Construction Of USP22Shrna Lentiviral Vector And The Study Of It’s Interference Of Human Nasopharyngeal Carcinoma Cells CNE-2

OBJECTIVE: An USP22shRNA lentiviral vector is constructed and transfected intohuman nasopharyngeal carcinoma cells in this work. The inhibition efficiency ofexpression of USP22gene by this shRNA lentiviral vector is also carefullyinvestigated. This study establishes an USP22shRNA lentiviral vector stableinterferential cell line for investigating the mechanisms of USP22in nasopharyngealcarcinoma.METHODS: Two specific interfere sequences were designed according to USP22gene sequences. Both of the sequences contain the restriction enzyme sites HpaI andXhoI. After annealing and phosphorylation, these interfere sequences were linked topLL3.7lentiviral expression vector. Through transformation and cultivation, weextracted plasmid by restriction enzyme digestion electrophoresis with HpaI andXhoI.The correct plasmid was analysis by sequencing. Resulting lentiviral expressionvector was co-transfect in293T cells along with the packaging plasmids (pGag/Pol,pRev and pVSV-G). The titer and infection efficiency were determined by observationof the expression of GFP under fluorescence microscopy. With appropriatemultiplicity of infection, human nasopharyngeal carcinoma cells were infected bylentivirus. We examine inhibitory efficiency of the expression of USP22gene byqRT-PCR and Western blot, and get the rate of the inhibition.RESULTS: The lentiviral expression vector of pLL-USP22-shRNA was successfullyconstructed and identified by using restriction enzyme digestion electrophoresis withHpaI and XhoI and sequencing analysis. The results proved that the correct vector wasobtained. The titer of concentrated virus measured by293T cells was4×107TU/ml,which showed that the the lentivirus is suitable for infecting target cells. When themultiplicity of infection was50, the infection efficiency of USP22shRNA lentiviral vector to human nasopharyngeal carcinoma cells CNE-2was＞95%. The results ofqRT-PCR and Western blot showed that the lentiviral vector can successfully inhibitthe expression of USP22gene in CNE-2, and the inhibition rates was64.03%.CONCLUSIONS: The USP22shRNA lentiviral vector was successfully constructed,and it was able to inhibit the expression of USP22gene of human nasopharyngealcarcinoma cells CNE-2. This study established the USP22shRNA lentiviral vectorstable interference CNE-2cell lines, which provide the basis for future establishmentof biological function of USP22gene in human nasopharyngeal carcinoma cells.