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Caracterização dos diferentes transcritos produzidos pelo locus HJURP (HollidayJunctionRecognizing-Protein) em células de glioblastoma

  • Petitto Netto, Renato
Publication Date
Jan 30, 2018
Repositório Institucional UNESP
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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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