半滑舌鳎miR-200a和miR-200b前体克隆及其免疫应答分析

沙珍霞陈学杰,陈亚东颜 慧

(1. 青岛大学生命科学学院, 青岛 266071; 2. 中国水产科学研究院黄海水产研究所, 农业部海洋渔业可持续发展重点实验室,青岛 266071; 3. 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室, 青岛 266200; 4. 上海海洋大学水产与生命学院, 上海 201306)

半滑舌鳎miR-200a和miR-200b前体克隆及其免疫应答分析

沙珍霞1,2,3陈学杰2,3,4陈亚东2,3颜 慧2

(1. 青岛大学生命科学学院, 青岛 266071; 2. 中国水产科学研究院黄海水产研究所, 农业部海洋渔业可持续发展重点实验室,青岛 266071; 3. 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室, 青岛 266200; 4. 上海海洋大学水产与生命学院, 上海 201306)

为了探究半滑舌鳎(Cynoglossus semilaevis) miR-200a和miR-200b在免疫应答中的作用, 采用PCR方法克隆了半滑舌鳎miR-200家族的miR-200a和miR-200b的前体序列, 长度分别为82和88 bp; 用The mfold Web Server和Clustalx1.83软件对其前体序列进行了二级结构和同源性分析, miR-200a和miR-200b都具有典型的颈环结构, 与其他物种具有较高的同源性。qRT-PCR分析结果显示, miR-200a和miR-200b在健康半滑舌鳎13种组织(肝脏、肠、脾脏、头肾、后肾、鳃、血液、脑、皮肤、肌肉、胃、心脏和卵巢)中均有表达, miR-200a在头肾中表达量最高, 在血液中表达量最低, miR-200b在肝脏中表达量最高, 在肌肉中表达量最低; miR-200a和miR-200b在鳗弧菌(Vibrio anguillarum)感染半滑舌鳎后不同时间点的4种免疫相关组织(肝脏、肠、脾脏和头肾)中的表达呈现出先上调后下降的规律, 但表达达到峰值的时间点有所不同。miR-200a在肝脏和脾中的表达峰值出现在鳗弧菌感染后6h, 在肠和头肾中则是鳗弧菌感染后12h, miR-200b在肠、脾和头肾中均在鳗弧菌感染后12h达到表达高峰; 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种病原模拟物刺激后的半滑舌鳎肝脏细胞系中呈现出上调表达趋势, 其中Poly I:C刺激半滑舌鳎肝脏细胞系后miR-200a上调表达趋势明显, 6h的表达量为0h的9倍, 在WGP刺激半滑舌鳎肝脏细胞后miR-200b上调表达趋势明显, 2h的表达量为0的9倍。研究结果为揭示miRNA在半滑舌鳎免疫应答中的作用提供了科学依据。

半滑舌鳎; miR-200a; miR-200b; 前体克隆; 荧光实时定量PCR; 免疫应答

半滑舌鳎(Cynoglossus semilaevis)属于鲽形目、舌鳎科、舌鳎属, 是我国重要的经济鱼类[1], 但其养殖业受到各种病原菌的严重威胁。从分子层面揭示半滑舌鳎的免疫调控机制, 将为健康养殖提供新思路。

microRNA(miRNA)是一类非编码的、长度约18—25 nt的内源性单链型小RNA分子[2,3], 在动植物、病毒等多种有机体的生物学过程中发挥着重要作用, 如个体发育[4,5]、细胞增殖或凋亡[6,7]、细胞分化[8,9]、免疫应答[10]和肿瘤发生[11,12]等。为了寻找半滑舌鳎免疫相关的miRNA, 本课题组进行了半滑舌鳎病原刺激前后免疫组织miRNA的高通量测序[13]和miRNA芯片研究[14], 获得了包括miR-200a和miR-200b在内的99个表达差异显著的miRNA。miR-200a和miR-200b同属于miR-200家族, 其成员还包括miR-200c、miR-141和miR-429[15,16]。miR-200家族通过作用于两个重要的细胞转录抑制因子ZEB1 (Zinc finger E-box-binding homeobox 1)和ZEB2 (Zinc finger E-box-binding homeobox 2), 调节上皮间质的转化过程, 从而抑制肿瘤的发生和侵袭。在哺乳动物中miR-200家族在鼻咽癌[17]、肝癌[18]、肾透明细胞癌[19]、脑膜瘤[20]等多种肿瘤中差异表达。目前关于miR-200a和miR-200b的研究主要集中在与人类肿瘤相关基因的相互调控上, 而其在鱼类免疫调节机制中如何发挥作用未见报道。

本研究旨在获得半滑舌鳎miR-200a及miR-200b的前体序列、探究其组织表达特征及响应鳗弧菌感染和病原模拟物刺激后在免疫组织及体外培养肝脏细胞的时空表达规律, 为揭示miRNA在半滑舌鳎免疫应答中的作用提供科学依据。

1 材料与方法

1.1 实验材料

半滑舌鳎购自日照东鑫现代渔业技术研究所,鱼龄在1.5龄左右, 平均体长(30.5±2.5) cm, 平均体重(220±6.4) g。感染前在实验室水族箱中暂养7d,水温控制在(25±1)℃, 每天更换新鲜灭菌海水。

本研究所用半滑舌鳎肝脏细胞系(Liver cell line of half-smooth tongue-sole, HTLC)[21]已传至27代。细胞在含有1%青链霉素和15%胎牛血清的DMEM培养基(Invitrogen, 美国)中于24℃培养。

1.2 样品处理和采集

健康组织的收集随机选取3条健康的半滑舌鳎, 分别采集每条鱼的肝脏、肠、脾脏、头肾、后肾、鳃、血液、脑、皮肤、肌肉、胃、心脏和卵巢13种组织, 并立即投入液氮中冷冻, 然后转移至-80℃保存, 以备RNA提取。

鳗弧菌感染组和PBS对照组组织收集实验注射用的鳗弧菌为本实验分离获得的菌种, 进行培养后用磷酸缓冲液(PBS)重新悬浮至浓度为2.864×108CFU/mL。鳗弧菌感染参照Sha等[22]实验方法略作修改, 采用腹腔注射的方法对实验组进行鳗弧菌感染, 感染剂量(半致死剂量)为3.18×105CFU/g鱼体, 对照组注射相应剂量的PBS。在0、6h、12h、24h、48h和72h共6个时间点, 分别收集鳗弧菌感染组和PBS对照组的肝脏、肠、脾脏和头肾4种组织, 每个时间点取3条鱼。将所取组织立即投入液氮研磨, 然后转移至-80℃保存, 以备RNA提取。

HTLC细胞样品收集将HTLC细胞接种到1 2孔细胞培养板中, 待细胞长满培养板的70%—80%, 分别用终浓度为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]刺激细胞, 每个实验设置3个重复。刺激过程持续30min, 然后去除刺激物, PBS清洗细胞, 继续培养细胞。用PBS同时处理HTLC作为对照组。分别收集感染后0(感染开始时)、2h、6h、12h和24h共5个时间点的细胞, 每个时间点取3个重复, 低温离心后去除离心管中上清液, 加入1 mL的裂解液(RZ), 然后转移至-80℃保存, 以备RNA提取。

1.3 RNA的提取和cDNA模板的制备

RNA的提取按照RNA提取试剂盒(天根, 北京)的说明书进行操作。miRNA cDNA第一条链的合成按照miRcute miRNA cDNA第一链合成试剂盒(天根, 北京)说明书进行。最终得到的cDNA在-20℃保存待用。

1.4 半滑舌鳎miR-200a和miR-200b前体克隆及序列分析

将半滑舌鳎miR-200a和miR-200b的成熟体序列比对到半滑舌鳎基因组[24], 结合其相近物种的前体序列及二级结构的稳定性得到半滑舌鳎miR-200a和miR-200b前体的基因组序列, 根据获得的序列设计特异性引物miR-200a-F、miR-200a-R和miR-200b-F、miR-200b-R(表 1), 用于扩增miR-200a和miR-200b的前体序列, 将克隆得到的序列连接在T1载体(全式金, 北京)上, 转化至Top10感受态细胞中, 37℃、200 r/min培养1h, 取200 μL菌液均匀涂在含有Amp+抗性的LB平板上, 37℃培养过夜,挑取单克隆于含Amp+的液体LB培养基中在37℃、200 r/min培养4—6h, 进行菌液PCR检测, PCR产物用1%的琼脂糖凝胶电泳进行检测, 将含有阳性克隆的菌液送至苏州金唯智生物技术有限公司测序。

将测序结果去除载体序列后用The mfold Web Server (http://mfold.rna.albany.edu/？q=mfold/ RNAFolding-Form)在线软件对miR-200a和miR-200b进行二级结构预测。再将序列在miRBase (http:// www.mirbase.org/)上进行BLAST分析, 并利用Clustalx1.83 (http://www.genome.jp/tools/clustalx/)进行同源性分析。

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

1.5 半滑舌鳎miR-200a和miR-200b实时定量(qRT-PCR)分析

根据半滑舌鳎miR-200a和miR-200b的成熟体序列设计qRT-PCR的正向引物qmiR-200a-F和qmiR-200b-F, 反向引物为miRcute miRNA qPCR Detection Kit (天根, 北京)自带引物, 用半滑舌鳎U6基因作为内参, 设计内参引物U6-F和U6-R(引物序列见表 1)。按照miRcute miRNA qPCR Detection Kit的说明在ABI PRISM 7500 Fast实时定量扩增仪上进行qRT-PCR, 程序设置为94℃, 2min, 1次循环; 94℃, 20s, 60℃, 34s, 40次循环。

1.6 统计学分析

使用SPSS 19.0统计软件对qRT-PCR检测结果进行分析。实时定量数据用3组重复平均值±标准误(SE)表示, 使用2-ΔΔCt法计算相对表达量。采用单因素方差分析法中的Duncan法对多组样本均数进行两两比较分析, P＜0.05时, 认为存在显著性差异。

2 结果

2.1 半滑舌鳎miR-200a和miR-200b的前体克隆及序列分析

采用PCR方法对半滑舌鳎miR-200a和miR-200b前体进行了克隆, 测序结果去除载体序列后显示miR-200a前体序列长度为82 bp, miR-200b前体序列长度为88 bp。通过The mfold Web Server在线软件对半滑舌鳎miR-200a和miR-200b前体序列进行了二级结构的分析(图 1), 显示miR-200a和miR-200b都具有典型的颈环结构特征, miR-200a的最小折叠自由能为-34.60 kJ/mol, miR-200b的最小折叠自由能-33.00 kJ/mol。将miR-200a和miR-200b的前体序列在miBase21.0数据库中进行BLAST比对,结果显示miR-200a和miR-200b在斑马鱼(Danio rerio)、鲤(Cyprinus carpio)、红鳍东方鲀(Fugu rubripes)、青斑河鲀(Tetraodon nigroviridis)、人(Homo sapiens)、小家鼠(Mus muscuLus)等10个物种中有同源序列, 用Clustalx1.83进行同源性分析发现miR-200a和miR-200b前体与其他物种具有较高的同源性, 特别是颈部为miRNA成熟体所在的部位,保守性更高。

2.2 半滑舌鳎miR-200a和miR-200b在组织中的表达

miR-200a和miR-200b在健康半滑舌鳎组织中的表达miR-200a和miR-200b在健康半滑舌鳎13种组织(肝脏、肠、脾、头肾、后肾、鳃、血液、脑、皮肤、肌肉、胃、心脏和卵巢)中均有表达, 但相对表达量存在较大差异(图 2)。miR-200a在头肾中表达量最高(58.07), 其次是肝脏(57.97)和脾(22.56), 在血液中表达量最低(1.00), 其次是肌肉(1.16)和脑(1.35)。miR-200b在肝脏中的表达量最高(93.92), 其次是头肾(67.45)和鳃(30.10), 在肌肉中表达量最低(1.00), 其次是血液(1.17)和卵巢(1.22)。

图 1 半滑舌鳎miR-200a和miR-200b前体序列的颈环结构Fig. 1 Hairpin structures formed by precursor sequence of miR-200a and miR-200b in C. semilaevis

图 2 miR-200a和miR-200b在健康半滑舌鳎组织中的相对表达量Fig. 2 The expression of miR-200a and miR-200b in tissues of C. semilaevis字母“a, b, c, d”代表SPSS多重分析的不同分组; 不同字母表示组间差异显著(P＜0.05); 下同; b. 脑; bl. 血液; g. 鳃; h. 心脏; hk.头肾; in. 肠; l. 肝脏; m. 肌肉; me. 后肾; o. 卵巢; s. 皮肤; sp. 脾脏; 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

鳗弧菌感染半滑舌鳎后miR-200a和miR-200b在四种免疫组织中的相对表达鳗弧菌感染半滑舌鳎后miR-200a在肝脏、肠、脾脏和头肾4种组织中都呈现先上升后下降的表达趋势(图 3):其中在肝脏和脾中的表达峰值出现在鳗弧菌感染后6h, 分别为是0表达量的6.30倍和9.80倍, 随后表达量逐步下降, 感染后48h肝脏的表达量降为0的1/3, 在脾脏中则是在72h达到表达的最低值; 在肠和头肾中表达的峰值则是在鳗弧菌感染后12h, 分别为0表达量的6.52倍和7倍, 随后下调表达, 至72h,接近0的表达水平。

鳗弧菌感染半滑舌鳎后miR-200b在4种免疫组织中也都呈现先上升后下降的表达趋势(图 4), 在肝脏中表达峰值出现在感染后6h, 为0表达量的14倍, 在12h和24h开始出现下调, 并在48h和72h恢复到表达的初始值; 而在肠、脾和头肾中则是感染后12h出现表达峰值, 在肠中鳗弧菌感染后6h开始出现上调, 直到12h出现表达的峰值, 为0表达量的8.5倍, 到24h和48h开始逐渐下调, 并在72h降至0表达水平。在脾中鳗弧菌感染后6h较0变化不大, 但在12h表达量上升为0的10.12倍, 在24h开始表达下调, 在48h和72h保持0h的表达水平。在头肾中鳗弧菌感染后12h出现表达峰值为21.13, 到72h下调到0的表达水平。

图 3 鳗弧菌感染半滑舌鳎后miR-200a在不同时间点4种免疫组织中的相对表达量Fig. 3 The relative expression levels of miR-200a in four tissues at different time points after C. semilaevis infected with Vibrio anguillarum每个时间点miR-200a的表达量为感染组除以PBS组的相对表达量The expression levels of miR-200a at each point was presented relative expression levels which infected group divided by the PBS group

图 4 鳗弧菌感染半滑舌鳎后miR-200b在不同时间点4种免疫组织中的相对表达量Fig. 4 The relative expression levels of miR-200b in four tissues at different time points after C. semilaevis infected with Vibrio anguillarum每个时间点miR-200b的表达量为感染组除以PBS组的相对表达量The expression levels of miR-200b at each point was presented relative expression levels which infected group divided by the PBS group

2.3 四种病原模拟物刺激体外培养的HTLC细胞后miR-200a和miR-200b的相对表达

4种病原模拟物刺激后miR-200a在HTLC细胞中呈现上调表达的趋势(图 5), 除了LPS刺激HTLC细胞12h后miR-200a表达量达到最大值, miR-200a在PGN、WGP和Poly I:C刺激HTLC细胞后均在6h出现表达的最高峰, 其中最大表达量是Poly I:C刺激后6h, 为0的9倍, miR-200a在WGP刺激后2—12h, 表达量都在0的4倍以上, 在24h也达到0的2倍, LPS、PGN和Poly I:C刺激后miR-200a表达变化趋势基本一致, 即在6h或12h出现较高的上调表达峰值外, 在其他时间点的表达量与0相比变化不大。

miR-200b在4种病原模拟物刺激的HTLC细胞中同样呈现上调表达的趋势(图 6), 但不同病原模拟物刺激后miR-200b出现上调表达峰值的时间点和变化倍数有所不同, 其中, LPS刺激后2h表达开始出现上调, 在12h出现表达的最高峰为0的4.2倍, 到24h恢复表达的初始水平; 而PGN和Poly I:C刺激后都是在6h出现表达的最大值, 均为0的6倍左右; 在WGP刺激后, miR-200b表达变化的峰值出现2h为0表达量的9倍, 而后表达量逐渐下调, 但在24h的表达量仍为0h的2倍。

图 5 LPS、PGN、WGP和poly I:C刺激半滑舌鳎肝脏细胞后miR-200a的相对表达量Fig. 5 The relative expression levels of miR-200a in HTLC response to the challenge of LPS, PGN, WGP and polyl: C

图 6 LPS, PGN, WGP和poly I:C刺激半滑舌鳎肝脏细胞后miR-200b的相对表达量Fig. 6 The relative expression levels of miR-200b in HTLC response to the challenge of LPS, PGN, WGP and polyl: C

3 讨论

miRNA广泛存在于不同进化层次的生物体中,其成熟体序列存在种间高度保守的特征, 在生物体胚胎形成、组织器官发育、细胞分化凋亡、免疫功能调控及疾病发生等各种生命活动过程中发挥着重要作用, 是一种新的免疫调控因子[25,26]。在哺乳动物方面的研究证据表明具有侵染能力的小鼠乳腺肿瘤细胞中miR-200家族具有较高的表达[27]; Lee等[28]发现, miR-200家族成员的差异表达出现在了子宫内膜癌的组织中; Hiroki等[29]报道了子宫内膜腺癌组织中miR-200a表达与癌旁正常组织相比明显增高。鱼类在进化上与哺乳动物差别较大, miRNA在结构上虽然与哺乳动物较为相似, 但在功能上可能还是存在较大差异, 由于鱼类miRNA的研究刚刚起步, 在免疫应答和疾病产生等领域目前尚缺乏表达和功能相关的研究积累。

本研究首次克隆了半滑舌鳎miR-200a和miR-200b的前体序列, 比对结果显示它们的颈部在不同物种之间具有较高的同源性, 因为此部位经过Dicer酶的剪切加工后可以形成miRNA成熟体, 进而与其靶基因结合, 在转录后水平发挥抑制作用。miR-200a和miR-200b广泛表达于各组织中, 但在不同组织中存在较大差异, 这可能与其在不同组织中的生物学功能不同有关, 现有研究表明miR-200家族参与了不同的信号通路, miR-200a主要作用于蛋白激酶C/转化生长因子β (Protein Kinase C/Transforming growth facter-β, PKC/Tgf-β)信号通路[30], 而miR-200b主要参与磷酸肌醇3激酶/蛋白激酶B (Phosphatidyl-inositol 3-Kinase/Protein KinaseB, PI3K/AKT)信号通路抑制癌细胞的扩散和迁移[31]。miR-200a和miR-200b表达量最高的两个组织都是肝脏和头肾, 肝脏和头肾都是鱼类重要的免疫器官,该结果表明, miR-200a和miR-200b可能在调控肝脏和头肾免疫细胞生成和功能上具有作用。Joseph等[32]研究了miR-122a、miR-21和miR-200b这3个miRNA在肝脏、胰脏和胃中的表达, miR-200b在肝脏中表达量最高。

鳗弧菌感染半滑舌鳎后miR-200a和miR-200b在4种免疫相关组织中都表现出上调表达的趋势,说明miR-200a和miR-200b在病原入侵后做出了免疫应答反应。Lv等[33]用LPS刺激了仿刺参的体腔细胞, 发现miR-200a和miR-200b主要通过参与TLR (toll-like receptor)信号通路使体腔细胞发挥抗菌作用。TLR是迄今为止研究最为透彻的重要模式受体, 能够选择性的识别来自细菌、病毒和真菌的刺激[34], 通过接头蛋白等将细胞外刺激信号传递至细胞内, 从而诱导促炎症反应相关因子的产生, 上调协同刺激因子的表达, 迅速激活天然免疫, 为激活机体适应性免疫应答、形成免疫记忆提供必要条件[35]。

综上所述, miR-200a和miR-200b参与了免疫应答反应。本研究为半滑舌鳎miRNA的免疫相关性提供了科学依据, 为后续半滑舌鳎miRNA的研究奠定了理论基础。

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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

国家自然科学基金(31572644和31172439); 鳌山科技创新计划(2015ASKJ02-03)资助 [Supported by the National Natural Science Foundation of China (31572644和31172439); Aoshan Science and Technology Innovation Project (2015ASKJ02-03)]

沙珍霞, 女, 研究员, 博士; 主要从事鱼类分子免疫学、基因组学和细胞生物学研究。E-mail: shazhenxia@163.com