半滑舌鰨miR-200a和miR-200b前體克隆及其免疫應(yīng)答分析

沙珍霞陳學(xué)杰,陳亞?wèn)|顏 慧

(1. 青島大學(xué)生命科學(xué)學(xué)院, 青島 266071; 2. 中國(guó)水產(chǎn)科學(xué)研究院黃海水產(chǎn)研究所, 農(nóng)業(yè)部海洋漁業(yè)可持續(xù)發(fā)展重點(diǎn)實(shí)驗(yàn)室,青島 266071; 3. 青島海洋科學(xué)與技術(shù)國(guó)家實(shí)驗(yàn)室海洋漁業(yè)科學(xué)與食物產(chǎn)出過(guò)程功能實(shí)驗(yàn)室, 青島 266200; 4. 上海海洋大學(xué)水產(chǎn)與生命學(xué)院, 上海 201306)

半滑舌鰨miR-200a和miR-200b前體克隆及其免疫應(yīng)答分析

沙珍霞1,2,3陳學(xué)杰2,3,4陳亞?wèn)|2,3顏 慧2

(1. 青島大學(xué)生命科學(xué)學(xué)院, 青島 266071; 2. 中國(guó)水產(chǎn)科學(xué)研究院黃海水產(chǎn)研究所, 農(nóng)業(yè)部海洋漁業(yè)可持續(xù)發(fā)展重點(diǎn)實(shí)驗(yàn)室,青島 266071; 3. 青島海洋科學(xué)與技術(shù)國(guó)家實(shí)驗(yàn)室海洋漁業(yè)科學(xué)與食物產(chǎn)出過(guò)程功能實(shí)驗(yàn)室, 青島 266200; 4. 上海海洋大學(xué)水產(chǎn)與生命學(xué)院, 上海 201306)

為了探究半滑舌鰨(Cynoglossus semilaevis) miR-200a和miR-200b在免疫應(yīng)答中的作用, 采用PCR方法克隆了半滑舌鰨miR-200家族的miR-200a和miR-200b的前體序列, 長(zhǎng)度分別為82和88 bp; 用The mfold Web Server和Clustalx1.83軟件對(duì)其前體序列進(jìn)行了二級(jí)結(jié)構(gòu)和同源性分析, miR-200a和miR-200b都具有典型的頸環(huán)結(jié)構(gòu), 與其他物種具有較高的同源性。qRT-PCR分析結(jié)果顯示, miR-200a和miR-200b在健康半滑舌鰨13種組織(肝臟、腸、脾臟、頭腎、后腎、鰓、血液、腦、皮膚、肌肉、胃、心臟和卵巢)中均有表達(dá), miR-200a在頭腎中表達(dá)量最高, 在血液中表達(dá)量最低, miR-200b在肝臟中表達(dá)量最高, 在肌肉中表達(dá)量最低; miR-200a和miR-200b在鰻弧菌(Vibrio anguillarum)感染半滑舌鰨后不同時(shí)間點(diǎn)的4種免疫相關(guān)組織(肝臟、腸、脾臟和頭腎)中的表達(dá)呈現(xiàn)出先上調(diào)后下降的規(guī)律, 但表達(dá)達(dá)到峰值的時(shí)間點(diǎn)有所不同。miR-200a在肝臟和脾中的表達(dá)峰值出現(xiàn)在鰻弧菌感染后6h, 在腸和頭腎中則是鰻弧菌感染后12h, miR-200b在腸、脾和頭腎中均在鰻弧菌感染后12h達(dá)到表達(dá)高峰; miR-200a和miR-200b在脂多糖(LPS)、肽聚糖(PGN)、葡聚糖(WGP)、聚肌胞苷酸(poly I:C) 4種病原模擬物刺激后的半滑舌鰨肝臟細(xì)胞系中呈現(xiàn)出上調(diào)表達(dá)趨勢(shì), 其中Poly I:C刺激半滑舌鰨肝臟細(xì)胞系后miR-200a上調(diào)表達(dá)趨勢(shì)明顯, 6h的表達(dá)量為0h的9倍, 在WGP刺激半滑舌鰨肝臟細(xì)胞后miR-200b上調(diào)表達(dá)趨勢(shì)明顯, 2h的表達(dá)量為0的9倍。研究結(jié)果為揭示miRNA在半滑舌鰨免疫應(yīng)答中的作用提供了科學(xué)依據(jù)。

半滑舌鰨; miR-200a; miR-200b; 前體克隆; 熒光實(shí)時(shí)定量PCR; 免疫應(yīng)答

半滑舌鰨(Cynoglossus semilaevis)屬于鰈形目、舌鰨科、舌鰨屬, 是我國(guó)重要的經(jīng)濟(jì)魚類[1], 但其養(yǎng)殖業(yè)受到各種病原菌的嚴(yán)重威脅。從分子層面揭示半滑舌鰨的免疫調(diào)控機(jī)制, 將為健康養(yǎng)殖提供新思路。

microRNA(miRNA)是一類非編碼的、長(zhǎng)度約18—25 nt的內(nèi)源性單鏈型小RNA分子[2,3], 在動(dòng)植物、病毒等多種有機(jī)體的生物學(xué)過(guò)程中發(fā)揮著重要作用, 如個(gè)體發(fā)育[4,5]、細(xì)胞增殖或凋亡[6,7]、細(xì)胞分化[8,9]、免疫應(yīng)答[10]和腫瘤發(fā)生[11,12]等。為了尋找半滑舌鰨免疫相關(guān)的miRNA, 本課題組進(jìn)行了半滑舌鰨病原刺激前后免疫組織miRNA的高通量測(cè)序[13]和miRNA芯片研究[14], 獲得了包括miR-200a和miR-200b在內(nèi)的99個(gè)表達(dá)差異顯著的miRNA。miR-200a和miR-200b同屬于miR-200家族, 其成員還包括miR-200c、miR-141和miR-429[15,16]。miR-200家族通過(guò)作用于兩個(gè)重要的細(xì)胞轉(zhuǎn)錄抑制因子ZEB1 (Zinc finger E-box-binding homeobox 1)和ZEB2 (Zinc finger E-box-binding homeobox 2), 調(diào)節(jié)上皮間質(zhì)的轉(zhuǎn)化過(guò)程, 從而抑制腫瘤的發(fā)生和侵襲。在哺乳動(dòng)物中miR-200家族在鼻咽癌[17]、肝癌[18]、腎透明細(xì)胞癌[19]、腦膜瘤[20]等多種腫瘤中差異表達(dá)。目前關(guān)于miR-200a和miR-200b的研究主要集中在與人類腫瘤相關(guān)基因的相互調(diào)控上, 而其在魚類免疫調(diào)節(jié)機(jī)制中如何發(fā)揮作用未見報(bào)道。

本研究旨在獲得半滑舌鰨miR-200a及miR-200b的前體序列、探究其組織表達(dá)特征及響應(yīng)鰻弧菌感染和病原模擬物刺激后在免疫組織及體外培養(yǎng)肝臟細(xì)胞的時(shí)空表達(dá)規(guī)律, 為揭示miRNA在半滑舌鰨免疫應(yīng)答中的作用提供科學(xué)依據(jù)。

1 材料與方法

1.1 實(shí)驗(yàn)材料

半滑舌鰨購(gòu)自日照東鑫現(xiàn)代漁業(yè)技術(shù)研究所,魚齡在1.5齡左右, 平均體長(zhǎng)(30.5±2.5) cm, 平均體重(220±6.4) g。感染前在實(shí)驗(yàn)室水族箱中暫養(yǎng)7d,水溫控制在(25±1)℃, 每天更換新鮮滅菌海水。

本研究所用半滑舌鰨肝臟細(xì)胞系(Liver cell line of half-smooth tongue-sole, HTLC)[21]已傳至27代。細(xì)胞在含有1%青鏈霉素和15%胎牛血清的DMEM培養(yǎng)基(Invitrogen, 美國(guó))中于24℃培養(yǎng)。

1.2 樣品處理和采集

健康組織的收集隨機(jī)選取3條健康的半滑舌鰨, 分別采集每條魚的肝臟、腸、脾臟、頭腎、后腎、鰓、血液、腦、皮膚、肌肉、胃、心臟和卵巢13種組織, 并立即投入液氮中冷凍, 然后轉(zhuǎn)移至-80℃保存, 以備RNA提取。

鰻弧菌感染組和PBS對(duì)照組組織收集實(shí)驗(yàn)注射用的鰻弧菌為本實(shí)驗(yàn)分離獲得的菌種, 進(jìn)行培養(yǎng)后用磷酸緩沖液(PBS)重新懸浮至濃度為2.864×108CFU/mL。鰻弧菌感染參照Sha等[22]實(shí)驗(yàn)方法略作修改, 采用腹腔注射的方法對(duì)實(shí)驗(yàn)組進(jìn)行鰻弧菌感染, 感染劑量(半致死劑量)為3.18×105CFU/g魚體, 對(duì)照組注射相應(yīng)劑量的PBS。在0、6h、12h、24h、48h和72h共6個(gè)時(shí)間點(diǎn), 分別收集鰻弧菌感染組和PBS對(duì)照組的肝臟、腸、脾臟和頭腎4種組織, 每個(gè)時(shí)間點(diǎn)取3條魚。將所取組織立即投入液氮研磨, 然后轉(zhuǎn)移至-80℃保存, 以備RNA提取。

HTLC細(xì)胞樣品收集將HTLC細(xì)胞接種到1 2孔細(xì)胞培養(yǎng)板中, 待細(xì)胞長(zhǎng)滿培養(yǎng)板的70%—80%, 分別用終濃度為50 ng/mL的脂多糖(LPS)、100 μg/mL的肽聚糖(PGN)、50 ng/mL的葡聚糖(WGP)、50 μg/mL的聚肌胞苷酸(poly I:C)[23]刺激細(xì)胞, 每個(gè)實(shí)驗(yàn)設(shè)置3個(gè)重復(fù)。刺激過(guò)程持續(xù)30min, 然后去除刺激物, PBS清洗細(xì)胞, 繼續(xù)培養(yǎng)細(xì)胞。用PBS同時(shí)處理HTLC作為對(duì)照組。分別收集感染后0(感染開始時(shí))、2h、6h、12h和24h共5個(gè)時(shí)間點(diǎn)的細(xì)胞, 每個(gè)時(shí)間點(diǎn)取3個(gè)重復(fù), 低溫離心后去除離心管中上清液, 加入1 mL的裂解液(RZ), 然后轉(zhuǎn)移至-80℃保存, 以備RNA提取。

1.3 RNA的提取和cDNA模板的制備

RNA的提取按照RNA提取試劑盒(天根, 北京)的說(shuō)明書進(jìn)行操作。miRNA cDNA第一條鏈的合成按照miRcute miRNA cDNA第一鏈合成試劑盒(天根, 北京)說(shuō)明書進(jìn)行。最終得到的cDNA在-20℃保存待用。

1.4 半滑舌鰨miR-200a和miR-200b前體克隆及序列分析

將半滑舌鰨miR-200a和miR-200b的成熟體序列比對(duì)到半滑舌鰨基因組[24], 結(jié)合其相近物種的前體序列及二級(jí)結(jié)構(gòu)的穩(wěn)定性得到半滑舌鰨miR-200a和miR-200b前體的基因組序列, 根據(jù)獲得的序列設(shè)計(jì)特異性引物miR-200a-F、miR-200a-R和miR-200b-F、miR-200b-R(表 1), 用于擴(kuò)增miR-200a和miR-200b的前體序列, 將克隆得到的序列連接在T1載體(全式金, 北京)上, 轉(zhuǎn)化至Top10感受態(tài)細(xì)胞中, 37℃、200 r/min培養(yǎng)1h, 取200 μL菌液均勻涂在含有Amp+抗性的LB平板上, 37℃培養(yǎng)過(guò)夜,挑取單克隆于含Amp+的液體LB培養(yǎng)基中在37℃、200 r/min培養(yǎng)4—6h, 進(jìn)行菌液PCR檢測(cè), PCR產(chǎn)物用1%的瓊脂糖凝膠電泳進(jìn)行檢測(cè), 將含有陽(yáng)性克隆的菌液送至蘇州金唯智生物技術(shù)有限公司測(cè)序。

將測(cè)序結(jié)果去除載體序列后用The mfold Web Server (http://mfold.rna.albany.edu/？q=mfold/ RNAFolding-Form)在線軟件對(duì)miR-200a和miR-200b進(jìn)行二級(jí)結(jié)構(gòu)預(yù)測(cè)。再將序列在miRBase (http:// www.mirbase.org/)上進(jìn)行BLAST分析, 并利用Clustalx1.83 (http://www.genome.jp/tools/clustalx/)進(jìn)行同源性分析。

表 1 半滑舌鰨miR-200a和miR-200b前體克隆及定量引物Tab. 1 Primers used in the current study

1.5 半滑舌鰨miR-200a和miR-200b實(shí)時(shí)定量(qRT-PCR)分析

根據(jù)半滑舌鰨miR-200a和miR-200b的成熟體序列設(shè)計(jì)qRT-PCR的正向引物qmiR-200a-F和qmiR-200b-F, 反向引物為miRcute miRNA qPCR Detection Kit (天根, 北京)自帶引物, 用半滑舌鰨U6基因作為內(nèi)參, 設(shè)計(jì)內(nèi)參引物U6-F和U6-R(引物序列見表 1)。按照miRcute miRNA qPCR Detection Kit的說(shuō)明在ABI PRISM 7500 Fast實(shí)時(shí)定量擴(kuò)增儀上進(jìn)行qRT-PCR, 程序設(shè)置為94℃, 2min, 1次循環(huán); 94℃, 20s, 60℃, 34s, 40次循環(huán)。

1.6 統(tǒng)計(jì)學(xué)分析

使用SPSS 19.0統(tǒng)計(jì)軟件對(duì)qRT-PCR檢測(cè)結(jié)果進(jìn)行分析。實(shí)時(shí)定量數(shù)據(jù)用3組重復(fù)平均值±標(biāo)準(zhǔn)誤(SE)表示, 使用2-ΔΔCt法計(jì)算相對(duì)表達(dá)量。采用單因素方差分析法中的Duncan法對(duì)多組樣本均數(shù)進(jìn)行兩兩比較分析, P＜0.05時(shí), 認(rèn)為存在顯著性差異。

2 結(jié)果

2.1 半滑舌鰨miR-200a和miR-200b的前體克隆及序列分析

采用PCR方法對(duì)半滑舌鰨miR-200a和miR-200b前體進(jìn)行了克隆, 測(cè)序結(jié)果去除載體序列后顯示miR-200a前體序列長(zhǎng)度為82 bp, miR-200b前體序列長(zhǎng)度為88 bp。通過(guò)The mfold Web Server在線軟件對(duì)半滑舌鰨miR-200a和miR-200b前體序列進(jìn)行了二級(jí)結(jié)構(gòu)的分析(圖 1), 顯示miR-200a和miR-200b都具有典型的頸環(huán)結(jié)構(gòu)特征, miR-200a的最小折疊自由能為-34.60 kJ/mol, miR-200b的最小折疊自由能-33.00 kJ/mol。將miR-200a和miR-200b的前體序列在miBase21.0數(shù)據(jù)庫(kù)中進(jìn)行BLAST比對(duì),結(jié)果顯示miR-200a和miR-200b在斑馬魚(Danio rerio)、鯉(Cyprinus carpio)、紅鰭東方鲀(Fugu rubripes)、青斑河鲀(Tetraodon nigroviridis)、人(Homo sapiens)、小家鼠(Mus muscuLus)等10個(gè)物種中有同源序列, 用Clustalx1.83進(jìn)行同源性分析發(fā)現(xiàn)miR-200a和miR-200b前體與其他物種具有較高的同源性, 特別是頸部為miRNA成熟體所在的部位,保守性更高。

2.2 半滑舌鰨miR-200a和miR-200b在組織中的表達(dá)

miR-200a和miR-200b在健康半滑舌鰨組織中的表達(dá)miR-200a和miR-200b在健康半滑舌鰨13種組織(肝臟、腸、脾、頭腎、后腎、鰓、血液、腦、皮膚、肌肉、胃、心臟和卵巢)中均有表達(dá), 但相對(duì)表達(dá)量存在較大差異(圖 2)。miR-200a在頭腎中表達(dá)量最高(58.07), 其次是肝臟(57.97)和脾(22.56), 在血液中表達(dá)量最低(1.00), 其次是肌肉(1.16)和腦(1.35)。miR-200b在肝臟中的表達(dá)量最高(93.92), 其次是頭腎(67.45)和鰓(30.10), 在肌肉中表達(dá)量最低(1.00), 其次是血液(1.17)和卵巢(1.22)。

圖 1 半滑舌鰨miR-200a和miR-200b前體序列的頸環(huán)結(jié)構(gòu)Fig. 1 Hairpin structures formed by precursor sequence of miR-200a and miR-200b in C. semilaevis

圖 2 miR-200a和miR-200b在健康半滑舌鰨組織中的相對(duì)表達(dá)量Fig. 2 The expression of miR-200a and miR-200b in tissues of C. semilaevis字母“a, b, c, d”代表SPSS多重分析的不同分組; 不同字母表示組間差異顯著(P＜0.05); 下同; b. 腦; bl. 血液; g. 鰓; h. 心臟; hk.頭腎; in. 腸; l. 肝臟; m. 肌肉; me. 后腎; o. 卵巢; s. 皮膚; sp. 脾臟; st. 胃The letters of ‘a(chǎn), b, c and d’ indicated the Duncan grouping in SPSS. Different letters indicate significant difference between groups (P＜0.05); The same applies below. b. brain; bl. blood; g. gill; h. heart; hk. head kidney; in. intestine; l. liver; m. muscle; me. metanephros; o. ovary; s. skin; sp. spleen; st. stomach

鰻弧菌感染半滑舌鰨后miR-200a和miR-200b在四種免疫組織中的相對(duì)表達(dá)鰻弧菌感染半滑舌鰨后miR-200a在肝臟、腸、脾臟和頭腎4種組織中都呈現(xiàn)先上升后下降的表達(dá)趨勢(shì)(圖 3):其中在肝臟和脾中的表達(dá)峰值出現(xiàn)在鰻弧菌感染后6h, 分別為是0表達(dá)量的6.30倍和9.80倍, 隨后表達(dá)量逐步下降, 感染后48h肝臟的表達(dá)量降為0的1/3, 在脾臟中則是在72h達(dá)到表達(dá)的最低值; 在腸和頭腎中表達(dá)的峰值則是在鰻弧菌感染后12h, 分別為0表達(dá)量的6.52倍和7倍, 隨后下調(diào)表達(dá), 至72h,接近0的表達(dá)水平。

鰻弧菌感染半滑舌鰨后miR-200b在4種免疫組織中也都呈現(xiàn)先上升后下降的表達(dá)趨勢(shì)(圖 4), 在肝臟中表達(dá)峰值出現(xiàn)在感染后6h, 為0表達(dá)量的14倍, 在12h和24h開始出現(xiàn)下調(diào), 并在48h和72h恢復(fù)到表達(dá)的初始值; 而在腸、脾和頭腎中則是感染后12h出現(xiàn)表達(dá)峰值, 在腸中鰻弧菌感染后6h開始出現(xiàn)上調(diào), 直到12h出現(xiàn)表達(dá)的峰值, 為0表達(dá)量的8.5倍, 到24h和48h開始逐漸下調(diào), 并在72h降至0表達(dá)水平。在脾中鰻弧菌感染后6h較0變化不大, 但在12h表達(dá)量上升為0的10.12倍, 在24h開始表達(dá)下調(diào), 在48h和72h保持0h的表達(dá)水平。在頭腎中鰻弧菌感染后12h出現(xiàn)表達(dá)峰值為21.13, 到72h下調(diào)到0的表達(dá)水平。

圖 3 鰻弧菌感染半滑舌鰨后miR-200a在不同時(shí)間點(diǎn)4種免疫組織中的相對(duì)表達(dá)量Fig. 3 The relative expression levels of miR-200a in four tissues at different time points after C. semilaevis infected with Vibrio anguillarum每個(gè)時(shí)間點(diǎn)miR-200a的表達(dá)量為感染組除以PBS組的相對(duì)表達(dá)量The expression levels of miR-200a at each point was presented relative expression levels which infected group divided by the PBS group

圖 4 鰻弧菌感染半滑舌鰨后miR-200b在不同時(shí)間點(diǎn)4種免疫組織中的相對(duì)表達(dá)量Fig. 4 The relative expression levels of miR-200b in four tissues at different time points after C. semilaevis infected with Vibrio anguillarum每個(gè)時(shí)間點(diǎn)miR-200b的表達(dá)量為感染組除以PBS組的相對(duì)表達(dá)量The expression levels of miR-200b at each point was presented relative expression levels which infected group divided by the PBS group

2.3 四種病原模擬物刺激體外培養(yǎng)的HTLC細(xì)胞后miR-200a和miR-200b的相對(duì)表達(dá)

4種病原模擬物刺激后miR-200a在HTLC細(xì)胞中呈現(xiàn)上調(diào)表達(dá)的趨勢(shì)(圖 5), 除了LPS刺激HTLC細(xì)胞12h后miR-200a表達(dá)量達(dá)到最大值, miR-200a在PGN、WGP和Poly I:C刺激HTLC細(xì)胞后均在6h出現(xiàn)表達(dá)的最高峰, 其中最大表達(dá)量是Poly I:C刺激后6h, 為0的9倍, miR-200a在WGP刺激后2—12h, 表達(dá)量都在0的4倍以上, 在24h也達(dá)到0的2倍, LPS、PGN和Poly I:C刺激后miR-200a表達(dá)變化趨勢(shì)基本一致, 即在6h或12h出現(xiàn)較高的上調(diào)表達(dá)峰值外, 在其他時(shí)間點(diǎn)的表達(dá)量與0相比變化不大。

miR-200b在4種病原模擬物刺激的HTLC細(xì)胞中同樣呈現(xiàn)上調(diào)表達(dá)的趨勢(shì)(圖 6), 但不同病原模擬物刺激后miR-200b出現(xiàn)上調(diào)表達(dá)峰值的時(shí)間點(diǎn)和變化倍數(shù)有所不同, 其中, LPS刺激后2h表達(dá)開始出現(xiàn)上調(diào), 在12h出現(xiàn)表達(dá)的最高峰為0的4.2倍, 到24h恢復(fù)表達(dá)的初始水平; 而PGN和Poly I:C刺激后都是在6h出現(xiàn)表達(dá)的最大值, 均為0的6倍左右; 在WGP刺激后, miR-200b表達(dá)變化的峰值出現(xiàn)2h為0表達(dá)量的9倍, 而后表達(dá)量逐漸下調(diào), 但在24h的表達(dá)量仍為0h的2倍。

圖 5 LPS、PGN、WGP和poly I:C刺激半滑舌鰨肝臟細(xì)胞后miR-200a的相對(duì)表達(dá)量Fig. 5 The relative expression levels of miR-200a in HTLC response to the challenge of LPS, PGN, WGP and polyl: C

圖 6 LPS, PGN, WGP和poly I:C刺激半滑舌鰨肝臟細(xì)胞后miR-200b的相對(duì)表達(dá)量Fig. 6 The relative expression levels of miR-200b in HTLC response to the challenge of LPS, PGN, WGP and polyl: C

3 討論

miRNA廣泛存在于不同進(jìn)化層次的生物體中,其成熟體序列存在種間高度保守的特征, 在生物體胚胎形成、組織器官發(fā)育、細(xì)胞分化凋亡、免疫功能調(diào)控及疾病發(fā)生等各種生命活動(dòng)過(guò)程中發(fā)揮著重要作用, 是一種新的免疫調(diào)控因子[25,26]。在哺乳動(dòng)物方面的研究證據(jù)表明具有侵染能力的小鼠乳腺腫瘤細(xì)胞中miR-200家族具有較高的表達(dá)[27]; Lee等[28]發(fā)現(xiàn), miR-200家族成員的差異表達(dá)出現(xiàn)在了子宮內(nèi)膜癌的組織中; Hiroki等[29]報(bào)道了子宮內(nèi)膜腺癌組織中miR-200a表達(dá)與癌旁正常組織相比明顯增高。魚類在進(jìn)化上與哺乳動(dòng)物差別較大, miRNA在結(jié)構(gòu)上雖然與哺乳動(dòng)物較為相似, 但在功能上可能還是存在較大差異, 由于魚類miRNA的研究剛剛起步, 在免疫應(yīng)答和疾病產(chǎn)生等領(lǐng)域目前尚缺乏表達(dá)和功能相關(guān)的研究積累。

本研究首次克隆了半滑舌鰨miR-200a和miR-200b的前體序列, 比對(duì)結(jié)果顯示它們的頸部在不同物種之間具有較高的同源性, 因?yàn)榇瞬课唤?jīng)過(guò)Dicer酶的剪切加工后可以形成miRNA成熟體, 進(jìn)而與其靶基因結(jié)合, 在轉(zhuǎn)錄后水平發(fā)揮抑制作用。miR-200a和miR-200b廣泛表達(dá)于各組織中, 但在不同組織中存在較大差異, 這可能與其在不同組織中的生物學(xué)功能不同有關(guān), 現(xiàn)有研究表明miR-200家族參與了不同的信號(hào)通路, miR-200a主要作用于蛋白激酶C/轉(zhuǎn)化生長(zhǎng)因子β (Protein Kinase C/Transforming growth facter-β, PKC/Tgf-β)信號(hào)通路[30], 而miR-200b主要參與磷酸肌醇3激酶/蛋白激酶B (Phosphatidyl-inositol 3-Kinase/Protein KinaseB, PI3K/AKT)信號(hào)通路抑制癌細(xì)胞的擴(kuò)散和遷移[31]。miR-200a和miR-200b表達(dá)量最高的兩個(gè)組織都是肝臟和頭腎, 肝臟和頭腎都是魚類重要的免疫器官,該結(jié)果表明, miR-200a和miR-200b可能在調(diào)控肝臟和頭腎免疫細(xì)胞生成和功能上具有作用。Joseph等[32]研究了miR-122a、miR-21和miR-200b這3個(gè)miRNA在肝臟、胰臟和胃中的表達(dá), miR-200b在肝臟中表達(dá)量最高。

鰻弧菌感染半滑舌鰨后miR-200a和miR-200b在4種免疫相關(guān)組織中都表現(xiàn)出上調(diào)表達(dá)的趨勢(shì),說(shuō)明miR-200a和miR-200b在病原入侵后做出了免疫應(yīng)答反應(yīng)。Lv等[33]用LPS刺激了仿刺參的體腔細(xì)胞, 發(fā)現(xiàn)miR-200a和miR-200b主要通過(guò)參與TLR (toll-like receptor)信號(hào)通路使體腔細(xì)胞發(fā)揮抗菌作用。TLR是迄今為止研究最為透徹的重要模式受體, 能夠選擇性的識(shí)別來(lái)自細(xì)菌、病毒和真菌的刺激[34], 通過(guò)接頭蛋白等將細(xì)胞外刺激信號(hào)傳遞至細(xì)胞內(nèi), 從而誘導(dǎo)促炎癥反應(yīng)相關(guān)因子的產(chǎn)生, 上調(diào)協(xié)同刺激因子的表達(dá), 迅速激活天然免疫, 為激活機(jī)體適應(yīng)性免疫應(yīng)答、形成免疫記憶提供必要條件[35]。

綜上所述, miR-200a和miR-200b參與了免疫應(yīng)答反應(yīng)。本研究為半滑舌鰨miRNA的免疫相關(guān)性提供了科學(xué)依據(jù), 為后續(xù)半滑舌鰨miRNA的研究奠定了理論基礎(chǔ)。

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THE PRECURSOR CLONING AND IMMUNE RESPONSE ANALYSIS OF MIR-200A AND MIR-200B IN CYNOGLOSSUS SEMILAEVIS

SHA Zhen-Xia1,2,3, CHEN Xue-Jie2,3,4, CHEN Ya-Dong2,3and YAN Hui2
(1. College of Life Science, Qingdao University, Qingdao 266071, China; 2. Key Lab for Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; 3. Laboratory for Marine Fisheries and Aquaculture, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266200, China; 4. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China)

In this study, we cloned the precursor sequences of miR-200a and miR-200b from C. semilaevis miR-200 family by PCR. The precursor sequences of miR-200a and miR-200b were 82 bp and 88bp in length respectively. We analyzed the secondary structure and homology of the precursor sequence using the mfold Web Server and Clustalx1.83. The results showed that both precursors had typical stem-ring structure and shared highly homology with other species. The qRT-PCR results indicated that miR-200a and miR-200b were expressed in all 13 tissues (liver, intestine, spleen, head kidney, metanephros, gill, blood, brain, skin, muscle, stomach, heart and ovary) of C. semilaevis, and that miR-200a highly expressed in head kidney and lowly in blood, and miR-200b highly expressed in liver and lowly in muscle. Vibro anguillarum infection initially induced and then decreased the expression f miR-200a and miR-200b in 4 immune tissues (liver, intestine, spleen and head kidney) of C. semilaevis with various peak at each time point. miR-200a has the highest level in the liver and spleen at 6h and in intestine and head kidney at 12h respectively after infecttion by V. anguillarum. miR-200b had the highest level in the intestine, spleen and head kidney at 12h after infection by V. anguillarum. miR-200a and miR-200b were expressed up-regulated in liver cell of C. semilaevis (HTLC) when stimulated using pathogen analogs (LPS, PGN, WGP and PolyI:C). miR-200a was obviously up-regulated in HTLC after treatment by PolyI:C and the expression level achieved 9 times at 6h than at 0 h. miR-200b was obviously up-regulated quickly in HTLC after stimulated by WGP, compared with 0 h, the expression level increased 9 times at 2h. In a word, the results of this study would provide the theory basis for the research of immune-related miRNA in C. semilaevis.

Cynoglossus semilaevis; miR-200a; miR-200b; Precursor cloning; qRT-PCR; Immune response

Q522

A

1000-3207(2017)02-0314-07

10.7541/2017.38

2016-05-28;

2016-07-14

國(guó)家自然科學(xué)基金(31572644和31172439); 鰲山科技創(chuàng)新計(jì)劃(2015ASKJ02-03)資助 [Supported by the National Natural Science Foundation of China (31572644和31172439); Aoshan Science and Technology Innovation Project (2015ASKJ02-03)]

沙珍霞, 女, 研究員, 博士; 主要從事魚類分子免疫學(xué)、基因組學(xué)和細(xì)胞生物學(xué)研究。E-mail: shazhenxia@163.com