Tumor Biology

, Volume 37, Issue 8, pp 11339–11348 | Cite as

PCA3 long noncoding RNA modulates the expression of key cancer-related genes in LNCaP prostate cancer cells

  • Ana Emília Goulart Lemos
  • Luciana Bueno Ferreira
  • Nadia Maria Batoreu
  • Paula Priscilla de Freitas
  • Martin Hernan Bonamino
  • Etel Rodrigues Pereira Gimba
Original Article

Abstract

Prostate cancer antigen 3 (PCA3) is a prostate-specific long noncoding RNA (lncRNA) involved in the control of prostate cancer (PCa) cell survival, through modulating androgen receptor (AR) signaling. To further comprehend the mechanisms by which PCA3 modulates LNCaP cell survival, we characterized the expression patterns of several cancer-related genes, including those involved in epithelial-mesenchymal transition (EMT) and AR cofactors in response to PCA3 silencing. We also aimed to develop a strategy to stably silence PCA3. Small interfering RNA (siRNA) or short hairpin RNA (shRNA) was used to knock down PCA3 in LNCaP cells. The expression of 84 cancer-related genes, as well as those coding for AR cofactors and EMT markers, was analyzed by quantitative real-time PCR (qRT-PCR). LNCaP-PCA3 silenced cells differentially expressed 16 of the 84 cancer genes tested, mainly those involved in gene expression control and cell signaling. PCA3 knockdown also induced the upregulation of several transcripts coding for AR cofactors and modulated the expression of EMT markers. LNCaP cells transduced with lentivirus vectors carrying an shRNA sequence targeting PCA3 stably downregulated PCA3 expression, causing a significant drop (60 %) in the proportion of LNCaP cells expressing the transgene. In conclusion, our data provide evidence that PCA3 silencing modulates the expression of key cancer-related genes, including those coding for AR cofactors and EMT markers. Transducing LNCaP cells with an shRNA sequence targeting PCA3 led to loss of viability of the cells, supporting the proposal of PCA3 knockdown as a putative therapeutic approach to inhibit PCa growth.

Keywords

PCA3 Long noncoding RNA Gene expression Cancer-related genes Prostate cancer 

Notes

Acknowledgments

This study was supported by grants from the Fundação de Amparo à Pesquisa do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Instituto Nacional de Ciência e Tecnologia (INCT) para Controle do Câncer, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Ministério da Saúde (MS)/Fundação do Câncer, Instituto Nacional de Câncer (INCa), Fundação Oswaldo Cruz (FIOCRUZ)/Bio-Manguinhos, and Swiss Bridge Foundation. We are especially grateful to Dr. Marcos Freire and Dr. Marco Medeiros for kindly supporting this study. We also thank Dr. Marcelo Ribeiro Alves for his kind support with statistical analyses.

Conflicts of interest

None

Supplementary material

13277_2016_5012_MOESM1_ESM.pdf (222 kb)
Online Resource 1 Oligonucleotide sequences used for EMT marker and AR cofactor expression level analysis by qRT-PCR. β-actin was used as the constitutive gene to normalize gene expression (PDF 222 kb)
13277_2016_5012_MOESM2_ESM.pdf (191 kb)
Online Resource 2 PCA3 shRNA and Scramble shRNA sequences used for PCA3 stable downregulation. A specific shRNA sequence for PCA3 was designed based on the siPCA3 sequence that was used for the previously described siRNA experiments. Similarly, the scrambled shRNA sequence was designed based on the siScrbl sequence (PDF 191 kb)
13277_2016_5012_MOESM3_ESM.pdf (156 kb)
Online Resource 3 Real-time PCR validation assays to demonstrate PCA3 downregulation. Relative PCA3 expression was analyzed by qRT-PCR after transfecting LNCaP cells with siPCA, when compared to cells transfected to siScrbl, is represented on the bar graphs. Data shown represent biological replicates: (a) PCA3 silencing for PCR array assays (b) PCA3 silencing for assays to evaluate EMT makers and AR coregulators (PDF 156 kb)
13277_2016_5012_MOESM4_ESM.pdf (21 kb)
Online Resource 4 Genes differentially expressed in response to PCA3 downregulation in LNCaP cells. Multiple genes related to DNA damage repair, apoptosis, signal transduction and gene regulation, cell adhesion, angiogenesis, invasion, and metastasis were evaluated for expression levels using the RT2 Profiler PCR Array system. This table lists genes that showed significant delta CT (p < 0.05) values and genes with at least a 2.0-fold change in expression levels in LNCaP cells in response to PCA3 downregulation, in relation to siPCA3-transfected LNCaP cells. The functional class of each gene is shown in this additional file. Positive values indicate up-regulation, and negative values indicate down-regulation of individual genes. The data were evaluated by two-tailed Student’s t test (PDF 21 kb)
13277_2016_5012_MOESM5_ESM.pdf (374 kb)
Online Resource 5 Representative FACS dot plots of GFP expression 82 days after LNCaP cell transduction. Flow-cytometry analyses for percentage of GFP-positive cells after LNCaP cell transduction with a lentiviral empty vector, or a lentiviral vector containing a shPCA3 sequence or a shScrbl PCA3 sequence were performed with the software CellQuest (BD Biosciences) or with FLOWJO. A dot plot of non-transduced LNCaP cells (LNCaP wild-type) is also provided (PDF 373 kb)
13277_2016_5012_MOESM6_ESM.pdf (5.4 mb)
Online Resource 6 Percentage of LNCaP GFP+ cells obtained using an inverted fluorescence microscope. Total numbers of LNCaP cells (red) and GFP+ cells (green) counted in five different gates of images were obtained 72 h after LNCaP cell transduction with lentiviral vector containing an shPCA3 and a GFP sequence, using an inverted fluorescence microscope. The Adobe Photoshop CS6 program was used for counting (PDF 5562 kb)

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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Ana Emília Goulart Lemos
    • 1
  • Luciana Bueno Ferreira
    • 2
  • Nadia Maria Batoreu
    • 1
  • Paula Priscilla de Freitas
    • 3
    • 4
  • Martin Hernan Bonamino
    • 4
    • 5
  • Etel Rodrigues Pereira Gimba
    • 4
    • 6
  1. 1.Fundação Oswaldo CruzRio de JaneiroBrazil
  2. 2.Instituto de Patologia Molecular e Imunologia da Universidade do PortoPortoPortugal
  3. 3.Fundação Centro Universitário Estadual da Zona OesteRio de JaneiroBrazil
  4. 4.Instituto Nacional de Câncer José Alencar Gomes da Silva, Coordenação de PesquisaRio de JaneiroBrazil
  5. 5.Fundação Oswaldo Cruz, Vice-presidência de Pesquisa e Laboratórios de ReferênciaRio de JaneiroBrazil
  6. 6.Universidade Federal FluminenseInstituto de Humanidades e Sáude, Departamento de Ciências da NaturezaRio de JaneiroBrazil

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