Caracterização do perfil de expressão e avaliação funcional de microRNAs em carcinomas de vulva / Characterization of microRNA expression profiles and their functional evaluation in vulvar carcinomas

INTRODUÇÃO: O carcinoma de vulva é um tumor ginecológico raro, cuja escassez de informações biológicas torna veemente a necessidade de se identificar moléculas que possibilitem a compreensão dos mecanismos atuantes neste tipo de tumor, tais como os microRNAs, cuja expressão desregulada tem sido demonstrada com relevância na tumorigênese. OBJETIVOS: Realizar a caracterização do perfil de expressão de microRNAs em amostras de carcinomas de vulva na presença e ausência de HPVs de alto risco, correlacionar a expressão de microRNAs com características clinicopatológicas das pacientes, bem avaliar o papel destes microRNAs e seus alvos no câncer vulvar através de estudos funcionais. MÉTODOS: Quarenta amostras de carcinomas de células escamosas de vulva, sendo 20 amostras HPV positivas e 20 HPV negativas foram selecionadas para análise de expressão diferencial de microRNAs. Sete amostras de bordas normais foram utilizadas para a construção de um pool usado como controle não tumoral global. A identificação dos microRNAs diferencialmente expressos nas diferentes amostras foi realizada por qRT-PCR através do sistema de cartões TaqMan human microRNA array 2.0 A+B. Foi, ainda, realizada a busca in silico de possíveis alvos gênicos dos microRNAs diferencialmente expressos por meio do algoritmo MAGIA. MicroRNAs diferencialmente expressos em tumores HPV negativos foram correlacionados com dados clinicopatológicos importantes das pacientes e aqueles de potencial interesse tiveram sua atividade biológica avaliada por meio de ensaios celulares ­ proliferação, migração e invasão ­ em uma cultura de células de vulva (SW962), antes e após a transfecção de oligonucleotídeos miméticos destes microRNAs. Foi realizada, ainda, a avaliação da expressão gênica de um importante alvo de um destes microRNAs por qRT-PCR, e proteica por western blot e imunofluorescência. Finalmente, foi realizada a avaliação do alvo selecionado em amostras parafinadas de dezoito pacientes e a expressão proteica destes alvos foi associada com dados clinicopatológicos. RESULTADOS: Foram observados 25 microRNAs diferencialmente expressos na comparação dos grupos de tumores HPV positivos versus HPV negativos; e 79 microRNAs na comparação entre tumores versus controle não tumoral. Uma rede de interação entre os perfis de expressão dos microRNAs diferencialmente expressos e mRNAs-alvo previamente descritos com relevância nos carcinomas de vulva foi obtida, como por exemplo, TP53, RB, PTEN e EGFR. A diminuição da expressão dos microRNAs miR-223-5p e miR-19-b1-5p foi associada com a presença de metástase linfonodal nas pacientes, enquanto a diminuição da expressão dos microRNAs miR-100-3p e, novamente, miR-19-b1-5p foi associada com a presença de invasão vascular; já o aumento de expressão dos microRNAs miR-519b e miR-133a associou-se com estadiamentos FIGO mais avançados. Em um segundo momento, miR-223-5p foi selecionado para as subsequentes análises in vitro. Quando adicionados miR-223-5p miméticos às células SW962 por meio de transfecçãotransiente, observou-se uma diminuição das taxas de proliferação (p<0.01) e migração (p<0.001) celulares e, em contrapartida, um aumento do potencial invasivo destas células (p<0.004).Após cuidadosa análise in silico de potenciais alvos do miR-223-5p, TP63 foi selecionado e o miR-223-5p foi demonstrado por afetar a expressão de p63, tanto a nível de proteína quanto mRNA (p<0.001 e p<0.0001, respectivamente). Foi demonstrado, ainda, que a diminuição da expressão de p63 em amostras de pacientes foi associada com maior invasão tumoral (p=0.0345) e menor sobrevida global (p=0.0494). CONCLUSÕES: Nosso estudo demonstrou que microRNAs podem ser clinicamente importantes em carcinomas vulvares e que sua desregulação parece ser importante na carcinogênese vulvar. Além disso, nossos resultados mostram um microRNA em particular, miR-223-5p, como tendo papel importante na regulação de expressão gênica relacionada a valor prognóstico em carcinoma de vulva.

INTRODUCTION: Vulvar carcinoma is a rare gynecological tumor, and the lack of biological information regarding this tumor strongly incites the need to identify molecules that enable the understanding of the mechanisms acting in this type of tumor, such as microRNAs, which dysregulated expression has been demonstrated with relevance in tumorigenesis. OBJECTIVES: To characterize microRNA expression profile in vulvar carcinoma samples in the presence and absence of highrisk HPVs, to correlate the expression of microRNAs with clinicopathological characteristics of the patients and to evaluate the role of these microRNAs in vulvar cancer through functional studies. METHODS: Forty samples of squamous cell carcinoma of the vulva, being 20 positive and 20 negative for HPV were selected for the analysis of differentially expressed microRNAs. Seven samples of normal vulva were used for the construction of a non-tumor control. The identification of microRNAs differentially expressed between the samples was performed by qRTPCR using TaqMan human microRNA array 2.0 A+B. Were also performed validation of the selected microRNAs using fresh-frozen tissue and an in silico search of potential target genes of these microRNAs through MAGIA algorithm. Differentially expressed microRNAs in the HPV negative tumor group were correlated with important clinicopathological data from patients and those of potential interest had their biological activity assessed by cellular assays ­ proliferation, migration and invasion ­ in a vulvar cell culture (SW962) before and after transfection with mimetic oligonucleotides of these microRNAs. We also performed the evaluation of gene expression of an important target of one of these microRNAs by qRT-PCR, and protein by western blot and immunofluorescence. Finally, protein expression of the selected target was evaluated on eighteen paraffined samples and the expression was associated with clinicopathological data of the patients. RESULTS: There were 25 differentially expressed microRNAs in the comparison of HPV-positive versus HPV negative tumors group; and 79 microRNAs when comparing tumors versus normal samples. An interaction network between the expression profiles of the differentially expressed microRNAs and mRNAs target previously described with relevance in vulva carcinoma, such as TP53, RB, EGFR and PTEN was obtained. Downregulation of miR-223-5p, and miR-19-b1-5p were correlated with lymph node metastasis in patients, while the reduction of the expression of miR-100-3p, and again, miR-19-b1-5p were correlated with the presence of vascular invasion; overexpression of microRNA miR-133a and miR-519b were associated with advanced FIGO staging. In a second moment, miR223-5p was selected for subsequent in vitro analysis. When miR-223-5p mimetics were added to SW962 cells by transient transfection, we observed a decrease of the proliferation rates (p<0.01) and migration (p<0.001), and in contrast, an increased invasive potential of these cells (p<0.004). After a careful in silico analysis for potential targets of miR-223-5p, TP63 has been selected and was shown to affected by the chosen microRNA, at both protein and mRNA level (p<0.001 and p<0.0001, respectively). It has been demonstrated also that a decreased expression of p63 in samples of patients was associated with increased tumor invasion (p=0.0345) and shorter overall survival (p=0.0494). CONCLUSIONS: Our study demonstrated that microRNAs may be clinically important in vulvar carcinomas and that their deregulation appears to be important in the vulvar carcinogenesis. Furthermore, our results demonstrate a particular microRNA, miR-223-5p, as having an important role on the regulation of genic expression related to a prognostic value in vulvar carcinomas.

ROCK1 as a novel prognostic marker in vulvar cancer.

BACKGROUND: Vulvar carcinoma is an infrequent tumour, accounting for fewer than 3% of all malignant tumours that affect women, but its incidence is rising in the past few decades. In young women, the manifestation of the vulvar carcinoma is often linked to risk factors such as smoking and HPV infection, but most cases develop in women aged over 50 years through poorly understood genetic mechanisms. Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) has been implicated in many cellular processes, but its function in vulvar cancer has never been examined. In this study, we aimed to determine the prognostic value of ROCK1 gene and protein analysis in vulvar squamous cell carcinoma (VSCC). METHODS: ROCK1 expression levels were measured in 16 vulvar tumour samples and adjacent normal tissue by qRT-PCR. Further, 96 VSCC samples were examined by immunohistochemistry (IHC) to confirm the involvement of ROCK1 in the disease. The molecular and pathological results were correlated with the clinical data of the patients. Sixteen fresh VSCC samples were analyzed by array-based comparative genomic hybridization (aCGH). RESULTS: In each pair of samples, ROCK1 levels were higher by qRT-PCR in normal tissue compared with the tumour samples (p = 0.016). By IHC, 100% of invasive front areas of the tumour and 95.8% of central tumour areas were positive for ROCK1. Greater expression of ROCK1 was associated with the absence of lymph node metastasis (p = 0.022) and a lower depth of invasion (p = 0.002). In addition, higher ROCK1 levels correlated with greater recurrence-free survival (p = 0.001). Loss of ROCK1 was independently linked to worse cancer-specific survival (p = 0.0054) by multivariate analysis. This finding was validated by IHC, which demonstrated enhanced protein expression in normal versus tumour tissue (p < 0.001). By aCGH, 42.9% of samples showed a gain in copy number of the ROCK1 gene. CONCLUSIONS: ROCK1 is lower expressed in tumour tissue when compared with adjacent normal vulvar epithelia. In an independent sample set of VSCCs, lower expression levels of ROCK1 correlated with worse survival rates and a poor prognosis. These findings provide important information for the clinical management of vulvar cancer.