Estabelecimento de modelo in vitro para estudo de efeitos de miRBARTs do EBV em células humanas de epitélio nasofaríngeo
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Data
2022-05-06
Autores
Rocha, Viviana Loureiro
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Universidade Estadual Paulista (Unesp)
Resumo
A infecção pelo vírus de Epstein-Barr (EBV) está associada a alguns tipos de cânceres, como o linfoma de Burkitt (BL) e o carcinoma de nasofaringe (NPC). O EBV é ubíquo e infecta, de forma latente, a maioria da população adulta. A latência biológica do EBV é caracterizada por elevada restrição na expressão de produtos virais, sendo limitada a poucas proteínas e RNAs não codificantes, como os miR-BARTs. A superexpressão de miR-BART 7 no NPC regula a expressão do produto do gene supressor tumoral PTEN. O miR-BART 9, por sua vez, regula a expressão da oncoproteína viral LMP1 e de caderina E celular. A regulação da expressão destas proteínas sugere papel dos miR-BARTs 7 e 9 no fenômeno de transição epitelial-mesenquimal (EMT), importante para progressão de cânceres de origem epitelial. Dessa forma, este estudo tem como objetivo a criação de um modelo experimental para verificar o papel exercido por miR-BART 7 e miR-BART 9 na aquisição de características favoráveis a carcinogênese. O modelo estabelecido baseia-se em células de epitélio de nasofaringe infectadas com partículas virais selvagens ou mutadas para estes miRNAs. A construção deste modelo emprega a tecnologia CRISPR/Cas9 para edição do genoma do EBV em células Akata-EBV+. As células Akata-EBV+ com genoma selvagem ou mutado foram quimicamente tratadas para ativação de ciclo lítico, e produção de partículas virais. A validação foi realizada a partir da expressão de marcadores de ciclo lítico, BZLF1 e gp350, enquanto a quantificação das partículas produzidas foi realizada a partir de curva-padrão com o genoma B95-8. A infecção de células epiteliais de nasofaringe foi avaliada a partir da estimativa do número de cópias do genoma viral. As células Akata-EBV+ contendo os genomas com comprometimento da expressão dos miR-BARTs 7 ou 9 apresentaram deficiência na produção de partículas virais quando comparadas as células contendo o genoma selvagem, enquanto na infecção de células epiteliais de nasofaringe, o comprometimento da expressão dos miR-BARTs 7 ou 9 influenciou positivamente, aumentando o número de cópias virais nas células infectadas com as versões editadas. Os dados apresentados demonstram estabelecimento de modelo experimental de estudo dos miR-BARTs do EBV, e indicam uma possível relação com eventos relacionados ao ciclo lítico do vírus.
Epstein-Barr virus infection is associated with some types of cancers, such as Burkitt lymphoma (BL), and nasopharyngeal carcinoma (NPC). EBV is a ubiquitous virus, and infects, latently, the majority of adult population. The EBV latency is characterized by limited viral expression, expressing a few proteins and non-coding RNAs, such as miR-BARTs. The overexpression of EBV miR-BART 7 in NPC was associated with regulation of protein levels of PTEN suppressor gene. EBV miR-BART 9, on the other hand, regulates the expression of the LMP1 viral oncoprotein and the cellular E-cadherin. The regulation of these proteins suggests a role of miR-BARTs 7 and 9 in epithelial-mesenchymal transition (EMT), which is important for progression of epithelial cancers. This study aims to create an experimental model to verify the role played by miR-BART7 and miR-BART9 in acquisition of features favorable to carcinogenesis. The model established is based on immortalized nasopharynx epithelial cells infected with wild type or edited viral particles for these miRNAs. The construction of this model employs the CRISPR/Cas9 technology for the EBV genome editing in Akata-EBV+ cells. The EBV+ Akata cells with wild type or mutated genome were chemically treated to activate lytic cycle and viral particles production. The validation was performed evaluating the expression of lytic cycle markers, BZLF1 and gp350, while the quantification of the produced particles was performed using a B95-8 genome standard curve. The nasopharynx epithelial cells infection was evaluated from the estimate of the viral genome copy number. The Akata-EBV+ cells containing genomes with impaired expression of miR-BARTs 7 or 9 showed deficiency in the production of viral particles when compared to cells containing the wild type genome, while in nasopharynx epithelial cells infection, the impairment of expression of miR-BARTs 7 or 9 positively influenced, increasing the viral copy number in cells infected with the edited versions. The data presented demonstrate the establishment of an experimental model for the study of EBV miR-BARTs, and indicate a possible relationship with events related to the lytic cycle of the virus.
Epstein-Barr virus infection is associated with some types of cancers, such as Burkitt lymphoma (BL), and nasopharyngeal carcinoma (NPC). EBV is a ubiquitous virus, and infects, latently, the majority of adult population. The EBV latency is characterized by limited viral expression, expressing a few proteins and non-coding RNAs, such as miR-BARTs. The overexpression of EBV miR-BART 7 in NPC was associated with regulation of protein levels of PTEN suppressor gene. EBV miR-BART 9, on the other hand, regulates the expression of the LMP1 viral oncoprotein and the cellular E-cadherin. The regulation of these proteins suggests a role of miR-BARTs 7 and 9 in epithelial-mesenchymal transition (EMT), which is important for progression of epithelial cancers. This study aims to create an experimental model to verify the role played by miR-BART7 and miR-BART9 in acquisition of features favorable to carcinogenesis. The model established is based on immortalized nasopharynx epithelial cells infected with wild type or edited viral particles for these miRNAs. The construction of this model employs the CRISPR/Cas9 technology for the EBV genome editing in Akata-EBV+ cells. The EBV+ Akata cells with wild type or mutated genome were chemically treated to activate lytic cycle and viral particles production. The validation was performed evaluating the expression of lytic cycle markers, BZLF1 and gp350, while the quantification of the produced particles was performed using a B95-8 genome standard curve. The nasopharynx epithelial cells infection was evaluated from the estimate of the viral genome copy number. The Akata-EBV+ cells containing genomes with impaired expression of miR-BARTs 7 or 9 showed deficiency in the production of viral particles when compared to cells containing the wild type genome, while in nasopharynx epithelial cells infection, the impairment of expression of miR-BARTs 7 or 9 positively influenced, increasing the viral copy number in cells infected with the edited versions. The data presented demonstrate the establishment of an experimental model for the study of EBV miR-BARTs, and indicate a possible relationship with events related to the lytic cycle of the virus.
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Carcinogênese, MicroRNAs, Herpesvírus humano, Técnicas in vitro