Analysis of lncRNAs expression in monocytes and osteoclasts of rheumatoid arthritis patients

Detalhes bibliográficos
Autor(a) principal: Amaral, Inês Filipa Pereira Abrunhosa
Data de Publicação: 2019
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10773/27039
Resumo: Rheumatoid arthritis (RA) is a chronic inflammatory immune-mediated rheumatic disease. Bone erosion is one of the hallmarks of RA pathophysiology and is associated with disease severity. Bone erosions in RA patients result from an imbalanced bone metabolism due, in part, to excessive osteoclastogenesis (the process by which precursor cells of the monocyte lineage differentiate into osteoclasts) and osteoclastic activity. Variable expression of long non-coding RNAs (lncRNAs) has been observed during mouse osteoclastogenesis, suggesting a physiologic role for lncRNAs in the process. In RA patients, lncRNA expression has been further shown to be altered in cellular types critical for its pathophysiology, like peripheral blood mononuclear leukocytes and activated fibroblast-like synoviocytes, in comparison to healthy controls. However, no study has yet examined lncRNA expression in monocytes and osteoclasts of RA patients. This work aims to address this question by analyzing the expression of a panel of 8 lncRNAs (GAS5, NEAT1, Meg3, DANCR, HOTAIR, Meg9, H19, and Sox2OT) in monocytes and osteoclasts of early arthritis patients, established RA patients and healthy controls. Both groups of patients were recruited at the Rheumatology Department, Hospital de Santa Maria, Lisbon Academic Medical Centre, Portugal. Age and sex matched donors were used as healthy controls. Heparinized blood was collected from each participant. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples by density gradient centrifugation. Adherent monocytes were in vitro differentiated into osteoclasts by a 21-day differentiation protocol. Total RNA was extracted from both monocytes and osteoclasts, for further analysis of lncRNA expression by real-time quantitative PCR (RT-qPCR). LncRNA expression analysis was performed for seventeen subjects, including 7 established rheumatoid arthritis patients, 3 early arthritis patients and 7 healthy donors. In vitro osteoclastogenesis produced a substantially higher number of osteoclasts in early arthritis patients, when compared to healthy controls. From our lncRNA panel, only GAS5, NEAT1, and DANCR presented a measurable expression in all tested samples. Our results showed an increased expression of NEAT1 and GAS5 along with a decreased expression of DANCR in monocytes of established RA patients, in comparison to those of healthy controls. An increased expression of NEAT1 along with a decreased expression of GAS5 and DANCR was observed in osteoclasts of early arthritis patients, when compared to those of healthy controls. No statistically significant differences were found for both analyses. Our data are consistent with an increased osteoclastogenic potential of peripheral monocytes of early arthritis patients, and thus, with an imbalanced bone metabolism in this pathological condition. Overall, our results prompted us to suggest that the lncRNAs here analyzed may in fact play a role in osteoclastogenesis in RA, as an altered lncRNA expression was observed in monocytes of early arthritis and established RA patients, when compared to healthy controls.
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spelling Analysis of lncRNAs expression in monocytes and osteoclasts of rheumatoid arthritis patientsRheumatoid arthritisBone erosionMonocytesOsteoclastsOsteoclastogenesisLong non-coding RNAs (lncRNAs).Rheumatoid arthritis (RA) is a chronic inflammatory immune-mediated rheumatic disease. Bone erosion is one of the hallmarks of RA pathophysiology and is associated with disease severity. Bone erosions in RA patients result from an imbalanced bone metabolism due, in part, to excessive osteoclastogenesis (the process by which precursor cells of the monocyte lineage differentiate into osteoclasts) and osteoclastic activity. Variable expression of long non-coding RNAs (lncRNAs) has been observed during mouse osteoclastogenesis, suggesting a physiologic role for lncRNAs in the process. In RA patients, lncRNA expression has been further shown to be altered in cellular types critical for its pathophysiology, like peripheral blood mononuclear leukocytes and activated fibroblast-like synoviocytes, in comparison to healthy controls. However, no study has yet examined lncRNA expression in monocytes and osteoclasts of RA patients. This work aims to address this question by analyzing the expression of a panel of 8 lncRNAs (GAS5, NEAT1, Meg3, DANCR, HOTAIR, Meg9, H19, and Sox2OT) in monocytes and osteoclasts of early arthritis patients, established RA patients and healthy controls. Both groups of patients were recruited at the Rheumatology Department, Hospital de Santa Maria, Lisbon Academic Medical Centre, Portugal. Age and sex matched donors were used as healthy controls. Heparinized blood was collected from each participant. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples by density gradient centrifugation. Adherent monocytes were in vitro differentiated into osteoclasts by a 21-day differentiation protocol. Total RNA was extracted from both monocytes and osteoclasts, for further analysis of lncRNA expression by real-time quantitative PCR (RT-qPCR). LncRNA expression analysis was performed for seventeen subjects, including 7 established rheumatoid arthritis patients, 3 early arthritis patients and 7 healthy donors. In vitro osteoclastogenesis produced a substantially higher number of osteoclasts in early arthritis patients, when compared to healthy controls. From our lncRNA panel, only GAS5, NEAT1, and DANCR presented a measurable expression in all tested samples. Our results showed an increased expression of NEAT1 and GAS5 along with a decreased expression of DANCR in monocytes of established RA patients, in comparison to those of healthy controls. An increased expression of NEAT1 along with a decreased expression of GAS5 and DANCR was observed in osteoclasts of early arthritis patients, when compared to those of healthy controls. No statistically significant differences were found for both analyses. Our data are consistent with an increased osteoclastogenic potential of peripheral monocytes of early arthritis patients, and thus, with an imbalanced bone metabolism in this pathological condition. Overall, our results prompted us to suggest that the lncRNAs here analyzed may in fact play a role in osteoclastogenesis in RA, as an altered lncRNA expression was observed in monocytes of early arthritis and established RA patients, when compared to healthy controls.A artrite reumatóide (AR) é uma doença reumática crónica, inflamatória e imuno-mediada. A erosão óssea é uma das características da fisiopatologia da AR e está associada à gravidade da doença. As erosões ósseas em pacientes com AR resultam de um metabolismo ósseo alterado devido, em parte, a osteoclastogénese (o processo pelo qual as células precursoras da linhagem de monócitos se diferenciam em osteoclastos) e a atividade osteoclástica excessivas. Num estudo recente, observou-se uma expressão variável de RNAs longos não codificantes (lncRNAs) durante a osteoclastogénese no ratinho, sugerindo um papel fisiológico dos lncRNAs neste processo. Em pacientes com AR, vários estudos observaram até à data uma expressão alterada de lncRNAs em tipos celulares críticos da fisiopatologia da doença, como leucócitos mononucleares do sangue periférico e sinoviócitos ativados tipo-fibroblastos. No entanto, nenhum estudo examinou, até ao momento, a expressão de lncRNAs em monócitos e osteoclastos de pacientes com AR. Este trabalho visou abordar esta questão analisando a expressão de um painel de 8 lncRNAs (GAS5, NEAT1, Meg3, DANCR, HOTAIR, Meg9, H19 e Sox2OT) em monócitos e osteoclastos de pacientes com AR, artrite inicial e controlos saudáveis. Os doentes foram recrutados no Serviço de Reumatologia do Hospital de Santa Maria, Centro Académico de Medicina de Lisboa. Dadores emparelhados por idade e sexo foram usados como controlos saudáveis. Foi colhido sangue heparinizado, a partir do qual, se isolaram células mononucleadas de sangue periférico (PBMCs) por centrifugação em gradiente de densidade. Os monócitos aderentes foram depois diferenciados in vitro em osteoclastos por um protocolo de diferenciação de 21 dias. RNA total foi extraído de monócitos e osteoclastos, para posterior análise da expressão de lncRNAs por PCR quantitativo em tempo real (RT-qPCR). Estudou-se a expressão de lncRNAs para dezassete indivíduos, incluindo 7 pacientes com artrite reumatóide estabelecida, 3 pacientes com artrite inicial e 7 dadores saudáveis. Observou-se um número substancialmente superior de osteoclastos em pacientes com artrite inicial face ao obtido para controlos saudáveis. Observámos um aumento da expressão de NEAT1 e GAS5, e uma diminuição da expressão de DANCR, em monócitos de pacientes com AR estabelecida. Por outro lado, observou-se uma expressão aumentada de NEAT1 e diminuída de GAS5 e DANCR em osteoclastos de pacientes com artrite inicial. Não foram encontradas diferenças estatisticamente significativas. Os resultados obtidos são consistentes com um potencial osteoclastogénico aumentado em monócitos periféricos de pacientes com artrite inicial e, portanto, com um metabolismo ósseo alterado. Globalmente, os resultados de expressão de lncRNAs sugerem que os lncRNAs testados possam ter um papel na osteoclastogénese na AR, atendendo aos seus níveis de expressão alterados em monócitos de pacientes com artrite inicial e com AR.2020-07-23T00:00:00Z2019-07-18T00:00:00Z2019-07-18info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10773/27039engAmaral, Inês Filipa Pereira Abrunhosainfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-02-22T11:52:25Zoai:ria.ua.pt:10773/27039Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T02:59:55.809543Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Analysis of lncRNAs expression in monocytes and osteoclasts of rheumatoid arthritis patients
title Analysis of lncRNAs expression in monocytes and osteoclasts of rheumatoid arthritis patients
spellingShingle Analysis of lncRNAs expression in monocytes and osteoclasts of rheumatoid arthritis patients
Amaral, Inês Filipa Pereira Abrunhosa
Rheumatoid arthritis
Bone erosion
Monocytes
Osteoclasts
Osteoclastogenesis
Long non-coding RNAs (lncRNAs).
title_short Analysis of lncRNAs expression in monocytes and osteoclasts of rheumatoid arthritis patients
title_full Analysis of lncRNAs expression in monocytes and osteoclasts of rheumatoid arthritis patients
title_fullStr Analysis of lncRNAs expression in monocytes and osteoclasts of rheumatoid arthritis patients
title_full_unstemmed Analysis of lncRNAs expression in monocytes and osteoclasts of rheumatoid arthritis patients
title_sort Analysis of lncRNAs expression in monocytes and osteoclasts of rheumatoid arthritis patients
author Amaral, Inês Filipa Pereira Abrunhosa
author_facet Amaral, Inês Filipa Pereira Abrunhosa
author_role author
dc.contributor.author.fl_str_mv Amaral, Inês Filipa Pereira Abrunhosa
dc.subject.por.fl_str_mv Rheumatoid arthritis
Bone erosion
Monocytes
Osteoclasts
Osteoclastogenesis
Long non-coding RNAs (lncRNAs).
topic Rheumatoid arthritis
Bone erosion
Monocytes
Osteoclasts
Osteoclastogenesis
Long non-coding RNAs (lncRNAs).
description Rheumatoid arthritis (RA) is a chronic inflammatory immune-mediated rheumatic disease. Bone erosion is one of the hallmarks of RA pathophysiology and is associated with disease severity. Bone erosions in RA patients result from an imbalanced bone metabolism due, in part, to excessive osteoclastogenesis (the process by which precursor cells of the monocyte lineage differentiate into osteoclasts) and osteoclastic activity. Variable expression of long non-coding RNAs (lncRNAs) has been observed during mouse osteoclastogenesis, suggesting a physiologic role for lncRNAs in the process. In RA patients, lncRNA expression has been further shown to be altered in cellular types critical for its pathophysiology, like peripheral blood mononuclear leukocytes and activated fibroblast-like synoviocytes, in comparison to healthy controls. However, no study has yet examined lncRNA expression in monocytes and osteoclasts of RA patients. This work aims to address this question by analyzing the expression of a panel of 8 lncRNAs (GAS5, NEAT1, Meg3, DANCR, HOTAIR, Meg9, H19, and Sox2OT) in monocytes and osteoclasts of early arthritis patients, established RA patients and healthy controls. Both groups of patients were recruited at the Rheumatology Department, Hospital de Santa Maria, Lisbon Academic Medical Centre, Portugal. Age and sex matched donors were used as healthy controls. Heparinized blood was collected from each participant. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples by density gradient centrifugation. Adherent monocytes were in vitro differentiated into osteoclasts by a 21-day differentiation protocol. Total RNA was extracted from both monocytes and osteoclasts, for further analysis of lncRNA expression by real-time quantitative PCR (RT-qPCR). LncRNA expression analysis was performed for seventeen subjects, including 7 established rheumatoid arthritis patients, 3 early arthritis patients and 7 healthy donors. In vitro osteoclastogenesis produced a substantially higher number of osteoclasts in early arthritis patients, when compared to healthy controls. From our lncRNA panel, only GAS5, NEAT1, and DANCR presented a measurable expression in all tested samples. Our results showed an increased expression of NEAT1 and GAS5 along with a decreased expression of DANCR in monocytes of established RA patients, in comparison to those of healthy controls. An increased expression of NEAT1 along with a decreased expression of GAS5 and DANCR was observed in osteoclasts of early arthritis patients, when compared to those of healthy controls. No statistically significant differences were found for both analyses. Our data are consistent with an increased osteoclastogenic potential of peripheral monocytes of early arthritis patients, and thus, with an imbalanced bone metabolism in this pathological condition. Overall, our results prompted us to suggest that the lncRNAs here analyzed may in fact play a role in osteoclastogenesis in RA, as an altered lncRNA expression was observed in monocytes of early arthritis and established RA patients, when compared to healthy controls.
publishDate 2019
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2019-07-18
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