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
We have previously analysed the invasion capacity of different melanoma cell lines in the three-dimensional dermal equivalent. The melanoma cell line M4Beu acquired invasive behaviour upon changing its cultivation conditions before the seeding on top of the collagen lattice from single cell suspension to spheroid. Based on this phenomenon SSH was used to search for the genes related to the invasive phenotype of melanoma cells. From differentially expressed clones we focused on four: fibronectin, RhoA, COXII, and H-ras-like protein. By RT-PCR the expression of these genes were tested in different populations (monolayer, spheroids on dermal equivalent) of melanoma cell line M4Beu and three additional melanoma cell lines. The expression of fibronectin was also examined by immunohistochemistry staining of co-culture spheroids-dermal equivalent.