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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 2017-09-27
地点 东南大学医学院基一楼103会议室 论文选题来源 国家自然科学基金项目     论文字数 5.2 (万字)
题目 癌基因TBC1D3诱导人乳腺癌细胞迁移的机制
主题词 前列腺癌基因17,氧化低密度脂蛋白受体1,肿瘤坏死因子,β-肌动蛋白,核纤层蛋白
摘要 目的: 肿瘤细胞转移是乳腺癌患者死亡的最主要原因,涉及多个步骤包括肿瘤细胞迁移。本实验室近来研究发现,TBC1D3促进人乳腺癌细胞迁移,但TBC1D3诱导乳腺癌细胞迁移的机制依然未知。本研究拟用转录组高通量测序和免疫共沉淀结合质谱分析等等方法,分别筛选和鉴定TBC1D3的下游靶基因和细胞核内相互作用蛋白,阐明TBC1D3诱导乳腺癌细胞迁移的机制。 方法: 以Flag载体和Flag-TBC1D3分别转染MCF-7细胞,然后提取细胞总RNA,由广州锐博生物公司进行转录组高通量测序及分析,以筛选TBC1D3调控的潜在下游靶基因;用定量RT-PCR及Western blot等方法验证筛选的潜在下游靶基因包括乳腺癌细胞迁移相关的OLR1。 用shRNA慢病毒载体,分别构建TBC1D3下游靶基因OLR1敲低的MCF-7和BT549细胞稳转株,用细胞划痕实验和transwell细胞迁移实验等方法,观察OLR1敲低对TBC1D3诱导人乳腺癌细胞迁移的影响,从而明确OLR1在TBC1D3诱导人乳腺癌细胞迁移中的作用。 用TNFα抑制剂pomalidomide (Pom)下调OLR1表达,观察TNFα信号途径在TBC1D3诱导人乳腺癌细胞迁移中的作用;制备MCF-7和BT549细胞浆和细胞核蛋白,观察TBC1D3高表达对TNFα信号途径下游主要效应分子NF-κB活性的影响;用Pom抑制TNFα信号途径,观察该信号途径在TBC1D3调节NF-κB活性中的作用;用NF-κB抑制剂caffeic acid phenethyl ester (CAPE)抑制NF-κB活性,观察NF-κB在TBC1D3诱导OLR1表达和人乳腺癌细胞迁移中的作用,从而明确TNFα/NF-κB信号途径在TBC1D3诱导OLR1表达和人乳腺癌细胞迁移中的作用。 用ELISA方法,观察TBC1D3高表达对MCF-7和BT549细胞TNFα分泌的影响;用Western blot方法,观察TBC1D3高表达对MCF-7和BT549细胞TNFα受体(TNFR1和TNFR2)和该受体相关的多种衔接蛋白如TRAFs表达的影响;用蛋白降解分析及蛋白泛素化分析等方法,观察TBC1D3高表达对MCF-7细胞内TNFR1和多种TRAFs蛋白稳定性的影响及蛋白泛素化在其中的作用,从而从多方面探讨TBC1D3调节TNFα信号途径的机制。 制备MCF-7细胞浆和细胞核蛋白,用免疫共沉淀结合质谱分析等方法,筛选TBC1D3的细胞核内相互作用蛋白;用免疫共沉定结合Western blot方法,对可能的相互作用蛋白进行初步鉴定,从而为深入探讨TBC1D3的细胞核内功能及其调节TNFR1和多种TRAFs在乳腺癌细胞内表达的机制奠定基础。 结果: 转录组高通量测序结果发现,在所检测到的37364个基因中,TBC1D3显著上调人乳腺癌细胞43个基因,显著下调56个基因。定量RT-PCR(qRT-PCR)验证实验结果表明,TBC1D3上调或下调差异倍数最高的10个基因中,绝大多数基因,两种方法所得结果一致。鉴于多个实验室的研究结果表明,在上调基因中的OLR1基因能促进肿瘤细胞包括乳腺癌细胞迁移和转移,故随后主要研究了OLR1基因在TBC1D3诱导人乳腺癌细胞迁移中的作用及其机制。 TNFα和NF-κB的抑制剂Pom,CAPE处理细胞,发现Pom和 CAPE处理细胞后,TBC1D3诱导的乳腺癌MCF-7和MDA-MB-231细胞OLR1表达上调和细胞迁移增强被显著抑制。相比于Flag空载体,转染Flag-TBC1D3 的MCF-7和BT549细胞,胞核中NF-κB p65显著增加,而TNFα抑制剂Pom处理MCF-7和BT549细胞后,TBC1D3诱导的NF-κB p65核转位被显著抑制。Flag-TBC1D3高表达的MCF-7和BT549细胞的TNF分泌量分别是Flag对照转染细胞的1.4倍和2.5倍;用Flag-TBC1D3转染使MCF-7和BT549细胞中TNFR1、TRAF1、TRAF5、TRAF6的蛋白水平显著升高,而TNFR2和TRAF3蛋白的水平没有明显变化。与蛋白表达结果一致,实时定量PCR结果也显示,高表达TBC1D3的MCF-7细胞中,TNFR1、TRAF1、TRAF5、TRAF6的mRNA水平显著升高,而TRAF3的mRNA水平无明显变化。转染空载体的MCF-7细胞中TNFR1蛋白降解迅速,在TNFα刺激30分钟后,近一半的TNFR1蛋白被降解,在刺激2小时后,仅剩有约40%,相比之下,转染Flag-TBC1D3的MCF-7细胞中,TNFR1蛋白的降解明显延迟,在TNFα刺激30分钟时,仅仅1/4的TNFR1蛋白降解,在TNFα刺激2小时后,大约2/3的TNFR1持续存在。于对TNFR1的作用不同,TBC1D3并不能延迟降解乳腺癌细胞中的TRAF1、TRAF3、TRAF5和TRAF6蛋白。在TNFR1蛋白降解被用蛋白酶体抑制剂MG132阻断后,TNFα诱导的泛素化TNFR1蛋白大量存在于Flag对照载体的MCF-7细胞中,然而,Flag-TBC1D3转染的MCF-7细胞中TNFα诱导的TNFR1泛素化显著降低。 MCF-7细胞分别转染Flag和Flag-TBC1D3,制备细胞核蛋白进行免疫共沉淀,发现两条TBC1D3的可能结合蛋白条带,分别在大约42kDa和66kDa, 质谱分析结果显示差异蛋白条带分别匹配核纤层蛋白B和β-肌动蛋白。随后进行验证,通过用抗Flag抗体来进行免疫共沉淀,免疫沉淀复合物中可检测到与Flag-TBC1D3而不是Flag对照结合的是核纤层蛋白B和β-肌动蛋白;用同种属lgG为阴性对照,用抗核纤层蛋白B和β-肌动蛋白分别进行免疫共沉淀,沉淀复合物中可检测到与核纤层蛋白B和β-肌动蛋白结合的是TBC1D3。 结论: 1. OLR1介导癌蛋白TBC1D3诱导的人乳腺癌细胞迁移; 2.癌蛋白TBC1D3通过激活TNFα/NF-κB 信号途径调控诱导OLR1表达和人乳腺癌细胞迁移; 3.TBC1D3通过多种方式包括促进人乳腺癌细胞TNFα释放,增强TNFR1、TRAF1、 TRAF5、和 TRAF6转录以及抑制TNFR1降解等激活TNF信号途径; 4. TBC1D3与β-肌动蛋白在乳腺癌MCF-7细胞浆和细胞核内均可能相存在相互作用; 5.TBC1D3与核纤层蛋白B在乳腺癌MCF-7细胞核内可能存在相互作用。
英文题目 Mechanisms underlying TBC1D3 oncogene-induced migration of human breast cancer cells
英文主题词 TBC1D3; OLR1; TNFα; β-actin; Lamin B;
英文摘要 Objective: We recently found that the TBC1D3 oncogene promotes the migration of breast cancer cells, however, litter is known regarding the mechanism by which TBC1D3 induces the migration of cancer cells. Here. we demonstrated that TBC1D3 stimulated the oxidized low density lipoprotein receptor 1(OLR1), a stimulator of cell migration, in breast cancers at the transcritional level. TBC1D3 is a Hominoid-specific oncogene highly expressed in tumor cells and tissue, which interacts with a variety of protein. It is necessary to find new protein interact with TBC1D3 and explore the biological significance between them Methods: In order to exprole the mechanism by which TBC1D3 induces the migration of cancer cells, MCF-7 cells were transfected with Flag-TBC1D3 or control Flag vector, then total RNA was extracted, and subjected to high-throughput sequencing. Q-RT-PCR was used to validate the RNA level and Western blot was used to validate the protein level. In order to define the role of OLR1 in where TBC1D3 affect the migration of MCF-7 cells, we build OLR1 shRNA lentivirus and control lentivirus NC shRNA. MCF-7 and BT549 cells were infected with NC and OLR1 shRNA lentiviruses, OLR1 gene expression was detected by Western blot to determine the effectiveness of specific shRNA. To perform transwell migration experiment, stable OLR1 knockdown MCF-7 were transfected with Flag-TBC1D3. In order to explore the role of TNF signaling pathway in the TBC1D3-induced up-regulation of OLR1, TNF inhibitor pomalidomide and NF-κB inhibition using its specific inhibitor caffeic acid phenethyl ester were chosen. Since activation of TNF signaling pathway is intricately regulated by TNFα itself and TNFα receptors as well as a variety of adaptor proteins such as TRAFs, we examined the release of TNFα from MCF-7 ad BT549 cells using the enzyme-linked immunosorbent assay. We also examined the expression of TNFR and multiple TRAFs by Western blotting. Since protein levels in cells are regulated y the rate of both their synthesis and degradation, we next sought to determine whether TBC1D3 protects TNFR1 and TRAFs from intracellular protein degradation. Since ubiquitination has been shown to play a vital role in TNF-induced degradation of TNFR1 via the proteasome pathway, we examined the ubiquitination fo TNFR1 in MCF-7 cells transfected with Flag-TBC1D3. In order to find new proteins interacting with TBC1D3, MCF-7 cells were transfected with Flag-TBC1D3 or control Flag vector, then seperation of cytoplasm and nuclear protein were subjected to immunoprecipitation using anti-Flag and beads, Precipitated was separated by SDS-PAGE and colored by coomassie brilliant blue dyestuff. Then, proteins that probably associated with TBC1D3 was analyzed by MS/MS. Results: we demonstrated that TBC1D3 stimulated the expression of OLR1, a stimulator of cell migration, in breast cancer cells at the transcriptional level. Depletion of OLR1 by siRNA or down-regulation of OLR1 expression using pomalidomide, significantly decreased TBC1D3-induced migration of these cells. Notably, TBC1D3 overexpression activated NF-κB , a major effector of TNFα signalling suppressed the effects of TBC1D3. Consistent with this, NF-κB inhibition using its specific inhibitor caffeic acid phenethyl ester decreased both TBC1D3-induced OLR1 expression and cell migration. TBC1D3-overexpressing MCF-7 and BT549 cells exhibited approximately 1.4- and 2.5-fold increases in TNF secretion, respectively. We also examined the expression of TNFR and TRAFs by Western blotting. Transfection with Flag-TBC1D3 considerably increased the expression of TNFR1, TRAF1, TRAF5 and TRAF6 proteins in MCF-7 and BT549 cells, while there was no significant change in the expression of TNFR2 and TRAF3. Consistent with these results, overexpression of TBC1D3 stimulated the Mrna expression of TNFR1, TRAF1, TRAF5 and TRAF6, but no TRAF3 in MCF-7 cells. we next sought to determine whether TBC1D3 protects TNFR1 and TRAFs from intracellular protein degradation. we foud MCF-7 cells transfected with control Flag vector showed a rapid degradation of TNFR. In contrast, TNFR1 degradation was significantly delayed in MCF-7 cells expressing TBC1D3. However, TBC1D3 failed to delay the degradation of TRAF1, TRAF3, TRAF5 and TRAF6 in MCF-7 cells. Afer cytoplasm and nuclear protein were subjected to immunoprecipitation using anti-Flag and beads, some possible proteins binding to TBC1D3 were found. Among them, 42 and 66 KDa proteins were obvious and repeatable. On the contrary, there were no obvious protein bands with the same molecular weight for negative control. Then, the 42 and 55 KDa proteins were cut and sent for MS/MS assay. According to the results, two bands were Lamin B and Beta-actin respectively. TBC1D3 and β-actin interact in the cytoplasm and nuclear of MCF-7 cells and TBC1D3 interacts with Lamin B in the nuclear of MCF-7 cells by co-immunoprecitation. Conclusion: 1. TBC1D3 induces the up-regulation of OLR1 to promote the migration of breast cancer cells MCF-7 and BT549 2. TNF signaling contributes to TBC1D3-induced OLR1 expression and cell migration 3. TBC1D3 induces activation of TNF signaling on multiple levels, including by increasing the release of TNF, elevating the transcription of TNFR1, TRAF1, TRAF5 and TRAF6, and decreasing the degradation of TNFR1 4. TBC1D3 interacts with Beta-actin in the cytoplasm and nuclear of MCF-7 cells 5. TBC1D3 interacts with Lamin B in the nuclear of MCF-7 cells
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代表作
论文名称
Up-regulation of OLR1 expression by TBC1D3 through activation of TNFα/NF-κB pathway promotes the mig
 
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
胡守友 正高 主任医师 博导 江苏省中医院
王大勇 正高 博导 东南大学生命科学研究院
曹志刚 正高 主任医师 硕导 南京市第一医院
姜藻 正高 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