Flavia Maziero Andreghetto

MicroRNA expression profile in head and neck cancer: HOX-cluster embedded microRNA-196a and microRNA-10b dysregulation implicated in cell proliferation

BackgroundCurrent 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.MethodsMicroRNA 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.ResultsAltered 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.ConclusionsFunctional 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.

Small RNAs in metastatic and non-metastatic oral squamous cell carcinoma

BackgroundSmall non-coding regulatory RNAs control cellular functions at the transcriptional and post-transcriptional levels. Oral squamous cell carcinoma is among the leading cancers in the world and the presence of cervical lymph node metastases is currently its strongest prognostic factor. In this work we aimed at finding small RNAs expressed in oral squamous cell carcinoma that could be associated with the presence of lymph node metastasis.MethodsSmall RNA libraries from metastatic and non-metastatic oral squamous cell carcinomas were sequenced for the identification and quantification of known small RNAs. Selected markers were validated in plasma samples. Additionally, we used in silico analysis to investigate possible new molecules, not previously described, involved in the metastatic process.ResultsGlobal expression patterns were not associated with cervical metastases. MiR-21, miR-203 and miR-205 were highly expressed throughout samples, in agreement with their role in epithelial cell biology, but disagreeing with studies correlating these molecules with cancer invasion. Eighteen microRNAs, but no other small RNA class, varied consistently between metastatic and non-metastatic samples. Nine of these microRNAs had been previously detected in human plasma, eight of which presented consistent results between tissue and plasma samples. MiR-31 and miR-130b, known to inhibit several steps in the metastatic process, were over-expressed in non-metastatic samples and the expression of miR-130b was confirmed in plasma of patients showing no metastasis. MiR-181 and miR-296 were detected in metastatic tumors and the expression of miR-296 was confirmed in plasma of patients presenting metastasis. A novel microRNA-like molecule was also associated with non-metastatic samples, potentially targeting cell-signaling mechanisms.ConclusionsWe corroborate literature data on the role of small RNAs in cancer metastasis and suggest the detection of microRNAs as a tool that may assist in the evaluation of oral squamous cell carcinoma metastatic potential.

High-throughput sequencing of small RNA transcriptomes reveals critical biological features targeted by microRNAs in cell models used for squamous cell cancer research

BackgroundThe implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery.ResultsTen miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer.ConclusionsCritical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.

Septic Shock in Advanced Age: Transcriptome Analysis Reveals Altered Molecular Signatures in Neutrophil Granulocytes.

Sepsis is one of the highest causes of mortality in hospitalized people and a common complication in both surgical and clinical patients admitted to hospital for non-infectious reasons. Sepsis is especially common in older people and its incidence is likely to increase substantially as a population ages. Despite its increased prevalence and mortality in older people, immune responses in the elderly during septic shock appear similar to that in younger patients. The purpose of this study was to conduct a genome-wide gene expression analysis of circulating neutrophils from old and young septic patients to better understand how aged individuals respond to severe infectious insult. We detected several genes whose expression could be used to differentiate immune responses of the elderly from those of young people, including genes related to oxidative phosphorylation, mitochondrial dysfunction and TGF-β signaling, among others. Our results identify major molecular pathways that are particularly affected in the elderly during sepsis, which might have a pivotal role in worsening clinical outcomes compared with young people with sepsis.

MicroRNAs and cell proliferation in head and neck squamous cell carcinoma

Background Functional in vitro studies are essential for the understanding of the role of microRNAs, small noncoding RNA molecules that function as posttranscriptional regulators, in cancer. In this study we investigated the effect of the over-expression of microRNAs previously identified as deregulated in squamous cell carcinomas of the head and neck (miRNA-1, miRNA-7, miRNA-10b e miRNA-196a) in cell proliferation.

Avaliação da expressão de microRNAs em linhagens celulares de carcinoma epidermoide de cabeça e pescoço e em cultura primária de queratinócitos orais

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 sao essenciais para a compreensao do papel de microRNAs, pequenas moleculas de RNA que desempenham papel importante na regulacao genica, no câncer. Neste estudo, analisamos a viabilidade de linhagens celulares derivadas de carcinoma epidermoide de cabeca e pescoco, queratinocitos orais provenientes de culturas primarias e queratinocitos imortalizados, como modelos para estudos funcionais de microRNAs previamente identificados como desregulados nesse tipo de carcinoma. Metodos: Avaliamos a expressao de quatro microRNAs em linhagens celulares e em cultura primaria de queratinocitos orais por meio de reacoes em cadeia da polimerase em tempo real especifica. As linhagens celulares de carcinoma epidermoide de boca foram previamente caracterizadas quanto ao seu perfil de sequencias de DNA do tipo STR (do ingles short tandem repeats ou repeticoes curtas em sequencia) com o objetivo de confirmar a identidade da linhagem. Avaliamos ainda a possivel influencia da expressao genica detectada na camada de sustentacao usada no cultivo de queratinocitos no resultado global obtido. Resultados: Nossos resultados apontam diferencas significativas na expressao dos microRNAs entre linhagens celulares passiveis de serem utilizadas como modelos para estudos funcionais em carcinoma epidermoide de cabeca e pescoco. Ressaltam-se diferencas entre linhagens de carcinoma de lingua e de faringe, bem como diferencas expressivas entre a linhagem de queratinocitos orais imortalizados e queratinocitos orais normais provenientes de culturas primarias. Conclusao: Culturas primarias de queratinocitos orais bem como linhagens tumorais sao obtidas de forma relativamente simples. Entretanto, cada modelo celular possui caracteristicas particulares que os tornam mais ou menos adequados para um determinado estudo. Conclui-se que a selecao cuidadosa das linhagens e fundamental para estudos funcionais sobre câncer.

Evaluation of microRNA expression in head and neck squamous cell carcinoma cell lines and in primary culture of oral keratinocytes.

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.

Abstract 1087: Circulating microRNAs as metastasis biomarkers in oral cavity and oropharyngeal squamous cell carcinomas

MicroRNAs are small non-coding RNA molecules with roles in gene expression regulation and implications in cancer initiation and progression. Cell-free microRNAs detected in body fluids, and in particular in plasma, are of clinical interest due to their possible use as biomarkers. Despite the fact that their reliable quantification depends on several technical parameters, there is great clinical interest in this approach due to its simplicity and low risk to the patient. Squamous cell carcinoma of the head and neck is one of the most common cancer types worldwide, with survival rates of about 50% in 5 years. Tobacco and alcohol consumption are the most important risk factors and, currently, the presence of cervical lymph node metastases remain the strongest prognostic factor for this cancer type. In this study we identified plasma microRNAs associated with squamous cell carcinomas of the oral cavity and oropharynx as well as with the presence of cervical lymph node metastases. We evaluated the expression of 179 microRNAs in plasma from 45 patients (28 presenting cervical lymph node metastases at diagnosis and 17 with no lymph node metastasis) and 15 healthy controls. Samples were matched according to age, sex, drinking and smoking habits. Among microRNAs mostly expressed in tumor samples, some had been already detected in oral squamous cell carcinoma tissues, such as miR-210, while others, such as miR-573, are still poorly studied. Comparisons between oral and oropharyngeal carcinomas yielded several differences, in accordance with molecular distinctions between these cancer types. MicroRNAs possibly involved in the metastatic process were identified when comparing plasma samples from individuals presenting lymph node metastasis at diagnosis and plasma from patients who were metastasis-free. Among these molecules, miR-192 and miR-574 were identified as more expressed in plasma from individuals with metastatic oropharyngeal cancer. We conclude that plasma microRNAs associated with specific clinical conditions or cancer sub-sites may help in the diagnosis and prognosis assessment of head and neck squamous cell carcinomas. Citation Format: Flavia Maziero Andreghetto, Mariana Barbosa de Souza, Raquel Ajub Moyses, Tatiana Natasha Toporcov, Fabio Daumas Nunes, Marcos Brasilino de Carvalho, Patricia Severino. Circulating microRNAs as metastasis biomarkers in oral cavity and oropharyngeal squamous cell carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1087.

Abstract 3983: MicroRNAs and other small RNA molecules expressed in metastatic and non-metastatic oral squamous cell carcinoma

Small noncoding RNAs are regulatory molecules that play important roles in several aspects of cellular biology. They vary from 18 to 30 nucleotides in length and often act through the inactivation of complementary sequences. A variety of small RNA classes have been identified to date and the list is constantly growing, owing to the advent of new sequencing technologies. Among these molecules, microRNAs are the most extensively studied. They are known to play important roles in human diseases, including cancer. More recently, genome-wide analyses have showed that changes in the expression levels of other small noncoding RNAs are also associated with cancer, with correlations between RNA abundance and disease status. Oral squamous cell carcinoma, associated with chronic tobacco and alcohol consumption, is among the leading cancers in the world. The presence of cervical lymph node metastases is currently its strongest prognostic factor. The possibility to evaluate its metastatic potential is relevant to the clinical and molecular oncologist due to frequent asymptomatic development of such cancer in its early stages. In this study small RNA libraries from 30 oral squamous cell carcinoma samples were sequenced for the identification and quantification of known small RNAs. Samples were divided in two groups: those presenting lymph node metastasis at the time of diagnosis and those that did not present this characteristic. Additionally, plasma of 30 patients was accessed for the presence of microRNAs identified as differentially expressed between metastatic and non-metastatic tumor samples as a means to identify circulating molecules that could be considered potential biomarkers for lymph node metastasis. Global expression patterns of small RNA molecules were not associated with cervical metastases. MiR-21, miR-203 and miR-205 were highly expressed throughout samples, in agreement with their role in epithelial cell biology, but disagreeing with studies correlating these molecules with cancer invasion. Nineteen microRNAs, but no other small RNA class, varied consistently between metastatic and non-metastatic cancer samples, some of which were also detected at corresponding levels in plasma. MiR-31 and miR-130b, known to inhibit several steps in the metastatic process, were over-expressed in non-metastatic samples and the expression of miR-130b was confirmed in plasma of patients presenting non-metastatic oral squamous cell carcinoma. MiR-181 and miR-296 were associated with metastasis but were not detected in plasma. In conclusion, we identified microRNAs linked to the metastatic status in a group of patients diagnosed with oral squamous cell carcinoma, both in tissue and plasma samples. We also demonstrate that other small RNA classes are expressed and should, therefore, be involved and contribute to the metastatic phenotype of this disease. Citation Format: Patricia Severino, Liliane Santana Oliveira, Flavia Maziero Andreghetto, Natalia Torres, Otavio Curioni, Patricia Maluf Cury, Tatiana Natasha Toporcov, Alexandre Rossi Paschoal, Alan Mitchell Durham. MicroRNAs and other small RNA molecules expressed in metastatic and non-metastatic oral squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3983. doi:10.1158/1538-7445.AM2015-3983