Desenvolvimento e caracterização de aptâmeros ligantes específicos ao gene PCA3 e implicações na modulação da expressão gênica de células tumorais transfectadas

Abstract

CHAPTER II:The prostate cancer antigen 3 (PCA3) mRNA expression has been used as a prostate cancer (PCa) biomarker in tissue and urine clinical samples, but its function is unknown. Analysis of the PCA3 sequence has predicted a significant tertiary configuration based on a complex double-stranded fold-back structure formed through the complementary base pairing of highly repetitive sequences. RNA aptamers were selected against the partial structure of the PCA3 transcript encompassing exons 1, 3 and 4. Six PCA3-ligand aptamers with high affinity were selected and one of the most repetitive aptamer motifs was reduced and synthesized for validation. The novel biotinylated RNA aptamer (CG3) was used to detect the transcript on prostate histopathological sections in tissue microarrays (TMA) through immunohistochemical analysis. Biotin-labeled CG3 aptamer stained the cytoplasm and nucleus of PCa and BPH tissue and is absent in stromal cells, corroborating previous findings of the PCA3 RNA cellular localization. Additional immunoassays provided final evidences that the CG3 aptamer has specific binding to the PCA3 transcript, and may be used as an auxiliary biomarker in PCa diagnosis. Its detection in the peripheral blood and interference in the transcript function still remain to be demonstrated. CHAPTER III: Currently, the non-coding prostate cancer antigen 3 (PCA3) is one of the most specific and overexperessed prostate cancer (PCa) genes, and considered a potential biomarker with unknown function. It forms hairpin-loop structures that may be required either for editing or processing. We hypothesized that disruption of its secundary strucuture by an RNA aptamer ligand probably modulates the expression of genes that may be associated with PCA3 and consequently with PCa development. An RNA aptamer with high affinity to PCA3 (CG3) was transfected into LNCaP and PC3 cell lines. A reverse binding assay and immunhistochemistry with tissue microarrays confirmed its specificity to the PCA3 transcript and to PCa cells, respectively. Differential gene expression profiles of both transfected cells suggest a multigenic modulation by the PCA3. PCa and benign prostatic hyperplasia patients were further evaluated for the PCA3 expression in the prostatic tissue, and besides confirming its diagnostic potential (AUC=0.80), it showed a similar pattern to the LNCaP cell. Finally, the opposite behavior of prostate cancer cell lines indicates that PCA3, AR and TLR signaling pathways genes are connected and their combination may be responsible for the tumor development and the transition from the hormonal to the refractory response profile.