The Study Of Long Non-coding RNA As A Novel Plasma Biomarker For Early Diagnosis Of Prostate Cancer

Objective To determine whether lncRNAs derived from plasma can serve as apotential biomarker for prostate cancer (PCa) detection by means of the existence andstability of plasma lncRNA, to explore the source of plasma lncRNA and itsfragments through cellular, xenograft and clinical experiments, and to explore the roleof using MALAT-1derived (MD) miniRNA as a biomarker for PCa detection usingclinical samples.Methods The study was divided into three parts:(1) the existence pattern ofplasma lncRNA was studied in8PCa plasma samples; primers for amplicons thatwere found every1000bp over the complete transcript of PCA3and MALAT-1weredesigned, and all of the PCA3and MALAT-1fragments expression levels weredetermined from8PCa plasma samples. The characteristics of PCa-related lncRNAfragments were systematically studied in plasma or serum of25patients; the plasmawas treated under harsh circumstances including multiple freeze-thaw cycles,incubation at room temperature for up to24h, incubation at minus80degrees,acid-base treatment, and RNAses.(2) The source of the circulating MD-miniRNA wasexplored in vitro and in vivo.6human prostate cell lines (PWR-1E,22RV1,LNCap-AD, LNCap-AI, C4-2, and PC3) were selected and the MD-miniRNA plasmalevel was assessed in the cell culture medium which was collected1day,2days and3days after cell passage to demonstrate that the MD-miniRNA was secreted by PCa celllines. A mouse subcutaneous22RV1xenograft model system was used and theMD-miniRNA plasma level was assessed in the blood samples which were collectedonce the tumors were well established to demonstrate that the presence of cancer canlead to a significant increase in plasma MD-miniRNA expression. The MD-miniRNAplasma level was assessed in PCa patients (n=10) before (pre-Op) and7days after (7days post-Op) surgical removal of the tumor to indicate that plasma MD-miniRNA isderived from prostate tumors and was correlated with serum PSA levels.(3) thediagnostic performance of MALAT-1derived (MD) miniRNA was further validated inan independent cohort of192patients, including87PCa plasma samples (positive biopsy),82BPH plasma samples (negative biopsy) and23healthy controls (PSA<4ng/ml). The expression levels of lncRNAs and their fragments were measured byqRT-PCR. The MD-miniRNA copies were calculated using a standard curve. AnAUC-ROC analysis was applied to assess the predictive power.Results Genome-wide profiling revealed that MALAT-1and DD3are markedlyoverexpressed in PCa tissues. Plasma lncRNAs probably exist in the form offragments with different expression levels, and LncRNA fragments are present inhuman plasma in a remarkably stable form. MD-miniRNA, the MALAT-1fragmentswith the highest plasma expression levels, enters cell culture medium at levelsadequate to be measurable, and MD-miniRNA derived from human PCa xenograftsactually enters the circulation, which can be readily measured in plasma and canobviously distinguish xenografted mice from controls. In addition, plasmaMD-miniRNA levels are significantly elevated in PCa patients compared to non-PCapatients (p<0.0001). At a cut-off of532MD-miniRNA copies per microliter of plasma,the sensitivity is58.6%,58.6%,43.5%and the specificity is84.8%,84.8%,81.6%in discriminating PCa from non-PCa, positive biopsy from negative biopsy(PSA>4ng/ml) and positive biopsy from negative biopsy (PSA=4-10ng/ml),respectively.Conclusion We conclude that MD-miniRNA can serve as a novel plasma-basedbiomarker for PCa detection and can improve diagnostic accuracy by predictingprostate biopsy outcomes. Further large-scale studies are needed to confirm ourfindings.