HPV16 E6 Gene Polymorphisms and the Functions of the Mutation Site in Cervical Cancer Among Uygur and Han Women in Xinjiang

Background: To investigate the genotype distribution of human papillomavirus (HPV) in infected Uygur and Han women in Xinjiang; analyze the HPV16 E6 gene polymorphism site and relationship with the development of cervical cancer.Methods: The HPV16 E6 sequence was analyzed using the European standard prototype to perform an evolutionary tree. HPV16 E6-295T/350T, 295G/350G, and 295T/350G GV230 vectors were stably transfected into cervical cancer C33A cells to analyze the cell proliferation, migration and invasion, apoptosis by CCK8 and clonogenic assays, transwell and cell scratch assays, FACS experiments. Results: The total HPV infection rate was 26.390% (760/2879), whereas the Uygur 22.87% (196/857) and the Han was 27.89% (564/2022) (P < 0.05). Among 110 mutations, 65 cases of E6 genes were mutated at nucleotide 350 (T350G) with the leucine changing to valine (L83V). Moreover, there were 7 cases of E6 gene mutated at nucleotide 295 (T295G) with aspartic changing to glutamic (D64E). When E6 vector(s) of mutations sites were transfected into C33A cells, they were found to promote cellular proliferation, migration, invasion, and inhibit apoptosis. The 295T/350G had the strongest effect on C33A cells and 295G/350G was significantly stronger than 295T/350T (P < 0.05).Conclusions: The positive HPV infection rates differed between the Uygur and Han in Xinjiang, and the genotype distribution of infection was different. After transfecting C33A cells with different eukaryotic expression vectors, the 295T/350G mutation site promoted the proliferation，migration, and invasion of C33A cells to a greater extent than 295G/350G; however, 295G/350G had a stronger effect than 295T/350T.

Molecular Insights Into the Interaction of HPV-16 E6 Variants Against MAGI-1 PDZ1 Domain

Oncogenic protein E6 from Human Papilloma Virus 16 (HPV-16) mediates the degradation of Membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1), throughout the interaction of its protein binding motif (PBM) with the Discs-large homologous regions 1 (PDZ1) domain of MAG1-1. Generic variation in the E6 gene that translates to changes in the protein’s amino acidic sequence modifies the interaction of E6 with the cellular protein MAGI-1. MAGI-1 is a scaffolding protein found at tight junctions of epithelial cells, where it interacts with a variety of proteins regulating signaling pathways. MAGI-1 is a multidomain protein containing two WW (rsp-domain-9), one guanylate kinase-like, and six PDZ domains. PDZ domains played an important role in the function of MAGI-1 and served as targets for several viral proteins including the HPV-16 E6. The aim of this work was to evaluate, with an in silico approach, employing molecular dynamics simulation and protein-protein docking, the interaction of the intragenic variants E-G350 (L83V), E-C188/G350 (E29Q/L83V), E-A176/G350 (D25N/L83V), E6-AAa (Q14H/H78Y/83V) y E6-AAc (Q14H/I27RH78Y/L83V) and E6-reference of HPV-16 with MAGI-1. We found that variants E-G350, E-C188/G350, E-A176/G350, AAa and AAc increase their affinity to our two models of MAGI-1 compared to E6-reference.

Insights in the Interaction of HPV-16 E6 and Its Variants (E-G350, E-A176/G350, E-C188/G350, AAa and AAc) with MAGI-1

Oncogenic protein E6 from Human Papilloma Virus 16 (HPV-16) mediates the degradation of Membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1), throughout the interaction of its protein binding motif (PBM) with the Discs-large homologous regions 1 (PDZ1) domain of MAG1-1. Generic variation in the E6 gene that translates to changes in the protein&rsquo;s amino acidic sequence modifies the interaction of E6 with the cellular protein MAGI-1. MAGI-1 is a scaffolding protein found at tight junctions of epithelial cells, where it interacts with a variety of proteins regulating signaling pathways. MAGI-1 is a multidomain protein containing two WW (rsp-domain-9), one guanylate kinase-like, and six PDZ domains. PDZ domains played an important role in the function of MAGI-1 and served as targets for several viral proteins including the HPV-16 E6. The aim of this work was to evaluate, with an in silico approach, employing molecular dynamics simulation and protein-protein docking, the interaction of the intragenic variants E-G350 (L83V), E-C188/G350 (E29Q/L83V), E-A176/G350 (D25N/L83V), E6-AAa (Q14H/H78Y/83V) y E6-AAc (Q14H/I27RH78Y/L83V) and E6-reference of HPV-16 with MAGI-1. We found that variants E-G350, E-C188/G350, E-A176/G350, AAa and AAc increase their affinity to our two models of MAGI-1 compared to E6-reference.

Lineage analysis of human papillomavirus type 39 in cervical samples of Iranian women

Background The data with regards to the regional variants of distinct HPV types is of great value. Accordance with this, this study aimed to investigate the sequence variations of E6 gene and long control region of HPV 39 among normal, premalignant and malignant cervical samples in order to characterize the frequent HPV 39 variants circulating in Tehran, Iran. Methods In total, 70 cervical samples (45 normal, 16 premalignant, and 9 malignant samples) infected with HPV 39 were analyzed by nested-PCR and sequencing. Results Our results revealed that all samples belonged to A lineage. Almost all sequences (98.6%) were classified in A1 sublineage and only one sample (1.4%) was A2 sub lineage. Conclusions Our findings showed that lineages A, sublineage A1, is dominant in Tehran, Iran. However, the small sample size was the most important limitations of this study. Further studies with larger sample size from different geographical regions of Iran are necessary to estimate the pathogenicity risk of HPV 39 variants in this population.

Antiproliferative Effects of AAV-Delivered CRISPR/Cas9-Based Degradation of the HPV18-E6 Gene in HeLa Cells

Human papillomavirus infections are associated with most cervical cancers, which are the fourth most common cancer in women. HPV-E6 protein binds to protein p53 and inhibits its function, leading to the switching of normal cells toward cancer cells. Here, we disrupted the HPV-E6 gene and investigated its effects on the proliferation and apoptosis of HeLa cells. The HPV18-E6 gene was targeted with two designed sgRNAs cloned into an AAV-CRISPR-based plasmid. The AAV-E6-CRISPR/Cas9 virions were prepared and titrated in HEK293t cells. The cleavage created in the HPV-E6 gene was detected using the T7E1 assay. Cell cycle profiling, MTT assay, and annexin V/PI staining were performed. Also, the p53 protein level was measured by Western blotting. Our data showed that disruption of the HPV-E6 gene led to increased cell apoptosis and decreased cell proliferation. A significant accumulation of infected cells in sub-G1 phase was observed in the cell profiling assay. Also, HPV-E6 gene disruption resulted in a significant increase in the level of P53 protein. Our findings indicated that AAV-mediated delivery of CRISPR/Cas9 can effectively target the HPV-E6 gene in HeLa cells, and its antiproliferative effects may provide therapeutic benefits of local administration of this gene-editing system for HPV-related cervical cancers.

Low frequency of human cytomegalovirus in cancerous and precancerous cervical samples of Iranian women

Background: HPV-16 has a significant role in cervical cancers; co-infection with human cytomegalovirus (HCMV) as an oncomodulatory pathogen may increase the risk of carcinogenesis. This study aimed to investigate the frequencies of HCMV and HPV-16 in cervical samples. Materials & methods: A total of 102 cancerous and precancerous cervical samples were examined by real-time PCR targeting the HPV-16 E6 gene, and HCMV immediate-early gene. Results: In total, 65 samples (63.7%) were positive for HPV-16. HCMV was found in seven samples (6.9%). Both HPV-16 and HCMV were present in four samples (cervical intraepithelial neoplasia-3 and squamous cell carcinoma groups with two samples each). Conclusion: HCMV can infect cervical tissues at a low frequency, suggesting that HCMV is unlikely to play a role in the cervical carcinogenesis.

Sequence variation analysis of the E1 and E2 genes of human papillomavirus type 16 in cervical lesions in women from the south of Poland

The E1 and E2 genes of the human papillomavirus encode the so-called early proteins, their sequences are conserved, and regulatory functions are associated with the viral oncoproteins. The purpose of this study is to determine the HPV16 E1 and E2 mutations appearing in the female population of southern Poland, depending on the severity of cervical pathological changes. We also take into account the number of E1 and E2 mutations detected in the E6 gene variant (350G or 350T). This publication is one of the first in the Central and Eastern Europe to deal with this topic. We identified 4 mutations in the E1 gene and 24 mutations in the E2 gene that have not been described so far. In three cases of squamous cell carcinoma a C3409T mutation occurred, which is widely described as oncogenic. This mutation lies in the 3243-3539 area of the E2 hinge region. Statistical analyses show a possible relationship of mutations in this area with oncogenesis. The discovered dependencies may be important in the context of oncogenesis, however, a study with a larger group of patients is needed in order to confirm this view.

Epithelial to Mesenchymal Transition (EMT) in a Laryngeal Squamous Cell Carcinoma of a Horse: Future Perspectives

Squamous cell carcinoma (SCC) is one of the most frequent tumors of skin and muco-cutaneous junctions in the horse. Equine papillomavirus type 2 (EcPV2) has been detected in equine SCC of the oral tract and genitals, and recently also in the larynx. As human squamous cell carcinoma of the larynx (SCCL), it is strongly etiologically associated with high-risk papillomavirus (h-HPV) infection. This study focuses on tumor cells behavior in a naturally occurring tumor that can undergo the so-called epithelial to mesenchymal transition (EMT). A SCCL in a horse was investigated by immunohistochemistry using antibodies against E-cadherin, pan-cytokeratin AE3/AE1, β-catenin, N-cadherin, vimentin, ZEB-1, TWIST, and HIF-1α. EcPV2 DNA detection and expression of oncogenes in SCC were investigated. A cadherin switch and an intermediate filaments rearrangement within primary site tumor cells together with the expression of the EMT-related transcription factors TWIST-1, ZEB-1, and HIF-1α were observed. DNA obtained from the tumor showed EcPV2 positivity, with E2 gene disruption and E6 gene dysregulation. The results suggest that equine SCCL might be a valuable model for studying EMT and the potential interactions between EcPV2 oncoproteins and the EMT process in SCCL.

HPV16 E6 gene polymorphisms and the functions of the mutation site in cervical cancer among Uygur and Han women in Xinjiang

Objective : This study aimed to: 1) investigate the status and genotype distribution of human papillomavirus (HPV) in infected Uygur and Han women in Xinjiang; 2) elucidate the variation of the HPV16 E6 gene sequence in the cervix of Uygur and Han women in Xinjiang; and 3) analyze the HPV16 E6 gene polymorphism site and relationship with the development of cervical cancer. Methods : A total of 2879 samples of cervical mucus from the exfoliated cells of Uygur and Han women were collected for an epidemiological analysis of HPV. Genomic DNA was extracted from the cervical HPV16-positive tissues of 110 Uygur and Han women, and E6 was amplified by PCR and sequenced. The HPV16 E6 sequence was analyzed using the European standard as the prototype, and an evolutionary tree analysis was performed. HPV16 E6-295T/350T-GV230, HPV16 E6-295G/350G-GV230, and HPV16 E6-295T/350G-GV230 were stably transfected into human cervical cancer C33A cells. HPV16 E6 protein expression was confirmed using a direct immunofluorescence assay. CCK8 and clonogenic assays were used to analyze C33A cell proliferation. Both a transwell and cell scratch assay were used to study C33A cell migration and invasion. C33A cell apoptosis was analyzed using FACS experiments. SPSS17.0 statistical software was used for statistical data processing. P < 0.05 was considered statistically significant. Results : The total HPV infection rate was 26.390% (760/2879), whereas the Uygur infection rate was 22.87% (196/857) and the Han infection rate was 27.89% (564/2022) (P < 0.05). HPV16, HPV 52, and HPV 53 were associated with higher detection rates in Uygur, whereas HPV16, HPV52, and HPV58 exhibited a higher detection rate in Han. HPV-infected women from Uygur and Han commonly exhibited a single infection. A total of 14 mutation sites were identified in the HPV16 E6 gene by sequencing 110 HPV16-positive samples, including eight missense and six synonymous mutations. Among these, 65 cases of E6 genes were mutated at nucleotide 350 (T350G) with the corresponding amino acids changing from leucine to valine (L83V) and a mutation rate of 59.09%. Moreover, there were seven cases of an E6 gene mutation at nucleotide 295 (T295G) with corresponding amino acid changes from aspartic to glutamic (D64E) and a mutation rate of 6.36%. It is important to note that these seven cases of HPV16 E6 T295G mutations were accompanied by the E6 T350G mutation. Phylogenetic analysis showed that there were HPV16 European (Ep), European variant (E), and three Asian (As) types in Uygur and Han women. No African (Af) and Asian American (AA) types were observed. When HPV16 E6 295T/350T, 295G/350G, and 295T/350G GV230 eukaryotic expression vector(s) were stably transfected into cervical cancer C33A cells, they were found to promote cellular proliferation, migration, invasion, and inhibit apoptosis. The 295T/350G-GV230 had the strongest effect on C33A cells and 295G/350G-GV230 was significantly stronger than 295T/350T-GV230 (P < 0.05). Conclusions : The positive HPV infection rates differed between the Uygur and Han groups in Xinjiang, and the genotype distribution of HPV infection was different. Between the Uygur and Han women in Xinjiang, the main types of HPV16 infection were European (E) and Asian (As). After stably transfecting C33A cells with a eukaryotic expression vector for different polymorphism sites (295T/350T, 295G/350G, and 295T/350G), the 295T/350G mutation site promoted the proliferation，migration, and invasion of C33A cells to a greater extent than 295G/350G; however, 295G/350G had a stronger effect than 295T/350T.