CN115927336B - 一种miRNA及其在调控C5aR1基因中的应用 - Google Patents
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Abstract
本发明提供一种半滑舌鳎miRNA及其应用，所述miRNA的序列为SEQID NO:1，对应的靶基因为半滑舌鳎C5aR1，并且提供了所述半滑舌鳎RNA在调控半滑舌鳎C5aR1基因表达中的应用。本发明发现了调控半滑舌鳎C5aR1基因的miRNA，利用所述的miRNA能够调控C5aR1基因在半滑舌鳎体内的表达，从而降低炎症因子的表达，减少半滑舌鳎在细菌性炎症中的死亡。
Description
技术领域
本发明属于水产养殖生物免疫技术领域，具体涉及一种miRNA及其在调控C5aR1基因中的应用。
背景技术
半滑舌鳎(Cynoglossus semilaevis)是一种暖温性近海大型底栖鱼类，为我国重要的经济海水鱼类。随着养殖规模的扩大，细菌性疾病成为养殖过程中的一大难题。鳗弧菌(Vibro anguillarum)作为其主要致病菌会造成养殖过程中的高死亡率，感染后会伴有出血，败血症等症状，导致严重的细菌性炎症。因此减少细菌性炎症对半滑舌鳎带来的危害至关重要。
发明内容
本发明是提供一种miRNA及其在调控半滑舌鳎C5aR1基因中的应用，从而有效的降低炎症因子的表达，减少半滑舌鳎在细菌性炎症中的死亡率。
本发明首先提供一种miRNA，其核酸序列为TTTTGCAGAAACGTTTCAGATT(SEQ ID NO:1)；
本发明再一个方面还提供所述的miRNA的衍生物，是在上述的SEQ ID NO:1的核酸片段上取代、缺失、添加一个或几个核苷酸，由其所衍生的miRNA。
本发明再一个方面还提供所述的miRNA在调控半滑舌鳎C5aR1基因表达中的应用；
本发明再一个方面还提供上述的miRNA的另一种用途，是在制备用于调控半滑舌鳎C5aR1基因表达制品中的应用。
本发明还提供上述的miRNA在制备用于半滑舌鳎在细菌性炎症治疗的制品中的应用。
本发明发现了调控C5aR1基因的miRNA，利用miRNA能够调控C5aR1基因在半滑舌鳎体内的表达，从而减轻半滑舌鳎在细菌感染后的炎症反应。
附图说明
图1为PGLO-C5aR1载体的结构示意图。
图2为人工合成miRNA转染72h后双荧光素酶实验结果图。
图3为人工合成miRNA转染后对C5aR1的调控水平验证结果图。
图4为注射人工合成miRNA模拟物、抑制剂后，进一步注射鳗弧菌后对照组和处理组肝脏中炎症因子的表达情况。
图5：鳗弧菌感染后模拟物组(mimic)、随机对照组(NC)、抑制剂组(inhibitor)的半滑舌鳎死亡率曲线图；
图6：鳗弧菌感染后模拟物组(mimic)、随机对照组(NC)、抑制剂组(inhibitor)半滑舌鳎肠组织病理切片分析图，其中A：正常组B：mimic组C：NC组D：inhibitor组↑：肠绒毛断裂▲：黏膜层和黏膜固有层解离；
图7：LPS刺激后模拟物组、随机对照组、抑制剂组炎症因子在半滑舌鳎肝细胞中的表达分析图。
具体实施方式
MicroRNA(miRNAs)是一种小型非编码RNA，一般由22个核苷酸组成，通过调节mRNA的稳定性参与许多细胞和生物学过程。miRNA与细胞分化、先天免疫、凋亡和许多其他细胞命运决定有关，在生物的发生发展中扮演着重要的角色。综上所述，对miRNA及其靶基因以及其调控机制进行研究，对于进一步了解生物内源性调控代谢机制有着重要意义。
半滑舌鳎C5aR1是C5a的主要功能受体，与C5a结合后可以诱导分泌IL-1、IL-6、IL-8及TNF-α等细胞因子。C5a是补体活化的主要效应产物,通过与其受体C5aR结合发挥强大的致炎效应。C5aR1被认为是功能最强的炎症驱动因子，一直被认为是治疗炎症的主要靶点。大量研究表明，可以通过抑制C5a/C5aR1通路来降低炎症感染带来的机体损伤。因此，C5aR1在炎症反应中的作用至关重要。
结合以下具体实例对本发明的技术方案作进一步详细的说明，实施方案中未注明的具体实验条件，按照常规条件或制造商说明书中建议的条件进行。
实施例1
申请人筛选获得了核酸序列为TTTTGCAGAAACGTTTCAGATT(SEQ ID NO:1)的miRNA，命名为miR-722，根据miRNA种子序列的与靶基因的互补性及miRNA与靶基因所形成的二级结构的稳定性，进行靶基因的预测。靶基因预测结果显示miR-722的靶基因可能为C5aR1。
构建一种半滑舌鳎C5aR1基因双荧光素酶报告基因载体，包含双荧光素酶报告基因载体和C5aR1基因的3’UTR区核苷酸序列片段。
根据半滑舌鳎转录组数据，设计带有NheⅠ和SalⅠ限制性内切酶酶切位点特异性引物C5aR1-eF、C5aR1-eR用于扩增C5aR1基因的3’-UTR，并通过双酶切后连接的方式插入到双荧光素酶报告基因载体中,带有NheⅠ和SalⅠ两种限制性内切酶酶切位点的PCR扩增正反引物如下：
C5aR1-eF:CTAGCTAGCCATTGATTGGTCAGTTGTCTGT；
C5aR1-eR:ACGCGTCGACTTCATTTGTGGGTAAAT
然后将阳性单克隆进行测序验证，结果如图1所示，结果表明成功构建PGLO-C5aR1重组载体。
进行细胞转染,本实施例转染的细胞为HEK 293T细胞，按照细胞培养的常规条件，37℃恒温，5％的CO2浓度，在12孔板中培养24h后开始进行转染。本方案共设置4个转染组：①共转染PGLO-C5aR1重组载体和miR-722模拟物；②共转染空的双荧光素酶载体GLO和miR-722；③单独转染PGLO-C5aR1重组载体；④单独转染空的双荧光素酶载体GLO。
使用lip2000转染试剂(Invitrogen公司，美国)将上述载体转染至细胞中，将细胞培养板放置在恒温培养箱中培养48h，进行荧光素酶检测，采用碧云天双荧光酶报告基因检测试剂盒，相对荧光表达量是以海肾荧光素酶为内参。检测结果使用SPSS19.0软件进行分析。实验数据用3组重复平均值±标准误(SE)表示。
结果如图2表明，将空的双荧光素酶载体GLO和miR-722模拟物共同转染HEK293细胞后其荧光素酶活性与GLO单独转染细胞荧光素酶活性相差不大，将PGLO-C5aR1重组载体和miR-722模拟物共同转染HEK293细胞后其荧光素酶活性是GLO-C5aR1单独转染细胞荧光素酶活性的1/4。据此可以证明miR-722模拟物对重组双荧光载体的表达产生了影响，进一步验证了半滑舌鳎补体相关基因C5aR1为该miRNA的靶基因。
实施例2
实施例以体外培养的半滑舌鳎巨噬细胞为载体，采用miR-722模拟物转染体外培养巨噬细胞6h后，通过实时定量PCR技术检测CsC5aR1不同位置mRNA的相对表达量(设计了6对不同位置的定量引物(R1-R6)，引物扩增区域存在重叠)，结果显示不同位置的CsC5aR1表达差异不显著，说明靶基因未出现降解，即mRNA水平CsemiR-722未影响靶基因CsC5aR1的表达。
提取体外培养单核/巨噬细胞中的CsC5aR1蛋白，与制备的效价为512K的抗体进行杂交，通过western blot检测，发现miR-722模拟物转染体外培养巨噬细胞中CsC5aR1蛋白表达量下降，在miRNA模拟物刺激6小时后，表达量下降为对照组的0.32倍，下降显著。体外实验证明miR-722抑制了靶基因的翻译过程，通过转录后调控靶基因蛋白表达量，结果如图3表明。
实施例3
本实施例通过人工合成miR-722模拟物(mimic)和抑制物(inhibitor)注射到半滑舌鳎体内，进一步注射鳗弧菌建立细菌炎症模型，发现与注射随机合成序列相比，注射抑制剂后，在肝、肠、脾中，CsIL1-β，CsIL6，CsIL8，CsTNFα相关炎症因子的表达量呈上调趋势：而注射miR-722模拟物后在肝、肠、脾中四种炎症因子呈下调趋势，结果如图4表明。该实验结果表明可以通过调控miR-722的表达进一步调控鳗弧菌感染半滑舌鳎后炎症因子的表达以及免疫基因的表达，说明miR-722在免疫反应中起着重要的调控作用。
实施例4
本实施例通过人工合成miR-722模拟物和抑制物注射到半滑舌鳎体内，进一步注射鳗弧菌建立细菌炎症模型，根据半滑舌鳎死亡数量统计分析发现，12hmimic组的死亡率为20％，NC对照组的死亡率为21％，inhibitor组的死亡率为42％；24h mimic组的死亡率为56.6％，NC对照组的死亡率为68％，inhibitor组的死亡率为96％。inhibitor组的死亡率明显高于其他两组，且在鳗弧菌感染24h内出现集中的爆发性死亡。鳗弧菌感染36h后半滑舌鳎死亡趋于稳定，不再死亡，结果如图5表明。该实验结果表明可以通过调控miR-722的表达进一步调控鳗弧菌感染半滑舌鳎后的死亡率。
实施例5
本实施例通过人工合成miR-722模拟物和抑制物注射到半滑舌鳎体内，进一步注射鳗弧菌建立细菌炎症模型，根据半滑舌鳎肠组织HE染色结果发现,抑制剂组的鱼明显出现肠内结构疏松，黏膜层和黏膜固有层组织分离，小肠绒毛断裂现象；NC对照组的鱼出现黏膜层和黏膜固有层组织分离，肠内结构疏松的现象；模拟物组出现轻微结构疏松的现象，损伤程度明显轻于抑制剂组，结果如图6表明。该实验结果表明调控miR-722的表达可以影响鳗弧菌感染半滑舌鳎后的组织损伤情况。
实施例6
本实施例通过人工合成miR-722模拟物和抑制物转染到半滑舌鳎肝细胞内，进一步加入LPS刺激建立炎症模型，发现与注射随机合成序列相比，转染抑制剂后，CsIL1-β，CsIL6，CsIL8，CsTNFα相关炎症因子的表达量呈上调趋势。而注射miR-722模拟物后四种炎症因子呈下调趋势，结果如图7表明。该结果与体内感染实验结果一致，说明miR-722在免疫反应中起着重要的调控作用。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
Claims (3)
1.核酸序列为SEQ ID NO:1的miRNA在调控半滑舌鳎C5aR1基因表达中的应用。
2.核酸序列为SEQ ID NO:1的miRNA在制备用于调控半滑舌鳎C5aR1基因表达的制品中的应用。
3.核酸序列为SEQ ID NO:1的miRNA在制备用于半滑舌鳎的鳗弧菌感染治疗的制品中的应用。
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| CN109136388A (zh) * | 2018-09-03 | 2019-01-04 | 天津渤海水产研究所 | 半滑舌鳎差异表达的microRNA标签及应用 |
| CN114517198A (zh) * | 2022-03-09 | 2022-05-20 | 中国水产科学研究院黄海水产研究所 | 一种半滑舌鳎miRNA及其在调控tgfb2基因表达中的应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109136388A (zh) * | 2018-09-03 | 2019-01-04 | 天津渤海水产研究所 | 半滑舌鳎差异表达的microRNA标签及应用 |
| CN114517198A (zh) * | 2022-03-09 | 2022-05-20 | 中国水产科学研究院黄海水产研究所 | 一种半滑舌鳎miRNA及其在调控tgfb2基因表达中的应用 |
Non-Patent Citations (4)
| Title |
|---|
| A Conserved MicroRNA Regulatory Circuit Is Differentially Controlled during Limb/Appendage Regeneration;Benjamin L. King等;《 PLoS ONE》;第11卷(第6期);文章编号：e0157106 * |
| Identification and characterization of Cynoglossus semilaevis microRNA response to Vibrio anguillarum infection through high-throughput sequencing;Zhenxia Sha等;《Developmental & Comparative Immunology》;第44卷(第1期);第59-69页 * |
| 半滑舌鳎miRNA前体预测及靶基因分析;郭林豪;《中国优秀硕士学位论文全文数据库 农业科技辑》(第第12期期);第D052-56页 * |
| 应用高通量测序及基因芯片技术鉴定鳗弧菌感染后半滑舌鳎microRNA及其特征;公光业等;《中国水产学会鱼病专业委员会2013年学术研讨会论文摘要汇编》;第122页 * |
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