| Backgrounds and objectiveTumor 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.Results1. 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.Conclusions1. 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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