Proteomic Profiling Between Low-and High-invasion-migration Nasopharyngeal Carcinoma Cells And Functional Study Of Metastasis Related Genes

Nasopharyngeal Carcinoma (NPC) is one of the ten most common malignant tumors in China. Eighty percent of NPC cases in all over the world exist in China. The morbility of NPC in South China is almost100times than in Europe. The etiology of NPC involves infection of Epstein Barr Virus, heredity, ingestion of nitrosamine and environmental factors (such as high concentration of Cadmium or Niccolum in drinking water). Lymph nodes metastasis might occur even in the early stage of NPC. Whether accompanied with lymph nodes metastasis is one of the most important indicators determining the prognosis of NPC. Metastasis is the chief reason for failure in NPC treatment. Despite the diagnostic and therapeutic techniques is continuously developed,60-85%patients had suffered metastasis when they got the final diagnosis, so the5-year survival rate of NPC patients is hard to be enhanced. It is a huge challenge we have to face that to explore the molecular mechanism of NPC matastasis, to search new therapy target and to prevent NPC spread from situ.It is an important approach in the modern medical research to probe the biological property of tumor cells by using cell strain. The first high differentiated NPC epithelial cell strain, CNE-1, was established in1976, then the low differentiated one, CNE-2, was established in1980in China. In2006, Qian isolated29subclones from CNE-2by limited dilution methods. And it was found that clone18(S-18) possesses stronger capacity in invasion and migration comparing with the parental cell line and other subclones. Since S-18is derived from CNE-2, except the different metastastic ability, it has similar property in heredity, pathology and biology with CNE-2. The individual variant will be the minimum when investigation is performed between these two cell lines. So the isolation of S-18provides a convenient and reliable platform for exploring the underlying mechanism between NPC cells with different metastatic capacity.Proteomics is one of the basis and components of the systemic biology. Its role becomes more and more important with the coming of post genomic era. Two-dimensional gel electrophoresis (2-DE) is a classic method in proteomic researches, by which the protein mix detracted from cells, tissues or other bio-samples can be analyzed. So far,2-DE is the only technique which can isolate and indicate thousands of proteins from samples. It has higher resolution, better reproducibility and more feasible for micropreparation than other methods and is especially necessary for the expression proteomics.2-DE, computer based image analysis and large scale datamation are the supporting techniques in proteomics researches. By utilizing these advanced techniques in the system biology and proteomics, we hope to disclose the underlying differences between S-18cells and their parental cells on the level of "discovery science".After proteins significantly expressed are identified by proteomics screening, it is necessary to explore the functions of these proteins so that the underlying molecular mechanisms can be revealed. Gene-overexpression technique, also well known as gene-transfection technique, is a method that to study the functions of genes (proteins) or to perform gene therapy by transduction of exogenous genes into target cells so that the corresponding proteins are expressed. The lentivector based on HIV-1is an prospective gene-transfer vector for gene function study and gene therapy as it can transfect non-dividing cells, can stably and constantly express candidate proteins by integrating target gene into the host genome, and can avoid strong immune response. RNA interference (RNAi) is a phenomenon of post-transcriptional gene silencing (PTGS) which is induced by double-stranded RNA which is sequence-homologous with target gene. Since it was discovered by Fire in1998, RNAi has been proved to be a specific, efficient and economic approach to inhibit gene expression. Combined with gene-overexpression technique, RNAi can be used to further demonstrate functions of genes and corresponding proteins in an opposite direction.In the present study, proteomic technique was used to profile the differently expressed proteins between NPC cell line CNE-2and its high metastatic subclone S-18, and eighteen proteins were found (10proteins are up-regulated and8proteins are down-regulated in S-18cell line). Through bioinformatics analysis, seven of the eighteen proteins were believed concerning closely with cancer metastasis. And the expression levels of four proteins in the two cell lines were further verified by Western Blot. That is, compared with CNE-2, S-18has higher heat shock protein27(HSP27) and Ezrin level, and lower Keratin18and vasolin containing protein (VCP) level. Previous studies indicated that HSP27and Ezrin could significantly promote metastatic ability of cancer cells, and Keratinl8had the property to stabilize cell skeleton and cell morphology. The abnormal expression of these genes may be the important internal factors that result in the high metastatic potential of S-18.HSP27is the chief member of small HSP family, and is also the most obvious and extensive induced one of the chaperons. HSP27perform protective role in normal cells, however, it is usually a marker for bad prognostic in cancer patients. More and more researchers pay attention to the carcinogenetic and metastasis-enhancing potential of HSP27. It is commonly regarded that the carcinogenetic property of HSP27is associated with its anti-apoptosis capacity. But we know very little about the underlying mechanism of metastasis-enhancing potential of HSP27. In order to further the study about the molecular mechanism of HSP27in cancer metastasis, we utilized lentivector to over-expressed HSP27in NP-460, which is a normal nasopharyngeal epithelial cell line with low endogenous HSP27level; and used RNA interference technique to decrease the high HSP27level in S-18cell line. Then we used transwell test to determine the invasive and migratory ability overexpressed or silenced cells, and utilized real time PCR to detect the transcription levels of NF-κ B, MMP2, MMP9and MMP11in HSP27-silenced S-18cells. Data indicated that the invasive and migratory capacity of NP-460was significantly enhanced after HSP27was overexpressed, and the invasive and migratory capacity of S-18was significantly attenuated accompanying with the levels of NF-κ B, MMP9and MMP11were significantly downregulated after HSP27was inhibited. Interestingly, the transcriptional level of MMP2was not influenced with inhibition of HSP27.Based on the above data, we believe that HSP27plays a vital role in metastatic process of NPC cells, and we hypothesize it is via the following three probable mechanisms that HSP27promotes the metastatic ability of NPC cells. Firstly, HSP27can activate the NF-κ B signal pathway by enhancing the transcriptional level of NF-K B, by increasing the activity of IKK or by directly promoting the degradation of I κ B, then MMP9and MMP11will be upregulated and activated, so that the invasive capacity of NPC cells are enhanced. Secondly, HSP27can promote the activity of MMP2without influencing its transcriptional level. Thirdly, HSP27can enhance the deformability and motility of NPC cells by interacting with F-actin, Keratin8and Keratin18, which are cytoskeleton proteins.Altogether, for the goal to explore the molecular mechanisms of metastasis of NPC cells, NPC cell line CNE-2and its high metastatic subclone S-18were selected as the researching objects. Proteomics was utilized to profile the proteins differently expressed between the two cell lines and eighteen proteins were identified. Among these proteins, HSP27and Ezrin can promote metastasis of tumor cells, Keratin18has the property to stabilize cytoskeleton and cell shape. By using lentivectors and RNA interference technique, the metastasis-enhancing function of HSP27in NPC cells was verified. The molecular mechanisms of HSP27in promoting metastasis may be closely associated to NF-κ B signal pathway, activity of MMPs and its interaction with the cytoskeleton-related proteins such as F-actin, Keratin8and Keratin18. These findings will provide useful clues for further studying mechanisms of metastasis, finding new therapy targets and reducing metastasis of NPC.