Efeitos dos miRNAs humanos miR-100-5p, miR-192-5p e miR-574-3p na expressão gênica e migração em célula imortalizada de nasofaringe
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Data
2022-02-21
Autores
Müller-Coan, Bárbara Grasiele
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Os cânceres são doenças neoplásicas malignas com etiopatogenia complexa. As células transformadas adquirem diversas propriedades que propiciam alterações em taxas de migração, invasão e sobrevivência celular, facilitando a formação de metástases. Esse processo sofre a influência de diversos fatores, inclusive de pequenos RNAs não codificantes, como os miRNAs. Os miRNAs são moléculas que canonicamente impedem a tradução de um mRNA, podendo agir em diversos processos celulares, incluindo na carcinogênese e progressão do câncer, como o carcinoma de nasofaringe (NPC). O NPC é um câncer agressivo com alta taxa de invasão em tecidos adjacentes, como base do crânio, seios nasais e órbitas, além de alta taxa de metástase para linfonodos regionais. Sua forma não queratinizante compreende mais de 95% dos casos e quando indiferenciado, é fortemente associado ao vírus de Epstein-Barr (EBV). Em um estudo recente de nosso grupo de pesquisa, observamos que células imortalizadas de nasofaringe (NP69) apresentavam um aumento na expressão do miR-100-5p, miR-192-5p e miR-574-3p quando transfectadas com a proteína latente de membrana 1 (Latent Membrane Protein 1 – LMP1) do EBV proveniente da variante viral M81, em comparação com a LMP1 proveniente da variante viral B95-8A. Portanto, este estudo visa elucidar os efeitos do miR-100-5p, miR-192-5p e miR-574-3p na viabilidade, proliferação e migração de células imortalizadas de nasofaringe (NP69) cultivadas in vitro. Para tanto, foi realizada a predição dos alvos dos respectivos miRNAs e o enriquecimento de vias para inferir quais vias de sinalização intracelular poderiam ser moduladas por cada miRNA, seguida pela análise dos transcritos dos genes alvos selecionados. Adicionalmente, células NP69 foram transfectadas com os miméticos do miR-100-5p, miR-192-5p ou miR-574-3p para a análise de comportamento celular. Quando essas foram transfectadas com o mimético do miR-192-5p, foi observada a redução de 40% no transcrito do gene RAB2A e redução de 43% na sua migração celular. No enriquecimento de vias, o miR-192-5p teve sua ação predita em vias como MAPK, GPCRs, TKR e TGF-β. Células NP69 transfectadas com os miméticos do miR-100-5p e miR-574-3p reduziram significativamente os transcritos dos genes FZD8 e STC1, respectivamente. Entretanto, nos ensaios funcionais realizados, não foram observadas alterações significativas no comportamento celular. Com esses resultados pode-se observar que o miR-192-5p possui uma função de supressor tumoral, elucidando parte de sua função em células de nasofaringe. Portanto, isso demonstra sua importância no contexto do NPC e a necessidade de seu estudo adicional.
Cancers are malignant neoplastic diseases with complex etiopathogenesis. Transformed cells acquire diverse biological properties that induce alteration in the migration, invasion, and cell survival rates, favoring metastasis formation. This process is under the influence of many factors, including miRNAs. MiRNAs are molecules that canonically prevent mRNA translation, influencing many cellular processes, carcinogenesis and progression of cancers, like nasopharyngeal carcinoma (NPC). NPC is an aggressive cancer with high invasion rates to adjacent tissues, such as the base of the cranium, face sinuses, and orbits, in addition to a high metastatic rate to local lymph nodes. The nonkeratinizing NPC subtype comprehends over 95% of total cases and, when undifferentiated, is highly related to Epstein-Barr virus (EBV) infection. In a recent study of our group, we observed that immortalized nasopharyngeal cells NP69SV40T transfected with the EBV oncoprotein, Latent membrane protein 1 (LMP1), from M81 strain showed increased expression of human miR-100-5p, miR-192-5p, and miR-574-3p compared to NP69SV40T cells transfected with LMP1 from EBV strain B95.8A. Thus, this study aimed to assess the putative effects of each of the identified microRNAs on the viability, migration, and proliferation of human immortalized nasopharyngeal cells (NP69SV40T) cultivated in vitro. Therefore, target prediction and pathway enrichment analysis were performed to infer which pathways could be modulated by each miRNA followed by expression analysis of selected predicted target genes. Additionally, NP69SV40T cells were transfected with miR-100-5p, miR-192-5p, or miR-574-3p mimic to analyze cell behavior. After their transfection with miR-192-5p mimic, we observed a 40% decrease in RAB2A transcript level and a 43% reduction in cell migration. During the pathway enrichment analysis, miR-192-5p showed predicted involvement in pathways such as MAPK, GPCRs, TKR, and TGF-β. NP69SV40T cells transfected with miR-100-5p or miR574-3p reduced transcriptional levels of its targets FZD8 and STC1, respectively. However, in the performed cellular assays, they did not alter cell behavior. The results demonstrate that miR-192-5p acts as a tumor suppressor, answering part of its function in nasopharyngeal cells. This shows its importance in the NPC context and the necessity of further studies of this miRNA.
Cancers are malignant neoplastic diseases with complex etiopathogenesis. Transformed cells acquire diverse biological properties that induce alteration in the migration, invasion, and cell survival rates, favoring metastasis formation. This process is under the influence of many factors, including miRNAs. MiRNAs are molecules that canonically prevent mRNA translation, influencing many cellular processes, carcinogenesis and progression of cancers, like nasopharyngeal carcinoma (NPC). NPC is an aggressive cancer with high invasion rates to adjacent tissues, such as the base of the cranium, face sinuses, and orbits, in addition to a high metastatic rate to local lymph nodes. The nonkeratinizing NPC subtype comprehends over 95% of total cases and, when undifferentiated, is highly related to Epstein-Barr virus (EBV) infection. In a recent study of our group, we observed that immortalized nasopharyngeal cells NP69SV40T transfected with the EBV oncoprotein, Latent membrane protein 1 (LMP1), from M81 strain showed increased expression of human miR-100-5p, miR-192-5p, and miR-574-3p compared to NP69SV40T cells transfected with LMP1 from EBV strain B95.8A. Thus, this study aimed to assess the putative effects of each of the identified microRNAs on the viability, migration, and proliferation of human immortalized nasopharyngeal cells (NP69SV40T) cultivated in vitro. Therefore, target prediction and pathway enrichment analysis were performed to infer which pathways could be modulated by each miRNA followed by expression analysis of selected predicted target genes. Additionally, NP69SV40T cells were transfected with miR-100-5p, miR-192-5p, or miR-574-3p mimic to analyze cell behavior. After their transfection with miR-192-5p mimic, we observed a 40% decrease in RAB2A transcript level and a 43% reduction in cell migration. During the pathway enrichment analysis, miR-192-5p showed predicted involvement in pathways such as MAPK, GPCRs, TKR, and TGF-β. NP69SV40T cells transfected with miR-100-5p or miR574-3p reduced transcriptional levels of its targets FZD8 and STC1, respectively. However, in the performed cellular assays, they did not alter cell behavior. The results demonstrate that miR-192-5p acts as a tumor suppressor, answering part of its function in nasopharyngeal cells. This shows its importance in the NPC context and the necessity of further studies of this miRNA.
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Palavras-chave
miR-100-5p, miR-192-5p, miR-574-3p, RAB2A, Nasopharyngeal carcinoma