The establishment of in vitro model will provide optimal conditions for the study of human papillomavirus (HPV)-associated cervical cancer. In this study, E6 and E7 gens of HPV31 were cloned and expressed in E. coli. The recombinant proteins were purified and used as antigens to immunize mice for the production of polyclonal antibody. Mammalian expression plasmid pBudCE4. 1-HPV31-E6/E7 was also constructed and transfected into C33A cells. The transfected cells were then selected by Zeocin. The expressions of the E6 and E7 mRNAs and proteins were detected by RT-PCR and Western blot respectively. A stable cervical cancer cell line was established as an in vitro model for the study of human papillomavirus type 31(HPV31) associated cervical cancer.
Animals ; Cell Line ; virology ; Female ; Human papillomavirus 31 ; genetics ; metabolism ; Humans ; Mice ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomavirus E7 Proteins ; genetics ; metabolism ; Papillomavirus Infections ; virology ; Recombinant Proteins ; genetics ; metabolism ; Transfection

We wished to screen the cell line that stably expresses the HPV18E5 protein, and to ascertain the influence of HPV18E5 protein on cell proliferation and the cell cycle. The HPV18E5 gene was amplified by the polymerase chain reaction. Then, the His-tag pSecTag-HPV18E5 eukaryotic expression vector was constructed by digestion ligation and connection. The recombinant plasmid was transfected into Balb/c3T3 cells with lipofectamine, and positive cell lines were screened by a culture medium containing bleomycin. HPV18E5 expression in cells was confirmed by western blotting and immuno-enzymatic methods. The influence of HPV18E5 on cell proliferation and the cell cycle were detected by Cell Counting Kit-8 and flow cytometry, respectively. The pSecTag-HPV18E5 eukaryotic expression vector was constructed. After 21-day selection in a culture medium containing 400 μg/mL bleomycin, stably expressing HPV18E5 protein cells were harvested. Compared with control groups, cell proliferation in HPV18E5 stably expressed cells was obviously increased, as was the S phase in the cell cycle. Our results suggested that HPV18E5 influences cell proliferation and the cell cycle. Our study has laid the foundation of the biologic properties of HPV18E5 protein, which will aid further studies on the mechanism of action of carcinogenesis.
Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Human papillomavirus 18 ; genetics ; metabolism ; Humans ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomavirus Infections ; physiopathology ; virology ; Transfection

OBJECTIVETo explore the in vitro inhibitive effect and underlying mechanisms of Brucea Javanica oil emulsion (BJOE) on human papilloma virus (HPV) type 16 infected cells.

METHODSThe HPV16 E61E7 immortalized human ectocervical Ect1/E6E7 cell line and the CaSki cell line were selected as the in vitro models of premalignant cervical lesion and cervical cancer respectively. After treated with BJOE at different concentrations (5, 10, 20, and 40 microg/mL) at the operation time points (24, 48, and 72 h), the effects of BJOE on proliferative activities were measured by MTT assay. The morphologic changes of cell apoptosis stained with Hochest 33,258 were observed by fluorescence microscope. The effect on the cell apoptosis rate was analyzed by Annexin V-FITC/PI double-labeled flow cytometry. The mRNA expressions of HPV16 E6 and E7 were determined by semi-quantitative RT-PCR. The protein expressions of HPV16 E6, E7 oncogene, and specifically interacted p53, Rb antioncogene were stained by immunocytochemical staining (Elivison two-step procedure).

RESULTS(1) The proliferative activities of the Ect1/E6E7 cell and the CaSki cell treated with BJOE at different concentrations (5, 10, 20, and 40 p g/mL) at the operation time points (24, 48, and 72 h) were obviously inhibited, showing dose- and time-dependent manners (P <0.05). (2) Typical changes of apoptosis were observed in both HPV 16 positive cell lines after treated with BJOE. The cell apoptosis rates increased markedly after being cultured with BJOE at different concentrations (5, 10, and 20 microg/mL) for 48 h (P < 0.05). (3) After treated with BJOE at different concentrations (5, 10, and 20 microg/mL) for 48 h, the HPV16 E6 and E7 mRNA relative expressions in both HPV 16 positive cell lines decreased significantly (P < 0.05). (4) After treated with BJOE at different concentrations (5, 10, and 20 microg/ mL), the expressions of HPV16 E6, E7, and mutant p53 protein decreased gradually (P < 0.05), while the Rb protein expression increased gradually (P < 0.05).

CONCLUSIONSBJOE showed obvious in vitro inhibitory effects on HPV type 16 infected cells. Its underlying mechanisms might be possibly associated with down-regulating expressions of HPV16 E6 and E7 oncogenes.

Apoptosis ; drug effects ; Brucea ; chemistry ; Cell Line, Tumor ; Drugs, Chinese Herbal ; pharmacology ; Female ; Human papillomavirus 16 ; drug effects ; pathogenicity ; Humans ; Oncogene Proteins, Viral ; metabolism ; Papillomavirus E7 Proteins ; metabolism ; Papillomavirus Infections ; Plant Oils ; pharmacology ; Repressor Proteins ; metabolism

OBJECTIVETo analyze how the infection of human papillomavirus 16 (HPV16) affects expression of DNA polymerase beta (DNA polB) with the aim of probing the mechanism of over-expression of DNA polB in human cancers.

METHODSFour fragments of human DNA polB promoter were amplified and constructed into luciferase reporter vector pGL-Basic, generating pGL-BP, pGL-BMH, pGL-BMS, and pGL-BAT constructs respectively, and co-transfected with HPV16 or HPV6 into Hep2 cells. Luciferase activity was assayed 48 hours after transfection. Semi-quantitative RT-PCR was used to measure mRNA expression of endogenous DNA polB. Immunohistochemistry and in situ hybridization were used to analyze DNA polB expression and HPV16 or HPV6 infection in 38 cases of cervical lesions respectively.

RESULTSWith co-transfection of HPV16 and DNA polB promoter-driving reporters into Hep2 cells, pGL-BP reporter in full-length DNA polB promoter presented markedly elevated luciferase activities (P < 0.05). However, the other three mutant reporters: pGL-BMH, pGL-BMS, and pGL-BAT, generated no reporting activities in the presence of HPV16 (P > 0.05). On the contrary, all of polB promoter reporters were little stimulated in co-transfection of HPV6 (P > 0.05). The transfection of HPV16 could enhance the endogenous polB mRNA expression compared with that of HPV6 (3.42 vs. 0.80, P < 0.05). The DNA polB expression was found in 8 of 10 HPV16-positive cervical intraepithelial neoplasia grade III (CIN III) cases, while was only found in 3 of 11 HPV6-positive condyloma accuminatum cases, but was negative in all chronic cervicitis cases. The correlation of DNA polB expression with HPV16 infection in cervical lesions was significant (P < 0.05).

CONCLUSIONSHPV16 is able to specifically stimulate the expression of DNA polB in human epithelial cells through interaction with the core upstream regulatory sequences of DNA polB promoter. Over-expression of DNA polB might be an explanation for the molecular mechanism underlying HPV-related human cancers.

Cell Line ; DNA Polymerase beta ; genetics ; metabolism ; Female ; Gene Expression Regulation, Enzymologic ; Human papillomavirus 16 ; genetics ; metabolism ; Human papillomavirus 6 ; genetics ; metabolism ; Humans ; Papillomavirus Infections ; Promoter Regions, Genetic ; Uterine Cervical Neoplasms ; genetics ; pathology ; virology

This study was aimed to identify pET21b-HPV16E2/BL21(DE3) strain and to optimize the expression of human papillomavirus type 16 (HPV16) E2 protein by orthogonal analysis. Four influence factors on two levels were selected to increase the target protein quantity. The four factors were induction time, induction temperature, inductor concentration and cell density. The quantity of HPV16 E2 protein was used as the evaluation parameter. Induced by IPTG, HPV16 E2 protein was analyzed by SDS-PAGE and Western Blot. Target protein was analyzed by GIS imaging system to quantify the protein level. SPSS13. 0 software was applied to analyze the result. Data showed that the expression strain pET211rHPV16 E2/BL21(DE3) was identified correctly. HPV16 E2 protein expressed mainly at insoluble form. The 42KD protein band was identified by SDS-PAGE and Western blot. Orthogonal test was applied on influence factor analysis and expression optimization successfully. Main influence factors were inductor concentration and induction temperature. The optimimum condition of maximum expression quantity was 37 degrees C, 7h, 1.0 mmol/L IPTG and OD600 1.0. In this experiment, orthogonal test could not only be used to analyze the influential factors and promote the target protein expression, but also be used to provide a better experiment method for molecular biological study.
DNA-Binding Proteins ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Human papillomavirus 16 ; metabolism ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Papillomavirus Infections ; virology ; Recombinant Proteins ; biosynthesis ; genetics

High-risk human papillomavirus (HPV) is the principal cause of various cancers including cervical cancer, anal cancer, vulvar cancer, and some head and neck cancers. In the viral life cycle, by interacting with both viral and host DNA and proteins, the HPV E2 protein plays a pivotal role in viral transcriptional regulation and DNA replication, and it is also associated with modification of various cellular processes, including host gene transcription, RNA processing, apoptosis, ubiquitination, and intracellular trafficking, to create a convenient environment for a replicative cycle of the virus and contribute to the HPV pathogenesis. Elucidating the roles of E2 protein throughout the viral life cycle will improve our understanding of the viral life cycle and pathogenesis and help us identify novel antiviral agents with therapeutic potential. This article reviews the research progress in the structure, roles, and activity of high-risk HPV E2 protein, particularly that of HPV-16.
Animals ; DNA-Binding Proteins ; genetics ; metabolism ; Female ; Gene Expression Regulation, Viral ; Human papillomavirus 16 ; genetics ; metabolism ; Humans ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomavirus Infections ; genetics ; metabolism ; virology ; Uterine Cervical Neoplasms ; genetics ; metabolism ; virology

Human papilloma virus (HPV) is believed to be an essential factor for the development of cervical cancer. Early diagnosis and treatment of cervical intraepithelial neoplasia can effectively inhibit the future progression. HPV late 1 protein possesses epitope that can identify and adhere to host cells, and thus may play an important role in HPV infection and cervical carcinogenesis.
Capsid Proteins ; Cervix Uteri ; metabolism ; virology ; Female ; Humans ; Oncogene Proteins, Viral ; Papillomavirus Infections ; complications ; Uterine Cervical Neoplasms ; virology

OBJECTIVE: To explore the differential expression of microRNA (miRNA) in HPV16-positive squamous carcinoma of the cervix in the Uygur of southern Xinjiang and to predict the target genes of the miRNAs.
 METHODS: Samples of HPV16-positive squamous carcinoma of the cervix from 5 Uygurs were collected for miRNA microarray assay. The differentially expressed miRNAs were selected for further verification by real-time quantitative RT-PCR. The software, including targetscan, miRwalk, miRanda and pictar, were used to predict the target genes of the verified miRNAs.
 RESULTS: Eighteen differentially expressed miRNAs were identified by miRNA microarray assay. The significantly differentially expressed miRNA-138 and miRNA-720 were verified by real-time quantitative RT-PCR. According to the prediction, the target genes for miRNA-138 were EZH2, LYPLA1, ARHGEF3, CLNS1A, EIF4EBP1, GNAI2, LIMK1, RHOC, ROCK2, SLC20A1, TERT, and H2AFX, while for miRNA-720 were EZH2, AGAP2, SPOCK2, FGF14, HNRNPA2B1, QKI, FOXG1, ACVR1B, DNMT3A, EPHB2, LATS2, KRAS, CCND2, NBN, ENAM, AMELX, PRNP, and CALB1.
 CONCLUSION miR-138 and miR-720 are the down-regulated target miRNAs in HPV16-positive squamous carcinoma of the cervix in the Uygur of southern Xinjiang. The common target gene for miR-138 and miR-720 is EZH2, which might be related to cervical squamous carcinoma invasion and metastasis.
Carcinoma, Squamous Cell ; metabolism ; virology ; China ; Down-Regulation ; Female ; Human papillomavirus 16 ; isolation & purification ; Humans ; MicroRNAs ; metabolism ; Papillomavirus Infections ; metabolism ; Real-Time Polymerase Chain Reaction ; Uterine Cervical Neoplasms ; metabolism ; virology

The relationship between Human papillomavirus (HPV) 16 infection and the natural course of colorectal adenocarcinoma has not been fully defined. In this study, the HPV 16 E7 DNA was detected in 82 patients with primary colorectal adenocarcinoma to study the relationship between HPV 16 infection and colorectal carcinoma. Samples were taken from both the tumors and the adjacent normal mucosa in the same patient. Polymerase chain reaction (PCR) was used to amplify the HPV16 E7 DNA fragment. The PCR products were gel-purified and sequenced for HPV genotyping. DNA sequence analysis indicated that PCR product was 297 bp. It was the equivalent of 562-858th pairs in the HPV 16 primitive sequences. Our results showed HPV16 E7 DNA expression was significantly higher in colorectal carcinoma (42/82) than in adjacent normal mucosa (4/82). The correlation was found between HPV16 E7 expression and tumor's location; the positive rate was 18.18% in the ascending colon carcinoma group and 64.10% in the rectal carcinoma group. High HPV16 E7 expression was also associated with lower Dukes stage (P < 0.01). These results indicated that there was correlation between colorectal adenocarcinoma and HPV 16 infection. HPV16 infection was relatively higher in the colorectal carcinoma and rare in the adjacent normal mucosa. Specimens expressing higher levels of HPV 16 E7 DNA were associated with lower Dukes stage and more distal location.
Adenocarcinoma ; metabolism ; virology ; Adult ; Aged ; Aged, 80 and over ; Colorectal Neoplasms ; metabolism ; virology ; DNA, Viral ; analysis ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Papillomaviridae ; isolation & purification ; Papillomavirus E7 Proteins ; Papillomavirus Infections ; metabolism ; virology

OBJECTIVETo detect the infection of human papillomavirus (HPV) 6/11, 16/18, 31/33 in patients with head and neck squamous cell carcinoma and explore the relationship between HPV infection and expression of p16 and EGFR in the tumor tissue and their clinical significance.

METHODSThe infection of HPV6/11, 16/18, 31/33 was detected by in situ hybridization (ISH), and expression of p16 and EGFR was assessed by immunohistochemistry in biopsy or surgical specimens of 43 cases of head and neck squamous cell carcinoma, and analyzed its impact on the prognosis. Spearman rank correlation method was used for analysis of the relationship. Overall survival rate of the patients was estimated by Kaplan-Meier analysis. Cox regression model was used for multivariate analysis.

RESULTSHPV6/11, 16/18, 31/33 were detected in 25.6% (11/43) of this group of patients, among them, HPV16/18 accounted for 63.6%, HPV31/33 accounted for 27.3%, and HPV6/11 accounted for 0. EGFR was expressed in 69.8% and p16 was expressed in 53.5% of the patients. The difference was statistically significant between the HPV-positive and HPV-negative groups in ethnicity, smoking, alcohol consumption (P = 0.045, 0.040, 0.011, respectively). HPV infection was found to be positively correlated with p16 expression and inversely correlated with EGFR expression (P = 0.029, P = 0.009). The expression of p16 protein was negatively correlated with EGFR protein expression (r = -0.447, P = 0.003). The 3-year overall survival rate was 60.0% in the HPV-positive group and 59.7% in the negative group (P = 0.789); 72.2% in the p16-positive patients and 43.9% in the p16-negative patients (P = 0.012); 48.8% in the EGFR-positive patients and 81.8% in the EGFR-negative patients (P = 0.037).

CONCLUSIONSThe results of our study suggest that the HPV infection rate, HPV subtypes and clinicopathological features of HPV-positive SCCHN are in accordance with those reported in Western literatures. There may be differences between the HPV infections in Uygur and Han nationalities. HPV infection is positively correlated with p16 and negatively correlated with EGFR expressions. The prognosis of p16-positive patients is significantly better than that of negative cases, and p16 is an independent prognostic factor for head and neck squamous cell carcinoma.

Carcinoma, Squamous Cell ; mortality ; virology ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Head and Neck Neoplasms ; mortality ; virology ; Human papillomavirus 16 ; Humans ; Papillomavirus Infections ; metabolism ; Prognosis ; Receptor, Epidermal Growth Factor ; metabolism ; Survival Analysis