| Background and objectiveThe incidence rate of cervical cancer is only second to that of breast cancer among those of female malignant tumor.HPV(human papillomavirus)infection is not only the primary initiating factor of cervical cancer,but also the key factor of carcinogenesis,among which HPV16 is most common and likely to cause cancer.HPV16 genome consists of early gene region,late gene region and long regulatory region.The early gene region encodes six early proteins,namely E1,E2,E4,E5,E6 and E7 proteins.HPV16 E2 is the major transcriptional regulator of viral gene expression,and also the initiation protein of viral replication.HPV16 E6 is one of the major oncogenes of HPV16,which involves in cell cycle arrest,gene transcription activation and cell apoptosis by inhibiting the biological activities of p53,which is conducive to the development of cancer.HPV16 E2 can inhibit the transcription of oncogene E6/E7,thus reduce their expression products and weaken the transformation ability of HPV as a negative regulator of HPV oncogene, and also can activate p53 to inhibit cell proliferation.Meanwhile,HPV E2 can affect cell proliferation independently of the other HPV proteins,which may be an apoptotic inducing factor by activating exogenous apoptosis pathway.Death domain associated protein(Daxx)is a highly conserved multifunctional nuclear protein,which plays an important role in transcriptional regulation,apoptosis and signaling pathway.It can induce apoptosis by activating Fas-Daxx-ASK1-JNK signal pathway.The biological activity of Daxx is closely related to its subcellular localization.As an adaptor interacting with proteins,Daxx can regulate the transcription of various cytokines.The interaction of Daxx with HDAC1,p53,Pax3,Axin or other proteins such as some viral proteins can affect its own localization and normal function.Our previous studies showed that HPV16 E6 could interact with Daxx in He La cells,suggesting that it may affect cell apoptosis;HPV18E2 can co-locate with Daxx in the nucleus of He La cells,but HPV16 E2 and Daxx were co-located in the cytoplasm of Caski cells;Daxx expression transferred its location gradually from the nucleus to the nuclear membrane,cytoplasm and cell membrane after CIN Ⅱstage.Totally,it is speculated that the different localization of Daxx in cervical cancer cells may be related to HPV16 E2;and HPV16 E2 may be involved in Daxx-induced apoptosis.Therefore,this study aims to understand the most common genotype for cervical cancer prevention and its pathogenic mechanism by investigating the current status of HPV infection and its genotype distribution among women in Hengyang,then to clarify the interaction between HPV16 E6 and Daxx and determine the effects of HPV16 E6 on Daxx-induced apoptosis in HPV negative cervical cancer cell line C33 A cells so as to analyze whether or not HPV16 E2 can affect Daxx regulating apoptosis by down-regulate the transcription and expression of HPV16 E6 in a p53-independent manner in non HPV transformed cells,and further to explore the apoptosis regulation mechanism of HPV16 E2 and Daxx in HPV infected cells.It is of great significance for the study of analyzing the epidemic status of HPV and approaching the pathogenic mechanism of cervical cancer in order to provide an experimental basis for the treatment of cervical cancer.Methods1.The clinical data of 1 2053 female patients with suspected gynecological diseases were retrospectively analyzed.The cervical exfoliated cells of patients were detected for HPV positive by polymerase chain reaction(PCR),and then those positive were amplified for HPV genotype by HPV PCR flow fluorescence method.Meanwhile,a series of medical examinations such as pathological histology were performed to diagnose intraepithelial neoplasia or tumor related diseases about cervix,uterus or pelvis.SPSS version 18.0 was used to analyze the statistical data.The binomial distribution was used to estimate the HPV infection rate,single or multiple HPV infection and HPV genotype.Chi square test was used to compare the difference of the infection rate of HPV and HR-HPV and the positive ratio of HR-HPV and LR-HPV.2.HPV16 positive cervical cancer tissues and its adjacent tissues were collected and then fixed with formaldehyde to make pathological sections.The expression and localization of HPV16 E2 in cervical tissue cells were detected by immunohistochemical test.The expressions of HPV16 E2 protein in cervical tissue cells were identified by SDS-PAGE and Western blot.The effects of HPV16 E2 on HPV16 E6 m RNA level in C33 A cells transfected with pcDNA3.1(+)/HPV16 E2 and pcDNA3.1(+)/HPV16 E6 plasmid were detected by QPCR.The effects of HPV16 E2 on E6 protein expression by SDS-PAGE and Western blot.3.The total protein was extracted from C33 A cells transfected with pcDNA3.1(+)/HPV16 E6 eukaryotic expression plasmid,and then the interaction between Daxx and E6 was assessed by immunocoprecipitation tests.The distribution and co-localization of Daxx and HPV16 E6 were observed in C33 A cells transfected with p Ds Red-C1/HPV16 E6 plasmid by direct and indirect immunofluorescence.The effects of HPV16 E6 on Daxx m RNA level were detected by QPCR.The effects of HPV16 E6 on Daxx protein expression by SDS-PAGE and Western blot.4.The inhibitory effects of HPV16 E6 on the cell proliferation were detected in C33 A cells transfected with pcDNA3.1(+)/HPV16 E6 eukaryotic expression plasmid by MTT method.The apoptotic cells were observed with Hoechst staining and the effects of HPV16 E6 on cell apoptosis were assessed by flow cytometry.The effects of HPV16E6 on Caspase-8 activities in C33 A cells were determined by spectrophotometry.Then,the flow cytometry was used to verify the effects of HPV16 E2 on HPV16 E6 inhibiting Daxx-induced apoptosis in C33 A cells.5.HPV16 positive cervical cancer tissues and its adjacent tissues were collected and then fixed with formaldehyde to make pathological sections.The expression and localization of Daxx in cervical tissue cells were detected by immunohistochemical test.The expressions of Daxx protein in cervical tissue cells were identified by SDS-PAGE and Western blot.The Daxx m RNA level was detected by QPCR.Meanwhile,the whole Daxx gene in cervical tissue cells was amplified by PCR,and then the PCR products were sent to Shanghai Shenggong for sequencing.The interaction between Daxx and HPV16 E2 was assessed by immunocoprecipitation test.6.The inhibitory effects of HPV16 E2 on the cell proliferation were detected in C33 A cells transfected with pcDNA3.1(+)/HPV16 E2 eukaryotic expression plasmid by MTT method.The apoptotic cells were observed with Hoechst staining and the effects of HPV16 E2 on the cell apoptosis were determined by the flow cytometry.7.The distribution and localization of Daxx in C33 A,Si Ha,Caski and He La cells were observed by indirect immunofluorescence,and the distribution and co-localization of Daxx and HPV16 E2 were observed in He La cells transfected with p Ds Red-C1/HPV16 E2 eukaryotic expression plasmid by direct and indirect immunofluorescence.The distribution and co-localization of Daxx and PML were observed in He La cells transfected with pcDNA3.1(+)/HPV16 E2 eukaryotic expression plasmid by indirect immunofluorescence.The effects of HPV16 E2 on the cell apoptosis under the treatment of LMB were determined by the flow cytometry,so as to identify the pro-apoptosis effects of HPV16 E2 by changing the nuclear localization of Daxx.8.The total RNA was extracted from the cells transfected with pcDNA3.1(+)/HPV16 E2 plasmid to synthesize the c DNA strand.The effects of HPV16 E2 on Daxx m RNA level were detected by QPCR.The total protein extracted from the transfected cells was used to be analyzed the effects of HPV16 E2 on Daxx protein expression by SDS-PAGE and Western blot.Meanwhile,using c DNA as the template,the whole Daxx gene was amplified by PCR,and then the PCR products were sent to Shanghai Shenggong for sequencing.9.The total protein of transfected cells with pcDNA3.1(+)/Daxx eukaryotic expression plasmid was extracted,and then the effects of Daxx with different concentrations on JNK and p-JNK protein expression were analyzed by SDS-PAGE and Western blot.The effects of Daxx on Caspase-8 activity in the transfected cells were determined by spectrophotometry.The effects of HPV16 E2 with different concentrations on JNK and p-JNK protein expression and Caspase-8activity in cells transfected with pcDNA3.1(+)/HPV16 E2 plasmid were analyzed the same as the above treatments.The effects of HPV16 E2 on Daxx-induced apoptosis were determined by the flow cytometry.10.The effects of HPV16 E2 on JNK and p-JNK protein expression under the low expression of Daxx were confirmed in He La cells transfected with pcDNA3.1(+)/HPV16 E2 plasmid and p LVX-sh RNA1-Daxx plasmid by SDS-PAGE and Western blot and the Caspase-8 activities by spectrophotometry,respectively.The apoptosis effects of HPV16 E2 with lower expression of Daxx were detected in He La cells transfected with p LVX-sh RNA1-Daxx lentivirus recombinant expression plasmid and pcDNA3.1(+)/HPV16 E2 plasmid by the flow cytometry.Results1.Among 12 053 women cases,1 224 cases were HPV positive, including 1 003 cases with single infection and 221 cases with multiple infection.The total HPV infection rate was 10.16% and the multiple genotype infection rate was 1.83%.HPV16 was the most common and its infection rate was 2.19%.The other five most common were HPV58,HPV52,HPV39,HPV51 and HPV53,respectively.The infection rate of HR-HPV was 8.52%.Among 1 224 HPV positive patients,HR-HPV positive patients accounted for 83.91%,which was more than LR-HPV patients(16.09%).The HPV infection rate and HR-HPV infection rate(26.32%,22.63%)of the group over 60 years old were both the highest,of which HR-HPV-infected patients accounted for 86%.The HPV infection rate of patients under 21 years old was 23.21%,ranking the second,but the proportion of HR-HPV-infected patients was relatively low(16.07%).The infection rates of HPV and HR-HPV in inpatients(12.52%,10.40%)were significantly higher than those in outpatients(8.96%,7.57%),respectively.Among 4048 hospitalized patients,the HPV infection rate and HR-HPV infection rate of 50～60 years old group(42%,34%)were both the highest in all age groups,respectively.The infection rates of HPV and HR-HPV in over 60 years old group were26.58% and 24.05% respectively.Among the 507 hospitalized patients with HPV infection,HR-HPV positive inpatients occupied 90.49% in284 patients with histopathological examination,which was significantly higher than that proportion in 223 patients without histopathological examination(78.03%)(p < 0.01).Also,HR-HPV positive patients accounted for 95.71% in the patients with cervical cancer or precancerous lesions,which was the highest proportion among HPV-infected hospitalized patients(p < 0.05).In 52 cases of HPV positive cervical squamous carcinoma,the first six genotypes of HPV infection were HPV16,HPV52,HPV39,HPV51,HPV58,HPV18 and HPV53,among which HPV16 infection accounted for 61.54%.2.HPV16 E2 was expressed in the cytoplasm and nucleus of epithelial cells from HPV16 positive cervical tissues,but it expressed absent or little in cervical cancer tissues.In C33 A cells with the expression of HPV16 E2,HPV16 E6 m RNA level and its protein expression were down-regulated(p < 0.05),indicating that HPV16 E2 could suppress the transcription and expression of HPV16 E6.3.In C33 A cells,the expressed HPV16 E6 protein could co-locate and interact with Daxx.Although the over-expression of HPV16 E6 could down-regulate Daxx m RNA level to a certain extent when Daxx was highly expressed p < 0.05),it had no significant effect on the expression of Daxx protein(p > 0.05).4.HPV16 E6 had no significant effect on the amount of living cells and the proliferation of C33 A cells(p > 0.05).However,HPV16 E6 significantly increased the amount of living cells and decreased the proliferation inhibition rate of over-expressed Daxx on cells(p < 0.05), suggesting that HPV16 E6 could down-regulate the growth inhibition induced by the over-expression of Daxx.And,HPV16 E6 significantly reduced the apoptotic rate of those cells with the over-expression of Daxx(p < 0.05),which also suggested that HPV16 E6 might inhibit cell apoptosis induced by the over-expressed Daxx.The further expression of HPV16 E2 inhibited the down-regulation of HPV16 E6 on cell apoptosis induced by the over-expression of Daxx(p < 0.05).5.In HPV16 positive cervical cancer cells,Daxx was distributed in not only the nucleus,but also the nuclear membrane,cytoplasm and membrane.The m RNA levels and protein expression of Daxx in HPV16 positive cervical cancer tissues were significantly higher than those in the adjacent tissues of cervical cancer(p < 0.01;p < 0.05),and there was the missing of Daxx gene fragment in cervical cancer.HPV16 E2 antibody can detect the compounds precipitated by Daxx or HPV16 E2antibodies;Daxx antibodies also can detect these compounds,indicating that HPV16 E2 could interact with Daxx in HPV16 positive cervical cells.6.He La cells expressed HPV16 E2 with different concentration all had the apparent growth peaks after 48 h,and then became dropped(p < 0.05).Among the HPV16 E2 transfection group from 2 μg group began to repress cell proliferation(p < 0.05).It was confirmed that HPV16 E2 could inhibit He La cells proliferation and induce cells apoptosis by the flow cytometry.7.In non HPV transformed C33 A cells,Daxx was expressed mainly in the nucleus and rarely in the cytoplasm.In HPV16 transformed Si Ha cells,Daxx expressed in cytoplasm and nucleus,and the expression in nucleus was stronger than that in cytoplasm.In HPV16 transformed Caski cells,Daxx was mainly expressed around the nuclear membrane and in the cytoplasm.In HPV18 transformed He La cells,Daxx was mainly concentrated in the nucleus,but there was little cytoplasmic expression in few cells.It was suggested that the different localization of Daxx may be related to HPV16 or HPV16 E2.In He La and C33 A cells,whether HPV16 E2 and Daxx existed in cytoplasm or nucleus,there was co-localization between them,suggesting the expression of HPV16 E2 could lead to the nuclear expression trend of Daxx.In He La cells expressed with HPV16 E2,Daxx and PML were still largely expressed in the nucleus,but there were also a little expression in the cytoplasm of some cells,and they were still co-located with each other.When He La cells were treated with LMB,the up-regulation of HPV16 E2 on Daxx-induced apoptosis was down-regulate(p < 0.05),which suggested that HPV16 E2 maybe induce cell apoptosis through changing the location of Daxx or other proteins from nucleus to cytoplasmic.8.Meanwhile,HPV16 E2 could partly up-regulate the m RNA level and protein expression of Daxx(p < 0.05),although it did not increase in a concentration-dependent manner.However,there were no effect of HPV16 E2 on Daxx gene sequence.9.Daxx could activate JNK and promote the activation of Caspase-8,which were increased with a concentration-dependent manner.HPV16 E2 can also activate JNK and promote Caspase-8activity in a concentration-dependent manner.HPV16 E2 could up-regulate cell apoptosis induced by the over-expression of Daxx(p <0.01).It was suggested that HPV16 E2 could promote cell apoptosis by activating JNK and Caspase-8 activity via Daxx.10.After interfering with Daxx expression,HPV16 E2 had no significant effect on p-JNK protein level and Caspase-8 activity(p >0.05),which was certified that the interference with Daxx expression did not reduce HPV16 E2-induced apoptosis(p > 0.05).It further suggested that HPV16 E2 induced apoptosis by inducing the change of Daxx localization and also indicate Daxx was not the only pathway for HPV16E2 activating JNK.Conclusions1.HPV16 is the most common genotype of HPV infection among women in Hengyang,and HPV16 infection is the most in the patients with cervical cancer.2.HPV16 E2 can down-regulate E6 expression in a p53-independent manner and promote Daxx-induced apoptosis of cervical cancer cells.3.HPV16 E2 can promote Daxx expression and its nuclear translocation to induce apoptosis through Daxx/JNK pathway in cervical cancer cells.4.HPV16 E2 can promote cervical cancer cells apoptosis through JNK pathway in a Daxx-independent manner. |
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