ABSTRACT
BACKGROUND: MicroRNAs (miRNAs or miRs) serve crucial roles in the progression of osteoporosis. This study investigated the role and specific molecular mechanism of miR-135-5p in regulating osteoblast differentiation and calcification. METHODS: Bone morphogenetic protein 2 (BMP2) was employed to interfere with the differentiation of MC3T3-E1. Then, miR-135-5p mimic or miR-135-5p inhibitor was transfected into MC3T3-E1, and quantitative RT-PCR was used to measure the expression of miR-135-5p. The expressions of runt-related transcription factor 2 (Runx2), osterix (OSX), osteopontin (OPN), and osteocalcin (OCN) were determined using western blot. Alkaline phosphatase (ALP) activity was measured using an appropriate kit assay. Calcium nodule staining was evaluated with alizarin red staining. A luciferase reporter assay was used to verify the target of miR-135-5p. Hypoxia-inducible factor 1 α inhibitor (HIF1AN) overexpression was applied to investigate its own role in the mechanism and a miR-135-5p rescue experiment was also performed. RESULTS: Overexpression of miR-135-5p promoted osteogenic differentiation and calcification, as shown by the increase in ALP activity, calcification and osteogenic marker levels, including Runx2, OSX, OPN and OCN. Knockdown of miR-135-5p yielded the opposite results. HIF1AN was confirmed as a direct target of miR-135-5p. HIF1AN overexpression inhibited osteogenic differentiation and calcification while miR-135-5p reversed these effects. CONCLUSIONS: These results indicate that miR-135-5p might have a therapeutic application related to its promotion of bone formation through the targeting of HIF1AN.
Subject(s)
MicroRNAs/genetics , Mixed Function Oxygenases/genetics , Osteoblasts/cytology , Osteogenesis , Animals , Cell Differentiation , Cell Line , Gene Expression Regulation, Developmental , Mice , Osteoblasts/metabolism , Up-RegulationABSTRACT
The novel supramolecular complexes were prepared with a water-insoluble anticancer drug camptothecin (CPT) loading onto functionalized multiwalled carbon nanotubes via pi-stacking, in order to improve their solubility and antitumor activity. The multiwalled carbon nanotubes were firstly coated with the tri-block copolymer (Pluronic P123) to render high aqueous solubility. The copolymer-coated multiwalled carbon nanotubes can effectively form non-covalent supramolecular complexes with camptothecin. The supramolecular assembly of the complexes (f-MWNTs-CPT) were systematically characterized by transmission electron microscopy (TEM), UV-vis spectrophotometry (UV), fluorescence spectrophotometry, atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). Furthermore, in vitro cytotoxicity studies of f-MWNTs-CPT supramolecular complexes using the MTT assay exhibit enhanced antitumor activity, suggesting that the functionalized multiwalled carbon nanotubes can facilitate intracellular delivery of anticancer drug and improve drug activity.
Subject(s)
Antineoplastic Agents, Phytogenic/administration & dosage , Camptothecin/administration & dosage , Nanotubes, Carbon/chemistry , Nanotubes, Carbon/ultrastructure , Drug Delivery Systems , Electrochemical Techniques , HeLa Cells , Humans , Macromolecular Substances/chemistry , Macromolecular Substances/ultrastructure , Microscopy, Atomic Force , Microscopy, Confocal , Microscopy, Electron, Transmission , Models, Molecular , Nanomedicine , Particle Size , Poloxalene , Solubility , Surface PropertiesABSTRACT
The chitosan-coated magnetic nanoparticles (CS MNPs) were prepared as carriers of 5-Fluorouracil (CS-5-Fu MNPs) through a reverse microemulsion method. The characteristics of CS-5-Fu MNPs were determined by using transmission electron microscopy (TEM), FTIR spectroscopy and vibrating-sampling magnetometry (VSM). It was found that the synthesized CS-5-Fu MNPs were spherical in shape with an average size of 100+/-20 nm, low aggregation and good magnetic responsivity. Meanwhile, the drug content and encapsulation rate of the nanoparticles was 16-23% and 60-92%, respectively. These CS-5-Fu MNPs also demonstrated sustained release of 5-Fu at 37 degrees C in different buffer solutions. The cytotoxicity of CS-5-Fu MNPs towards K562 cancer cells was investigated. The result showed that CS-5-Fu MNPs retained significant antitumor activities. Additionally, it was observed that the FITC-labeled CS-5-Fu MNPs could effectively enter into the SPCA-1 cancer cells and induced cell apoptosis.
Subject(s)
Chitosan/chemistry , Fluorouracil/pharmacology , Magnetics , Nanoparticles/chemistry , Apoptosis/drug effects , Cell Line, Tumor , Drug Carriers/chemistry , Fluorouracil/chemistry , HumansABSTRACT
The purpose of this study was to synthesize the complex of magnetic nanoparticles and antibody, and to apply them to isolate the CD34(+) cells from umbilical cord blood, then to evaluate its separation efficiency. The complex of magnetic nanoparticles and antibody was used to separate CD34(+) cells from umbilical cord blood in the outer magnetic field because of its superparamagnetism, specific identification and function of combination with CD34(+) cells. Scanning electron microscopy was used to observe the morphology of the separated CD34(+) cells. Flow Cytometer was applied to evaluate the sorting efficiency of magnetic nanoparticles, liquid culture and colony culture were taken to assay proliferation and differentiation capacity of the separated CD34(+) cells. The results showed that the CD34(+) cells from umbilical cord blood were isolated fast and effectively by the complex of magnetic nanoparticles and monoclonal antibody. Moreover, the isolated CD34(+) cells still maintained its normal morphology, highly proliferative and differentiative capacity. It is concluded that the complex of magnetic nanoparticles and monoclonal antibody has been successfully synthesized and developed as a technique which efficiently and quickly isolates CD34(+) cells from umbilical cord blood.
Subject(s)
Antigens, CD34/metabolism , Fetal Blood/cytology , Hematopoietic Stem Cells/cytology , Immunomagnetic Separation/methods , Flow Cytometry , Hematopoietic Stem Cells/immunology , Humans , Magnetics , NanoparticlesABSTRACT
Magnetic force microscopy (MFM) has been employed to observe antisense oligonucleotides (ASOs)-coupled silica-coated magnetic iron oxide nanoparticles (SMNPs) internalized into human leukemia (HL-60) cells. The experiment demonstrated that the ASOs-coupled SMNPs delivery into the cells really occurred. The nanoparticles were internalized into the cells and the apoptotic topography can be directly visualized simultaneously with MFM technology. These present observations offer direct morphology evidence on studying the apoptosis of tumor cells and provide useful information for better design of new diagnostic and therapeutic tools in tumor treatment.
