KLF4的泛素化调节在乳腺癌发生发展中的作用研究

发布时间：2018-03-24 17:14

本文选题：KLF4　切入点：FBXO32　出处：《北京协和医学院》2015年博士论文

【摘要】：锌指转录因子KLF4主要参与细胞增殖、分化、凋亡和自我更新在内的多种生理过程,在个体发育、应答细胞内外压力和多种疾病的病理过程中决定着细胞的命运。因此KLF4的调控机制就显得尤为重要。作为一个与细胞周期和分化密切相关的基因,KLF4在转录、转录后和翻译后水平都受到精密的调节。泛素化修饰是KLF4的翻译后修饰十分重要的一个方面,KLF4的蛋白量在细胞内受到泛素-蛋白酶体途径的严密控制,然而影响KLF4泛素化的机制目前仍未十分明确。我们的工作即着眼于参与KLF4泛素化调节的E3连接酶系统。首先我们购买了E3泛素连接酶siRNA文库,该文库主要包括F-box家族、SOCS-box家族和Cullin家族等约200个基因的特异性siRNA。利用该文库,我们在乳腺癌细胞中进行了针对E3泛素连接酶相关基因的siRNA敲降,以Western blotting方法筛选敲降后会影响KLF4蛋白水平的E3连接酶。通过筛选文库我们首先发现了17个敲降后导致KLF4蛋白水平上升的基因作为候选E3连接酶,然后我们从cDNA中通过PCR扩增得到了五个候选E3连接酶基因,接着我们利用放线菌酮追踪实验发现FBXO32可以显著促进外源KLF4蛋白的降解。于是我们选择了FBXO32作为研究对象进行了进一步的研究工作,证明了FBXO32可以特异性泛素化KLF4并促进KLF4通过泛素-蛋白酶体途径降解。我们又对FBXO32泛素化KLF4的具体机制进行了研究,发现FBXO32的羧基端能够与KLF4的氨基端直接结合,并泛素化修饰KLF4的羧基末端,并且F-box结构域是FBXO32促进KLF4泛素化降解的关键结构域。此外FBXO32介导的KLF4泛素化降解很有可能依赖于p38通路对KLF4的磷酸化。随后,我们对FBXO32泛素化降解KLF4在乳腺癌发生发展中的作用进行了初步的研究和探讨。KLF4在肿瘤发生发展中的作用自该基因被发现以来一直是人们关注的热点。但由于KLF4在细胞生命活动中的广泛参与——比如KLF4在抑制增殖的同时也具有抑制凋亡的特性,又如它在高分化的结肠隐窝上皮细胞中表达,但又具有诱导成年人成纤维细胞恢复干性的能力——使得KLF4在肿瘤中的功能因组织、细胞遗传背景和外界环境而异。由于细胞遗传背景的异质性,KLF4在乳腺癌中的功能更加扑朔迷离,KLF4促进或者抑制乳腺癌的能力对基因表达背景和压力刺激的依赖性更大。我们的工作发现,在正常状态下,FBXO32敲降导致的KLF4蛋白积累抑制了永生化乳腺上皮细胞的迁移能力,但在亚致死剂量DNA损伤的情况下,FBXO32过表达导致的KLF4降低则促进了乳腺癌细胞的凋亡。我们的研究着眼于KLF4的翻译后修饰,发现FBXO32能够直接促进KLF4的多聚泛素化,并以此促进KLF4通过蛋白酶体途径降解。FBXO32介导的KLF4降解调节了乳腺癌细胞的侵袭转移能力和对DNA损伤类化疗药物的敏感性,为进一步阐释乳腺癌发生发展的机制提供了线索。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

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