Efeitos da expressão do gene RPMS1 do vírus de Epstein-Barr em células humanas cultivadas in vitro
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Data
2020-11-26
Autores
Jorge, Beatrice Adrianne Silva
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Universidade Estadual Paulista (Unesp)
Resumo
O vírus Epstein-Barr (EBV) - formalmente gammaherpesvirus humano 4 (HHV-4) - é um herpesvírus associado ao desenvolvimento de uma variedade de malignidades humanas. Entre elas, destacam-se o linfoma de Burkitt (BL) e o carcinoma nasofaríngeo (NPC), além de subconjuntos de casos de linfoma de Hodgkin e não-Hodgkin (particularmente em pacien-tes imunocomprometidos) e carcinomas gástricos. A RPMS1 é um produto viral do EBV gerado por processamento alternativo (splicing) do segmento transcrito BamHI A direito (BART) e pouco estudado. Sabe-se que essa proteína viral está implicada na modulação da via Notch e possivelmente contribui para a transformação celular induzida pelo EBV. Digno de nota, um polimorfismo de nucleotídeo único (SNP) A/G na posição 155391 na ORF de RPMS1 foi associado com maior risco de NPC. Assim, esse trabalho teve como hipótese de que a expressão de RPMS1 do EBV poderia influenciar o fenótipo de linhagens de células humanas imortalizadas e neoplásicas in vitro. Para isso, foi feita a montagem de um vetor lentiviral contendo a ORF da RPMS1 que seria empregado para futuros experimentos. Por problemas na transdução, um vetor com expressão constitucional de RPMS1 foi empregado para avaliar as células NP69 quanto a migração e viabilidade celular. Foi observado que as células que expressavam mais taxa de RNAm de RPMS1 apresentavam menor viabilidade celular. Quanto a migração, nenhuma alteração foi notada. Adicionalmente, células linfoides EBV positivas foram genotipadas quanto ao SNP 155391 e toda apresentaram a variante G. Por fim, o nível de expressão de RPMS1 pelas células linfoides foram analisados, mostrando que cada linhagem expressa esse gene em quantidades diferentes.
The Epstein-Barr virus (EBV) – formally human gammaherpesvirus 4 (HHV-4) – is a herpes virus associated with the development of a variety of human malignancies, notably Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC), but also subsets of cases for Hodgkin and non-Hodgkin lymphomas (particularly in immunocompromised patients) and gastric carcinomas. RPMS1 is an EBV viral product generated by splicing of the BamHI A transcribed segment (BART) and with only a few studies published about. It is known that this viral protein is involved in the modulation of the Notch pathway and contributes to EBV-induced cell transformation. Of note, a single nucleotide polymorphism (SNP) A / G at position 155391 in the ORF of RPMS1 was associated with an increased risk of NPC. Thus, this work hypothesized that the expression of EBV RPMS1 could influence the phenotype of immortalized and neoplastic human cell lines in vitro. For this, a lentiviral vector containing the ORF of RPMS1 was assembled for future experiments. Due to transduction problems, a vector with constitutional expression of RPMS1 was used to evaluate NP69 cells for cell migration and viability. It was observed that cells that expressed a higher rate of RPMS1 mRNA had lower cell viability. As for the migration, no changes were noticed. In addition, positive EBV lymphoid cells were genotyped for SNP 155391 and all presented the G variant. Finally, the level of RPMS1 expression by lymphoid cells was analyzed, showing that each cell line expresses this gene in different rates.
The Epstein-Barr virus (EBV) – formally human gammaherpesvirus 4 (HHV-4) – is a herpes virus associated with the development of a variety of human malignancies, notably Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC), but also subsets of cases for Hodgkin and non-Hodgkin lymphomas (particularly in immunocompromised patients) and gastric carcinomas. RPMS1 is an EBV viral product generated by splicing of the BamHI A transcribed segment (BART) and with only a few studies published about. It is known that this viral protein is involved in the modulation of the Notch pathway and contributes to EBV-induced cell transformation. Of note, a single nucleotide polymorphism (SNP) A / G at position 155391 in the ORF of RPMS1 was associated with an increased risk of NPC. Thus, this work hypothesized that the expression of EBV RPMS1 could influence the phenotype of immortalized and neoplastic human cell lines in vitro. For this, a lentiviral vector containing the ORF of RPMS1 was assembled for future experiments. Due to transduction problems, a vector with constitutional expression of RPMS1 was used to evaluate NP69 cells for cell migration and viability. It was observed that cells that expressed a higher rate of RPMS1 mRNA had lower cell viability. As for the migration, no changes were noticed. In addition, positive EBV lymphoid cells were genotyped for SNP 155391 and all presented the G variant. Finally, the level of RPMS1 expression by lymphoid cells was analyzed, showing that each cell line expresses this gene in different rates.
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Palavras-chave
Epstein-Barr, Carcinogenese, Linfomas, Carcinoma de Nasofaringe, NPC, EBV, RPMS1