Expeirmartal Studies On The Inhibition Of AD-ING4-OSM On Proliferation Of Human Nasopharyngeal Carcinoma Cell Line CNE-2in Vitro

Objective:To investigate the inhibitory effect and mechanisms of adenovirus vector-mediated gene co-expression (AD-ING4-OSM) in nasopharyngeal carcinoma cell line CNE-2in vitro.Methods:Recombinant adenoviral vectors were transfected into HEK293t cells for high-tilter amplification. Different doses of empty adenovirus vector (Ad-GFP) carrying a green fluorescent protein (GFP) infected nasopharyngeal carcinoma CNE-2cells to determine the optimal multiplicity of infection (MOI)..then the transcription of ING4and OSM genes were detected by semi-quantitative RT-PCR; Apoptotic effect of CNE-2cells were evaluated by FCM(dyed by PI);Growth inhibitory effect of CNE-2cells were assessed by MTT; Nuclear morphological changes in CNE-2cells apoptosis were detected by Hoechst33258.The expression of P21, P27, P53and Survivin were detected by semi-quantitative RT-PCR.Results:Recombinant adenoviral vectors could be amplified in HEK293t cells successfully, and after repeated infection HEK293t cells, the virus titer of approximately1.3-2.7×109pfu/ml.The most suitable dose of Ad-GFP used to infect the human nasopharyngeal carcinoma cell line CNE-2was100MOI.Exogenous ING4and OSM were proved successful transcribed in human nasopharyngeal carcinoma cell line CNE-2by semi-quantitative RT-PCR, And endogenous ING4and OSM genes were not detected in the control group and the blank group. PI single staining flow cytometry (FCM) to detect apoptotic effect of nasopharyngeal carcinoma CNE-2double gene co-expression group was superior to the single gene ING4and OSM groups,which have no significant differences each other, but better than the blank group and empty virus group (P<0.05);MTT assay found that co-expression of the two genes group human nasopharyngeal carcinoma CNE-2cell growth inhibitory effect was significantly better than the single gene of ING4and OSM groups (P<0.05),no significant difference between the single-genome groups(P>0.05),but better than the blank group and empty virus group(P<0.05). The human nasopharyngeal carcinoma CNE-2cells apoptotic nuclear morphology changes more significantly in double gene co-expression group than others were detected by Hoechst33285. RT-PCR analysis showed that double gene co-expressed can obviously up-regulated P53, P27, P21and downward Survivin cell cycle and expression of apoptosis-related genes.Conclusion:1.Compared with the single-gene the AD-ING4-OSM double gene co-expression can be significantly inhibit of human nasopharyngeal carcinoma CNE-2cell growth and promote apoptosis in vitro, Which mechanism of action may be up-regulated P53, P27, P21, and downward Survivin cell cycle and apoptosis-related gene expression.2.The adenovirus can be used as safe and efficient vector for nasopharyngeal gene therapy.