半滑舌鰨miR-200a和miR-200b前體克隆及其免疫應答分析

沙珍霞陳學杰,陳亞東顏 慧

(1. 青島大學生命科學學院, 青島 266071; 2. 中國水產科學研究院黃海水產研究所, 農業部海洋漁業可持續發展重點實驗室,青島 266071; 3. 青島海洋科學與技術國家實驗室海洋漁業科學與食物產出過程功能實驗室, 青島 266200; 4. 上海海洋大學水產與生命學院, 上海 201306)

半滑舌鰨miR-200a和miR-200b前體克隆及其免疫應答分析

沙珍霞1,2,3陳學杰2,3,4陳亞東2,3顏 慧2

(1. 青島大學生命科學學院, 青島 266071; 2. 中國水產科學研究院黃海水產研究所, 農業部海洋漁業可持續發展重點實驗室,青島 266071; 3. 青島海洋科學與技術國家實驗室海洋漁業科學與食物產出過程功能實驗室, 青島 266200; 4. 上海海洋大學水產與生命學院, 上海 201306)

為了探究半滑舌鰨(Cynoglossus semilaevis) miR-200a和miR-200b在免疫應答中的作用, 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, 長度分別為82和88 bp; 用The mfold Web Server和Clustalx1.83軟件對其前體序列進行了二級結構和同源性分析, miR-200a和miR-200b都具有典型的頸環結構, 與其他物種具有較高的同源性。qRT-PCR分析結果顯示, 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, miR-200a在頭腎中表達量最高, 在血液中表達量最低, miR-200b在肝臟中表達量最高, 在肌肉中表達量最低; miR-200a和miR-200b在鰻弧菌(Vibrio anguillarum)感染半滑舌鰨后不同時間點的4種免疫相關組織(肝臟、腸、脾臟和頭腎)中的表達呈現出先上調后下降的規律, 但表達達到峰值的時間點有所不同。miR-200a在肝臟和脾中的表達峰值出現在鰻弧菌感染后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達到表達高峰; 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胞系中呈現出上調表達趨勢, 其中Poly I:C刺激半滑舌鰨肝臟細胞系后miR-200a上調表達趨勢明顯, 6h的表達量為0h的9倍, 在WGP刺激半滑舌鰨肝臟細胞后miR-200b上調表達趨勢明顯, 2h的表達量為0的9倍。研究結果為揭示miRNA在半滑舌鰨免疫應答中的作用提供了科學依據。

半滑舌鰨; miR-200a; miR-200b; 前體克隆; 熒光實時定量PCR; 免疫應答

半滑舌鰨(Cynoglossus semilaevis)屬于鰈形目、舌鰨科、舌鰨屬, 是我國重要的經濟魚類[1], 但其養殖業受到各種病原菌的嚴重威脅。從分子層面揭示半滑舌鰨的免疫調控機制, 將為健康養殖提供新思路。

microRNA(miRNA)是一類非編碼的、長度約18—25 nt的內源性單鏈型小RNA分子[2,3], 在動植物、病毒等多種有機體的生物學過程中發揮著重要作用, 如個體發育[4,5]、細胞增殖或凋亡[6,7]、細胞分化[8,9]、免疫應答[10]和腫瘤發生[11,12]等。為了尋找半滑舌鰨免疫相關的miRNA, 本課題組進行了半滑舌鰨病原刺激前后免疫組織miRNA的高通量測序[13]和miRNA芯片研究[14], 獲得了包括miR-200a和miR-200b在內的99個表達差異顯著的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家族通過作用于兩個重要的細胞轉錄抑制因子ZEB1 (Zinc finger E-box-binding homeobox 1)和ZEB2 (Zinc finger E-box-binding homeobox 2), 調節上皮間質的轉化過程, 從而抑制腫瘤的發生和侵襲。在哺乳動物中miR-200家族在鼻咽癌[17]、肝癌[18]、腎透明細胞癌[19]、腦膜瘤[20]等多種腫瘤中差異表達。目前關于miR-200a和miR-200b的研究主要集中在與人類腫瘤相關基因的相互調控上, 而其在魚類免疫調節機制中如何發揮作用未見報道。

本研究旨在獲得半滑舌鰨miR-200a及miR-200b的前體序列、探究其組織表達特征及響應鰻弧菌感染和病原模擬物刺激后在免疫組織及體外培養肝臟細胞的時空表達規律, 為揭示miRNA在半滑舌鰨免疫應答中的作用提供科學依據。

1 材料與方法

1.1 實驗材料

半滑舌鰨購自日照東鑫現代漁業技術研究所,魚齡在1.5齡左右, 平均體長(30.5±2.5) cm, 平均體重(220±6.4) g。感染前在實驗室水族箱中暫養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胞系(Liver cell line of half-smooth tongue-sole, HTLC)[21]已傳至27代。細胞在含有1%青鏈霉素和15%胎牛血清的DMEM培養基(Invitrogen, 美國)中于24℃培養。

1.2 樣品處理和采集

健康組織的收集隨機選取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, 然后轉移至-80℃保存, 以備RNA提取。

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

HTLC細胞樣品收集將HTLC細胞接種到1 2孔細胞培養板中, 待細胞長滿培養板的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]刺激細胞, 每個實驗設置3個重復。刺激過程持續30min, 然后去除刺激物, PBS清洗細胞, 繼續培養細胞。用PBS同時處理HTLC作為對照組。分別收集感染后0(感染開始時)、2h、6h、12h和24h共5個時間點的細胞, 每個時間點取3個重復, 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)的說明書進行操作。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)說明書進行。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的成熟體序列比對到半滑舌鰨基因組[24], 結合其相近物種的前體序列及二級結構的穩定性得到半滑舌鰨miR-200a和miR-200b前體的基因組序列, 根據獲得的序列設計特異性引物miR-200a-F、miR-200a-R和miR-200b-F、miR-200b-R(表 1), 用于擴增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, 轉化至Top10感受態細胞中, 37℃、200 r/min培養1h, 取200 μL菌液均勻涂在含有Amp+抗性的LB平板上, 37℃培養過夜,挑取單克隆于含Amp+的液體LB培養基中在37℃、200 r/min培養4—6h, 進行菌液PCR檢測, PCR產物用1%的瓊脂糖凝膠電泳進行檢測, 將含有陽性克隆的菌液送至蘇州金唯智生物技術有限公司測序。

將測序結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miR-200a和miR-200b進行二級結構預測。再將序列在miRBase (http:// www.mirbase.org/)上進行BLAST分析, 并利用Clustalx1.83 (http://www.genome.jp/tools/clustalx/)進行同源性分析。

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實時定量(qRT-PCR)分析

根據半滑舌鰨miR-200a和miR-200b的成熟體序列設計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基因作為內參, 設計內參引物U6-F和U6-R(引物序列見表 1)。按照miRcute miRNA qPCR Detection Kit的說明在ABI PRISM 7500 Fast實時定量擴增儀上進行qRT-PCR, 程序設置為94℃, 2min, 1次循環; 94℃, 20s, 60℃, 34s, 40次循環。

1.6 統計學分析

使用SPSS 19.0統計軟件對qRT-PCR檢測結果進行分析。實時定量數據用3組重復平均值±標準誤(SE)表示, 使用2-ΔΔCt法計算相對表達量。采用單因素方差分析法中的Duncan法對多組樣本均數進行兩兩比較分析, P＜0.05時, 認為存在顯著性差異。

2 結果

2.1 半滑舌鰨miR-200a和miR-200b的前體克隆及序列分析

采用PCR方法對半滑舌鰨miR-200a和miR-200b前體進行了克隆, 測序結果去除載體序列后顯示miR-200a前體序列長度為82 bp, miR-200b前體序列長度為88 bp。通過The mfold Web Server在線軟件對半滑舌鰨miR-200a和miR-200b前體序列進行了二級結構的分析(圖 1), 顯示miR-200a和miR-200b都具有典型的頸環結構特征, 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數據庫中進行BLAST比對,結果顯示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個物種中有同源序列, 用Clustalx1.83進行同源性分析發現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

miR-200a和miR-200b在健康半滑舌鰨組織中的表達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, 但相對表達量存在較大差異(圖 2)。miR-200a在頭腎中表達量最高(58.07), 其次是肝臟(57.97)和脾(22.56), 在血液中表達量最低(1.00), 其次是肌肉(1.16)和腦(1.35)。miR-200b在肝臟中的表達量最高(93.92), 其次是頭腎(67.45)和鰓(30.10), 在肌肉中表達量最低(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前體序列的頸環結構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表達量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, 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表達鰻弧菌感染半滑舌鰨后miR-200a在肝臟、腸、脾臟和頭腎4種組織中都呈現先上升后下降的表達趨勢(圖 3):其中在肝臟和脾中的表達峰值出現在鰻弧菌感染后6h, 分別為是0表達量的6.30倍和9.80倍, 隨后表達量逐步下降, 感染后48h肝臟的表達量降為0的1/3, 在脾臟中則是在72h達到表達的最低值; 在腸和頭腎中表達的峰值則是在鰻弧菌感染后12h, 分別為0表達量的6.52倍和7倍, 隨后下調表達, 至72h,接近0的表達水平。

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

圖 3 鰻弧菌感染半滑舌鰨后miR-200a在不同時間點4種免疫組織中的相對表達量Fig. 3 The relative expression levels of miR-200a in four tissues at different time points after C. semilaevis infected with Vibrio anguillarum每個時間點miR-200a的表達量為感染組除以PBS組的相對表達量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在不同時間點4種免疫組織中的相對表達量Fig. 4 The relative expression levels of miR-200b in four tissues at different time points after C. semilaevis infected with Vibrio anguillarum每個時間點miR-200b的表達量為感染組除以PBS組的相對表達量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的HTLC細胞后miR-200a和miR-200b的相對表達

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

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

圖 5 LPS、PGN、WGP和poly I:C刺激半滑舌鰨肝臟細胞后miR-200a的相對表達量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胞后miR-200b的相對表達量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、組織器官發育、細胞分化凋亡、免疫功能調控及疾病發生等各種生命活動過程中發揮著重要作用, 是一種新的免疫調控因子[25,26]。在哺乳動物方面的研究證據表明具有侵染能力的小鼠乳腺腫瘤細胞中miR-200家族具有較高的表達[27]; Lee等[28]發現, miR-200家族成員的差異表達出現在了子宮內膜癌的組織中; Hiroki等[29]報道了子宮內膜腺癌組織中miR-200a表達與癌旁正常組織相比明顯增高。魚類在進化上與哺乳動物差別較大, miRNA在結構上雖然與哺乳動物較為相似, 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, 在免疫應答和疾病產生等領域目前尚缺乏表達和功能相關的研究積累。

本研究首次克隆了半滑舌鰨miR-200a和miR-200b的前體序列, 比對結果顯示它們的頸部在不同物種之間具有較高的同源性, 因為此部位經過Dicer酶的剪切加工后可以形成miRNA成熟體, 進而與其靶基因結合, 在轉錄后水平發揮抑制作用。miR-200a和miR-200b廣泛表達于各組織中, 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功能不同有關, 現有研究表明miR-200家族參與了不同的信號通路, miR-200a主要作用于蛋白激酶C/轉化生長因子β (Protein Kinase C/Transforming growth facter-β, PKC/Tgf-β)信號通路[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)信號通路抑制癌細胞的擴散和遷移[31]。miR-200a和miR-200b表達量最高的兩個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,該結果表明, miR-200a和miR-200b可能在調控肝臟和頭腎免疫細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miRNA在肝臟、胰臟和胃中的表達, miR-200b在肝臟中表達量最高。

鰻弧菌感染半滑舌鰨后miR-200a和miR-200b在4種免疫相關組織中都表現出上調表達的趨勢,說明miR-200a和miR-200b在病原入侵后做出了免疫應答反應。Lv等[33]用LPS刺激了仿刺參的體腔細胞, 發現miR-200a和miR-200b主要通過參與TLR (toll-like receptor)信號通路使體腔細胞發揮抗菌作用。TLR是迄今為止研究最為透徹的重要模式受體, 能夠選擇性的識別來自細菌、病毒和真菌的刺激[34], 通過接頭蛋白等將細胞外刺激信號傳遞至細胞內, 從而誘導促炎癥反應相關因子的產生, 上調協同刺激因子的表達, 迅速激活天然免疫, 為激活機體適應性免疫應答、形成免疫記憶提供必要條件[35]。

綜上所述, miR-200a和miR-200b參與了免疫應答反應。本研究為半滑舌鰨miRNA的免疫相關性提供了科學依據, 為后續半滑舌鰨miRNA的研究奠定了理論基礎。

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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

國家自然科學基金(31572644和31172439); 鰲山科技創新計劃(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、基因組學和細胞生物學研究。E-mail: shazhenxia@163.com