RESUMEN
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.
Asunto(s)
Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Papillomavirus Humano 16/genética , MicroARNs/genética , Infecciones por Papillomavirus/genética , ARN Largo no Codificante/genética , Neoplasias del Cuello Uterino/genética , Factor de Transcripción YY1/genética , Empalme Alternativo , Secuencia de Bases , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Cuello del Útero/metabolismo , Cuello del Útero/patología , Cuello del Útero/virología , Factores de Transcripción E2F/genética , Factores de Transcripción E2F/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Femenino , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/patogenicidad , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/metabolismo , Papillomavirus Humano 18/patogenicidad , Humanos , Queratinocitos/metabolismo , Queratinocitos/patología , Queratinocitos/virología , MicroARNs/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Regiones Promotoras Genéticas , ARN Largo no Codificante/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virología , Factor de Transcripción YY1/metabolismoRESUMEN
BACKGROUND: Radix Euphorbiae Ebracteolatae (REE) was recently reported to be significantly superior to vitamin A acid ointment in treating multiple plantar warts. However, the effects of REE on HPV18 remain unclear. Therefore, the current study aimed to investigate the effects of REE on the proliferation of HPV18, and explore possible molecular mechanisms underlying the effects. METHODS: HFK and HFK-HPV18 were treated with water-extracted single or compound REE, ethanol-extracted single or compound REE, TNF-α and IFN for 3 days, respectively. In addition, the organotypic rafts containing HFK-HPV18 and HFK were treated with REE, IFN and TNF-α for 7 days, respectively. Cell proliferation rates were measured with Brdu. mRNA expression of E6, L1, p53 and Rb was detected by qPCR. Protein expression of p53, Rb and L1 was detected by Western blot. RESULTS: Compared to HFK group, HFK-HPV18 group had significantly higher expression of E6 and L1. Compared to the control group, HFK-HPV18 treated with REE, TNF-α and IFN displayed significantly lower proliferation rates. The mRNA expression of E6 was markedly lower, and mRNA expression of p53 and Rb was significantly higher after treatment of REE in HFK-HPV18 or in organotypic rafts containing HFK-HPV18. Treatment with REE markedly increased the protein expression of p53 and Rb, and decreased the protein expression of L1 in HFK-HPV18 or in organotypic rafts containing HFK-HPV18. Among all formula of REE, the inhibition of proliferation rates and expression of E6 and L1, and the increase in expression of p53 and Rb in HFK-HPV18 was highest in ethanol-extracted compound REE group. CONCLUSIONS: The proliferation rates are significantly lower in HFK-HPV18 treated with REE. The expression of E6 and L1 is markedly lower, and expression of p53 and Rb is significantly higher after REE treatment in HFK-HPV18 or organotypic rafts containing HFK-HPV18. Among all formula of REE, ethanol-extracted compound REE displays the highest protection against HPV18.