RESUMEN
BACKGROUND: Cervical cancer, primarily caused by HPV infection, remains a global health concern. Current treatments face challenges including drug resistance and toxicity. This study investigates combining E5-siRNA with chemotherapy drugs, Oxaliplatin and Ifosfamide, to enhance treatment efficacy in HPV-16 positive cervical cancer cells, targeting E5 oncoprotein to overcome limitations of existing therapies. METHODS: The CaSki cervical cancer cell line was transfected with E5-siRNA, and subsequently treated with Oxaliplatin/Ifosfamide. Quantitative real-time PCR was employed to assess the expression of related genes including p53, MMP2, Nanog, and Caspases. Cell apoptosis, cell cycle progression, and cell viability were evaluated using Annexin V/PI staining, DAPI staining, and MTT test, respectively. Furthermore, stemness ability was determined through a colony formation assay, and cell motility was assessed by wound healing assay. RESULTS: E5-siRNA transfection significantly reduced E5 mRNA expression in CaSki cells compared to the control group. The MTT assay revealed that monotherapy with E5-siRNA, Oxaliplatin, or Ifosfamide had moderate effects on cell viability. However, combination therapy showed synergistic effects, reducing the IC50 of Oxaliplatin from 11.42 × 10-8 M (45.36 µg/ml) to 6.71 × 10-8 M (26.66 µg/ml) and Ifosfamide from 12.52 × 10-5 M (32.7 µg/ml) to 8.206 × 10-5 M (21.43 µg/ml). Flow cytometry analysis demonstrated a significant increase in apoptosis for combination treatments, with apoptosis rates rising from 11.02 % (Oxaliplatin alone) and 16.98 % (Ifosfamide alone) to 24.8 % (Oxaliplatin + E5-siRNA) and 34.9 % (Ifosfamide + E5-siRNA). The sub-G1 cell population increased from 15.7 % (Oxaliplatin alone) and 18 % (Ifosfamide alone) to 21.9 % (Oxaliplatin + E5-siRNA) and 27.1 % (Ifosfamide + E5-siRNA), indicating cell cycle arrest. The colony formation assay revealed a substantial decrease in the number of colonies following combination treatment. qRT-PCR analysis showed decreased expression of stemness-related genes CD44 and Nanog, and migration-related genes MMP2 and CXCL8 in the combination groups. Apoptosis-related genes Casp-3, Casp-9, and pP53 showed increased expression following combination therapy, while BAX expression increased and BCL2 expression decreased relative to the control. CONCLUSION: The study demonstrates that combining E5-siRNA with Oxaliplatin or Ifosfamide enhances the efficacy of chemotherapy in HPV-16 positive cervical cancer cells. This synergistic approach effectively targets multiple aspects of cancer cell behavior, including proliferation, apoptosis, migration, and stemness. The findings suggest that this combination strategy could potentially allow for lower chemotherapy doses, thereby reducing toxicity while maintaining therapeutic efficacy. This research provides valuable insights into targeting HPV E5 as a complementary approach to existing therapies focused on E6 and E7 oncoproteins, opening new avenues for combination therapies in cervical cancer treatment.
Asunto(s)
Apoptosis , Papillomavirus Humano 16 , Ifosfamida , Oxaliplatino , ARN Interferente Pequeño , Neoplasias del Cuello Uterino , Humanos , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , Oxaliplatino/farmacología , Femenino , ARN Interferente Pequeño/genética , Línea Celular Tumoral , Ifosfamida/farmacología , Apoptosis/efectos de los fármacos , Papillomavirus Humano 16/genética , Infecciones por Papillomavirus/tratamiento farmacológico , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/virología , Supervivencia Celular/efectos de los fármacos , Proteínas Oncogénicas Virales/genética , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacosRESUMEN
Background: In the general population, primary human papillomavirus (HPV) testing is advocated for cervical cancer (CC) screening. HPV E6/E7 mRNA (Aptima HPV, AHPV) assays have garnered considerable traction due to their higher specificity when compared with HPV DNA assays. Here, we investigated age-specific primary AHPV screening assays and different triage strategies versus cytology to identify the best approach. Methods: Between April 2018 and December 2021, we recruited female participants from 34 communities across Liaoning province and Qingdao City, China. Primary cervical screening protocols included liquid-based cytology (LBC) and AHPV assays, with females positive for any assays undergoing colposcopy. Genotyping (AHPV-GT) was conducted on all HPV-positive samples. Our primary outcomes were the identification of age-specific detection rates, colposcopy referral rates, and sensitivity and specificity values for high-grade squamous intraepithelial lesions or worse (HSIL+). AHPV and different triage strategy performances were also examined across different age cohorts. Results: Our investigation included 9911 eligible females. Age-specific abnormal cytology rates were in the 6.1%-8.0% range, and were highest in 45-54-year olds. When compared with 35-44-or 45-54-year olds, HPV prevalence was highest in 55-64-year olds (12.2% or 11.6% vs.14.1%, P = 0.048 and P = 0.002, respectively). In 35-44-year olds, AHPV sensitivity for detecting HSIL+ was 96.6 (95% confidence interval [CI]: 89.7-100) - significantly higher than LBC sensitivity (65.5 [95% CI: 48.3-82.8], P < 0.001). When compared with LBC, HSIL+ detection rates by AHPV-GT using reflex LBC triage increased by 31.5% (9.6 vs. 7.3), and colposcopy referral rates decreased by 16.4% (5.1% vs. 6.1%). In 45-54-year olds, HSIL+ detection rates for AHPV-GT using reflex LBC triage were lower than LBC rates (6.2 vs. 6.6). In 55-64-year olds, AHPV sensitivity (97.2 [95% CI: 91.7-100.0]) was higher than LBC sensitivity (66.7 [95% CI: 50.0-80.6], P = 0.003). The area under the curve (AUC) value was not significantly different between AHPV-GT with reflex LBC triage and LBC (0.845 [95% CI: 0.771-0.920] vs. 0.812 [95% CI: 0.734-0.891], P = 0.236). Conclusions: Primary AHPV screening using different triage strategies were different across different age cohorts. Thus, AHPV may be an appropriate primary screening method for 35-44 and 55-64 year old females, while AHPV-GT with reflex LBC triage may be more apt for 35-44 year old females.
Asunto(s)
Detección Precoz del Cáncer , Infecciones por Papillomavirus , Sensibilidad y Especificidad , Triaje , Neoplasias del Cuello Uterino , Humanos , Femenino , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/virología , Persona de Mediana Edad , China/epidemiología , Adulto , Detección Precoz del Cáncer/métodos , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/virología , Triaje/métodos , Anciano , Factores de Edad , Colposcopía , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , ARN Mensajero/genética , Proteínas Oncogénicas Virales/genética , Adulto Joven , Genotipo , Tamizaje Masivo/métodos , Virus del Papiloma Humano , CitologíaRESUMEN
High-risk human papilloma virus (HR-HPV) persistent infection is closely associated with the development of cervical cancer and squamous intraepithelial lesion (SIL).The α-9 HPVs, which is predominantly composed of HR-HPV types, account for 75% of HR-HPV infection in Sichuan. The oncoproteins E6 and E7 of HPV play a crucial role in tumor initiation and progression. Notably, HPV-35 is the only HR-HPV type within the α-9 genus that is not included in the nine-valent HPV prophylactic vaccine. Cervical cell samples obtained from Sichuan were collected for HPV detection and genotyping. Among the 406 HPV-positive samples, 31 HPV-35 were detected, 24 HPV-35 E6 and 26 E7 were successfully amplified and sequenced, five nucleotide mutations in E6 and three in E7 were detected, T232C, T434G of E6 (W78R, I145R) and C67T, G84T of E7 (H23Y, L28F) were non-synonymy mutation. PAML 4.8 server was used to detect positive selection sites of HPV-35 E6, E7, and E6 is W78R. Phyre2 were used to predict and analyze protein structures, W78R made influences on protein structure. IEDB were used to screen epitopes vaccine target for HPV-35 affection therapy, and 5 HPV-35 E6 and 3 HPV-35 E7 most potential epitopes were obtained, the most potential peptides for therapy vaccine design were 79-91YRYSVYGETLEKQ, 45-60FACYDLCIVREGQPY, 124-135RFHNIGGRWTGR of E6; 3-19GEITTLQDYVLDLEPEA, 38-47TIDGPAGQAK, 70-88VQSTHIDIRKLEDLLMGTF of E7 and W78R mainly decreased the epitopes affinity.Conclusions Amino acid substitution in the positive selection sites of HPV-35 E6 and E7 genes have been found to influence protein structure and to decrease the overall affinity of antigen epitopes. This observation aligns with the evolutionary significance of positive selection site, which may confer advantages to the virus by making infected cells more challenging for the immune system to detect, thereby enhancing HPV's adaptability to the host environment. The polymorphism analysis of HPV-35 E6, E7 contributes to the enrichment of α-9 HPV data in Sichuan China, which is instrumental in improving the effectiveness of clinical detection. Furthermore, these findings provide a relevant theoretical foundation for the prevention and treatment of HPV-related diseases.
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Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Vacunas contra Papillomavirus , Humanos , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/inmunología , Femenino , China , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/prevención & control , Vacunas contra Papillomavirus/inmunología , Vacunas contra Papillomavirus/genética , Polimorfismo Genético , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/inmunología , Genotipo , Adulto , Neoplasias del Cuello Uterino/virología , Neoplasias del Cuello Uterino/prevención & control , Epítopos/inmunología , Epítopos/genética , Alphapapillomavirus/genética , Alphapapillomavirus/inmunología , Alphapapillomavirus/clasificación , Persona de Mediana Edad , Mutación , Virus del Papiloma HumanoRESUMEN
The natural history of cervical cancer is closely linked to that of high-risk human papillomaviruses (HPV) infection. It is recognized that upon HPV DNA integration, partial or complete loss of the E2 open reading frame precludes expression of the corresponding protein, resulting in upregulation of the E6 and E7 viral oncoproteins. To better characterize HPV16 infection at the cervical level, viral load, viral DNA integration, and viral early transcript expression (E2, E5, and E6) were analyzed in a series of 158 cervical specimens representative of the full spectrum of cervical disease. Overall, the frequency of early transcript detection varied from 45% to 90% and tended to increase with lesion severity. In addition, the levels of E2, E5, and E6 transcript expression were slightly higher in high-grade lesions than in cervical specimens without abnormalities. Notably, early transcript expression was clearly associated with viral load, and no inverse correlation was found between the expression of E2 and E6 transcripts. No clear association was found between early transcript expression and HPV16 DNA integration, with the exception that samples with a fully integrated HPV16 genome did not harbor E2 or E5 transcripts. In conclusion, early HPV16 transcript expression appears to be associated with viral load rather than lesion grade. From a practical point of view, quantification of HPV16 early transcripts is difficult to translate into a relevant biomarker for cervical cancer screening.
Asunto(s)
Papillomavirus Humano 16 , Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Carga Viral , Humanos , Femenino , Papillomavirus Humano 16/genética , Infecciones por Papillomavirus/virología , Proteínas Oncogénicas Virales/genética , Neoplasias del Cuello Uterino/virología , Integración Viral , Adulto , Persona de Mediana Edad , ADN Viral/genética , Anciano , Cuello del Útero/virología , Cuello del Útero/patologíaRESUMEN
E6 and E7 oncogenes are pivotal in the carcinogenic transformation in HPV infections and efficient diagnostic methods can ensure the detection and differentiation of HPV genotype. This study describes the development and validation of an electrochemical, label-free genosensor coupled with a microfluidic system for detecting the E6 and E7 oncogenes in cervical scraping samples. The nanostructuring employed was based on a cysteine and graphene quantum dots layer that provides functional groups, surface area, and interesting electrochemical properties. Biorecognition tests with cervical scraping samples showed differentiation in the voltammetric response. Low-risk HPV exhibited a lower biorecognition response, reflected in ΔI% values of 82.33 % ± 0.29 for HPV06 and 80.65 % ± 0.68 for HPV11 at a dilution of 1:100. Meanwhile, high-risk, HPV16 and HPV18, demonstrated ΔI% values of 96.65 % ± 1.27 and 93 % ± 0.026, respectively, at the same dilution. Therefore, the biorecognition intensity followed the order: HPV16 >HPV18 >HPV06 >HPV11. The limit of detection and the limit of quantification of E6E7 microfluidic LOC-Genosensor was 26 fM, and 79.6 fM. Consequently, the E6E7 biosensor is a valuable alternative for clinical HPV diagnosis, capable of detecting the potential for oncogenic progression even in the early stages of infection.
Asunto(s)
Técnicas Biosensibles , Proteínas Oncogénicas Virales , Técnicas Biosensibles/métodos , Humanos , Proteínas Oncogénicas Virales/genética , Femenino , Límite de Detección , Proteínas E7 de Papillomavirus/genética , Cuello del Útero/virología , Grafito/química , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/virología , Técnicas Electroquímicas/métodos , Proteínas Represoras/genética , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Puntos Cuánticos/química , Dispositivos Laboratorio en un Chip , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificaciónRESUMEN
BACKGROUND: Human papillomavirus 16 (HPV16) is an oncogenic virus responsible for the majority of invasive cervical cancer cases worldwide. Due to genetic modifications, some variants are more oncogenic than others. We analysed the HPV16 phylogeny in HPV16-positive cervical Desoxyribonucleic Acid (DNA) samples collected from South African and Mozambican women to detect the circulating lineages. METHODS: Polymerase chain reaction (PCR) amplification of the long control region (LCR) and 300 nucleotides of the E6 region was performed using HPV16-specific primers on HPV16-positive cervical samples collected in women from South Africa and Mozambique. HPV16 sequences were obtained through Next Generation Sequencing (NGS) methods. Geneious prime and MEGA 11 software were used to align the sequences to 16 HPV16 reference sequences, gathering the A, B, C, and D lineages and generating the phylogenetic tree. Single nucleotide polymorphisms (SNPs) in the LCR and E6 regions were analysed and the phylogenetic tree was generated using Geneious Prime software. RESULTS: Fifty-eight sequences were analysed. Of these sequences, 79% (46/58) were from women who had abnormal cervical cytology. Fifteen SNPs in the LCR and eight in the E6 region were found to be the most common in all sequences. The phylogenetic analysis determined that 45% of the isolates belonged to the A1 sublineage (European variant), 34% belonged to the C1 sublineage (African 1 variant), 16% belonged to the B1 and B2 sublineage (African 2 variant), two isolates belonged to the D1-3 sublineages (Asian-American variant), and one to the North American variant. CONCLUSIONS: The African and European HPV16 variants were the most common circulating lineages in South African and Mozambican women. A high-grade squamous intraepithelial lesion (HSIL) was the most common cervical abnormality observed and linked to European and African lineages. These findings may contribute to understanding molecular HPV16 epidemiology in South Africa and Mozambique.
Asunto(s)
Papillomavirus Humano 16 , Infecciones por Papillomavirus , Filogenia , Neoplasias del Cuello Uterino , Humanos , Femenino , Mozambique/epidemiología , Sudáfrica/epidemiología , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/aislamiento & purificación , Papillomavirus Humano 16/clasificación , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/epidemiología , Neoplasias del Cuello Uterino/virología , Adulto , Cuello del Útero/virología , Cuello del Útero/patología , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , ADN Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas Oncogénicas Virales/genética , Adulto Joven , CitologíaRESUMEN
Human papillomaviruses (HPVs) account for more than 30% of cancer cases, with definite identification of the oncogenic role of viral E6 and E7 genes. However, the identification of high-risk HPV genotypes has largely relied on lagged biological exploration and clinical observation, with types unclassified and oncogenicity unknown for many HPVs. In the present study, we retrieved and cleaned HPV sequence records with high quality and analyzed their genomic compositional traits of dinucleotide (DNT) and DNT representation (DCR) to overview the distribution difference among various types of HPVs. Then, a deep learning model was built to predict the oncogenic potential of all HPVs based on E6 and E7 genes. Our results showed that the main three groups of Alpha, Beta, and Gamma HPVs were clearly separated between/among types in the DCR trait for either E6 or E7 coding sequence (CDS) and were clustered within the same group. Moreover, the DCR data of either E6 or E7 were learnable with a convolutional neural network (CNN) model. Either CNN classifier predicted accurately the oncogenicity label of high and low oncogenic HPVs. In summary, the compositional traits of HPV oncogenicity-related genes E6 and E7 were much different between the high and low oncogenic HPVs, and the compositional trait of the DCR-based deep learning classifier predicted the oncogenic phenotype accurately of HPVs. The trained predictor in this study will facilitate the identification of HPV oncogenicity, particularly for those HPVs without clear genotype or phenotype.
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Aprendizaje Profundo , Genoma Viral , Papillomaviridae , Infecciones por Papillomavirus , Humanos , Infecciones por Papillomavirus/virología , Papillomaviridae/genética , Genoma Viral/genética , Genotipo , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , Carcinogénesis/genéticaRESUMEN
Human papillomavirus (HPV) 11/16 E6/E7 proteins have been recognized to be pivotal in viral pathogenesis. This study sought to uncover the potential mechanisms of how HPV11/16 E6/E7-transfected keratinocytes inhibit cytokine secretion in peripheral blood mononuclear cells (PBMC). Upon co-culturing HPV11/16 E6/E7-transfected keratinocytes with PBMC in a non-contact manner, we observed a marked decrease in various cytokines secreted by PBMC. To determine if this suppression was mediated by specific common secreted factors, we conducted transcriptomic sequencing on these transfected cells. This analysis identified 53 common differentially secreted genes in all four HPV-transfected cells. Bioinformatics analysis demonstrated these genes were predominantly involved in immune regulation. Results from quantitative PCR (qPCR) and an extensive literature review suggested the downregulation of 12 genes (ACE2, BMP3, BPIFB1, CLU, CST6, CTF1, HMGB2, MMP12, PDGFA, RNASE7, SULF2, TGM2), and upregulation of 7 genes (CCL17, CCL22, FBLN1, PLAU, S100A7, S100A8, S100A9), may be crucial in modulating tumor immunity and combating pathogenic infections, with genes S100A8 and S100A9, and IL-17 signaling pathway being particularly noteworthy. Thus, HPV11/16 E6/E7 proteins may inhibit cytokine secretion of immune cells by altering the expression of host-secreted genes. Further exploration of these genes may yield new insights into the complex dynamics of HPV infection.
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Citocinas , Leucocitos Mononucleares , Proteínas Oncogénicas Virales , Humanos , Citocinas/metabolismo , Citocinas/genética , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Leucocitos Mononucleares/metabolismo , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Proteínas Oncogénicas Virales/inmunología , Queratinocitos/virología , Queratinocitos/inmunología , Queratinocitos/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/inmunología , Papillomavirus Humano 11/genética , Papillomavirus Humano 11/inmunología , Perfilación de la Expresión Génica , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/genética , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas E7 de Papillomavirus/inmunología , Técnicas de Cocultivo , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/genéticaRESUMEN
High-risk human papillomaviruses (HPVs) cause most cases of cervical cancer, a disease with an increasing impact worldwide. Recent studies have shown that the synthesis of viral oncoproteins is strongly subject to translational control. Thus, targeting the protein synthesis machinery might open novel avenues to develop innovative therapies aiming to improve patients' survival.
Asunto(s)
Papillomaviridae , Biosíntesis de Proteínas , ARN Mensajero , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/virología , ARN Viral/genética , ARN Viral/metabolismo , Neoplasias del Cuello Uterino/virología , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/genética , Regulación Viral de la Expresión Génica , FemeninoRESUMEN
Three-stranded DNA-RNA structures known as R-loops that form during papillomavirus transcription can cause transcription-replication conflicts and lead to DNA damage. We found that R-loops accumulated at the viral early promoter in human papillomavirus (HPV) episomal cells but were greatly reduced in cells with integrated HPV genomes. RNA-DNA helicases unwind R-loops and allow for transcription and replication to proceed. Depletion of the RNA-DNA helicase senataxin (SETX) using siRNAs increased the presence of R-loops at the viral early promoter in HPV-31 (CIN612) and HPV-16 (W12) episomal HPV cell lines. Depletion of SETX reduced viral transcripts in episomal HPV cell lines. The viral E2 protein, which binds with high affinity to specific palindromes near the promoter and origin, complexes with SETX, and both SETX and E2 are present at the viral p97 promoter in CIN612 and W12 cells. SETX overexpression increased E2 transcription activity on the p97 promoter. SETX depletion also significantly increased integration of viral genomes in CIN612 cells. Our results demonstrate that SETX resolves viral R-loops to proceed with HPV transcription and prevent genome integration.IMPORTANCEPapillomaviruses contain small circular genomes of approximately 8 kilobase pairs and undergo unidirectional transcription from the sense strand of the viral genome. Co-transcriptional R-loops were recently reported to be present at high levels in cells that maintain episomal HPV and were also detected at the early viral promoter. R-loops can inhibit transcription and DNA replication. The process that removes R-loops from the PV genome and the requisite enzymes are unknown. We propose a model in which the host RNA-DNA helicase senataxin assembles on the HPV genome to resolve R-loops in order to maintain the episomal status of the viral genome.
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ADN Helicasas , Enzimas Multifuncionales , Regiones Promotoras Genéticas , Estructuras R-Loop , ARN Helicasas , Humanos , ARN Helicasas/genética , ARN Helicasas/metabolismo , Enzimas Multifuncionales/genética , Enzimas Multifuncionales/metabolismo , ADN Helicasas/metabolismo , ADN Helicasas/genética , Estructuras R-Loop/genética , Plásmidos/genética , Replicación Viral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Papillomaviridae/genética , Genoma Viral , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Transcripción Genética , Línea Celular , ADN Viral/genéticaRESUMEN
BACKGROUND: High-risk human papillomavirus (HR-HPV) infection is an important factor for the development of cervical cancer. HPV18 is the second most common HR-HPV after HPV16. METHODS: In this study, MEGA11 software was used to analyze the variation and phylogenetic tree of HPV18 E6-E7 and L1 genes. The selective pressure to E6, E7 and L1 genes was estimated using pamlX. In addition, the B cell epitopes of L1 amino acid sequences and T cell epitopes of E6-E7 amino acid sequences in HPV18 were predicted by ABCpred server and IEDB website, respectively. RESULTS: A total of 9 single nucleotide variants were found in E6-E7 sequences, of which 2 were nonsynonymous variants and 7 were synonymous variants. Twenty single nucleotide variants were identified in L1 sequence, including 11 nonsynonymous variants and 9 synonymous variants. Phylogenetic analysis showed that E6-E7 and L1 sequences were all distributed in A lineage. In HPV18 E6, E7 and L1 sequences, no positively selected site was found. The nonconservative substitution R545C in L1 affected hypothetical B cell epitope. Two nonconservative substitutions, S82A in E6, and R53Q in E7, impacted multiple hypothetical T cell epitopes. CONCLUSION: The sequence variation data of HPV18 may lay a foundation for the virus diagnosis, further study of cervical cancer and vaccine design in central China.
Asunto(s)
Variación Genética , Papillomavirus Humano 18 , Proteínas Oncogénicas Virales , Proteínas E7 de Papillomavirus , Filogenia , Proteínas Oncogénicas Virales/genética , China , Humanos , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/clasificación , Proteínas E7 de Papillomavirus/genética , Proteínas de la Cápside/genética , Femenino , Epítopos de Linfocito T/genética , Infecciones por Papillomavirus/virología , Proteínas Represoras/genética , Epítopos de Linfocito B/genética , Proteínas de Unión al ADNRESUMEN
BACKGROUND: There is a high incidence of cervical cancer in Xinjiang. Genetic variation in human papillomavirus may increase its ability to invade, spread, and escape host immune response. METHODS: HPV16 genome was sequenced for 90 positive samples of HPV16 infection. Sequences of the E4, E5 and L2 genes were analysed to reveal sequence variation of HPV16 in Xinjiang and the distribution of variation among the positive samples of HPV16 infection. RESULTS: Eighty-one of the 90 samples of HPV16 infection showed variation in HPV16 E4 gene with 18 nucleotide variation sites, of which 8 sites were synonymous variations and 11 missense variations. 90 samples of HPV16 infection showed variation in HPV16 E5 and L2 genes with 16 nucleotide variation sites (6 synonymous, 11 missense variations) in the E5 gene and 100 nucleotide variation sites in L2 gene (37 synonymous, 67 missense variations). The frequency of HPV16 L2 gene missense variations G3377A, G3599A, G3703A, and G3757A was higher in the case groups than in the control groups. CONCLUSIONS: Phylogenetic tree analysis showed that 87 samples were European strains, 3 cases were Asian strains, there were no other variations, and G4181A was related to Asian strains. HPV16 L2 gene missense variations G3377A, G3599A, G3703A, and G3757A were significantly more frequent in the case groups than in the control groups.
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Variación Genética , Papillomavirus Humano 16 , Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Filogenia , Humanos , Femenino , China , Papillomavirus Humano 16/genética , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/genética , Proteínas Oncogénicas Virales/genética , Neoplasias del Cuello Uterino/virología , Neoplasias del Cuello Uterino/genética , Adulto , Persona de Mediana Edad , Mutación MissenseRESUMEN
The available prophylactic vaccines for human papillomavirus (HPV) in the market are only effective against specific types of HPV, rendering them ineffective for other types of HPV infections. The objective of this research is to investigate the stability of the recombinant protein constructed, namely chimeric L1/L2 protein from HPV type 52, with improved cross-neutralization ability. The 3D model, predicted using Alphafold, Robetta, I-Tasser, and refined with Galaxy Refinement, is validated using Ramachandran plot analysis. The stability is verified through molecular dynamics simulations, considering parameters such as RMSD, RMSF, Rg, and SASA, where stable conditions are observed. The chimeric L1/L2 protein from HPV type 52 is purified using affinity chromatography, and the His-tag is cleaved using SUMO protease to obtain pure chimeric protein with the size of ~ 55 kDa. Western blot analysis confirms binding to anti-L1 HPV type 52 polyclonal antibody. The obtained vaccine candidate can be utilized as an effective prophylactic vaccine against HPV.
Asunto(s)
Escherichia coli , Simulación de Dinámica Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/aislamiento & purificación , Proteínas de la Cápside/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Oncogénicas Virales/inmunología , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/aislamiento & purificación , Virus del Papiloma Humano , AlphapapillomavirusRESUMEN
Human papilloma virus (HPV) is an etiological factor of head and neck squamous cell carcinoma (HNSCC). To investigate the role of HPV antigen in anti-tumor immunity, we established mouse models by expressing HPV16 E6 and E7 in a SCC tumor cell line. We obtained two HPV antigen-expressing clones (C-225 and C-100) transplantable into C57BL/6 recipients. We found that C-225 elicited complete eradication in C57BL/6 mice (eradicated), whereas C-100 grew progressively (growing). We examined immune tumor microenvironment (TME) using flow cytometry and found that eradicated or growing tumors exhibited differential immune profiles that may influence the outcome of anti-tumor immunity. Surprisingly, the percentage of CD8 and CD4 tumor-infiltrating lymphocytes (TILs) was much higher in growing (C-100) than eradicated (C-225) tumor. However, the TILs upregulated PD-1 and LAG-3 more potently and exhibited impaired effector functions in growing tumor compared to their counterparts in eradicated tumor. C-225 TME is highly enriched with myeloid cells, especially polymorphonuclear (PMN) myeloid-derived suppressor cells (MDSC), whereas the percentage of M-MDSC and tumor-associated macrophages (TAMs) was much higher in C-100 TME, especially M2-TAMs (CD206+). The complete eradication of C-225 depended on CD8 T cells and elicited anti-tumor memory responses upon secondary tumor challenge. We employed DNA sequencing to identify differences in the T cell receptor of peripheral blood lymphocytes pre- and post-secondary tumor challenge. Lastly, C-225 and C-100 tumor lines harbored different somatic mutations. Overall, we uncovered differential immune TME that may underlie the divergent outcomes of anti-tumor immunity by establishing two SCC tumor lines, both of which express HPV16 E6 and E7 antigens. Our experimental models may provide a platform for pinpointing tumor-intrinsic versus host-intrinsic differences in orchestrating an immunosuppressive TME in HNSCCs and for identifying new targets that render tumor cells vulnerable to immune attack.
Asunto(s)
Modelos Animales de Enfermedad , Linfocitos Infiltrantes de Tumor , Ratones Endogámicos C57BL , Proteínas Oncogénicas Virales , Proteínas E7 de Papillomavirus , Infecciones por Papillomavirus , Microambiente Tumoral , Animales , Microambiente Tumoral/inmunología , Ratones , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Línea Celular Tumoral , Proteínas Oncogénicas Virales/inmunología , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/inmunología , Proteínas E7 de Papillomavirus/genética , Infecciones por Papillomavirus/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/virología , Proteínas Represoras/genética , Proteína del Gen 3 de Activación de Linfocitos , Humanos , Progresión de la Enfermedad , Linfocitos T CD8-positivos/inmunología , Receptor de Muerte Celular Programada 1 , Femenino , Papillomavirus Humano 16/inmunología , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/virologíaRESUMEN
This study was designed to investigate the expression of HPV16 L1-protein in biopsies of oral mucosa samples. The expression of HPV16 L1 protein was investigated in biopsies taken from oral mucosa from patients who required pathological diagnosis of oral lesions. Seventy-two samples were incubated with anti-L1 protein monoclonal antibodies and protein detection was revealed with diaminobenzidine. Expression of L1 protein was performed by a pathologist blinded for tissue diagnosis under light microscopy. Most of the lesions of oral mucosa were present in lining mucosa (75 %) and the most frequent lesion were mucocele (n = 17, 23.6 %), epithelial hyperplasia (n = 6, 8.33 %), fibroma (n = 5, 6.9 %) and inflammatory hyperplasia (n = 5, 6.9 %). L1 protein expression was observed only in five (6.9 %) samples (two squamous cell carcinomas, two epithelial hyperplasia, and one gingival hyperplasia). We concluded that L1 expression in oral biopsies presented a low frequency in oral mucosal biopsies samples.
Asunto(s)
Proteínas de la Cápside , Mucosa Bucal , Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Humanos , Biopsia , Femenino , Mucosa Bucal/virología , Mucosa Bucal/patología , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/virología , Adulto , Masculino , Proteínas Oncogénicas Virales/genética , Persona de Mediana Edad , Ecuador/epidemiología , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Adulto Joven , Adolescente , Anciano , Prevalencia , Enfermedades de la Boca/virología , Enfermedades de la Boca/patología , Enfermedades de la Boca/epidemiología , Papillomavirus Humano 16/genética , Inmunohistoquímica , Neoplasias de la Boca/virología , Neoplasias de la Boca/patología , Neoplasias de la Boca/diagnósticoRESUMEN
Oncogenic HPV E6 proteins have a PDZ-binding motif (PBM) which plays important roles in both the viral life cycle and tumor development. The PBM confers interaction with a large number of different PDZ domain-containing substrates, one of which is Sorting Nexin 27. This protein is part of the retromer complex and plays an important role in endocytic sorting pathways. It has been shown that at least two SNX27 interacting partners, GLUT1 and TANC2, are aberrantly trafficked due to the E6 PBM-dependent interaction with SNX27. To investigate further which other components of the endocytic trafficking pathway might be affected by the SNX27-HPV E6 interaction, we analyzed the SNX27 proteome interaction profile in a previously described HeLa cell line expressing GFP-SNX27, both in the presence and absence of the HPV-18 E6 oncoprotein. In this study, we identify a novel interacting partner of SNX27, secreted glycoprotein EMILIN2, whose release is blocked by HPV18 E6 in a PBM-dependent manner. Mechanistically, E6 can block EMILIN2 interaction with the WNT1 ligand, thereby enhancing WNT1 signaling and promoting cell proliferation. IMPORTANCE: This study demonstrates that HPV E6 blocks EMILIN2 inhibition of WNT1 signaling, thereby enhancing cell proliferation in HPV-positive tumor cells. This involves a novel mechanism whereby the E6 PBM actually contributes toward enhancing the interaction between SNX27 and EMILIN2, suggesting that the mode of recognition of SNX27 by E6 and EMILIN2 is different. This is the first example of the E6 PBM altering a PDZ domain-containing protein to enhance potential substrate recognition.
Asunto(s)
Papillomavirus Humano 18 , Proteínas Oncogénicas Virales , Nexinas de Clasificación , Vía de Señalización Wnt , Humanos , Proteínas de Unión al ADN , Células HEK293 , Células HeLa , Papillomavirus Humano 18/metabolismo , Papillomavirus Humano 18/genética , Proteínas Oncogénicas Virales/metabolismo , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/metabolismo , Dominios PDZ , Unión Proteica , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/genéticaRESUMEN
Despite epidermal turnover, the skin is host to a complex array of microbes, including viruses, such as HPV, which must infect and manipulate skin keratinocyte stem cells (KSCs) to survive. This crosstalk between the virome and KSC populations remains largely unknown. Here, we investigated the effect of HPV8 on KSCs using various mouse models. We observed that the HPV8 early region gene E6 specifically caused Lrig1+ hair follicle junctional zone KSC proliferation and expansion, which would facilitate viral transmission. Within Lrig1+ KSCs specifically, HPV8 E6 bound intracellular p300 to phosphorylate the STAT3 transcriptional regulatory node. This induced ΔNp63 expression, resulting in KSC expansion into the overlying epidermis. HPV8 was associated with 70% of human actinic keratoses. Together, these results define the "hit-and-run" mechanism for HPV8 in human actinic keratosis as an expansion of KSCs, which lack melanosome protection and are thus susceptible to sun light-induced malignant transformation.
Asunto(s)
Proliferación Celular , Queratinocitos , Queratosis Actínica , Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Factor de Transcripción STAT3 , Células Madre , Factor de Transcripción STAT3/metabolismo , Queratinocitos/virología , Queratinocitos/metabolismo , Queratinocitos/patología , Humanos , Queratosis Actínica/patología , Queratosis Actínica/metabolismo , Queratosis Actínica/virología , Animales , Ratones , Células Madre/metabolismo , Células Madre/virología , Proteínas Oncogénicas Virales/metabolismo , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/complicaciones , Modelos Animales de Enfermedad , FemeninoRESUMEN
Human papillomavirus (HPV), a common cause of sexually transmitted diseases, may cause warts and lead to various types of cancers, which makes it important to understand the risk factors associated with it. HPV is the leading risk factor and plays a crucial role in the progression of cervical cancer. Viral oncoproteins E6 and E7 play a pivotal role in this process. Beyond cervical cancer, HPV-associated cancers of the mouth and throat are also increasing. HPV can also contribute to other malignancies like penile, vulvar, and vaginal cancers. Emerging evidence links HPV to these cancers. Research on the oncogenic effect of HPV is still ongoing and explorations of screening techniques, vaccination, immunotherapy and targeted therapeutics are all in progress. The present review offers valuable insight into the current understanding of the role of HPV in cancer and its potential implications for treatment and prevention in the future.
Asunto(s)
Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Humanos , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/complicaciones , Femenino , Neoplasias del Cuello Uterino/virología , Neoplasias del Cuello Uterino/etiología , Papillomaviridae/patogenicidad , Neoplasias/virología , Neoplasias/terapia , Proteínas Oncogénicas Virales/metabolismo , Proteínas Oncogénicas Virales/genética , Factores de Riesgo , MasculinoRESUMEN
Human papillomavirus (HPV) type 81 has recently become one of the most common low-risk HPV types; however, literature focusing on it is limited. This study aimed to analyze the reasons for the increased detection rate of HPV81 and investigate its evolving pathogenicity. We analyzed the detection rates and trends of HPV81 in 229 061 exfoliated cervical cell samples collected from 2014 to 2023; collected samples of HPV81 single infections from two different time periods; and analyzed the allele frequencies, positive selection, viral load, persistent infection capacity, and pathogenicity of E6 and E7 genotypes. We found that the detection rate of HPV81 ranked first among the low-risk types in exfoliated cervical cells and exhibited a significantly increasing trend (p < 0.001). The frequency of the E6 prototype allele of HPV81 (n = 317) was significantly increased (p = 0.018) and demonstrated the strongest adaptive capacity. The viral load and persistent infection capacity of the E6 prototype were significantly higher than those of the mutants, thus serving as key drivers for increasing the detection rate of HPV81 and enhancing its pathogenicity. The viral load was positively correlated with persistent infection capacity and pathogenicity. Persistent infection was a crucial factor in the pathogenicity of HPV81. Successful adaptive evolution of HPV81 is accompanied by enhanced pathogenicity.
Asunto(s)
Genotipo , Infecciones por Papillomavirus , Infección Persistente , Polimorfismo Genético , Carga Viral , Humanos , Infecciones por Papillomavirus/virología , Femenino , Infección Persistente/virología , Cuello del Útero/virología , Cuello del Útero/patología , Adulto , Papillomaviridae/genética , Papillomaviridae/patogenicidad , Papillomaviridae/clasificación , Papillomaviridae/aislamiento & purificación , Frecuencia de los Genes , Proteínas Oncogénicas Virales/genética , Virulencia/genética , Alphapapillomavirus/genética , Alphapapillomavirus/patogenicidad , Alphapapillomavirus/clasificación , Alphapapillomavirus/aislamiento & purificación , Virus del Papiloma HumanoRESUMEN
Integration of the human papillomavirus (HPV) genome into the cellular genome is a key event that leads to constitutive expression of viral oncoprotein E6/E7 and drives the progression of cervical cancer. However, HPV integration patterns differ on a case-by-case basis among related malignancies. Next-generation sequencing technologies still face challenges for interrogating HPV integration sites. In this study, utilizing Nanopore long-read sequencing, we identified 452 and 108 potential integration sites from the cervical cancer cell lines (CaSki and HeLa) and five tissue samples, respectively. Based on long Nanopore chimeric reads, we were able to analyze the methylation status of the HPV long control region (LCR), which controls oncogene E6/E7 expression, and to identify transcriptionally-active integrants among the numerous integrants. As a proof of concept, we identified an active HPV integrant in between RUNX2 and CLIC5 on chromosome 6 in the CaSki cell line, which was supported by ATAC-seq, H3K27Ac ChIP-seq, and RNA-seq analysis. Knockout of the active HPV integrant, by the CRISPR/Cas9 system, dramatically crippled cell proliferation and induced cell senescence. In conclusion, identifying transcriptionally-active HPV integrants with Nanopore sequencing can provide viable targets for gene therapy against HPV-associated cancers.