RT-qPCR based screening of blood-circulating microRNAs for the identification of biomarkers and novel drug targets

Abstract
Introduction: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and are known to take part in the control of various biological processes. Recently, miRNAs have been identified to be secreted into the bloodstream from cells of various tissues, thus, possibly indicating local pathological processes. Therefore, there is a growing interest in circulating miRNAs as minimal-invasive diagnostic targets or as potential targets for drug development.
Methods: We have implemented a commercial LNA-based RT-qPCR method for screening of hundreds of miRNAs in cell-free blood samples. Pre-analytical variability in terms of sample storage, type, and freeze/thawing was assessed, and analytical variability was controlled using a combination of 5 spike-in controls added at various stages during the analysis workflow. Based on pilot studies, a discovery panel for circulating miRNAs analysis with 375 primer pairs was designed and used in the course of two research studies.
Results: The first case study was designed to identify changes in circulating miRNAs in response to recent osteoporotic fractures at the hip, a major health burden in aging populations of developed countries. In an exploratory analysis of 7 fracture and 7 control patients, six miRNAs with significant (adj. p < 0.05) changes in serum levels were identified. Clustering and ROC analyses showed that these miRNA together provided excellent discrimination of case and control samples. Subsequent validation in an independent cohort of 23 patients (11 control, 12 fracture) confirmed significant regulation for three miRNAs. These miRNAs were selected for in vitro testing using an in-house developed bioassay for bone formation, in order to evaluate correlative or causative relation to fracture-risk. The second case study aimed to identify circulating miRNAs specific to elite HIV controllers. These patients are known to maintain HIV-1 viral loads below the limit of detection, albeit being infected with HIV-1. For that purpose EDTA-Plasma from HIV elite controllers (n=9) and chronically infected individuals (n=9) was compared against healthy donors (n=6). Unsupervised cluster analysis and statistical comparison identified strong difference in circulating miRNA patterns between chronic HIV infected patients and healthy controls, while no differential expression between elite patients and healthy controls could be observed. Three miRNAs, hsa-miR-29b-3p, hsa-miR-33a-5p and hsa-miR-146a-5p were found to be significantly higher in plasma from elite controllers than chronic infected and therefore selected for in vitro testing. The results clearly show that hsa-miR-29b-3p and hsa-miR-33a-5p overexpression reduced the viral production in MT2 and primary T CD4+ cells. Conclusion: These data clearly show that the analysis of circulating miRNAs using a robust RT-qPCR pipeline is not only suitable for biomarker identification but also a viable approach for drug target identification.