Experimental Study On Nasopharyngeal Carcinoma HNE-1 Cell Line By SiRNA Interfering HPV16E6 Gene

BackgroundNasopharyngeal carcinoma(NPC) is a common malignant tumour of head and neck in the Southern China.Radiotherapy is an effective treatment of early NPC patients.But for advanced NPC patients,the effect is poor,and recurrence and metastasis often happen.Therefore, it is extremely urgent to look for a new therapeutic method.New RNA interference (RNAi) is very valuable in the treatment of tumors.It is some small double-stranded RNA(dsRNA) that can be used to block the expression of particular gene, impel the mRNA to degrade and get the result of the'gene silencing'and inhibiting the cell proliferation and invasion.It has been confirmed that Human papilloma virus(HPV)has close relation to head and neck malignant tumor,and high-risk type 16/18 plays an important role in the HPV infection.Protein expression levels of HPV16 E6 is the necessary condition of malignant phenotype in cancer.It has been reported that RNAi technology interfering HPV16E6 gene can inhibit the cell proliferation and induce the cell apoptosis.However,little report about nasopharyngeal carcinoma HNE-1 cell line can be read.This research aims to investigate the effect of RNAi technology interfering HPV16E6 gene on nasopharyngeal carcinoma HNE-1 cell line. ObjectiveIn this study,with the siRNA against HPV16E6 transfected cells,we interfered HPV16E6 gene transcription and protein expression on nasopha- ryngeal carcinoma HNE-1 cell lines,and investigated the cell proliferation, apoptosis and invasion on HNE-1 cells after transfection.Methords1.Transfecting siRNA into HNE-1 cell:we designed four pairs of E6 siRNA after looking up HPV16E6 gene sequence.After transfecting siRNA into HNE-1 cell,we observed the transfection efficiency using fluorescence microscopy.2.Picking out the effective siRNA.Groups in experiment:four pairs of E6 siRNA groups and negative control group.RT-PCR was used to detect the expression of HPV16 E6 and protein level of HPV16 E6 was measured by Immunohistochemical staining and Western blot.Then,the effective siRNA against HPV16E6 was picked out.We selected blank group,the effective siRNA group and negative control group to following experiment.3.Testing the cell proliferation after transfection using MTT.4.Measuring the cell cycle by Flow cytometry(FCM).5.Detecting apoptosis by FCM.6.Determining invasion by transwell experiment.Results1.Under the fluorescence microscope,the green fluorescence with the cells could be seen after transfection,suggesting the transfection was successful.The transfection efficiency was 60 percent.2.After 48hours of transfection,RT-PCR showed that after transfecting siRNA-3 into HNE-1 cell,the HPV16E6 mRNA inhibition rate was 66.3 percent.Compared with other groups,the difference was statistically significant.Western blot analysis showed that siRNA-3 could significantly inhibit the expression of HPV16E6 protein and the inhibition rate was 56.7 percent(p<0.05).Immunohistochemical assay showed that the number of HPV16E6 positive cells in the negative control group is 65.2 percent, compared with 20.1 percent in the siRNA-3 group,suggesting siRNA-3 could decreased the expression of HPV16E6 protein.3.MTT showed that the inhibition ratio of cell proliferation were 24%, 23%, 30% in 24h, 48h, 72h respectively after transfection.The result was statistically significant.4.The cell cycle was detected by FCM.The study showed that the ratio of G0-G1 phase was 80.46 percent after siRNA-3 transfection and negative control group rate of 65.55 percent.The difference was statistically signify- cant.5.Though FCM,the cell apoptosis rate is about 16.82 percent after siRNA-3 transfection.Compared with negative control group, there was a significant difference.6.Transwell experiment showed that the number of cells which crossed the membrane was 45.3 in siRNA-3 transfection group,146.7 in blank group and 147.7 in negative control group(p<0.05).Conclusion1.siRNA against HPV16E6 could effectively inhibit the expression of HPV16E6 and the protein level of HPV16E6 on nasopharyngeal carcinoma HNE-1 cell line.2.siRNA interfering HPV16E6 could significantly reduce the cell pro- liferation on nasopharyngeal carcinoma HNE-1 cell line.3.siRNA interfering HPV16E6 could decrease the DNA synthesis(the proportion of cell in S phase reduced)on nasopharyngeal carcinoma HNE-1 cell line.The cell cycle was blocked in the G0-G1 phase. 4.siRNA interfering HPV16E6 could induce the cell apoptosis on nasopharyngeal carcinoma HNE-1 cell line.5.siRNA interfering HPV16E6 could downregulate the invasion on nasopharyngeal carcinoma HNE-1 cell line.