OncomiR detection in circulating body fluids: a PDMS microdevice perspective.

There is an increasing interest in circulating microRNAs (miRNAs) as potential minimally invasive diagnostic biomarkers in oncology. Considerable efforts are being made in the development of lab-on-a-chip devices for biomedical applications to purify and detect miRNAs from biological fluids. Here, we report the development of an innovative polydimethylsiloxane (PDMS)-based parallel device whose internal surface can opportunely be functionalized with positively charged 3-aminopropyltriethoxysilane (APTES) alone or mixed with two different neutral poly(ethylene glycol) silanes (PEG-s). The differently functionalized internal surfaces of the PDMS chip were characterized with s-SDTB (sulfosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate) and the portion of the surface able to adsorb a synthetic fluorescently labeled miRNA was determined. Interestingly, the adsorbed miRNA (both synthetic and cell supernatant-derived) was found mainly on the bottom surface of the chip and could be reverse transcribed into cDNA directly on the same PDMS chip used for its purification, saving hours with respect to the use of standard purification kits. We identified 0.1% APTES/0.9% PEG-silane as the most efficient PDMS functionalization to capture both synthetic and extracellular miRNA. Moreover, the amount of captured miRNA was increased by treating the cell supernatant with a commercially available lysis buffer for RNA extraction. We assessed that the available miRNA binding sites on the functionalized surface were efficiently saturated with only one incubation, shortening the time and greatly simplifying the protocol for miRNA purification from biological samples. Finally, the extracellular miRNA purification efficiency of the PDMS functionalized multichip determined via real-time quantitative polymerase chain reaction (RT-qPCR) was confirmed by droplet digital PCR (ddPCR) quantification. This work shows an innovative, rapid and easy to use microdevice for the purification and reverse transcription of circulating miRNAs, approaching the realization of diagnostic and prognostic oncomiR-based assays.

Effect of the HDAC inhibitor vorinostat on the osteogenic differentiation of mesenchymal stem cells in vitro and bone formation in vivo.

AIM: Vorinostat, a histone deacetylase (HDAC) inhibitor currently in a clinical phase III trial for multiple myeloma (MM) patients, has been reported to cause bone loss. The purpose of this study was to test whether, and to what extent, vorinostat influences the osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and bone formation in vivo. METHODS: Bone marrow-derived MSCs were prepared from both normal donors and MM patients. The MSCs were cultured in an osteogenic differentiation induction medium to induce osteogenic differentiation, which was evaluated by alkaline phosphatase (ALP) staining, Alizarin Red S staining and the mRNA expression of osteogenic markers. Naïve mice were administered vorinostat (100 mg/kg, ip) every other day for 3 weeks. After the mice were sacrificed, bone formation was assessed based on serum osteocalcin level and histomorphometric analysis. RESULTS: Vorinostat inhibited the viability of hMSCs in a concentration-dependent manner (the IC50 value was 15.57 µmol/L). The low concentration of vorinostat (1 µmol/L) did not significantly increase apoptosis in hMSCs, whereas pronounced apoptosis was observed following exposure to higher concentrations of vorinostat (10 and 50 µmol/L). In bone marrow-derived hMSCs from both normal donors and MM patients, vorinostat (1 µmol/L) significantly increased ALP activity, mRNA expression of osteogenic markers, and matrix mineralization. These effects were associated with upregulation of the bone-specifying transcription factor Runx2 and with the epigenetic alterations during normal hMSCs osteogenic differentiation. Importantly, the mice treated with vorinostat did not show any bone loss in response to the optimized treatment regimen. CONCLUSION: Vorinostat, known as a potent anti-myeloma drug, stimulates MSC osteogenesis in vitro. With the optimized treatment regimen, any decrease in bone formation was not observed in vivo.