Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition

MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies

Gender differences in gait kinematics for patients with knee osteoarthritis

BACKGROUND: Females have a two-fold risk of developing knee osteoarthritis (OA) as compared to their male counterparts and atypical walking gait biomechanics are also considered a factor in the aetiology of knee OA. However, few studies have investigated sex-related differences in walking mechanics for patients with knee OA and of those, conflicting results have been reported. Therefore, this study was designed to examine the differences in gait kinematics (1) between male and female subjects with and without knee OA and (2) between healthy gender-matched subjects as compared with their OA counterparts. METHODS: One hundred subjects with knee OA (45 males and 55 females) and 43 healthy subjects (18 males and 25 females) participated in this study. Three-dimensional kinematic data were collected during treadmill-walking and analysed using (1) a traditional approach based on discrete variables and (2) a machine learning approach based on principal component analysis (PCA) and support vector machine (SVM) using waveform data. RESULTS: OA and healthy females exhibited significantly greater knee abduction and hip adduction angles compared to their male counterparts. No significant differences were found in any discrete gait kinematic variable between OA and healthy subjects in either the male or female group. Using PCA and SVM approaches, classification accuracies of 98–100 % were found between gender groups as well as between OA groups. CONCLUSIONS: These results suggest that care should be taken to account for gender when investigating the biomechanical aetiology of knee OA and that gender-specific analysis and rehabilitation protocols should be developed