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类型 基础研究 预答辩日期 2017-11-29
开始(开题)日期 2015-12-28 论文结束日期 2017-05-31
地点 丁家桥校区基一楼103会议室 论文选题来源 国家自然科学基金项目     论文字数 5.4 (万字)
题目 钙调蛋白调节人乳腺癌细胞内核浆癌蛋白TBC1D3的稳定性及生物学意义
主题词 前列腺癌基因17,钙调蛋白,泛素-蛋白酶体途径,基质金属蛋白酶9,迁移
摘要 目的: TBC1D3又称PRC17(前列腺癌基因17),是人科动物特有的癌基因,高表达于多种肿瘤细胞,如前列腺癌、膀胱癌、乳腺癌和骨髓增生异常综合征等。TBC1D3的表达能使正常成纤维细胞NIH 3T3发生恶性转化在裸鼠体内形成肿瘤,也能通过延迟EGFR和IRS-1的降解增强GFs信号,促进细胞的增殖和肿瘤的发生。GFs信号负反馈调节TBC1D3的稳定性,在E3泛素连接酶CUL7的介导下泛素化和降解TBC1D3,使得TBC1D3的蛋白水平维持动态平衡。钙调蛋白(CaM)参与众多蛋白的稳定性调节,如雌激素受体?(ER-?)和TRE17/USP6等,我们的预实验显示CaM通过抑制TBC1D3的降解提高其稳定性。因此,探讨CaM调节TBC1D3稳定性的机制,将有助于阐明CaM和TBC1D3在肿瘤发生发展中的作用,为肿瘤的诊断和防治提供新线索。 方法: 1.为了研究CaM对TBC1D3降解的调节作用,我们通过转染GST-CaM使CaM高表达或使用CaM抑制剂W7抑制内源性CaM功能,应用Western blot和免疫共沉淀实验分别观察MCF-7和BT-549细胞内CaM对TBC1D3降解和泛素化的调节作用。通过抽提细胞浆、细胞核蛋白,观察TBC1D3降解的亚细胞部位及CaM的调节作用。 2.为了探讨CaM抑制TBC1D3降解的机制是否通过相互作用,以GST载体为对照,将GST-CaM和Flag-TBC1D3复合转染MCF-7细胞中进行表达,用抗GST抗体进行免疫共沉淀,检测CaM与TBC1D3之间的相互作用;以Flag载体为对照,将Flag-TBC1D3和GST-CaM复合转染MCF-7细胞中进行表达,用抗Flag抗体进行免疫共沉淀,反向检测CaM与TBC1D3之间的相互作用;使用Ca2+螯合剂EGTA处理,通过免疫共沉淀分别观察MCF-7和BT-549细胞内CaM与TBC1D3的相互作用;over-lap PCR构建TBC1D3内在缺失突变体TBC1D3(Δ157-171)和TBC1D3(Δ303-312),以Flag为对照,分别将GST-CaM和Flag-TBC1D3及其突变体复合转染MCF-7细胞,抗Flag抗体做免疫共沉淀检测它们与CaM的相互作用,以分析确定TBC1D3氨基酸序列中与CaM相互作用的功能部位;以GST载体为对照,Flag-TBC1D3及其突变体与GST-CaM分别复合转染转染MCF-7细胞,抗GST抗体做免疫共沉淀,反向验证TBC1D3氨基酸序列中与CaM相互作用的功能部位。 3.为了分析TBC1D3内与CaM结合部位的功能,用over-lap PCR构建TBC1D3内点突变体Flag-TBC1D3(Y163A-T165A)和Flag-TBC1D3(K166R)。分别转染Flag-TBC1D3、Flag-TBC1D3(Δ157-171)及点突变体,通过Western Blot和CO-IP检测TBC1D3及点突变体的降解水平和泛素化水平;以Flag为对照,将Flag-TBC1D3及点突变体分别和GST-CaM复合转染MCF-7细胞,抗Flag抗体做免疫共沉淀检测它们与CaM的相互作用。 4. 为了检测TBC1D3的稳定性对细胞迁移的作用,以Flag空载体为对照,和Flag-TBC1D3分别转染MCF-7和BT-549细胞,进行划痕和Transwell迁移实验。为了探讨TBC1D3影响细胞迁移的机制,Flag空载体和Flag-TBC1D3分别转染MCF-7细胞,无血清饥饿24h后,明胶酶谱法检测培养液内MMP-9活性;实时荧光定量PCR和Western blot分别检测细胞裂解物内MMP-9的mRNA和蛋白水平。复合转染GST-CaM,观察CaM对TBC1D3稳定性的调节作用对细胞迁移、MMP-9活性和蛋白水平的影响。 结果: 1.Western blot结果表明10%FCS可诱导TBC1D3降解,复合转染CaM并高表达时,10%FCS诱导的TBC1D3降解受到明显抑制,说明CaM能够抑制TBC1D3的降解;使用W7抑制内源性CaM功能后,TBC1D3的降解趋势更加明显,说明内源性CaM在TBC1D3降解中的抑制作用;免疫共沉淀实验显示10%FCS可有效诱导TBC1D3的泛素化,CaM的表达可大大降低TBC1D3的泛素化水平,说明CaM可以降低TBC1D3的泛素化水平;细胞浆、细胞核蛋白抽提实验显示在MCF-7细胞中,10%FCS刺激诱导TBC1D3降解既发生于细胞浆、也发生于细胞核且细胞核内降解趋势更加明显;复合转染CaM并高表达时,TBC1D3细胞浆、细胞核内降解受到明显的抑制,均大大减少。在BT-549细胞中,10%FCS刺激诱导的TBC1D3降解在细胞浆、细胞核内均被检测到且细胞浆内降解趋势更加明显;复合转染CaM并高表达时,TBC1D3细胞浆、细胞核内降解均受到明显的抑制。 2.用抗GST抗体进行免疫共沉淀,结果显示GST对照组无TBC1D3结合条带,GST-CaM组检测到TBC1D3结合条带,说明TBC1D3与CaM存在相互作用;以抗Flag抗体做反向免疫共沉淀,结果显示Flag对照组无CaM结合条带,Flag-TBC1D3组检测到CaM结合条带,说明CaM与TBC1D3存在相互作用;使用EGTA螯合Ca2+后,以抗Flag抗体或抗GST抗体做免疫共沉淀,均未检测到TBC1D3与CaM的相互作用,提示CaM与TBC1D3的相互作用依赖于Ca2+环境。Over-lap PCR构建的TBC1D3缺失突变体Flag-TBC1D3(Δ157-171)和Flag-TBC1D3(Δ303-312),经Bam HI和Xho I双酶切和测序鉴定,序列正确符合预期。以抗Flag抗体或抗GST抗体做免疫共沉淀,结果均显示157位-171位氨基酸缺失后TBC1D3不能与CaM结合,提示TBC1D3蛋白序列上与CaM相互作用的部位可能在N端的157到171位氨基酸之间。 3.Over-lap PCR构建的TBC1D3点突变体Flag-TBC1D3(Y163A-T165A)和Flag-TBC1D3(K166R),经Bam HI和Xho I双酶切和测序鉴定,序列正确符合预期。Western blot结果显示10%FCS可诱导Flag-TBC1D3(Y163A-T165A)降解,而Flag-TBC1D3(Δ157-171) 和Flag-TBC1D3(K166R) 无明显降解降解;以抗Flag抗体做免疫共沉淀检测泛素水平,结果显示Flag-TBC1D3(Y163A-T165A)与Flag-TBC1D3均检测到明显泛素,Flag-TBC1D3(K166R)泛素水平大大降低。以抗Flag抗体做免疫共沉淀,结果显示TBC1D3和TBC1D3(K166R)与CaM存在相互作用,Flag对照和TBC1D3(Y163A-T165A)无CaM结合条带,提示CaM抑制TBC1D3的降解是通过与TBC1D3相互作用抑制K166的泛素化。 4.细胞划痕和Transwell迁移实验结果显示,转染Flag-TBC1D3的MCF-7和BT-549细胞迁移能力明显强于转染Flag空载体;实时荧光定量PCR结果显示TBC1D3可上调MMP-9mRNA水平,Western blot检测显示TBC1D3可上调MMP-9蛋白水平,明胶酶谱实验显示TBC1D3可增强MMP-9活性,提示TBC1D3促进乳腺癌细胞迁移可能是通过上调MMP-9的蛋白和活性水平。复合转染CaM时,TBC1D3上调的MMP-9的蛋白和活性水平以及诱导的细胞迁移均被增强。 结论: 1. CaM增强人乳腺癌细胞浆、细胞核TBC1D3蛋白的稳定性。 2. Ca2+/CaM与TBC1D3的N端157位-171位氨基酸序列相互作用。 3. 泛素化位点K166突变增强人乳腺癌细胞内TBC1D3蛋白的稳定性。 4.CaM增强TBC1D3诱导的人乳腺癌细胞MMP-9活性和细胞迁移。
英文题目 Camodulin regalutes the stability and Biological significance of Nucleocytoplasmic Protein TBC1D3 in Human Breast cancer cells
英文主题词 Prostate cancer gene 17,Camodulin,Ubiquitin-proteasome pathway,Matrix metalloproteinases 9,Migration
英文摘要 Objective TBC1D3 (also referred to as prostate cancer gene 17, PRC17) was identified as a hominoid-specific gene, which widely expressed in human tissues and overexpressed in prostate, breast, bladder and pancreatic cancer as well as in myelodysplastic syndrome (MDS).Ectopic expression of TBC1D3 confers tumorigenicity to mouse NIH 3T3 embryonic fibroblast cells, indicating that TBC1D3 functions as an oncogene. Moreover, TBC1D3 inhibits the ubiquitination of epidermal growth factor receptor (EGFR) and insulin receptor substrate-1 (IRS-1), and their subsequent degradation, thereby enhancing EGF and insulin signaling and consequential cell proliferation. Nevertheless, growth factor (GF) signaling promotes SCF-FBXW8 E3 ubiquitin ligases CUL7-mediated TBC1D3 ubiquitination and proteasomal degradation. Camodulin(CaM)has been demonstrated that regulates stability of a body of proteins, such as estrogen receptor-α (ER-α) and TRE17/USP6. Furthermore, our preliminary experiment indicated that CaM inhibits the degradation of TBC1D3, and then enhances its stability. Therefore, to explore the mechanisms by which CaM regulates the stability of TBC1D3 may be beneficial to elucidate the role of CaM and TBC1D3 in tumor development and progression, and to provide new clues for the diagnosis and prevention of tumor. Methods: 1. In order to study the effect of CaM on the degradation of TBC1D3, MCF-7 and BT-549cells were transfected with GST-CaM for ectopic expression or treated with CaM antagonist W7 to inhibit function of endogenous CaM, then the role of CaM in the process of TBC1D3’s ubiquitination and degradation were observed by western blot and co-immunoprecipitation. And then the impact of CaM on the subcellular location of TBC1D3’s degradation was observed by extracting and analyzing cytoplasmic and nuclear protein. 2. In order to investigate whether the interaction between CaM and TBC1D3 inhibits the ubiquitination and degradation of TBC1D3, MCF-7 cells were co-transfected with Flag-TBC1D3 and GST Vector or GST-CaM plasmids and performed the co-immunoprecipitation with anti-GST antibody; then revised co-immunoprecipitation was performed used anti-Flag antibody in MCF-7 cells co-transfected with GST-CaM and Flag Vector or Flag-TBC1D3 plasmids. And then, the interaction between CaM and TBC1D3 was observed by immunoprecipitation with anti-Flag antibody and anti-GST antibody treated with or not Ca2 + chelator EGTA in MCF-7 and BT-549 cells, respectively. Furthermore,TBC1D3 deletion mutants Flag-TBC1D3(Δ157-171) and Flag-TBC1D3(Δ303-312) were constructed by over-lap PCR, and they were all co-transfected with GST-CaM into MCF-7 cells, as well as Flag-TBC1D3. Then cells were lysed and performed the co-immunoprecipitation with anti-GST antibody; then revised co-immunoprecipitation was performed in MCF-7 cells co-transfected with GST-CaM and Flag Vector or Flag-TBC1D3 plasmids 3. In order to explore the function of interacting motif between TBC1D3, TBC1D3 internal point mutant Flag-TBC1D3 (Y163A-T165A) and Flag-TBC1D3 (K166R) were constructed by over-lap PCR. Then Flag-TBC1D3、Flag-TBC1D3 (Δ157-171) and point mutants were transfected into MCF-7 cells and their ubiquitination and degradation level were with detected by Western Blot and CO-IP. Finally, Flag-TBC1D3, point mutants were co-transfected with GST-CaM into MCF-7 cells and were performed immunoprecipitation with anti-Flag antibody to perform to detect their interaction with CaM. 4. In order to detect the effect of TBC1D3 on cell migration, Flag vector and Flag-TBC1D3 plasmids were transfected into MCF-7 and BT-549 cells, then were observed by cell scratch and transwell migration assay. As asking for the mechanisms by which TBC1D3 affects cell migration, MCF-7 cells were respectively transfected with Flag vector and Flag-TBC1D3 plasmids, and then were starved with serum-free medium for 24 hours. The activity of MMP-9 in culture medium was detected by gelatin zymography, while the mRNA and protein levels of MMP-9 in cell lysates were measured by real-time fluorescence quantitative PCR and western blot simultaneously. Finally, MCF-7 cells were co-transfected with GST-CaM to enhance the stability of TBC1D3 and observe the impacts of enhanced TBC1D3’s stability on the cell migration, MMP-9 activity and protein level. Results: 1. Western blot analysis showed that 10% FCS induced the degradation of TBC1D3, while ectopic expression of GST-CaM inhibited TBC1D3’s degradation, indicating that CaM could inhibit the degradation of TBC1D3. When the function of endogenous CaM was inhibited by W7, TBC1D3 degraded much obviously by 10%FCS stimulation, indicating that endogenous CaM could inhibit TBC1D3’s degradation. The results in immunoprecipitation experiments showed that 10% FCS stimulation could induce the ubiquitination of TBC1D3, however, over-expression of CaM significantly reduce the TBC1D3’s ubiquitination, suggesting that CaM could decrease the ubiquitination level of TBC1D3. The cytoplasmic and nuclear protein extraction showed that TBC1D3 degradation induced by 10% FCS in MCF-7 and BT-549 cells occurred both in the cytoplasm and the nucleus. What’s more, there’s much degradation of nuclear TBC1D3 in MCF-7cells, while much decrease of cytoplasmic TBC1D3 in BT-549 cells. Moreover, over-expression of CaM significantly reduces the TBC1D3’s degradation both in the cytoplasm and the nucleus. 2. Immunoprecipitation was performed with anti-GST antibody and the results showed that TBC1D3 binding band was detected in GST-CaM group but not GST control group, indicating that TBC1D3 interacted with CaM. When the immunoprecipitation was performed with anti-Flag antibody, the CaM binding band was detected in Flag-TBC1D3 group but not Flag control group. However, when Ca2+ was chelated by EGTA, the interaction between TBC1D3 and CaM was abolished both in co-immunoprecipitation with anti-Flag antibody and anti-GST antibody, suggesting that the interaction between CaM and TBC1D3 in a Ca2 + manner. TBC1D3 deletion mutants Flag-TBC1D3 (Δ157-171) and Flag-TBC1D3 (Δ303-312) constructed by Over-lap PCR were correctly sequenced and the results co-immunoprecipitated with anti-Flag antibody or anti-GST antibody showed that deletion mutants TBC1D3 (Δ157-171) abolished the interaction between TBC1D3 and CaM, suggesting that the CaM-interacting site in TBC1D3 resides at or near amino acids 157–171. 3. TBC1D3 point mutants Flag-TBC1D3 (Y163A-T165A) and Flag-TBC1D3 (K166R) constructed by Over-lap PCR were correctly sequenced. Western blot analysis showed that 10% FCS stimulation induces the degradation of TBC1D3 (Y163A-T165A), but not TBC1D3 (Δ157-171) and TBC1D3 (K166R). Immunoprecipitation experiments with anti Flag antibody was performed, the results showed that significant ubiquitin was detected in Flag-TBC1D3 (Y163A-T165A) and Flag-TBC1D3, while hardly in Flag-TBC1D3 (K166R). Moreover, CaM binds with TBC1D3 and TBC1D3 (K166R), but not Flag control and TBC1D3 (Y163A-T165A), suggesting that CaM interacts with TBC1D3 and inhibits GF signaling-induced ubiquitination at K166 and subsequent degradation of the oncoprotein in human breast cancer cells. 4. The results of cell scratches and transwell migration showed that the migrated number of MCF-7 and BT-549 cells transfected with Flag-TBC1D3 was much more than that of the cells transfected Flag vector. Real-time fluorescence quantitative PCR showed that TBC1D3 could up-regulate the level of MMP-9 mRNA; Western blot displayed that TBC1D3 enhances the expression of MMP-9; Gelatin zymogram experiments showed that TBC13 enhances the activity of MMP-9, suggesting that TBC1D3 promotes the migration of human breast cancer cells in a manner involving the expression and activation of MMP-9. Moreover, TBC13 up-regulates the level and activity of MMP-9 and induces cell migration were all enhanced by ectopic expressed CaM, suggesting that CaM enhances the TBC1D3-induced migration of human breast cancer cells by a mechanism involving the expression and activation of MMP-9. Conclusion: 1. CaM enhances the stability of TBC1D3 both in the cytoplasm and the nucleus. 2. CaM interacts with TBC1D3 at or near amino acids 157-171 resides in a Ca2+ dependent manner. 3. The lysine residue 166 within the CaM-interacting motifs of TBC1D3 was the actual site for the GF signaling-induced ubiquitination. 4. CaM enhances the TBC1D3-induced migration of human breast cancer cells by a mechanism involving the expression and activation of MMP-9.
学术讨论
主办单位时间地点报告人报告主题
东南大学病理生理学系 2014-05-09 基一楼114病理生理办公室 赵虎子 Importin-7 Mediates Nuclear Trafficking of DNA in Mammalian Cells
东南大学病理生理学系 2015-04-17 基一楼114病理生理办公室 赵虎子 Calmodulin binds HER2 and modulates HER2 signaling
东南大学病理学与病理生理学系 2016--09-14 基一楼203会议室 赵虎子 Ubiquitination and Degradation of the Hominoid-Specific Oncoprotein TBC1D3 Is Mediated by CUL7 E3 Ligase
东南大学病理学与病理生理学系 2016-11-24 基一楼203会议室 赵虎子 Cytoplasmic Retention of a Nucleocytoplasmic Protein TBC1D3 by Microtubule Network Is Required for Enhanced EGFR Signaling
东南大学病理生理学系 2015-05-29 基一楼114病理生理办公室 万青 Kinesin Family Deregulation Coordinated by Bromodomain Protein ANCCA and Histone Methyltransferase MLL for Breast Cancer Cell Growth, Survival, and Tamoxifen Resistance
东南大学病理生理学系 2015-04-03 基一楼114病理生理办公室 王北 An N-terminal Nuclear Export Signal Regulates Trafficking and Aggregation of Huntingtin (Htt) Protein Exon 1
东南大学病理生理学系 2015-04-30 基一楼114病理生理办公室 赵蕾 LIFR is a breast cancer metastasis suppressor upstream of the Hippo-YAP pathway and a prognostic marker
东南大学病理生理学系 2015-05-15 基一楼114病理生理办公室 张丽娜 TRE17/USP6 oncogene translocated in aneurysmal bone cyst induces matrix metalloproteinase production via activation of NF-kB
     
学术会议
会议名称时间地点本人报告本人报告题目
江苏省病理生理学会第三届代表大会暨学术研讨会 2015年5月8-10号 苏州大学 钙调蛋白(CaM)调节TBC1D3的稳定性 及生物学意义的研究
欧洲肿瘤内科年会亚洲分会(ESMO Asia 2016) 2016年12月16-19号 新加坡 TC-1 is required for TBC1D3-induced Wnt/beta-catenin accumulation and cell migration in MCF-7 breast cancer cells
     
代表作
论文名称
Calmodulin promotes matrix metalloproteinase 9 production and cell migration by inhibiting the ubiqu
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
郑杰 正高 教授 博导 东南大学医学院
王大勇 正高 博导 东南大学生命科学院
胡守友 正高 主任医师 博导 江苏省中医院
曹志刚 其他 主任医师 硕导 南京市第一医院
姜藻 其他 主任医师 硕导 东南大学附属中大医院
      
答辩秘书信息
姓名职称工作单位备注
廖凯 其他 讲师 东南大学医学院