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
Menaquinone (MK) was an attractive membrane-bound intracellular chemical. To enhance its production, we tried to find the relationship between its synthesis and the state of cell membrane in producing strain. Due to non-ionic surfactant-polyoxyethylene oleyl ether (POE) and plant oil-cedar wood oil (CWO) can typically increase extracellular secretion and intracellular synthesis of MK respectively, the effect of these two substances on cell morphology, physical properties of cell membrane was investigated. Finally, two engineering strains were constructed to verify whether the state of cell membrane can enhance MK synthesis. The result showed that the edge of cells was broken when POE added in the medium. Other physical properties such as total fatty acid content decreased by 40.7% and the ratio of saturated fatty acids to unsaturated fatty acids decreased from 1.58 ± 0.05 to 1.31 ± 0.04. Meanwhile, cell membrane leakage was enhanced from 7.14 to 64.31%. Different from POE group, cell membrane was intact in CWO group. Moreover, the ratio of saturated fatty acids to unsaturated fatty acids increased from 1.58 ± 0.05 to 1.78 ± 0.04 and the average lipid length decreased from 16.05 ± 0.08 to 15.99 ± 0.10. Two constructed strains, especially Escherichia coli DH5α FatB, exhibited strong MK secretion ability and the extracellular MK reached 10.71 ± 0.19 mg/L. An understanding of these functionary mechanisms could not only provide a new idea for the synthesis of MK, but also provide a reference to increase the yield of intracellular membrane-bound metabolites.