microRNA-210通过靶向NF-κB1负性调节LPS诱导的炎症反应

发布时间：2018-04-16 21:32

本文选题：microRNA + NF-κB1　；参考：《山东大学》2012年博士论文

【摘要】：固有免疫是机体抵抗病原微生物感染的第一道防线。巨噬细胞作为固有免疫中的一个重要成分,在清除病毒和细菌感染中发挥着重要的作用。它们通过其表面的模式识别受体(PRR)识别细菌和病毒结构上高度保守的病原相关分子模式(PAMP),然后经过一系列的信号转导机制最终激活NF-κB、IRF3和AP-1等转录因子,这些转录因子与相应的反应原件结合,产生大量的促炎性细胞因子、趋化因子等,从而促进炎症反应的发生。尽管巨噬细胞活化后,产生的促炎性细胞因子和趋化因子在清除微生物感染中发挥着重要的作用,但是巨噬细胞过度活化,促炎性细胞因子和趋化因子的过度产生,将引发一些与炎症相关的疾病或自身免疫性疾病。因此,严格的控制促炎性细胞因子的产生显得尤为重要。 MicroRNA (miRNA)是近年来发现的进化上高度保守的一类长度约为21-25个核苷酸(nt)的内源性非编码小RNA分子,它通过与靶mRNA的3’-UTR互补结合,使靶mRNA被剪切或转录抑制。然而,miRNA是否可以负性调控LPS诱导的巨噬细胞的炎症反应尚知之甚少。因此,本研究拟从以下几个方面进行：研究目的： 1、检测LPS诱导巨噬细胞活化后,那些microRNA的表达被上调或下调； 2、研究被上调或下调的microRNA对LPS诱导的巨噬细胞活化发挥什么样的作用； 3、探讨上调或下调的microRNA对LPS诱导的巨噬细胞活化发挥作用的机制。研究方法： 1、在LPS诱导活化的巨噬细胞中,检测表达变化的microRNA。 1.1LPS诱导巨噬细胞活化情况的检测分离了小鼠骨髓来源的巨噬细胞(Bone marrow derived macrophages, BMDM),经LPS刺激24h后,mRNA水平检测iNOS、TNF-a和IL-12p40的表达,以确定巨噬细胞是否被LPS活化。 1.2microRNA Assay分析表达变化的microRNA 将上述经过鉴定的样本进行microRNA Assay分析,与未刺激组相比,观察那些microRNA被上调或下调。 1.3Real-time验证上调或下调的microRNA 用real-time的方法验证microRNA Assay分析的数据,进一步的确定表达变化的microRNA。 2、表达变化的microRNA对LPS诱导的巨噬细胞活化的影响。 2.1转染相应microRNA mimics后,检测巨噬细胞促炎性细胞因子的表达和分泌情况将microRNA mimics转染入腹腔巨噬细胞48h后,再用LPS刺激巨噬细胞使其活化,PCR分析炎性细胞因子mRNA水平上的变化,ELISA检测炎性细胞因子蛋白水平的变化。 2.2转染相应microRNA inhibitor后,检测巨噬细胞促炎性细胞因子的表达和分泌情况将microRNA inhibitor转染入腹腔巨噬细胞48h后,再用LPS刺激巨噬细胞使其活化,PCR分析炎性细胞因子mRNA水平上的变化,ELISA检测炎性细胞因子蛋白水平的变化。 3、表达变化的microRNA对LP8诱导的巨噬细胞活化的作用机制研究。 3.1转染microRNA mimics和不同的TLR信号通路接头分子以及NF-κB报告质粒后,检测NF-κB报告基因的活性将：microRNA mimics和不同的接头分子转染HEK293细胞后,用报告基因的方法检测microRNA mimics对NF-κB报告质粒活性的影响。 3.2报告基因分析确定表达变化的microRNA对LPS诱导的巨噬细胞活化发挥作用的靶点通过microRNA靶基因预测软件分析microRNA有可能发挥作用的靶点,构建相应靶点的野生型或突变型3’-UTR报告基因质粒,转染HEK293细胞后,分析荧光素酶活性的变化,确定microRNA的作用靶点。 3.3WB检测表达变化的microRNA对内源性靶蛋白表达的影响将microRNA mimics转染巨噬细胞48h后,western blot检测靶基因表达的变化,确定microRNA对内源性靶基因的影响。 3.4表达变化的microRNA对LPS诱导的巨噬细胞活化发挥作用的方式将microRNA mimics转染巨噬细胞48h后,再用LPS刺激1h,染色质免疫共沉淀(Chromatin immunoprecipitation, CHIP)方法检测靶基因与促炎性细胞因子启动子区的结合情况,揭示microRNA影响巨噬细胞促炎性细胞因子分泌的机制。研究结果： 1、LPS诱导巨噬细胞活化后,microRNA-210的表达被显著上调。 1.1LPS刺激BMDM24h后,促炎性细胞因子的表达显著增加我们用LPS刺激BMDM24h后,在mRNA水平上检测了iNOS、TNF-a和IL-12p40的表达水平,发现与未刺激组相比,这些炎性细胞因子的表达被明显的上调。由此也证明了我们样本的收集是非常完美的。 1.2microRNA Assay分析发现microRNA-210的表达被上调了大约6倍我们用上述经过验证的LPS未刺激和刺激组的总RNA进行了microRNA Assay分析,发现miR-155、miR-147和miR-146的表达分别被上调了344、18和4倍。这与以前的报道是非常一致的,从而也证明了我们实验结果的可信性。未报道的microRNA,如miR-210被上调了大约6倍。 1.3Real-time PCR证实了miR-210的表达情况与芯片结果一致,其表达被显著上调我们合成了针对miR-210的特异性茎环引物,用real-time PCR方法分析了miR-210的表达,结果与芯片分析相符,与对照组相比,LPS刺激可显著上调miR-210的表达。 2、m i R-210对LPS诱导的巨噬细胞活化起到一定的抑制作用。 2.1miR-210mimics抑制促炎性细胞因子的分泌我们将miR-210mimics转染入原代腹腔巨噬细胞后,用real-time PCR方法检测了miR-210的表达情况,发现miR-210被上调了800倍左右。然后,我们再对转染过的细胞进行LPS刺激,发现巨噬细胞促炎性细胞因子的分泌显著下调。 2.1miR-210inhibitor促进促炎性细胞因子的分泌我们将miR-210inhibitor转染入原代腹腔巨噬细胞后,用real-time PCR方法检测了miR-210的表达情况,发现miR-210被显著下调。然后,我们再对转染过的细胞进行LPS刺激,发现巨噬细胞促炎性细胞因子的分泌显著增强。 3、miR-210通过靶向于NF-κ B1的3‘-UTR来发挥它的抑制效果。 3.1miR-210抑制NF-κ报告基因的活性我们将control mimics或miR-210mimics和不同的接头分子以及NF-κB报告质粒共转染HEK293细胞后,发现与对照组相比,miR-210mimics组可显著降低MyD88、TRAF6、TAK1和IKK-P所诱导的NF-κB报告质粒的荧光素酶活性。这也揭示了miR-210的作用靶点位于NF-κB本身或其下游。 3.2miR-210可与NF-κ B1的3’-UTR相互作用经过软件预测分析,我们发现miR-210可与NF-κB1的3’-UTR发生相互作用。所以我们构建了NF-κB13'-UTR的野生型和突变型报告质粒。miR-210mimics和报告质粒共转染实验证实了miR-210确实可作用于NF-κB1的3’-UTR,影响报告基因的活性。这与我们前期的预测结果也是非常一致的。 3.3miR-210抑制内源性NF-κ B1的表达基于以上的实验结果,我们用Western blot方法检测了转染miR-210mimics后内源性NF-κB1的表达情况,发现NF-κB1的表达被显著抑制。 3.4miR-210抑制p50/p65异二聚体与促炎性细胞因子启动子区的结合我们用CHIP的方法分析了p50/p65异二聚体与炎性细胞因子启动子区的结合情况,发现在转染miR-210mimics后,p50/p65异二聚体向启动子区的结合显著减少。结论： 1、在LPS诱导活化的巨噬细胞中,miR-210的表达显著上调； 2、miR-210抑制LPS诱导的巨噬细胞活化； 3、miR-210通过与NF-κB1的3’-UTR相互作用,从而抑制其蛋白的表达,最终使p50/p65异二聚体与促炎性细胞因子启动子区的结合减少。创新点及意义： 1、本研究首次证实了miR-210可抑制LPS诱导的巨噬细胞活化,而且阐明了这种抑制作用主要是miR-210通过它的"seed sequence"作用于NF-κB1的3’-UTR影响p105/p50的表达,最终导致了p50/p65异二聚体与促炎性细胞因子启动子区的结合下降。 2、本研究为负性调控巨噬细胞提供新的实验证据,为巨噬细胞参与的炎症反应性疾病的研究、诊断和治疗提供新的思路。
[Abstract]:As an important component of innate immunity , the innate immunity plays an important role in removing viruses and bacterial infections . These transcription factors play an important role in removing viruses and bacterial infections . These transcription factors play an important role in clearing microbial infections through a series of signal transduction mechanisms .

MicroRNA ( miRNA ) is an endogenous non - coding small RNA molecule of approximately 21 - 25 nucleotides ( nt ) , which has been found to be highly conserved in recent years , which binds the target mRNA by cleavage or transcription by complementary binding to the 3 ' - untranslated region of the target mRNA . However , it is unknown whether the miRNA can negatively regulate the inflammatory response of LPS - induced macrophages . Therefore , the present study is to be conducted from the following aspects :

Purpose of study :

1 . After LPS - induced activation of macrophages , the expression of those microRNA was up - regulated or down regulated ;

2 . The effect of the up - regulated or down - regulated microRNA on LPS - induced activation of macrophages was studied .

3 . To investigate the mechanism of up - regulated or down - regulated microRNA on LPS - induced activation of macrophages .

Study method :

1 . In LPS - induced activated macrophages , the expression change of microRNA was detected .

1.1 Detection of LPS - induced activation of macrophages

The expression of iNOS , TNF - a and IL - 12p40 in bone marrow derived macrophages ( BMDM ) and bone marrow derived macrophages ( BMDM ) were isolated from mouse bone marrow .

1.2 microRNA Assay Analysis of MicroRNAs Expressing Change

The identified samples were subjected to microRNA assay analysis and observed to be up - regulated or down - regulated as compared to unstimulated groups .

1.3 Real - time verification of up - regulated or down - regulated microRNA

A real - time method was used to validate the data of the microRNA assay and further determine the microRNA with the change of expression .

2 . The effect of microRNA on LPS - induced activation of macrophages .

2.1 After transfection of the corresponding microRNA , the expression and secretion of pro - inflammatory cytokines in macrophages were detected .

The expression of inflammatory cytokine protein was detected by ELISA , and the level of inflammatory cytokine protein was detected by ELISA .

2.2 After transfection of the corresponding microRNA inhibitor , the expression and secretion of pro - inflammatory cytokines in macrophages were detected .

After transfection of microRNA inhibitor into peritoneal macrophages for 48 hours , macrophages were stimulated by LPS to activate the macrophages , and the changes of inflammatory cytokine mRNA levels were analyzed by PCR , and the changes of inflammatory cytokine protein levels were detected by ELISA .

3 . The effect of microRNA on the activation of LP8 - induced macrophages was studied .

3.1 The activity of NF - 魏B reporter gene was detected after transfection of microRNA microarray and different TLR signaling pathway linker molecules and NF - 魏B reporter plasmid .

The effect of microRNA expression on the activity of NF - 魏B reporter plasmid was investigated by using the reporter gene method after the microRNA molecules and the different linker molecules were transfected into 293 cells .

3.2 Report gene analysis determines the target point for the activation of LPS - induced macrophages by microRNA expression - varying microRNA

The microRNA target gene prediction software is used for predicting the target point that the microRNA has the potential to play a role , constructing a wild - type or mutant 3 & # x2032 ; & # x2032 ; & # x2032 ;

3 . Effect of microRNA on expression of endogenous target protein in detection of expression changes in 3WB

After 48h , the expression of target gene was detected by western blot , and the effect of microRNA on endogenous target gene was determined .

3.4 Expression of varying microRNA in LPS - induced activation of macrophages

After 48 hours of transfection , the binding of the target gene and the promoter region of pro - inflammatory cytokines was detected by using the method of LPS - stimulated 1h and chromatocytes ( CHIP ) , and the mechanism of microRNA influencing the secretion of pro - inflammatory cytokines was revealed .

Results of the study :

1 . After LPS - induced activation of macrophages , the expression of microRNA - 210 was significantly increased .

1 . After LPS stimulated BMDM24h , the expression of pro - inflammatory cytokines increased significantly .

After stimulated BMDM24h with LPS , the levels of iNOS , TNF - a and IL - 12p40 were detected at mRNA level . It was found that the expression of these inflammatory cytokines was up - regulated in comparison with unstimulated group .

1.2 microRNA Assay found that microRNA - 210 expression was up - regulated by about 6 times

We analyzed the total RNA of the non - stimulated and stimulated groups of LPS , and found that the expression of miR - 155 , miR - 147 and miR - 146 was up - regulated 344 , 18 and 4 times , respectively . This was consistent with previous reports , thus demonstrating the credibility of our experimental results . Unreported microRNA , such as miR - 210 , were up - regulated by about 6 times .

1.3Real - time PCR confirmed that the expression of miR - 210 was consistent with the results of the chip , and its expression was up - regulated .

We synthesized a specific stem - loop primer for miR - 210 , analyzed the expression of miR - 210 by real - time PCR , and the results were consistent with the analysis of the chip . Compared with the control group , LPS stimulation could significantly increase the expression of miR - 210 .

2 . m i R - 210 inhibited LPS - induced activation of macrophages .

2.1 . 1miR - 210D3 inhibits the secretion of pro - inflammatory cytokines

The expression of miR - 210 was detected by real - time PCR , and miR - 210 was increased by about 800 fold after transfected into the primary peritoneal macrophages by real - time PCR . Then , we stimulated the transfected cells and found that the secretion of pro - inflammatory cytokines was down - regulated .

2.1 miR - 210inhibitor promotes the secretion of pro - inflammatory cytokines

After transfection of miR - 210 inhibitor into the primary peritoneal macrophages , the expression of miR - 210 was detected by real - time PCR , and it was found that miR - 210 was downregulated . Then , we stimulated the transfected cells and found that the secretion of pro - inflammatory cytokines was significantly enhanced .

3 . miR - 210 exerts its inhibitory effect by targeting the 3 ' - untranslated region of NF - . kappa . B1 . 3.1 miR - 210 Inhibition of NF - 魏B Activity After co - transfection of the control kinase or miR - 210bp with different linker molecules and the NF - . kappa . B reporter plasmid , it was found that the miR - 210bp group significantly reduced the luciferase activity of the NF - . kappa . B reporter plasmid induced by MyD88 , TRAF6 , TAK1 and IKK - P compared to the control group , which also revealed that the target point of the miR - 210 is located at or downstream of the NF - . kappa . B itself . 3 . 2miR - 210 can interact with the 3 ' - untranslated region of NF - 魏B

We have found that miR - 210 can interact with the 3 ' - untranslated region of NF - 魏B , so we constructed the wild - type and mutant reporter plasmid of NF - kappa B - 1 . The co - transfection of miR - 210 and reporter plasmid confirmed that miR - 210 can act on the 3 ' - untranslated region of NF - 魏B , which affects the activity of the reporter gene . This is also very consistent with the results of our earlier prediction .

3.3miR - 210 inhibits the expression of endogenous NF - 魏B

Based on the above experimental results , we detected the expression of endogenous NF - 魏B in transfected miR - 210bp by Western blot , and found that the expression of NF - 魏B was significantly inhibited .

3 . 4miR - 210 Inhibits the Binding of p50 / P65 Heterodimer with pro - inflammatory cytokine promoter region

We analyzed the binding of p50 / P50 heterodimer and the promoter region of inflammatory cytokines by using CHIP method , and found that after transfected with miR - 210bp , the binding of p50 / P65 heterodimer to the promoter region was significantly reduced .

Conclusion :

1 . In LPS - induced activated macrophages , the expression of miR - 210 was significantly increased .

2 . miR - 210 inhibits LPS - induced macrophage activation ;

3 . miR - 210 inhibits the expression of its protein by interacting with the 3 ' - untranslated region of NF - . kappa . B1 , resulting in a reduction in the binding of the p50 / p50 heterodimer to the pro - inflammatory cytokine promoter region .

Innovation point and significance :

1 . For the first time , the study demonstrated that miR - 210 can inhibit LPS - induced activation of macrophages , and that this inhibitory action is mainly the expression of miR - 210 by its " seed sequence " in the 3 ' - untranslated region of NF - . kappa . B1 , which ultimately leads to a decrease in the binding of the p50 / p50 heterodimer with the pro - inflammatory cytokine promoter region .

2 . This study provides new experimental evidence for negative regulatory macrophages , and provides a new idea for the study , diagnosis and treatment of inflammatory response diseases involving macrophages .

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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