本文選題：KLF4 + 熱休克反應��； 參考：《中南大學》2006年博士論文

【摘要】： 熱休克反應(heat shock response，HSR)是機體中普遍存在的一種應激反應。在熱休克反應中，細胞內(nèi)一組熱休克蛋白(heat shock proteins，HSPs)誘導表達增加。HSPs作為重要的分子伴侶(molecular chaperone)，具有幫助蛋白質(zhì)的折疊與移位，防止蛋白質(zhì)聚集，幫助變性蛋白質(zhì)的解聚及復性，促進嚴重受損蛋白質(zhì)的降解等功能。熱休克時HSPs的誘導表達主要在轉錄水平受熱休克因子1(heat shock factor 1，HSF1)的調(diào)控。在熱休克反應中，HSF1被活化并從胞漿移位到細胞核，從轉錄水平激活HSPs的表達。除HSPs外，HSF1還能直接調(diào)控多種其它基因的表達。此外，，采用HSF1基因敲除小鼠研究還發(fā)現(xiàn)HSF1在非活化狀態(tài)下能夠調(diào)控HSPs的組成型表達，但其機制目前尚不清楚。 Kruppel樣因子4(Kruppel-like factor 4，KLF4)是Sp1／KLF鋅指轉錄因子家族的成員之一。作為一個核轉錄因子，它能夠與下游基因啟動子區(qū)的GC盒，CACCC盒和基礎轉錄元件(basic transcription elements)三種結合元件相結合，從而直接調(diào)控這些基因的表達。通過對下游基因表達的調(diào)控，KLF4在正常細胞的增殖和分化、胚胎發(fā)育以及腫瘤的發(fā)生發(fā)展過程中起著重要的作用。但是，KLF4與熱休克反應、HSF1及HSPs之間有何關系，至今尚未見報道。 為了探討KLF4與熱休克反應之間的關系，本研究第一部分采用RT-PCR和Western blot分別檢測了KLF4 mRNA和蛋白質(zhì)在熱休克反應中的表達情況，結果發(fā)現(xiàn)熱休克導致整體小鼠的多種組織以及多種離體培養(yǎng)細胞中KLF4的表達顯著增加。根據(jù)上述結果，我們首次提出：KLF4可能是一個熱休克反應相關基因。由于熱休克反應中多個基因的表達都受到HSF1的調(diào)控，我們進一步采用HSF1基因敲除小鼠探討了HSF1對KLF4 mRNA表達的可能影響，結果發(fā)現(xiàn)HSF1既可以促進KLF4的基礎表達，又可以促進其在熱休克時的誘導表達。此外，我們還在KLF4基因啟動子區(qū)找到了HSF1結合元件(heat shock element，HSE)。由此可見，KLF4是HSF1調(diào)控的下游基因。 HSPs是熱休克反應中受HSF1直接調(diào)控的重要功能蛋白。作為熱休克反應中表達增加的轉錄因子，KLF4對HSPs基因表達的可能影響引起了我們的關注。以前的研究發(fā)現(xiàn)，Sp1／KLF家族的某些成員既可以調(diào)控HSPs的組成型表達，又可以調(diào)控其在熱休克時的誘導表達。因此，本研究第二部分首先構建了KLF4過表達細胞株，并采用反義寡核苷酸技術建立了內(nèi)源性KLF4表達抑制的模型；接著分別采用RT-PCR和Western blot在mRNA和蛋白質(zhì)水平檢測了KLF4過表達和內(nèi)源性KLF4表達抑制對小分子HSPs家族的αB晶狀體蛋白和HSP25，HSP70家族的HSP72和HSP73以及HSP90家族的HSP84和HSP86的組成型表達和誘導表達的影響。結果發(fā)現(xiàn)，KLF4過表達能夠上調(diào)上述HSPs的組成型表達，而KLF4表達抑制則下調(diào)上述HSPs的組成型表達；但KLF4過表達或表達抑制均不影響上述HSPs的誘導表達。為了進一步闡明KLF4調(diào)控α晶狀體蛋白、HSP25和HSP84組成型表達的機制，本研究采用EMSA和熒光素酶報告基因方法進行了深入探討。結果發(fā)現(xiàn)，KLF4能夠通過其DNA結合結構域與α晶狀體蛋白、HSP25和HSP84基因啟動子區(qū)的KLF4結合元件相結合而直接促進其轉錄，從而維持其組成型表達。為了更全面地了解KLF4能夠調(diào)節(jié)哪些HSPs的組成型表達，我們進一步采用cDNA芯片檢測了KLF4過表達對HSPs基因表達的影響，結果發(fā)現(xiàn)有9個HSPs的組成型表達可能受到KLF4的調(diào)控。此外，本研究還發(fā)現(xiàn)熱休克反應中KLF4與HSPs基因啟動子的結合顯著減少，這可能是熱休克時KLF4對HSPs失去調(diào)控作用的主要原因。 綜上所述，本研究首次發(fā)現(xiàn)KLF4是一個受HSF1調(diào)控的熱休克反應相關基因。KLF4的組成型表達及熱休克導致的誘導表達均受到HSF1的調(diào)控。KLF4通過其DNA結合結構域與αB晶狀體蛋白、HSP25及HSP84等基因啟動子區(qū)的KLF4結合元件相結合而直接調(diào)控上述HSPs的組成型表達，即KLF4能夠介導HSF1對HSPs組成型表達的促進作用。而熱休克時，由于KLF4與HSPs基因啟動子的結合減少而導致其失去對HSPs的調(diào)控作用。上述研究揭示了KLF4在HSPs表達調(diào)控方面的新功能，完善了HSPs的表達調(diào)控機制。有關KLF4在生理狀態(tài)下及熱休克反應和其它應激反應中的功能和生物學意義值得進一步深入研究。
[Abstract]:Heat shock response (HSR) is a common stress response in the body. In the heat shock response, a group of heat shock proteins (heat shock proteins, HSPs) can induce the expression of.HSPs as an important molecular chaperone (molecular chaperone), which helps the folding and displacement of protein and prevents protein aggregation. Help the depolymerization and renaturation of denatured proteins and promote the degradation of severely damaged proteins. The induction of HSPs in heat shock is mainly regulated at the transcriptional level of heat shock factor 1 (heat shock factor 1, HSF1). In the heat shock response, HSF1 is activated and moved from the cytoplasm to the nucleus and activates the expression of HSPs from the transcriptional level. Except HS, the expression of HSPs is activated. HSF1 can also directly regulate the expression of a variety of other genes. In addition, the study of HSF1 knockout mice also found that HSF1 can regulate the expression of HSPs in the non activated state, but its mechanism is not yet clear.
Kruppel like factor 4 (Kruppel-like factor 4, KLF4) is one of the members of the Sp1 / KLF zinc finger transcription factor family. As a nuclear transcription factor, it can be combined with the three binding elements of the GC box, CACCC box and the basic transcriptional element (basic transcription elements) in the promoter region of the downstream gene, which directly regulates the expression of these genes. By regulating the expression of the downstream genes, KLF4 plays an important role in the proliferation and differentiation of normal cells, the development of embryo and the development of tumor. However, the relationship between KLF4 and heat shock response and the relationship between HSF1 and HSPs has not yet been reported.
In order to investigate the relationship between KLF4 and heat shock response, the first part of this study used RT-PCR and Western blot to detect the expression of KLF4 mRNA and protein in the heat shock response. The results showed that the heat shock resulted in a significant increase in the expression of KLF4 in various tissues of the whole mice and in a variety of isolated cultured cells. For the first time, we suggest that KLF4 may be a related gene for heat shock response. Because the expression of multiple genes in the heat shock response is regulated by HSF1, we further use HSF1 knockout mice to explore the possible influence of HSF1 on the expression of KLF4 mRNA. The results show that HSF1 can promote both the basic expression of KLF4 and the promotion of the expression of the KLF4. In addition, we also found the HSF1 binding element (heat shock element, HSE) in the promoter region of the KLF4 gene. Thus, KLF4 is a downstream gene for HSF1 regulation.
HSPs is an important functional protein regulated by HSF1 in the heat shock response. As a transcriptional factor expressed in the heat shock response, the possible influence of KLF4 on the expression of HSPs gene has aroused our attention. Previous studies have found that some members of the Sp1 / KLF family can regulate the composition of HSPs and regulate their thermal rest in the heat shock. Therefore, the second part of this study first constructed the KLF4 overexpressed cell lines, and established a model of endogenous KLF4 expression inhibition by antisense oligonucleotides, and then used RT-PCR and Western blot to detect KLF4 overexpression and endogenous KLF4 expression inhibition to small molecule HSPs at mRNA and protein levels, respectively. The family's alpha B crystallin and HSP25, the HSP72 and HSP73 of the HSP70 family, and the expression and induction of HSP84 and HSP86 in the HSP90 family. The results showed that the overexpression of KLF4 overexpressed the expression of the above-mentioned HSPs, while the inhibition of KLF4 expression downregulated the expression of the above-mentioned HSPs. In order to further elucidate the mechanism of KLF4 regulation of the expression of alpha crystallin, HSP25 and HSP84, this study uses EMSA and luciferase reporter gene method to further explore the mechanism of the expression of alpha crystallin, HSP25 and HSP84. The results show that KLF4 can be used in the promoter region of the alpha crystallin, HSP25 and HSP84 genes through its DNA binding domain. In order to understand the composition expression of which HSPs can be regulated by KLF4, we further use cDNA chip to detect the effect of KLF4 overexpression on the expression of HSPs gene, and we found that the expression of 9 HSPs components may be regulated by KLF4 in order to understand more fully the expression of which HSPs expression can be regulated in a more comprehensive way. In addition, this study also found that the combination of KLF4 and HSPs gene promoter in the heat shock response decreased significantly, which may be the main cause of the loss of KLF4 on HSPs during heat shock.
To sum up, this study for the first time found that KLF4 is a HSF1 regulated heat shock response related gene.KLF4 and the induced expression of heat shock induced by HSF1 is regulated by.KLF4 through its DNA binding domain and the binding of KLF4 binding elements in the promoter region of the alpha B lens protein, HSP25 and HSP84. The component expression of the above HSPs, that is, KLF4 can mediate the promotion of HSF1 on the expression of HSPs, and the decrease in the binding of KLF4 and HSPs gene promoters to the regulation of HSPs. These studies reveal the new functions of KLF4 in the regulation of HSPs expression, and improve the mechanism of the expression and regulation of HSPs. The functional and biological significance of LF4 in physiological state, heat shock response and other stress responses deserves further study.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2006
【分類號】：R363

【參考文獻】

相關期刊論文 前2條

1 劉瑛,袁燦,張華莉,王秋鵬,肖獻忠;從基因敲除小鼠心肌組織中篩選熱休克反應中受HSF1調(diào)控的靶基因[J];中南大學學報(醫(yī)學版);2004年01期

2 袁開宇,趙震宇,劉瑛,鄒江,劉梅冬,陳廣文,尢家

本文編號：2022079