MiR-322负调控LPS诱导炎症反应的机制研究

发布时间：2018-04-19 00:34

本文选题：miR-322 + NF-κB1　；参考：《华中农业大学》2015年硕士论文

【摘要】：炎症反应是机体抵抗病原入侵,消灭病原体的正常自我保护机制,然而过度的炎症反应会导致机体损伤,导致相关免疫性疾病的发生,对炎症反应的精确调控显得尤为重要。微小RNA(mi RNA)是一段长为19~24nt的非编码小RNA分子,在进化过程中高度保守。mi RNA通过与靶基因m RNA 3'UTR完全或部分结合,对靶m RNA剪切降解或抑制其翻译,mi RNA介导的转录后水平在炎症反应和免疫调节中发挥重要作用。mmu-mi R-322与人mi R-424同源,是mi R-322家族的重要成员,mi R-322在多种疾病情况下差异表达,且在不同组织细胞中具有高度的保守性。研究表明,mi R-322在细胞增殖、细胞分化等生物学过程中发挥重要作用,但mi R-322在免疫调节中的作用及生物学功能尚待进一步研究。因此,本研究通过分析炎症反应与mi R-322的关系;研究mi R-322在巨噬细胞增殖分化中的作用;预测mi R-322靶基因并揭示其相互作用,阐明mi R-322在炎性反应中的调控作用,为揭示其免疫调控功能奠定基础。具体研究方法如下:q PCR检测mi R-322的表达变化:LPS刺激RAW264.7细胞,q PCR检测mi R-322的表达,结果显示,LPS刺激后mi R-322表达下调,且具有时间依赖性,提示mi R-322可能参与了炎症反应的调控。mi R-322对炎症反应的影响:将mi R-322 mimics转染RAW264.7细胞,转染24h后LPS刺激,q PCR检测IL-1β、IL-6、TNF-α的表达,结果显示,LPS刺激后IL-1β、IL-6、TNF-α大量表达,而在转染mi R-322后IL-1β、IL-6、TNF-α的表达被显著抑制。由此表明,mi R-322负调控LPS诱导的炎症反应。mi R-322对LPS介导的巨噬细胞增殖的影响:将mi R-322 mimics转染RAW264.7细胞,转染24h后LPS刺激,q PCR检测细胞周期蛋白cyclin D、cyclin E、P21、P27的表达,结果显示,LPS刺激后cyclin D、cyclin E显著下调,P21、P27显著上调,说明LPS刺激导致细胞周期紊乱,抑制了细胞增殖,而在转染mi R-322后,LPS诱导的cyclin D、cyclin E下调以及P21、P27上调被明显抑制,表明mi R-322能够影响细胞周期进程,随后利用流式细胞技术进一步验证发现mi R-322能够促进巨噬细胞周期进程,进而促进细胞增殖,缓解细胞损伤。利用MTT法检测活细胞数,同样证明了该结果,即LPS刺激后,活细胞数显著减少,细胞损伤严重,而在转染mi R-322后细胞损伤得到缓解,活细胞数明显增加。以上结果共同表明,mi R-322能够促进巨噬细胞增殖,缓解炎性损伤。靶基因的预测:利用mi RNA靶基因预测软件(mi Randa、targetscan)预测mi R-322靶基因,发现mi R-322能够与NF-κB1互补结合,提示NF-κB1可能是mi R-322的靶基因。mi R-322靶基因的验证:将NF-κB1 3'UTR克隆至双荧光素酶质粒上,构建双荧光素酶报告载体以及突变体,将其与mi R-322 mimics(模拟物)以及mi R-322 inhibitors(抑制剂)共转染至293T细胞,检测荧光素酶活性,结果显示,mi R-322转染组荧光素酶活性明显下调,且差异显著(P0.0001),mi R-322 inhibitors组荧光素酶活性显著上调,差异显著(P0.05),表明mi R-322能够与NF-κB1相互作用。随后利用q PCR以及western blot技术检测mi R-322对NF-κB1 m RNA及蛋白水平表达的影响,结果显示转染mi R-322 mimics后NF-κB1 m RNA及蛋白水平均明显下调,转染mi R-322 inhibitors后结果相反。表明NF-κB1是mi R-322的靶基因,并且通过mi R-322通过降解NF-κB1 m RNA来实现对NF-κB1的表达抑制。结论:mi R-322通过靶向抑制NF-κB1表达负调控LPS介导的炎症反应,并且mi R-322能促进细胞增殖,缓解炎性损伤。
[Abstract]:Inflammation is the body's resistance to pathogen invasion, destroy pathogens normal self protection mechanism, but the excessive inflammatory reaction will cause body damage, lead to autoimmune diseases, precise regulation of inflammatory responses is particularly important. The tiny RNA (MI RNA) is a 19~24nt encoding RNA in non small molecules. The evolutionary process is highly conserved.Mi RNA binding with target gene m RNA 3'UTR completely or partially, translation of target m RNA shear degradation or inhibit the transcription of MI, RNA mediated level in inflammation and immune regulation play an important role in.Mmu-mi R-322 and MI R-424 homology, is an important member of MI R-322 family mi R-322, in a variety of diseases under the condition of differential expression, and is highly conserved in different tissues. The results show that MI R-322 play an important role in cell proliferation, cell differentiation and other biological processes, but Mi R-322 still needs further research on the effects of immune regulation and biological function. Therefore, this research through the analysis on the relationship between inflammation and MI R-322; study the role of MI R-322 in the proliferation and differentiation of macrophages; MI prediction of R-322 target genes and reveal the interaction, clarify the regulatory role of MI R-322 in inflammatory reaction. Lay a foundation for revealing its immune regulation function. Specific research methods are as follows: to examine the expression of MI R-322 Q PCR: LPS stimulation of RAW264.7 cells, the expression of MI, R-322 Q PCR detection showed that LPS stimulated mi R-322 expression, which is time-dependent effect of R-322 Mi regulation of.Mi R-322 may be involved in inflammation in response to inflammatory reaction: Mi R-322 mimics was transfected into RAW264.7 cells. After transfection of 24h LPS stimulation, Q PCR detection of IL-1 beta, IL-6, expression of TNF- alpha showed that after LPS stimulation of IL-1 beta, IL-6, TNF- a large number of tables Da, IL-1 in transfected mi R-322 beta, IL-6, TNF- expression was significantly inhibited. The effect of.Mi R-322 mi R-322 negatively regulates the inflammatory response induced by LPS on LPS mediated macrophage proliferation: Mi R-322 mimics was transfected into RAW264.7 cells. After transfection of 24h LPS stimulation, Q PCR detection of cell cycle protein cyclin D, cyclin E, P21, P27 expression showed that LPS stimulated cyclin D, cyclin P21, P27 E were down regulated and up-regulated, indicating that LPS stimulation leads to the disorder of cell cycle, inhibiting cell proliferation, while transfection of MI R-322, LPS induced cyclin D, cyclin and E down P21, P27 upregulation was inhibited obviously, show that MI R-322 can affect cell cycle progression, followed by a further verify that MI R-322 can promote macrophage cycle process of flow cytometry, thus promoting cell proliferation, relieve cell injury. The MTT was used to detect the number of live cells, The results also proved that, after LPS stimulation, the number of live cells was significantly reduced, serious cell damage, and relieve cell injury in MI R-322 after transfection was significantly increased, the number of live cells. These results show that MI R-322 can promote macrophage proliferation, alleviate inflammatory injury. The predicted target gene prediction software: using mi the target gene of RNA (MI Randa targetscan) mi R-322 target gene prediction, found that MI R-322 can combine with complementary NF- kappa B1, suggesting that NF- kappa B1 may be verified the target gene.Mi R-322 target gene mi R-322: the NF- kappa B1 3'UTR was cloned into the dual luciferase plasmid, construct dual luciferase reporter vector and the mutant, with the MI R-322 mimics and MI R-322 (mimics) inhibitors (inhibitor) were transfected into 293T cells. Luciferase activity was detected, results showed that MI transfected with R-322 luciferase activity was significantly reduced, and the difference was significant (P0. 0001), MI R-322 group inhibitors luciferase activity was significantly increased, significant difference (P0.05), R-322 and NF- showed that MI kappa B1 interaction. Then the use of Q PCR and western of blot mi R-322 technique to detect the expression of NF- K B1 m RNA and protein level, results showed that the R-322 mimics NF- after transfection of MI kappa B1 m RNA and protein levels were significantly reduced after inhibitors R-322, transfection of MI in contrast to the results. The results indicated that NF- K B1 is the target gene of MI R-322, and Mi through the degradation of R-322 through the RNA to achieve m NF- kappa B1 on the expression of NF- kappa B1. Conclusion: Mi inhibited the expression of R-322 negatively regulates the inflammatory reaction mediated by LPS to inhibit NF- K B1 by target, MI and R-322 can promote cell proliferation, alleviate inflammatory injury.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.3

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