RNA Interference C-Met Pro-oncogene Effects On The Nasopharyngeal Carcinoma And Its Mechanism

Part 1 The Expression of c-Met in Nasopharyngeal Carcinoma and its clinical significanceObjective:To investigate the expression of proto-oncogene c-Met in nasopharyngeal carcinoma and its clinical significance, and to explore the role of c-Met on invasion and metastasis in nasopharyngeal carcinoma.Material and Methods:Immunohistochemical SP method and RT-PCR were detected in 31 cases of NPC and 10 cases of chronic nasopharyngitis tissues of c-Met protein and c-Met mRNA expression levels, and to contact their clinical and pathological information.Results:c-Met protein in NPC biopsies positive expression was significantly higher than nasopharyngitis organization, the difference was significant (P<0.01). c-Met expression and nasopharyngeal carcinoma clinical stage, lymph node metastasis (P<0.05); with age, gender, histological type were not related (P>0.05). c-Met mRNA in nasopharyngeal biopsy tissues were significantly higher than nasopharyngitis organization, the difference was significant (P<0.05). Conclusion:Nasopharyngeal carcinoma c-Met protein and mRNA levels were highly expressed, c-Met and its high expression of invasion and metastasis are closely related, c-Met can be used as nasopharyngeal carcinoma and the prognosis of treatment. Part 2 pGP-silencer-U6/Neo/GFP/c-Met eukaryotic expression plasmid and identification of biological activityObjective:Design for the human c-Met gene targeting shRNA, the shRNA construct the recombinant plasmid carrying detect the expression of the inhibitory effect of c-Met, the most powerful screening shRNA interference RNA fragments.Methods:Gene for c-Met mRNA sequence of three interfering targets, were constructed three shRNA plasmid expression vector and a random sequence of plasmid expression vector, the E. coli amplification, restriction enzyme digestion, PCR, DNA sequencing, using liposomes Mediated method to transfer to the NPC CNE-2 cells, using real-time PCR and western blot detection of c-Met gene expression at the mRNA and protein were compared before and after transfection the expression of differences, the judge of the shRNA Interference effect.Results:Confirmed by sequencing, pGP-silencer-U6/Neo/GFP/c-Met successfully constructed the eukaryotic expression plasmid. Plasmid transfected CNE-2 cells, c-Met gene mRNA levels and protein levels were significantly reduced, in which the strongest effect of recombinant plasmid 2, mRNA levels decreased 90%,75% lower protein levels.Conclusion:Successfully constructed for the c-Met gene RNAi plasmid for further research on c-Met gene in nasopharyngeal carcinoma CNE-2 cells the function of the biological behavior of the foundation. Part 3 c-Met gene silencing on the role of the biological behavior of NPC cell researchObjective:To investigate the effects of RNAi to c-Met gene on proliferation, invasion, metastasis and apoptosis of human NPC cell line CNE-2.Methods:Transfection pGP-silencer-U6/Neo/GFP/c-Met established eukaryotic expression vector and control plasmid stability in random sequence monoclonal cells were used to CCK-8, matrix adhesive with experiments, in vitro migration assay, transwell invasion in vitro experiments, flow cytometry before and after transfection cell proliferation, cell adhesion, cell motility, cell invasion and cell apoptosis rate.Results:After downregulation of c-Met, CNE-2 cell proliferation, cell adhesion on Matrigel was significantly lower (P<0.05), CNE-2 cells in vitro migration, invasion was significantly lower (P<0.01), CNE-2 cell apoptosis rate was increased (P<0.05).Conclusion:Proto-oncogene c-Met was significantly promoted the NPC CNE-2 cell lines in vitro proliferation, adhesion, migration and invasion, inhibition of nasopharyngeal carcinoma CNE-2 cell apoptosis. Suggest c-Met in the NPC plays an important role in malignant progression, is a potential therapeutic target in nasopharyngeal carcinoma. Part 4 Effects of c-Met gene silencing on human nasopharyngeal carcinoma xnograft model of nude miceObjective:To explore the effects Of c-Met shRNA eukaryotic expression vector on human nasopharyngeal carcinoma CNE-2 cell growth of nude mice.Methods:Establishment of nasopharyngeal carcinoma xenograft in nude mice to observe the c-Met shRNA eukaryotic expression vector of anti-tumor efficacy, respectively, in CNE-2 tumor cells in the body formed the base of multi-department and c-Met shRNA microinjection of eukaryotic Plasmid expression vector, injected with a random vector and saline as a control group, tumor volume growth curves recorded, and mice were killed at 4 weeks, remove the tumor, using western blot and TUNEL were used to detect tumor oncogenes c-Met protein expression and apoptosis of tumor cells in situ.Results:None of 36 mice died, weight evenly increased tumor formation rate of 100%. In the injection group, c-Met shRNA eukaryotic expression vector group of the growth of tumor volume than the corresponding random vector injection and the saline group slowly. Tumor removed in the c-Met protein expression and in situ TUNEL results suggest: transplanted tumors relative to normal saline control group and random plasmid control group, c-Met shRNA eukaryotic expression of c-Met containing protein group significantly decreased (P<0.05);transplanted tumors and normal saline control group and random plasmid compared apoptosis, c-Met shRNA eukaryotic expression contained in the apoptosis rate increased (P<0.05).Conclusion:c-Met shRNA eukaryotic expression vector significantly inhibited human nasopharyngeal carcinoma CNE-2 cells in nude mice after tumor growth, shRNA eukaryotic expression vector-mediated gene therapy promises to be targeting c-Met nasopharyngeal carcinoma treatment The new strategy.