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Objective: To investigate the clinical significance and predictive value of human papillomavirus (HPV) 16/18 E6 protein in the different cervical intraepithelial neoplasia. Methods: The expression of HPV16/18 E6 in 10 normal cervical tissues, 33 cervical intraepithelial neoplasia I (CIN I ), 31 CIN II-III, 30 cervical cancers was detected by immunohistochemistry, explored the expression difference and the relationship with the clinicopathological characteristics of cervical cancer and the prognosis of different CIN. Results: The positive expression rates of HPV16/18 E6 in normal cervical tissues, CIN I, CIN II-III and cervical cancer group were up-regulated (χ2=19.82, P=0.000). HPV16/18 E6 increased positive expression rates in the low grade and the big size tumors of cervical cancer tissues were detected (P=0.033, P=0.011). There were positive correlations between the overexpression and the pathological grade, tumor size, poor prognosis of cervical cancers respectively (r=0.456, P=0.011; r=0.578, P=0.000; r=0.645, P=0.000). The sensitivity, specificity, and diagnostic accuracy rates of HPV16/18 E6 positive expression to the progression of CIN I, CIN II-III and cervical cancer were respectively 100.00%, 62.50%, 43.75%; 96.77%, 91.30%, 92.86%; 96.97%, 83.87%, 66.67%. Conclusion: HPV16/18 E6 overexpression plays an important role in the generation, development and the poor prognosis of cervical cancer. HPV16/18 E6 has a good predictive value for the prognosis and hierarchical management of cervical diseases.
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Objective·To investigate the clinical significance and predictive value of human papillomavirus (HPV) 16/18 E6 protein in the different cervical intraepithelial neoplasia. Methods·The expression of HPV16/18 E6 in 10 normal cervical tissues, 33 cervical intraepithelial neoplasiaⅠ (CINⅠ ), 31 CINⅡ- Ⅲ, 30 cervical cancers was detected by immunohistochemistry, explored the expression difference and the relationship with the clinicopathological characteristics of cervical cancer and the prognosis of different CIN. Results·The positive expression rates of HPV16/18 E6 in normal cervical tissues, CINⅠ , CINⅡ - Ⅲ and cervical cancer group were up-regulated (χ2=19.82, P=0.000). HPV16/18 E6 increased positive expression rates in the low grade and the big size tumors of cervical cancer tissues were detected (P=0.033, P=0.011). There were positive correlations between the overexpression and the pathological grade, tumor size, poor prognosis of cervical cancers respectively (r=0.456, P=0.011; r=0.578, P=0.000; r=0.645, P=0.000).The sensitivity,specificity,and diagnostic accuracy rates of HPV16/18 E6 positive expression to the progression of CINⅠ,CINⅡ-Ⅲand cervical cancer were respectively 100.00%, 62.50%, 43.75%; 96.77%, 91.30%, 92.86%; 96.97%, 83.87%, 66.67%. Conclusion·HPV16/18 E6 overexpression plays an important role in the generation, development and the poor prognosis of cervical cancer. HPV16/18 E6 has a good predictive value for the prognosis and hierarchical management of cervical diseases.
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Objective To prepare a peptide monoclonal antibody (McAb)against human papillomavirus 18E6 separately,and identify its specificity and pathogenicity.Metheds The advantage epitope peptide was designed and synthesized by ABCpred and Bcepred,and then used to immunize BALB/c mice after coupling with bovine serum albumin (BSA).And the McAb was prepared by hybridoma technique.HPV18E6 gene was amplified from cervical swab specimen containing HPV18 and insert-ed into expression vector pET-28a.The constructed recombinant plasmid was transformed to E.coli BL21(DE3)for expres-sion under induction of isopropyl thio-β-D-galactoside.The expressed protein was used to identified the McAb had been pre-pared.Results The hybridoma cell lines could constantly produce MAbs against HPV18E6 peptides.Sequencing proved that recombinant plasmid pET-28a-HPV18E6 was constructed correctly.Western blotting showed that the anti-HPV18E6 pep-tides antibody could specifically recognize HPV18E6.Conclusion A monoclonal antibody against the advantage epitope pep-tide of human papillomavirus 18E6 prepared could specifically recognize HPV18E6 specifically.
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Objective To explore the relationship of signal transduction among human papillomavirus 18 E6 oncoprotein (HPV18E6), signal transducers and activators of transcription 1 (STAT1), protein kinase R( PKR )/α subunit of eukaryotic initiation factor 2 ( eIF2α ), nuclear factor-kappa Bp65 ( NF-κBp65 ), mitogen-activated protein kinase( MAPK)/c-Jun N-terminal kinase(JNK) ,and possible molecular mechanism. Methods Construct two lentiviral vectors which contain shRNA interfering sequence aiming at the targets of HPV18E6 oncogene and NC sequence( HPV18E6-RNAi-LV, NC-GFP-LV), based on the transduction with HPV18E6-RNAi-LV and NC-GFP-LV into HeLa cell to interfere the expression of HPV18E6 oncogene and NC sequence,the expressions of mRNA and protein( including phosphating patem)of HPV18E6, STATI, PKR, eIF2α, NF-κBp65, MAPK, JNK are measured with RT-PCR and Western blot, the difference of proliferation and sensitivity to carboplatin of HeLa cell are determined with Transwell cell methods and MTT among every groups. Results The expression of HPV18E6 oncogene can affect the expression level of mRNA and protein of NF-κBp65 and PKR genes, also affect phosphating levels of phosphating protein p-STAT1, p-PKR and p-eIF2α;the restraining rates of proliferation and sensitivity to carboplatin of HeLa cell are higher in HPV18E6-RNAi-LV group than the other groups( P＜0. 05 or P＜0.01 ). Conclusion HPV18E6 oncoprotein not only reduces the expression of PKR but dephosphorylates p-STAT1, pPKR and p-eIF2α to restrain activation of PKR/eIF2α signal transduction passage, maintain the proliferation and invading ability of HeLa cell and restrain apoptosis. The signal transduction among HPV18E6, MAPK/JNK are not clear.
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Objective To elucidates the effects of HPV18 E6 siRNA targeting at human papillomavirus(HPV)18 E6 gene on the proliferative activity of HeLa cells and chemotherapy sensitivity.Methods HPV18 E6 expression of HeLa cells was inhibited by siRNA interference,the change of P53 and P21 proteins expression level was measured by Western blot.MTT assay was used to detected proliferative activity and sensitivity to paclitaxel liposome of HeLa cells.Results After inhibition of E6 expression,P53 and P21 proteins increased and the growth of HeLa cells was decreased(P ＜0.01).The inhibition rate of HeLa was markedly increased after transfection of HPV18 E6 siRNA and paclitaxel liposome.Conclusion HPV18 E6 siRNA can effectively silence gene expression of E6 and inhibit proliferation of HeLa cells.HeLa cells are more sensitive to combine HPV18 E6 siRNA with paclitaxel liposome than that of control groups.
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By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of reporter genes were investigated.Total mRNA extracted from Hela cells was reversely transcribed into cDNA.Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector.The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing.The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector were transformed into the yeast cell AH109,respectively.After they were cultured respectively in YPDA liquid medium and nutrition-deficient culture medium,their toxicity and transcriptional activation were tested by both the phenotype assay and the color assay.The bait plasmid HPV18 E6 was successfully obtained.After being cultured in YPDA liquid medium for 16h,the A600 nm values of two yeast fluids were 0.98±0.03 and 0.99±0.02,respectively.The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector could grow to white colonies on SD/-Trp/X-α-gal plates,while no colony could survive on SD/-His/-Trp/X-α-gal,SD/-Ade/-Trp/X-α-gal plates,indicating that the bait plasmid pGBKT7-HPV18 E6 was constructed successfully and expressed correctly,and could not activate the transcription of reporter gene alone.The yeast two-hybrid GAL4 system 3 can be utilized to find HPV18 E6 interacting proteins.
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Background and purpose: It has been reported that activation of Notch1 could strongly inhibit proliferation of HPV (human papilloma virus)-positive HeLa cells by down-regulation of the E6 and E7 genes. The aim of this paper was to investigate the role of the Notch signaling pathway in growth arrest of EC109 cells in vitro and the molecular mechanism. Methods: EC109 cell lines, a well differentiated human ESCC (esophageal squamous cell carcinoma) cell line with HPV18-positive, was used in the study. Exogenous intracellular domain of Notch1(ICN) was transfected into cultured EC109 cells by lipofectamine transfection, the proliferation of the transfected cells was measured by an MTT assay. Cell cycle distribution was analyzed by flow cytometry. Human papilloma virus type 18 (HPV18) E6/E7 mRNA expression was detected by RT-PCR, and p53 protein expression was detected by Western blot.Results: Activation of Notchl signaling resulted in inhibition of EC109 cell proliferation with the induction of G_2/ M arrest. There was a significant difference in terms of the percentage of G_2/M phase cells among the ICN-transfected group (42.57±1.57)% and the non-transfected group (1.88±0.66)% or the empty plasmid transfected group (1.99±1.02)% (P<0.01). Down modulation of HPV18 E6/E7 gene expression and upregulation of p53 expression was (2.15±0.23) in ICN-transfected group higher than non- transfected group (0.45±0.07) and empty plasmid transfected group (0.46±0.02) (P<0.01). Conclusion: Repression of HPV18 E6/E7 expression by Notch1 signaling results in growth suppression of HPV18-positive EC109 cells with concomitant activation of p53-mediated pathways.
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To screen for novel binding proteins interacting with high-risk HPV 18 E6 oncogene, the strain AH109 was transformed with pGBKT7-HPV18 E6 plasmid, and subsequent transference was utilized to screen for interacting proteins with HPV 18 E6 in human Hela cDNA library. HPVl8 E6 mRNA was expressed in yeast and there was no self-activation and toxicity in AH109. Seven proteins that interacted with HPV18 E6, including transmembrane protein 87B, phosphonoformate im- muno-associated protein 5, vimentin, KM-HN-1 protein, dedicator of cytokinesis 7, vaccinia related kinase 2 and a hypothetical protein, were identified. It was suggested that yeast two-hybrid system is an efficient for screening interacting proteins. The high-risk HPV 18 E6 oncogene may interact with the proteins, which may be associated with signal transduction and transeriptional control, epithelial cell invasion and migration, as well as humoral and cellular immune etc. This investigation provides functional clues for further exploration of potential oncogenesis targets for cancer biotherapy.
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Objective To study the effect of small interfering RNA(siRNA) of human papillomavirus(HPV) 18(E6) gene on apoptosis of HPV-related cervical HeLa cell line.Methods siRNA targeting HPV18 E6 mRNA was designed and generated by PCR amplification.The PCR products containing U6 promoter and the siRNA sequence were then transfected into HeLa cells via Lipofectamine()~(TM)2000.Cell viability was determined by MTT assay.Apoptosis was detected by morphological observation and flow cytometry analysis.The expression level of HPV18 E6 mRNA was assayed by RT-PCR.Results The cell growth and viability of(siRNA) transfected group were significantly inhibited(P
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Objective In order to study the pathogenesis of human papillomavirus(HPV) and seek for a therapeutic approach of the diseases caused by HPV, the construction of HPV18 E6E7 antisense RNA expressing recombinants was studied. Methods We amplified the HPV18 E6E7 816bp by PCR with HPV18 plasmid DNA as the template. pLNSX retroviruses were used as vectors,the HPV18 E6E7 retrovirus recombinants were constructed. And then the recombinants were cleaved with restriction endonuclease and hybridized with Southern blot for identifying the inserting direction and special check respectively. Results and conclusion The HPV18 E6E7 antisense RNA retrovirus expressing recombinants were screened and obtained,which had laid the foundation of studying the function of E6E7 genes further and explore whether the antisense technique can adjust and control the expression of E6E7 genes.
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Objective:To evaluate the role of human papillomavirus(HPV)16/18E6,cyclin D1,and human telomerase transcriptase(hTERT)in the development of laryngeal carcinoma by detecting the expression of these three genes.Methods:Immunohistochemistry was used to detect the levels of HPV16/18E6,cyclin D1,and hTERT in 42 cases of paraffin-embedded tissues.Results:(1)The positive expression ratios of HPV16/18E6,cyclinD1,and hTERT in the laryngeal carcinoma group were 42.9%(18/42),50%(21/42),and 83.3%(35/42),respectively.These values were significantly higher than that in the control group.(2)The increase in the expression of HPV16/18E6 and cyclinD1 was associated with T stage advance and lymph node metastases.The expression of hTERT increased along with T stage advance,but not with lymph nodes metastases.The changes in the expression of HPV16/18E6,cyclinD1,and hTERT were not associated with gender and age.(3)Positive correlation was observed between HPV16/18E6 and cyclinD1 as well as between HPV16/18E6 and hTERT in laryngeal carcinoma.Conclusion:(1)The expression of HPV16/18E6,cyclin D1,and hTERT may be associated with the development of laryngeal carcinoma.(2)There may be interactions between HPV16/18E6 and cyclinD1,between HPV16/18E6 and hTERT.These interactions may induce the development of laryngeal carcinoma.The information provided by this study may be important for further investigation into the relationship between HPV16/18 and laryngeal carcinoma.