金梅，張麗娟，曹倩，郭鑫英

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MT和FGF5調(diào)控遼寧絨山羊絨毛生長(zhǎng)相關(guān)LncRNA的篩選及鑒定

金梅，張麗娟，曹倩，郭鑫英

（遼寧師范大學(xué)生命科學(xué)學(xué)院/遼寧省生物技術(shù)與分子藥物研發(fā)重點(diǎn)實(shí)驗(yàn)室，遼寧大連 116029）

【目的】篩選出遼寧絨山羊皮膚成纖維細(xì)胞中與絨毛生長(zhǎng)相關(guān)的LncRNA，為絨毛生長(zhǎng)相關(guān)LncRNA的功能及機(jī)制研究提供基礎(chǔ)性數(shù)據(jù)。【方法】提取MT和FGF5處理的遼寧絨山羊皮膚成纖維細(xì)胞總RNA，通過(guò)樣品總RNA電泳檢測(cè)、測(cè)序數(shù)據(jù)質(zhì)量評(píng)估、Mapping比對(duì)、樣品間相關(guān)性檢查對(duì)提取的總RNA進(jìn)行質(zhì)量檢測(cè)。篩選出差異表達(dá)的LncRNA并預(yù)測(cè)其靶基因，通過(guò)GO和KEGG富集分析，篩選出與絨毛生長(zhǎng)相關(guān)的LncRNA，并通過(guò)Real-time PCR對(duì)目標(biāo)LncRNA進(jìn)行表達(dá)驗(yàn)證。【結(jié)果】（1）樣品總RNA質(zhì)量檢測(cè)結(jié)果顯示：RNA 完整性良好、GC含量相對(duì)較高，序列較穩(wěn)定、樣品間表達(dá)水平相關(guān)性均較高、符合測(cè)序要求。（2）差異表達(dá)LncRNA的篩選結(jié)果顯示：1.0g?L-124h組差異表達(dá)LncRNA有32個(gè)，其中4個(gè)表達(dá)上調(diào)，28個(gè)表達(dá)下調(diào)；0.2g?L-124h組差異表達(dá)LncRNA 有10個(gè)，其中4個(gè)表達(dá)上調(diào)，6個(gè)表達(dá)下調(diào)；0.2g?L-172h組差異表達(dá)LncRNA有113個(gè)，其中5個(gè)表達(dá)上調(diào)，108個(gè)表達(dá)下調(diào)。10-4g?L-124 h組差異表達(dá)LncRNA有164個(gè)，其中有70個(gè)上調(diào)，94個(gè)下調(diào)；10-4g?L-172 h差異表達(dá)LncRNA 有189個(gè)，其中有78個(gè)上調(diào)，111個(gè)下調(diào)；10-6g?L-124 h組差異表達(dá)的LncRNA有123個(gè)，其中有27個(gè)上調(diào)，96個(gè)下調(diào) 。（3） 靶基因GO富集分析結(jié)果顯示：1.0g?L-124h組差異表達(dá)LncRNA靶基因富集在GO的negative regulation of transcription from RNA polymerase II promoter；0.2g?L-124h組無(wú)差異表達(dá)LncRNA靶基因富集的GO term；0.2g?L-172h組差異表達(dá)LncRNA靶基因富集在GO的cellular metabolic process biological_process，binding molecular_function，F(xiàn)GF5處理組中只有10-4g?L-172 h組差異表達(dá)LncRNA靶基因富集在cell cellular_component、cell part cellular_component、intracellular cellular_component、binding molecular_function等6個(gè)條目。（4）靶基因KEGG富集分析結(jié)果顯示：1.0g?L-124h組差異表達(dá)LncRNA靶基因富集在Steroid biosynthesis pathway；0.2g?L-124h組無(wú)差異表達(dá)LncRNA靶基因富集的Pathway term；0.2g?L-172h組差異表達(dá)LncRNA靶基因富集在Cell cycle，DNA replication，Steroid biosynthesis，TNF，Nod-like receptor，NF-kappa B等信號(hào)通路，其中TNF和NF-kappa B信號(hào)通路與絨毛生長(zhǎng)相關(guān)。FGF5處理組中，10-4g?L-172 h組差異表達(dá)的LncRNA靶基因顯著富集到Fanconi anemia pathway，Huntington's disease，Metabolic pathway，Aminoacyl-tRNA biosynthesis等9個(gè)pathway term，其中Metabolic信號(hào)通路與絨毛生長(zhǎng)相關(guān)；10-4g?L-124 h組差異表達(dá)的LncRNA靶基因無(wú)顯著富集的pathway term；10-6g?L-124 h組差異表達(dá)的LncRNA靶基因只富集在Taste transduction pathway。（5）NF-κB和TNF兩個(gè)信號(hào)通路中富集的靶基因TNFα、TNFAIP3（A20）、NFKBIA（IkBα）、NFKB2、IL8所對(duì)應(yīng)的LncRNA有2個(gè)，分別為（Gene ID）：XLOC_005914；XLOC_018763；Metabolic信號(hào)通路中靶基因所對(duì)應(yīng)的LncRNA有4個(gè)，分別為（Gene ID）：XLOC_011424、XLOC_009522、XLOC_009063、XLOC_01115。Real-time PCR結(jié)果顯示：LncRNA XLOC_011424、XLOC_011157、LncRNA XLOC_005914和XLOC_018763與高通量測(cè)序結(jié)果一致?！窘Y(jié)論】 LncRNA XLOC_011424、XLOC_011157、LncRNA XLOC_005914和XLOC_018763可能通過(guò)調(diào)控與絨毛生長(zhǎng)相關(guān)的NF-κB 、TNF或Metabolic信號(hào)通路，提高羊絨密度和長(zhǎng)度，進(jìn)而提高遼寧絨山羊絨產(chǎn)量及品質(zhì)。

遼寧絨山羊；褪黑激素；FGF5；LncRNA；RNA-seq; 信號(hào)通路

0 引言

【研究意義】遼寧絨山羊是中國(guó)代表性的絨山羊品種，其體型較大，適應(yīng)能力強(qiáng)，遺傳穩(wěn)定性好。遼寧絨山羊是絨肉兼用的地方良種，該種絨山羊產(chǎn)絨量高，絨毛品質(zhì)好，其絨毛是珍貴的紡織原料。因此，研究如何提高遼寧絨山羊羊絨產(chǎn)量及品質(zhì)尤為重要?！厩叭搜芯窟M(jìn)展】毛囊是皮膚中的附屬結(jié)構(gòu)，可分為初級(jí)毛囊和次級(jí)毛囊，初級(jí)毛囊產(chǎn)毛，次級(jí)毛囊產(chǎn)絨[3]。哺乳動(dòng)物被毛的生長(zhǎng)替換是一個(gè)復(fù)雜的生理過(guò)程，被毛的替換與毛囊的周期性生長(zhǎng)密切相關(guān)，一般一個(gè)生長(zhǎng)周期內(nèi)毛囊要經(jīng)歷生長(zhǎng)期、退行期和休止期三個(gè)階段[4]。環(huán)境、代謝水平和基因調(diào)控等因素都可影響絨山羊毛囊的周期性生長(zhǎng)過(guò)程，有研究表明，很多的信號(hào)分子都在毛囊的形態(tài)發(fā)生過(guò)程中具有很重要的作用，褪黑激素(melatonin，MT)、催乳素(prolactin)、成纖維細(xì)胞生長(zhǎng)因子5（fibroblast growth factor 5, FGF5）、甲狀腺素(thyroxine)等[5-6]。MT是由松果體分泌出來(lái)的一種高度保守的吲哚類激素[7]。它在很多細(xì)胞、組織和器官中都起到重要作用[8]。IBRAHEEM 等研究發(fā)現(xiàn)，催乳素和褪黑激素對(duì)次級(jí)毛囊的毛干伸長(zhǎng)具有刺激作用[9]。Logan等發(fā)現(xiàn)，褪黑激素能夠抑制由α-黑素細(xì)胞刺激素（MSH）引起的黑素生成的增加[10]。有學(xué)者發(fā)現(xiàn)，皮膚組織是除松果體外，MT合成與代謝的又一重要的場(chǎng)所[11]。近年來(lái)，很多研究表明MT可能在毛發(fā)生理學(xué)中起重要作用，其受體（MT2和RORα）以毛發(fā)周期依賴性方式在小鼠皮膚中表達(dá)，抑制角質(zhì)形成細(xì)胞凋亡[12-13]。另外，MT能夠改變山羊中羊絨生長(zhǎng)周期的時(shí)間，外源性的MT能夠促進(jìn)毛囊從休止期向生長(zhǎng)初期轉(zhuǎn)變，延長(zhǎng)生長(zhǎng)初期[14-15]。有學(xué)者發(fā)現(xiàn)，褪黑激素可作為自由基清除劑和DNA修復(fù)誘導(dǎo)劑，代謝和增殖活性高的毛發(fā)生長(zhǎng)初期毛球也可以利用褪黑素合成作為自身細(xì)胞保護(hù)策略[16]。FGF5是目前所發(fā)現(xiàn)的一種與絨毛生長(zhǎng)有直接關(guān)系的基因之一。最早發(fā)現(xiàn)的安哥拉鼠被毛增長(zhǎng)就是由于FGF5基因突變所致[17]。此后，很多研究者們開(kāi)始了一系列有關(guān)FGF5與毛發(fā)生長(zhǎng)的研究。2007年JAMES等人利用家貓作為實(shí)驗(yàn)動(dòng)物進(jìn)行研究，結(jié)果顯示FGF5是影響家貓毛發(fā)長(zhǎng)度的主要因素[18]。SUZUKI等利用體外注射蛋白產(chǎn)物的方法進(jìn)行了驗(yàn)證實(shí)驗(yàn)，結(jié)果表明FGF5的蛋白產(chǎn)物在毛囊生長(zhǎng)的不同時(shí)期都具有影響[19]。此外，有些學(xué)者發(fā)現(xiàn)FGF5在毛發(fā)生長(zhǎng)及某些老鼠的脫毛過(guò)程中也具有調(diào)節(jié)作用[20]。KREGE等人發(fā)現(xiàn)FGF5能夠通過(guò)影響Sox2的表達(dá)從而對(duì)皮膚毛囊的再生起到非常重要的影響[21]。筆者一直致力于如何提高遼寧絨山羊的絨毛產(chǎn)量及品質(zhì)的研究，通過(guò)大量的試驗(yàn)，最終篩選出了MT和FGF5這兩種藥物，其中MT處理組中1.0g?L-124h、0.2g?L-124h、0.2g?L-172h三個(gè)處理?xiàng)l件最有利于遼寧絨山羊皮膚成纖維細(xì)胞的增殖與生長(zhǎng)，F(xiàn)GF5處理組中10-4g?L-124 h、10-4g?L-172 h、10-6g?L-124 h三個(gè)處理?xiàng)l件最適宜遼寧絨山羊皮膚成纖維細(xì)胞的增殖與生長(zhǎng)。長(zhǎng)鏈非編碼RNA(long noncoding RNA, LncRNA)是由RNA聚合酶Ⅱ轉(zhuǎn)錄的無(wú)蛋白質(zhì)編碼功能的調(diào)節(jié)性非編碼RNA，是一類新型的真核生物轉(zhuǎn)錄物[22]。目前，人們將其分為：正義LncRNA、反義LncRNA、雙向LncRNA、基因內(nèi)LncRNA和基因間LncRNA 5種主要類型[23]。很多學(xué)者研究發(fā)現(xiàn)lncRNA在多種生命過(guò)程中發(fā)揮重要作用，并且在細(xì)胞及生物體中的調(diào)節(jié)方式有多種，例如某些LncRNA可作為轉(zhuǎn)錄調(diào)控因子（或共調(diào)控因子）上調(diào)或下調(diào)某些基因的表達(dá)[24]。某些LncRNA在一定程度上對(duì)細(xì)胞的分化與增殖、生長(zhǎng)發(fā)育、器官生成、免疫應(yīng)答及腫瘤發(fā)生等多個(gè)生命活動(dòng)都有一定的調(diào)節(jié)作用[25-26]。Ren等通過(guò)將幽州黑山羊和渝東白山羊作為實(shí)驗(yàn)動(dòng)物進(jìn)行研究，他們對(duì)色素沉著早期階段皮膚細(xì)胞中的LncRNA進(jìn)行結(jié)構(gòu)和表達(dá)分析，篩選出差異表達(dá)的LncRNA，并對(duì)LncRNA進(jìn)行順式和反式靶基因的預(yù)測(cè)[27]。ZHU等發(fā)現(xiàn)，長(zhǎng)鏈非編碼RNA H19轉(zhuǎn)錄物與毛囊重建的真皮乳頭細(xì)胞的誘導(dǎo)能力有關(guān)，在次生毛囊中，lncRNA-H19轉(zhuǎn)錄物在毛發(fā)生長(zhǎng)初期階段的相對(duì)表達(dá)顯著高于毛發(fā)生長(zhǎng)終期階段和毛發(fā)生長(zhǎng)中期階段，表明lncRNA-H19轉(zhuǎn)錄物可能在山羊絨絨纖維的形成和生長(zhǎng)中起重要作用[28]。LIN等在毛乳頭細(xì)胞中發(fā)現(xiàn)了有助于毛發(fā)生長(zhǎng)相關(guān)基因表達(dá)的LncRNA的表達(dá)[29]。有學(xué)者通過(guò)高通量測(cè)序技術(shù)對(duì)綿羊的LncRNA進(jìn)行了生物信息學(xué)分析, 對(duì)綿羊基因組的研究有重大幫助[30]?！颈狙芯壳腥朦c(diǎn)】近年來(lái)，關(guān)于如何提高羊絨產(chǎn)量及品質(zhì)的研究越來(lái)越多。但是，有關(guān)MT和FGF5兩種藥物能否通過(guò)影響相關(guān)LncRNA的表達(dá)進(jìn)而提高羊絨產(chǎn)量及品質(zhì)的研究非常少。【擬解決的關(guān)鍵問(wèn)題】本試驗(yàn)以遼寧絨山羊?yàn)檠芯繉?duì)象，分別用MT和FGF5處理遼寧絨山羊皮膚成纖維細(xì)胞，通過(guò)高通量測(cè)序技術(shù)篩選出差異表達(dá)的LncRNA，預(yù)測(cè)差異表達(dá)LncRNA的靶基因，并通過(guò)GO和KEGG富集分析篩選出與絨毛生長(zhǎng)相關(guān)的LncRNA并進(jìn)行Real-time PCR驗(yàn)證。

1 材料與方法

1.1 細(xì)胞培養(yǎng)與藥物處理

試驗(yàn)于2016年在遼寧師范大學(xué)生命科學(xué)學(xué)院，遼寧省生物技術(shù)與分子藥物研發(fā)重點(diǎn)實(shí)驗(yàn)室進(jìn)行。遼寧絨山羊皮膚成纖維細(xì)胞原代培養(yǎng)，用不同濃度及時(shí)間的MT和FGF5進(jìn)行處理, 處理的條件分別為：MT處理組 1.0g?L-124h（M1_24H)、0.2g?L-124h（M2_24H)、0.2g?L-172h（M2_72H)； FGF5處理組，10-4g?L-124 h（F4_24H）、10-4g?L-172 h（F4_72H）、10-6g?L-124 h（F6_24H）。

1.2 提取RNA

參照寶生物工程(大連)有限公司的 DNase I(RNase Free)使用說(shuō)明進(jìn)行操作。

1.3 RNA-Seq文庫(kù)測(cè)序

由北京諾禾致源生物信息科技有限公司提供測(cè)序服務(wù)，測(cè)序平臺(tái)為 Illumina HiseqTM2500，以PE125的測(cè)序策略進(jìn)行測(cè)序。利用fastx_toolkit(v0.0.14)軟件對(duì)得到的Raw reads進(jìn)行分析。應(yīng)用Illumina Casva1.8軟件通過(guò)QPhred=-10log10(e)公式對(duì)堿基的質(zhì)量進(jìn)行檢測(cè)。

1.4 Mapping以及樣品相關(guān)性檢驗(yàn)

利用Tophat 2(V2.0.9)對(duì)過(guò)濾后的測(cè)序序列與山羊參考基因組進(jìn)行比對(duì)分析，應(yīng)用cufflinks和scripture軟件進(jìn)行轉(zhuǎn)錄本的拼接。

1.5 差異表達(dá)LncRNA的篩選

首先通過(guò)五步篩選法進(jìn)行基本的篩選，然后利用CPC、CNCI、pfam蛋白結(jié)構(gòu)域及PhyloCSF分析方法進(jìn)行編碼潛能的篩選，這幾種方法篩選的交集為候選的LncRNA。然后從候選LncRNA中篩選出差異表達(dá)的LncRNA

1.6 差異表達(dá)的LncRNA靶基因預(yù)測(cè)

LncRNA是通過(guò)與其靶基因mRNA相互作用來(lái)發(fā)揮作用的，因此采用Pearson相關(guān)系數(shù)法分析各樣本中LncRNA與蛋白編碼基因的表達(dá)量相關(guān)性和共表達(dá)分析方法來(lái)預(yù)測(cè)其靶基因。使用cuffdiff（http:// cufflinks.cbcb.umd.edu/manual.html#cuffdiff）軟件對(duì)篩選所得到的LncRNA進(jìn)行定量分析，從而得到各樣品中LncRNA的FPKM信息。

1.7 差異表達(dá)LncRNA靶基因的功能富集分析

將差異表達(dá)LncRNA的cis和trans靶基因分別做GO和KEGG富集分析。

1.8 目標(biāo)LncRNA的表達(dá)驗(yàn)證

首先培養(yǎng)遼寧絨山羊皮膚成纖維原代細(xì)胞，用0.2g?L-1的MT和10-4g?L-1的FGF5分別處理細(xì)胞72h后，提取RNA并反轉(zhuǎn)錄成cDNA，接下來(lái)通過(guò)Real-time PCR對(duì)差異表達(dá)的LncRNA進(jìn)行驗(yàn)證，引物見(jiàn)表1。

2 結(jié)果

2.1 樣品總RNA電泳檢測(cè)

經(jīng)過(guò) DNase I(RNase Free)處理，得到遼寧絨山羊皮膚成纖維細(xì)胞總RNA樣品，然后對(duì)各組樣品進(jìn)行瓊脂糖凝膠電泳。結(jié)果顯示試驗(yàn)中提取的遼寧絨山羊皮膚成纖維細(xì)胞樣品的總RNA 瓊脂糖凝膠電泳檢測(cè)條帶清晰，無(wú)拖尾和降解現(xiàn)象，表明提取的 RNA 完整性良好，可以用于后續(xù)分析（圖1）。

表1 RT-PCR引物序列

2.2 測(cè)序數(shù)據(jù)質(zhì)量評(píng)估

對(duì)RNA-Seq測(cè)序得到的數(shù)據(jù)質(zhì)量進(jìn)行檢測(cè)的結(jié)果如表2.1所示。通過(guò)表中數(shù)據(jù)可知，Error rate表示測(cè)序錯(cuò)誤率，它與堿基質(zhì)量有關(guān)，同時(shí)也受測(cè)序儀本身、測(cè)序試劑、樣品等多個(gè)因素共同影響，由表中數(shù)據(jù)可知堿基錯(cuò)誤率較低。Q = -10log10p，其中p值是由Phred計(jì)算出，它表示一個(gè)堿基被識(shí)別錯(cuò)誤的可能性，Q 值為 10 表示這個(gè)堿有90%的概率是正確的, 20 就是 99%。各個(gè)樣品中絕大部分reads的Q值均大于20?；鵊C含量相對(duì)較高，表明測(cè)序序列較穩(wěn)定。綜上，我們認(rèn)為各個(gè)樣品的測(cè)序序列都具有較高的質(zhì)量，可以進(jìn)行后續(xù)分析。

圖A為MT處理組，圖B為FGF5處理組。圖A中泳道1、2、3、4分別為M2_72H組、M2_24H組、M1_24H組和對(duì)照組C；圖B中泳道1、2、3、4分別為F4_24H組、F6_24H組、F4_72H和對(duì)照組C

2.3 Mapping

通過(guò)Tophat 2軟件將試驗(yàn)中樣品的clean reads分別與NCBI中山羊參考基因組進(jìn)行比對(duì)分析，發(fā)現(xiàn)試驗(yàn)中所產(chǎn)生的測(cè)序序列定位百分比均低于70%，其中具有多個(gè)定位的測(cè)序序列占總體的百分比也均低于10%，表明試驗(yàn)中參考基因組選擇合適，不存在污染。而且所有樣品中Unique Mapping Rate均為80%以上，因此可進(jìn)行下一步分析。從圖2中可以更加直觀看出染色體長(zhǎng)度和reads總數(shù)的關(guān)系，染色體的長(zhǎng)度與定位到該染色體內(nèi)reads總數(shù)呈正相關(guān)，MT處理組和FGF5處理組樣品比對(duì)到山羊1號(hào)、2號(hào)染色體上的reads相對(duì)來(lái)說(shuō)都比較多。

2.4 樣品間相關(guān)性檢查

樣品間表達(dá)水平相關(guān)性是檢驗(yàn)試驗(yàn)可靠性和樣本選擇是否合理的重要指標(biāo)。如圖3所示，可知各處理組與對(duì)照組相比較，2值均大于0.8, 表明樣品間表達(dá)水平相關(guān)性均較高。因此本試驗(yàn)所選擇的樣品均符合測(cè)序要求，可以繼續(xù)進(jìn)行下一步分析。

2.5 差異表達(dá)的LncRNA的篩選

篩選的條件是P-adjust <0.05，log2(Fold change) >1。由圖4可知，在M1_24H與C進(jìn)行比較，獲得32個(gè)差異表達(dá)的LncRNA，其中有4個(gè)LncRNA上調(diào)，28個(gè)下調(diào)；M2_24H與C組進(jìn)行比較，獲得10個(gè)差異表達(dá)的LncRNA，其中有4個(gè)LncRNA上調(diào)，6個(gè)下調(diào)；M2_72H與C組中進(jìn)行比較，獲得113個(gè)差異表達(dá)的LncRNA，其中有5個(gè)LncRNA上調(diào)，108個(gè)下調(diào)；F6_24H與C進(jìn)行比較，獲得123個(gè)差異表達(dá)的LncRNA，27個(gè)上調(diào)，96個(gè)下調(diào)；F4_24H與C進(jìn)行比較，獲得164個(gè)差異表達(dá)的LncRNA，70個(gè)上調(diào)，94個(gè)下調(diào)；F4_72H與C組中進(jìn)行比較，獲得189個(gè)差異表達(dá)的LncRNA，其中有78個(gè)上調(diào)，111個(gè)下調(diào)。

根據(jù)不同樣品中差異表達(dá)的LncRNA表達(dá)水平的高低，進(jìn)行層次聚類（hierarchical clustering）分析，從而判斷這些LncRNA在不同試驗(yàn)條件下的表達(dá)模式（圖5）。結(jié)果顯示在F4_24H組和M2_72H中差異表達(dá)的LncRNA表達(dá)水平相對(duì)較高。

橫坐標(biāo)：染色體的長(zhǎng)度信息（以百萬(wàn)堿基為單位）；縱坐標(biāo)：log2（reads的密度的中位數(shù)）；綠色為正鏈，紅色為負(fù)鏈

表2 RNA-Seq數(shù)據(jù)一覽表

樣品名稱：_1代表左端的reads，_2代表右端的reads；Raw reads：統(tǒng)計(jì)原始序列數(shù)據(jù)；Clean reads：過(guò)濾后的測(cè)序數(shù)據(jù)；Clean bases：測(cè)序序列個(gè)數(shù)與其長(zhǎng)度的積；Error rate：堿基錯(cuò)誤率；Q20、Q30：Phred 數(shù)值大于20、30的堿基與總堿基數(shù)之比；GC含量：含有G和C的堿基數(shù)量與占總堿基數(shù)之比

Sample_name: _1 represents reads of the left side, _2 represents reads of the right side; Raw reads: The original sequence data; Clean reads: The filtered sequencing data; Clean bases: The product of the number of sequencing sequence and the length of sequencing sequence; Error rate: Base error rate; Q20, Q30: The percentage of the bases that phred values are greater than 20 or 30 and all the bases; GC content: The percentage of the number of G and C bases and all the bases

圖3 各處理組中樣品間相關(guān)性檢查

有顯著性差異表達(dá)的轉(zhuǎn)錄本用紅色點(diǎn)（上調(diào)LncRNA）和綠色點(diǎn)（下調(diào)LncRNA）表示；橫坐標(biāo)代表LncRNA表達(dá)水平變化；縱坐標(biāo)代表LncRNA表達(dá)變化的統(tǒng)計(jì)學(xué)意義

2.6 差異表達(dá)的LncRNA靶基因Gene Ontology功能顯著性富集分析

分別根據(jù)LncRNA臨近位置的（上下游10k /100k）蛋白編碼基因和LncRNA與蛋白編碼基因的表達(dá)量相關(guān)性分析或共表達(dá)分析方法來(lái)預(yù)測(cè)其cis/trans靶基因。再對(duì)靶基因分別進(jìn)行cis和trans的GO富集分析，分別從生物過(guò)程（biological process，BP）、分子功能（molecular function，MF）和細(xì)胞組分（cellular component，CC）三個(gè)層面對(duì)靶基因進(jìn)行GO注釋。結(jié)果如表3、4所示：M2_24H vs C、F4_24H vs C和 F6_24H vs C組中差異表達(dá)的LncRNA均無(wú)顯著富集的條目；M1_24H vs C組中差異表達(dá)LncRNA的trans靶基因無(wú)顯著富集的GO term，其cis靶基因只在BP中有一個(gè)顯著富集的GO term，即negative regulation of transcription from RNA polymerase II promoter；M2_72H vs C和F4_72H vs C組中差異表達(dá)的LncRNA的cis靶基因均無(wú)顯著富集的GO term，而M2_72H vs C組trans靶基因主要富集在BP的cellular metabolic process biological_process、nitrogen compound metabolic process biological_process；MF中的binding molecular_function、catalytic activity molecular_function；CC中的membrane- bounded organelle cellular_component、F4_72H vs C組中差異表達(dá)的LncRNA的 rans靶基因主要富集在BP的cellular metabolic process biological_process、cellular macromolecule metabolic process biological_process，CC的cell cellular_component、cell part cellular_component、intracellular cellular_component，MF的binding molecular_ function中。

2.7 差異表達(dá)的LncRNA靶基因Pathway顯著性富集分析

通過(guò)KEGG數(shù)據(jù)庫(kù)，對(duì)樣品中差異表達(dá)LncRNA的靶基因進(jìn)行通路富集分析，得到各比較組合中顯著性富集（Qvalue<0.05）的pathway term。結(jié)果如表5—9所示：M2_24H vs C和F4_24H vs C組中均無(wú)顯著富集的pathway term；F6_24H vs C組中差異表達(dá)的LncRNA的trans靶基因無(wú)顯著富集的pathway term，其cis靶基因只富集在Taste transduction通路；M2_72H vs C、M1_24H vs C和F4_72H vs C組中差異表達(dá)的LncRNA的cis靶基因均無(wú)顯著富集的pathway term，M2_72H vs C 組差異表達(dá)的LncRNA的trans靶基因顯著富集到15個(gè)pathway term，其中TNF和NF-kappa B 信號(hào)通路與毛囊發(fā)育及絨毛周期性生長(zhǎng)相關(guān)，其通路中差異表達(dá)的靶基因、M1_24H vs C組差異表達(dá)的LncRNA的trans靶基因只富集在Steroid biosynthesis通路、F4_72H vs C組中差異表達(dá)的LncRNA的trans靶基因顯著富集到9個(gè)pathway term，其中只有Metabolic信號(hào)通路與毛囊發(fā)育及絨毛周期性生長(zhǎng)相關(guān)，其通路中差異表達(dá)的靶基因。

每列代表一個(gè)樣品，每行代表一種基因；紅色代表高表達(dá)LncRNA，藍(lán)色代表低表達(dá)LncRNA

2.8 目標(biāo)LncRNA的表達(dá)驗(yàn)證

M2_72H組LcRNA靶基因富集的NF-κB信號(hào)通路中靶基因所對(duì)應(yīng)的LncRNA有兩個(gè)，分別為（Gene ID）：XLOC_005914；XLOC_018763。F4_72H 組靶基因富集的Metabolic 信號(hào)通路中靶基因所對(duì)應(yīng)的LncRNA有4個(gè)，分別為（Gene ID）：XLOC_011424、XLOC_009522、XLOC_009063、XLOC_01115。通過(guò)Real-time PCR對(duì)篩選出的6個(gè)LncRNA進(jìn)行驗(yàn)證，結(jié)果如圖7所示，MT作用相關(guān)的兩個(gè)LncRNA在RNA- Seq中的表達(dá)量上調(diào)，Real-time PCR檢測(cè)結(jié)果與RNA- Seq測(cè)序結(jié)果一致，進(jìn)一步驗(yàn)證了RNA-Seq測(cè)序結(jié)果的準(zhǔn)確性。FGF5處理遼寧絨山羊皮膚成纖維細(xì)胞后，Real-time PCR結(jié)果為L(zhǎng)ncRNA XLOC_011424和XLOC_011157表達(dá)量下調(diào)；LncRNAXLOC_009063表達(dá)量上調(diào); LncRNA XLOC_009522表達(dá)量無(wú)顯著性差異。結(jié)合RNA-Seq測(cè)序中4個(gè)LncRNA表達(dá)量均下調(diào)的結(jié)果，表明LncRNA XLOC_011424、LncRNA XLOC_011157與前期結(jié)果一致。

表3 M2_72H vs C組差異表達(dá)LncRNA靶基因GO term分類

?*：P<0.05

表4 F4_72H vs C組差異表達(dá)LncRNA靶基因的GO term分類

表5 M2_72H vs C組Pathways富集數(shù)據(jù)表

樣本編號(hào)：已注釋到該條通路中，同時(shí)在表達(dá)水平上有統(tǒng)計(jì)學(xué)意義的基因總數(shù)；背景編號(hào)：該條通路中所有的基因數(shù)

Sample number: The number of comment the differentially expressed genes in this pathway；Background number: The number of all genes in this pathway

表6 M2_72H vs C組差異表達(dá)基因富集的絨毛生長(zhǎng)相關(guān)信號(hào)通路

表7 FGF處理組，KEGG富集分析

表8 FGF處理組，Pathways顯著性富集數(shù)據(jù)表

樣本編號(hào)：已注釋到該條通路中，同時(shí)在表達(dá)水平上有統(tǒng)計(jì)學(xué)意義的基因總數(shù)；背景編號(hào)：該條通路中所有的基因數(shù)

Sample number: The number of comment the differentially expressed genes in this pathway；Background number: The number of all genes in this pathway

表9 F4_72H vs C組差異表達(dá)基因富集的絨毛生長(zhǎng)相關(guān)信號(hào)通路

3 討論

本試驗(yàn)通過(guò)KEGG富集分析，共篩選出了3個(gè)與絨毛生長(zhǎng)相關(guān)的信號(hào)通路，分別為T(mén)NF、NF-κB和Metabolic信號(hào)通路。

KLOEPPERT等研究發(fā)現(xiàn)，NF-κB在維持人類毛囊的生長(zhǎng)期階段具有功能重要性。人類毛發(fā)生長(zhǎng)初期，頭皮毛囊快速增殖的毛發(fā)基質(zhì)上皮中，NF-κB活性非常顯著，關(guān)鍵的毛發(fā)生長(zhǎng)調(diào)節(jié)劑如TNFα和IL-1通過(guò)調(diào)控NF-κB信號(hào)通路從而影響絨毛生長(zhǎng)[35]。核因子（NF）-κB途徑參與毛囊的形態(tài)發(fā)生，Gilon M、Sher N、Cohen S和 Gat U通過(guò)瞬時(shí)轉(zhuǎn)染技術(shù)分析了p65 / RelA（一種NF-kB效應(yīng)子）對(duì)毛發(fā)角蛋白（HK）調(diào)節(jié)區(qū)的影響，結(jié)果表明p65能夠誘導(dǎo)人和小鼠來(lái)源的幾種酸性毛發(fā)角蛋白5（Ha5）的轉(zhuǎn)錄激活，p65與Ha5基因調(diào)節(jié)區(qū)域中的NF-κB/ RelA結(jié)合位點(diǎn)直接結(jié)合[36]。NF-κB/ Rel轉(zhuǎn)錄因子和IkappaB激酶（IKK）參與骨形態(tài)發(fā)生，皮膚增殖和分化等過(guò)程。另外，Schmidt-Ullrich等研究發(fā)現(xiàn)，抑制NF-κB的小鼠會(huì)出現(xiàn)毛囊缺陷[37]。皮膚干細(xì)胞可以再生表皮附屬物。然而，由于受傷而損失的毛囊?guī)缀鯖](méi)有再生。Wang等研究顯示，傷口中的巨噬細(xì)胞激活毛囊干細(xì)胞，導(dǎo)致傷口周圍的毛囊在休止期向生長(zhǎng)期過(guò)渡，毛囊再生等過(guò)程，主要通過(guò)TNF信號(hào)傳導(dǎo)調(diào)控[38]。LAURIKKALA等發(fā)現(xiàn)，外異蛋白（ED1）和外異蛋白A受體（EDAR）作為新的TNF配體-受體對(duì)的鑒定表明，TNF信號(hào)在胚胎形態(tài)發(fā)生中的作用，另外他們認(rèn)為ED1/EDAR信號(hào)傳導(dǎo)也調(diào)節(jié)毛囊的形態(tài)發(fā)生[39]。

蛋白質(zhì)是生命活動(dòng)的主要承擔(dān)者，而氨基酸是構(gòu)成蛋白質(zhì)分子的基本單位。L-半胱氨酸目前被認(rèn)為是條件必需的硫氨基酸，不僅是角蛋白的關(guān)鍵組分，還可以促進(jìn)許多生物途徑[40]。角蛋白相關(guān)蛋白8.1基因（KAP8.1）是一種負(fù)責(zé)羊絨的結(jié)構(gòu)基因。KAP8.1蛋白含有高甘氨酸和酪氨酸，參與基質(zhì)結(jié)構(gòu)纖維的調(diào)節(jié)。ZHAO等認(rèn)為KAP8.1基因的多態(tài)性可能與纖維直徑有關(guān)[41]。TONG等發(fā)現(xiàn)，原代培養(yǎng)物中的角蛋白17（K17）無(wú)效時(shí)，皮膚角質(zhì)形成細(xì)胞對(duì)TNFα選擇性更敏感。K17與TNF受體1（TNFR1）相關(guān)的死亡域蛋白（TRADD）相互作用，這是一種必需的死亡適配體TNFR1依賴性信號(hào)傳遞，而且NF-κB（TNFα的下游靶標(biāo)）的活性在K17無(wú)效皮膚中增加[42]。Wnt信號(hào)通路是毛囊發(fā)育中重要的途徑之一，次級(jí)毛囊中成纖維細(xì)胞生長(zhǎng)因子21和酪蛋白激酶是Wnt途徑中β-連環(huán)蛋白的重要調(diào)節(jié)因子。天冬酰胺和絲氨酸可能在初級(jí)毛囊生長(zhǎng)過(guò)程中具有重要作用[43]。由此可見(jiàn)，Metabolic、NF-kB 、TNF三個(gè)信號(hào)通路對(duì)絨毛生長(zhǎng)發(fā)育有著十分重要的作用。

LncRNA是影響絨毛生長(zhǎng)的重要因素之一。近幾年，關(guān)于LncRNA影響絨毛生長(zhǎng)的研究越來(lái)越多。BAIA等研究表明，LncRNA（LncRNA-599618、-599556、-599554、-599547、-599531和-599509）在毛發(fā)生長(zhǎng)初期階段的表達(dá)量顯著高于毛發(fā)生長(zhǎng)終期階段[31]。CAI 等發(fā)現(xiàn)，LncRNA5322能夠通過(guò)靶向毛囊干細(xì)胞中miR-21介導(dǎo)的PI3K-AKT信號(hào)傳導(dǎo)途徑來(lái)促進(jìn)毛囊干細(xì)胞的增殖和分化[32]。ZHOU 等在山羊皮膚中鑒定了1 122種已知的和403種新的LncRNA，其中173種在毛發(fā)生長(zhǎng)初期和退化期之間差異表達(dá)。另外他們發(fā)現(xiàn)，LncRNA和miRNA在毛囊生長(zhǎng)轉(zhuǎn)變中協(xié)同作用，并且退行期誘導(dǎo)因子（TGFβ1和BDNF）在miRNA-miRNA-mRNA網(wǎng)絡(luò)中由miR-873和Lnc108635596調(diào)節(jié)[33]。Song等研究表明，LncRNA XLOC_539599，XLOC_556463，XLOC_015081，XLOC_1285606，XLOC_297809和XLOC_764219對(duì)原發(fā)性羊毛毛囊誘導(dǎo)具有潛在的重要性，且差異表達(dá)的LncRNA的潛在靶基因在NF-κB信號(hào)通路顯著富集[34]。

本試驗(yàn)，利用高通量測(cè)序和Real-time PCR技術(shù)在Metabolic、NF-kB 、TNF 3個(gè)信號(hào)通路靶基因?qū)?yīng)的LncRNA中共篩選出4個(gè)與遼寧絨山羊絨毛生長(zhǎng)相關(guān)的LncRNA，分別為：LncRNA XLOC_ 011424、XLOC_011157、XLOC_005914、XLOC_ 018763。因此，可以認(rèn)為MT和FGF5兩種藥物處理，可通過(guò)影響某些相關(guān)LncRNA的表達(dá)，進(jìn)而影響絨毛生長(zhǎng)。

4 結(jié)論

LncRNA XLOC_011424、XLOC_011157、XLOC_ 005914和XLOC_018763可能通過(guò)增加羊絨密度及長(zhǎng)度，進(jìn)而提高遼寧絨山羊羊絨產(chǎn)量及品質(zhì)。其中，前兩個(gè)LncRNA通過(guò)調(diào)節(jié)其上游或下游的靶基因，調(diào)節(jié)TNF或NF-kB信號(hào)通路，進(jìn)而影響絨毛的生長(zhǎng)。LncRNA XLOC_011424和XLOC_011157通過(guò)調(diào)節(jié)其與Metabolic pathway相關(guān)的靶基因從而影響絨毛生長(zhǎng)。筆者所選擇的兩種藥物中，MT更能引起某些與絨毛生長(zhǎng)相關(guān)LncRNA的差異表達(dá)從而影響絨毛生長(zhǎng)。但是這4種LncRNA具體的功能和作用機(jī)制尚不清楚，后續(xù)試驗(yàn)研究將集中探討LncRNA促進(jìn)絨毛纖維生長(zhǎng)的作用機(jī)制。

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（責(zé)任編輯 林鑒非）

The Screening and Identification of Lncrna Related to Villus Growth in Liaoning Cashmere Goats by MT and FGF5

Jin Mei, Zhang LiJuan, Cao Qian, Guo XinYing

(Liaoning Normal University School of Life Sciences, Liaoning Provincial Key Laboratory of Biotechnology and Molecular Drug Development, Dalian 116029, Liaoning)

【Objective】 The aim of this study was to screen out the LncRNA associated with villus growth in Liaoning cashmere goat skin fibroblasts, and provide basic data for the study of the function and mechanism of LncRNA related to villus growth. 【Method】The total RNA of MT and FGF5 treated Liaoning cashmere goat skin fibroblasts was extracted, and the total RNA extracted was detected by total RNA electrophoresis detection, sequencing data quality evaluation, mapping comparison and inter-sample correlation test. The differentially expressed LncRNA was screened and its target gene was predicted. The LncRNA related to villus growth was screened by GO and KEGG enrichment analysis, and the target LncRNA was verified by Real-time PCR. 【Result】(1) The total RNA quality of the sample showed that the RNA was in good integrity, the GC content was relatively high, the sequence was stable, and the expression level between samples was high, which met the sequencing requirements.(2) Screening of differentially expressed LncRNA showed that there were 32 differentially expressed LncRNA in 1.0g?L-124h group, 4 of which were up-regulated and 28 of which were down-regulated. There were 10 differentially expressed LncRNA in 0.2g?L-124h group, 4 of which were up-regulated and 6 were down-regulated. There were 113 differentially expressed LncRNA in the 0.2g?L-172h group, of which 5 were up-regulated and 108 were down-regulated. There were 164 differentially expressed LncRNA in the 10-4g?L-124 h group, of which 70 were up-regulated and 94 were down-regulated. There were 189 differentially expressed LncRNA in the10-4g?L-172 h group, of which 78 were up-regulated and 111 were down-regulated. There were 123 LncRNA differentially expressed in the 10-6g?L-124 h group, among which 27 up and 96 down.(3) Target gene GO enrichment analysis showed that the 1.0g?L-124h group differentially expressed LncRNA target gene enrichment in GO's negative regulation of transcription from RNA polymerase II promoter; 0.2g?L-124h group did not differentially express LncRNA target gene enriched GO term;0.2g?L-172h group Differentially expressed LncRNA target gene enrichment in GO's cellular metabolic process biological_process, binding molecular_function, FGF5 treatment group only10-4g?L-172 h group differentially expressed LncRNA target gene enriched in cell cellular_component, cell part cellular_component, intracellular cellular_component, binding molecular_function and other six items. (4) Target gene KEGG enrichment analysis showed that the differential expression of LncRNA target gene in 1.0g?L-124h group was enriched in Steroid biosynthesis pathway; in 0.2g?L-124h group, there was no differential expression of LncRNA target gene enrichment Pathway term; 0.2g?L-172h group differentially expressed LncRNA target gene enrichment in Cell cycle, DNA replication, Steroid biosynthesis, TNF, Nod-like receptor, NF-kappa B and other signaling pathways, in which TNF and NF-kappa B signaling pathways are involved in villus growth. In FGF5-treated group, differentially expressed LncRNA targets in 10-4g?L-172 h group The gene was significantly enriched into nine path termes such as Fanconi anemia pathway, Huntington's disease, Metabolic pathway, Aminoacyl-tRNA biosynthesis, among which Metabolic pathway was associated with villus growth; the differentially expressed LncRNA target gene in 10-4g?L-124 h group had no significant enriched pathway term;10-6g?L-124 h The differentially expressed LncRNA target genes were only enriched in the Taste transduction pathway. (5) There are two LncRNA corresponding to the target genes TNFα, TNFAIP3 (A20), NFKBIA (IkBα), NFKB2 and IL8 enriched in NF-κB and TNF signaling pathways, respectively (Gene ID): XLOC_005914; XLOC_018763; There are four LncRNA corresponding to the target genes in the Metabolic pathway, namely (Gene ID): XLOC_011424, XLOC_009522, XLOC_009063, XLOC_01115. Real-time PCR results showed that LncRNA XLOC_011424, XLOC_011157, LncRNA XLOC_005914 and XLOC_018763 were consistent with high-throughput sequencing results. 【Conclusion】 LncRNA XLOC_011424, XLOC_011157, LncRNA XLOC_005914 and XLOC_018763 may increase the density and length of cashmere by regulating NF-κB, TNF or Metabolic signaling pathways related to villus growth, and thus improve the yield and quality of cashmere in Liaoning cashmere goat.

Liaoning cashmere goat; melatonin; FGF5; LncRNA; RNA-seq; signaling pathway

10.3864/j.issn.0578-1752.2019.04.014

2018-09-03；

2018-12-03

國(guó)家自然科學(xué)基金（31772557）、遼寧省自然科學(xué)基金（20170540577）

金梅，E-mail：jm6688210@163.com