MicroRNA-155在T-2毒素诱导下对炎症因子的调控机制研究
发布时间：2018-08-31 13:07
【摘要】：T-2毒素由镰刀菌产生,是单端孢霉烯族类毒素中毒性最强的毒素。通过污染饲料,T-2毒素对动物和动物性食品消费者的健康造成严重危害。研究表明,T-2毒素主要损伤动物机体的免疫系统。该类毒素能使机体在低剂量的暴露下引起免疫刺激,但在高剂量的暴露下引起免疫抑制,导致免疫应答水平被严重干扰,机体免疫功能紊乱。这类毒素细胞毒性的发挥主要是作用于核糖体,一方面可以与肽酰转移酶催化位点结合来阻碍生物大分子如蛋白质等的生成,另一方面通过核糖体应激反应快速激活丝裂原活化蛋白激酶(MAPK)通路,使相关免疫应答的下游因子的表达在转录和转录后水平受到调控。其中被诱导的促炎性细胞因子的基因表达上调是促使T-2毒素发挥各种毒性效应的主要介质。MicroRNA-155作为一个典型的多功能的microRNA,同众多microRNA一样,也是通过与靶基因的3’UTR结合,抑制基因的表达。MicroRNA-155抑制超过60种靶基因,可以通过靶向不同的基因而发挥不同的作用。研究表明它不仅在炎症反应中可以被快速激活,而且受到刺激差异性表达的microRNA-155在免疫调节反应中起着非常关键的作用。但是关于microRNA-155是否在T-2毒素介导的炎症反应中发挥作用仍未见报道。本研究以RAW264.7细胞为模型,初步探究了microRNA-155在T-2毒素诱导下对炎症因子的调控作用机制。本研究首先用qRT-PCR的方法检测了T-2毒素处理下microRNA-155是否差异性表达,结果显示,14nM的T-2毒素处理RAW264.7细胞1h后microRNA-155上调,2h时上调最明显(p0.01),后续虽有下调,但与空白组相比仍明显上调。这种上调也出现在不同浓度(7nM、14nM、28nM)的T-2毒素处理RAW264.7细胞12h后。其中7nM时,microRNA-155上调最显著(p0.01)。说明T-2毒素可以促进RAW264.7细胞中microRNA-155表达。其次利用microRNA-155 mimic和抑制剂(inhibitor)分别过表达和抑制细胞中microRNA-155的表达水平,T-2毒素处理后发现,IL-6(p0.01)、IL-1β(p0.05)和TNF-α(p0.01)的表达水平随microRNA-155的上调而显著上升,随microRNA-155下调而下降(p0.05),说明microRNA-155参与并促进T-2毒素诱导的促炎症因子的表达。SOCS1是JAK/STAT通路的负性调节因子,被验证是microRNA-155的靶标。microRNA-155在多种情况下都可以通过直接抑制SOCS1发挥促炎的作用。但是在T-2毒素的诱导下这种调节作用是否存在却未可知。于是分别通过mimic和inhibitor上调和下调了microRNA-155的表达,发现SOCS1的表达在microRNA-155上调时显著下降(p0.01),在其下调时显著上升(p0.01)。说明在T-2毒素的诱导下,microRNA-155通过靶向抑制SOCS1发挥了促炎的作用。每一个microRNA都有多个靶基因,通过靶向不同的基因发挥不同的作用。通过生物信息学方法确定了两个与炎症相关的microRNA-155的靶标rheb和atg3。Rheb是Ras同源基因,广泛存在于多种生物中,是一个高度保守的GTP结合蛋白,并且是mTOR的上游关键调控因子,参与调节机体的如细胞生长、分化、氧化应激等众多生物学功能。Atg3是一种类似于泛素化过程中E2酶的一种催化蛋白,作为自噬家族中的一员,连接LC3和磷脂酰乙醇胺,通过脂化蛋白质,降解长寿命蛋白质和已损坏或不必要的细胞器,在囊泡或溶酶体系统进行抗原提呈、抗感染、细胞分化、细胞发育等生物学过程中发挥重要作用。本实验分别通过mimic和inhibitor上调和下调了microRNA-155的表达,发现rheb和atg3的mRNA水平和蛋白水平均在microRNA-155上调时显著下降,在其下调时显著上升。分别构建了含rheb和atg3 3’UTR结合位点序列的pGL3野生质粒和突变质粒,将microRNA-155 mimic和NC分别与这两个质粒组合转染进RAW264.7细胞内进行了双荧光素酶报告基因实验。结果显示:microRNA-155对p GL3-3’UTR野生型的荧光素酶活性的表达有极显著的抑制作用(p0.01),荧光素酶的活性均下调60%左右,说明了microRNA-155分别通过与rheb和atg3的3’UTR相互作用,抑制了rheb和atg3的表达,rheb和atg3是microRNA-155的直接靶标。通过构建pcDNA-rheb显著上调细胞内rheb表达水平(p0.01)后发现,IL-6、IL-1β和TNF-α的表达水平显著上升,说明rheb在T-2毒素诱导下可以促进炎症因子的表达。通过siRNA方法干扰细胞中rheb表达后,炎症因子的表达变化却不明显,猜测可能原因rheb只是T-2毒素诱导的促进炎症因子表达的其中一种因素,当rheb表达减少时,其他因素或许代偿性增加,导致对炎症因子的影响并不大。最后得出microRNA-155在T-2毒素的诱导下通过抑制rheb发挥抗炎的作用。通过构建pcDNA-atg3显著上调细胞内atg3表达水平(p0.01)后发现,IL-6极显著下调(p0.01),IL-1β和TNF-α略微下调,说明上调的atg3可以抑制T-2毒素诱导的促炎症因子的表达。细胞内atg3在经过siRNA下调之后,T-2毒素再处理12h,IL-6(p0.05)、IL-1β(p0.01)和TNF-α(p0.01)明显上调,说明下调的atg3可以促进T-2毒素诱导的促炎症因子的表达。由此证明了microRNA-155通过直接靶向调节atg3来促进T-2毒素诱导的炎症因子的产生。综上,本文发现在T-2毒素诱导下,rheb具有促进炎症因子表达的作用,atg3具有抑制炎症因子表达的作用,并且是microRNA-155的新靶标。MicroRNA-155在T-2毒素的诱导下发挥了双重的作用。它一方面通过靶向抑制SOCS1和atg3发挥促炎的作用,另一面通过靶向抑制rheb发挥抗炎的作用,防止炎症的进一步过大。进一步完善了microRNA-155的功能研究,有助于深入阐释T-2毒素的毒性机制。为T-2毒素的功能研究、发展有效的安全评估和开发出高效的拮抗剂奠定科学基础。
[Abstract]:T-2 toxin, produced by Fusarium spp., is the most poisonous toxin of the monotetramethylene toxoid. By contaminating feedstuffs, T-2 toxin causes serious harm to the health of animals and consumers of animal food. Studies have shown that T-2 toxin mainly damages the immune system of animals. This toxin can cause immunity in low doses of exposure. The cytotoxicity of these toxins mainly acts on the ribosome. On the one hand, they can bind to peptidyltransferase catalytic sites to inhibit the production of biological macromolecules such as proteins, on the other hand, they can interfere with the level of immune response and disturb the immune function of the body. Ribosomal stress reacts rapidly to activate the mitogen-activated protein kinase (MAPK) pathway, which regulates the expression of downstream factors associated with immune responses at both transcriptional and post-transcriptional levels. Increased expression of inducible pro-inflammatory cytokines is the main mediator for T-2 toxins to exert various toxic effects. MicroRNA-155 acts as one of the major mediators. MicroRNA-155 inhibits more than 60 target genes and plays different roles by targeting different genes. Studies have shown that it can not only be activated rapidly in inflammation, but also be poorly stimulated. Heterosexual expression of microRNA-155 plays a key role in the immune regulatory response. However, it has not been reported whether microRNA-155 plays a role in T-2 toxin-mediated inflammatory response. The results showed that the expression of microRNA-155 in RAW264.7 cells treated with 14nM T-2 toxin was up-regulated after 1 hour and up-regulated most significantly after 2 hours (p0.01). Although down-regulated, the expression of microRNA-155 was still up-regulated in different concentrations (7nM, 14nM, 28nM). Twelve hours after treatment with T-2 toxin, the expression of microRNA-155 in RAW264.7 cells was up-regulated most significantly at 7 nM (p0.01). This indicated that T-2 toxin could promote the expression of microRNA-155 in RAW264.7 cells. Secondly, microRNA-155 mic and inhibitor were used to overexpress and inhibit the expression of microRNA-155 in RAW264.7 cells. After treatment with T-2 toxin, IL-6 (p0.0) was found. 1), IL-1 beta (p0.05) and TNF-alpha (p0.01) expression levels increased significantly with the increase of microRNA-155, but decreased with the decrease of microRNA-155 (p0.05), indicating that microRNA-155 participated in and promoted the expression of pro-inflammatory factors induced by T-2 toxin. SOCS1 is a negative regulator of JAK/STAT pathway, and has been proved to be a target of microRNA-155. It was found that the expression of microRNA-155 was up-regulated and down-regulated by mimic and inhibitor respectively, and the expression of SOCS1 was down-regulated significantly when the expression of microRNA-155 was up-regulated (p0.01). MicroRNA-155 plays an inflammatory role by targeting SOCS1. Each microRNA has multiple target genes and plays different roles by targeting different genes. Two inflammation-related microRNA-155 targets, rheb and atg3, were identified by bioinformatics. Atg3 is a highly conserved GTP binding protein and a key regulator upstream of mTOR. It is involved in many biological functions such as cell growth, differentiation, oxidative stress and so on. Atg3 is a catalytic protein similar to E2 enzymes during ubiquitination, and is one of the autophagy families. Members, linked to LC3 and phosphatidylethanolamine, degrade long-lived proteins and damaged or unnecessary organelles by lipoylation of proteins, play important roles in the biological processes of antigen presentation, anti-infection, cell differentiation, and cell development in vesicle or lysosome systems. The expression of oRNA-155 showed that both the mRNA and protein levels of rheb and atg3 decreased significantly when microRNA-155 was up-regulated and increased significantly when microRNA-155 was down-regulated. The results showed that microRNA-155 inhibited the expression of luciferase in wild-type P GL3-3'UTR significantly (p0.01). The activity of luciferase was down by about 60%, indicating that microRNA-155 inhibited rheb and ATG 3 by interacting with 3'UTR of rheb and ATG 3, respectively. The expression of rheb and atg3 was the direct target of microRNA-155. After constructing pcDNA-rheb, the expression of rheb was up-regulated significantly (p0.01). The expression of IL-6, IL-1beta and TNF-alpha was up-regulated significantly, indicating that rheb could promote the expression of inflammatory factors induced by T-2 toxin. It is speculated that rheb is only one of the factors that promote the expression of inflammatory factors induced by T-2 toxin. When the expression of rheb decreases, other factors may increase compensatively, resulting in little effect on inflammatory factors. Finally, it is concluded that microRNA-155 plays an anti-inflammatory role by inhibiting rheb induced by T-2 toxin. After constructing pcDNA-atg3, the expression of atg3 was significantly up-regulated (p0.01), IL-6 was significantly down-regulated (p0.01), IL-1 beta and TNF-a were slightly down-regulated, indicating that the up-regulated atg3 could inhibit the expression of pro-inflammatory factors induced by T-2 toxin. In conclusion, rheb can promote the expression of inflammatory factors induced by T-2 toxin, and atg3 can inhibit the expression of inflammatory factors induced by T-2 toxin. MicroRNA-155 plays a dual role in the induction of T-2 toxin. On the one hand, it plays a pro-inflammatory role by targeting the inhibition of SOCS1 and atg3, on the other hand, it plays an anti-inflammatory role by targeting the inhibition of rheb to prevent inflammation further. The functional study of A-155 will help to elucidate the toxic mechanism of T-2 toxin and lay a scientific foundation for the functional study of T-2 toxin, the development of effective safety assessment and the development of effective antagonists.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.8
本文编号：2215077
[Abstract]:T-2 toxin, produced by Fusarium spp., is the most poisonous toxin of the monotetramethylene toxoid. By contaminating feedstuffs, T-2 toxin causes serious harm to the health of animals and consumers of animal food. Studies have shown that T-2 toxin mainly damages the immune system of animals. This toxin can cause immunity in low doses of exposure. The cytotoxicity of these toxins mainly acts on the ribosome. On the one hand, they can bind to peptidyltransferase catalytic sites to inhibit the production of biological macromolecules such as proteins, on the other hand, they can interfere with the level of immune response and disturb the immune function of the body. Ribosomal stress reacts rapidly to activate the mitogen-activated protein kinase (MAPK) pathway, which regulates the expression of downstream factors associated with immune responses at both transcriptional and post-transcriptional levels. Increased expression of inducible pro-inflammatory cytokines is the main mediator for T-2 toxins to exert various toxic effects. MicroRNA-155 acts as one of the major mediators. MicroRNA-155 inhibits more than 60 target genes and plays different roles by targeting different genes. Studies have shown that it can not only be activated rapidly in inflammation, but also be poorly stimulated. Heterosexual expression of microRNA-155 plays a key role in the immune regulatory response. However, it has not been reported whether microRNA-155 plays a role in T-2 toxin-mediated inflammatory response. The results showed that the expression of microRNA-155 in RAW264.7 cells treated with 14nM T-2 toxin was up-regulated after 1 hour and up-regulated most significantly after 2 hours (p0.01). Although down-regulated, the expression of microRNA-155 was still up-regulated in different concentrations (7nM, 14nM, 28nM). Twelve hours after treatment with T-2 toxin, the expression of microRNA-155 in RAW264.7 cells was up-regulated most significantly at 7 nM (p0.01). This indicated that T-2 toxin could promote the expression of microRNA-155 in RAW264.7 cells. Secondly, microRNA-155 mic and inhibitor were used to overexpress and inhibit the expression of microRNA-155 in RAW264.7 cells. After treatment with T-2 toxin, IL-6 (p0.0) was found. 1), IL-1 beta (p0.05) and TNF-alpha (p0.01) expression levels increased significantly with the increase of microRNA-155, but decreased with the decrease of microRNA-155 (p0.05), indicating that microRNA-155 participated in and promoted the expression of pro-inflammatory factors induced by T-2 toxin. SOCS1 is a negative regulator of JAK/STAT pathway, and has been proved to be a target of microRNA-155. It was found that the expression of microRNA-155 was up-regulated and down-regulated by mimic and inhibitor respectively, and the expression of SOCS1 was down-regulated significantly when the expression of microRNA-155 was up-regulated (p0.01). MicroRNA-155 plays an inflammatory role by targeting SOCS1. Each microRNA has multiple target genes and plays different roles by targeting different genes. Two inflammation-related microRNA-155 targets, rheb and atg3, were identified by bioinformatics. Atg3 is a highly conserved GTP binding protein and a key regulator upstream of mTOR. It is involved in many biological functions such as cell growth, differentiation, oxidative stress and so on. Atg3 is a catalytic protein similar to E2 enzymes during ubiquitination, and is one of the autophagy families. Members, linked to LC3 and phosphatidylethanolamine, degrade long-lived proteins and damaged or unnecessary organelles by lipoylation of proteins, play important roles in the biological processes of antigen presentation, anti-infection, cell differentiation, and cell development in vesicle or lysosome systems. The expression of oRNA-155 showed that both the mRNA and protein levels of rheb and atg3 decreased significantly when microRNA-155 was up-regulated and increased significantly when microRNA-155 was down-regulated. The results showed that microRNA-155 inhibited the expression of luciferase in wild-type P GL3-3'UTR significantly (p0.01). The activity of luciferase was down by about 60%, indicating that microRNA-155 inhibited rheb and ATG 3 by interacting with 3'UTR of rheb and ATG 3, respectively. The expression of rheb and atg3 was the direct target of microRNA-155. After constructing pcDNA-rheb, the expression of rheb was up-regulated significantly (p0.01). The expression of IL-6, IL-1beta and TNF-alpha was up-regulated significantly, indicating that rheb could promote the expression of inflammatory factors induced by T-2 toxin. It is speculated that rheb is only one of the factors that promote the expression of inflammatory factors induced by T-2 toxin. When the expression of rheb decreases, other factors may increase compensatively, resulting in little effect on inflammatory factors. Finally, it is concluded that microRNA-155 plays an anti-inflammatory role by inhibiting rheb induced by T-2 toxin. After constructing pcDNA-atg3, the expression of atg3 was significantly up-regulated (p0.01), IL-6 was significantly down-regulated (p0.01), IL-1 beta and TNF-a were slightly down-regulated, indicating that the up-regulated atg3 could inhibit the expression of pro-inflammatory factors induced by T-2 toxin. In conclusion, rheb can promote the expression of inflammatory factors induced by T-2 toxin, and atg3 can inhibit the expression of inflammatory factors induced by T-2 toxin. MicroRNA-155 plays a dual role in the induction of T-2 toxin. On the one hand, it plays a pro-inflammatory role by targeting the inhibition of SOCS1 and atg3, on the other hand, it plays an anti-inflammatory role by targeting the inhibition of rheb to prevent inflammation further. The functional study of A-155 will help to elucidate the toxic mechanism of T-2 toxin and lay a scientific foundation for the functional study of T-2 toxin, the development of effective safety assessment and the development of effective antagonists.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.8
【参考文献】
相关期刊论文 前1条
1 ;MicroRNA signatures in liver diseases[J];World Journal of Gastroenterology;2009年14期
相关博士学位论文 前1条
1 吴庆华;T-2毒素的代谢和毒作用信号转导研究[D];华中农业大学;2014年
相关硕士学位论文 前1条
1 柳芹;JAK/STAT信号通路在DON和T-2毒素对RAW264.7细胞毒性作用中的机制研究[D];华中农业大学;2011年
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