RESUMO
Long noncoding RNAs (lncRNAs) play diverse roles in biological processes, but their expression profiles and functions in cervical carcinogenesis remain unknown. By RNA-sequencing (RNA-seq) analyses of 18 clinical specimens and selective validation by RT-qPCR analyses of 72 clinical samples, we provide evidence that, relative to normal cervical tissues, 194 lncRNAs are differentially regulated in high-risk (HR)-HPV infection along with cervical lesion progression. One such lncRNA, lnc-FANCI-2, is extensively characterized because it is expressed from a genomic locus adjacent to the FANCI gene encoding an important DNA repair factor. Both genes are up-regulated in HPV lesions and in in vitro model systems of HR-HPV18 infection. We observe a moderate reciprocal regulation of lnc-FANCI-2 and FANCI in cervical cancer CaSki cells. In these cells, lnc-FANCI-2 is transcribed from two alternative promoters, alternatively spliced, and polyadenylated at one of two alternative poly(A) sites. About 10 copies of lnc-FANCI-2 per cell are detected preferentially in the cytoplasm. Mechanistically, HR-HPVs, but not low-risk (LR)-HPV oncogenes induce lnc-FANCI-2 in primary and immortalized human keratinocytes. The induction is mediated primarily by E7, and to a lesser extent by E6, mostly independent of p53/E6AP and pRb/E2F. We show that YY1 interacts with an E7 CR3 core motif and transactivates the promoter of lnc-FANCI-2 by binding to two critical YY1-binding motifs. Moreover, HPV18 increases YY1 expression by reducing miR-29a, which targets the 3' untranslated region of YY1 mRNA. These data have provided insights into the mechanisms of how HR-HPV infections contribute to cervical carcinogenesis.
Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Papillomavirus Humano 16/genética , MicroRNAs/genética , Infecções por Papillomavirus/genética , RNA Longo não Codificante/genética , Neoplasias do Colo do Útero/genética , Fator de Transcrição YY1/genética , Processamento Alternativo , Sequência de Bases , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Colo do Útero/metabolismo , Colo do Útero/patologia , Colo do Útero/virologia , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Feminino , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/patogenicidade , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/metabolismo , Papillomavirus Humano 18/patogenicidade , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinócitos/virologia , MicroRNAs/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Fator de Transcrição YY1/metabolismoAssuntos
Antivirais/farmacologia , Guanina/análogos & derivados , Organofosfonatos/farmacologia , Papillomaviridae/efeitos dos fármacos , Pró-Fármacos/farmacologia , Animais , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , DNA Viral , Avaliação Pré-Clínica de Medicamentos , Guanina/farmacologia , Humanos , Replicação Viral/efeitos dos fármacosRESUMO
Rapid detection of single nucleotide polymorphisms (SNPs) has potential applications in both genetic screening and pharmacogenomics. Planar waveguide fluorescent biosensor technology was employed to detect SNPs using a simple hybridization assay with the complementary strand ("capture oligo") immobilized on the waveguide. This technology allows real-time measurements of DNA hybridization kinetics. Under normal conditions, both the wild-type sequence and the SNP-containing sequence will hybridize with the capture oligo, but with different reaction kinetics and equilibrium duplex concentrations. A "design of experiments" approach was used to maximize the differences in the kinetics profiles of the two. Nearly perfect discrimination can be achieved at short times (2 min) with temperatures that destabilize or melt the heteroduplex while maintaining the stability of the homoduplex. The counter ion content of the solvent was shown to have significant effect not only on the melting point of the heteroduplex and the homoduplex but also on the hybridization rate. Changes in both the stability and the difference between the hybridization rates of the hetero- and homoduplex were observed with varying concentrations of three different cations (Na(+), K(+), Mg(2+)). With the difference in hybridization rates maximized, discrimination between the hetero- and the homoduplex can be obtained at lower, less rigorous temperatures at hybridization times of 7.5 min or longer.