RESUMEN
Piwi-interacting RNAs (piRNAs) are a distinct group of small noncoding RNAs (sncRNAs) that silence transposable genetic elements to protect genome integrity. Because of their limited expression in gonads and sequence diversity, piRNAs remain the most mysterious class of small RNAs. Studies have shown piRNAs are present in somatic cells and dysregulated in gastric, breast and liver cancers. By deep sequencing 24 frozen benign kidney and clear cell renal cell carcinoma (ccRCC) specimens and using the publically available piRNA database, we found 26,991 piRNAs present in human kidney tissue. Among 920 piRNAs that had at least two copies in one specimen, 19 were differentially expressed in benign kidney and ccRCC tissues, and 46 were associated with metastasis. Among the metastasis-related piRNAs, we found three piRNAs (piR-32051, piR-39894 and piR-43607) to be derived from the same piRNA cluster at chromosome 17. We confirmed the three selected piRNAs not to be miRNAs or miRNA-like sncRNAs. We further validated the aberrant expression of the three piRNAs in a 68-case formalin-fixed and paraffin-embedded (FFPE) ccRCC tissue cohort and showed the up-regulation of the three piRNAs to be highly associated with ccRCC metastasis, late clinical stage and poor cancer-specific survival.
Asunto(s)
Carcinoma de Células Renales/genética , Carcinoma de Células Renales/mortalidad , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/genética , Neoplasias Renales/mortalidad , ARN Interferente Pequeño/genética , Anciano , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Estudios de Cohortes , Femenino , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genómica , Humanos , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Familia de Multigenes , Clasificación del Tumor , Metástasis de la Neoplasia , Estadificación de Neoplasias , Pronóstico , Reproducibilidad de los ResultadosRESUMEN
Renal cell carcinoma (RCC) is one of the leading causes of cancer mortality. Characterization of microRNA (miRNA) expression of RCC will help disclose new pathogenic pathways in tumourigenesis and progression and may lead to the development of molecular biomarkers and target-specific therapies for diagnosis, prognostication and treatment. With limitations in test specificity and the ability to detect novel miRNA and other small non-coding RNAs (smRNAs), microarray and RT-PCR techniques are being replaced by the evolving deep-sequencing technologies, at least in the discovery phase. Until now, cancer miRNA profiling of human benign and tumour specimen sets, using smRNA deep-sequencing (smRNA-seq), has not been reported. Specifically, due to concern over possible poor RNA quality/integrity, formalin-fixed paraffin-embedded (FFPE) samples have not been used for such studies. Here, we performed whole-genome smRNA-seq analysis using a benign and RCC specimen set and have successfully profiled the miRNA expression. Studies performed on paired frozen and FFPE specimens showed very similar results. Moreover, a comparison study of microarray, deep-sequencing and RT-PCR methodologies also showed a high correlation among the three technologies. To our knowledge, this is the first study to demonstrate that FFPE specimens can be used reliably for miRNA deep-sequencing analysis, making future large-scale clinical cohort/trial-based studies possible.
Asunto(s)
Carcinoma de Células Renales/genética , Neoplasias Renales/genética , Análisis por Conglomerados , Criopreservación , Formaldehído , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Adhesión en Parafina , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Fijación del Tejido/métodosRESUMEN
Renal cell carcinoma (RCC) metastasis portends a poor prognosis and cannot be reliably predicted. Early determination of the metastatic potential of RCC may help guide proper treatment. We analyzed microRNA (miRNA) expression in clear cell RCC (ccRCC) for the purpose of developing a miRNA expression signature to determine the risk of metastasis and prognosis. We used the microarray technology to profile miRNA expression of 78 benign kidney and ccRCC samples. Using 28 localized and metastatic ccRCC specimens as the training cohort and the univariate logistic regression and risk score methods, we developed a miRNA signature model in which the expression levels of miR-10b, miR-139-5p, miR-130b and miR-199b-5p were used to determine the status of ccRCC metastasis. We validated the signature in an independent 40-sample testing cohort of different stages of primary ccRCCs using the microarray data. Within the testing cohort patients who had at least 5 years follow-up if no metastasis developed, the signature showed a high sensitivity and specificity. The risk status was proven to be associated with the cancer-specific survival. Using the most stably expressed miRNA among benign and tumorous kidney tissue as the internal reference for normalization, we successfully converted his signature to be a quantitative PCR (qPCR)-based assay, which showed the same high sensitivity and specificity. The 4-miRNA is associated with ccRCC metastasis and prognosis. The signature is ready for and will benefit from further large clinical cohort validation and has the potential for clinical application.