【作者】 董愫；

【导师】 柳忠辉； 赵宇彤；

【作者基本信息】 吉林大学 ， 免疫学， 2014， 博士

【摘要】 研究背景Rac3是一种多功能的小GTP酶蛋白，可以调节细胞的粘附、迁移和分化。Rac3蛋白被认为是乳腺癌的致癌基因之一，然而该蛋白在食道癌中的表达以及其稳定性的调节方式尚未被研究。F-box蛋白是泛素蛋白酶水解系统中泛素连接Skp1-Cullin-1-F-box（SCF）E3的重要亚单位，参与蛋白质的泛素化和蛋白酶降解。前期研究中，我们发现F-box蛋白家族中的SCFFBXL19作用于Rho-GTP酶Rac1和RhoA，介导其泛素化和降解。本论文研究中，将探讨FBXL19调节Rac3的泛素化和稳定性。转化生长因子β1（TGFβ1）与食道癌的预后不良密切相关，它可以降低多种上皮源性肿瘤细胞中钙黏连蛋白（E-cadherin）的表达，进而导致肿瘤细胞的转移。本研究中，我们将探讨在食道癌细胞中，FBXL19通过介导Rac3蛋白的降解和泛素化进而抑制TGFβ1诱导的食道癌细胞钙黏连蛋白下调。实验方法本研究采用免疫印迹法和免疫共沉淀法测定F-box介导Rac3蛋白的稳定性；采用免疫印迹法和免疫染色法探讨转化生长因子β1（TGFβ1）介导食道癌细胞（OE19和OE33）中钙黏连蛋白（E-cadherin）的下调。实验结果1. FBXL19降低Rac3蛋白的表达量。（1）构建了FBXL19质粒（FBXL19-V5，FBXL19-HA），转染人胚肾细胞（HEK293），均可以导致HEK293细胞内源性Rac3蛋白表达下降；（2）检测不同细胞系中Rac3蛋白的表达量，食道癌细胞系（OE19）、人胚肾细胞系（HEK293）中Rac3蛋白高表达，非小细胞肺癌细胞系（A549）中Rac3蛋白低表达，鼠肺泡上皮细胞（MLE12）中Rac3蛋白未见表达；（3）构建Rac3质粒（Rac3-V5），将其与FBXL19质粒共同转染MLE12细胞，结果表明FBXL19降低Rac3蛋白的表达呈剂量依赖性；（4）RT-PCR证实在蛋白水平上FBXL19调节Rac3的稳定性，而非mRNA水平。2. FBXL19介导Rac3降解位于蛋白酶体系统。（1）饥饿状态可以导致Rac3蛋白呈时间依赖性降解，蛋白酶体抑制剂（MG-132）可以减少饥饿状态下Rac3蛋白的降解；（2）蛋白酶体抑制剂（MG-132）可以减少FBXL19介导的Rac3蛋白的降解，证实FBXL19介导Rac3降解位于蛋白酶体系统。3. Rac3蛋白赖氨酸166位点是FBXL19介导Rac3泛素化的靶位点。4. Rac3蛋白与FBXL19相互作用位于FBXL19的羧基端（COOH）。5. Rac3蛋白调节转化生长因子β1（TGFβ1）介导的钙黏连蛋白（E-cadherin）下调。（3）TGFβ1介导食道癌细胞系（OE19和OE33）中E-cadherin下调；（4）构建Rac3失活质粒（Rac3N17-V5）和敲除质粒（shRac3），转染食道癌细胞均可抑制TGFβ1介导的E-cadherin下调；（5）食道癌细胞中转染Rac3增加Snail的表达。6. FBXL19调节TGFβ1介导的E-cadherin下调。（1）FBXL19介导食道癌细胞系（OE19和OE33）内源性Rac3蛋白泛素化和降解；（2）FBXL19质粒转染食道癌细胞，阻抑肿瘤细胞TGFβ1介导的E-cadherin下调。结论1.发现并确认了FBXL19与Rac3蛋白间存在相互作用，FBXL19介导Rac3蛋白泛素化和降解；确定了FBXL19与Rac3的赖氨酸166位点结合，Rac3蛋白与FBXL19的COOH末端结合。2. Rac3蛋白是调节TGFβ1介导肿瘤细胞钙黏连蛋白下调的重要蛋白。3. FBXL19通过介导食道癌细胞Rac3蛋白的泛素化和降解，进而抑制肿瘤细胞TGFβ1介导的钙黏连蛋白（E-cadherin）下调。更多还原

【Abstract】 BackgroundRac3is a small GTPase multifunctional protein that regulates cell adhesion, migration,and differentiation. It has been considered as an oncogene in breast cancer; however, its rolein esophageal cancer and the regulation of its stability have not been studied. F-box proteinsare major subunits within the Skp1-Cullin-1-F-box (SCF) E3ubiquitin ligases that recognizeparticular substrates for ubiquitination and proteasomal degradation. Recently, we have shownthat SCFFBXL19targets Rac1and RhoA, thus regulating Rac1and RhoA ubiquitination anddegradation. Here, we demonstrate the role of FBXL19in the regulation of Rac3site-specificubiquitination and stability. Expression of TGFβ1is associated with poor prognosis ofesophageal cancer. TGFβ1reduces tumor suppressor, E-cadherin, expression in variousepithelial-derived cancers. Here we investigate the role of FBXL19-mediated Rac3degradation in TGFβ1-induced E-cadherin down-regulation in esophageal cancer cells.MethodsFBXL19-regulated endogenous and over-expressed Rac3stability were determined byimmunoblotting and co-immunoprecipitation. Esophageal cancer cells (OE19and OE33) wereused to investigate TGFβ1-induced E-cadherin down-regulation by Immunoblotting andImmunostaining.ResultsOverexpression of FBXL19decreased endogenous and over-expressed Rac3expressionby interacting and polyubiquitinating Rac3, while down-regulation of FBXL19suppressedRac3degradation. Lysine166within Rac3was identified as an ubiquitination acceptor site.The FBXL19variant with truncation at the N-terminus resulted in an increase in Rac3degradation; however, the FBXL19variant with truncation at the C-terminus lost its ability tointeract with Rac3and ubiquitinate Rac3protein. Further, we found that Rac3plays a criticalrole in TGFβ1-induced E-cadherin down-regulation in esophageal cancer cells. Over- expression of FBXL19attenuated TGFβ1-induced E-cadherin down-regulation andesophageal cancer cells elongation phenotype.ConclusionsCollectively these data unveil that FBXL19functions as an antagonist of Rac3byregulating its stability and regulates the TGFβ1-induced E-cadherin down-regulation. Thisstudy will provide a new potential therapeutic strategy to regulate TGFβ1signaling, thussuppressing esophageal tumorgenesis.更多还原