In Vitro Antiviral and Anticancer Effects of Tanacetum sinaicum Essential Oil on Human Cervical and Breast Cancer.

BACKGROUND: Cervical cancer has been linked to human papillomavirus (HPV) types 16 and 18. Essential oils (EOs) are vital natural products of plants with various therapeutic and biological properties. OBJECTIVES: The purpose of this study is to investigate and assess Tanacetum sinaicum essential oil's possible antiviral and anticancer properties, with a focus on its in vitro effects on human cervical cancer and human breast adenocarcinoma cell lines. MATERIALS AND METHODS: Tanacetum sinaicum EO was extracted via hydrodistillation (HD) and characterized using gas chromatography-mass spectrometry (GC-MS). MTT assay was used to determine the cell viability of Hela (a human epithelial cervical cancer) and MCF-7 (human breast adenocarcinoma) cell lines. Quantitative real-time polymerase chain reaction (PCR) was utilized to assess the antiviral efficacy of EO against HPV-16 and 18, and anti-metastatic characteristics. The biological activity of EO was assessed using Autophage and Cell genotoxicity via the comet assay. RESULTS: EO is mostly composed of chrysanthenyl acetate, thujone, and verbenol. The cell viability was reduced after 24 hours of incubation at doses from 100 to 400 µg/ml. Concentrations of 800 to 3,200 µg/ml significantly inhibit cell growth. After a 24-hour incubation period, doses ranging from 100 to 400 µg/ml reduced cell viability from 62 to 72%. Concentrations of 800 to 3,200 µg/ml significantly suppress cell growth by over 95%. In MCF7 and HeLa cell lines, EO lowered virus copy numbers in a dose-dependent manner, with higher concentrations of the oil inhibiting virus replication more effectively. EO treatment increased the number of autophagosomes/autolysosomes and acidic vesicular organelles in both cell lines. On the HeLa and MCF7 cell lines, EO demonstrated antiproliferative and antimetastatic effects. The results demonstrated that EO had dose-dependent genotoxic effects on both cancer cell lines, as evidenced by DNA damage. CONCLUSION: Tanacetum sinaicum EO is a prospective source of natural bioactive compounds that can be employed in pharmaceutical and medicinal applications due to its antiviral, antiproliferative, anti-metastatic and genotoxic properties.

Human papillomavirus type 16 E7 promotes cell viability and migration in cervical cancer by regulating the miR-23a/HOXC8 axis.

BACKGROUND: Human papillomavirus (HPV) is a risk factor for the occurrence of cervical cancer (CC). Here, we aimed to explore the role of HPV16 in CC and identify the underlying mechanism. METHODS: The expression of miR-23a, HPV16 E6/E7 and homeobox C8 (HOXC8) was measured by quantitative real-time PCR or western blot. Cell viability and migration were evaluated using cell counting kit-8, Transwell and wound healing assays. The targeting relationship between miR-23a and HOXC8 was revealed by dual-luciferase reporter assay. RESULTS: miR-23a was downregulated in HPV16-positive (HPV16+) CC tissues and HPV16+ and HPV18+ cells. Additionally, E6/E7 expression was increased in CC cells. Then, we found that E7, rather than E6, positively regulated miR-23a expression. miR-23a suppressed cell viability and migration, whereas E7 overexpression abrogated this suppression. miR-23a targeted HOXC8, which reversed miR-23a-mediated cell viability and migration. CONCLUSIONS: HPV16 E7-mediated miR-23a suppressed CC cell viability and migration by targeting HOXC8, suggesting a novel mechanism of HPV-induced CC.

Cervical cancer (CC) is a common gynaecological malignancy, and persistent human papillomavirus (HPV) infection, especially HPV16, is a main cause of CC. In this study, we explored the role of HPV16 in CC and the molecular mechanism. We used in vitro study to measure CC cell biological behaviours mediated by HPV16 E7, miR-23a and homeobox C8 (HOXC8). We found that HPV16 E7 promotes CC cell viability and migration. miR-23a expression is decreased in CC cells and inhibits cell viability and migration. HOXC8 is a target of miR-23a that reversed the effects on cellular processes caused by miR-23a. These results showed that miR-23a and HOXC8 may be the therapeutic targets of HPV16 E7-infected CC. What is more, our findings provide new insights into the progression of CC.

Genetic variation of E6 and E7 genes of human papillomavirus type 16 from central China.

BACKGROUND: Persistent high-risk human papillomavirus (HR-HPV) infection is an important factor in the development of cervical cancer, and human papillomavirus type 16 (HPV-16) is the most common HR-HPV type worldwide. The oncogenic potential of HPV-16 is closely related to viral sequence variation. METHODS: In order to clarify the variant characteristics of HPV-16 E6 and E7 genes in central China, E6 and E7 sequences of 205 HPV-16 positive samples were amplified by polymerase chain reaction. PCR products of E6 and E7 genes were further sequenced and subjected to variation analysis, phylogenetic analysis, selective pressure analysis and B-cell epitope prediction. RESULTS: Twenty-six single nucleotide variants were observed in E6 sequence, including 21 non-synonymous and 5 synonymous variants. Twelve single nucleotide variants were identified in E7 sequence, including 6 non-synonymous and 6 synonymous variants. Four new variants were found. Furthermore, nucleotide variation A647G (N29S) in E7 was significantly related to the higher risk of HSIL and cervical cancer. Phylogenetic analysis showed that the E6 and E7 sequences were all distributed in A lineage. No positively selected site was found in HPV-16 E6 and E7 sequences. Non-conservative substitutions in E6, H31Y, D32N, D32E, I34M, L35V, E36Q, L45P, N65S and K75T, affected multiple B-cell epitopes. However, the variation of E7 gene had little impact on the corresponding B-cell epitopes (score < 0.85). CONCLUSION: HPV-16 E6 and E7 sequences variation data may contribute to HR-HPV prevention and vaccine development in Jingzhou, central China.

Distribution and Clinical Significance of HPV16 Variants in Head and Neck Squamous Cell Carcinomas: Data from a Portuguese Cohort and Systematic Review.

INTRODUCTION: Genomic variants of the human papillomavirus type 16 (HPV16) are thought to play differential roles in the susceptibility to head and neck squamous cell carcinomas (HNSCC) and its biological behaviour. This study aimed to establish the prevalence of HPV16 variants in an HNSCC cohort and associate them with clinical pathological characteristics and patient survival. METHODS: We retrieved samples and clinical data from 68 HNSCC patients. DNA samples were available from tumour biopsy at the time of the primary diagnosis. Targeted next-generation sequencing was used to obtain whole-genome sequences, and variants were established based on phylogenetic classification. RESULTS: 74% of samples clustered in lineage A, 5.7% in lineage B, 2.9% in lineage C, and 17.1% in lineage D. Comparative genome analysis revealed 243 single nucleotide variations. Of these, one hundred were previously reported, according to our systematic review. No significant associations with clinical pathological variables or patient survival were observed. The E6 amino acid variations E31G, L83V, and D25E and E7 N29S, associated with cervical cancer, were not observed, except for N29S in a single patient. CONCLUSION: These results provide a comprehensive genomic map of HPV16 in HSNCC, highlighting tissue-specific characteristics which will help design tailored therapies for cancer patients.

Deteccion del virus del papiloma humano en biopsias de cancer de mama mediante PCR e inmunohistoquimica en el Hospital Santa Rosa durante el 2019 / Detection of human papilloma virus in breast cancer biopsies using PCR and immunohistochemistry at the Santa Rosa Hospital during 2019

El objetivo de este estudio fue determinar la presencia del Virus Papiloma Humano (VPH) tipo 16 y 18 en biopsias de tejido mamario parafinado de pacientes con diagnóstico clínico de cáncer de mama. Se analizaron 32 biopsias de cáncer de mama embebidas en parafina para detectar el ADN de VPH mediante PCR en tiempo real, los iniciadores estuvieron dirigidos al gen E6. Se evaluaron el tipo histológico, grado histológico y la sobreexpresión de C-erB2 y Ki-67 mediante inmunohistoquímica. El 84,38% (27) fueron positivos para VPH, el 25% (8) fueron positivos para VPH-16 y el 59,38% (19) para VPH-18. El 15,63% (5) de las muestras presentaron infección mixta. Se evidenció la sobrexpresión de C-erbB2 y Ki-67 en 6,25% (2) de las muestras positivas para VPH-16 y 15,63% (5) de las muestras positivas para VPH-18. Se detectó ADN de VPH-16 y VPH-18 en las muestras de biopsias analizadas mediante PCR en tiempo real.

The aim of this study was to determine the presence of Human Papillomavirus (HPV) type 16 and 18 in biopsies of paraffin-embedded breast tissue from patients with clinically diagnosed breast cancer. 32 paraffin-embedded breast cancer biopsies were analyzed in order to detect HPV DNA by real-time PCR, the primers were directed at the E6 gene. The histological type, histological grade and overexpression of C-erB2 and Ki-67 were evaluated by immunohistochemistry. 84.38% (27) of the samples were positive for HPV, 25% (8) were positive for HPV-16 and 59.38% (19) were positive for HPV-18. Mixed infection was found in 15.63% (5) of the samples. Overexpression of C-erbB2 and Ki-67 was seen in 6.25% (2) of the samples positive for HPV-16 and in 15.63% (5) samples positive for HPV-18. HPV-16 and HPV-18 DNA was detected in the biopsy samples analyzed by real-time PCR.

Deep Sequencing of HPV16 E6 Region Reveals Unique Mutation Pattern of HPV16 and Predicts Cervical Cancer.

The genetic diversity of human papillomavirus (HPV) 16 within cervical cells and tissue is usually associated with persistent virus infection and precancerous lesions. To explore the HPV16 mutation patterns contributing to the cervical cancer (CC) progression, a total of 199 DNA samples from HPV16-positive cervical specimens were collected and divided into high-grade squamous intraepithelial lesion (HSIL) and the non-HSIL(NHSIL) groups. The HPV16 E6 region (nt 7125-7566) was sequenced using next-generation sequencing. Based on HPV16 E6 amino acid mutation features selected by Lasso algorithm, four machine learning approaches were used to establish HSIL prediction models. The receiver operating characteristic was used to evaluate the model performance in both training and validation cohorts. Western blot was used to detect the degradation of p53 by the E6 variants. Based on the 13 significant mutation features, the logistic regression (LR) model demonstrated the best predictive performance in the training cohort (AUC = 0.944, 95% CI: 0.913-0.976), and also achieved a high discriminative ability in the independent validation cohort (AUC = 0.802, 95% CI: 0.601-1.000). Among these features, the E6 D32E and H85Y variants have higher ability to degrade p53 compared to the E6 wildtype (P < 0.05). In conclusion, our study provides evidence for the first time that HPV16 E6 sequences contain vital mutation features in predicting HSIL. Moreover, the D32E and H85Y variants of E6 exhibited a significantly higher ability to degrade p53, which may play a vital role in the development of CC. IMPORTANCE The study provides evidence for the first time that HPV16 E6 sequences contain vital mutation features in predicting the high-grade squamous intraepithelial lesion and can reduce even more unneeded colposcopies without a loss of sensitivity to detect cervical cancer. Moreover, the D32E and H85Y variants of E6 exhibited a significantly higher ability to degrade p53, which may play a vital role in the development of cervical cancer.

CRISPR-Cas9‒Based Genomic Engineering in Keratinocytes: From Technology to Application.

CRISPR-Cas9 is the most straightforward genome-editing tool to date. However, its implementation across disciplines is hampered by variable genome-editing efficiencies, reduced cell viability, and low success rates in obtaining clonal cell lines. This review aims to recognize all CRISPR-Cas9‒related work within the experimental dermatology field to identify key factors for successful strategies in the different keratinocyte (KC) cell sources available. On the basis of these findings, we conclude that most groups use immortalized KCs for generating knockout KCs. Our critical considerations for future studies using CRISPR-Cas9, both for fundamental and clinical applications, may guide implementation strategies of CRISPR-Cas9 technologies in the (experimental) dermatology field.

Construction and immune effect of an HPV16/18/58 trivalent therapeutic adenovirus vector vaccine.

OBJECTIVE: This study aims to prepare candidate vaccines for cervical cancer immunotherapy by inserting the fused genes of human papillomavirus (HPV)16/18/58 mE6E7 lacking transforming activity into an adenovirus vector and to verify its efficiency in model mice with tumor expressing the associated HPV genes. METHODS: The E6/E7 genes of HPV16/18/58 were point-mutated to abolish their transforming activity, and adenovirus (AD)-HPV16/18/58 mE6E7 adenovirus vaccine was constructed. The immune effect of the adenovirus vaccine against HPV16/18/58-type tumors was analyzed by tumor morphology, enzyme linked immunosorbent assay, enzyme-linked immunospot and specific cytotoxic T lymphocyte (CTL) and T lymphocyte subsets. RESULTS: The HPV16/18/58 mE6E7 plasmid containing point mutations was verified by quantitative real-time polymerase chain reaction (qRT-PCR), enzyme digestion and electrophoresis, and gene sequencing. qRT-PCR and Western blots verified that AD-HPV16/18/58 mE6E7 could express the HPV16 mE6E7, HPV18 mE6E7 and HPV58 mE6E7 fusion genes and proteins in cells. The results of animal experiments were as follows: In the vaccine group, the tumors formed later, the incubation period was longer, the growth was slower, growth was inhibited, and the survival period was significantly prolonged. The immunological results all showed that the vaccine could induce effective humoral and cellular immunity in mice with three types of tumors, compared with the phosphate buffered saline (PBS) group and the adenovirus-negative control (AD-NC) group, the differences were statistically significant (P < 0.05). CONCLUSION: We successfully constructed the HPV16/18/58 trivalent therapeutic adenovirus vaccine AD-HPV16/18/58 mE6E7. The AD-HPV16/18/58 mE6E7 adenovirus vaccine can protect immunized mice to a certain extent from TC-1, U14/LV-HPV18 E6E7 and U14/LV-HPV58 E6E7 cells, which contain HPV16, 18 and 58 E6 and/or E7 genes, respectively.

Risk Assessment of Human Papillomavirus-Positive Cytology-Negative Cervical Cancer Screening in Black and White Women.

OBJECTIVES: As we move toward human papillomavirus (HPV) only as the preferred cervical cancer screening method, we performed a retrospective analysis of Black and White women with negative cytology (Papanicolaou negative [PAPneg]) and positive high-risk HPV (hrHPV) (HPVpos) results and determined follow-up. METHODS: We searched our pathology data system for patients with PAPneg/HPVpos results (2017-2019). Follow-up data were reviewed (39 months), and a comparison among race was performed. RESULTS: In total, 1,728 patients were identified (Black, 53%; White, 47%). Twenty-nine percent of the patients had no follow-up with no difference among the races. HPV 16 was more common among Whites (P < .01), while non-16/18 hrHPV was more common among Black patients (P = .01). A total of 30 (3.3%) Black and 26 (3.2%) White patients were diagnosed with cervical intraepithelial neoplasia grade 2/3 (CIN 2/3). More White women were diagnosed on biopsy alone (negative endocervical curettage) compared with Black women (20 vs 9, P < .01). Meanwhile, there were 21 Black and 6 White women with CIN 2/3 on endocervical curettage (P = .01). CONCLUSIONS: Follow-up of women with PAPneg/HPVpos remains a challenge. There was no disparity in follow-up when cohorts were compared. However, Black women had higher numbers of high-grade intraepithelial lesions on endocervical curettage.

HPV-16 E2/E6 and POU5F1B as Biomarkers to Determine Cervical High-Grade Squamous Lesions and More.

PURPOSE: Human papillomavirus-16 (HPV-16) is the most carcinogenic HPV genotype. This study aimed to evaluate the clinical value of POU5F1B and HPV-16-E2/E6 by cervical cytology specimens to predict the cervical intraepithelial neoplasia two grade and more (CIN2+). METHODS: Finally, 248 patients with HPV-16 single infection were enrolled. Using cytology specimen by real-time quantitative PCR (qPCR), POU5F1B mRNA and HPV-16-E2/E6 were detected. The relationship of POU5F1B, HPV-16-E2/E6 and CIN2+ were analyzed, and the optimal cut-off values of POU5F1B and HPV-16-E2/E6 to predict CIN2+ were calculated. RESULTS: The mean HPV-16-E2/E6 decreased significantly with cervical lesions development, especially compared with CIN2+ (p<0.05). And the POU5F1B demonstrated higher expression in CIN2+ than that of normal cervical tissue and CIN1 (p<0.05). What is more, POU5F1B was negatively correlated with HPV-16-E2/E6. It demonstrated that the area under the receiver operating characteristic curve (AUC) for POU5F1B (0.9058) was higher than that for HPV-16-E2/E6 (0.8983), and the sensitivity and specificity of POU5F1B in the diagnosis of CIN2+ were higher than HPV-E2/E6. Furthermore, it demonstrated that the POU5F1B had the highest odds ratio (OR= 16.84; 95% CI (8.00-35.46)) for the detection of CIN 2+. CONCLUSION: HPV-16-E2/E6≤0.6471 or POU5F1B≥1.0310 in cervical exfoliated cells can be used as a reliable predictor of CIN2+. POU5F1B can be used as a new auxiliary biomarker to determine the HPV infection status and a reliable predictor of CIN2+. The expression of POU5F1B≥1.0310 had the highest OR for the detection of CIN2+.

Anti-Retroviral Protease Inhibitors Regulate Human Papillomavirus 16 Infection of Primary Oral and Cervical Epithelium.

Epidemiology studies suggest that Human Immunodeficiency Virus (HIV)-infected patients on highly active anti-retroviral therapy (HAART) may be at increased risk of acquiring opportunistic Human Papillomavirus (HPV) infections and developing oral and cervical cancers. Effective HAART usage has improved survival but increased the risk for HPV-associated cancers. In this manuscript, we report that Protease Inhibitors (PI) treatment of three-dimensional tissues derived from primary human gingiva and cervical epithelial cells compromised cell-cell junctions within stratified epithelium and enhanced paracellular permeability of HPV16 to the basal layer for infection, culminating in de novo biosynthesis of progeny HPV16 as determined using 5-Bromo-2'-deoxyuridine (BrdU) labeling of newly synthesized genomes. We propose that HAART/PI represent a novel class of co-factors that modulate HPV infection of the target epithelium. Our in vitro tissue culture model is an important tool to study the mechanistic role of anti-retroviral drugs in promoting HPV infections in HAART-naïve primary epithelium. Changes in subsequent viral load could promote new infections, create HPV reservoirs that increase virus persistence, and increase the risk of oral and cervical cancer development in HIV-positive patients undergoing long-term HAART treatment.

Effects of HPV16 E6 protein on Daxx-induced apoptosis in C33A cells.

AIMS: Daxx is a highly conserved nuclear protein with an important role in transcription, apoptosis and other cell processes. We investigated the role of HPV16 E6 in Daxx-induced apoptosis through their interactions in C33A cells. METHODS: The binding of HPV16 E6 and Daxx was confirmed in C33A cells using co-immunoprecipitation and indirect immunofluorescence assays. Quantitative PCR and western blotting were performed to determine the RNA and protein expressions of Daxx, respectively. Automatic cell count and MTT assays were performed to investigate the proliferation of C33A cells. The apoptosis rate of C33A cells was determined via flow cytometry using Annexin V-FITC/PI staining. The relative activity of caspase-8 was tested using ELISA. RESULTS: HPV16 E6 can bind with Daxx and cause its translocation in C33A cells. The transfected HPV16 E6 can cause a decrease in relative quantification for Daxx in Daxx-overexpressing cells. After Daxx transfection, cell proliferation was found to decrease sharply and cell apoptosis to increase sharply. However, when HPV16 E6 was co-transfected with Daxx, this decrease and increase both became gentle. Similarly, HPV16 E6 made the Daxx-induced increase in caspase-8 activity milder. CONCLUSIONS: HPV16 E6 is involved in inhibiting apoptosis through deregulation of Daxx-induced caspase-8 activities.

Digital RNA Sequencing of Human Epidermal Keratinocytes Carrying Human Papillomavirus Type 16 E7.

High-risk human papillomavirus (HPV) infections are the predominant cause of cervical cancer and its early gene E7 plays an important role in cellular proliferation and cell-cycle progression. While tremendous progress has been made in exploring the molecular mechanisms in late tumorigenesis, many pathways showing how HPV deregulates host gene expression in early inapparent infections and early tumorigenesis still remain undefined. Digital RNA sequencing was performed and a total of 195 differentially expressed genes were identified between the HPV16 E7-transfected NHEKs and control cells (p < 0.05, fold-change > 2). GO enrichment showed that HPV16 E7 primarily affected processes involved in anti-viral and immune responses, while KEGG pathway analysis showed enrichment of gene clusters of associated with HPV infection and MAPK signaling. Of the differentially expressed genes, IFI6, SLC39A9 and ZNF185 showed a strong correlation with tumor progression and patient survival in the OncoLnc database while roles for AKAP12 and DUSP5 in carcinogenesis and poor prognosis have previously been established for other cancer types. Our study identified several novel HPV16 E7-regulated candidate genes with putative functions in tumorigenesis, thus providing new insights into HPV persistence in keratinocytes and early onset of tumorigenesis.

Association of Human Papillomavirus Type 16 Long Control Region Variations with Cervical Cancer in a Han Chinese Population.

Objective: High-risk human papillomavirus (HPV) E6 and E7 proteins are the major oncoproteins involved in the tumorigenesis of cervical cancer. The long control region (LCR) in HPV plays an important role in regulating the expression of the E6 and E7 oncogenes. In the current study, we investigated the association of HPV16 LCR variations with cervical cancer. Methods: A total of 139 HPV16-positive cervical cancer patients (case group) and 116 HPV16-positive asymptomatic individuals (control group) were enrolled in the current study. Then, the HPV16 LCR was sequenced to determine the association between LCR variations and cervical cancer. Results: In the current study, HPV16 A1-A3 (19.4%), A4 (78.4%) and D3 (2.2%) variants were found in the case group. However, only A1-A3 (34.5%) and A4 variants (65.5%) were found in the control group. The distribution of the HPV16 variants between the case and control groups was significantly different (P=0.009). Moreover, a total of eleven variations (A7167G, A7173C, C7176T, C7200T, T7269C, C7286A, C7729A, C7763T, A7841G, G7867A and T24C) were significantly different between the case and control groups (P<0.05). For the sub-lineage analysis, only C7873G variations were significantly different between the case and control groups in the A4 (As) variant (P=0.039). Conclusion: Our results showed that specific variations in the HPV16 LCR were associated with cervical cancer. Our study will provide a good reference for further understanding of the relationship between HPV16 LCR variation and cervical cancer.

Frequency of Epstein-Barr virus and human papilloma virus in patients with nasopharyngeal carcinoma.

PURPOSE: Nasopharyngeal cancer is a type of malignancy originating from the epithelial cells lining the nasopharynx. In genetic and environmental factors, infection with Epstein-Barr virus is one of the particular factors held accountable for the etiopathogenesis. Human papillomavirus has been associated with cervical, anogenital, and oropharyngeal cancers. The aim of the present study is to demonstrate the presence and incidence of Epstein-Barr virus and human papillomavirus in patients with nasopharyngeal cancer. METHODS: The information collected for these patients included age at the time of biopsy, gender, alcohol consumption and smoking, and histopathological type of nasopharyngeal cancer. Only patients for whom nasopharyngeal biopsy was performed as punch biopsy were included in the study. In situ hybridization was performed with formalin-fixed, paraffin-embedded tissue sections for Human Papillomavirus and Epstein-Barr virus nucleic acids obtained by means of automated Ventana BenchMark Medical system RESULTS: Utilizing in situ hybridization with samples obtained from 56 patients diagnosed with nasopharyngeal cancer. Epstein-Barr virus was positive in 41 out of the 56 (73.2%) patients, while human papillomavirus was positive in 3 (5.4%), and 1 patient (1.8%) had co-infection. Thirty seven (90.2%) of the 41 patients positive for Epstein-Barr virus were Type-2 according to WHO, while 4 (9.8%) were Type-1. All three patients (100%) with Human Papillomavirus positivity were Type-2 according to WHO. CONCLUSIONS: This study shows the close association between nasopharyngeal cancer and Epstein-Barr virus whereas such an association is not shown for Human Papillomavirus.

HPV16 E6 regulates the proliferation, invasion, and apoptosis of cervical cancer cells by downregulating miR-504.

BACKGROUND: Human papillomavirus (HPV) 16 infection is a necessary condition for the pathogenesis and development of cervical cancer. The E6 protein is expressed by the HPV16 E6 gene and promotes malignant phenotype transformation, which is an important mechanism for the occurrence and development of cervical cancer. MicroRNA-504 (miR-504) has been reported as an oncogene or tumor suppressor gene; the expression of miR-504 in cervical cancer has been found to be negatively correlated with HPV infection. However, the relationship between HPV16 E6 and miR-504 and the role of miR-504 in cervical cancer are not clear. In the current study, we observed the effect of HPV16 E6 on the expression of miR-504 in cervical cancer cells, and analyzed whether HPV16 E6 affects proliferation, invasion, and apoptosis in cervical cancer cells by regulating the expression of miR-504. METHODS: Cervical cancer cells (SiHa) were divided into four groups: the empty vector group, E6 overexpression group, E6 overexpression + miR-NC group, and E6 overexpression+miR-504 group. The expressions levels of HPV16 E6 mRNA and miR-504 were detected by real-time polymerase chain reaction (PCR), and the expression level of HPV16 E6 protein was detected by Western blot. Cell proliferation, invasion, and apoptosis were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Tastelessly, and flow cytometry, respectively. RESULTS: The expression level of miR-504 was significantly decreased in E6 overexpression cells compared to the control cells (P<0.05); the overexpression of miR-504 with miR-504 mimic significantly reversed the downregulation of miR-504 in E6 overexpression SiHa cells (P<0.05). MTT and Transwell assays showed that the overexpression of E6 significantly increased proliferation and invasion of SiHa cells (P<0.05). The overexpression of miR-504 reversed the role of HPV16 E6 on the proliferation and invasion in E6 overexpression SiHa cells, and the difference was statistically significant (P<0.05). Further analysis showed that the overexpression of E6 significantly reduced apoptosis of SiHa cells (P<0.05). The overexpression of miR-504 reversed the role of HPV16 E6 on apoptosis in E6 overexpression SiHa cells, and the difference was statistically significant (P<0.05). CONCLUSIONS: HPV16 E6 may promote the proliferation and invasion, and inhibit the apoptosis, of cervical cancer SiHa cells by downregulating miR-504 expression.

Genetic characterization of variants of HPV­16, HPV­18 and HPV­52 circulating in Italy among general and high­risk populations.

Viral factors, such as high­risk human papillomavirus variants, can increase the risk of viral persistence and influence the progression to cancer. In the present study, the long control region (LCR) of human papillomavirus (HPV)­16 and HPV­52, and the L1 region of HPV­16 and HPV­18, identified from subjects belonging to both general and high­risk populations (migrants, HIV+ subjects and adolescent/young people) residing in Italy, were characterized using molecular and phylogenetic techniques. To the best of our knowledge, this is the first Italian study to analyze a large number of sequences (n=458) and report phylogenetic data on the HPV­52 variants. The phylogenetic analysis showed that 90% of the LCR variants of HPV­16 and HPV­52 clustered within lineage A (European lineage) and only sequences identified from subjects belonging to high­risk populations fell into the non­European lineages. Analysis of the LCRs revealed a high genomic diversity with a large number of changes. Several mutations in the binding sites for viral and cellular transcription factors characterized the HPV­16 LCR variants belonging to the African lineages B and C, were observed in subjects with cytological abnormalities (high squamous intraepithelial lesions). The HPV­16 and HPV­18 L1 molecular characterization identified 30% of changes in the immune­dominant epitope loops. These data give a clear picture of the situation in Italy, and a starting point for understanding the molecular pathogenesis and developing molecular diagnostics for HPV, vaccines and other therapeutic approaches in order to control and/or eliminate virus­induced diseases.

Human Papillomavirus Type 16 E1 Mutations Associated with Cervical Cancer in a Han Chinese Population.

Human papillomavirus type 16 (HPV16) is a high-risk HPV type and a potent carcinogen. HPV E1 is one of the most highly conserved proteins and it plays a central role in initiating HPV DNA replication. In current study, we enrolled 161 HPV16-positive cervical cancer patients (case group) and 171 HPV16-positive asymptomatic individuals (control group) in a study to analyse the association between HPV16 E1 genetic mutations and cervical cancer. The samples of case group were cervical cancer tissues and the samples of control group were cervical exfoliated cells. Three variants (A4, A1-A3 and D3) were found in the case group, 68.3% of the HPV16 E1 sequences belonged to the A4 (As) sub-lineage, 29.2% belonged to the A1-A3 (EUR) sub-lineage, and 2.5% belonged to the D3 (AA1) sub-lineage. Two variants (A4 and A1-A3) occurred in the control group. The A4 (As) sub-lineage was predominant in this group as well (66.1%), followed by the A1-A3 (EUR) sub-lineage (33.9%), but the D3 (AA1) sub-lineage was not found in the control group. The distribution of the HPV16 variants between the case and control groups was significantly different (P<0.05). When the distribution of the HPV16 E1 gene mutations was compared, the distribution of twenty-seven mutations was significantly different between the case and control groups (P<0.05), and twenty-two mutations occurred only in the D3 (AA1) sub-lineage, two were found only in the A4 (As) sub-lineage, one was found in the A1-A3 (EUR) sub-lineage, two was found in both the A4 (As) and A1-A3 (EUR) sub-lineages. In the sub-lineage analysis, the differences in the T933A (A23A), T1014G (D50E) and G2160A (R432R) mutations were statistically significant between the case and control groups for the A4 (As) sub-lineage (P<0.05), and the differences in the T2232C (F456F), G2337A (M491I) and A2547G (P561P) mutations were statistically significant between the case and control groups for the A1-A3 (EUR) sub-lineage (P<0.05). In the current study, we describe specific mutations in the HPV16 E1 gene associated with cervical cancer, and our study will provide a good reference for further functional studies of the relationship between cervical cancer carcinogenesis and HPV genes.

Human papillomavirus type 16 E6 and E7 gene variations associated with cervical cancer in a Han Chinese population.

BACKGROUD: Human papillomavirus type 16 (HPV16) is a high-risk HPV subtype and a potent carcinogen. The HPV16 E6 and E7 genes are considered oncogenes that play a core role in the development of cervical cancer. METHODS: In the current study, we enrolled 97 HPV16-positive cervical cancer patients (case group) and 136 HPV16-positive asymptomatic individuals (control group) in a study to analyse the association between HPV16 E6 and E7 gene variations and cervical cancer. RESULTS: Our results showed that three HPV16 sub-lineages (A1-A3, A4 and D3) were present; the distribution of these variants between the case and control group was not significantly different (P = 0.178). When the distribution of the HPV16 E6 and E7 gene variations was compared, the distribution of only A131C (R10R) in the E6 gene showed a different trend between the case and control groups and C749T (S63F) in the E7 gene was significantly different between the case and control groups (P = 0.071 and P = 4.861 × 10-10, respectively). Regarding the sub-lineages, no variations in the E6 gene were significantly different between the case and control group for the A4 (As) and A1-A3 (EUR) sub-lineages. However, the distribution of C749T (S63F) in the E7 gene was significantly different between the case and control groups for the A4 (As) and A1-A3 (EUR) sub-lineages (P = 1.815 × 10-8 and P = 0.008). In the current study, we found that the C749T (S63F) variation in the HPV16 E7 gene was associated with cervical cancer not only in the A4 (As) sub-lineage but also in the A1-A3 (EUR) sub-lineage. CONCLUSION: Our study will provide a good reference for further functional studies of the relationship between cervical cancer carcinogenesis and the HPV16 E6 and E7 genes.