ABSTRACT
BACKGROUND: Current evidence implicates aberrant microRNA expression patterns in human malignancies; measurement of microRNA expression may have diagnostic and prognostic applications. Roles for microRNAs in head and neck squamous cell carcinomas (HNSCC) are largely unknown. HNSCC, a smoking-related cancer, is one of the most common malignancies worldwide but reliable diagnostic and prognostic markers have not been discovered so far. Some studies have evaluated the potential use of microRNA as biomarkers with clinical application in HNSCC. METHODS: MicroRNA expression profile of oral squamous cell carcinoma samples was determined by means of DNA microarrays. We also performed gain-of-function assays for two differentially expressed microRNA using two squamous cell carcinoma cell lines and normal oral keratinocytes. The effect of the over-expression of these molecules was evaluated by means of global gene expression profiling and cell proliferation assessment. RESULTS: Altered microRNA expression was detected for a total of 72 microRNAs. Among these we found well studied molecules, such as the miR-17-92 cluster, comprising potent oncogenic microRNA, and miR-34, recently found to interact with p53. HOX-cluster embedded miR-196a/b and miR-10b were up- and down-regulated, respectively, in tumor samples. Since validated HOX gene targets for these microRNAs are not consistently deregulated in HNSCC, we performed gain-of-function experiments, in an attempt to outline their possible role. Our results suggest that both molecules interfere in cell proliferation through distinct processes, possibly targeting a small set of genes involved in cell cycle progression. CONCLUSIONS: Functional data on miRNAs in HNSCC is still scarce. Our data corroborate current literature and brings new insights into the role of microRNAs in HNSCC. We also show that miR-196a and miR-10b, not previously associated with HNSCC, may play an oncogenic role in this disease through the deregulation of cell proliferation. The study of microRNA alterations in HNSCC is an essential step to the mechanistic understanding of tumor formation and could lead to the discovery of clinically relevant biomarkers.
Subject(s)
Head and Neck Neoplasms/genetics , MicroRNAs/genetics , Transcriptome , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Cell Proliferation , Disease Progression , Female , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/pathology , Homeodomain Proteins/genetics , Humans , Male , Middle Aged , Mouth Neoplasms/genetics , Mouth Neoplasms/metabolism , Mouth Neoplasms/pathology , Multigene Family , Neoplasm Grading , Neoplasm Staging , Reproducibility of Results , Signal Transduction , Squamous Cell Carcinoma of Head and NeckABSTRACT
Objective: Functional in vitro studies are fundamental to understand the role of microRNAs, small non coding RNA molecules that function as post-transcriptional regulators, in cancer. The objective of this study was to determine the applicability of head and neck squamous cell carcinoma cell lines and human oral keratinocytes as models for functional studies on microRNAs previously identified as deregulated in head and neck squamous cell carcinomas. Methods: The expression level of four microRNAs was assessed in cell lines and in primary cultures of oral keratinocytes using specific real-time polymerase chain reactions. The identity of oral squamous cell carcinoma cell lines was confirmed by means of STR (short tandem repeats) profiling. The possible impact of feeder-layer gene expression in global microRNA expression results from keratinocyte primary culture was also evaluated. Results: Significant differences in microRNA gene expression were observed among squamous cell carcinoma cell lines, particularly among cells lines from distinct subsites, as well as between primary culture of human keratinocytes and immortalized keratinocyte cell lines. Conclusions: Primary cultures of human keratinocytes and diverse tumor cell lines are relatively easy to obtain. However, each cell model possesses a characteristic phenotype; whereas one may be useful for a specific study, it may be inappropriate for another. Therefore, it is imperative that suitable cell lines are cautiously selected for functional studies in cancer.
Objetivo: Estudos funcionais in vitro são essenciais para a compreensão do papel de microRNAs, pequenas moléculas de RNA que desempenham papel importante na regulação gênica, no câncer. Neste estudo, analisamos a viabilidade de linhagens celulares derivadas de carcinoma epidermoide de cabeça e pescoço, queratinócitos orais provenientes de culturas primárias e queratinócitos imortalizados, como modelos para estudos funcionais de microRNAs previamente identificados como desregulados nesse tipo de carcinoma. Métodos: Avaliamos a expressão de quatro microRNAs em linhagens celulares e em cultura primária de queratinócitos orais por meio de reações em cadeia da polimerase em tempo real específica. As linhagens celulares de carcinoma epidermoide de boca foram previamente caracterizadas quanto ao seu perfil de sequências de DNA do tipo STR (do inglês short tandem repeats ou repetições curtas em sequência) com o objetivo de confirmar a identidade da linhagem. Avaliamos ainda a possível influência da expressão gênica detectada na camada de sustentação usada no cultivo de queratinócitos no resultado global obtido. Resultados: Nossos resultados apontam diferenças significativas na expressão dos microRNAs entre linhagens celulares passíveis de serem utilizadas como modelos para estudos funcionais em carcinoma epidermoide de cabeça e pescoço. Ressaltam-se diferenças entre linhagens de carcinoma de língua e de faringe, bem como diferenças expressivas entre a linhagem de queratinócitos orais imortalizados e queratinócitos orais normais provenientes de culturas primárias. Conclusão: Culturas primárias de queratinócitos orais bem como linhagens tumorais são obtidas de forma relativamente simples. Entretanto, cada modelo celular possui características particulares que os tornam mais ou menos adequados para um determinado estudo. Conclui-se que a seleção cuidadosa das linhagens é fundamental para estudos funcionais sobre câncer.
Subject(s)
Carcinoma, Squamous Cell , Gene Expression , Head and Neck Neoplasms , KeratinocytesABSTRACT
OBJECTIVE: Functional in vitro studies are fundamental to understand the role of microRNAs, small non coding RNA molecules that function as post-transcriptional regulators, in cancer. The objective of this study was to determine the applicability of head and neck squamous cell carcinoma cell lines and human oral keratinocytes as models for functional studies on microRNAs previously identified as deregulated in head and neck squamous cell carcinomas. METHODS: The expression level of four microRNAs was assessed in cell lines and in primary cultures of oral keratinocytes using specific real-time polymerase chain reactions. The identity of oral squamous cell carcinoma cell lines was confirmed by means of STR (short tandem repeats) profiling. The possible impact of feeder-layer gene expression in global microRNA expression results from keratinocyte primary culture was also evaluated. RESULTS: Significant differences in microRNA gene expression were observed among squamous cell carcinoma cell lines, particularly among cells lines from distinct subsites, as well as between primary culture of human keratinocytes and immortalized keratinocyte cell lines. CONCLUSIONS: Primary cultures of human keratinocytes and diverse tumor cell lines are relatively easy to obtain. However, each cell model possesses a characteristic phenotype; whereas one may be useful for a specific study, it may be inappropriate for another. Therefore, it is imperative that suitable cell lines are cautiously selected for functional studies in cancer.
