蟲劑耐藥性中研究較多[20-21]。Yilmaz等[22]通過熒光定量PCR對(duì)阿苯達(dá)唑不同敏感程度的捻轉(zhuǎn)血矛線蟲敏感株和耐藥株P(guān)450家族進(jìn)行檢測，結(jié)果表明該家族基因在阿苯達(dá)唑耐藥株中的表達(dá)量比敏感株高2.4–3.7倍，與本研究結(jié)果一致。差異表達(dá)lncRNA的靶基因除細(xì)胞色素P450與捻轉(zhuǎn)血矛線蟲阿苯達(dá)唑耐藥性相關(guān)外，筆者未發(fā)現(xiàn)其他的靶基因與捻轉(zhuǎn)血矛線蟲耐藥性有相關(guān)聯(lián)系性的研究。但是鋅指結(jié)構(gòu)域含有蛋白(Zinc finger domain-containing protein)、脫氧核苷激酶(Deoxynucleoside kinase) 等基因同樣參與了細(xì)胞凋亡、多重耐藥的過程。有關(guān)捻轉(zhuǎn)血矛線蟲阿苯達(dá)唑耐藥性中部分信號(hào)通路及耐藥基因的研究仍屬未知，下一步可對(duì)樣本間差異表達(dá)的lncRNA進(jìn)行生物功能注釋分析及生物學(xué)功能驗(yàn)證，進(jìn)一步對(duì)差異表達(dá)的lncRNA進(jìn)行功能及代謝通路富集分析，以闡述捻轉(zhuǎn)血矛線蟲lncRNA對(duì)丙硫咪唑耐藥性產(chǎn)生的分子調(diào)控機(jī)制。

4 結(jié)論

本研究采用RNA-Seq測序技術(shù)對(duì)捻轉(zhuǎn)血矛線蟲敏感株和耐藥株進(jìn)行轉(zhuǎn)錄組學(xué)測序，篩選出差異的lncRNAs，并對(duì)差異顯著的lncRNAs與其共表達(dá)的差異cis和trans調(diào)控靶基因進(jìn)行生物信息學(xué)分析，推測lncRNA可能對(duì)捻轉(zhuǎn)血矛線蟲丙硫咪唑耐藥性具有重要的調(diào)節(jié)作用，為從lncRNA角度分析捻轉(zhuǎn)血矛線蟲敏感株和耐藥株的差 異及丙硫咪唑耐藥機(jī)制的進(jìn)一步研究提供理論基礎(chǔ)。

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Transcriptome sequencing and analysis of lncRNA related to albendazole resistant strain of

Xueliang Zhao, Wenlong Wang, and Huhebateer

Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease, Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, Inner Mongolia, China

The objective of this study was to explore the link between long non-coding RNAs (lncRNAs) and resistance mechanism of albendazole inby analyzing the gene expression profile of lncRNA in sensitive and resistant strains. This study provided the base for the resistance mechanism of. In this experiment,the cDNA sequencing libraries were collected constructed for the sensitive and resistant strains of, and the sequencing was performed using Illumina HiSeq 4000 platform, and differentially expressed lncRNAs (DEIncRNAs) were screened. Then the-targeted and-targeted genes of DElncRNAs were predicted, and the GO enrichment, KEGG pathway enrichment analysis were also made. The results displayed that 6 377 and 6 356 candidate lncRNA transcripts of sensitive and resistive strains were respectively obtained, 168 DElncRNAs of which were selected. Compared with resistive strains, there were 92 up-regulated and 76 down-regulated lncRNAs in sensitive strains. Meanwhile, 416 candidate target genes of DElncRNAs were obtained. The analytical results indicated that these genes participated in 641 GO terms and 92 signal pathways. The pathways involved in drug resistance are drug metabolism-other enzymes, drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450 and so on. In conclusion, these findings inferred that some lncRNA-mediated target genes were associated with the resistance of, and lncRNA may play an important role in the resistance of. This study explored the expression profile of lncRNA in theof sensitive and resistant strains. It was found that DElncRNAs in the sensitive and resistant strains, which helped to find out theresistance mechanism of albendazole and provided a scientific basis for exploring the mechanism of resistance to.

, albendazole, drug resistance, transcriptome, long non-coding RNA

趙學(xué)亮, 王文龍, 呼和巴特爾. 捻轉(zhuǎn)血矛線蟲丙硫咪唑耐藥相關(guān)lncRNA的轉(zhuǎn)錄組學(xué)測序與分析. 生物工程學(xué)報(bào), 2020, 36(2): 250–258.

Zhao XL, Wang WL, Huhebateer. Transcriptome sequencing and analysis of lncRNA related to albendazole resistant strain of. Chin J Biotech, 2020, 36(2): 250–258.

June 7, 2019;

September 2, 2019

Supported by: National Natural Science Foundation of China (No. 31760731), Science and Technology Plan Project of Inner Mongolia Autonomous Region (No. 201702074).

Wenlong Wang. Tel:+86-471-4303726; E-mail: wwl.imau@163.com

Huhebateer. Tel:+86-471-4303726; E-mail: hhbte@163.com

10.13345/j.cjb.190238

國家自然科學(xué)基金 (No. 31760731)，內(nèi)蒙古自治區(qū)科技計(jì)劃項(xiàng)目 (No. 201702074) 資助。

2019-09-18

http://kns.cnki.net/kcms/detail/11.1998.Q.20190918.1016.003.html

(本文責(zé)編 郝麗芳)