Caracterização dos diferentes transcritos produzidos pelo locus HJURP (HollidayJunctionRecognizing-Protein) em células de glioblastoma
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2018-01-30
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Universidade Estadual Paulista (Unesp)
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RESUMO O glioblastoma (GBM) é o tipo mais comum e mortal de tumor cerebral, sendo que os pacientes apresentam uma sobrevida média de apenas 14 meses. O tratamento é realizado com remoção cirúrgica da massa tumoral, seguida de radioterapia e quimioterapia, porém sua eficácia é muito reduzida devido a acelerada atividade proliferativa e elevada resistência das células tumorais aos agentes genotóxicos. Dados do nosso grupo sustentam a hipótese de que este quadro pode estar associado em parte ao ganho de competência na atividade de reparo de DNA. Demonstramos anteriormente que, a HJURP (HollidayJunctionRecognizing Protein), uma proteína envolvida em reparo de DNA e montagem da cromatina centromérica, está superexpressa nos astrocitomas e que seus altos níveis de expressão estão correlacionados com o pior prognóstico dos pacientes. Dados da literatura evidenciam a existência de três RNAs mensageiros para o locus HJURP. O maior deles, denominado hjurp1, apresenta 9 éxons e codifica a isoformaprotéica A, que contém 748 aminoácidos. Os transcritos 2 e 3 apresentam deleção de alguns éxons, codificando proteínas hipoteticamente menores. Embora existam vários cDNAs depositados no GenBank que corroboram a expressão destes três mRNAs, não há dados na literatura sobre seu padrão de expressão ou a funcionalidade destas isoformas. Sendo assim, neste projeto avaliamos a expressão dos diferentes transcritos do locus HJURP em células de GBM, amostras de diferentes graus de astrocitoma, pares de linhagens de diferentes tipos não tumorais/tumorais e amostra de diferentes tecidos não tumorais. Através de PCR convencional detectamos as três isoformas nos astrócitos não tumorais (ACBRI371) e nas linhagens tumorais U87MG, U138MG e U251MG. Porém, as linhagens T98G e U343MG apresentaram variações, sendo que nas células T98G foram detectadas somente as isoformas 1 e 3, e nas células U343MG apenas a isoforma 1. Através de análise por PCR quantitativo, observamos que as linhagens de GBM apresentamum aumento marcante na expressão dos três transcritos de HJURP quando comparados aos astrócitos não tumorais ACBRI371. Como esperado, as células T98G apresentaram os maiores níveis de HJURP, sendo que a expressão dos três transcritos está aumentada. Além disso, percebemos que o transcrito hjurp1 é de fato bem mais abundante que as isoformas 2 e 3 nas linhagens celulares. Embora tenha sido observado um significativo aumento global para todas as isoformasnas amostras tumorais (tumores versus substância branca), percebemos que a isoforma 2 é mais abundante que a 1 no tecido não tumoral e astrocitomas de baixo grau (AST2), porém há um ganho de expressão da isoforma 1, que não é acompanhado pela isoforma 2, nas amostras de astrocimotas de grau 3 (AST3) e GBM, criando perfis de expressão característicos da progressão tumoral. Análises de expressão de um painel de tecidos não tumorais e pares de linhagens tumorais e não tumorais de outras origens, mostraram resultados similares, consolidando a existência de uma desregulação nos transcritos de HJURP que favorece a expressão da isoforma 1 nas células tumorais. Ensaios recentes demonstraram que o silenciamento dirigido ao transcrito 1 de HJURP nas linhagens U87MG e T98G, parece estimular a expressãoda HJURP 2 e mostra um impacto reduzido sobre a viabilidade celular, quando comparado ao silenciamento da proteína HJURP total. Esses dados sugerem que ao isoformas 2 e 3 estão ativas e auxiliam na manutenção da viabiliadde celular em linhagens de GMB. Palavras-chave: Glioblastoma. HJURP.Transcritos.Isoformas.
ABSTRACT Glioblastoma (GBM) is the most common and lethal brain tumor in humans, with a median survival rate of ~ 14 months. Standard optimized treatment implies in surgery, followed by radiation and chemotherapy, but it has low efficacy due to tumor fast proliferative activity and high resistance against genotoxic agents. Preliminary data from our group showed that HJURP (Holliday Junction Recognizing Protein), a protein which plays a central role in chromatin assembly and is related to take part in double-stranded DNA breaks (DSBs) repair, is overexpressed in astrocytomas. High levels of this protein have been associated with patient pour prognosis. According to the literature, three transcript variants are predicted to encode HJURP gene. The variant (2) and (3) differ from the main splice variant (1) by the exclusion of some exons, generated by exon skipping. Although many cDNA sequences have been deposited on GeneBank database, it is still unclear their functions and expression pattern among tumors. Thus, in this work, we aimed to evaluate the expression of the three variants of HJURP gene in GBM cells, patient samples of different grades of astrocytoma, and different tumor and non-tumor cell lines. Expression of all transcripts was confirmed by conventional PCR in non-tumoral astrocytes (ACBRI371) and GBM cell lines (U87MG, U138MG, and U251MG). However, T98G and U343MG showed a distinct pattern. The variant (1) was detected in both cell lines, but the variant (3) was only detectable in T98G cells. Using quantitative PCR (q-PCR) we demonstrated that all transcripts were expressed at higher levels in GBM cells than in non-tumoral astrocytes. As we expected, T98G showed the highest levels of HJURP expression (variants 1,2 and 3). We also observed that variant (1) is more abundant in the cell lines. Albeit, there was an overall increase of all variants in tumor samples when compared to the brain white matter. We demonstrated that variant (2) is more prevalent than variant (1) in non-tumoral tissues and low-grade astrocytomas (AST2). In contrast, anaplastic astrocytoma (AST3) and GBM showed a considerable increase in variant (1) levels, arising a characteristic profile of tumor progression. Expression analyses of a panel of tumor and non-tumoral cell lines from distinct origins confirmed our previously described finding. Recent data demonstrated that transcript (1) silencing of U87MG and T98G may stimulate transcrip (2) expression and it shows the low impact on cell viability when compared to total HJURP silencing. These data suggest that variants (2) and (3) are functional and assist in GBM cell viability maintenance. Keywords: Glioblastoma. HJURP. Transcripts.Isoforms.
ABSTRACT Glioblastoma (GBM) is the most common and lethal brain tumor in humans, with a median survival rate of ~ 14 months. Standard optimized treatment implies in surgery, followed by radiation and chemotherapy, but it has low efficacy due to tumor fast proliferative activity and high resistance against genotoxic agents. Preliminary data from our group showed that HJURP (Holliday Junction Recognizing Protein), a protein which plays a central role in chromatin assembly and is related to take part in double-stranded DNA breaks (DSBs) repair, is overexpressed in astrocytomas. High levels of this protein have been associated with patient pour prognosis. According to the literature, three transcript variants are predicted to encode HJURP gene. The variant (2) and (3) differ from the main splice variant (1) by the exclusion of some exons, generated by exon skipping. Although many cDNA sequences have been deposited on GeneBank database, it is still unclear their functions and expression pattern among tumors. Thus, in this work, we aimed to evaluate the expression of the three variants of HJURP gene in GBM cells, patient samples of different grades of astrocytoma, and different tumor and non-tumor cell lines. Expression of all transcripts was confirmed by conventional PCR in non-tumoral astrocytes (ACBRI371) and GBM cell lines (U87MG, U138MG, and U251MG). However, T98G and U343MG showed a distinct pattern. The variant (1) was detected in both cell lines, but the variant (3) was only detectable in T98G cells. Using quantitative PCR (q-PCR) we demonstrated that all transcripts were expressed at higher levels in GBM cells than in non-tumoral astrocytes. As we expected, T98G showed the highest levels of HJURP expression (variants 1,2 and 3). We also observed that variant (1) is more abundant in the cell lines. Albeit, there was an overall increase of all variants in tumor samples when compared to the brain white matter. We demonstrated that variant (2) is more prevalent than variant (1) in non-tumoral tissues and low-grade astrocytomas (AST2). In contrast, anaplastic astrocytoma (AST3) and GBM showed a considerable increase in variant (1) levels, arising a characteristic profile of tumor progression. Expression analyses of a panel of tumor and non-tumoral cell lines from distinct origins confirmed our previously described finding. Recent data demonstrated that transcript (1) silencing of U87MG and T98G may stimulate transcrip (2) expression and it shows the low impact on cell viability when compared to total HJURP silencing. These data suggest that variants (2) and (3) are functional and assist in GBM cell viability maintenance. Keywords: Glioblastoma. HJURP. Transcripts.Isoforms.
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Glioblastoma, HJURP, Transcritos, Isoformas, Transcripts, Isoforms