Effect And Mechanisms Of Interaction Of Human Papillomavirus Type16E6Protein And Daxx

Objective: To provide the experimental basis for further studying effects of Daxx oncarcinogenic mechanisms of human papillomavirus type16（HPV16）E6protein,thetranscriptional regulation of HPV16E6protein interacting with Daxx on its DNA promoterand impact of Daxx on E6DNA replication in the Hela cells were explored.Methods:(1) Caski cells fixed with formalin were cut with sonication into chromatin fragments,andthe centrifuge supernatant received were divided into four groups: They were respectivelyGroup A incubated with rabbit anti-HPV16E6IgG, GroupB incubated with Anti-RNApolymeraseⅡ,Group C as input control,Group D incubated with mouse IgG.After everysupernatant group was incubated with antibody for overnight,antigen-antibody complexesprecipitated wih Protein G Agarose were washed with eluent. Then DNA productions ofevery supernatant were purified and their Daxx gene fragments were detected usingPolymerase chain reaction (PCR).(2) The specific primers including NcoI or HindⅢ restriction enzyme site were designedby the PRIMER5.0software.PCR was respectively used to amplify Daxx promoterfragments involving Daxx-1~-161nt,-161~-695nt and-1~-695nt.After the PCR productionswere digested with the restriction enzymes, the fragments were respectively inserted intothe same enzyme sites of pGL3-basic.Then the recombinants were transformed intoescherichia coli. Positive cloneswere screened and identified by DNA sequencing anddigestion of restriction enzymes. The recombinants plasmid of pGL3-basic/Daxx-1~-695nt,Daxx-1~-161nt,Daxx-161~-695ntwere respectively constructed.(3) pGL3-basic,pGL3-basic/Daxx-1~-695nt,pGL3-basic/Daxx-1~-161nt,pGL3-basic/Daxx-161~-695ntor with pcDNA3.1(-)/HPV16E6were respectively transfected into Hela cells.Twenty-four and forty-eight hours later,Hela cells were lysed and luciferase reagents were added into the supernatants. Their relative fluoreccence intensities were detected byfluorescence analyzer.(4) The plasmid of pEGFP-c1or pEGFP-c1/Daxx were transiently transfected into Helacells. Twenty-four hours and forty-eight hours later, Hela cells were lysed and thetotal RNA was extracted. Absolute quantitative PCR assay was used to detect E6genecopy number. The plasmid of pcDNA3.1(-)/HPV16E6was as an internal standard.Results:(1) Daxx DNA promoter fragments were observed by PCR amplificationin the chromatinimmune complexes precipitated with anti-HPV16E6antibody.(2) Every fragment (-1~-161nt,-161~-695nt or-1~-695nt) of Daxx gene promoter wasrespectively correctly inserted in to pGL3-basic. The luciferase reporter vectors of pGL3-basic,pGL3-basic/Daxx-1~-695nt,pGL3-basic/Daxx-1~-161nt,pGL3-basic/Daxx-161~-695nt,weresuccessfully constructed.(3) After pGL3-basic/Daxx-1~-695nt,pGL3-basic/Daxx-1~-161ntor pGL3-basic/Daxx-161~-695ntwas transfected into Hela cells, expressions and promoter activities of promoter fragmentsDaxx-1~-695nt,Daxx-1~-161nt or Daxx-161~-695nt were detected.The activities of thesepromoter fragments were respectively decreased in Hela cells co-transfected withpcDNA3.1/HPV16E6. The activity of Daxx-161~-695nt fragment was the lowest.(4) After pEGFP-c1or pEGFP-c1/Daxxwas transfected into Hela cells, thegreenfluorescent proteins were observed at24hours and48hours. The levels of HPV16E6gene replication were not significant change in Hela cells over-expressing GFP,but theyobviously decreased in Hela cells over-expressing GFP-Daxx.Conclusions:(1) HPV16E6protein can bindDaxx promoter DNA fragment-1~-161nt.(2) The plasmid of pGL3-basic/Daxx-1~-695nt, pGL3-basic/Daxx-1~-161ntor pGL3-basic/Daxx-161~-695ntcan actively expressin Hela cells. Overexpression of HPV16E6proteinsdownregulate the transcriptional activity of Daxx promoter, and the critical region is thepromoter fragment of Daxx-161nt~-695nt.(3) Overexpression of Daxx can reduce the levels of HPV16E6gene duplication.