| BackgroundNasopharyngeal carcinoma (NPC) is a malignance originated from epithelium of nasopharynx with distinct ethnic and geographic distributions. It occurs with high frequency in Southereast Asia and southern China, with an incidence of 25-50 per 100,000 populations. NPC is also called as "Cantonese cancer" due to its high prevalence among the people in Guangdong province of southern China. Even in overseas Chinese who were born in Guangdong, the incidence of NPC is much higher than the local population. NPCs are usually undifferentiated squamous cell carcinomas and may metastasize in the early clinical stage. Worse of all, NPC is usually insensitive to radio- and chemotherapy. Genetic predisposition, EBV viralinfection, and environmental factors, play a synergistic role in the development of NPC. Mounting evidence suggestes that activation of oncogenes and inactivation of tumor suppressor genes and their unbalanced interaction is the molecular basis of NPC development. It is well known that uncontrolled proliferation is a hallmark of malignant tumor cells.. Eukaryotic cell division is a complex process that is regulated by a coordinated series of biochemical events to achieve accuracy and integrity of DNA replication. However, this process is frequently deregulated in tumor cells due to alterations of oncogenes and tumor suppressor genes that are involved in cell cycle regulation.. Perturbations of cell cycle lead to deregulated proliferation and drive tumorigenesis Resistance to apoptosis is another hallmark of tumor cells and responsible not only for tumor development but also for cancer cells’resistance to radiotherapy and chemotherapy..Apoptosis is controlled by two major regulatory pathways:one is that the bcl-2/bax apoptosis-related proteins induce the release of cytochrome C, and then activate the apoptotic effector protein caspase-3, caspase-9, PARP, and finally lead to apoptotic DNA breaks. The other pathway is through interaction of the death receptor Fas with its ligand Fas-L on the cell surface. Binding of FasL causes trimerization of Fas, which in turn activates caspase-8, caspase-3, PPAR and finally make cells undergo apoptosis.Invasion and metastasis is not only important biological characteristics of cancer, but also a key factor responsible for the clinical treatment failure. The processes of invasion and metastasis of cancer involve multiple factors. Cancer cells must pass through the blood or lymphatic vessels and then transfer to other sites of the body and eventually establish secondary tumors. Metastasis is associatyed with epithelial-mesenchymal transition (EMT). Epithelial cells are closely connected to each other by tight junctions, gap junctions and adherens junctions. They have an apico-basal polarity, polarization of the actincytoskeleton and are bound by a basal lamina at their basal surface. Mesenchymal cells, on the other hand, lack this polarization. During EMT process, epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties to become mesenchymal cells. EMT is essential for numerous developmental processes including mesoderm formation and neural tube formation. EMT has also been shown to occur in wound healing, in organ fibrosis and in the initiation of metastasis for cancer progression. Initiation of metastasis requires invasion, which is enabled by EMT. Carcinoma cells in primary tumor lose cell-cell adhesion mediated by E-cadherin repression and break through the basement membrane with increased invasive properties, and enter the bloodstream through intravasation. Later, when these circulating tumor cells (CTCs) exit the bloodstream to form micrometastases, they undergo (Mesenchymal-epithelial transition, MET) for clonal outgrowth at these metastatic sites. In the process of EMT, the mutual adhesion between cells is reduced, the epithelial cell polarity is gradually losed. Meanwhile, the capacity of cell migration and invasion is enforced. In addition, the cell phenotype also changes.For example:the loss of epithelial phenotype of cells, such as E. Cadherin (E-cadherin) and keratin filaments, and acquire of interstitial phenotypes, such as fibronectin, vimentin (Vimentin), N. Cadherin (N-cadherin), etc. Among the EMT process is a critical event of a decrease of E-cadherin levels, which makes the cells acquire the characteristics of invasion and metastasis.Because the lost of E-cadherin leads to decreased adhesion between the abnormal tumor cells and subsequently enhanced invasiveness of tumor cells. Mounting evidences demonstrated that loss of E-cadherin is closely associated with metastasis of nasopharyngeal carcinoma. Abnormal Vimentin expression is another feature of EMT. Vimentinis an important component of the cytoskeleton and plays an important role in maintaining normal cell morphology and organelles involved in cell differentiation and wound healing process.During the EMT progression, Vimentin is highly expressed, and is positively correlated with the extent of tumor invasion, high metastatic ability and poor prognostic factors in patients. Studies with tumor tissues from NPC patients showed that high expression of Vimentin is associated with poor prognosis and can be regarded as an independent factor of NPC. The progression of EMT can be induced by a variety of regulatory proteins including transcription factors Snail, Slug, ZEB1, ZEB2, Twist, etc.Their transcriptional activity can be regulated by a variety of protein kinases.The development of cancer involves a range of deregulated signaling pathways.PI3K/AKT (phosphatidylinositol 3-kinase/serine-threonine kinase) participatein a variety of growth factor signaling transduction and exert many biological functions.PI3K can be activated in two ways:The p85 regulatory subunit of PI3K binds to phosphorylated RTK(Receptor Tyrosine Kinase) such as IGF-1R and EGFR leads to conformational changes in the p110 catalytic domain of PI3K and consequent kinase activation. The other PI3K activation pathway is through binding of Ras to p110 which leads to PI3K activation directly.Activated PI3Kthen phosphorylates membrane bound PIP2 to generate PIP3 that anchors Akt to the plasma membrane and enables its phosphorylation at Ser308 by PDK1 which resultes in activation of Akt.It has been well documented that activation of PI3K/AKT pathway plays critical roles in the pathogenesis of human tumors. Active Akt can exert its anti-apoptotic role by affecting its downstream target proteins. For example: ① AKT has been shown to phosphorylate the transcription factor Forkhead family member FKHR and inhibit its nuclear translocation. In addition, active AKT can inhibit the activation of apoptosis gene target BIM and FAS ligand.②Upon activation, AKT directly phosphorylates Bcl-2 family member BAD and Caspase-9.AKT can maintain cells’ survival through a variety of pathways, and finally affect cell proliferation. For example: ①Activated P13K/AKT pathway promotes inhibitory factor (1KB) degradation, which allowes NF-κB complexes dissociate from it. The NF-κB then translocates into the nucleus, resulting in transcriptional activation, promoting cell proliferation.②AKT can also reguale the p53 tumor suppressor, thus affecting cell viability.Akt phosphorylates MDM2, a negative regulator of p53. The phosphorylated MDM2 translocates into nucleus to bind with p53, which leads to the degradation of p53.③Activation of AKT can also directly regulate cyclin-dependent kinase (CDK), thus regulating cell cycle.AKT can phosphorylate activity of GSK3β kinase, thereby preventing degradation of Cyclin D1,. AKT regulates cell cycle also through repressing CDK inhibitors (CDIs), such as p21 and p27activity. Ecotropic viral integration site-1 (Evi-1) was first identified as a common retro viral integration site in AKXD murine myeloid tumors in 1988. Evi-1 is located on human chromosome 3q26.2, with gene length of 60000 bp. It has 16 exons and is a member of the transcription factor SET/PR domain family. Murine and human Evi-1 gene is highly homologous to in nucleic acid sequences and amino acid sequence, with the rate of 91% and 94%, respectively. As a nuclear transcription factor, Evi-1 binds to GACAAGATA-like motifs in the regulated genes, thereby inhibiting or promoting the downstream gene expression.It has been demonstrated that Evi-1 promotes the growth of oocytes during early embryonic development, and promote cell proliferation and angiogenesis during interim.Meanwhile, Evi-1 transcription factor can also raise GATA-2 to promote hematopoietic stem cell proliferation.Mounting studies revealed that high expression of Evi-1 are associated with tumor development. For example, in acute myeloid leukemia, multiple myeloma and chronic leukemia cells, Evi-1 was shown to be a poor prognostic factor and is linked to patients’shorter overall survival time.Evi-1 not only plays an important role in hematopoietic malignancies but also is closely related to the development of solid tumors.Forexample, Evi-1 is often highly expressed in cancers of ovarian, colon, pancreas, and lung. The role of Evil in the pathogenesis of NPC has not been studied. Recently, our group has found that human chromosome region 3q26.2 where Evil gene resides is highly amplified in NPC. This finding prompted us to address whether Evi-1 gene is overexpressed in NPC and how Evi-1 might affect NPC development.Objective1. To identify Evi-1 expression characteristics in nasopharyngeal carcinoma tissues and cell lines, respectively.2. To explore the role of Evi-1 in regulating proliferation of NPC cells.3. To explore the role of Evi-1 in regulating migration and invasion of NPC cells.4. To explore the role of Evi-1 in regulating apoptosis of NPC cells.5. To explore whether Evi-1 modulates proliferation, apoptosis and invasion through activating AKT signaling pathway.Contents and methods1.To identify Evi-1 expression characteristics in nasopharyngeal carcinoma tissues and cell lines respectively①Detection of Evi-1 gene expression in NP-69 and NPC cell lines(SUNE-1、5-8F、 CNE-1、HONE-1、6-10B) by western blot assay.②To dectect the Evi-1 gene expression in non-NPC tissues and NPC tissues by IHC assay.2. To explore Evi-1 effect on NPC cell proliferation and investigate the molecular mechanisms①Knock down of Evi-1 in SUNE-1 and HONE-1 cells by sh-RNA. Western blot assay was performed to evaluate the knockdown efficiency.②Evaluate the effect of knockdown of Evi-1 on proliferation with the use of MTT、 EdU、colony formation assay.③Detect the proteins that regulate cell proliferation with the use of western blot assay.④Evaluate the effect of knockdown Evi-1 in nude mice.3. To find the influence of Evi-1 on cell migration and invasion and its molecular mechanisms①Transwell assay was performed to evaluate the migration ability.②Boyden assay was performed to evaluate the invasion ability.③The change of some proteins related to invasion was identified by western blot.④To identify whether or not knockdown of Evi-1 decreases the invasion ability in vivo.4. To find the influence of Evi-1 on cell apoptosis and its molecular mechanisms①Evaluate the effect of knockdown Evi-1 on cell apoptosis by the flow cytometry assay.②The change of some proteins related to apoptosis was identified by western blot.③Identify whether knockdown of Evi-1 can sensitive NPC cells to cisplatin treatment. 5. To find out Evi-1 modulates PI3K/AKT pathway through activating Ras 6. Statistical analysisAnalyze the data with the use of SPSS 16.0.The results of measurement data are represented by mean± SD.Cross table χ2 test was used to evaluate the rates between the two different groups.Independent samples t test was used to evaluate the ratio between the two different treatment groups.Cox regression model was used to evaluate Survival analysis. P<0.05 was considered as statistical different.Results1. The expression level of Evi-1 was elevated in NPC cell lines as compared to NP-69 cells. In addition, the expression level of Evi-1 was elevated in NPC tissues as compared to non-NPC tissues. The elevated expression of Evi-1 was positive associated with N and M classification, and has no correlation with sex, age, T classification, and clinical stage. Further analysis found that Evi-1 was negative associated with patients’overall survival.①Evaluate the expression level of Evi-1 in NP-69 and NPC cell lines. The results showed that when compared with NP-69, the expression level of Evi-1 was elevated in NPC cell lines. HONE-1 and SUNE-1, which have the highest level of Evi-1, were chosen for subsequent experiment.②The IHC assay showed that the Evi-1 gene was dominantly expressed in nuclear. The expression level of Evi-1 was significantly higher in NPC tissues, as compared with non-NPC tissues. The expression level of Evi-1 has no correlation with sex, age, T classification, and clinical stage, but was positive associated with N and M classification.③Survival assay showed that the expression level of Evi-1 gene was negatively associated with patients’ overall survival. 2. Evi-1 promotes NPC cells survival ability①shRNA was used to knock down the Evi-1 expression in NPC cells. In addition, Evi-1-expressing vector was used to over-expresses Evi-1 in NPC cells.②Knock down of Evi-1 impedes NPC cells proliferation ability as assessed by EdU, colony formation assays, while over-expression of Evi-1 increases NPC cells proliferation ability.③Knock down of Evi-1 decreased the CDK1 and CDK4 expression level, as detected by western blot assay. Over-expression of Evi-1 has the opponent effect, while this effect can be blocked by LY294002.④Knock down of Evi-1 impedes tumor growth in vivo. 3. Evi-1 promotes NPC cells migration and invasion①Transwell assay showed that after knock down of Evi-1, the migration ability of NPC cells was decreased. However, over-expression of Evi-1 increased the migration of NPC cells. The similar phenomena were observed in cell invasion as evaluated by Boyden assay.②After knock down of Evi-1, the protein associated with invasion was down-regulated as determined by Western blot.③Knock down of Evi-1 inhibited invasion in vivo.4. Evi-1 makes NPC cells resistant to apoptosis①Knock down of Evi-1 sensitizes NPC cells to apoptosis, while over-expression of Evi-1 leads NPC cells resistant to apoptosis.②Knock down of Evi-1 decreases the expression level of bcl-2, while increases the expression level of active caspase-3. Over-expression of Evi-1 has the opponent effect, while this effect can be blocked by LY294002.③Knock down of Evi-1 sensitizes NPC cells to cisplatin treatment, while over-expression of Evi-1 render NPC cells resistant to cisplatin. 5.Evi-1 modulates PI3K/AKT pathway through avtivating Ras①Knock down of Evi-1 decreases the expression level of K-ras, while over-expression of Evi-1 promotes the expression of K-ras.②In Evi-1 knockdowned cells, over-expression of K-ras can actives PI3K/AKT pathway. In Evi-1 over-expressed cells, knock-down of K-ras reverses the effect of Evi-1.Conclusion1. Evi-1 gene is highly expressed in NPC cell lines and NPC tissues. High expression of Evi-1 is associated with N classification and M classification of NPC patients. In addition, high expression of Evi-1 is negative associated with patients’ overall survival.2. Evi-1 gene can promote NPC cell proliferation and invasion, but lead to apoptosis resistance.3. Evi-1 exerts its function through modulating K-as/PI3K/AKT signaling pathway. |
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