IOCAS-IR  > 实验海洋生物学重点实验室
miRNA在长牡蛎环境胁迫应答中的调控作用
其他题名miRNA-mediated Modulation in the Stress Response of Oyster Crassostrea gigas
陈浩1,2
学位类型博士
导师宋林生研究员
2017-06
学位授予单位中国科学院大学
学位授予地点北京
学位专业marine biology
关键词长牡蛎 表观遗传 潮间带适应机制 神经内分泌免疫调节网络 免疫应答 干露胁迫
摘要潮间带区域环境复杂多变,长牡蛎作为典型潮间带生物,终生营固着方式生存于潮间带区域,进化形成了较强的抗病原侵染、抗干露胁迫等抗环境胁迫机制。miRNA作为重要的表观调控手段,能够在转录后水平调控基因表达,参与免疫应答等生理过程。然而目前对miRNA的研究主要集中于高等动物或其他模式生物中,在无脊椎非模式生物中的研究还处于起步阶段。本研究以长牡蛎为材料,通过分子生物学、细胞生物学与生物信息学的手段,鉴定了长牡蛎血淋巴细胞miRNA,并概述了其在神经内分泌免疫调节系统中的作用;基于长牡蛎免疫过程,筛查了关键代表性miRNA,并解析了其在病原胁迫下介导的免疫应答调控机制;通过多组学整合分析,筛选了干露胁迫相关miRNA,探究了其介导的干露适应调节机制。
通过二代测序技术结合生物信息学分析,对长牡蛎miRNA进行了全基因组范围的筛查及鉴定,在长牡蛎中发现了331条miRNA,其中76条为保守miRNA,255条为全新miRNA,长牡蛎中鉴定出的miRNA数量为已知贝类中最多。有38条长牡蛎miRNA同时被发现响应免疫调质乙酰胆碱ACh或去甲肾上腺素NE刺激,并能够靶定355个长牡蛎基因,其中包括免疫识别受体,免疫通路信号分子,免疫效应分子及神经内分泌相关分子等。
从长牡蛎中鉴定出保守miRNA cgi-miR-92d,其表达水平在免疫应答早期显著下调,在免疫应答后期迅速升高。CgLITAF3作为长牡蛎LITAF同源基因及cgi-miR-92d的靶基因,其表达水平在免疫应答早期迅速上升,在免疫应答后期显著下降。体外双荧光实验结果表明,cgi-miR-92d能够通过结合CgLITAF3编码区抑制其表达。体外过表达实验表明,CgLITAF3能诱导HEK293T细胞中HsTNF-α表达,且诱导效果受cgi-miR-92d抑制。CgTNF作为长牡蛎TNF-α同源基因,其表达水平在免疫应答早期显著升高,而在免疫应答后期显著下降,且趋势晚于CgLITAF3。研究发现,病原刺激后,敲减长牡蛎体内cgi-miR-92d能够显著降低CgLITAF3及CgTNF表达水平。
scaffold42648_5080是长牡蛎特有miRNA,编码于长牡蛎基因内含子区域。scaffold42648_5080能响应LPS刺激且在免疫应答早期显著下调。同时能够靶定包括CgILK在内的31个长牡蛎基因,参与细胞通讯、定位、应激反应等调控过程。其中,CgILK作为长牡蛎integrin通路中核心信号转导分子,同样能响应LPS刺激且显著上调。通过双荧光素酶报告基因实验证实,scaffold42648_5080能够在体外结合CgILK 3’-UTR,并抑制其表达。在免疫应答过程中,长牡蛎血细胞迁移能力随CgILK表达量的升高或降低而发生相应变化,在体内敲减scaffold42648_5080后,CgILK表达水平显著升高,长牡蛎血淋巴细胞迁移能力显著升高。
免疫细胞吞噬作用是机体清除外来病原的重要机制,受NF-κB等通路调控。灿烂弧菌刺激后长牡蛎血淋巴细胞吞噬能力显著升高而NF-κB通路中核心抑制因子CgIκB2表达水平显著降低。体内敲减CgIκB2基因后,长牡蛎血淋巴细胞吞噬能力显著升高。长牡蛎能够编码一个无脊椎动物特有的miRNA cgi-miR-2d。灿烂弧菌刺激后,其表达水平迅速升高,趋势与CgIκB2基因相反。生物信息学分析及双荧光素酶报告基因实验显示,cgi-miR-2d能够结合CgIκB2 3’-UTR并抑制其表达。同时,在长牡蛎体内过表达cgi-miR-2d后,CgIκB2基因表达水平显著降低,细胞吞噬能力显著升高;在长牡蛎体内敲减cgi-miR-2d后,CgIκB2基因表达水平略有升高,细胞吞噬能力迅速降低。
乙酰胆碱ACh是胆碱能神经系统主要的神经递质,也是重要的免疫调质。贝类具有原始的胆碱能神经免疫调节系统,并能参与调控其免疫反应。研究发现,灿烂弧菌刺激12小时后,长牡蛎血淋巴细胞中ACh显著升高,同时长牡蛎胆碱转运体同源基因CgCTL1表达量在免疫应答前期降低,在免疫应答后期恢复正常。敲减CgCTL1后,长牡蛎血淋巴细胞内胆碱及细胞外ACh含量显著降低,CgTNFs表达水平显著升高。生物信息学分析及双荧光实验表明,cgi-miR-2d能够结合CgCTL1 3’-UTR并抑制其表达。体内过表达cgi-miR-2d后CgCTL1表达水平显著降低,血淋巴细胞内胆碱转运与ACh合成释放迅速下降,CgTNFs表达水平升高;体内敲减cgi-miR-2d后,CgCTL1表达水平显著升高,血淋巴细胞内胆碱含量显著增加,细胞外ACh含量及CgTNFs表达水平保持不变,细胞抗菌能力显著降低。
去甲肾上腺素NE是高等动物儿茶酚胺能系统中主要的神经递质,也是重要的应激激素。长牡蛎具有原始的儿茶酚胺能系统,并在干露胁迫后迅速激活。干露胁迫后cgi-miR-365表达水平显著升高,且在NE受体封闭后降低表达。靶基因分析表明,cgi-miR-365能够靶定CgHSP90AA1在内15个长牡蛎基因,参与调控物质代谢及胁迫应激等生理过程。其中,CgHSP90AA1能响应NE刺激并在干露胁迫后表达升高。双荧光实验表明,cgi-miR-365能够结合CgHSP90AA1 3’-UTR并促进其表达。在长牡蛎原代细胞中过表达cgi-miR-365后,CgHSP90AA1表达水平显著升高。在干露胁迫的长牡蛎中敲减cgi-miR-365后,CgHSP90AA1表达水平则显著降低。
长牡蛎钙调蛋白CgCaM是细胞内重要的钙离子结合蛋白,能够激活一氧化氮合酶NOS,促进NO合成释放。NO是细胞内重要的炎症调控因子,参与调节细胞因子表达。干露胁迫后,长牡蛎血淋巴中CgCaM与细胞因子CgTNF等表达水平、NOS活性、NO含量呈现相似的先降低后升高的趋势,而钙离子浓度持续升高。对CgCaM进行抑制剂注射或体内敲减后,发现NOS酶活、NO含量、CgTNF等细胞因子表达水平均显著降低。研究发现scaffold631_909、cgi-miR-7、scaffold43988_2479、cgi-miR-92a、cgi-miR-92c、cgi-miR-92e、cgi-miR-92f、scaffold1600_212等8个长牡蛎miRNA能够调控CgCaM。与此同时, scaffold43988_2479、cgi-miR-92a、cgi-miR-92c及scaffold1600_212等6个miRNA在干露后迅速上调表达,后期表达回落。双荧光实验表明,以上miRNA能够在体外结合CgCaM 3’-UTR并抑制其表达。同时,干露胁迫后,在长牡蛎体内过表达单一miRNA,CgCaM表达水平显著降低,NOS酶活、NO含量、CgTNF表达水平显著降低;而敲减单一miRNA,CgCaM表达水平、NOS酶活、NO含量、CgTNF表达水平则无显著变化。干露胁迫中同时敲减scaffold43988_2479、cgi-miR-92a、cgi-miR-92c及scaffold1600_212,CgCaM表达水平、NOS酶活、NO含量、CgTNF表达水平则显著升高。干露胁迫中持续注射CgCaM或NOS的抑制剂CDZ或L-NMMA后,长牡蛎肝胰腺损伤情况显著好转。
本研究的结果表明,长牡蛎具有复杂的miRNA介导的环境胁迫应答调控机制,能够通过靶定免疫识别受体,免疫通路信号分子,免疫效应分子及神经内分泌相关分子等系统地调控机体免疫应答反应及干露胁迫应答反应, miRNA调控机制对于长牡蛎适应潮间带环境具有重要生理意义。
其他摘要The oyster Crassostrea gigas is a dominant sessile mollusc in intertidal zone throughout the world and weights greatly in aquaculture. The life span of adult oysters, unlike other intertidal organisms, is geologically limited to rock surfaces after their settlement at spat stage. Therefore, the oysters have evolved a complex mechanism to respond against the vigorously changing environments and were regarded as an ideal model in stress adaptation, especially the adaptation against pathogen infection and desiccation. As an important class of gene regulators at post-transcriptional level, miRNAs have been identified in numbers of organisms and proved indispensable in almost all biological processes. However, less was known in invertebrate miRNAs especially their role in the adaptation mechanism of oysters living in intertidal zone.
“Neural-endocrine-immune (NEI) system” is a major modulation network among the nervous system, endocrine system and immune system and plays vital roles in organisms during stress and infection. The oyster C. gigas is an intertidal mollusc and believed to evolve a primary NEI system. Six small RNA libraries from haemocytes of oyster stimulated with acetylcholine (ACh) and norepinephrine (NE) were sequenced to survey the immunomodulation role of neurotransmitter-responsive miRNAs. A total number of 132 new miRNA were identified in the present study. Along with 199 miRNAs identified previously, 331 miRNAs were identified in oyster haemocytes so far. Among those miRNAs, a sum of 21 and 16 miRNAs expressed differentially (FDR < 0.05) after ACh and NE stimulation, respectively. Besides, 21 miRNAs expressed differently between ACh stimulation group and NE stimulation group. A total of 570 pairs of neurotransmitter-responsive miRNAs and target genes were predicted, representing 35 miRNAs and 355 genes. Immune-related GO terms, including death, immune system process and response to stimulus were annotated with those targets genes. Moreover, immune-related genes, including PRRs, intracellular receptors, signaling transducers and immune effectors, were also predicted to be modulated by those NeurimmiRs.
Tumor necrosis factor alpha (TNF-α) mediated inflammatory response plays indispensable roles in organisms defending against the invaded bacteria, during which microRNAs have been found crucial by controlling multiple TNF-α-related genes. In the present study, cgi-miR-92d was annotated as a member of miR-17~92 family and could target the CDS region of lipopolysaccharide (LPS)-induced TNF-α factor (CgLITAF3) in oyster C. gigas. It was observed that cgi-miR-92d could be vigorously modulated by Vibrio splendidus or LPS stimulation while CgLITAF3 altered oppositely. Two putative binding sites of cgi-miR-92d were then found at CDS region of CgLITAF3. The interaction between cgi-miR-92d and CgLITAF3 was subsequently verified both in vitro and in vivo. As a result, a significant decrease of cellular luminescence was observed in CgLITAF3 luciferase reporter assay when cgi-miR-92d was overexpressed. The luminescent decrease was then recuperated when cgi-miR-92d inhibitor was co-transfected with miRNA mimics. Besides, CgLITAF3 transcripts were significantly down-regulated when cgi-miR-92d was overexpressed in vivo during V. splendidus challenge. Gain-of-function assay of CgLITAF3 was then conducted in HEK293T cells to verify its function. Consequently, a significant increase of TNF-α was observed during the assay. At the meantime, CgTNF was also down-regulated in gain-of-function assay of cgi-miR-92 in vivo, which was a member of TNF superfamily in oysters which could be robustly induced after pathogen stimulation. Together, these results verify the interaction between CgLITAF3 and cgi-miR-92d, which might dedicate crucially in the repaid activation of CgTNF expression during inflammatory response of oysters.
Dozens of species-specific miRNAs have been identified in oyster C. gigas while their functions remain largely unknown. In the present study, an oyster species-specific miRNA scaffold42648_5080 was found responsive to LPS stimulation and might target a total of 31 genes possibly involved in cell communication, cellular localization and cellular response to stimulus of oysters. Besides, in gain-of-function assay of scaffold42648_5080 in vivo, the phagocytosis (30.90% in miRNA group verse 23.20% in miRNA control group), apoptosis (3.10% in miRNA group verse 5.30% in miRNA control group) and migration rate (13.88% in miRNA group verse 21.03% in miRNA control group) of oyster haemocytes were found significantly altered after the injection of scaffold42648_5080 mimics. Among the target genes, integrin-linked kinase (CgILK) was considered crucial in cell migration and its interaction with scaffold42648_5080 was then verified both in vitro and in vivo. Consequently, a significant decrease of relative luciferase ratio was observed in CgILK 3’-UTR luciferase reporter assay after transfection of scaffold42648_5080 mimics (0.70-fold of that in blank group, p < 0.01). Meanwhile, when scaffold42648_5080 was overexpressed in vivo (5.41-fold of miRNA control group, p < 0.01), the expression of CgILK declined significantly to 0.25-fold of miRNA control group (p < 0.01). Comparatively, a significant decrease of the haemocyte migration rate (19.76% verse 34.82% in siEGFP control group, p < 0.01) was observed after knock-down of CgILK in vivo. The present study, as far as we know, for the first time revealed the immunomodulation role of an oyster species-specific miRNA, which might provide new insights into miRNA-mediated adaptation mechanism of oysters. 
Nuclear factor (NF)-κB pathway is an evolutionally conserved pathway in activating immune response, in which IκBs can repress the activation. In the present study, cgi-miR-2d, an invertebrate-specific miRNA, was proved to regulate CgIκB2 expression and haemocyte phagocytosis during bacterial infection in oyster C. gigas. The expression of cgi-miR-2d was significantly up-regulated after V. splendidus challenge, while CgIκB2 transcripts decreased. Significant decreases in both luminescence and CgIκB2 3’UTR level was observed after transfection of cgi-miR-2d in CgIκB2 3’UTR luciferase reporter assay. CgIκB2 mRNA level decreased significantly (0.51-fold of control group, p < 0.05) in gain-of-function assay of cgi-miR-2d in vivo while it increased markedly (1.27-fold, p < 0.05) when cgi-miR-2d was repressed (0.10-fold, p < 0.01). A significant increase of haemocyte phagocytosis rate was observed in cgi-miR-2d overexpression group (p < 0.01), which was consistent with results in CgIκB2 knock-down group (p < 0.01). Moreover, the apoptosis rate of haemocytes was found significantly declined (28.57%, p < 0.01) in gain-of-function assay of cgi-miR-2d. Together, those results not only depicted the functional conservation of miR-2d family in anti-apoptosis of oysters but also highlighted its interaction with phagocytosis by modulating NF-κB pathway, which might dedicate critically to the well-balance of host immune response.
Acetylcholine (ACh) is the main neurotransmitter in cholinergic neuroendocrine system and plays indispensable role in modulating diverse immune response. As an important transporter in choline uptake, choline transporter-like proteins (CTLs) can control ACh synthesis and release indirectly in multiple organisms. In the present study, cgi-miR-2d, an invertebrate-specific miRNA in oyster C. gigas, is proved to repress the synthesis/release of ACh by targeting CgCTL1 and choline uptake in haemocytes during the early-stage of pathogen infection. In short, an opposite expression pattern between CgCTL1 and cgi-miR-2d is observed during V. splendidus infection, accompanied with changes in haemolymph ACh. And expression level of CgCTL1 is found significantly repressed after cgi-miR-2d overexpression in vivo. Meanwhile, gain-of-function of cgi-miR-2d also leads to a strong decrease in both haemocyte choline and haemolymph ACh in vivo, which is similar with that in CgCTL1 knock-down assay. As a result, the expression of two tumor necrosis factor like proteins and the bacteriostatic activity of oyster haemocytes, are found altered significantly by either gain-of-function cgi-miR-2d or knock-down of CgCTL1. To our knowledge, this is the first miRNA identified in invertebrates that can target ancient cholinergic system and augment immune response during infection.
The adaptation mechanism of environmental stress in oyster involves multiple levels, and miRNA is one of the most important regulators in post-transcriptional level. In the present study, an oyster norepinephrine-responsive miRNA cgi-miR-365 was proved to contribute to the host adaptation against desiccation by directly promoting the expression of CgHSP90AA1. Briefly, a significant increase of cgi-miR-365 was observed from the first day after aerial exposure and the up-regulation was vigorously repressed when oysters were injected with adrenoceptors antagonists. A total of 15 genes involved in biological regulation, metabolic process and response to stimulus were predicted to be modulated by cgi-miR-365. Among these genes, CgHSP90AA1 was up-regulated significantly during desiccation and could be down-regulated after simultaneous injection of adrenoceptors antagonists. The interaction between cgi-miR-365 and CgHSP90AA1 was subsequently verified in vitro, and a significant promotion of CgHSP90AA1 transcripts was observed after overexpressing cgi-miR-365 in either in vitro luciferase reporter assay or primarily cultured haemocytes. Meanwhile CgHSP90AA1 transcripts decreased in vivo when cgi-miR-365 was repressed by its inhibitor during desiccation. Collectively, it was suggested that cgi-miR-365 could be induced by norepinephrine during desiccation and promote CgHSP90AA1 expression directly after binding to its 3’-UTR. This study provided new evidences in miRNA-mediated adaptation mechanism in oysters against intertidal stresses. 
Oysters were found tolerant against desiccation and could survive for days before succumbed in overwhelming inflammation. In the present study, the expression levels of CgTNFs were surveyed during desiccation and also found vigorously up-regulated at the late-stage of stress. As reported, NO could modulate the expression of cytokines in multiple organisms. Here the relative activity of NOS and concentration of NO were surveyed simultaneously during desiccation and found altered synchronously with CgTNFs or CgIL17. CgNOS was then blocked by L-NMMA injection, and expression levels of CgTNF6440 and CgIL17-4 were found inhibited at the same time. CaM was an important class of Ca2+ sensor and could activate NOS. Here CgCaMs were found modulated during aerial exposure in a post-transcriptional way. Meanwhile, NOS activity and NO concentration could be significantly repressed by inhibition of CgCaMs. Expression levels of CgTNFs were also remarkably down-regulated during the inhibition of CgCaMs. To figure out the underlying modulation mechanism of CgCaMs, miRNAs targeting CgCaMs were searched globally. It was found that eight miRNAs could target CgCaM24243 while six of them shared opposite expression pattern during stress. Gain- and loss-of function assay of above six miRNAs were then conducted in HEK293T cells or oysters. As a result, CgCaM24243 could be significantly repressed by the coordination of miRNAs. Meanwhile, NOS activity and NO concentration could also be modulated. Tissue injuries of oyster hepatopancreas were then found greatly relieved by continuous inhibition of either CgCaMs or NOS during desiccation. miRNAs are likely to play a vital role in the intertidal life of oysters especially during their responses against pathogen infection or desiccation stress.
学科领域海洋生物学 ; 分子生物学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136543
专题实验海洋生物学重点实验室
作者单位1.中国科学院海洋研究所
2.中国科学院大学
第一作者单位中国科学院海洋研究所
推荐引用方式
GB/T 7714
陈浩. miRNA在长牡蛎环境胁迫应答中的调控作用[D]. 北京. 中国科学院大学,2017.
条目包含的文件
文件名称/大小 文献类型 版本类型 开放类型 使用许可
提交-miRNA在长牡蛎环境胁迫应答中的(18735KB)学位论文 限制开放CC BY-NC-SA浏览
个性服务
推荐该条目
保存到收藏夹
查看访问统计
导出为Endnote文件
谷歌学术
谷歌学术中相似的文章
[陈浩]的文章
百度学术
百度学术中相似的文章
[陈浩]的文章
必应学术
必应学术中相似的文章
[陈浩]的文章
相关权益政策
暂无数据
收藏/分享
文件名: 提交-miRNA在长牡蛎环境胁迫应答中的调控作用-0522.pdf
格式: Adobe PDF
所有评论 (0)
暂无评论
 

除非特别说明,本系统中所有内容都受版权保护,并保留所有权利。