热休克刺激诱导NFKBIA基因表达的转录后调控机制研究
发布时间：2018-09-07 09:03
【摘要】：研究背景1962年,Ferruccio Ritossa观察到基础体温为25 ℃的果蝇(Drosophila)暴露在37℃高温时,其唾液腺可合成一组相应的蛋白质。经过深入研究,发现该组蛋白质对细胞功能和内环境的稳定具有广泛的保护作用,因而将这些蛋白质命名为热休克蛋白(heat shock protein,HSP),而将果蝇处于高温时的基因表达和生理保护这一现象称为热休克反应(heat shock response,HSR)。热休克反应,这一所有应激反应中最原始和保守的反应,可诱导合成HSP,作为“分子伴侣”维持蛋白质的正确折叠状态,保护细胞抵御包括热休克、缺氧、缺血-再灌注损伤、内毒素、重金属和乙醇的毒性刺激等一系列的应激损伤,增强机体细胞对损害的耐受程度,维持细胞的正常功能代谢,提高细胞生存率,具有强大的保护作用。在应激反应中HSP基因首先表达,而其他基因的表达则暂时被抑制。在高温、亚砷酸钠、前列腺素-A1诱导的HSR中,HSP的表达可抑制促炎症因子的表达,其主要作用是通过抑制NF-κκB的激活来防止过度炎症反应对细胞的损伤。HSP抑制NF-κB的可能途径为:抑制IκBα的磷酸化和IKK的活性;增强IκBαmRNA的表达;竞争性抑制NF-κB经核孔复合体进入细胞核。有研究证实,在转录水平,IκBα的启动子内含有热休克元件(heat shock element, HSE),HSP基因转录的同时也有NFKBIA基因的转录,近年来又发现HSR在转录后水平也诱导NFKBIA基因的表达,本研究的创新点是探讨热休克过程中转录后水平诱导NFKBIA基因表达的机制。NF-κB是1986年首先发现于B细胞Igκ轻链转录调控中而得名,是将信号从胞浆传至胞核引起相应基因表达的一种诱导型、高效真核细胞核转录因子,对许多免疫调节基因的表达具有重要的调节功能,其中最重要的是调节细胞因子、粘附分子和其他免疫介质的基因转录。NF-κκB的激活主要使前炎症介质表达增加,导致急性炎症反应,而促炎因子如TNF-α等又可以激活NF-κB,因此在炎症反应过程中,有时很难区分两者的因果关系,它们之间的相互作用导致了炎症反应的扩大。NF-κB是由Rel蛋白家族的成员以同源或异源二聚体的形式组成的一组转录因子,一般意义上的NF-κB通常是指p50/p65二聚体。静息状态下,NF-κB与κB抑制因子(IκB)结合存在于细胞胞浆内,处于无活性状态。在κB家族中IκBα是目前发现最早和研究最清楚的成员。当细胞遇到刺激因子TNF-α、IL-18、内毒素时,NF-κB诱导激酶(NIK)激活IκB激酶复合物(IKK-1、2),IKK-1、2使IκBα在其32、36位点上的丝氨酸磷酸化、泛素化,然后由蛋白酶体降解使NF-κB游离进入细胞核,在核内NF-κB与基因上的κB序列结合,激活下游的靶基因转录,由于IκBα基因在其转录启动区含有3个NF-κB结合位点,因此当NF-κB激活的同时诱导IκBα的大量表达,这一途径构成了 NF-κB激活的负反馈途径。生物体是一个有机的统一整体,各种基因根据机体生长、发育、繁殖的需要,随着环境的变化,有规律的、程序性地表达,以适应环境,发挥其正常的生理功能。基因表达调控是在多级水平上进行的:基因活化、转录、转录后加工、翻译、翻译后加工等。基因转录起始的调控是大多数基因的主要调控形式,但是其他的调控能够在DNA到蛋白质途径时发挥作用,以调节所产生的基因产物数量,进一步保证了体内基因调控准确和精确的发生。基因表达的调控是一个复杂而有序的过程,包括有多级水平的调控。越来越多的研究表明,基因表达转录后水平的调控起着不可忽视的作用,主要通过影响mRNA稳定性及翻译效率来发挥作用。基因表达的转录后调控一般指在RNA聚合酶结合到基因启动子后,对转录产物进行的一系列修饰,主要包括:转录的提前终止、剪接、mRNA通过核孔和胞质内定位、RNA编辑、翻译起始和翻译效率,mRNA的稳定性等多个环节。研究发现转录本中调控mRNA稳定性等的元件存在于3'非翻译区(3'UTR),大部分转录后调控机制的靶向部位都位于转录本mRNA的3'UTR。现已发现约5-8%的人类基因编码的mRNA其3'UTR含有ARE序列,ARE序列是一段长度约为50-150nt富含腺嘿呤(adenine)和尿嘧啶(uridine)的序列,与mRNA的稳定性有密切关系。ARE序列能够招募许多ARE结合蛋白(ARE-binding protein,ARE-BP),这些ARE-BP相互协作或竞争地与mRNA的ARE结构域结合,共同调节mRNA的稳定性和翻译,NFKBIA基因编码的mRNA中也存在着ARE序列。利用 Streptavidin-biotin进行RNA-pulldown得到与NFKBIA mRNA 3'UTR相互作用的蛋白复合物,经SDS-PAGE凝胶电泳分离、银染分析和蛋白质谱鉴定后,得到热休克过程中与NFKBIA基因的3'UTR相互作用蛋白质hnRNP Q。hnRNP Q是不均一性核糖核蛋白(heterogeneous nuclear ribonucleo-protein, hnRNP)家族的成员之一,主要定位于细胞核内,可与新合成的不均一性RNA (heterogeneous nuclear RNA, hnRNA)相结合,从而调控 mRNA前体(pre-mRNA)的剪接、mRNA核质转运、翻译和降解等一系列重要过程,是一种重要的RNA结合蛋白,本研究将进一步探讨其在热休克过程中参与NFKBIA mRNA 3'UTR功能调节的机制。研究目的本课题主要探讨热休克刺激诱导NFKBIA基因表达的转录后调控机制,重点探讨NFKBIA mRNA 3'UTR结合蛋白hnRNP Q在热休克刺激诱导NFKBIA基因表达的转录后调控中的作用及机制。研究内容1.采用蛋白免疫印迹技术检测热休克刺激后IκBα蛋白表达及半衰期改变;2.采用荧光定量PCR技术检测热休克刺激后NFKBIA mRNA表达及半衰期改变;3.构建NFKBIA mRNA 3'UTR双荧光素酶报告基因质粒,利用双荧光素酶报告基因系统研究NFKBIA mRNA 3 'UTR在热休克过程中的作用;4.利用Streptavidin-biotin进行RNA-pulldown,筛选与NFKBIAmRNA3'UTR相互作用的蛋白复合物,并经过SDS-PAGE凝胶电泳分离、银染分析和蛋白质谱鉴定与NFKBIA mRNA 3'UTR相互作用蛋白及验证;5.热休克过程中hnRNP Q参与调节NFKBIA mRNA 3'UTR功能及其机制。研究方法采用荧光定量PCR检测mRNA的相对表达量,蛋白免疫印迹技术检测蛋白表达,免疫荧光观察蛋白及mRNA共定位,双荧光素酶报告基因系统间接反映NFKBIA mRNA 3'UTR功能。统计分析软件为SPSS 13.0,首先采用Levene进行方差齐性检验,文中样本方差均齐,则采用单因素方差分析(One-way ANOVA),若总体而言各组间有差异,则进一步进行多重比较,以α=0.05为检验水准,作图软件为OriginPr08.0。通过以上实验研究,得到以下结果:1.正常对照组RAW细胞IκBα蛋白质半衰期大约为3 h,给予热休克刺激后其半衰期延长至约4 h,正常对照组NFKBIA mRNA的半衰期约为40 min,给予热休克刺激后其半衰期明显延长至约85 min。同时检测了热休克后细胞恢复正常培养的不同时间点NFKBIA mRNA及蛋白质表达量,,发现NFKBIA mRNA的表达量与对照组相比,在恢复培养240 min前的各个时间点变化不明显,而NFKBIA蛋白的表达量呈现先减少后增加再减少的趋势,通过以上数据不难发现,RAW细胞给予43℃C热休克处理1 h后,其NFKBIA mRNA及蛋白质表达量呈现不一致的变化,说明存在转录后调控机制。2.将NFKBIA mRNA 3'UTR构建在荧光素酶报告基因(简称pLuNB )之后,通过荧光素酶报告基因的检测来间接反映热休克诱导的NFKBIA mRNA 3'UTR的作用。结果显示,将pLuNB转染NIH 3T3细胞48 h后给予热休克刺激(43℃1 h),其报告基因检测值与对照组相比在各时间点均升高,说明热休克过程中NFKBIA mRNA 3'UTR参与了 NFKBIA基因转录后调控。3.通过 Streptavidin-biotin 进行 RNA-pull down 得到与 NFKBIA mRNA 3'UTR相互作用的蛋白复合物,并进行SDS-PAGE凝胶电泳分离、蛋白银染、蛋白质谱鉴定、生物学分析,确定了本研究中热休克诱导后与NFKBIA mRNA 3'UTR相互作用蛋白hnRNP Q的存在。通过Biotin-RNA pull down证明了 hnRNP Q确实可以与NFKBIA mRNA 3'UTR特异性结合,并进一步证实,当过表达及干扰hnRNPQ后,IκBα蛋白表达分别出现上调及下调现象,初步证实hnRNPQ参与了 NFKBIA基因的转录后调控。4.当共转染hnRNP Q1及pLuNB 48 h后给予热休克刺激43℃C 1 h,恢复至正常培养条件,分别在0、1、2、3 h收细胞进行荧光素酶报告基因检测。与对照组相比,恢复正常培养后的各个时间点报告基因数值均降低。当共转染hnRNPQ与NFKBIA特异性siRNA(SY23)48 h后,qPCR检测其干扰效率约为47%,并给予热休克处理后,在以上各个时间点收细胞进行荧光素酶报告基因检测,其数值与对照组相比均升高。以上数据表明,hnRNP Q1参与了热休克诱导的NFKBIA基因表达的转录后调控。5.NIH 3T3细胞过表达hnRNP Q1及hnRNP Q2 24 h后给予热休克刺激,与不刺激对照组同时检测NFKBIA mRNA表达量及IκBα蛋白表达变化,结果显示过表达组与空质粒载体组热休克处理前后NFKBIA mRNA的表达量变化均不明显,但对IκBα蛋白表达有下调作用。6.为进一步揭示hnRNP Q在热休克诱导的NFKBIA基因转录后水平调控中的作用,本研究引入了 MS2 vector系统,构建了 pMS2-cherry融合表达蛋白,其可以表达特异性与茎环结构结合的蛋白质,当构建的带有8个茎环结构的NFKBIA mRNA 3'UTR质粒表达后,与红色荧光融合蛋白相互结合来间接反映NFKBIA mRNA 3'UTR 细胞内定位。结果显示在未给予热休克处理时hnRNP Q蛋白弥散分布于胞浆与细胞核内,与NFKBIA mRNA 3'UTR有明显共定位,可能hnRNP Q参与NFKBIA mRNA 3'UTR出核、成熟及细胞内定位等过程;当受到热休克刺激之后,恢复0-2 h浆中hnRNP Q明显形成颗粒状小体,恢复4 h颗粒有所减少,且这些颗粒小体与NFKBIA mRNA 3'UTR存在共定位;实验组NFKBIA mRNA 3'UTR在未受到热休克刺激时,与P小体(P body)主要组成成分hDcP1a蛋白存在明显的共定位,热休克刺激1h,恢复正常培养0h DcP1a颗粒明显增多,且与NFKBIA mRNA 3'UTR有共定位,恢复2 h粒逐渐减少;实验组G3Bp1蛋白在未受到热休克刺激时,在胞浆中成弥散性分布,与NFKBIA mRNA 3'UTR无明显共定位,当给予热休克刺激1h后,恢复正常培养2hG3Bp1蛋白分布于包浆并形成明显的颗粒状小体,分布于胞浆,且与NFKBIA mRNA 3'UTR存在共定位,恢复4 颗粒开始减少,作为上述实验的对照组在各时间点均未观察到p8MS2空质粒组mRNA与hnRNPQ、hDcP1a、G3Bp1蛋白存在共定位。
[Abstract]:Background In 1962, Ferruccio Ritossa observed that the salivary glands of Drosophila (Drosophila) exposed to basal body temperature of 25 C could synthesize a set of corresponding proteins when exposed to high temperature of 37 C. Heat shock protein (HSP), which is called heat shock response (HSR), is the most primitive and conserved response in all stress reactions that induce the synthesis of HSP as a "molecular chaperone" to maintain the correct folding of proteins. Protecting cells against a series of stress injuries including heat shock, hypoxia, ischemia-reperfusion injury, endotoxin, toxic stimuli of heavy metals and ethanol, enhance the tolerance of the body cells to damage, maintain the normal functional metabolism of cells, improve cell survival rate, has a strong protective effect. In HSR induced by hyperthermia, sodium arsenite, and prostaglandin-A1, the expression of HSP can inhibit the expression of pro-inflammatory factors. The main effect of HSP is to prevent excessive inflammation by inhibiting the activation of NF-kappa B. Acidification and IKK activity; enhancement of I-kappa B alpha mRNA expression; competitive inhibition of NF-kappa B entry into the nucleus via the nuclear pore complex. Studies have shown that at the transcriptional level, the promoter of I-kappa B alpha contains heat shock element (HSE), and the transcription of HSP gene is accompanied by the transcription of NFKBIA gene. In recent years, HSR has also been found at the post-transcriptional level. The innovation of this study is to explore the mechanism of inducing NFKBIA gene expression at post-transcriptional level during heat shock. NF-kappa B was first discovered in the regulation of Ig-kappa light chain transcription in B cells in 1986. It is an inducible, highly efficient eukaryotic cell nuclear transduction in which signal is transmitted from cytoplasm to nucleus to induce corresponding gene expression. The activation of NF-kappa B mainly increases the expression of pro-inflammatory mediators, leading to acute inflammation, and pro-inflammatory factors such as TNF-a can activate NF-kappa B. Therefore, pro-inflammatory factors such as TNF-a can activate NF-kappa B. NF-kappa B is a group of transcription factors composed of members of the Rel protein family in the form of homologous or heterodimers. In general, NF-kappa B is usually referred to as p50/p65 dimer. In resting state, NF-kappa B is a member of the Rel protein family. In the kappa B family, I-kappa B alpha is the earliest and most well-known member. When cells encounter the stimulating factors TNF-alpha, IL-18 and endotoxin, NF-kappa B-induced kinase (NIK) activates I-kappa B kinase complex (IKK-1,2), IKK-1,2 causes I-kappa B alpha to be on its 32,36 sites. Serine phosphorylation, ubiquitination, and then proteasome degradation free NF-kappa B into the nucleus, NF-kappa B in the nucleus and gene kappa B sequence binding, activation of downstream target gene transcription, because the Ikappa B alpha gene in its transcription initiation region contains three NF-kappa B binding sites, so when NF-kappa B activation at the same time induce a large number of expression of I-kappa B alpha, this way. The pathway constitutes a negative feedback pathway for the activation of NF-kappa B. The organism is an organic whole. Various genes are expressed regularly and programmatically according to the needs of the organism's growth, development and reproduction in order to adapt to the environment and play their normal physiological functions. Gene transcription initiation is the main regulation form of most genes, but other regulation can play a role in the DNA-protein pathway to regulate the number of gene products produced, further ensuring the accuracy and accuracy of gene regulation in vivo. The regulation of gene expression at post-transcriptional level is a complex and orderly process, including multi-level regulation. More and more studies have shown that the regulation of gene expression at post-transcriptional level plays an important role, mainly by affecting the stability of mRNA and translation efficiency. After the promoter, a series of modifications have been made to the transcript products, mainly including early termination of transcription, splicing, localization of mRNA through nuclear pores and cytoplasm, RNA editing, translation initiation and translation efficiency, stability of mRNA and so on. The target sites of post-transcriptional regulation mechanisms are located at 3'UTR of the transcript mRNA. Around 5-8% of human gene-encoded mRNAs have been found to contain ARE sequences. The ARE sequence is a 50-150 NT long sequence rich in adenine and uridine and is closely related to the stability of the mRNA. ARE sequences can recruit many ARE junctions. ARE-binding protein (ARE-BP), these ARE-BP cooperate or compete with the ARE domain of the mRNA to regulate the stability and translation of the mRNA. The ARE sequence also exists in the mRNA encoded by the NFKBIA gene. The protein hnRNP Q. hnRNP Q, which interacts with the 3'UTR of NFKBIA gene during heat shock, is one of the members of the heterogeneous nuclear ribonucleo-protein (hnRNP) family. It is located mainly in the nucleus of the cell and can be inhomogeneously synthesized. Sexual RNA (hnRNA) binds to regulate a series of important processes, such as splicing of pre-mRNA, nuclear-cytoplasmic transport, translation and degradation of mRNA. It is an important RNA-binding protein. This study will further explore its role in regulating the function of NFKBIA mRNA 3'UTR during heat shock. The present study was designed to investigate the post-transcriptional regulation of NFKBIA gene expression induced by heat shock, with emphasis on the role and mechanism of NFKBIA mRNA 3'UTR binding protein hnRNP Q in the post-transcriptional regulation of NFKBIA gene expression induced by heat shock. Content 1. Detection of I-kappa B-alpha protein expression and its mechanism by Western blotting after heat shock. The expression of NFKBIA mRNA and the change of half-life were detected by fluorescence quantitative PCR. 3. The plasmid of NFKBIA mRNA 3'UTR double luciferase reporter gene was constructed and the role of NFKBIA mRNA 3'UTR in the process of heat shock was studied by double luciferase reporter gene system. 4. Streptavidin-biotin was used for RNA-pull. Down, screening the protein complexes interacting with NFKBI AmRNA 3'UTR, and then isolating them by SDS-PAGE gel electrophoresis, silver staining and protein profiling to identify the protein interacting with NFKBIA mRNA 3'UTR; 5. hnRNP Q is involved in regulating the function and mechanism of NFKBIA mRNA 3'UTR during heat shock. The relative expression of NFKBIA mRNA was detected by Western blot, co-localized by immunofluorescence and mRNA, and the function of NFKBIA mRNA 3'UTR was indirectly reflected by the dual luciferase reporter gene system. One-way ANOVA), if there are differences between the groups as a whole, then further multiple comparisons were carried out. Taking alpha=0.05 as the test level, the drawing software was OriginPr08.0. Through the above experimental study, the following results were obtained: 1. The half-life of I-kappa B-alpha protein in RAW cells of normal control group was about 3 hours, and its half-life was prolonged to about 4 hours after heat shock stimulation. The half-life of NFKBIA mRNA in the control group was about 40 minutes, and the half-life of NFKBIA mRNA was prolonged to about 85 minutes after heat shock stimulation. The expression of NFKBIA mRNA and protein was detected at different time points after heat shock, and it was found that the expression of NFKBIA mRNA was higher than that in the control group at different time points before 240 minutes of recovery culture. The changes of NFKBIA mRNA and protein expression were not obvious, but the expression of NFKBIA protein decreased first, then increased and then decreased. It was not difficult to find that the expression of NFKBIA mRNA and protein in RAW cells after heat shock treatment at 43 C for 1 hour showed inconsistent changes, indicating that there was a post-transcriptional regulation mechanism. 2. NFKBIA mRNA 3'UTR was constructed in luciferin. After transfection of pLuNB into NIH 3T3 cells for 48 hours, heat shock stimulation (43 1 h) was given, and the detection value of reporter gene increased at all time points compared with the control group, indicating that heat shock was over. NFKBIA mRNA 3'UTR is involved in the post-transcriptional regulation of NFKBIA gene. 3. The protein complexes interacting with NFKBIA mRNA 3'UTR were obtained by RNA-pull down through Streptavidin-biotin. SDS-PAGE gel electrophoresis, protein silver staining, protein spectrum identification and biological analysis were carried out to determine the relationship between NFKBIA and heat shock-induced NFKB. Biotin-RNA pull down demonstrated that hnRNPQ could specifically bind to the 3'UTR of NFKBIA mRNA, and further confirmed that the expression of I-kappa B-alpha protein was up-regulated and down-regulated respectively after overexpression and interference with hnRNPQ. It was preliminarily confirmed that hnRNPQ was involved in the post-transcriptional regulation of NFKBIA gene. 4. After co-transfection of hnRNP Q1 and pLuNB for 48 hours, the cells were stimulated by heat shock at 43 C_ 1 h and then returned to normal culture conditions. Luciferase reporter gene was detected at 0, 1, 2, and 3 h respectively. Compared with the control group, the number of reporter gene decreased at each time point after recovery from normal culture. After h, the interfering efficiency of qPCR was about 47%. After heat shock treatment, the number of luciferase reporter gene in the cells collected at each time point was higher than that in the control group. The above data showed that hnRNP Q1 was involved in the post-transcriptional regulation of NFKBIA gene expression induced by heat shock. 5. NIH 3T3 cells overexpressed hnRNP Q1. The expression of NFKBIA mRNA and the expression of I-kappa B-alpha protein were detected simultaneously in the over-expression group and the blank plasmid carrier group. The results showed that the expression of NFKBIA mRNA was not significantly changed before and after heat shock treatment, but the expression of I-kappa B-alpha protein was down-regulated in the over-expression group and the blank plasmid carrier group. In this study, MS2 vector system was introduced to construct the pMS2-cherry fusion protein, which can express the protein specifically binding to the stem ring structure. When the constructed NFKBIA mRNA 3'UTR plasmid with eight stem ring structures was expressed, it was fused with the red fluorescent protein. The results showed that hnRNP Q protein was diffusely distributed in the cytoplasm and nucleus without heat shock treatment, and co-localized with NFKBIA mRNA 3'UTR. It was possible that hnRNP Q participated in the process of exocytosis, maturation and intracellular localization of NFKBIA mRNA 3'UTR. After 0-2 h of recovery, hnRNP Q significantly formed granular bodies, and the granular bodies decreased at 4 h of recovery, and these granular bodies co-localized with NFKBIA mRNA 3'UTR. In the experimental group, NFKBIA mRNA 3'UTR co-localized with hDcP1a protein, the main component of P body, without heat shock stimulation, and returned to normal after 1 h of heat shock stimulation. The number of DcP1a granules increased significantly at 0 h after culture and co-localized with NFKBIA mRNA 3'UTR. The number of granules decreased gradually at 2 h after recovery.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363
本文编号：2227811
[Abstract]:Background In 1962, Ferruccio Ritossa observed that the salivary glands of Drosophila (Drosophila) exposed to basal body temperature of 25 C could synthesize a set of corresponding proteins when exposed to high temperature of 37 C. Heat shock protein (HSP), which is called heat shock response (HSR), is the most primitive and conserved response in all stress reactions that induce the synthesis of HSP as a "molecular chaperone" to maintain the correct folding of proteins. Protecting cells against a series of stress injuries including heat shock, hypoxia, ischemia-reperfusion injury, endotoxin, toxic stimuli of heavy metals and ethanol, enhance the tolerance of the body cells to damage, maintain the normal functional metabolism of cells, improve cell survival rate, has a strong protective effect. In HSR induced by hyperthermia, sodium arsenite, and prostaglandin-A1, the expression of HSP can inhibit the expression of pro-inflammatory factors. The main effect of HSP is to prevent excessive inflammation by inhibiting the activation of NF-kappa B. Acidification and IKK activity; enhancement of I-kappa B alpha mRNA expression; competitive inhibition of NF-kappa B entry into the nucleus via the nuclear pore complex. Studies have shown that at the transcriptional level, the promoter of I-kappa B alpha contains heat shock element (HSE), and the transcription of HSP gene is accompanied by the transcription of NFKBIA gene. In recent years, HSR has also been found at the post-transcriptional level. The innovation of this study is to explore the mechanism of inducing NFKBIA gene expression at post-transcriptional level during heat shock. NF-kappa B was first discovered in the regulation of Ig-kappa light chain transcription in B cells in 1986. It is an inducible, highly efficient eukaryotic cell nuclear transduction in which signal is transmitted from cytoplasm to nucleus to induce corresponding gene expression. The activation of NF-kappa B mainly increases the expression of pro-inflammatory mediators, leading to acute inflammation, and pro-inflammatory factors such as TNF-a can activate NF-kappa B. Therefore, pro-inflammatory factors such as TNF-a can activate NF-kappa B. NF-kappa B is a group of transcription factors composed of members of the Rel protein family in the form of homologous or heterodimers. In general, NF-kappa B is usually referred to as p50/p65 dimer. In resting state, NF-kappa B is a member of the Rel protein family. In the kappa B family, I-kappa B alpha is the earliest and most well-known member. When cells encounter the stimulating factors TNF-alpha, IL-18 and endotoxin, NF-kappa B-induced kinase (NIK) activates I-kappa B kinase complex (IKK-1,2), IKK-1,2 causes I-kappa B alpha to be on its 32,36 sites. Serine phosphorylation, ubiquitination, and then proteasome degradation free NF-kappa B into the nucleus, NF-kappa B in the nucleus and gene kappa B sequence binding, activation of downstream target gene transcription, because the Ikappa B alpha gene in its transcription initiation region contains three NF-kappa B binding sites, so when NF-kappa B activation at the same time induce a large number of expression of I-kappa B alpha, this way. The pathway constitutes a negative feedback pathway for the activation of NF-kappa B. The organism is an organic whole. Various genes are expressed regularly and programmatically according to the needs of the organism's growth, development and reproduction in order to adapt to the environment and play their normal physiological functions. Gene transcription initiation is the main regulation form of most genes, but other regulation can play a role in the DNA-protein pathway to regulate the number of gene products produced, further ensuring the accuracy and accuracy of gene regulation in vivo. The regulation of gene expression at post-transcriptional level is a complex and orderly process, including multi-level regulation. More and more studies have shown that the regulation of gene expression at post-transcriptional level plays an important role, mainly by affecting the stability of mRNA and translation efficiency. After the promoter, a series of modifications have been made to the transcript products, mainly including early termination of transcription, splicing, localization of mRNA through nuclear pores and cytoplasm, RNA editing, translation initiation and translation efficiency, stability of mRNA and so on. The target sites of post-transcriptional regulation mechanisms are located at 3'UTR of the transcript mRNA. Around 5-8% of human gene-encoded mRNAs have been found to contain ARE sequences. The ARE sequence is a 50-150 NT long sequence rich in adenine and uridine and is closely related to the stability of the mRNA. ARE sequences can recruit many ARE junctions. ARE-binding protein (ARE-BP), these ARE-BP cooperate or compete with the ARE domain of the mRNA to regulate the stability and translation of the mRNA. The ARE sequence also exists in the mRNA encoded by the NFKBIA gene. The protein hnRNP Q. hnRNP Q, which interacts with the 3'UTR of NFKBIA gene during heat shock, is one of the members of the heterogeneous nuclear ribonucleo-protein (hnRNP) family. It is located mainly in the nucleus of the cell and can be inhomogeneously synthesized. Sexual RNA (hnRNA) binds to regulate a series of important processes, such as splicing of pre-mRNA, nuclear-cytoplasmic transport, translation and degradation of mRNA. It is an important RNA-binding protein. This study will further explore its role in regulating the function of NFKBIA mRNA 3'UTR during heat shock. The present study was designed to investigate the post-transcriptional regulation of NFKBIA gene expression induced by heat shock, with emphasis on the role and mechanism of NFKBIA mRNA 3'UTR binding protein hnRNP Q in the post-transcriptional regulation of NFKBIA gene expression induced by heat shock. Content 1. Detection of I-kappa B-alpha protein expression and its mechanism by Western blotting after heat shock. The expression of NFKBIA mRNA and the change of half-life were detected by fluorescence quantitative PCR. 3. The plasmid of NFKBIA mRNA 3'UTR double luciferase reporter gene was constructed and the role of NFKBIA mRNA 3'UTR in the process of heat shock was studied by double luciferase reporter gene system. 4. Streptavidin-biotin was used for RNA-pull. Down, screening the protein complexes interacting with NFKBI AmRNA 3'UTR, and then isolating them by SDS-PAGE gel electrophoresis, silver staining and protein profiling to identify the protein interacting with NFKBIA mRNA 3'UTR; 5. hnRNP Q is involved in regulating the function and mechanism of NFKBIA mRNA 3'UTR during heat shock. The relative expression of NFKBIA mRNA was detected by Western blot, co-localized by immunofluorescence and mRNA, and the function of NFKBIA mRNA 3'UTR was indirectly reflected by the dual luciferase reporter gene system. One-way ANOVA), if there are differences between the groups as a whole, then further multiple comparisons were carried out. Taking alpha=0.05 as the test level, the drawing software was OriginPr08.0. Through the above experimental study, the following results were obtained: 1. The half-life of I-kappa B-alpha protein in RAW cells of normal control group was about 3 hours, and its half-life was prolonged to about 4 hours after heat shock stimulation. The half-life of NFKBIA mRNA in the control group was about 40 minutes, and the half-life of NFKBIA mRNA was prolonged to about 85 minutes after heat shock stimulation. The expression of NFKBIA mRNA and protein was detected at different time points after heat shock, and it was found that the expression of NFKBIA mRNA was higher than that in the control group at different time points before 240 minutes of recovery culture. The changes of NFKBIA mRNA and protein expression were not obvious, but the expression of NFKBIA protein decreased first, then increased and then decreased. It was not difficult to find that the expression of NFKBIA mRNA and protein in RAW cells after heat shock treatment at 43 C for 1 hour showed inconsistent changes, indicating that there was a post-transcriptional regulation mechanism. 2. NFKBIA mRNA 3'UTR was constructed in luciferin. After transfection of pLuNB into NIH 3T3 cells for 48 hours, heat shock stimulation (43 1 h) was given, and the detection value of reporter gene increased at all time points compared with the control group, indicating that heat shock was over. NFKBIA mRNA 3'UTR is involved in the post-transcriptional regulation of NFKBIA gene. 3. The protein complexes interacting with NFKBIA mRNA 3'UTR were obtained by RNA-pull down through Streptavidin-biotin. SDS-PAGE gel electrophoresis, protein silver staining, protein spectrum identification and biological analysis were carried out to determine the relationship between NFKBIA and heat shock-induced NFKB. Biotin-RNA pull down demonstrated that hnRNPQ could specifically bind to the 3'UTR of NFKBIA mRNA, and further confirmed that the expression of I-kappa B-alpha protein was up-regulated and down-regulated respectively after overexpression and interference with hnRNPQ. It was preliminarily confirmed that hnRNPQ was involved in the post-transcriptional regulation of NFKBIA gene. 4. After co-transfection of hnRNP Q1 and pLuNB for 48 hours, the cells were stimulated by heat shock at 43 C_ 1 h and then returned to normal culture conditions. Luciferase reporter gene was detected at 0, 1, 2, and 3 h respectively. Compared with the control group, the number of reporter gene decreased at each time point after recovery from normal culture. After h, the interfering efficiency of qPCR was about 47%. After heat shock treatment, the number of luciferase reporter gene in the cells collected at each time point was higher than that in the control group. The above data showed that hnRNP Q1 was involved in the post-transcriptional regulation of NFKBIA gene expression induced by heat shock. 5. NIH 3T3 cells overexpressed hnRNP Q1. The expression of NFKBIA mRNA and the expression of I-kappa B-alpha protein were detected simultaneously in the over-expression group and the blank plasmid carrier group. The results showed that the expression of NFKBIA mRNA was not significantly changed before and after heat shock treatment, but the expression of I-kappa B-alpha protein was down-regulated in the over-expression group and the blank plasmid carrier group. In this study, MS2 vector system was introduced to construct the pMS2-cherry fusion protein, which can express the protein specifically binding to the stem ring structure. When the constructed NFKBIA mRNA 3'UTR plasmid with eight stem ring structures was expressed, it was fused with the red fluorescent protein. The results showed that hnRNP Q protein was diffusely distributed in the cytoplasm and nucleus without heat shock treatment, and co-localized with NFKBIA mRNA 3'UTR. It was possible that hnRNP Q participated in the process of exocytosis, maturation and intracellular localization of NFKBIA mRNA 3'UTR. After 0-2 h of recovery, hnRNP Q significantly formed granular bodies, and the granular bodies decreased at 4 h of recovery, and these granular bodies co-localized with NFKBIA mRNA 3'UTR. In the experimental group, NFKBIA mRNA 3'UTR co-localized with hDcP1a protein, the main component of P body, without heat shock stimulation, and returned to normal after 1 h of heat shock stimulation. The number of DcP1a granules increased significantly at 0 h after culture and co-localized with NFKBIA mRNA 3'UTR. The number of granules decreased gradually at 2 h after recovery.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363
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