Isolamento e caracterização de um RNA regulador, de Cantharellus cibarius, com atividade antitumoral
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2018-05-02
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Cantharellus cibarius é um cogumelo comestível de amplo consumo devido às suas propriedades sensoriais. Estudos prévios demonstraram que este cogumelo apresenta uma elevada atividade antiproliferativa contra várias linhas celulares cancerígenas, sendo o principal responsável por esta ação uma fração isolada por cromatografia de troca aniónica, que os dados disponíveis indicavam ser maioritariamente constituída por ácido ribonucleico (RNA). Os principais objetivos deste estudo foram a confirmação da natureza da molécula responsável pela atividade antitumoral, observação da sua ação, determinação da sequência de nucleótidos constituintes do RNA em causa e execução de estudos preliminares relacionados com potenciais locais da sua interação e ação na célula. Os biopolimeros do Cantharellus cibarius foram inicialmente extraídos, isolados e purificados, tendo-se obtido três frações: CC1, CC2 e CC3, sendo esta última a fração que apresentou capacidade antitumoral. De seguida, as frações foram caracterizadas quimicamente, com especial enfoque na fração CC3. Foi realizada a análise da sua composição em açúcares, ácidos gordos e fosfolípidos e realizou-se um estudo inicial da sua natureza química rica em ácidos nucleicos através de metodologias como eletroforese em gel de agarose, espectrofotometria UV-vis e FTIR. Após sugestão da presença de RNA, realizou-se a síntese de cDNA a partir do RNA isolado e a sua amplificação por DOP-PCR. De seguida procedeuse à clonagem dos produtos PCR obtidos e extração do DNA plasmídico dos clones positivos. Estes foram sequenciados e as sequências obtidas foram analisadas bioinformaticamente de modo a estudar o tipo de RNA presente. Adicionalmente, a fração CC3 foi colocada em contacto com a linha celular Caco-2 de modo a verificar a sua ação antitumoral sendo, de seguida, executada a técnica de RNA-FISH de modo a observar a sua possível incorporação no interior da célula. Com base na sequência mais conservada, e de forma ainda muito preliminar, efetuou-se a sua síntese artificial (mimics). O mimics tem como finalidade ser, posteriormente, utilizado em estudos celulares que permitam observar o fenótipo verificado diretamente a partir de CC3. A análise da composição em açúcares mostrou que o açúcar maioritário presente na fracção CC3 é a ribose, principal constituinte do RNA (também confirmado por espectrofotometria UV-vis, eletroforese e FTIR). Através da sequenciação e posterior análise bioinformática foi possível descobrir qual a sequência de RNA potencialmente responsável por esta atividade (CCTGCAGGTCGA-CTCTAGAGGATCCCCC-CGACTCGAGTCGG), sendouma sequência de pequeno tamanho e com elevada percentagem de guaninas e citosinas, formando, provavelmente, uma molécula com uma estrutura secundária estável. A fração em estudo mostrou possuir atividade antitumoral, provocando a morte celular das células Caco-2 ao fim de 48h. Através do RNA-FISH foi possível verificar que o RNA presente em CC3 se incorpora nas células atravessando tanto a membrana celular como nuclear, acumulando-se no citoplasma e no núcleo das mesmas. O C. cibarius revelou apresentar componentes com atividade anticancerígena e, uma vez descoberta a sequência de RNA alvo e demonstradas as suas interações com a célula, este trabalho torna-se bastante promissor para a futura síntese artificial deste RNA, sem recorrer ao cogumelo, uma vez que este não é cultivável, podendo, num futuro próximo, proceder-se à sua síntese artificial e posterior incorporação em fármacos.
Cantharellus cibarius is an edible mushroom of ample consumption due to its sensorial properties. Previous studies revealed that this mushroom has a high antiproliferative activity against several cancer cell lines. The main responsible for this action is a fraction, isolated by anion exchange chromatography, wich availablçe data indicated to be mainly composed by ribonucleic acid (RNA). The main objectives of this study were to confirm the nature of the molecule carrying antitumor activity, to observe its action, to determine its nucleotide sequence and to carry out preliminary studies related to potential sites of its interaction and action in the cell. Extraction, isolation and purification of Cantharellus cibarius biopolymers was performed, from which three fractions were obtained: CC1, CC2 and CC3. CC3 is the fraction that shows antitumor activity. Afterwards, the fractions were chemically characterized, with a special focus on the CC3. Its composition in sugars, fatty acids and phospholipids was analyzed. and a detailed analysis of nucleic acids was carried out by UV-vis spectrophotometry, agarose gel electrophoresis and FTIR. After the suggestion of RNA presence, the cDNA synthesis from the isolated RNA and its amplification by DOP-PCR was performed. Then, the obtained PCR products were cloned and the plasmid DNA from the positive clones was extracted. These were sequenced. The obtained sequences were analyzed bioinformatically in order to verify the type of this RNA. Moreover, the isolated fraction was placed in contact with cancer cell lines, namely colorectal cancer cells (Caco-2) followed by RNA-FISH methodology in order to detect and localize the RNAs within the cells. Based on the most conserved sequence, and still very preliminary, its artificial synthesis (mimics) was performed. Mimics is intended to be used in cellular studies that allow to observe the phenotype verified directly from CC3. The analysis of the composition in sugars showed that the major sugar present in CC3 is ribose, the main constituent of the RNA (also confirmed by UV-vis spectrophotometry, electrophoresis and FTIR). Through sequencing and subsequent bioinformatic analysis it was possible to find out which RNA sequence is responsible for this activity (CCTGCAGGTCGACTCTAGAGGATCCCCC-CGACTCGAGTCGG). It is a sequence with small size and high percentage of guanines and cytosines, probably forming a molecule with a stable secondary structure. The fraction under study shows antitumor activity since cell death of Caco-2 cells occurred in contact with it after 48h. Through RNA-FISH, it was possible to observe that the RNA presentin CC3 is incorporated into cells. This RNA is capable to crossing both cellular and nuclear membranes and accumulating in the cytoplasm and nucleus of the cells. C. cibarius revealed to have compounds with anticancer activity. Once the target RNA sequence has been discovered and its interactions with the cell demonstrated, this work becomes very promising for future artificial synthesis of this RNA (without resorting to the mushroom once that it is not cultivable). Thus, in the future, it will be possible to proceed to its artificial synthesis and subsequent incorporation into drugs.
Cantharellus cibarius is an edible mushroom of ample consumption due to its sensorial properties. Previous studies revealed that this mushroom has a high antiproliferative activity against several cancer cell lines. The main responsible for this action is a fraction, isolated by anion exchange chromatography, wich availablçe data indicated to be mainly composed by ribonucleic acid (RNA). The main objectives of this study were to confirm the nature of the molecule carrying antitumor activity, to observe its action, to determine its nucleotide sequence and to carry out preliminary studies related to potential sites of its interaction and action in the cell. Extraction, isolation and purification of Cantharellus cibarius biopolymers was performed, from which three fractions were obtained: CC1, CC2 and CC3. CC3 is the fraction that shows antitumor activity. Afterwards, the fractions were chemically characterized, with a special focus on the CC3. Its composition in sugars, fatty acids and phospholipids was analyzed. and a detailed analysis of nucleic acids was carried out by UV-vis spectrophotometry, agarose gel electrophoresis and FTIR. After the suggestion of RNA presence, the cDNA synthesis from the isolated RNA and its amplification by DOP-PCR was performed. Then, the obtained PCR products were cloned and the plasmid DNA from the positive clones was extracted. These were sequenced. The obtained sequences were analyzed bioinformatically in order to verify the type of this RNA. Moreover, the isolated fraction was placed in contact with cancer cell lines, namely colorectal cancer cells (Caco-2) followed by RNA-FISH methodology in order to detect and localize the RNAs within the cells. Based on the most conserved sequence, and still very preliminary, its artificial synthesis (mimics) was performed. Mimics is intended to be used in cellular studies that allow to observe the phenotype verified directly from CC3. The analysis of the composition in sugars showed that the major sugar present in CC3 is ribose, the main constituent of the RNA (also confirmed by UV-vis spectrophotometry, electrophoresis and FTIR). Through sequencing and subsequent bioinformatic analysis it was possible to find out which RNA sequence is responsible for this activity (CCTGCAGGTCGACTCTAGAGGATCCCCC-CGACTCGAGTCGG). It is a sequence with small size and high percentage of guanines and cytosines, probably forming a molecule with a stable secondary structure. The fraction under study shows antitumor activity since cell death of Caco-2 cells occurred in contact with it after 48h. Through RNA-FISH, it was possible to observe that the RNA presentin CC3 is incorporated into cells. This RNA is capable to crossing both cellular and nuclear membranes and accumulating in the cytoplasm and nucleus of the cells. C. cibarius revealed to have compounds with anticancer activity. Once the target RNA sequence has been discovered and its interactions with the cell demonstrated, this work becomes very promising for future artificial synthesis of this RNA (without resorting to the mushroom once that it is not cultivable). Thus, in the future, it will be possible to proceed to its artificial synthesis and subsequent incorporation into drugs.
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Cantharellus cibarius , atividade anticancerígena