Diversity and signature of small RNA in different bodily fluids using next generation sequencing.

BACKGROUND: Small RNAs are critical components in regulating various cellular pathways. These molecules may be tissue-associated or circulating in bodily fluids and have been shown to associate with different tumors. Next generation sequencing (NGS) on small RNAs is a powerful tool for profiling and discovery of microRNAs (miRNAs). RESULTS: In this study, we isolated total RNA from various bodily fluids: blood, leukocytes, serum, plasma, saliva, cell-free saliva, urine and cell-free urine. Next, we used Illumina's NGS platform and intensive bioinformatics analysis to investigate the distribution and signature of small RNAs in the various fluids. Successful NGS was accomplished despite the variations in RNA concentrations among the different fluids. Among the fluids studied, blood and plasma were found to be the most promising fluids for small RNA profiling as well as novel miRNA prediction. Saliva and urine yielded lower numbers of identifiable molecules and therefore were less reliable in small RNA profiling and less useful in predicting novel molecules. In addition, all fluids shared many molecules, including 139 miRNAs, the most abundant tRNAs, and the most abundant piwi-interacting RNAs (piRNAs). Fluids of similar origin (blood, urine or saliva) displayed closer clustering, while each fluid still retains its own characteristic signature based on its unique molecules and its levels of the common molecules. Donor urine samples showed sex-dependent differential clustering, which may prove useful for future studies. CONCLUSIONS: This study shows the successful clustering and unique signatures of bodily fluids based on their miRNA, tRNA and piRNA content. With this information, cohorts may be differentiated based on multiple molecules from each small RNA class by a multidimensional assessment of the overall molecular signature.

Potential Urinary miRNA Biomarker Candidates for the Accurate Detection of Prostate Cancer among Benign Prostatic Hyperplasia Patients.

MicroRNAs (miRNAs) are a class of short (~22nt), single stranded RNA molecules that function as post-transcriptional regulators of gene expression. MiRNAs can regulate a variety of important biological pathways, including: cellular proliferation, differentiation and apoptosis. Profiling of miRNA expression patterns was shown to be more useful than the equivalent mRNA profiles for characterizing poorly differentiated tumours. As such, miRNA expression "signatures" are expected to offer serious potential for diagnosing and prognosing cancers of any provenance. The aim of this study was to investigate the potential of using deregulation of urinary miRNAs in order to detect Prostate Cancer (PCa) among Benign Prostatic Hyperplasia (BPH). To identify the miRNA signatures specific for PCa, miRNA expression profiling of 8 PCa patients, 12 BPH patients and 10 healthy males was carried out using whole genome expression profiling. Differential expression of two individual miRNAs between healthy males and BPH patients was detected and found to possibly target genes related to PCa development and progression. The sensitivity and specificity of miR-1825 for detecting PCa among BPH individuals was found to be 60% and 69%, respectively. Whereas, the sensitivity and specificity of miR-484 were 80% and 19%, respectively. Additionally, the sensitivity and specificity for miR-1825/484 in tandem were 45% and 75%, respectively. The proposed PCa miRNA signatures may therefore be of great value for the accurate diagnosis of PCa and BPH. This exploratory study has identified several possible targets that merit further investigation towards the development and validation of diagnostically useful, non-invasive, urine-based tests that might not only help diagnose PCa but also possibly help differentiate it from BPH.

Potential Urinary Protein Biomarker Candidates for the Accurate Detection of Prostate Cancer among Benign Prostatic Hyperplasia Patients.

Globally, Prostate cancer (PCa) is the most frequently occurring non-cutaneous cancer, and is the second highest cause of cancer mortality in men. Serum prostate specific antigen (PSA) has been the standard in PCa screening since its approval by the American Food & Drug Administration (FDA) in 1994. Currently, PSA is used as an indicator for PCa - patients with a serum PSA level above 4ng/mL will often undergo prostate biopsy to confirm cancer. Unfortunately fewer than ~30% of these men will biopsy positive for cancer, meaning that the majority of men undergo invasive biopsy with little benefit. Despite PSA's notoriously poor specificity (33%), there is still a significant lack of credible alternatives. Therefore an ideal biomarker that can specifically detect PCa at an early stage is urgently required. The aim of this study was to investigate the potential of using deregulation of urinary proteins in order to detect Prostate Cancer (PCa) among Benign Prostatic Hyperplasia (BPH). To identify the protein signatures specific for PCa, protein expression profiling of 8 PCa patients, 12 BPH patients and 10 healthy males was carried out using LC-MS/MS. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR. This approach revealed that significant the down-regulation of Fibronectin and TP53INP2 was a characteristic event among PCa patients. Fibronectin mRNA down-regulation, was identified as offering improved specificity (50%) over PSA, albeit with a slightly lower although still acceptable sensitivity (75%) for detecting PCa. As for TP53INP2 on the other hand, its down-regulation was moderately sensitive (75%), identifying many patients with PCa, but was entirely non-specific (7%), designating many of the benign samples as malignant and being unable to accurately identify more than one negative.