Diagnostic significance of urinary long non-coding PCA3 RNA in prostate cancer

Abstract

Prostate cancer antigen 3(PCA3) is a long non-coding RNA, which was found increased expression in CaP patients than healthy individual. In this study, the individual nucleic acid of PCA3 and PSA was recombinant expressed as a reference reagent, and a quantitative RT-PCR with TaqMan assay was developed to examine the copies of PCA3 and PSA gene in urine. The results showed that the area under the receiver operating characteristic curve (AUROC) was 0.717, 0.444 and 0.916 for the number of PCA3 copy, PSA copy and for the score of PCA3/PSA RNA, respectively. Additionally, the AUROC for serum tPSA was 0.674 with a low specificity of 12.07%. Finally, the algorithm of PCA3 RNA versus PSA RNA was evaluated and corroborated as CaP biomarker by conducting a multicentric clinical trial. This study not only validated the developed technique of qRT-PCR with TaqMan assay for examination of urinary PCA3 and PSA RNA, it also demonstrated that the score of the PCA/PSA RNA was a reliable signature for CaP diagnosis.

Keywords: cell-free; diagnostic signature; prostate cancer; prostate cancer antigen 3; prostate-specific antigen.

Figure 1. Amplification of PCA3 fragments

The prostatic tissue from individual was collected and snap frozen immediately in -80°C. Total RNA was isolated and subjected to RT-PCR assay. Data was represented as a ratio of PCA3 RNA versus PSA RNA.

Figure 2. Recombinant expression of PCA 3 and PSA genes

Gene clone, plasmid construction and transcription in vitro were conducted to obtain individual PCA3 RNA and PSA RNA. The quality of isolated RNAs of PCA3(A) and PSA(B) were analyzed and confirmed using Agilent 2100 Bioanalyzer (USA).

Figure 3. Generation of PCA3 and PSA standard curve

(A) Recombinant expressed PCA3 RNA was serially diluted by ten-fold from 1.25 × 10⁷ to 1.25x 10³ (copy/ml). A qRT-PCR with TaqMan assay was conducted. The amplification curve (a) and the standard curve (b) were generated and presented. The PCA3 amplification efficiency was101.5%, the slope was calculated as -3.286, R² =1.00. (B) The similar procedure described as above was performed for PSA. PSA amplification curve (a) and the standard curve (b) were also generated and presented. The amplification efficiency was 113.6%, the slope was -3.034, and R²=1.

Figure 4. The receiver operating characteristic curve (ROC) for the copy of urinary PCA3 gene, the copy of urinary PSA gene and the score of urinary PCA3 / PSA RNA ratio (PCA3/PSA) in intended use of specimens (n=109)

Figure 5. The receiver operating characteristic curve (ROC) for the score of urinary PCA3/PSA RNA ratio and serum tPSA in intended use of specimens (n=88)