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1.
CRISPR J ; 3(6): 487-502, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33346711

RESUMO

Nucleic acid detection techniques are always critical to diagnosis, especially in the background of the present coronavirus disease 2019 pandemic. Simple and rapid detection techniques with high sensitivity and specificity are always urgently needed. However, current nucleic acid detection techniques are still limited by traditional amplification and hybridization. To overcome this limitation, here we developed CRISPR-Cas9-assisted DNA detection (CADD). In this detection, a DNA sample is incubated with a pair of capture single guide RNAs (sgRNAs; sgRNAa and sgRNAb) specific to a target DNA, dCas9, a signal readout-related probe, and an oligo-coated solid support beads or microplate at room temperature (RT) for 15 min. During this incubation, the dCas9-sgRNA-DNA complex is formed and captured on solid support by the capture sequence of sgRNAa, and the signal readout-related probe is captured by the capture sequence of sgRNAb. Finally, the detection result is reported by a fluorescent or colorimetric signal readout. This detection was verified by detecting DNA of bacteria, cancer cells, and viruses. In particular, by designing a set of sgRNAs specific to 15 high-risk human papillomaviruses (HPVs), the HPV infection in 64 clinical cervical samples was successfully detected by the method. All detections can be finished in 30 min at RT. This detection holds promise for rapid on-the-spot detection or point-of-care testing.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico/métodos , Animais , Sistemas CRISPR-Cas , DNA Viral/genética , Engenharia Genética/métodos , Humanos , Limite de Detecção , Infecções por Papillomavirus/genética , RNA Guia/genética , RNA Guia/metabolismo
2.
Zhonghua Liu Xing Bing Xue Za Zhi ; 41(12): 2112-2118, 2020 Dec 10.
Artigo em Chinês | MEDLINE | ID: mdl-33378825

RESUMO

Objective: To investigate the prevalence and genotype distribution of high-risk human papillomavirus (HR-HPV) and the correlation between cervical lesions and analyze the risk factors for HR-HPV infection. Methods: In June 2018, a population-based study for cervical cancer screening in Tuoli county of Xinjiang Uygur Autonomous Region was conducted. A total of 2 000 Kazak women aged 25-64 years were included in the study. Three cervical exfoliated cells samples were collected from them for careHPV, PCR HPV, p16(INK4a), and liquid-based cytology (LBC) tests. Women with any positive test were referred for colposcopy with biopsies taken at abnormal sites. Histo-pathological diagnoses were used as the gold standard. Results: The overall prevalence of HPV was 14.55%, among which the infection rate of HR-HPV was 12.90%, which was even higher in the 50-54 years age group. The most prevalent genotypes of HR-HPV were HPV16 (2.80%), HPV51(2.35%), HPV52 (1.70%), HPV56 (1.50%), and HPV39 (1.20%). The most common HPV infection was a single infection (71.48%). In the age group of 50-54 years, the multiple infection rates were higher, with the majority of double infection (69.88%), and HPV42 and 56 were the most common co-infection types. HPV16 (31.82%), HPV51 (27.27%) and HPV18 (13.64%) were higher in cervical intraepithelial neplasia grade 1, HPV16 (57.14%) was higher in cervical intraepithelial neplasia grade 2, and HPV16 (55.56%) and HPV18 (33.33%) were higher in cervical intraepithelial neplasia grade 3 or worse. Results from the multivariate logistic regression analysis showed that higher education, menopause, and syphilis infection increased the HPV infection. Conclusions: The most common prevalence genotypes of HR-HPV among Kazak women were HPV16, HPV51, and HPV52. The infection rate of HR-HPV among Kazak women was correlated with education level, menopausal status, and syphilis infection. Measures should be taken targeting high-risk factors. This result suggests that STD patients and women aged 50 and above should be encouraged for screening.


Assuntos
Grupos Étnicos , Papillomaviridae , Infecções por Papillomavirus , Adulto , China/epidemiologia , Grupos Étnicos/genética , Grupos Étnicos/psicologia , Grupos Étnicos/estatística & dados numéricos , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Papillomaviridae/genética , Infecções por Papillomavirus/etnologia , Infecções por Papillomavirus/genética , Fatores de Risco
3.
Anticancer Res ; 40(11): 6355-6366, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109573

RESUMO

BACKGROUND/AIM: p16 and PTEN are tumor suppressor genes. Loss of these molecules in oral squamous cell carcinoma (OSCC) has been studied worldwide. In this study, we explored whether p16 cooperates with inactive PTEN during the pathogenesis of OSCC, especially in regard to tumor aggressiveness and proliferation. MATERIALS AND METHODS: Immunocytochemistry and western blot analysis were used to examine the levels of p16 and PTEN. Sequencing analysis was performed to identify mutations in the PTEN gene and HPV infection. Fluorescence in situ hybridization was used to examine the presence of the PTEN locus. RESULTS: PTEN analysis showed high positivity in T4 samples. HPV-positive tumors correlated with tabagism, tumor size 3 and 4, disease stages 3 and 4, presence of lymph node metastasis (N1) and poor differentiation. Immunoexpression of p16 was strongly correlated with the presence of HPV. CONCLUSION: PTEN demonstrated a higher reactivity in advanced disease stages and p16 was strongly associated with HPV. Viral presence decreases tumor aggressiveness. Patients with advanced stage lesions demonstrated lower survival rate.


Assuntos
Carcinoma de Células Escamosas/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Neoplasias Bucais/genética , PTEN Fosfo-Hidrolase/genética , Adulto , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Hibridização in Situ Fluorescente , Perda de Heterozigosidade/genética , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Neoplasias Bucais/virologia , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia
4.
PLoS One ; 15(9): e0238497, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32986729

RESUMO

Human papilloma virus (HPV) causes a subset of head and neck squamous cell carcinomas (HNSCC) of the oropharynx. We combined targeted DNA- and genome-wide RNA-sequencing to identify genetic variants and gene expression signatures respectively from patients with HNSCC including oropharyngeal squamous cell carcinomas (OPSCC). DNA and RNA were purified from 35- formalin fixed and paraffin embedded (FFPE) HNSCC tumor samples. Immuno-histochemical evaluation of tumors was performed to determine the expression levels of p16INK4A and classified tumor samples either p16+ or p16-. Using ClearSeq Comprehensive Cancer panel, we examined the distribution of somatic mutations. Somatic single-nucleotide variants (SNV) were called using GATK-Mutect2 ("tumor-only" mode) approach. Using RNA-seq, we identified a catalog of 1,044 and 8 genes as significantly expressed between p16+ and p16-, respectively at FDR 0.05 (5%) and 0.1 (10%). The clinicopathological characteristics of the patients including anatomical site, smoking and survival were analyzed when comparing p16+ and p16- tumors. The majority of tumors (65%) were p16+. Population sequence variant databases, including gnomAD, ExAC, COSMIC and dbSNP, were used to identify the mutational landscape of somatic sequence variants within sequenced genes. Hierarchical clustering of The Cancer Genome Atlas (TCGA) samples based on HPV-status was observed using differentially expressed genes. Using RNA-seq in parallel with targeted DNA-seq, we identified mutational and gene expression signatures characteristic of p16+ and p16- HNSCC. Our gene signatures are consistent with previously published data including TCGA and support the need to further explore the biologic relevance of these alterations in HNSCC.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Neoplasias de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adulto , Idoso , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , DNA Viral/genética , Gerenciamento de Dados , Bases de Dados de Ácidos Nucleicos , Testes Diagnósticos de Rotina , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias Orofaríngeas/genética , Neoplasias Orofaríngeas/metabolismo , Neoplasias Orofaríngeas/virologia , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Transcriptoma
5.
APMIS ; 128(12): 637-646, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32911563

RESUMO

High-risk HPVs (HR-HPVs) are DNA viruses considered as primary etiologic factors in malignancies of the low female genital tract. Their presence has also been documented in oropharyngeal and laryngeal cancers. However, HPV infection is considered a necessary but not sufficient cause of tumoral development; meantime, increasing evidences on the tumorigenic role of cancer stem cells (CSCs) have been documented in the literature. CSCs represent a small subpopulation of neoplastic cells with self-renewal potential, capable of maintaining tumor growth and cell differentiation, also involved in metastatic process, recurrence, and resistance to chemotherapeutic agents. In the present study, performed on KB cell lines, we evaluated the tumor forming potential of CSCs, and their relationship with the HPV infection status. We started our study by identifying the most aggressive cell line on the minimal number of cells being able of growth in vivo in a model of athymic nude mice (BALB/c nu/nu). We used an oral-derived KB cell line separated in the KB-CD133+ and KB-CD133- populations, by using immunomagnetic beads and fluorescence-activated cell sorting (FACS). The separated populations were injected in athymic nude mice (BALB/c nu/nu). Xenograft tumors have been analyzed for tumor size, CD133 expression by immunohistochemistry (IHC) and for DNA HR-HPV integration by in situ hybridization (ISH), comparing CD133-enriched xenograft tumors versus the CD133 non-enriched ones. On standard conditions, the KB cell line has a poor population of glycosylated CD133 marker (<5.0%) when investigated with antibodies versus CD133, and more specifically its glycosylated epitope (AC133). Enriched CD133 KB cells possess a higher capacity of tumor growth in xenograft models of nude mice when compared to KB CD133-negative cells. We observed that the AC133 epitope, extensively used to purifying hematopoietic stem cells, is able to select an epithelial subpopulation of cancer stem cells with aggressive behavior. We retain that CD133 may be a useful target in anticancer strategies including pharmacological and immunological therapies.


Assuntos
Antígeno AC133/metabolismo , Papillomavirus Humano 18/fisiologia , Células-Tronco Neoplásicas/metabolismo , Infecções por Papillomavirus/metabolismo , Antígeno AC133/genética , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/patologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia
6.
Nat Genet ; 52(8): 800-810, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32747824

RESUMO

Cervical cancer is the most common cancer affecting sub-Saharan African women and is prevalent among HIV-positive (HIV+) individuals. No comprehensive profiling of cancer genomes, transcriptomes or epigenomes has been performed in this population thus far. We characterized 118 tumors from Ugandan patients, of whom 72 were HIV+, and performed extended mutation analysis on an additional 89 tumors. We detected human papillomavirus (HPV)-clade-specific differences in tumor DNA methylation, promoter- and enhancer-associated histone marks, gene expression and pathway dysregulation. Changes in histone modification at HPV integration events were correlated with upregulation of nearby genes and endogenous retroviruses.


Assuntos
Epigenoma/genética , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Transcriptoma/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Adulto , Idoso , Metilação de DNA/genética , Feminino , Humanos , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética , Uganda , Regulação para Cima/genética
7.
Virology ; 548: 174-181, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838940

RESUMO

The incidence of head and neck squamous cell carcinomas (HNSCCs) is rising in developed countries. This is driven by an increase in HNSCCs caused by high-risk human papillomavirus (HPV) infections or HPV + HNSCCs. Compared to HNSCCs not caused by HPV (HPV- HNSCCs), HPV + HNSCCs are more responsive to therapy and associated with better oncologic outcomes. As a result, the HPV status of an HNSCC is an important determinant in medical management. One method to determine the HPV status of an HNSCC is increased expression of p16 caused by the HPV E7 oncogene. We identified novel expression changes in HPV + HNSCCs. A comparison of gene expression among HPV+ and HPV- HNSCCs in The Cancer Genome Atlas demonstrated increased DNA repair gene expression in HPV + HNSCCs. Further, DNA repair gene expression correlated with HNSCC survival. Immunohistochemical analysis of a novel HNSCC microarray confirmed that DNA repair protein abundance is elevated in HPV + HNSCCs.


Assuntos
Alphapapillomavirus/metabolismo , Infecções por Papillomavirus/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adulto , Idoso , Alphapapillomavirus/genética , Alphapapillomavirus/isolamento & purificação , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Reparo do DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
8.
Life Sci ; 256: 118026, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32615187

RESUMO

AIM: We aimed to determine the biological processes and pathways involved in cervical carcinogenesis associated with high-risk human papillomavirus (HPV) infection. MATERIALS AND METHODS: Total RNA was extracted from three formalin-fixed paraffin-embedded (FFPE) samples each of normal cervix, HPV-infected low-grade squamous intraepithelial lesion (LSIL), high-grade SIL (HSIL) and squamous cell carcinoma (SCC). Transcriptomic profiling by microarrays was conducted followed by downstream Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. RESULTS: We examined the difference in GOs enriched for each transition stage from normal cervix to LSIL, HSIL, and SCC, and found 307 genes to be differentially expressed. In the transition from normal cervix to LSIL, the extracellular matrix (ECM) genes were significantly downregulated. The MHC class II genes were significantly upregulated in the LSIL to HSIL transition. In the final transition from HSIL to SCC, the immunoglobulin heavy locus genes were significantly upregulated and the ECM pathway was implicated. CONCLUSION: Deregulation of the immune-related genes including MHC II and immunoglobulin heavy chain genes were involved in the transitions from LSIL to HSIL and SCC, suggesting immune escape from host anti-tumour response. The extracellular matrix plays an important role during the early and late stages of cervical carcinogenesis.


Assuntos
Genes de Cadeia Pesada de Imunoglobulina/genética , Genes MHC da Classe II/genética , Infecções por Papillomavirus/complicações , Lesões Intraepiteliais Escamosas Cervicais/patologia , Neoplasias do Colo do Útero/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Regulação para Baixo , Matriz Extracelular/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Estadiamento de Neoplasias , Infecções por Papillomavirus/genética , Lesões Intraepiteliais Escamosas Cervicais/genética , Lesões Intraepiteliais Escamosas Cervicais/virologia , Transcriptoma , Regulação para Cima , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia
9.
J Med Microbiol ; 69(7): 960-970, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32510304

RESUMO

Introduction. Persistent human papillomavirus (HPV) type 16 infection is the main causal agent of cervical cancer. Most HPV infections clear spontaneously within 1-2 years. Although not all infected women develop detectable HPV antibodies, about 60-70 % seroconvert and retain their antibodies at low levels.Aim. We investigated if cervical HPV16 DNA positivity was associated with HPV16 seroreactivity measured with two different antigen formulations. We assessed if associations were influenced by co-infection with other HPV types and HPV16 viral load.Methodology. We used baseline data for women participating in the Ludwig-McGill cohort, a longitudinal investigation of the natural history of HPV infection and cervical neoplasia. The study enrolled 2462 Brazilian women from 1993 to 1997 (pre-vaccination). ELISA assays were based on L1-only or L1+L2 virus-like particles (VLPs). Seroreactivity was expressed as normalized absorbance ratios. HPV genotyping and viral load were evaluated by PCR protocols. Pearson's r was used to measure correlations between interval-scaled variables. Serological accuracy in HPV16 DNA detection was assessed using receiver operating characteristic (ROC) curves. We analysed the association between HPV DNA positivity and HPV16 seroreactivity by linear regression.Results. Correlations between L1+L2 and L1-only VLPs for detection of HPV16 were poor (r=0.43 and 0.44 for dilutions 1 : 10 and 1 : 50, respectively). The protocol with the best accuracy was L1+L2 VLPs at serum dilution 1 : 10 (ROC area=0.73, 95 % CI: 0.65-0.85). HPV16 DNA positivity was correlated with HPV16 seroreactivity and was not influenced by co-infection or viral load. To a lesser degree, HPV16 seroreactivity was correlated with infection by other Alpha-9 papillomavirus species.Conclusion. HPV16 DNA positivity and HPV16 seroreactivity are strongly correlated. L1+L2 VLPs perform better than L1-only VLPs for detecting IgG antibodies to HPV16 in women infected with HPV16 or other Alpha-9 HPV species. This study advances our understanding of humoral immune responses against HPV16 by providing insights about the influence of VLP antigen composition to measure humoral immune response against naturally acquired HPV infection.


Assuntos
Papillomavirus Humano 16/genética , Papillomavirus Humano 16/imunologia , Infecções por Papillomavirus/diagnóstico , Adulto , Anticorpos Antivirais/sangue , Brasil , Capsídeo/imunologia , Proteínas do Capsídeo/genética , Colo do Útero/virologia , Estudos de Coortes , Feminino , Papillomavirus Humano 16/patogenicidade , Humanos , Complexo Antígeno L1 Leucocitário/imunologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Carga Viral/métodos , Vírion/imunologia
10.
PLoS One ; 15(6): e0235065, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584870

RESUMO

INTRODUCTION: Human papillomavirus (HPV) infection is associated with the development of anogenital and head and neck cancers. In recent years a potential role of HPV in colorectal cancer (CRC) has been suggested. OBJECTIVE: To investigate the presence of HPV in colorectal carcinomas and to study the role of p16INK4a as a marker of transcriptionally active HPV infection. In addition, to investigate the correlation between these findings and the CRC prognostic factors. METHODS: Case control study with 92 cases of colorectal cancers, 75 controls of normal tissue adjacent to the tumor, and 30 controls of precursor lesions, including polyps and colorectal adenomas. Paraffinized samples were used, HPV detection and genotyping were performed by PCR and reverse hybridization by using the INNO LIPA kit, with SPF10 plus primers. The expression of the p16INK4a protein was investigated using immunohistochemistry. Data analysis was performed using descriptive, univariate statistics and survival curves were calculated by using the Kaplan Meier and log-rank method. RESULTS: HPV was detected in 13% of the cases and the most prevalent genotype was HPV 16. HPV DNA was not detected in either control groups. The high expression of p16INK4a was observed in 30% of the cases, but it was not associated to the presence of HPV. The overall survival was 53.3% and was influenced by prognostic factors such as later stage, lymph node and distant metastasis. CONCLUSIONS: Based on these results, HPV is unlikely to be involved in colorectal carcinogenesis and p16INK4a expression is not a relevant marker of transcriptionally active HPV infection in CRC.


Assuntos
Neoplasias Colorretais , Inibidor p16 de Quinase Dependente de Ciclina , Regulação Neoplásica da Expressão Gênica , Papillomavirus Humano 16 , Infecções por Papillomavirus , Adulto , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/virologia , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia
11.
PLoS Pathog ; 16(6): e1008624, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555725

RESUMO

Human papillomaviruses (HPV) are a major cause of malignancy worldwide. They are the aetiological agents of almost all cervical cancers as well as a sub-set of other anogenital and head and neck cancers. Hijacking of host cellular pathways is essential for virus pathogenesis; however, a major challenge remains to identify key host targets and to define their contribution to HPV-driven malignancy. The Hippo pathway regulates epithelial homeostasis by down-regulating the function of the transcription factor YAP. Increased YAP expression has been observed in cervical cancer but the mechanisms driving this increase remain unclear. We found significant down-regulation of the master Hippo regulatory kinase STK4 (also termed MST1) in cervical disease samples and cervical cancer cell lines compared with healthy controls. Re-introduction of STK4 inhibited the proliferation of HPV positive cervical cells and this corresponded with decreased YAP nuclear localization and decreased YAP-dependent gene expression. The HPV E6 and E7 oncoproteins maintained low STK4 expression in cervical cancer cells by upregulating the oncomiR miR-18a, which directly targeted the STK4 mRNA 3'UTR. Interestingly, miR-18a knockdown increased STK4 expression and activated the Hippo pathway, significantly reducing cervical cancer cell proliferation. Our results identify STK4 as a key cervical cancer tumour suppressor, which is targeted via miR-18a in HPV positive tumours. Our study indicates that activation of the Hippo pathway may offer a therapeutically beneficial option for cervical cancer treatment.


Assuntos
Transformação Celular Viral , MicroRNAs/metabolismo , Papillomaviridae/metabolismo , Infecções por Papillomavirus/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Neoplásico/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Neoplasias do Colo do Útero/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Proteínas Serina-Treonina Quinases/genética , RNA Neoplásico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia
12.
Biomol Concepts ; 11(1): 116-124, 2020 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-32417757

RESUMO

The single nucleotide polymorphism (SNP) of the promoter region of MMP-1 (at 1607 bp) and MMP-3 (at 1171 bp) create Ets binding sites. Correlations between these SNPs and sensitivity to several biological processes such as metastasis and recurrence of cancer have been reported in several studies. In this case-control study, we looked for these SNPs in women infected with or not with high-risk human papillomaviruses (HR-HPV). The frequency, distribution and correlation of these SNPs with the presence or absence of HR-HPV infection were evaluated. Genotypes 1G1G, 1G2G and 2G2G for MMP1 and genotypes 5A5A, 5A6A, 6A6A for MMP3 were found in our study population. In general, we noted that the 1G (40.8%) and 2G (64.8%) alleles were more frequent in non-infected women and infected women, respectively, and more specifically this difference was significant in women from Côte d'Ivoire. These results, although yet to be reaffirmed with assays for quantifying the mRNA of these genes, suggest that the SNP of the MMP-1 promoter could promote infection with HR-HPV.


Assuntos
Metaloproteinase 1 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , Papillomaviridae , Infecções por Papillomavirus/genética , Neoplasias do Colo do Útero/genética , Adolescente , Adulto , Idoso , Alelos , Burkina Faso , Estudos de Casos e Controles , Costa do Marfim , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Pessoa de Meia-Idade , Papillomaviridae/genética , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/enzimologia , Infecções por Papillomavirus/etiologia , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/virologia
13.
PLoS One ; 15(5): e0232474, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374757

RESUMO

BACKGROUND: In Brazil, penile cancer (PC) is not uncommon. The highest incidence of PC is in the North and Northeast of the country. In addition to phimosis, the Human Papillomavirus (HPV) and Epstein-Baar Virus (EBV) infections are also related as risk factors for PC. The overexpression of p16INK4a is a surrogate sensitive marker of HPV infection in PC. OBJECTIVES: To correlate p16INK4a overexpression and HPV infection status with EBV infection in a series of PC patients from the Amazon region. METHODS: Tumor tissues from 47 PC cases were analyzed for the presence of HPV and EBV DNA by PCR. All PC patients were diagnosed between 2013 and 2018 at a public reference cancer center hospital in Manaus, Amazonas-Brazil. HPV was genotyped using E7 HPV16/HPV18 type-specific real-time PCR and the PapilloCheck® HPV-Screening assay. p16INK4a expression was evaluated by immunohistochemistry using the automated Ventana® BenchMark Ultra. RESULTS: The mean age of patients at the time of diagnosis was 57.4 years ±SD 17.8 ranging from 20 to 90 years old. Most of the patients (64%) came from rural areas of the Amazonas State. Thirty patients had phimosis (64%). Among the patients with phimosis, 43% (13/30) underwent circumcision, three during childhood and 10 in adulthood. 60% of the patients were smokers or ex-smokers. HPV infection was observed in 45% (21/47) of cases. HPV16 was detected in 13 patients (61%). Other HPV types detected were HPV 6, 11, 42, 51, 53, 68 and 44/55. EBV infection was observed in 30% (14/47) of the patients with PC. Co-infection with HPV and EBV was observed in 28% (6/21) cases. p16INK4a was only investigated in 26 samples. The p16INK4a overexpression was observed exclusively in HPV 16 positive cases and four HPV negative cases. In the survival analysis, the follow-up time was 35.4 months/patient. The mortality rate during the follow up time was 38%. CONCLUSIONS: p16INK4a positivity presented a high correlation to HPV 16 DNA detection, reinforcing its use as a surrogate marker for HPV-driven cancers. Infection with EBV was quite frequent and its role in epithelial penile oncogenesis needs to be demonstrated.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Infecções por Vírus Epstein-Barr/complicações , Papillomaviridae , Infecções por Papillomavirus/complicações , Neoplasias Penianas/etiologia , Neoplasias Penianas/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Brasil/epidemiologia , DNA Viral/genética , DNA Viral/isolamento & purificação , Infecções por Vírus Epstein-Barr/virologia , Marcadores Genéticos , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/isolamento & purificação , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/patogenicidade , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/patogenicidade , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Papillomaviridae/genética , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Neoplasias Penianas/epidemiologia , Fatores de Risco , Regulação para Cima , Adulto Jovem
14.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32350070

RESUMO

The papillomavirus (PV) E2 protein is a critical regulator of viral transcription and genome replication. We previously reported that tyrosine (Y) 138 of HPV-31 E2 is phosphorylated by the fibroblast growth factor receptor 3 (FGFR3) kinase. In this study, we generated quasiviruses containing G418-selectable HPV-31 genomes with phosphodeficient phenylalanine mutant E2 Y138F and phosphomimetic glutamic acid mutant Y138E. We observed significantly fewer early viral transcripts immediately after infection with these Y138 mutant genomes even though E2 occupancy at the viral origin was equivalent to that of wild-type E2. Keratinocytes infected with Y138F quasiviruses formed stable colonies, and the genomes were maintained as episomes, while those infected with Y138E quasiviruses did not. We previously reported that the HPV-31 E2 Y138 mutation to glutamic acid did not bind to the Brd4 C-terminal motif (CTM). Here, we demonstrate that HPV-16 E2 Y138E bound to full-length Brd4 but not to the Brd4 CTM. We conclude that association of E2 with the Brd4 CTM is necessary for viral genome replication and suggest that this interaction can be regulated by phosphorylation of E2 Y138.IMPORTANCE Papillomavirus (PV) is a double-stranded DNA tumor virus infecting the cutaneous and mucosal epithelium. The PV E2 protein associates with a number of cellular factors to mediate replication of the HPV genome. Fibroblast growth factor receptor 3 (FGFR3) regulates HPV replication through phosphorylation of tyrosine 138 in the HPV E2 protein. Employing a quasivirus infection model and selection for G418 resistant genomes, we demonstrated that Y138 is a critical residue for Brd4 association and that inability to complex with Brd4 does not support episomal replication.


Assuntos
Papillomavirus Humano 31/metabolismo , Queratinócitos/metabolismo , Infecções por Papillomavirus/metabolismo , Plasmídeos/metabolismo , Proteínas do Envelope Viral/metabolismo , Substituição de Aminoácidos , Linhagem Celular , Humanos , Queratinócitos/patologia , Queratinócitos/virologia , Mutação de Sentido Incorreto , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Fosforilação , Plasmídeos/genética , Tirosina , Proteínas do Envelope Viral/genética
15.
Sci Rep ; 10(1): 7306, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350356

RESUMO

Several determining factors are involved in HPV infection outcomes; human leukocyte antigen (HLA) polymorphisms have been described as related factors. This study has ascertained the effect of genetic variation on HLA-DRB1 and DQB1 genes on HPV-16/-18/-31/-33/-45 and -58 clearance and redetection in Colombian women. PCR and qPCR were used for viral identification and the Illumina MiSeq system was used for HLA-typing of cervical samples (n = 276). Survival models were adjusted for identifying alleles/haplotypes related to HPV clearance/redetection; L1/L2 protein-epitope binding to MHC-II molecules was also predicted. Significant associations suggested effects favouring or hampering clearance/redetection events depending on the viral type involved in infection, e.g. just DRB1*12:01:01G favoured HPV-16 (coeff: 4.8) and HPV-45 clearance (coeff: 12.65) whilst HPV-18 (coeff: 2E-15), HPV-31 (coeff: 8E-17) and HPV-58 hindered elimination (coeff: 1E-14). An effect was only observed for some alelles when configured as haplotypes, e.g. DRB1*04:07:01G (having the greatest frequency in the target population) was associated with DQB1*02:01:1G or *03:02:03. Epitope prediction identified 23 clearance-related peptides and 29 were redetection-related; eight might have been related to HPV-16/-18 and -58 persistence and one to HPV-18 elimination. HLA allele/haplotype relationship with the course of HPV infection (clearance/redetection) depended on the infecting HPV type, in line with the specific viral epitopes displayed.


Assuntos
Alelos , Alphapapillomavirus , Epitopos , Cadeias beta de HLA-DQ , Cadeias HLA-DRB1 , Haplótipos , Infecções por Papillomavirus , Adulto , Alphapapillomavirus/genética , Alphapapillomavirus/imunologia , Intervalo Livre de Doença , Epitopos/genética , Epitopos/imunologia , Feminino , Seguimentos , Cadeias beta de HLA-DQ/genética , Cadeias beta de HLA-DQ/imunologia , Cadeias HLA-DRB1/genética , Cadeias HLA-DRB1/imunologia , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/mortalidade , Taxa de Sobrevida
16.
Hum Genet ; 139(6-7): 919-939, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32435828

RESUMO

Human papillomaviruses (HPVs) infect mucosal or cutaneous stratified epithelia. There are 5 genera and more than 200 types of HPV, each with a specific tropism and virulence. HPV infections are typically asymptomatic or result in benign tumors, which may be disseminated or persistent in rare cases, but a few oncogenic HPVs can cause cancers. This review deals with the human genetic and immunological basis of interindividual clinical variability in the course of HPV infections of the skin and mucosae. Typical epidermodysplasia verruciformis (EV) is characterized by ß-HPV-driven flat wart-like and pityriasis-like cutaneous lesions and non-melanoma skin cancers in patients with inborn errors of EVER1-EVER2-CIB1-dependent skin-intrinsic immunity. Atypical EV is associated with other infectious diseases in patients with inborn errors of T cells. Severe cutaneous or anogenital warts, including anogenital cancers, are also driven by certain α-, γ-, µ or ν-HPVs in patients with inborn errors of T lymphocytes and antigen-presenting cells. The genetic basis of HPV diseases at other mucosal sites, such as oral multifocal epithelial hyperplasia or juvenile recurrent respiratory papillomatosis (JRRP), remains poorly understood. The human genetic dissection of HPV-driven lesions will clarify the molecular and cellular basis of protective immunity to HPVs, and should lead to novel diagnostic, preventive, and curative approaches in patients.


Assuntos
Predisposição Genética para Doença , Papillomaviridae/genética , Infecções por Papillomavirus/patologia , Dermatopatias Infecciosas/patologia , Infecções Tumorais por Vírus/patologia , Humanos , Papillomaviridae/classificação , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Dermatopatias Infecciosas/genética , Dermatopatias Infecciosas/virologia , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/virologia
17.
Sci Rep ; 10(1): 7863, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398763

RESUMO

Equine penile squamous cell carcinoma (EpSCC) is a relatively common cutaneous neoplasm with a poor prognosis. In this study, we aimed to determine the protein expression and colocalisation of FRA1, c-Myc, Cyclin D1, and MMP7 in normal (NT), tumour (T), hyperplastic epidermis and/or squamous papilloma (Hyp/Pap), poorly-differentiated (PDSCC), or well-differentiated (WDSCC) EpSCC using a tissue array approach. Further objectives were to correlate protein expression to (i) levels of inflammation, using a convolutional neural network (ii) equine papillomavirus 2 (EcPV2) infection, detected using PCR amplification. We found an increase in expression of FRA1 in EpSCC compared to NT samples. c-Myc expression was higher in Hyp/Pap and WDSCC but not PDSCC whereas MMP7 was reduced in WDSCC compared with NT. There was a significant increase in the global intersection coefficient (GIC) of FRA1 with MMP7, c-Myc, and Cyclin D1 in EpSCC. Conversely, GIC for MMP7 with c-Myc was reduced in EpSCC tissue. Inflammation was positively associated with EcPV2 infection in both NT and EpSCC but not Hyp/Pap. Changes in protein expression could be correlated with EcPV2 for Cyclin D1 and c-Myc. Our results evaluate novel biomarkers of EpSCC and a putative correlation between the expression of biomarkers, EcPV2 infection and inflammation.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/genética , Neoplasias Penianas/genética , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/virologia , Ciclina D1/genética , Ciclina D1/metabolismo , Cavalos , Masculino , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 7 da Matriz/metabolismo , Papillomaviridae/fisiologia , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Neoplasias Penianas/diagnóstico , Neoplasias Penianas/virologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Curva ROC , Análise Serial de Tecidos/métodos
18.
Oncogene ; 39(24): 4741-4755, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32415241

RESUMO

Human papilloma virus (HPV)-associated oropharyngeal cancer (OPC) is an independent tumour type with regard to cellular, biological, and clinical features. The use of non-invasive biomarkers such as circulating tumour DNA (ctDNA) may be relevant in early diagnosis and eventually improve the outcomes of patients with head and neck squamous cell carcinoma (HNSCC). Genome-wide discovery using RNA sequencing and reduced representation bisulfite sequencing yielded 21 candidates for methylation-targeted genes. A verification study (252 HNSCC patients) using quantitative methylation-specific PCR (Q-MSP) identified 10 genes (ATP2A1, CALML5, DNAJC5G, GNMT, GPT, LY6D, LYNX1, MAL, MGC16275, and MRGPRF) that showed a significant increase recurrence in methylation groups with OPC. Further study on ctDNA using Q-MSP in HPV-associated OPC showed that three genes (CALML5, DNAJC5G, and LY6D) had a high predictive ability as emerging biomarkers for a validation set, each capable of discriminating between the plasma of the patients from healthy individuals. Among the 42 ctDNA samples, methylated CALML5, DNAJC5G, and LY6D were observed in 31 (73.8%), 19 (45.2%), and 19 (45.2%) samples, respectively. Among pre-treatment ctDNA samples, methylated CALML5, DNAJC5G, and LY6D were observed in 8/8 (100%), 7/8 (87.5%), and 7/8 (87.5%) samples, respectively. Methylated CALML5, DNAJC5G, and LY6D were found in 2/8 (25.0%), 0/8 (0%), and 1/8 (12.5%) of the final samples in the series, respectively. Here, we present the relationship between the methylation status of three specific genes and cancer recurrence for risk classification of HPV-associated OPC cases. In conclusion, ctDNA analysis has the potential to aid in determining patient prognosis and real-time surveillance for disease recurrences and serves as an alternative method of screening for HPV-associated OPC.


Assuntos
Biomarcadores Tumorais , Metilação de DNA , DNA de Neoplasias , Proteínas de Neoplasias , Neoplasias Orofaríngeas , Papillomaviridae , Infecções por Papillomavirus , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Orofaríngeas/genética , Neoplasias Orofaríngeas/metabolismo , Neoplasias Orofaríngeas/patologia , Neoplasias Orofaríngeas/virologia , Papillomaviridae/genética , Papillomaviridae/metabolismo , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia
19.
J Virol ; 94(15)2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32404527

RESUMO

Orf virus (ORFV) represents a suitable vector for the generation of efficient, prophylactic antiviral vaccines against different pathogens. The present study investigated for the first time the therapeutic application of ORFV vector-based vaccines against tumors induced by cottontail rabbit papillomavirus (CRPV). ORFV-CRPV recombinants were constructed expressing the early CRPV gene E1, E2, E7, or LE6. In two independent experiments we used in total 23 rabbits which were immunized with a mixture of the four ORFV-CRPV recombinants or empty ORFV vector as a control 5 weeks after the appearance of skin tumors. For the determination of the therapeutic efficacy, the subsequent growth of the tumors was recorded. In the first experiment, we could demonstrate that three immunizations of rabbits with high tumor burden with the combined four ORFV-CRPV recombinants resulted in significant growth retardation of the tumors compared to the control. A second experiment was performed to test the therapeutic effect of 5 doses of the combined vaccine in rabbits with a lower tumor burden than in nonimmunized rabbits. Tumor growth was significantly reduced after immunization, and one vaccinated rabbit even displayed complete tumor regression until the end of the observation period at 26 weeks. Results of delayed-type hypersensitivity (DTH) skin tests suggest the induction of a cellular immune response mediated by the ORFV-CRPV vaccine. The data presented show for the first time a therapeutic potential of the ORFV vector platform and encourage further studies for the development of a therapeutic vaccine against virus-induced tumors.IMPORTANCE Viral vectors are widely used for the development of therapeutic vaccines for the treatment of tumors. In our study we have used Orf virus (ORFV) strain D1701-V for the generation of recombinant vaccines expressing cottontail rabbit papillomavirus (CRPV) early proteins E1, E2, LE6, and E7. The therapeutic efficacy of the ORFV-CRPV vaccines was evaluated in two independent experiments using the outbred CRPV rabbit model. In both experiments the immunization achieved significant suppression of tumor growth. In total, 84.6% of all outbred animals benefited from the ORFV-CRPV vaccination, showing reduction in tumor size and significant tumor growth inhibition, including one animal with complete tumor regression without recurrence.


Assuntos
Vacinas Anticâncer/imunologia , Papillomavirus de Coelho Cottontail/imunologia , Neoplasias/terapia , Vírus do Orf/imunologia , Infecções por Papillomavirus/terapia , Vacinas Virais/imunologia , Animais , Vacinas Anticâncer/genética , Chlorocebus aethiops , Papillomavirus de Coelho Cottontail/genética , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/virologia , Vírus do Orf/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Coelhos , Células Vero , Proteínas Virais/genética , Proteínas Virais/imunologia , Vacinas Virais/genética
20.
Anticancer Res ; 40(4): 2117-2123, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32234904

RESUMO

BACKGROUND/AIM: The incidence of human papilloma virus (HPV)-related head and neck squamous cell carcinoma (HNSCC) has been increasing in the last decades. Analysis of oral brushing or rinsing samples for screening or stratification could potentially improve screening and prevention. PATIENTS AND METHODS: Oral brushes and mouthwashes were taken from 20 patients with HPV-associated HNSCC before definite therapy. HPV genotyping was performed for the detection of 14 high-risk HPV subtypes and correlated to DNA isolated from tumor tissue. RESULTS: Ten of 20 patients were tested HPV positive by using either method. There was a significant correlation between macroscopic visibility of tumor and positive HPV detection (p<0.001) and HPV detection and tumor size (p<0.001). HPV was detected in all macroscopically visible tumors. Half of the HPV cases who had macroscopically invisible tumors were missed by both methods. CONCLUSION: Both techniques are limited in the detection of macroscopically non-visible and small tumors. Therefore, the application of these techniques for screening or diagnosis of HNSCC is not recommended.


Assuntos
Neoplasias Orofaríngeas/genética , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Idoso , DNA Viral/genética , DNA Viral/isolamento & purificação , Feminino , Genótipo , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 16/patogenicidade , Humanos , Masculino , Pessoa de Meia-Idade , Antissépticos Bucais/análise , Neoplasias Orofaríngeas/patologia , Neoplasias Orofaríngeas/virologia , Papillomaviridae/isolamento & purificação , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia
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