Papel do complexo Ikaros-HDAC na regulação da expressão de micro-RNAs apoptóticos por linfócitos B-1
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Data
2023-11-01
Autores
Costa, Lucas Vasconcelos Soares [UNIFESP]
Orientadores
Popi, Ana Flavia [UNIFESP]
Tipo
Tese de doutorado
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Resumo
Os linfócitos B-1, um subtipo de células B, podem expressar o marcador CD5 e são responsáveis pela produção de IgM natural. Além disso, são capazes de autorrenovação e reconhecimento de autoantígenos, o que poderia facilitar sua diferenciação em células hiperproliferativas. De fato, estas células estão envolvidas no desenvolvimento da leucemia linfocítica crônica (LLC), caracterizada pelo aumento de células B-1 com expressão de CD5 e IgM, além de BCR responsivo a autoantígenos. A principal característica da LLC é a intensa atividade proliferativa de linfócitos B-1 CD5+CD23+ na medula óssea e órgãos linfoides e consequente acúmulo de células resistentes à morte no sangue periférico. Dentre as moléculas envolvidas na patogênese, os fatores de transcrição da família Ikaros (Ikzf1) encontram-se alterados. A principal forma de atividade de Ikaros se dá pela associação a complexos, que envolvem a presença de histonas deacetilases (HDAC) em especial a HDAC1 e HDAC2. No contexto da LLC, foi descrito que as HDACs se encontram, em sua maioria, super expressas e isto influencia na expressão de micro-RNAs (miRNAs) envolvidos na regulação da apoptose intrínseca. Assim, a proposta deste trabalho é investigar o papel de Ikaros na expressão de microRNAs e moléculas envolvidas na via de apoptose, a fim de fazer uma correlação com a LLC. As células B-1 murinas foram obtidas do peritônio de camundongos C57Bl/6 após o enriquecimento por cell sorting. Primeiramente foi confirmada a co-localização e interação de Ikaros e HDAC1 em células B-1 por imunofluorescência e co-imunoprecipitação respectivamente. A seguir, a expressão de Ikaros foi silenciada nestas células por siRNA e neste cenário, além de uma maior viabilidade celular, foi observado um aumento significativo na síntese de HDAC1 e c-Myc. Em nível proteico, observamos que a perda de Ikaros regula negativamente quase todas as proteínas apoptóticas analisadas. No estudo mais específico das moléculas da via, houve o aumento na expressão de Bcl-2 e da forma clivada da caspase-3, condizentes com o aumento da viabilidade. O array de miRNAs revelou a mudança na expressão de 37 miRNAS, dentre eles a baixa expressão dos miRNAs -29c e -145. O tratamento com um inibidor de HDACs, além de diminuir a viabilidade celular independente de Ikaros, restaurou os níveis do miR-29c na ausência de Ikaros mas não do miR-145. A super expressão do miR-29c foi capaz de reduzir os níveis de Bcl-2 e aumentar a expressão de Bax, enquanto a super expressão de miR-145, além de reduzir os níveis de c-Myc, aumentou a expressão do miR-29c e diminuiu Bcl-2 e MCL-1. No ensaio funcional, o miR-29c foi capaz de reverter os efeitos da ausência de Ikaros, mas a restauração do miR-145 e a combinação de ambos os miRNAs pareceu levar estas células à morte, além de modular a razão Bcl-2/Bax. Em um segundo modelo de estudo, investigamos a ausência de Ikaros em células B-1 no ambiente medular. Neste modelo, foi observado aumento significativo na viabilidade das células silenciadas sob co-cultura com linhagem células do estroma medular (OP9). Ainda, houve a mudança na expressão de 42 miRNAS, mas a baixa expressão dos miRNAs -29c e -145 se manteve. Os efeitos da inibição das HDACs e da restauração dos miRNAs se manteve como no modelo anterior mesmo com os estímulos fornecidos pelas células estromais. Assim, estes dados confirmam a participação de Ikaros na regulação de moléculas envolvidas na sobrevivência das células B-1, e os mecanismos incluem HDACs e o fator de transcrição c-Myc. Além disso, estes dados indicam que a baixa expressão dos miRNAs -145 e -29c, devido à desregulação de Ikaros, poderia ser uma das vias moleculares para induzir resistência à apoptose em células B leucêmicas. Por fim, foi postulado o miR-145 como um possível alvo terapêutico para a LLC.
B-1 lymphocytes, a subset of B cells, can express the CD5 marker and are responsible for the production of natural IgM. Furthermore, they are capable of self-renewal and recognizing self-antigens, which could facilitate their differentiation into hyperproliferative cells. In fact, these cells are involved in the development of chronic lymphocytic leukemia (CLL), disease that result in the increase of B-1 cells with expression of CD5 and IgM, in addition to BCR responsive to self-antigens. The main characteristic of CLL is the intense proliferative activity of CD5+CD23+ B-1 lymphocytes in the bone marrow and lymphoid sites with consequent accumulation of death-resistant cells in the peripheral blood. Among the molecules involved in pathogenesis, the transcription factors of the Ikaros family (Ikzf1) are altered. Most of Ikaros activity is through remodeling complexes, involving histone deacetylases (HDACs) 1 and 2. In CLL context, it has been described that HDACs are overexpressed and with consequences in the expression of intrinsic apoptosis regulating micro-RNAs (miRNAs). Therefore, this work purpose is to investigate the role of Ikaros in the expression of microRNAs and molecules involved in the apoptosis pathway, correlating with CLL. Murine B-1 cells were obtained from the peritoneum of C57Bl/6 mice after enrichment by cell sorting. Firstly, the co-localization and interaction of Ikaros and HDAC1 was confirmed by immunofluorescence and co-immunoprecipitation in B-1 cells. Next, Ikaros expression was silenced in these cells by siRNA and in this scenario, in addition to greater cell viability, we observed a significant increase in HDAC1 and c-Myc expression. In the protein level, we observed that Ikaros loss negatively regulates almost all apoptotic proteins analyzed. In a more specific study, there was an increase in the expression of Bcl-2 and the cleaved form of caspase-3, consistent with viability increase. The miRNA array revealed changes in the expression of 37 miRNAs, including the low expression of miRNAs -29c and -145. Treatment with an HDAC inhibitor, in addition to decreasing cell viability independently of Ikaros, restored miR-29c levels in Ikaros absence but not miR-145. Overexpression of miR-29c was able to reduce Bcl-2 levels and increase Bax, while overexpression of miR-145, in addition to reducing c-Myc levels, increased miR-29c expression and decreased Bcl-2 and MCL-1. In functional assay, miR-29c was able to reverse Ikaros absence effects, but miR-145 restoration and the combination of both miRNAs seemed to lead these cells to death, in addition with Bcl-2/Bax ratio modulation. In a second study model, we investigated Ikaros absence in B-1 cells submitted to a bone marrow environment. In this model, a significant viability increase of silenced cells was observed under co-culture with stromal cell lines (OP9). Furthermore, a change in 42 miRNAS expression was observed, but the low expression of miRNAs -29c and -145 remained. HDACs inhibition and miRNAs restoration remained effects as in the previous model, even with stromal cells stimuli. Thus, these data confirm the participation of Ikaros in the regulation of molecules involved in the survival of B-1 cells, and the mechanisms include HDAC1 and transcription factor c-Myc. Furthermore, these data indicate that low expression of miRNAs -145 and -29c, due to deregulation of Ikaros, could be one of the molecular pathways that induce resistance to apoptosis in leukemic B cells. Finally, miR-145 was postulated as a possible therapeutic target for CLL.
B-1 lymphocytes, a subset of B cells, can express the CD5 marker and are responsible for the production of natural IgM. Furthermore, they are capable of self-renewal and recognizing self-antigens, which could facilitate their differentiation into hyperproliferative cells. In fact, these cells are involved in the development of chronic lymphocytic leukemia (CLL), disease that result in the increase of B-1 cells with expression of CD5 and IgM, in addition to BCR responsive to self-antigens. The main characteristic of CLL is the intense proliferative activity of CD5+CD23+ B-1 lymphocytes in the bone marrow and lymphoid sites with consequent accumulation of death-resistant cells in the peripheral blood. Among the molecules involved in pathogenesis, the transcription factors of the Ikaros family (Ikzf1) are altered. Most of Ikaros activity is through remodeling complexes, involving histone deacetylases (HDACs) 1 and 2. In CLL context, it has been described that HDACs are overexpressed and with consequences in the expression of intrinsic apoptosis regulating micro-RNAs (miRNAs). Therefore, this work purpose is to investigate the role of Ikaros in the expression of microRNAs and molecules involved in the apoptosis pathway, correlating with CLL. Murine B-1 cells were obtained from the peritoneum of C57Bl/6 mice after enrichment by cell sorting. Firstly, the co-localization and interaction of Ikaros and HDAC1 was confirmed by immunofluorescence and co-immunoprecipitation in B-1 cells. Next, Ikaros expression was silenced in these cells by siRNA and in this scenario, in addition to greater cell viability, we observed a significant increase in HDAC1 and c-Myc expression. In the protein level, we observed that Ikaros loss negatively regulates almost all apoptotic proteins analyzed. In a more specific study, there was an increase in the expression of Bcl-2 and the cleaved form of caspase-3, consistent with viability increase. The miRNA array revealed changes in the expression of 37 miRNAs, including the low expression of miRNAs -29c and -145. Treatment with an HDAC inhibitor, in addition to decreasing cell viability independently of Ikaros, restored miR-29c levels in Ikaros absence but not miR-145. Overexpression of miR-29c was able to reduce Bcl-2 levels and increase Bax, while overexpression of miR-145, in addition to reducing c-Myc levels, increased miR-29c expression and decreased Bcl-2 and MCL-1. In functional assay, miR-29c was able to reverse Ikaros absence effects, but miR-145 restoration and the combination of both miRNAs seemed to lead these cells to death, in addition with Bcl-2/Bax ratio modulation. In a second study model, we investigated Ikaros absence in B-1 cells submitted to a bone marrow environment. In this model, a significant viability increase of silenced cells was observed under co-culture with stromal cell lines (OP9). Furthermore, a change in 42 miRNAS expression was observed, but the low expression of miRNAs -29c and -145 remained. HDACs inhibition and miRNAs restoration remained effects as in the previous model, even with stromal cells stimuli. Thus, these data confirm the participation of Ikaros in the regulation of molecules involved in the survival of B-1 cells, and the mechanisms include HDAC1 and transcription factor c-Myc. Furthermore, these data indicate that low expression of miRNAs -145 and -29c, due to deregulation of Ikaros, could be one of the molecular pathways that induce resistance to apoptosis in leukemic B cells. Finally, miR-145 was postulated as a possible therapeutic target for CLL.
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Citação
COSTA, Lucas Vasconcelos Soares. Papel do complexo Ikaros-HDAC na regulação da expressão de micro-RNAs apoptóticos por linfócitos B-1. 2024. 95 f. Tese (Doutorado em Microbiologia e Imunologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2024.