Establishment Of HaCaT Cell Line Stably Transfected By HPV16 E7 Oncogene And Its Effect On The HLA-â…  Antigen Expression

OBJECTIVETo transfect HPV16 E7 gene into HaCaT cell through pCMV-tag2B plasmid and construct the HaCaT-E7 cell lines.To determine whether HPV16 E7 oncogene could down-regulate surface HLA classâ… antigen in HPV16 E7 transfected cells,then explore possible mechanisms.METHODS1.Eatablishment of HaCaT-E7 cell lines HaCaT cells in logarithmic phase were stably transfected with pCMV-E7 using Lipofectamine 2000 according to the manufacturer's instructions.Following transfection,the cells were selected in DMEM containing 500μg/ml G418 for 21 days.After this time,G418-resistant colonies were marked,individually picked and expanded into clonal cell lines for analysis.HPV16 E7 expressed stable cell lines were maintained in 200μg/ml G418 containing medium. As a control,empty vector pCMV-Tag2B was transfected into HaCaT cells in the same condition.2.Identification of HaCaT-E7 cell lines Total RNA was isolated from normal HaCaT group,HaCaT-E7 group,HaCaT-pCMV group cells using Trizol and reverse transcribed into cDNA.The expression of HPV16 E7 mRNA was detected by real-time PCR,and a fragment of humanβ-actin was amplified as internal standard. Total protein was isolated from normal HaCaT group,HaCaT-E7 group, HaCaT-pCMV group cells.After SDS-PAGE,the expression of HPV16 E7 protein was detected by Western Blot.3.FACS analysis of HLA-â… expression The cells of normal HaCaT group, HaCaT-E7 group,HaCaT-pCMV group were detached from the flask,and then were washed and resuspended in PBS/1%bovine serum albumin(BSA,PBS-B)at a concentration of 10⁷ cells/ml.For the detection of surface HLA classâ… molecules,100μl of cells were aliquoted and incubated with 20μl FITC-Mouse Anti-Human HLA-ABC monoclonal antibody for 30 min at 4℃in the dark.The cells were then washed twice as above,resuspended in 400μl PBS-B and analyzed by a flow cytometer.4.Two-step real-time PCR analysis of HLA-â… mRNA expression Total RNA was isolated from normal HaCaT group,HaCaT-E7 group,HaCaT-pCMV group cells using Trizol and reverse transcribed into cDNA.The expression of HLA-A,B,C mRNA was detected by real-time PCR,and a fragment of humanβ-actin was amplified as internal standard.Comparative CT method(2^{-ΔΔCT})was used to analyze the relative changes in gene expression.RESULTS1.The monoclone was found after selecteded for 21days by 500ug/ml G418. Expression of HPV16 E7 mRNA was detected in HaCaT-E7 group using two-step real-time PCR,and the averageΔCT between HPV 16 E7 andβ-actin was 13.27±0.86. The amplificated fragment of HPV16 E7 PCR products was 93bp and coincided with the objected fragment.As shown of Western Blotting,expression of HPV16 E7 protein was detected in HaCaT-E7 cells and coincided with its expression in Caski cells.This indicated the HaCaT-E7 cell lines were sucessfully established.2.HLA-â… was measured in the normal HaCaT group,HaCaT-pCMV group and HaCaT-E7 group cells:the average mean fluorescence intensity(MFI)was 357.65±15.65,346.82±16.77,182.16±16.47,respectively.HLA-â… in HaCaT-E7 group was significantly down-regulated than in other two groups and there was statistical difference between them(P＜0.001).Membrane cofactor protein CD46 is ubiquitously expressed on the surface of human cells,and the expression of CD46 was not significantly altered among HPV16 E7 transfected cells,the empty vector control and parental cells(P=0.689).This data indicated that expression of HPV 16 E7 in stable transfected cells had no general downregulatory effects on cell-surface proteins,but only resulted in a specific decrease in cell-surface HLA classâ… molecules.3.Expression of HLA-A,B,C mRNA were detected in normal HaCaT group, HaCaT-pCMV group and HaCaT-E7 group:the level of HLA-A,B,C mRNA was determined relative toβ-actin by the 2^{-ΔΔCT}method,and there were no statistical difference of HLA-A,B,C mRNA expression among the three gourps(HLA-A, P=0.954;HLA-B,P=0.781;HLA-C,P=0.821).CONCLUSION1.Transfect HPV16 E7 gene into HaCaT cell through pCMV-tag2B plasmid,the HaCaT-E7 cell lines were sucessfully established.2.After selecteded for 21days by G418,HLA-â… in HaCaT-E7 group was significantly down-regulated,compared to other two groups.3.Expression of HLA-A,B,C mRNA of HaCaT-E7 cell lines were not down-regulated,indicating that the down-regulaion of HLA-â… happen in assemble phase,involving post-transcriptional mechanisms.