OBJECTIVETo explore the viral etiology of human breast cancer to determine whether there are novel molecular targets for gene therapy of breast cancer and provide evidence for the research of gene therapy and vaccine development for breast cancer.

METHODSPCR was used to screen HPV16 and HPV18 oncogenes E6 and E7 in the SKBR3 cell line and in 76 paraffin embedded breast cancer tissue samples. RNA interference was used to knock down the expression of HPV18 E6 and E7 in SKBR3 cells, then the changes in the expression of cell-cycle related proteins, cell viability, colony formation, metastasis, and cell cycle progression were determined.

RESULTSHPV18 oncogenes E6 and E7 were amplified and sequenced from the SKBR3 cells. Of the patient samples, 6.58% and 23.68% were tested to be positive for HPV18 E6 and HPV18 E7. In the cell culture models, the knockdown of HPV18 E6 and E7 inhibited the proliferation, metastasis, and cell cycle progression of SKBR3 cell. The knockdown also clearly affected the expression levels of cell cycle related proteins.

CONCLUSIONHPV was a contributor to virus caused human breast cancer, suggesting that the oncogenes in HPV were potential targets for gene therapy of breast cancer.

Adult ; Aged ; Aged, 80 and over ; Base Sequence ; Breast Neoplasms ; genetics ; therapy ; Female ; Genetic Therapy ; methods ; Humans ; Middle Aged ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomaviridae ; physiology ; Papillomavirus Infections ; genetics ; therapy ; Sequence Alignment

Human papillomaviruses (HPVs) including high-risk (HR) and low-risk (LR) subtypes have distinguishable variation on both genotypes and phenotypes. The co-infection of multiple HR-HPVs, headed by HPV16, is common in cervical cancer in female. Recently accumulating reports have focused on the interaction between virus and host, particularly the role of human microRNAs (miRNAs) in anti-viral defense by targeting viral genome. Here, we found a well-conserved target site of miRNAs in the genomes of most HR-HPVs, not LR-HPVs, by scanning all potential target sites of human miRNAs on 24 HPVs of unambiguous subtypes of risk. The site is targeted by two less common human miRNAs, miR-875 and miR-3144, and is located in E6 oncogene open reading frame (ORF) and overlap with the first alternative splice exon of viral early transcripts. In validation tests, miR-875 and miR-3144 were identified to suppress the target reporter activity markedly and inhibit the expression of both synthetically exogenous E6 and endogenous E6 oncogene. High level of two miRNAs can inhibit cell growth and promote apoptosis in HPV16-positive cervical cancer cells. This study provides a promising common target of miRNAs for most HR-HPVs and highlights the effects of two low expressed human miRNAs on tumour suppression.
Apoptosis ; genetics ; Base Sequence ; Binding Sites ; genetics ; Cell Line, Tumor ; Cell Proliferation ; genetics ; Female ; Gene Expression ; Host-Pathogen Interactions ; genetics ; Human papillomavirus 16 ; genetics ; physiology ; Humans ; MicroRNAs ; genetics ; Microscopy, Fluorescence ; Molecular Sequence Data ; Oncogene Proteins, Viral ; genetics ; Repressor Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Homology, Nucleic Acid ; Uterine Cervical Neoplasms ; genetics ; pathology ; virology

To identify the integration sites in the host genome for the hepatitis B virus (HBV)-encoded X protein (HBx) in hepatocellular carcinoma (HCC) biopsies that are positive for hepatitis B surface antigen (HBsAg). HCC biopsies were obtained from six patients that were HBV carriers, as demonstrated by the presence of HBsAg in their serum and sero-negativity for antibody to HBsAg. DNA was extracted from the tissue, fractionated, and circularized. Primers were designed according to the HBx sequence and used to amplify the circularized DNA templates by inverse polymerase chain reaction (IPCR). The amplified DNA fragments were checked by electrophoresis, cloned into the PMD18-T expression vector, and sequenced. Sequence alignment was performed by the Blast algorithms. Seven electrophoresis bands yielded 22 sequencing results, which represented a total of three HBx integration sites in the host genome: 19q12, 2q32.2, 22q12. The 19q12 integration site encompasses the CCNE1 gene, which encodes a G1/S-specific cyclin-E1. HBx-related integration sites exist in HBsAg-positive HCC biopsies. The CCNE1 gene may play a role in the development of HBx-related HCC.
Carcinoma, Hepatocellular ; blood ; genetics ; Cyclin E ; genetics ; DNA Primers ; DNA, Viral ; genetics ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B virus ; genetics ; physiology ; Humans ; Liver Neoplasms ; blood ; genetics ; Oncogene Proteins ; genetics ; Trans-Activators ; genetics ; Virus Integration

The recombined adenovirus DNA was transfected into 293 cells for packing and amplification of replication-deficient Ad-CMV-E6/E7, Ad-K14 -E6/E7 virus was purified by CsCl density gradient centrifugation , recombined adenovirus Ad-CMV-E6/E7, Ad-K14 -E6/E7 were used as experimental group, while pAd-CMV and pAdtrack-K14 were used as control group. Four of them were injected through one main vein of nude mice tail respectively. These mice were then treated with 0.05 mg 17beta-estradiol over 12 weeks. Mice were anaesthesiaed with 2.5% Avertint and the vagina, mammary gland, ovaries and uterus were dissected and fixed in 3.75% paraformaldehyde overnight at 4 degrees C. Paraffin-embedded sections, HE staining and identification of P53 and Bcl-2 protein via immunohistochemistry were performed. The expression of E6/E7 was verified by RT-PCR in different tissue of nude mice. HE staining showed evident hyperplasy in cervix-uterus transformation zone of experimental group 2. The expression of mutant P53 and Bcl-2 were higher than control group via immunohistochemical S-P method in uterus stroma-cell. Western blotting also showed that E6 protein was expressed. The expression of E6/E7 was higher than control group by human cytokeratin promoter 14 and hyperlasy changes were detected in epithelial tissue of cervix-uterus transformation zone.
Adenoviridae ; genetics ; Animals ; Blotting, Western ; Cell Line ; Female ; Genital Diseases, Female ; pathology ; virology ; Genitalia, Female ; pathology ; virology ; Humans ; Immunohistochemistry ; Mammary Glands, Animal ; metabolism ; pathology ; Mice ; Mice, Nude ; Oncogene Proteins, Viral ; genetics ; metabolism ; Ovary ; metabolism ; pathology ; Papillomaviridae ; metabolism ; physiology ; Papillomavirus E7 Proteins ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Suppressor Protein p53 ; metabolism ; Uterus ; metabolism ; pathology ; Vagina ; metabolism ; pathology

Common warts are close associated with HPVs infection. In this study, we amplified and sequenced the LCR fragment and E2 gene of HPV-2 that infected the patient of extensive common wart with cutaneous horns, and we constructed the recombinant CAT-reporter plasmids pBLCAT-LCR containing HPV-2 prototype or variant LCR and mammalian expression plasmids pcDNA3. 1-E2 containing prototype or variant E2 ORF individually. The promoter activities of HPV-2 variant and the transcriptional repression activities of the mutated E2 protein were evaluated by transient transfection into HeLa cells. The results showed that there were several mutations in LCR and E2 gene of HPV-2 variant. Compared with the prototype, the viral early promoter activity of variant was significantly increased uder the control of LCR. Compared with the wild type E2 protein, the transcriptional repression activities of the mutated E2 protein was abolished partially. We speculate herein that increased promoter activities and decreased repression effect of the mutated E2 protein are linked, at least partially, with the clinical phenotypes of the uncommon huge common wart.
DNA-Binding Proteins ; genetics ; physiology ; Humans ; Mutation ; Oncogene Proteins, Viral ; genetics ; physiology ; Papillomaviridae ; genetics ; Promoter Regions, Genetic ; Repressor Proteins ; physiology ; Warts ; virology

Amino Acid Sequence ; Animals ; Birds ; Cell Transformation, Viral ; Herpesvirus 2, Gallid ; genetics ; physiology ; Marek Disease ; virology ; Models, Biological ; Molecular Sequence Data ; Oncogene Proteins, Viral ; genetics ; physiology

Active Transport, Cell Nucleus ; Capsid Proteins ; physiology ; Heparan Sulfate Proteoglycans ; physiology ; Humans ; Oncogene Proteins, Viral ; physiology ; Papillomaviridae ; genetics ; physiology ; Papillomavirus Infections ; etiology ; Viral Structural Proteins ; physiology ; Virion ; physiology ; Virus Assembly

OBJECTIVETo investigate the association between HPV16 infection, E6/E7 variations and the cervical lesions.

METHODSHPV subtypes were detected by using flow-through hybridization technique, E6/E7 gene was extracted from cervical lesions in 80 patients with HPV16 infection, PCR amplified, cloned into plasmid pMD18-T and sequenced.

RESULTSHPV 16 was the most common type which accounted for 33.3% (154/463), the HPV16 infection rates increased with the severity of cervical lesions (P < 0.05). Totally in 72 cases the complete E6 and E7 regions were successfully sequenced, the DNA mutation rate of E6/E7 was 88.9% (64/72). A mutation, E6-D32E (T96G) coincided with a specific type of E7 mutation, N29S (A86G). D32E/N29S mutation rate was 38.9% (28/72), the detection rate increased with the severity of cervical lesions (P < 0.05).

CONCLUSIONHPV 16 was the most common type in women with cervical lesions in Beijing, D32E/N29S variant associated with the cervical lesions.

Adult ; Aged ; China ; epidemiology ; Female ; Gene Frequency ; Genotype ; Host-Pathogen Interactions ; Human papillomavirus 16 ; genetics ; physiology ; Humans ; Middle Aged ; Mutation ; Oncogene Proteins, Viral ; genetics ; Papillomavirus E7 Proteins ; Papillomavirus Infections ; epidemiology ; pathology ; virology ; Repressor Proteins ; genetics ; Severity of Illness Index ; Uterine Cervical Diseases ; epidemiology ; pathology ; virology ; Uterine Cervical Neoplasms ; epidemiology ; pathology ; virology ; Young Adult

Human papillomavirus (HPV) is a common small DNA tumor virus that specifically infects squamous epithelial cells and causes benign or malignant epithelial lesions such as genital warts and cervical cancer. High-risk HPV is detected in specimens of more than 90% of cervical cancer. In the 7. 9 kb genome of HPV, E6 and E7 are the crucial viral oncoproteins that consistently maintained after viral integration into host cell genome. These two proteins interfere with cell proliferation and differentiation through interacting with important tumor suppressors including p53 and pRb. High-risk HPV E6/E7 also induces genomic instability, facilitating cell transformation.
Cell Differentiation ; Cell Proliferation ; Cell Transformation, Neoplastic ; metabolism ; pathology ; Female ; Genomic Instability ; Host-Pathogen Interactions ; Humans ; Oncogene Proteins, Viral ; genetics ; physiology ; Papillomaviridae ; genetics ; physiology ; Papillomavirus Infections ; metabolism ; pathology ; Uterine Cervical Neoplasms ; metabolism ; pathology ; virology

The mitochondrial pathway of swine influenza virus (SIV)-induced apoptosis was investigated using porcine kidney (PK-15) cells, swine testicle (ST) cells, and HeLa cervical carcinoma cells which are known not to support viral replication. As judged by cell morphology, annexin V staining, and DNA fragmentation, PK-15 and ST cells infected with three different subtypes of SIV (H1N1, H3N2, and H1N2) were obviously killed by apoptosis, not necrosis. SIV infection in PK-15 and HeLa cells was shown to decrease the cellular levels of Bcl-2 protein compared to that of mock-infected control cells at 24 h post-infection, whereas expression levels of Bax protein increased in the PK-15 cells, but did not increase in HeLa cells by SIV infection. Cytochrome c upregulation was also observed in cytosolic fractions of the PK-15 and HeLa cells infected with SIV. Apoptosome (a multi-protein complex consisting of cytochrome c, Apaf-1, caspase-9, and ATP) formation was confirmed by immunoprecipitation using cytochrome c antibody. Furthermore, SIV infection increased the cellular levels of TAJ, an activator of the JNK-stressing pathway, and the c-Jun protein in the PK-15 and HeLa cells. Taken together, these results suggest that the mitochondrial pathway should be implicated in the apoptosis of PK-15 cells induced by SIV infection.
Animals ; Annexin A5/metabolism ; *Apoptosis ; Blotting, Western ; Cell Fractionation ; Cell Line ; Comparative Study ; Cytochrome c Group/metabolism ; Cytosol/chemistry ; DNA Fragmentation ; Enzyme Activation ; Gene Expression Regulation, Viral ; Hela Cells ; Humans ; Influenza A virus/*physiology ; Kinetics ; Mitochondria/metabolism/*physiology ; Precipitin Tests ; Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; Research Support, Non-U.S. Gov't ; Swine ; bcl-2-Associated X Protein/genetics/metabolism