MicroRNA-155在T-2毒素誘導下對炎癥因子的調控機制研究
發(fā)布時間：2018-08-31 13:07
【摘要】：T-2毒素由鐮刀菌產生,是單端孢霉烯族類毒素中毒性最強的毒素。通過污染飼料,T-2毒素對動物和動物性食品消費者的健康造成嚴重危害。研究表明,T-2毒素主要損傷動物機體的免疫系統(tǒng)。該類毒素能使機體在低劑量的暴露下引起免疫刺激,但在高劑量的暴露下引起免疫抑制,導致免疫應答水平被嚴重干擾,機體免疫功能紊亂。這類毒素細胞毒性的發(fā)揮主要是作用于核糖體,一方面可以與肽酰轉移酶催化位點結合來阻礙生物大分子如蛋白質等的生成,另一方面通過核糖體應激反應快速激活絲裂原活化蛋白激酶(MAPK)通路,使相關免疫應答的下游因子的表達在轉錄和轉錄后水平受到調控。其中被誘導的促炎性細胞因子的基因表達上調是促使T-2毒素發(fā)揮各種毒性效應的主要介質。MicroRNA-155作為一個典型的多功能的microRNA,同眾多microRNA一樣,也是通過與靶基因的3’UTR結合,抑制基因的表達。MicroRNA-155抑制超過60種靶基因,可以通過靶向不同的基因而發(fā)揮不同的作用。研究表明它不僅在炎癥反應中可以被快速激活,而且受到刺激差異性表達的microRNA-155在免疫調節(jié)反應中起著非常關鍵的作用。但是關于microRNA-155是否在T-2毒素介導的炎癥反應中發(fā)揮作用仍未見報道。本研究以RAW264.7細胞為模型,初步探究了microRNA-155在T-2毒素誘導下對炎癥因子的調控作用機制。本研究首先用qRT-PCR的方法檢測了T-2毒素處理下microRNA-155是否差異性表達,結果顯示,14nM的T-2毒素處理RAW264.7細胞1h后microRNA-155上調,2h時上調最明顯(p0.01),后續(xù)雖有下調,但與空白組相比仍明顯上調。這種上調也出現在不同濃度(7nM、14nM、28nM)的T-2毒素處理RAW264.7細胞12h后。其中7nM時,microRNA-155上調最顯著(p0.01)。說明T-2毒素可以促進RAW264.7細胞中microRNA-155表達。其次利用microRNA-155 mimic和抑制劑(inhibitor)分別過表達和抑制細胞中microRNA-155的表達水平,T-2毒素處理后發(fā)現,IL-6(p0.01)、IL-1β(p0.05)和TNF-α(p0.01)的表達水平隨microRNA-155的上調而顯著上升,隨microRNA-155下調而下降(p0.05),說明microRNA-155參與并促進T-2毒素誘導的促炎癥因子的表達。SOCS1是JAK/STAT通路的負性調節(jié)因子,被驗證是microRNA-155的靶標。microRNA-155在多種情況下都可以通過直接抑制SOCS1發(fā)揮促炎的作用。但是在T-2毒素的誘導下這種調節(jié)作用是否存在卻未可知。于是分別通過mimic和inhibitor上調和下調了microRNA-155的表達,發(fā)現SOCS1的表達在microRNA-155上調時顯著下降(p0.01),在其下調時顯著上升(p0.01)。說明在T-2毒素的誘導下,microRNA-155通過靶向抑制SOCS1發(fā)揮了促炎的作用。每一個microRNA都有多個靶基因,通過靶向不同的基因發(fā)揮不同的作用。通過生物信息學方法確定了兩個與炎癥相關的microRNA-155的靶標rheb和atg3。Rheb是Ras同源基因,廣泛存在于多種生物中,是一個高度保守的GTP結合蛋白,并且是mTOR的上游關鍵調控因子,參與調節(jié)機體的如細胞生長、分化、氧化應激等眾多生物學功能。Atg3是一種類似于泛素化過程中E2酶的一種催化蛋白,作為自噬家族中的一員,連接LC3和磷脂酰乙醇胺,通過脂化蛋白質,降解長壽命蛋白質和已損壞或不必要的細胞器,在囊泡或溶酶體系統(tǒng)進行抗原提呈、抗感染、細胞分化、細胞發(fā)育等生物學過程中發(fā)揮重要作用。本實驗分別通過mimic和inhibitor上調和下調了microRNA-155的表達,發(fā)現rheb和atg3的mRNA水平和蛋白水平均在microRNA-155上調時顯著下降,在其下調時顯著上升。分別構建了含rheb和atg3 3’UTR結合位點序列的pGL3野生質粒和突變質粒,將microRNA-155 mimic和NC分別與這兩個質粒組合轉染進RAW264.7細胞內進行了雙熒光素酶報告基因實驗。結果顯示:microRNA-155對p GL3-3’UTR野生型的熒光素酶活性的表達有極顯著的抑制作用(p0.01),熒光素酶的活性均下調60%左右,說明了microRNA-155分別通過與rheb和atg3的3’UTR相互作用,抑制了rheb和atg3的表達,rheb和atg3是microRNA-155的直接靶標。通過構建pcDNA-rheb顯著上調細胞內rheb表達水平(p0.01)后發(fā)現,IL-6、IL-1β和TNF-α的表達水平顯著上升,說明rheb在T-2毒素誘導下可以促進炎癥因子的表達。通過siRNA方法干擾細胞中rheb表達后,炎癥因子的表達變化卻不明顯,猜測可能原因rheb只是T-2毒素誘導的促進炎癥因子表達的其中一種因素,當rheb表達減少時,其他因素或許代償性增加,導致對炎癥因子的影響并不大。最后得出microRNA-155在T-2毒素的誘導下通過抑制rheb發(fā)揮抗炎的作用。通過構建pcDNA-atg3顯著上調細胞內atg3表達水平(p0.01)后發(fā)現,IL-6極顯著下調(p0.01),IL-1β和TNF-α略微下調,說明上調的atg3可以抑制T-2毒素誘導的促炎癥因子的表達。細胞內atg3在經過siRNA下調之后,T-2毒素再處理12h,IL-6(p0.05)、IL-1β(p0.01)和TNF-α(p0.01)明顯上調,說明下調的atg3可以促進T-2毒素誘導的促炎癥因子的表達。由此證明了microRNA-155通過直接靶向調節(jié)atg3來促進T-2毒素誘導的炎癥因子的產生。綜上,本文發(fā)現在T-2毒素誘導下,rheb具有促進炎癥因子表達的作用,atg3具有抑制炎癥因子表達的作用,并且是microRNA-155的新靶標。MicroRNA-155在T-2毒素的誘導下發(fā)揮了雙重的作用。它一方面通過靶向抑制SOCS1和atg3發(fā)揮促炎的作用,另一面通過靶向抑制rheb發(fā)揮抗炎的作用,防止炎癥的進一步過大。進一步完善了microRNA-155的功能研究,有助于深入闡釋T-2毒素的毒性機制。為T-2毒素的功能研究、發(fā)展有效的安全評估和開發(fā)出高效的拮抗劑奠定科學基礎。
[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.
【學位授予單位】：華中農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S859.8
本文編號：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.
【學位授予單位】：華中農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S859.8
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