| Nasopharyngeal carcinoma (NPC) is a common human malignancy in head and neck, which has a remarkably geographic distribution in south China and Southeast Asia. Study on etiology indicated that the infection of Epstein-Barr virus (EBV), hereditary susceptibility, eating habit and some environmental factor play an important role in the development of NPC, whose development is an complicated process with multiple gene and multiple procedure, in which EBV plays an important role. Among the EBV encoded proteins, latent membrane protein 2A (LMP2A) is involved in the long-term latence of EBV in the epithelial cell, and plays an important role in the development of NPC. Our research aimed to construct a lentivirus RNAi specific for human LMP2A gene, and to transfect nasopharyngeal carcinoma C666-1 cell, then to observe its effects on the biological behaviors and radiosensitivity of nasopharyngeal carcinoma C666-1 cell, in order to offer a new strategy for therapy of NPC.Part I Construction and identification of LMP2A-RNAi lentivirusObjective:To construct a lentivirus expressing RNAi targeted human LMP2A gene, which silences the LMP2A gene in human nasopharyngeal carcinoma C666-1 cell line. Methods:We first identified the mRNA sequence for human LMP2A, and designed 3 different LMP2A specific shRNA sequences and 1 negative control sequence, according to the principles of shRNA design. DNA oligos containing the target sequence were chemically synthesized, annealed, and inserted into the pGCSIL-GFP expression vector by double digestion with Age I/EcoR I enzymes, and ligation with T4 DNA ligase, then were transformed into competent cells. After the positive colonies were screened by using PCR technology and DNA sequencing, we obtained the recombinant plasmids. The most effective pGCSIL-LMP2A-shRNA recombinant plasmid was chose by detecting the LMP2A mRNA expression in the transfected C666-1 cells, using real-time PCR. The chosen pGCSIL-LMP2A-shRNA plasmid and package plasmids were infected into 293T cells by lipo2000, to produc pGCSIL-LMP2A-shRNA lentivirus. The titer of lentiviral vectors were calculated based on GFP expression in 293T cell. After the C666-1 cells were transfected by pGCSIL-LMP2A-shRNA lentivirus, the mRNA expression of LMP2A was determined by real-time PCR method, the expression of LMP2A protein in C666-1 cell was verified by Western blot analysis. Results:Digestion of pGCSIL-GFP vector with Age I/EcoR I produced 7.5 kb bands. The DNA fragments were ligated into pGCSIL-GFP vector. The titer of the constructed pGCSIL-LMP2A-shRNA lentivirus was about 3×108 TU/ml. The mRNA and protein expession of LMP2A were significantly decreased by the constructed lentivirus. Conclusion:We successfully constructed a recombinant lentivirus expressing RNAi targeted human LMP2A gene.Part Ⅱ Effects of lentivirus-mediated LMP2A-RNAi on biological behaviors of nasopharyngeal carcinoma cellsObjective:To study the effects of lentivirus-mediated LMP2A-RNAi on biological behaviors of human nasopharyngeal carcinoma C666-1 cell line. Methods:Human nasopharyngeal carcinoma C666-1 cell line was infected by pGCSIL-LMP2A-shRNA lentivirus, which was set as KD group. Cell infected with negative lentivirus was set as NC group, with the cultured cell set as CON group. Colony formation was determined by colony forming assay. Cell proliferation was tested by MTT assay. Cell cycle was observed by fluorescence activated cell sorting (FACS). The rate of apoptosis was evaluated by flow cytometry analysis for annexin V-APC staining. The proportion of SP cells was detected by SP flow cytometry. The mRNA and protein expression of Notchl in C666-1 cells were determined by real-time PCR and Western blot, respectively. Results:After infection of pGCSIL-LMP2A-shRNA lentivirus, the colony formation decreased significantly. After 72h, the OD value in the KD group was 0.238±0.002, which decreased significantly (P<0.05), compared with two control groups. The proportion of G0/G1 phase, S phase, G2/M phase in KD group were 52.57±1.09%,32.58±1.08%,14.85±0.97%, respectively. S phase of KD group was decreased, as compared with two controls. Flow cytometry analysis demonstrated that apoptosis rate in the KD group was 14.32±0.38%, which was increased as compared with two controls (P<0.05). After C666-1 cell line was infected by pGCSIL-LMP2A-shRNA lentivirus, the proportion of SP cells decreased, the mRNA and protein expression of Notchl gene were down regulated. Conclusion:Lentivirus-mediated RNAi knockdown of LMP2A inhibits the proliferation and colony formation of C666-1 cells, and increases apoptosis rate, in which down-regulating the Notchl and decreasing the SP cells play an important role.Part Ⅲ Effects of lentivirus-mediated LMP2A-RNAi on radiosensitivity of nasopharyngeal carcinoma cellsObjective:To study the effects of lentivirus-mediated LMP2A-RNAi on radiosensitivity of human nasopharyngeal carcinoma C666-1 cell line. Methods:Human nasopharyngeal carcinoma C666-1 cell line was infected by pGCSIL-LMP2A-shRNA lentivirus and then irradiated, which was set as KD group. Cell infected with negative lentivirus was set as NC group, with the cultured cell set as CON group. Colony formation was determined by colony forming assay. Cell proliferation was tested by MTT assay. Cell cycle was observed by fluorescence activated cell sorting (FACS). The rate of apoptosis was evaluated by flow cytometry analysis for annexin V-APC staining. Results:After irradiation, the colony formation of pGCSIL-LMP2A-shRNA lentivirus group decreased significantly. After 72h, the OD value in the KD group was 0.135±0.004, which decreased significantly (P<0.05), compared with two control groups. The proportion of GO/Gl phase, S phase, G2/M phase in KD group were 55.37±1.08%,31.48±1.07%,13.15± 12.94%, respectively. S phase of KD group was decreased, as compared with two controls. Flow cytometry analysis demonstrated that apoptosis rate in the KD group was 21.12±0.45%, which was increased as compared with two controls (P<0.05). Conclusion:Lentivirus-mediated LMP2A-RNAi enhances the radiosensitivity of NPC cell line C666-1.In this study, we successfully construct pGCSIL-LMP2A-shRNA lentivirus, using LMP2A as target gene, lentivirus as RNAi expression vector. The pGCSIL-LMP2A-shRNA lentivirus significantly decreases the mRNA and protein expression of LMP2A gene, and down-regulates the expression of Notch1, reduces the proportion of SP cells, furtherly inhibits colony formation and cell proliferation and induces cell apoptosis, and enhances the radiosensitivity in human nasopharyngeal carcinoma cell line C666-1, providing a new idea and method for therapy of nasopharyngeal carcinoma. |
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