本文選題：KLF4　切入點(diǎn)：FBXO32　出處：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：鋅指轉(zhuǎn)錄因子KLF4主要參與細(xì)胞增殖、分化、凋亡和自我更新在內(nèi)的多種生理過程,在個(gè)體發(fā)育、應(yīng)答細(xì)胞內(nèi)外壓力和多種疾病的病理過程中決定著細(xì)胞的命運(yùn)。因此KLF4的調(diào)控機(jī)制就顯得尤為重要。作為一個(gè)與細(xì)胞周期和分化密切相關(guān)的基因,KLF4在轉(zhuǎn)錄、轉(zhuǎn)錄后和翻譯后水平都受到精密的調(diào)節(jié)。泛素化修飾是KLF4的翻譯后修飾十分重要的一個(gè)方面,KLF4的蛋白量在細(xì)胞內(nèi)受到泛素-蛋白酶體途徑的嚴(yán)密控制,然而影響KLF4泛素化的機(jī)制目前仍未十分明確。我們的工作即著眼于參與KLF4泛素化調(diào)節(jié)的E3連接酶系統(tǒng)。首先我們購買了E3泛素連接酶siRNA文庫,該文庫主要包括F-box家族、SOCS-box家族和Cullin家族等約200個(gè)基因的特異性siRNA。利用該文庫,我們?cè)谌橄侔┘?xì)胞中進(jìn)行了針對(duì)E3泛素連接酶相關(guān)基因的siRNA敲降,以Western blotting方法篩選敲降后會(huì)影響KLF4蛋白水平的E3連接酶。通過篩選文庫我們首先發(fā)現(xiàn)了17個(gè)敲降后導(dǎo)致KLF4蛋白水平上升的基因作為候選E3連接酶,然后我們從cDNA中通過PCR擴(kuò)增得到了五個(gè)候選E3連接酶基因,接著我們利用放線菌酮追蹤實(shí)驗(yàn)發(fā)現(xiàn)FBXO32可以顯著促進(jìn)外源KLF4蛋白的降解。于是我們選擇了FBXO32作為研究對(duì)象進(jìn)行了進(jìn)一步的研究工作,證明了FBXO32可以特異性泛素化KLF4并促進(jìn)KLF4通過泛素-蛋白酶體途徑降解。我們又對(duì)FBXO32泛素化KLF4的具體機(jī)制進(jìn)行了研究,發(fā)現(xiàn)FBXO32的羧基端能夠與KLF4的氨基端直接結(jié)合,并泛素化修飾KLF4的羧基末端,并且F-box結(jié)構(gòu)域是FBXO32促進(jìn)KLF4泛素化降解的關(guān)鍵結(jié)構(gòu)域。此外FBXO32介導(dǎo)的KLF4泛素化降解很有可能依賴于p38通路對(duì)KLF4的磷酸化。隨后,我們對(duì)FBXO32泛素化降解KLF4在乳腺癌發(fā)生發(fā)展中的作用進(jìn)行了初步的研究和探討。KLF4在腫瘤發(fā)生發(fā)展中的作用自該基因被發(fā)現(xiàn)以來一直是人們關(guān)注的熱點(diǎn)。但由于KLF4在細(xì)胞生命活動(dòng)中的廣泛參與——比如KLF4在抑制增殖的同時(shí)也具有抑制凋亡的特性,又如它在高分化的結(jié)腸隱窩上皮細(xì)胞中表達(dá),但又具有誘導(dǎo)成年人成纖維細(xì)胞恢復(fù)干性的能力——使得KLF4在腫瘤中的功能因組織、細(xì)胞遺傳背景和外界環(huán)境而異。由于細(xì)胞遺傳背景的異質(zhì)性,KLF4在乳腺癌中的功能更加撲朔迷離,KLF4促進(jìn)或者抑制乳腺癌的能力對(duì)基因表達(dá)背景和壓力刺激的依賴性更大。我們的工作發(fā)現(xiàn),在正常狀態(tài)下,FBXO32敲降導(dǎo)致的KLF4蛋白積累抑制了永生化乳腺上皮細(xì)胞的遷移能力,但在亞致死劑量DNA損傷的情況下,FBXO32過表達(dá)導(dǎo)致的KLF4降低則促進(jìn)了乳腺癌細(xì)胞的凋亡。我們的研究著眼于KLF4的翻譯后修飾,發(fā)現(xiàn)FBXO32能夠直接促進(jìn)KLF4的多聚泛素化,并以此促進(jìn)KLF4通過蛋白酶體途徑降解。FBXO32介導(dǎo)的KLF4降解調(diào)節(jié)了乳腺癌細(xì)胞的侵襲轉(zhuǎn)移能力和對(duì)DNA損傷類化療藥物的敏感性,為進(jìn)一步闡釋乳腺癌發(fā)生發(fā)展的機(jī)制提供了線索。
[Abstract]:Zinc finger transcription factor KLF4 is involved in many physiological processes, such as cell proliferation, differentiation, apoptosis and self-renewal. The regulation mechanism of KLF4 is especially important. As a gene closely related to cell cycle and differentiation, KLF4 is transcribed. Both post-transcriptional and post-translational levels are precisely regulated. Ubiquitin modification is an important aspect of KLF4 posttranslational modification. The protein content of KLF4 is closely controlled by the ubiquitin proteasome pathway in cells. However, the mechanism affecting the ubiquification of KLF4 is still unclear. Our work is focused on the E3 ligase system involved in the regulation of KLF4 ubiquification. First, we purchased the E3 ubiquitin ligase siRNA library. The library mainly includes about 200 genes, such as F-box family, SOCS-box family and Cullin family. Using this library, we carried out siRNA knockout targeting E3 ubiquitin ligase related genes in breast cancer cells. Western blotting method was used to screen E3 ligases which affect the level of KLF4 protein after knockdown. By screening library, we first found 17 genes that caused KLF4 protein level to rise after knock down as candidate E3 ligase. Then we amplified five candidate E3 ligase genes from cDNA by PCR. Then we found that FBXO32 could significantly promote the degradation of exogenous KLF4 protein by using actinomycin tracing experiment. So we chose FBXO32 as the research object for further research. It has been proved that FBXO32 can specifically urinate KLF4 and promote the degradation of KLF4 by ubiquitin proteasome pathway. We also studied the mechanism of FBXO32 ubiquitin KLF4. We found that the carboxyl terminal of FBXO32 can bind directly with the amino terminal of KLF4. The carboxyl terminal of KLF4 was modified by Ubiquidization, and the F-box domain was the key domain of FBXO32 to promote the degradation of KLF4 ubiquiation. In addition, the FBXO32 mediated KLF4 ubiquification degradation probably depended on the phosphorylation of KLF4 by p38 pathway. We have preliminarily studied and discussed the role of FBXO32 ubiquitin degrading KLF4 in the carcinogenesis and development of breast cancer. The role of KLF4 in the carcinogenesis and development of breast cancer has been a hot topic since the discovery of the gene. However, the role of KLF4 in the development of breast cancer has always been a hot topic. The extensive involvement of cell life-such as KLF4, which inhibits proliferation as well as apoptosis, It is also expressed in well-differentiated epithelial cells of the crypt of the colon, but has the ability to induce adult fibroblasts to return to dry-making the function of KLF4 in tumor tissues. Because of the heterogeneity of cell genetic background, the function of KLF4 in breast cancer is more uncertain. The ability of KLF4 to promote or inhibit breast cancer is dependent on gene expression background and stress stimulation. Bigger. Our work found that, Under normal conditions, the accumulation of KLF4 protein induced by FBXO32 knockout inhibits the migration of immortalized breast epithelial cells. However, the decrease of KLF4 induced by overexpression of FBXO32 in sublethal dose of DNA could promote the apoptosis of breast cancer cells. Our study focused on the post-translational modification of KLF4 and found that FBXO32 can directly promote the polyubiquitin of KLF4. Thus, the degradation of KLF4 mediated by KLF4 via proteasome pathway regulated the ability of invasion and metastasis of breast cancer cells and the sensitivity to DNA damaging chemotherapeutic drugs, which provided clues for further elucidation of the mechanism of breast cancer occurrence and development.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 Chia-Hsin Chan;;Novel roles of Skp2 E3 ligase in cellular senescence,cancer progression,and metastasis[J];癌癥;2012年04期

2 ;Down-regulation of gut-enriched Kr，

本文編號(hào)：1659254