熱休克刺激誘導NFKBIA基因表達的轉錄后調控機制研究
發(fā)布時間：2018-09-07 09:03
【摘要】：研究背景1962年,Ferruccio Ritossa觀察到基礎體溫為25 ℃的果蠅(Drosophila)暴露在37℃高溫時,其唾液腺可合成一組相應的蛋白質。經(jīng)過深入研究,發(fā)現(xiàn)該組蛋白質對細胞功能和內環(huán)境的穩(wěn)定具有廣泛的保護作用,因而將這些蛋白質命名為熱休克蛋白(heat shock protein,HSP),而將果蠅處于高溫時的基因表達和生理保護這一現(xiàn)象稱為熱休克反應(heat shock response,HSR)。熱休克反應,這一所有應激反應中最原始和保守的反應,可誘導合成HSP,作為“分子伴侶”維持蛋白質的正確折疊狀態(tài),保護細胞抵御包括熱休克、缺氧、缺血-再灌注損傷、內毒素、重金屬和乙醇的毒性刺激等一系列的應激損傷,增強機體細胞對損害的耐受程度,維持細胞的正常功能代謝,提高細胞生存率,具有強大的保護作用。在應激反應中HSP基因首先表達,而其他基因的表達則暫時被抑制。在高溫、亞砷酸鈉、前列腺素-A1誘導的HSR中,HSP的表達可抑制促炎癥因子的表達,其主要作用是通過抑制NF-κκB的激活來防止過度炎癥反應對細胞的損傷。HSP抑制NF-κB的可能途徑為:抑制IκBα的磷酸化和IKK的活性;增強IκBαmRNA的表達;競爭性抑制NF-κB經(jīng)核孔復合體進入細胞核。有研究證實,在轉錄水平,IκBα的啟動子內含有熱休克元件(heat shock element, HSE),HSP基因轉錄的同時也有NFKBIA基因的轉錄,近年來又發(fā)現(xiàn)HSR在轉錄后水平也誘導NFKBIA基因的表達,本研究的創(chuàng)新點是探討熱休克過程中轉錄后水平誘導NFKBIA基因表達的機制。NF-κB是1986年首先發(fā)現(xiàn)于B細胞Igκ輕鏈轉錄調控中而得名,是將信號從胞漿傳至胞核引起相應基因表達的一種誘導型、高效真核細胞核轉錄因子,對許多免疫調節(jié)基因的表達具有重要的調節(jié)功能,其中最重要的是調節(jié)細胞因子、粘附分子和其他免疫介質的基因轉錄。NF-κκB的激活主要使前炎癥介質表達增加,導致急性炎癥反應,而促炎因子如TNF-α等又可以激活NF-κB,因此在炎癥反應過程中,有時很難區(qū)分兩者的因果關系,它們之間的相互作用導致了炎癥反應的擴大。NF-κB是由Rel蛋白家族的成員以同源或異源二聚體的形式組成的一組轉錄因子,一般意義上的NF-κB通常是指p50/p65二聚體。靜息狀態(tài)下,NF-κB與κB抑制因子(IκB)結合存在于細胞胞漿內,處于無活性狀態(tài)。在κB家族中IκBα是目前發(fā)現(xiàn)最早和研究最清楚的成員。當細胞遇到刺激因子TNF-α、IL-18、內毒素時,NF-κB誘導激酶(NIK)激活IκB激酶復合物(IKK-1、2),IKK-1、2使IκBα在其32、36位點上的絲氨酸磷酸化、泛素化,然后由蛋白酶體降解使NF-κB游離進入細胞核,在核內NF-κB與基因上的κB序列結合,激活下游的靶基因轉錄,由于IκBα基因在其轉錄啟動區(qū)含有3個NF-κB結合位點,因此當NF-κB激活的同時誘導IκBα的大量表達,這一途徑構成了 NF-κB激活的負反饋途徑。生物體是一個有機的統(tǒng)一整體,各種基因根據(jù)機體生長、發(fā)育、繁殖的需要,隨著環(huán)境的變化,有規(guī)律的、程序性地表達,以適應環(huán)境,發(fā)揮其正常的生理功能。基因表達調控是在多級水平上進行的:基因活化、轉錄、轉錄后加工、翻譯、翻譯后加工等。基因轉錄起始的調控是大多數(shù)基因的主要調控形式,但是其他的調控能夠在DNA到蛋白質途徑時發(fā)揮作用,以調節(jié)所產(chǎn)生的基因產(chǎn)物數(shù)量,進一步保證了體內基因調控準確和精確的發(fā)生。基因表達的調控是一個復雜而有序的過程,包括有多級水平的調控。越來越多的研究表明,基因表達轉錄后水平的調控起著不可忽視的作用,主要通過影響mRNA穩(wěn)定性及翻譯效率來發(fā)揮作用�；虮磉_的轉錄后調控一般指在RNA聚合酶結合到基因啟動子后,對轉錄產(chǎn)物進行的一系列修飾,主要包括:轉錄的提前終止、剪接、mRNA通過核孔和胞質內定位、RNA編輯、翻譯起始和翻譯效率,mRNA的穩(wěn)定性等多個環(huán)節(jié)。研究發(fā)現(xiàn)轉錄本中調控mRNA穩(wěn)定性等的元件存在于3'非翻譯區(qū)(3'UTR),大部分轉錄后調控機制的靶向部位都位于轉錄本mRNA的3'UTR。現(xiàn)已發(fā)現(xiàn)約5-8%的人類基因編碼的mRNA其3'UTR含有ARE序列,ARE序列是一段長度約為50-150nt富含腺嘿呤(adenine)和尿嘧啶(uridine)的序列,與mRNA的穩(wěn)定性有密切關系。ARE序列能夠招募許多ARE結合蛋白(ARE-binding protein,ARE-BP),這些ARE-BP相互協(xié)作或競爭地與mRNA的ARE結構域結合,共同調節(jié)mRNA的穩(wěn)定性和翻譯,NFKBIA基因編碼的mRNA中也存在著ARE序列。利用 Streptavidin-biotin進行RNA-pulldown得到與NFKBIA mRNA 3'UTR相互作用的蛋白復合物,經(jīng)SDS-PAGE凝膠電泳分離、銀染分析和蛋白質譜鑒定后,得到熱休克過程中與NFKBIA基因的3'UTR相互作用蛋白質hnRNP Q。hnRNP Q是不均一性核糖核蛋白(heterogeneous nuclear ribonucleo-protein, hnRNP)家族的成員之一,主要定位于細胞核內,可與新合成的不均一性RNA (heterogeneous nuclear RNA, hnRNA)相結合,從而調控 mRNA前體(pre-mRNA)的剪接、mRNA核質轉運、翻譯和降解等一系列重要過程,是一種重要的RNA結合蛋白,本研究將進一步探討其在熱休克過程中參與NFKBIA mRNA 3'UTR功能調節(jié)的機制。研究目的本課題主要探討熱休克刺激誘導NFKBIA基因表達的轉錄后調控機制,重點探討NFKBIA mRNA 3'UTR結合蛋白hnRNP Q在熱休克刺激誘導NFKBIA基因表達的轉錄后調控中的作用及機制。研究內容1.采用蛋白免疫印跡技術檢測熱休克刺激后IκBα蛋白表達及半衰期改變;2.采用熒光定量PCR技術檢測熱休克刺激后NFKBIA mRNA表達及半衰期改變;3.構建NFKBIA mRNA 3'UTR雙熒光素酶報告基因質粒,利用雙熒光素酶報告基因系統(tǒng)研究NFKBIA mRNA 3 'UTR在熱休克過程中的作用;4.利用Streptavidin-biotin進行RNA-pulldown,篩選與NFKBIAmRNA3'UTR相互作用的蛋白復合物,并經(jīng)過SDS-PAGE凝膠電泳分離、銀染分析和蛋白質譜鑒定與NFKBIA mRNA 3'UTR相互作用蛋白及驗證;5.熱休克過程中hnRNP Q參與調節(jié)NFKBIA mRNA 3'UTR功能及其機制。研究方法采用熒光定量PCR檢測mRNA的相對表達量,蛋白免疫印跡技術檢測蛋白表達,免疫熒光觀察蛋白及mRNA共定位,雙熒光素酶報告基因系統(tǒng)間接反映NFKBIA mRNA 3'UTR功能。統(tǒng)計分析軟件為SPSS 13.0,首先采用Levene進行方差齊性檢驗,文中樣本方差均齊,則采用單因素方差分析(One-way ANOVA),若總體而言各組間有差異,則進一步進行多重比較,以α=0.05為檢驗水準,作圖軟件為OriginPr08.0。通過以上實驗研究,得到以下結果:1.正常對照組RAW細胞IκBα蛋白質半衰期大約為3 h,給予熱休克刺激后其半衰期延長至約4 h,正常對照組NFKBIA mRNA的半衰期約為40 min,給予熱休克刺激后其半衰期明顯延長至約85 min。同時檢測了熱休克后細胞恢復正常培養(yǎng)的不同時間點NFKBIA mRNA及蛋白質表達量,,發(fā)現(xiàn)NFKBIA mRNA的表達量與對照組相比,在恢復培養(yǎng)240 min前的各個時間點變化不明顯,而NFKBIA蛋白的表達量呈現(xiàn)先減少后增加再減少的趨勢,通過以上數(shù)據(jù)不難發(fā)現(xiàn),RAW細胞給予43℃C熱休克處理1 h后,其NFKBIA mRNA及蛋白質表達量呈現(xiàn)不一致的變化,說明存在轉錄后調控機制。2.將NFKBIA mRNA 3'UTR構建在熒光素酶報告基因(簡稱pLuNB )之后,通過熒光素酶報告基因的檢測來間接反映熱休克誘導的NFKBIA mRNA 3'UTR的作用。結果顯示,將pLuNB轉染NIH 3T3細胞48 h后給予熱休克刺激(43℃1 h),其報告基因檢測值與對照組相比在各時間點均升高,說明熱休克過程中NFKBIA mRNA 3'UTR參與了 NFKBIA基因轉錄后調控。3.通過 Streptavidin-biotin 進行 RNA-pull down 得到與 NFKBIA mRNA 3'UTR相互作用的蛋白復合物,并進行SDS-PAGE凝膠電泳分離、蛋白銀染、蛋白質譜鑒定、生物學分析,確定了本研究中熱休克誘導后與NFKBIA mRNA 3'UTR相互作用蛋白hnRNP Q的存在。通過Biotin-RNA pull down證明了 hnRNP Q確實可以與NFKBIA mRNA 3'UTR特異性結合,并進一步證實,當過表達及干擾hnRNPQ后,IκBα蛋白表達分別出現(xiàn)上調及下調現(xiàn)象,初步證實hnRNPQ參與了 NFKBIA基因的轉錄后調控。4.當共轉染hnRNP Q1及pLuNB 48 h后給予熱休克刺激43℃C 1 h,恢復至正常培養(yǎng)條件,分別在0、1、2、3 h收細胞進行熒光素酶報告基因檢測。與對照組相比,恢復正常培養(yǎng)后的各個時間點報告基因數(shù)值均降低。當共轉染hnRNPQ與NFKBIA特異性siRNA(SY23)48 h后,qPCR檢測其干擾效率約為47%,并給予熱休克處理后,在以上各個時間點收細胞進行熒光素酶報告基因檢測,其數(shù)值與對照組相比均升高。以上數(shù)據(jù)表明,hnRNP Q1參與了熱休克誘導的NFKBIA基因表達的轉錄后調控。5.NIH 3T3細胞過表達hnRNP Q1及hnRNP Q2 24 h后給予熱休克刺激,與不刺激對照組同時檢測NFKBIA mRNA表達量及IκBα蛋白表達變化,結果顯示過表達組與空質粒載體組熱休克處理前后NFKBIA mRNA的表達量變化均不明顯,但對IκBα蛋白表達有下調作用。6.為進一步揭示hnRNP Q在熱休克誘導的NFKBIA基因轉錄后水平調控中的作用,本研究引入了 MS2 vector系統(tǒng),構建了 pMS2-cherry融合表達蛋白,其可以表達特異性與莖環(huán)結構結合的蛋白質,當構建的帶有8個莖環(huán)結構的NFKBIA mRNA 3'UTR質粒表達后,與紅色熒光融合蛋白相互結合來間接反映NFKBIA mRNA 3'UTR 細胞內定位。結果顯示在未給予熱休克處理時hnRNP Q蛋白彌散分布于胞漿與細胞核內,與NFKBIA mRNA 3'UTR有明顯共定位,可能hnRNP Q參與NFKBIA mRNA 3'UTR出核、成熟及細胞內定位等過程;當受到熱休克刺激之后,恢復0-2 h漿中hnRNP Q明顯形成顆粒狀小體,恢復4 h顆粒有所減少,且這些顆粒小體與NFKBIA mRNA 3'UTR存在共定位;實驗組NFKBIA mRNA 3'UTR在未受到熱休克刺激時,與P小體(P body)主要組成成分hDcP1a蛋白存在明顯的共定位,熱休克刺激1h,恢復正常培養(yǎng)0h DcP1a顆粒明顯增多,且與NFKBIA mRNA 3'UTR有共定位,恢復2 h粒逐漸減少;實驗組G3Bp1蛋白在未受到熱休克刺激時,在胞漿中成彌散性分布,與NFKBIA mRNA 3'UTR無明顯共定位,當給予熱休克刺激1h后,恢復正常培養(yǎng)2hG3Bp1蛋白分布于包漿并形成明顯的顆粒狀小體,分布于胞漿,且與NFKBIA mRNA 3'UTR存在共定位,恢復4 顆粒開始減少,作為上述實驗的對照組在各時間點均未觀察到p8MS2空質粒組mRNA與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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R363
本文編號：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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R363
【參考文獻】
相關期刊論文 前7條
1 楊建營;徐翔峰;向珍蛹;;NF-κB的研究進展[J];淮海醫(yī)藥;2011年01期
2 向靜;王寧;阿布力克木·吐爾地;;核因子-κB信號通路的研究進展[J];新疆醫(yī)科大學學報;2011年01期
3 焦義祖;;細胞因子mRNA轉錄后調控的研究進展[J];生命科學;2010年05期
4 耿東升;;NF-κB及其致炎作用和化學抑制劑[J];解放軍藥學學報;2009年03期
5 陳淑華;真核生物mRNA3′非翻譯區(qū)的調控功能[J];國外醫(yī)學(生理、病理科學與臨床分冊);2003年06期
6 江元清,凌毅,趙武玲;真核mRNA的3′非翻譯區(qū)轉錄后水平調控作用研究進展[J];植物學通報;2001年01期
7 杜光偉,周嚴,袁建剛,強伯勤;RRM RNA結合蛋白的結構與功能[J];生物化學與生物物理進展;1999年04期
，本文編號：2227811
本文鏈接：http://sikaile.net/xiyixuelunwen/2227811.html
最近更新
教材專著
