All analyses were performed using SPSS software, version 13

All analyses were performed using SPSS software, version 13.0 (IBM Corporation, Armonk, NY, USA). Results Expression patterns of miR-193b-3p and HDAC3 during hMSCs chondrogenesis hMSCs were induced to differentiate to chondrocytes were observed during the chondrogenic differentiation of hMSCs at 14-28 days, a finding that suggests miR-193b-3p may affect expression (Figure ?Physique1A-B,1A-B, I-J). nude mice, we found that miR-193b overexpression strongly enhanced cartilage formation compared to that found under control conditions. We also found that patients with OA had lower plasma exosomal miR-193b levels than control subjects. Conclusions: These findings indicate that miR-193b-3p directly targets HDAC3, promotes H3 acetylation, and regulates hMSC chondrogenesis and metabolism in PHCs. for 15 min), plasma was divided into aliquots and stored at -80 C until analyzed. All plasma samples were obtained prior to Kv3 modulator 2 any treatment and were analyzed within 3 months. Exosomes were isolated from 4 mL human plasma. Nanoparticle-tracking analysis (NTA) and transmission electron microscopy (TEM) were used to identify exosomes. Exosomal RNA was extracted using an miRNeasy Serum/Plasma Kit (Qiagen), and miR-39 was used as a reference gene according to the manufacturer’s instructions. Proteins were extracted from exosomes using a Total Exosome Protein Isolation Kit (Invitrogen, Carlsbad, USA) for further analysis. The experimental details are described in Supplementary Material. RNA extraction, reverse transcription, and qRT-PCR Cartilage and cell-seeded scaffolds were ground in liquid nitrogen prior to RNA isolation. Total RNA from cells, cartilage samples, and cell-seeded scaffolds was extracted using a miRNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Next, cDNA was synthesized Rabbit Polyclonal to MLH3 from miRNA and mRNA using a Mir-X? miRNA First-Strand Synthesis Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) and a PrimeScript? RT Grasp Mix (Takara, Shiga, Japan), respectively. qRT-PCR of target genes was performed using SYBR? Premix Ex Taq? II (Takara) and a CFX96 real-time qPCR instrument (Bio-Rad, Hercules, CA, USA), Kv3 modulator 2 according to the manufacturer’s instructions. Transcript levels were normalized to that of Kv3 modulator 2 the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for mRNA, the small U6 RNA for miRNA, or miR-39 for exosomal miRNA. The specific primers used for these analyses are shown in Supplementary Material. The mRQ 3 Primer (Clontech) was used as the reverse primer for Kv3 modulator 2 miRNA-193b-3p, and the miR-39 primer was supplied in the miRNeasy Serum/Plasma Kit. Gene expression was calculated using the 2-Ct method, and each experiment was performed in triplicate. Western blot analysis Western blotting of was performed as described previously 17. Total protein was isolated from hMSCs and PHCs. Thirty micrograms of protein from each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes Kv3 modulator 2 were incubated with primary antibodies specific for HDAC3, SOX9 (1:1,000 dilution, Cell Signaling Technology, Boston, MA, USA), COL2A1, AGGRECAN, MMP-13 (1:2,000 dilution, Abcam, Cambridge, MA, USA) acetylated histone H3 (ac-H3), total histone H3 (H3) (1:1,500 dilution, Millipore, Darmstadt, Germany), CD63, CD9 (1:1,000 dilution, System Biosciences, Palo Alto, CA, USA), and GAPDH (1:3,000 dilution, Cell Signaling Technology). The blots were then incubated with appropriate secondary antibodies conjugated with horseradish peroxidase (1:3,000 dilution, Cell Signaling Technology) at room temperature (22-26 oC) for 1 h, after which they were developed with an ECL Chemiluminescence Kit (Santa Cruz Biotech, Santa Cruz, CA, USA). Quantitative data were expressed by normalizing the densitometric units to the reference gene using Image J (http://imagej.nih.gov/ij/). Transfection of small-interfering RNA (siRNA) molecules, and miR-193b-3p mimics and inhibitors hMSCs and PHCs were transfected with an agomiR (50 nM) or an antagomiR (100 nM) (RiboBio, Guangzhou, China) of miR-193b-3p. PHCs were also transfected with siHDAC3 (100 nM) or siNC (RiboBio). Lipofectamine? 2000 Transfection Reagent (Life Technologies, Carlsbad, CA, USA) was used to transfect cells, according to the manufacturer’s instructions. Non-specific microRNA (miR-Control and anti-miR-Control; RiboBio) was used as a control. RNA-free nuclease water was used as a blank. For chondrogenic differentiation of hMSCs by micromass culture, hMSC monolayers were transfected twice, first on the day after plating and again after 3 days. hybridization, immunohistochemistry, and histology staining Samples were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA), decalcified (in the case of samples from human cartilage), embedded in paraffin, and cut into 5-m sections that were deparaffinized, rehydrated, and stained with Alcian blue and Safranin O to visualize the glycosaminoglycan (GAG) distribution. For human cartilage samples, following Safranin O staining, cartilage destruction was blindly scored by.