TRAP1基因沉默和基因过表达对A549细胞和MDA-MB-231细胞信号传导通路影响的研究
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摘要
研究背景和目的
肿瘤坏死因子α(Tumor necrosis factorα, TNF-α)是一种多功能细胞因子,由单核细胞和巨噬细胞等分泌产生,在生物机体的各个反应中具有重要作用,包括炎症反应、免疫调节、抗肿瘤与微生物感染等方面。TNF的生物学功能通过细胞表面相应的受体来实现。TNF受体家族主要是Ⅰ型膜蛋白受体,TNF-α有两类受体,分子量分别是55kD(TNFR-I)和75kD(TNFR-Ⅱ)。TNFR-Ⅰ广泛分布于正常细胞膜以及多种肿瘤细胞膜表面,TNFR-Ⅱ主要分布于造血细胞和内皮细胞。两类受体的胞外区有28%的同源性,而胞内区没有同源性,提示两者介导了不同的信号传递途径。TNFR-Ⅰ参与介导细胞活化信号和增殖信号,诱导一氧化氮合成酶和白介素8的活性,活化NF-κB,亦可诱导凋亡、介导炎症反应、抗病毒、促进成纤维细胞增殖等；而TNFR-Ⅱ主要调控胸腺细胞的增殖,抑制造血和调节内皮细胞或中性粒细胞活性等。TNF/TNFR系统不仅是重要的炎症因子,而且对细菌感染,某些自身免疫疾病和退化性疾病有保护功能。TNF与TNFR结合以后,一方面介导凋亡信号,在抗肿瘤和抗病毒感染中发挥重要作用,另一方面也参与自身免疫性疾病和败血症中对自身组织细胞的损伤。TNFR的生物学活性受诸多因素影响,包括遗传和环境因素,细胞分化以及细胞因子网络间的信息传递等。
TNF受体相关蛋白1(TNFR associated protein 1, TRAP 1)是在1995年鉴定出来的HSP90家族的一个成员,与肿瘤坏死因子1类受体的细胞内结构域结合。其结构与热休克蛋白75相似,主要定位于线粒体,在心脏肌原纤维、心脏细胞核、胰腺以及血管内皮细胞表面也有定位。近年来研究发现,TRAP1不但具有HSP90家族的共同特点,如在氧化应激时保护细胞,维持线粒体膜稳定、抑制活性氧的产生,避免凋亡；TRAP1还有不同于HSP90家族其他成员的独特特点,在于其特有的LxCxE模序,该模序可与Rb蛋白的SV40 T抗原结合结构域结合,在有丝分裂和热休克应激后,TRAP1可从线粒体转移至细胞核内,并借此结构域与Rb结合,参与辅助变性的Rb重新折叠,恢复其天然构象及功能,因此,TRAP1可做为Rb的分子伴侣,协助其调控细胞周期的进程,而HSPs其他成员则没有此模序及此功能。
热休克蛋白(Heat Shock Proteins, HSPs)又被称作“分子伴侣”(molecular chaperone),当细胞受到应激因素作用或处于病理生理状态下(如低氧、感染性炎症、缺血等)均可诱发热休克蛋白合成和表达增加。研究发现,许多肿瘤细胞可高度表达或特异表达不同亚族的HSP,认为HSP的表达参与了基因调控及细胞周期调控,与肿瘤细胞的增殖、分化、转移及凋亡有关,并与癌基因和抑癌基因有着密切关系,可能与肿瘤的发生、发展、治疗及预后及机体对抗肿瘤药物的耐药性等有关。我们实验室前期研究发现,TRAP1虽然在多数肿瘤中高表达,在某些肿瘤如非小细胞肺癌、非霍奇金淋巴瘤、胰腺癌中反而低表达。后继研究发现TRAP1在调节Rb/E2F1途径中发挥重要作用,影响细胞周期进程中G1／S期的转换。这一结果促使我们继续研究TRAP1在肿瘤细胞中的相关基因和信号通路,以期发现TRAP1不同于其他分子伴侣的独特特点,为治疗提供新的靶点。
本研究运用人类寡核苷酸芯片技术筛选TRAP1在肿瘤细胞中的相关基因,选用高表达TRAP1的A549肺腺癌细胞系和低表达TRAP1的MDA-MB-231乳腺癌细胞系,该两细胞系均为高度侵袭性,均表达Rb及E2F1。利用小干扰RNA技术和逆转录病毒载体在两个细胞系中分别敲低和过表达TRAP1,进行16小时的常氧或缺氧处理,在两个细胞系中分别比较在常氧和急性缺氧的初期,TRAP1表达高低不同所引起的相关基因的变化,将得到的差异表达基因列表上传至在线生物信息学分析软件进行基因功能注释、归纳聚类并建立基因相互作用网络图,研究TRAP1所影响的信号途径,探索其在肿瘤发病机制中的作用。
材料和方法
1.细胞培养：用siRNA技术转染A549细胞,同时以scramble RNA做为阴性对照；而用逆转录病毒载体在MDA-MB-231中构建长效稳定过表达TRAP1细胞株,同时以空载体转染该细胞做为阴性对照。即得到4组细胞株：A549/siRNA和A549/Scramble, MDA231/TRAP1+和MDA231/Mock。分别分组进行常氧或缺氧(0.1%02浓度)处理16小时。
2.收获细胞,提取总RNA,用Taqman实时荧光定量RT-PCR检测TRAP1表达水平,验证转染的效率。
3.体外扩增RNA并纯化,与荧光染料耦合,每个样本对应一个对照RNA,即人类通用参照总RNA。耦合后再次纯化,检测RNA浓度。
4.对芯片进行紫外偶联并预杂交、洗片、离心干燥备用。
5.将每个样本和一个对照RNA以相同质量混合加样于同一张芯片上,置于42℃杂交20小时。次日洗片、离心干燥。
6.在暗室用GenePix(?) 4000B芯片扫描仪扫描芯片,每个样本获得一个图像,用GenePix Pro 6.0软件进行图像分析,将图像转换为数据保存。用GeneSpringTM GX10软件对获得的图像数据进行分析。采用LOWESS方法对数据进行标准化、标记过滤、倍值过滤和表达水平过滤、采用对数值转换,去除每张芯片上变异系数(CV)大于25%的数据,设定比值改变(Fold change)>2.0为筛选标准,获得4个差异表达基因列表,即：常氧16小时后,A549/siRNA Vs A549/Scr, MDA231/TRAP 1+Vs MDA231/Mock;缺氧16小时后,A549/siRNA Vs A549/Scr, MDA231/TRAP1+Vs MDA231/Mock。
7.使用DAVID (Database for Annotation, Visualization and Integrated Discovery)在线数据库对差异表达基因的注解、功能聚类、通路分析,再用pSTIING在线数据库建立基因相互作用网络图。
8.挑选6个差异表达基因,用Taqman实时荧光定量RT-PCR验证芯片结果的可靠性。
结果
1. Taqman qRT-PCR显示,常氧和缺氧下,A549/siRNA组TRAP1的表达水平均明显低于A549/Scr组；而MDA231/TRAP1+组TRAP1的表达水平明显高于MDA231/Mock组,说明在两个细胞系中转染均成功。
2.常氧处理16小时后,A549细胞的两个比较组中有373个差异表达基因,其中A549／Scr组表达上调的基因198个,下调的175个；而MDA231的两个比较组中有163个差异表达基因,在MDA231/TRAP1+组上调的基因91个,下调的72个,A549细胞的差异表达基因数目大于MDA231细胞；而在缺氧处理16小时后,A549细胞系差异表达基因数目为377个,其中在A549／Scr组226个上调,151个下调；MDA231细胞系差异表达基因为421个,比在常氧时明显增多。
3.经DAVID在线功能聚类分析,敲低TRAP1基因时差异表达基因所参与的生物过程以及信号通路,与过表达TRAP1基因时差异表达基因所参与的生物过程以及信号通路基本相似,包括细胞周期调控、转录调节、免疫／炎症反应、血管生成、G蛋白偶联受体蛋白信号转导、细胞粘附、凋亡调节、磷酸化、离子转运和蛋白水解等方面。其中转录调节和细胞周期相关基因占的比例较大。
4.挑选的6个差异表达基因,用Taqman qRT-PCR实验验证了其表达水平与芯片结果一致,说明芯片结果基本可靠。
5.经pSTIING在线进行基因相互作用网络可视化,TNF途径、NF-κB途径、Rho激酶途径、JNK途径和MAPK级联反应途径受TRAP1的调节比较明显。
结论
1. TRAP1在两个细胞系中表达水平不同,其生理作用的重要性可能不同。在常氧下,TRAP1可能在正常表达有该蛋白的肿瘤细胞中发挥着更重要的作用,而低表达该蛋白的肿瘤细胞可能存在其他不依赖TRAP1的机制而促进肿瘤生长。
2.敲低TRAP1基因时差异表达基因所参与的生物过程以及信号通路,与过表达TRAP1基因时差异表达基因所参与的生物过程以及信号通路基本相似,证实了TRAP1所影响的生物通路。
3. TRAP1可能促进肿瘤细胞的细胞周期进程和增殖,与TNF途径、Rho激酶途径和G-蛋白偶联信号转导途径有关。
4. TRAP1可激活天然免疫反应,激活补体途径、调节巨噬细胞活性；也参与适应性免疫反应,参与T／B细胞活化、调节T细胞分化,影响Th1或Th2细胞亚群分泌细胞因子。
5. TRAP1促进细胞的磷酸化过程,其中MAPK级联反应途径发挥了重要作用。
6. TRAP1可能抑制肿瘤细胞转移,与细胞粘附相关基因的差异表达上调有关。
7. TRAP1可能促进肿瘤细胞血管生成,以满足或维持局部肿瘤组织的血供,以尽快使肿瘤细胞摆脱缺氧状态。
8. TRAP1对肿瘤细胞的凋亡无明显促进或抑制的趋势,有待进一步在具体肿瘤细胞中研究。
Backgrounds and objective
Tumor necrosis factor-α(TNF-α) is a multifunctional cytokine produced by monocytes/macrophages and lymphocytes, it is important in inflammatory and immune response, and for resistance to infection and cancers. TNF-αexerts many of its effects by binding to the cell membrane receptor of either 55 kDa termed TNFR-I or 75 kDa termed TNFR-Ⅱ. The two receptors share limited homology in the extracellular region, no homology is seen in the intracellular regions of the proteins, suggests the two receptors activate distinct signalling pathways. TNFR-I is involved in the activation of NF-κB, the induction of apoptosis, antivirus response, and activation of fibroblasts proliferation, etc, while TNFR-Ⅱis mainly involved in the proliferation of the thymus and regulation of the activity of endothelial cells and neutrophils. But the biological functions of the two receptors are not independent as many activities are completed by both of them, such as cytotoxicity and hepatotoxicity. The TNF/TNFR signaling pathway is not only important in inflammatory response, but also protective for bacterial infection and some autoimmune diseases. The binding of TNF to TNFR will initiate two opposite signaling pathways, which to some extent explain why TNFR can have contradictory biological functions. Therefore, the activity of TNFR depends on many factors that affect the balance of TNF/TNFR, it is also influenced by heredity, environment, stage of cell differentiation and many other signaling pathways among cytokines.
TNF receptor associated protein 1 (TRAP1) was first identified in 1995 as a member of HSP90 family, it binds to the intracellular domain of TNFR-I. TRAPl is identical to Hsp75 in its structure and locates mainly at the mitochondria but are also observed in certain tissues including cardiac sarcomeres, nuclei of pancreatic and heart cells, and on the cell surface of blood vessel endothelial cells. Recent studies reveal that TRAR1 has the general functions of HSPs, such as protecting cells from oxidative stress and apoptosis via maintaining the homeostasis of mitochondria membrane and inhibition of reactive oxygen species (ROS) generation. However, TRAP1 also has distinct functions from other HSP90 family members in that it has a unique LxCxE motif that allows it to bind to retinoblastoma protein (Rb) during mitosis and after heat shock when TRAP1 moves from its predominantly mitochondria location to the nucleus, to assist in the refolding of denatured Rb.
HSPs are also known as molecular chaperones, they can induce the change of the expression model of HSPs genes leading to the increasing generation of a series of HSPs when cells encounter stresses or are under pathophysiological conditions (i.e. hypoxia, inflammation and ischemia). Studies have shown that many tumor cells can express high level of different subgroups of HSPs, it is deemed that HSPs are implicated in the cell cycle regulation, the proliferation, differentiation, metastasis and apoptosis of tumor cells, also HSPs have close relationship with oncogenes and tumor suppressor genes and might be associated with the tumorigenesis and the tolerance of anticancer drugs. The preliminary study in our lab revealed that although TRAP1 is over-expressed in many tumors, its expression level is low in some tumors such as non-small cell lung cancer, non-Hodgkin lymphoma and pancreatic cancer. The preceding experiment discovered TRAPl plays key role in regulating Rb/E2F1 pathway and influences the G1/S progression of the cell cycle. This finding promoted us to further study the related genes and pathways of TRAP1 in tumor cells in order to find the distinct functions of TRAP1 and to find new therapeutic targets.
This study will use oligonucleotide microarray platform to detect the associated genes of TRAP1 in tumor cells, A549 lung adenocarcinoma cell line which expresses high level of TRAP 1 and MDA-MB-231 breast cancer cell line which expresses low level of TRAP 1 are used, both of them are highly invasive and express Rb and E2F1. Small interfering RNA (siRNA) and retroviral vector are used to construct the knock down model and the over-expression model of TRAP1, cells are treated under 16 hours of either normoxia or hypoxia. The gene expression profiles are compared between TRAP1 transfected group and control group, and the biological signaling pathways that TRAP1 affected are explored.
Materials and methods 1. Cell culture:Transient transfection of TRAP1 by siRNA in A549 cell line, scramble RNA transfected cells were negative control. Meanwhile using retroviral vector to construct stable over-expression model of TRAP 1 in MDA-MB-231 cell line, empty vector transfected cells were negative control. Thus four groups were constructed as following:A549/siRNA and A549/Scramble, MDA231/TRAP1+ and MDA231/Mock. Splited them into subgroups followed by 16 hours of either normoxic or hypoxic (0.1%O2) treatment.
2. Cells were harvested, total RNA was extracted and TRAP1 expression was verified by Taqman real time PCR.
3. Total RNA was amplified in vitro and purified followed by dye coupling of each sample. Universal Human Reference total RNA was used as reference for each sample. The coupled aRNA was again purified and the concentration was checked.
4. Microarray slides were UV-cross linked and pre-hybridized before use.
5. Each sample was mixed with same amount of reference RNA and then added onto one slide, the slides were placed into 42℃oven for 20 hours of hybridization. Slides were then washed and spinned to dry.
6. Slides were scanned using GenePix 4000B microarray scanner, the picture images were analyzed using GenePix 6.0 software, then the data were analyzed by GeneSpringTM GX10 software. Data were background subtracted and normalized by LOWESS and retrieved as the log transformed values. The filtering criteria were set as the coefficient of variation (CV)<25%and fold change>2.0 as cut-off. Thus 4 lists of differentially expressed genes were obtained, as:in normoxia, A549/siRNA Vs A549/Scr, MDA231/TRAP1+Vs MDA231/Mock; inhypoxia, A549/siRNA Vs A549/Scr, MDA231/TRAP1+Vs MDA231/Mock.
7. The gene lists were then analyzed using DAVID bioinformatics resources (Database for Annotation, Visualization and Integrated Discovery), finally genes of interest and their interaction networks were visualized by using pSTIING (Protein, Signalling, Transcriptional Interactions & inflammation Networks Gateway).
8. Taqman real time PCR was carried out for the expression of 6 genes chosen from the gene lists to verify the results of the microarray experiment.
Results
1. In either normoxia or hypoxia, the expression level of TRAP1 was obviously lower in A549/siRNA group than in A549/Scr group, and was obviously higher in MDA231/TRAP1+group than in MDA231/Mock group, indicating the high efficiency of TRAP 1 transfection.
2. After 16 hours of normoxic treatment, there were 373 differentially expressed genes in the A549 comparison groups, while in MDA231 comparison groups there were only 163 differentially expressed genes. After 16 hours of hypoxic treatment, the numbers of differentially expressed genes were 377 and 421 in the A549 comparison groups and MDA231 comparison groups respectively.
3. The analysis by using DAVID showed the affected biological pathways when TRAP1 was knocked down in A549 cells were similar to those when TRAP1 was restored in MDA231 cells, including the regulation of cell cycle, transcription, immune/inflammatory response, angiogenesis, G-protein coupled receptor protein pathway, cell adhesion, regulation of apoptosis, phosphorylation, etc. The percentage of genes involved in the regulation of cell cycle and transcription is relatively large.
4. The expression model of the 6 picked genes by Taqman real time PCR was the same as that of the microarray experiment, indicating the results of the microarray platform was trustable.
5. The analysis by pSTIING to visualize the interaction network of those differentially expressed genes showed that, TNF pathway, NF-κB pathway, Rho kinase pathway, JNK pathway and MAPK cascade pathway were influenced.
Conclusions
1. In normoxia, TRAP1 may be more important for tumor cells that normally express this protein rather than those that have lost it. Whereas at the beginning of a short period of hypoxia, tumor cells tend to start the mechanism of expressing this heat shock protein to survive through some common pathways.
2. The affected biological pathways when TRAP1 was knocked down in A549 cells were similar to those when TRAP1 was restored in MDA231 cells, which proved the role of TRAP 1 in these pathways.
3. TRAP1 may promote the cell cycle progression and proliferation of tumor cells, which is associated with TNF pathway and Rho kinase pathway and G-protein coupled receptor protein pathway.
4. TRAP1 may activate innate immune response, activate complement pathways and regulate microphage activity; it may also be involved in adaptive immunity, regulate T/B activation and T cell differentiation, and influence the cytokine production by Thl or Th2 subgroups.
5. TRAP1 promotes cell phosphorylation, in which MAPK cascade pathway plays a vital role.
6. TRAP1 might inhibit the metastasis of tumor cells, which is associated with the up-regulation of cell adhesion related genes.
7. TRAP1 may promote the angiogenesis of tumor cells, in order to maintain local blood supply and to avoid a hypoxic condition.
8. There was no obvious trend was found as for the promotion or inbition of apoptosis, which needs further study in specific tumors.
肿瘤坏死因子α(Tumor necrosis factorα, TNF-α)是一种多功能细胞因子,由单核细胞和巨噬细胞等分泌产生,在生物机体的各个反应中具有重要作用,包括炎症反应、免疫调节、抗肿瘤与微生物感染等方面。TNF的生物学功能通过细胞表面相应的受体来实现。TNF受体家族主要是Ⅰ型膜蛋白受体,TNF-α有两类受体,分子量分别是55kD(TNFR-I)和75kD(TNFR-Ⅱ)。TNFR-Ⅰ广泛分布于正常细胞膜以及多种肿瘤细胞膜表面,TNFR-Ⅱ主要分布于造血细胞和内皮细胞。两类受体的胞外区有28%的同源性,而胞内区没有同源性,提示两者介导了不同的信号传递途径。TNFR-Ⅰ参与介导细胞活化信号和增殖信号,诱导一氧化氮合成酶和白介素8的活性,活化NF-κB,亦可诱导凋亡、介导炎症反应、抗病毒、促进成纤维细胞增殖等；而TNFR-Ⅱ主要调控胸腺细胞的增殖,抑制造血和调节内皮细胞或中性粒细胞活性等。TNF/TNFR系统不仅是重要的炎症因子,而且对细菌感染,某些自身免疫疾病和退化性疾病有保护功能。TNF与TNFR结合以后,一方面介导凋亡信号,在抗肿瘤和抗病毒感染中发挥重要作用,另一方面也参与自身免疫性疾病和败血症中对自身组织细胞的损伤。TNFR的生物学活性受诸多因素影响,包括遗传和环境因素,细胞分化以及细胞因子网络间的信息传递等。
TNF受体相关蛋白1(TNFR associated protein 1, TRAP 1)是在1995年鉴定出来的HSP90家族的一个成员,与肿瘤坏死因子1类受体的细胞内结构域结合。其结构与热休克蛋白75相似,主要定位于线粒体,在心脏肌原纤维、心脏细胞核、胰腺以及血管内皮细胞表面也有定位。近年来研究发现,TRAP1不但具有HSP90家族的共同特点,如在氧化应激时保护细胞,维持线粒体膜稳定、抑制活性氧的产生,避免凋亡；TRAP1还有不同于HSP90家族其他成员的独特特点,在于其特有的LxCxE模序,该模序可与Rb蛋白的SV40 T抗原结合结构域结合,在有丝分裂和热休克应激后,TRAP1可从线粒体转移至细胞核内,并借此结构域与Rb结合,参与辅助变性的Rb重新折叠,恢复其天然构象及功能,因此,TRAP1可做为Rb的分子伴侣,协助其调控细胞周期的进程,而HSPs其他成员则没有此模序及此功能。
热休克蛋白(Heat Shock Proteins, HSPs)又被称作“分子伴侣”(molecular chaperone),当细胞受到应激因素作用或处于病理生理状态下(如低氧、感染性炎症、缺血等)均可诱发热休克蛋白合成和表达增加。研究发现,许多肿瘤细胞可高度表达或特异表达不同亚族的HSP,认为HSP的表达参与了基因调控及细胞周期调控,与肿瘤细胞的增殖、分化、转移及凋亡有关,并与癌基因和抑癌基因有着密切关系,可能与肿瘤的发生、发展、治疗及预后及机体对抗肿瘤药物的耐药性等有关。我们实验室前期研究发现,TRAP1虽然在多数肿瘤中高表达,在某些肿瘤如非小细胞肺癌、非霍奇金淋巴瘤、胰腺癌中反而低表达。后继研究发现TRAP1在调节Rb/E2F1途径中发挥重要作用,影响细胞周期进程中G1／S期的转换。这一结果促使我们继续研究TRAP1在肿瘤细胞中的相关基因和信号通路,以期发现TRAP1不同于其他分子伴侣的独特特点,为治疗提供新的靶点。
本研究运用人类寡核苷酸芯片技术筛选TRAP1在肿瘤细胞中的相关基因,选用高表达TRAP1的A549肺腺癌细胞系和低表达TRAP1的MDA-MB-231乳腺癌细胞系,该两细胞系均为高度侵袭性,均表达Rb及E2F1。利用小干扰RNA技术和逆转录病毒载体在两个细胞系中分别敲低和过表达TRAP1,进行16小时的常氧或缺氧处理,在两个细胞系中分别比较在常氧和急性缺氧的初期,TRAP1表达高低不同所引起的相关基因的变化,将得到的差异表达基因列表上传至在线生物信息学分析软件进行基因功能注释、归纳聚类并建立基因相互作用网络图,研究TRAP1所影响的信号途径,探索其在肿瘤发病机制中的作用。
材料和方法
1.细胞培养：用siRNA技术转染A549细胞,同时以scramble RNA做为阴性对照；而用逆转录病毒载体在MDA-MB-231中构建长效稳定过表达TRAP1细胞株,同时以空载体转染该细胞做为阴性对照。即得到4组细胞株：A549/siRNA和A549/Scramble, MDA231/TRAP1+和MDA231/Mock。分别分组进行常氧或缺氧(0.1%02浓度)处理16小时。
2.收获细胞,提取总RNA,用Taqman实时荧光定量RT-PCR检测TRAP1表达水平,验证转染的效率。
3.体外扩增RNA并纯化,与荧光染料耦合,每个样本对应一个对照RNA,即人类通用参照总RNA。耦合后再次纯化,检测RNA浓度。
4.对芯片进行紫外偶联并预杂交、洗片、离心干燥备用。
5.将每个样本和一个对照RNA以相同质量混合加样于同一张芯片上,置于42℃杂交20小时。次日洗片、离心干燥。
6.在暗室用GenePix(?) 4000B芯片扫描仪扫描芯片,每个样本获得一个图像,用GenePix Pro 6.0软件进行图像分析,将图像转换为数据保存。用GeneSpringTM GX10软件对获得的图像数据进行分析。采用LOWESS方法对数据进行标准化、标记过滤、倍值过滤和表达水平过滤、采用对数值转换,去除每张芯片上变异系数(CV)大于25%的数据,设定比值改变(Fold change)>2.0为筛选标准,获得4个差异表达基因列表,即：常氧16小时后,A549/siRNA Vs A549/Scr, MDA231/TRAP 1+Vs MDA231/Mock;缺氧16小时后,A549/siRNA Vs A549/Scr, MDA231/TRAP1+Vs MDA231/Mock。
7.使用DAVID (Database for Annotation, Visualization and Integrated Discovery)在线数据库对差异表达基因的注解、功能聚类、通路分析,再用pSTIING在线数据库建立基因相互作用网络图。
8.挑选6个差异表达基因,用Taqman实时荧光定量RT-PCR验证芯片结果的可靠性。
结果
1. Taqman qRT-PCR显示,常氧和缺氧下,A549/siRNA组TRAP1的表达水平均明显低于A549/Scr组；而MDA231/TRAP1+组TRAP1的表达水平明显高于MDA231/Mock组,说明在两个细胞系中转染均成功。
2.常氧处理16小时后,A549细胞的两个比较组中有373个差异表达基因,其中A549／Scr组表达上调的基因198个,下调的175个；而MDA231的两个比较组中有163个差异表达基因,在MDA231/TRAP1+组上调的基因91个,下调的72个,A549细胞的差异表达基因数目大于MDA231细胞；而在缺氧处理16小时后,A549细胞系差异表达基因数目为377个,其中在A549／Scr组226个上调,151个下调；MDA231细胞系差异表达基因为421个,比在常氧时明显增多。
3.经DAVID在线功能聚类分析,敲低TRAP1基因时差异表达基因所参与的生物过程以及信号通路,与过表达TRAP1基因时差异表达基因所参与的生物过程以及信号通路基本相似,包括细胞周期调控、转录调节、免疫／炎症反应、血管生成、G蛋白偶联受体蛋白信号转导、细胞粘附、凋亡调节、磷酸化、离子转运和蛋白水解等方面。其中转录调节和细胞周期相关基因占的比例较大。
4.挑选的6个差异表达基因,用Taqman qRT-PCR实验验证了其表达水平与芯片结果一致,说明芯片结果基本可靠。
5.经pSTIING在线进行基因相互作用网络可视化,TNF途径、NF-κB途径、Rho激酶途径、JNK途径和MAPK级联反应途径受TRAP1的调节比较明显。
结论
1. TRAP1在两个细胞系中表达水平不同,其生理作用的重要性可能不同。在常氧下,TRAP1可能在正常表达有该蛋白的肿瘤细胞中发挥着更重要的作用,而低表达该蛋白的肿瘤细胞可能存在其他不依赖TRAP1的机制而促进肿瘤生长。
2.敲低TRAP1基因时差异表达基因所参与的生物过程以及信号通路,与过表达TRAP1基因时差异表达基因所参与的生物过程以及信号通路基本相似,证实了TRAP1所影响的生物通路。
3. TRAP1可能促进肿瘤细胞的细胞周期进程和增殖,与TNF途径、Rho激酶途径和G-蛋白偶联信号转导途径有关。
4. TRAP1可激活天然免疫反应,激活补体途径、调节巨噬细胞活性；也参与适应性免疫反应,参与T／B细胞活化、调节T细胞分化,影响Th1或Th2细胞亚群分泌细胞因子。
5. TRAP1促进细胞的磷酸化过程,其中MAPK级联反应途径发挥了重要作用。
6. TRAP1可能抑制肿瘤细胞转移,与细胞粘附相关基因的差异表达上调有关。
7. TRAP1可能促进肿瘤细胞血管生成,以满足或维持局部肿瘤组织的血供,以尽快使肿瘤细胞摆脱缺氧状态。
8. TRAP1对肿瘤细胞的凋亡无明显促进或抑制的趋势,有待进一步在具体肿瘤细胞中研究。
Backgrounds and objective
Tumor necrosis factor-α(TNF-α) is a multifunctional cytokine produced by monocytes/macrophages and lymphocytes, it is important in inflammatory and immune response, and for resistance to infection and cancers. TNF-αexerts many of its effects by binding to the cell membrane receptor of either 55 kDa termed TNFR-I or 75 kDa termed TNFR-Ⅱ. The two receptors share limited homology in the extracellular region, no homology is seen in the intracellular regions of the proteins, suggests the two receptors activate distinct signalling pathways. TNFR-I is involved in the activation of NF-κB, the induction of apoptosis, antivirus response, and activation of fibroblasts proliferation, etc, while TNFR-Ⅱis mainly involved in the proliferation of the thymus and regulation of the activity of endothelial cells and neutrophils. But the biological functions of the two receptors are not independent as many activities are completed by both of them, such as cytotoxicity and hepatotoxicity. The TNF/TNFR signaling pathway is not only important in inflammatory response, but also protective for bacterial infection and some autoimmune diseases. The binding of TNF to TNFR will initiate two opposite signaling pathways, which to some extent explain why TNFR can have contradictory biological functions. Therefore, the activity of TNFR depends on many factors that affect the balance of TNF/TNFR, it is also influenced by heredity, environment, stage of cell differentiation and many other signaling pathways among cytokines.
TNF receptor associated protein 1 (TRAP1) was first identified in 1995 as a member of HSP90 family, it binds to the intracellular domain of TNFR-I. TRAPl is identical to Hsp75 in its structure and locates mainly at the mitochondria but are also observed in certain tissues including cardiac sarcomeres, nuclei of pancreatic and heart cells, and on the cell surface of blood vessel endothelial cells. Recent studies reveal that TRAR1 has the general functions of HSPs, such as protecting cells from oxidative stress and apoptosis via maintaining the homeostasis of mitochondria membrane and inhibition of reactive oxygen species (ROS) generation. However, TRAP1 also has distinct functions from other HSP90 family members in that it has a unique LxCxE motif that allows it to bind to retinoblastoma protein (Rb) during mitosis and after heat shock when TRAP1 moves from its predominantly mitochondria location to the nucleus, to assist in the refolding of denatured Rb.
HSPs are also known as molecular chaperones, they can induce the change of the expression model of HSPs genes leading to the increasing generation of a series of HSPs when cells encounter stresses or are under pathophysiological conditions (i.e. hypoxia, inflammation and ischemia). Studies have shown that many tumor cells can express high level of different subgroups of HSPs, it is deemed that HSPs are implicated in the cell cycle regulation, the proliferation, differentiation, metastasis and apoptosis of tumor cells, also HSPs have close relationship with oncogenes and tumor suppressor genes and might be associated with the tumorigenesis and the tolerance of anticancer drugs. The preliminary study in our lab revealed that although TRAP1 is over-expressed in many tumors, its expression level is low in some tumors such as non-small cell lung cancer, non-Hodgkin lymphoma and pancreatic cancer. The preceding experiment discovered TRAPl plays key role in regulating Rb/E2F1 pathway and influences the G1/S progression of the cell cycle. This finding promoted us to further study the related genes and pathways of TRAP1 in tumor cells in order to find the distinct functions of TRAP1 and to find new therapeutic targets.
This study will use oligonucleotide microarray platform to detect the associated genes of TRAP1 in tumor cells, A549 lung adenocarcinoma cell line which expresses high level of TRAP 1 and MDA-MB-231 breast cancer cell line which expresses low level of TRAP 1 are used, both of them are highly invasive and express Rb and E2F1. Small interfering RNA (siRNA) and retroviral vector are used to construct the knock down model and the over-expression model of TRAP1, cells are treated under 16 hours of either normoxia or hypoxia. The gene expression profiles are compared between TRAP1 transfected group and control group, and the biological signaling pathways that TRAP1 affected are explored.
Materials and methods 1. Cell culture:Transient transfection of TRAP1 by siRNA in A549 cell line, scramble RNA transfected cells were negative control. Meanwhile using retroviral vector to construct stable over-expression model of TRAP 1 in MDA-MB-231 cell line, empty vector transfected cells were negative control. Thus four groups were constructed as following:A549/siRNA and A549/Scramble, MDA231/TRAP1+ and MDA231/Mock. Splited them into subgroups followed by 16 hours of either normoxic or hypoxic (0.1%O2) treatment.
2. Cells were harvested, total RNA was extracted and TRAP1 expression was verified by Taqman real time PCR.
3. Total RNA was amplified in vitro and purified followed by dye coupling of each sample. Universal Human Reference total RNA was used as reference for each sample. The coupled aRNA was again purified and the concentration was checked.
4. Microarray slides were UV-cross linked and pre-hybridized before use.
5. Each sample was mixed with same amount of reference RNA and then added onto one slide, the slides were placed into 42℃oven for 20 hours of hybridization. Slides were then washed and spinned to dry.
6. Slides were scanned using GenePix 4000B microarray scanner, the picture images were analyzed using GenePix 6.0 software, then the data were analyzed by GeneSpringTM GX10 software. Data were background subtracted and normalized by LOWESS and retrieved as the log transformed values. The filtering criteria were set as the coefficient of variation (CV)<25%and fold change>2.0 as cut-off. Thus 4 lists of differentially expressed genes were obtained, as:in normoxia, A549/siRNA Vs A549/Scr, MDA231/TRAP1+Vs MDA231/Mock; inhypoxia, A549/siRNA Vs A549/Scr, MDA231/TRAP1+Vs MDA231/Mock.
7. The gene lists were then analyzed using DAVID bioinformatics resources (Database for Annotation, Visualization and Integrated Discovery), finally genes of interest and their interaction networks were visualized by using pSTIING (Protein, Signalling, Transcriptional Interactions & inflammation Networks Gateway).
8. Taqman real time PCR was carried out for the expression of 6 genes chosen from the gene lists to verify the results of the microarray experiment.
Results
1. In either normoxia or hypoxia, the expression level of TRAP1 was obviously lower in A549/siRNA group than in A549/Scr group, and was obviously higher in MDA231/TRAP1+group than in MDA231/Mock group, indicating the high efficiency of TRAP 1 transfection.
2. After 16 hours of normoxic treatment, there were 373 differentially expressed genes in the A549 comparison groups, while in MDA231 comparison groups there were only 163 differentially expressed genes. After 16 hours of hypoxic treatment, the numbers of differentially expressed genes were 377 and 421 in the A549 comparison groups and MDA231 comparison groups respectively.
3. The analysis by using DAVID showed the affected biological pathways when TRAP1 was knocked down in A549 cells were similar to those when TRAP1 was restored in MDA231 cells, including the regulation of cell cycle, transcription, immune/inflammatory response, angiogenesis, G-protein coupled receptor protein pathway, cell adhesion, regulation of apoptosis, phosphorylation, etc. The percentage of genes involved in the regulation of cell cycle and transcription is relatively large.
4. The expression model of the 6 picked genes by Taqman real time PCR was the same as that of the microarray experiment, indicating the results of the microarray platform was trustable.
5. The analysis by pSTIING to visualize the interaction network of those differentially expressed genes showed that, TNF pathway, NF-κB pathway, Rho kinase pathway, JNK pathway and MAPK cascade pathway were influenced.
Conclusions
1. In normoxia, TRAP1 may be more important for tumor cells that normally express this protein rather than those that have lost it. Whereas at the beginning of a short period of hypoxia, tumor cells tend to start the mechanism of expressing this heat shock protein to survive through some common pathways.
2. The affected biological pathways when TRAP1 was knocked down in A549 cells were similar to those when TRAP1 was restored in MDA231 cells, which proved the role of TRAP 1 in these pathways.
3. TRAP1 may promote the cell cycle progression and proliferation of tumor cells, which is associated with TNF pathway and Rho kinase pathway and G-protein coupled receptor protein pathway.
4. TRAP1 may activate innate immune response, activate complement pathways and regulate microphage activity; it may also be involved in adaptive immunity, regulate T/B activation and T cell differentiation, and influence the cytokine production by Thl or Th2 subgroups.
5. TRAP1 promotes cell phosphorylation, in which MAPK cascade pathway plays a vital role.
6. TRAP1 might inhibit the metastasis of tumor cells, which is associated with the up-regulation of cell adhesion related genes.
7. TRAP1 may promote the angiogenesis of tumor cells, in order to maintain local blood supply and to avoid a hypoxic condition.
8. There was no obvious trend was found as for the promotion or inbition of apoptosis, which needs further study in specific tumors.
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37. Chiosis Q Lucas B, Shtil A, et al:Development of a purine-scaffold novel class of Hsp90 binders that inhibit the proliferation of cancer cells and induce the degradation of Her2 tyrosine kinase. Bioorg Med Chem.2002; 10:3555-3564.
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44. Ahlander J, Bosco G:The RB/E2F pathway and regulation of RNA processing. Biochem Biophys Res Commun.2009;384:280-283.
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88. Liu X, Zhao D, Qin L, et al:Transcription enhancer factor 3 (TEF3) mediates the expression of Down syndrome candidate region 1 isoform 1 (DSCR1-1L) in endothelial cells. J Biol Chem.2008; 283:34159-34167.
89. Eto N, Miyagishi M, Inagi R, et al:Mitogen-activated protein 3 kinase 6 mediates angiogenic and tumongenic effects via vascular endothelial growth factor expression. Am J Pathol.2009;174:1553-1563.
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96. Jin H, Pan Y, He L, et al:p75 neurotrophin receptor inhibits invasion and metastasis of gastric cancer. Mol Cancer Res.2007;5:423-433.