HPV16 E7 Protein Antagonizes TNF-α-induced Apoptosis Of Cervical Cancer Cells Through Daxx/JNK Pathway

Objective: To study the interaction between Human papilloma virus type 16 E7(HPV16 E7)protein and death domain-associated protein(Daxx)and its colocalization,so as to explore the apoptosis-antagonizing of HPV16E7 on cervical cancer cells through Daxx/JNK pathway under the induce of TNF-α,which can provide the theoretical basis for the further study on HPV16 E7 in the pathogenesis of HPV.Method:1.According to the sequence of HPV16 E7 gene,the specific primers were designed,and then amplified through PCR by using the whole HPV16 gene as a template.After the double enzyme digestion,they were ligated with pcDNA3.1(+),pDsRed-monomer-C1 and pET30 a plasmids to construct the recombinant plasmids,which were sequenced for identification.2.The pcDNA3.1(+)/HPV16 E7 plasmid was extracted and transfected into HeLa and C33 A cells,respectively.After collecting the total cell protein,the co-immunoprecipitation technique was used to analyze whether HPV16E7 can interact with Daxx in the cell.3.The constructed pET30a/HPV16 E7 recombinant plasmid was induced to express protein by IPTG.Then the recombinant protein was purified with Ni-NTA resin and treated with polymyxin to remove endotoxin for later use.4.The pDsRed-monomer-C1/HPV16 E7 or pEGFP-C1/Daxx was transfected alone or co-transfected into HeLa and C33 A cells,respectively,and some cells transfected with pEGFP-C1/Daxx with the treation of HPV16E7 protein.Then the subcellular localization of HPV16 E7 or Daxx was analyzed by direct immunofluorescence under the excitation confocal microscope.5.HeLa and C33 A cells were treated with different concentrations of HPV16 E7 protein(0,5,10,15,20,25 μg/mL)for 24 hours,respectively.After collecting the total cell protein,the expression of Daxx,Bax,and Bcl2 proteins in the cells was analyzed by Western blot,respectively.HeLa and C33 A cells were treated with HPV16 E7 protein when the Daxx protein expression was the highest for different durations(0,6,12,18,24 h),respectively,and the expression of Daxx,Bax,and Bcl2 proteins in the cells as before.6.HeLa and C33 A cells were treated with HPV16 E7 protein at a concentration of 15 μg/mL for 18 h,and then TNF-α was used to induce cells apoptosis for 5 h.The expression of Daxx,Bax and Bcl2 protein was analyzed by Western blot,respectively,and the apoptosis of cells was analyzed by DAPI staining and flow cytometry.7.HeLa and C33 A cells were transfected with pGPU6/Neo-SiDaxx and pGPU6/Neo-Si empty vectors,respectively,and then treated with HPV16 E7 protein and TNF-α.The expression of Daxx,Bax and Bcl2 proteins was analyzed by Western blot,the apoptosis of cells was analyzed by DAPI staining and flow cytometry,and the influence of interference with Daxx expression on cell proliferation activity was analyzed by MTT.8.HeLa and C33 A cells were treated with HPV16 E7 protein for different durations(0,15,30,60,and 120 minutes).After collecting the total cell protein,the expression of t-JNK and p-JNK were detected by Western blot.HeLa and C33 A were pretreatment with different concentrations of JNK agonist anisomycin for 15 min,respectively.And then the cells were treated with HPV16 E7 protein and TNF-α.The expression of Bax and Bcl2 proteins was analyzed by Western blot,and the apoptosis of cells was analyzed by DAPI staining and flow cytometry.Result:1.The results of gene sequencing of the constructed vectors showed that pcDNA3.1(+)/HPV16 E7,pDsRed-monomer-C1/HPV16 E7 and pET30a/HPV16 E7 recombinant plasmids were successfully constructed.2.The results of co-immunoprecipitation analysis showed that HPV16E7 and Daxx can interact in HeLa and C33 A cells.3.SDS-PAGE and Western blot identification of HPV16 E7 protein expression found that the induced expression of HPV16 E7 protein showed a specific band at 28 k Da.4.Direct immunofluorescence and excitation confocal microscope were used to observe the subcellular localization of HPV16 E7 and Daxx protein.When pDsRed-monomer-C1/HPV16 E7 is co-transfected with pEGFPC1/Daxx,HPV16 E7 and Daxx fluorescent fusion protein can undergo cytoplasmic translocation.After treatment with HPV16 E7 protein,Daxx fluorescent fusion protein could also undergo cytoplasmic translocation.5.The results of expression of Daxx,Bax and Bcl2 detected by Western blot found that HPV16 E7 protein could up-regulate Daxx protein expression in a dose and time-dependent manner and produce anti-apoptotic effects in HeLa and C33 A cells.6.The results about effects of HPV16 E7 protein on cell apoptosis detected by Western blot,DAPI staining and flow cytometry was found that HPV16 E7 protein treated HeLa and C33 A cells could up-regulate of Daxx and Bcl2 protein expression,down-regulae of Bax protein,and increase the ratio of Bcl2/Bax,but decrease apoptosis.7.Western blot identification results of pGPU6/Neo-SiDaxx interference vector showed that transfection of pGPU6/Neo-SiDaxx can effectively interfere the Daxx protein expression in HeLa and C33 A cells.Western blot,DAPI staining and flow cytometry were used to detect the effects of Daxx on anti-apoptotic effect of HPV16 E7 protein,it was found that the interfering in Daxx expression can reduce the anti-apoptotic effect of HPV16 E7 protein.And the results of MTT analysis showed that the interference in Daxx expression could reduce the effect of HPV16 E7 protein in promoting cell proliferation.8.Western blot detection of JNK expression results show that HPV16E7 protein can inhibit the activation of JNK pathway.And when Western blot,DAPI staining and flow cytometry were used to detect the effect of JNK on the anti-apoptotic effect of HPV16 E7 protein,it was found that after pretreatment with anisomycin,the anti-apoptotic effect of HPV16 E7 protein was weakened,suggesting that the JNK signaling pathway is involved in HPV16 E7 antagonizing cell apoptosis.Conclusions: HPV16 E7 can interact with Daxx and affect their subcellular localization so as to antagonize the apoptosis of cervical cancer cells induced by TNF-α through Daxx/JNK pathway.