Disclosing the FA-SAT ncRNA role in cancer cells
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2022-06-03
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Ao longo dos anos, as sequências de DNA satélite (satDNA) têm atraído a atenção da
comunidade científica, como importantes contribuintes para a função celular e doença.
FA-SAT, o principal satDNA do genoma do gato doméstico, foi originalmente descoberto nesta
espécie, mas sabe-se que está altamente conservado e é transcrito sob a forma de RNA nãocodificante
(FA-SAT ncRNA) em várias espécies Bilateria, incluindo o Homem. Anteriormente,
a caracterização do perfil deste ncRNA, em diferentes linhas celulares de gato, revelou a
preservação da sua localização nuclear, existindo diferenças na sua distribuição ao longo do
ciclo celular: a sua transcrição foi definida como dependente de ciclo em células não tumorais
e a sua desregulação foi observada nas tumorais. Além disso, as funções deste ncRNA foram
descritas em células de gato e humanas, tendo sido demonstrada a sua interação nuclear com a
proteína PKM2, para regular a transição entre proliferação celular e apoptose.
Com este trabalho, pretendemos efetuar a caracterização celular destes transcritos, pela
primeira vez, no Homem. Para isso, examinámos o perfil do FA-SAT ncRNA em diferentes
linhas celulares humanas, analisando e comparando a sua localização, quantidade e aparência.
O seu comportamento ao longo do ciclo celular também foi avaliado numa dessas linhas
celulares. Seguidamente, desenvolvemos ensaios funcionais para desvendar o mecanismo pelo
qual o complexo FA-SAT ncRNA/PKM2 consegue efetuar a comunicação entre as vias
mitogénica e apoptótica, particularmente, o envolvimento de P53 na determinação desses
fenótipos (foram utilizadas células P53 wild-type-WT e nulas).
Os nossos resultados revelaram uma localização nuclear semelhante do ncRNA em todas
as linhas celulares humanas analisadas, existindo diferenças na sua quantidade e aparência,
dependendo do tipo celular. Um olhar mais atento sobre a localização deste ncRNA nas células
HeLa demostrou que este estava adicionalmente localizado no nucléolo (e na sua periferia).
Acerca da análise do seu comportamento ao longo do processo de divisão celular das células
HeLa, foi possível detetar transcritos FA-SAT em todas as fases do ciclo celular (com maior
acumulação na fase G1 e transição G1/S).
Relativamente aos ensaios funcionais do FA-SAT, o seu silenciamento resultou na
diminuição da quantidade de proteína nuclear PKM2 (também do seu RNA) e na inibição da
proliferação celular (com subexpressão de MYC), tanto em células P53 WT como nulas. Após
o silenciamento do FA-SAT, verificou-se também a ocorrência de apoptose nas duas linhas
celulares, mas a acumulação de P53 foi observada apenas nas células P53 WT. Assim, foi confirmada a associação FA-SAT ncRNA/PKM2 no núcleo dessas células humanas, para
regular a transição entre proliferação celular e apoptose, e definiu-se que a determinação desses
fenótipos era independente do estado de P53.
Em suma, este trabalho representa um avanço de conhecimento, uma vez que contribui
para melhor compreender como é que a expressão de FA-SAT (e a associação FA-SAT
ncRNA/PKM2) pode modular a progressão do ciclo celular/morte celular, nas células humanas.
Adicionalmente, promove a compreensão da maior fração dos nossos genomas (sequências
repetitivas de DNA), como elementos funcionais importantes e responsáveis pela
(des)regulação de diversos processos celulares e doenças, como o cancro.
Over the last years, satellite DNA (satDNA) sequences have attracted some attention from the scientific community, as important contributors to cell function and disease. FA-SAT, the major satDNA of the domestic cat genome, was originally discovered in this species, but it is known it is highly conserved and transcribed into non-coding RNA (FA-SAT ncRNA) in several Bilateria species, including humans. Earlier, the characterization of the ncRNA profile, in distinct cat cell lines, revealed the preservation of its nuclear location, with some differences in its distribution throughout the cell cycle: its expression was defined as cell-cycle dependent in non-tumor cells and the dysregulation of it was observed in the tumor ones. Moreover, the functions of this ncRNA were described in cat and human cells, having been stated its nuclear interaction with the PKM2 protein, to regulate the shift from cell proliferation to apoptosis. With this work, we intended to perform the cellular characterization of these transcripts, for the first time, in humans. For that, we scrutinized the FA-SAT ncRNA profile in distinct human cell lines, analyzing and comparing its location, amount and appearance. Its behavior across the cell cycle was also evaluated in one of these cell lines. Then, we developed functional assays to unveil the mechanism by which the FA-SAT ncRNA/PKM2 complex can cross-talk between the mitogenic and apoptotic pathways, particularly, the P53 involvement in determining these phenotypes (both P53 wild-type-WT and null cells were used). Our results revealed a similar nuclear location for the ncRNA in all the human cell lines analyzed, with some differences in its amount and appearance, depending on the cell type. A closer look at this ncRNA location in the HeLa cells showed that it was additionally located at the nucleolus (and its periphery). Regarding the analysis of its behavior along the cell division process of the HeLa cells, it was possible to detect FA-SAT transcripts in all the phases of the cell cycle (with major enrichment in the G1 phase and G1/S transition). Concerning the FA-SAT functional assays, its knockdown resulted in decreased amounts of PKM2 nuclear protein (also its RNA) and in inhibition of cell proliferation (with MYC downregulation), both in P53 wild-type and null cells. Upon FA-SAT silencing, an apoptosis outcome was also verified in both cell lines, but the P53 accumulation was only observed in the P53 wild-type cells. Therefore, it was confirmed the FA-SAT ncRNA/PKM2 association at the nucleus of these human cells, to regulate the shift from cell proliferation to apoptosis, and it was stated the independence from the P53 status of these phenotypes. In brief, this work represents an advance in knowledge, so it contributes to better understanding how the FA-SAT expression (and the FA-SAT ncRNA/PKM2 association) can modulate the cell-cycle progression/cell death, in the human cells. Additionally, it supports the comprehension of the largest fraction of our genomes (DNA repetitive sequences), as important functional elements responsible for the (dys)regulation of several cellular processes and diseases, such as cancer.
Over the last years, satellite DNA (satDNA) sequences have attracted some attention from the scientific community, as important contributors to cell function and disease. FA-SAT, the major satDNA of the domestic cat genome, was originally discovered in this species, but it is known it is highly conserved and transcribed into non-coding RNA (FA-SAT ncRNA) in several Bilateria species, including humans. Earlier, the characterization of the ncRNA profile, in distinct cat cell lines, revealed the preservation of its nuclear location, with some differences in its distribution throughout the cell cycle: its expression was defined as cell-cycle dependent in non-tumor cells and the dysregulation of it was observed in the tumor ones. Moreover, the functions of this ncRNA were described in cat and human cells, having been stated its nuclear interaction with the PKM2 protein, to regulate the shift from cell proliferation to apoptosis. With this work, we intended to perform the cellular characterization of these transcripts, for the first time, in humans. For that, we scrutinized the FA-SAT ncRNA profile in distinct human cell lines, analyzing and comparing its location, amount and appearance. Its behavior across the cell cycle was also evaluated in one of these cell lines. Then, we developed functional assays to unveil the mechanism by which the FA-SAT ncRNA/PKM2 complex can cross-talk between the mitogenic and apoptotic pathways, particularly, the P53 involvement in determining these phenotypes (both P53 wild-type-WT and null cells were used). Our results revealed a similar nuclear location for the ncRNA in all the human cell lines analyzed, with some differences in its amount and appearance, depending on the cell type. A closer look at this ncRNA location in the HeLa cells showed that it was additionally located at the nucleolus (and its periphery). Regarding the analysis of its behavior along the cell division process of the HeLa cells, it was possible to detect FA-SAT transcripts in all the phases of the cell cycle (with major enrichment in the G1 phase and G1/S transition). Concerning the FA-SAT functional assays, its knockdown resulted in decreased amounts of PKM2 nuclear protein (also its RNA) and in inhibition of cell proliferation (with MYC downregulation), both in P53 wild-type and null cells. Upon FA-SAT silencing, an apoptosis outcome was also verified in both cell lines, but the P53 accumulation was only observed in the P53 wild-type cells. Therefore, it was confirmed the FA-SAT ncRNA/PKM2 association at the nucleus of these human cells, to regulate the shift from cell proliferation to apoptosis, and it was stated the independence from the P53 status of these phenotypes. In brief, this work represents an advance in knowledge, so it contributes to better understanding how the FA-SAT expression (and the FA-SAT ncRNA/PKM2 association) can modulate the cell-cycle progression/cell death, in the human cells. Additionally, it supports the comprehension of the largest fraction of our genomes (DNA repetitive sequences), as important functional elements responsible for the (dys)regulation of several cellular processes and diseases, such as cancer.
Descrição
This master’s dissertation was specifically prepared to obtain the
MSc degree in Technologic, Comparative and Molecular Genetics
and it complies with the scientific and technical norms required by
the regulations of the University of Trás-os-Montes and Alto Douro
Palavras-chave
FA-SAT ncRNA , Linhas celulares de cancro humano