MiR-27a Targets sFRP1 in hFOB Cells to Regulate Proliferation, Apoptosis and Differentiation

<div><p>MicroRNAs (miRNAs) play a key role in the regulation of almost all the physiological and pathological processes, including bone metabolism. Recent studies have suggested that miR-27 might play a key role in osteoblast differentiation and bone formation. Increasing evidence indicates that the canonical Wnt signaling pathway contributes to different stages of bone formation. In this study, we identify miR-27a can promote osteoblast differentiation by repressing a new target, secreted frizzled-related proteins 1 (sFRP1) expression at the transcriptional level. Here, 21 candidate targets of miR-27a involved in canonical Wnt/β-catenin signaling were predicted, and a significant decrease in sFRP1 luciferase activity was observed both in 293T and MG63 cells co-transfected with the matched luciferase reporter constructs and miR-27a mimic. Furthermore, the presence of exogenous miR-27a significantly decreased sFRP1 mRNA and protein expression in hFOB1.19 cells during both proliferation and osteogenic differentiation. The over-expression of miR-27a or knockdown sFRP1 significantly increased the percentage of apoptotic hFOBs, the percentage of cells in the G2-M phase of the cell cycle and the expression of key osteoblastic markers, including ALP, SPP1, RUNX2 and ALP activity. Over-expression of miR-27a or knockdown of endogenous sFRP1 led to an accumulation of β-catenin in hFOBs. In the present study, we demonstrate that miR-27a induced gene silencing effect is a vital mechanism contributing to bone metabolism in hFOB cells in vitro, which is partly affected by the post-transcriptional regulation of sFRP1, during osteoblast proliferation, apoptosis and differentiation.</p></div

The effect of miR-27a/sFRP1 on osteoblast markers in osteogenic hFOBs.

<p>ALP (<b>A</b>), SPP1 and RUNX2 mRNA (<b>B</b>) expression was determined by real-time PCR, and ALP activity (<b>A</b>) was assessed in hFOBs cultured in the osteogenic medium at 39.4°C. BMP2 (<b>C</b>), RUNX2 (<b>D</b>) and ALP (<b>E</b>)mRNA expression in hFOBs cultured in the non-differentiation medium at 39.4°C was also evaluated by real-time PCR. N = 3; mean ± SD; *<i>p</i><0.05; **<i>p</i><0.01. NC was the matched negative control: miR NC for both miR-27a mimic and siR sFRP1; miR inhibitor NC for miR-27a inhibitor; siR sFRP1+ miR-27a inhibitor NC for siR sFRP1+ miR-27a inhibitor.</p

HFOB proliferation assay.

<p>The proliferation was assessed by the CCK-8 active cell number kit. (<b>A</b>), hFOBs at the same passage were seeded into 96-well plates at a density of 7, 000 cells/well and cultured for 1 to 5 days in the non-differentiation medium at 33.4°C (red curve) or in the osteogenic medium at 37°C (black curve) or 39.4°C (blue curve). At 33.4°C, the daily cell counts indicated exponential cell growth between days 2 and 3, and the doubling time was approximately 60 h. (<b>B</b> and <b>C</b>), the hFOBs were transfected with the miR-27a mimic, miR-27a inhibitor or siRNA sFRP1 or were co-transfected. After 8 h, the cells either remained in the non-differentiation medium at 33.4°C (<b>B</b>) or were transferred to the osteogenic medium at 39.4°C (<b>C</b>). The proliferative capability of the hFOB cells was expressed as the percentage of surviving cells in comparison to the matched control. N = 9 for each group. The values represent the mean ± SD. NC^# was the matched control: miR NC for miR-27a mimic or siRNA sFRP1; miR inhibitor NC for miR-27a inhibitor; siR-sFRP1+ miR-27a inhibitor NC for siR sFRP1+ miR-27a inhibitor.</p

Modulation of cell cycle progression by miR-27a and sFRP1.

<p>HFOBs were plated in 6-well microplates (2.6×10⁵ cells/well) and incubated in the non-differentiation medium at 33.4°C for 24 h to 75% confluence. hFOBs were then transfected with the miR-27a inhibitor (100 nM) (<b>A</b>), siRNA sFRP1 (100 nM) (<b>B</b>) or both (<b>C</b>), or the matched control (at the same concentration). The percentage of cells in each phase of the cell cycle was determined by FACS analysis, as described in the materials and methods, at 48 h after transfection. N = 6 for each group. The values represent the mean ± SD. **<i>p</i><0.01.</p

Relative expression of the endogenous miR-27a in different cell types.

<p>Relative miR-27a expression was determined after 48 h among three different cell types (<b>A</b>) and different time (<b>B</b> and <b>C</b>) under the conditions described as follows. MG63 and 293T cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C for up to 3 days (<b>A</b>). HFOB1.19 cells were maintained in the non-differentiation medium at 33.4°C (<b>B</b>) or in the osteogenic medium at 39.4 (<b>C</b>) for up to 3 days or 7 days. All data are shown as mean ± SD. The asterisk indicates significance (t-test, **<i>p</i><0.01).</p

The evaluation of the optimal proliferative and osteogenic cultural condition for hFOBs <i>in vitro</i>.

<p>(<b>A</b>), After 3 days in the proliferative culture, hFOBs became small, uniform, cuboidal or spindle-shaped and closely packed with scant cytoplasm, as observed under the inverted light microscope. After 7 days in the osteogenic culture, hFOBs became larger and uneven in size, with abundant cytoplasm and shortened processes. Compared with hFOBs in the non-differentiation medium at 33.4°C for 3 or 7 days, hFOBs cultured in the osteogenic medium at 39.4°C exhibited obvious increases in the mRNA expression of bone-related genes (including ALP, COL1A1, OPN, Osterix, RUNX2 and BMP2) (<b>B</b>), as well as increased ALP activity (<b>C</b>), ALP staining (<b>D</b> and <b>E</b>) and calcium deposition staining (<b>F</b>). N = 3; mean ± SD; <i>*p</i><0.05, <i>**p</i><0.01.</p

Iridium(III) Complex Radical and Corresponding Ligand Radical Functionalized by a Tris(2,4,6-trichlorophenyl)methyl Unit: Synthesis, Structure, and Photophysical Properties

Organic radical luminescent materials with doublet excited state character based on tris(2,4,6-trichlorophenyl)methyl (TTM) have attracted extensive attention in recent years. However, how they affect the phosphorescent iridium(III) complex characterized by the triplet excited state has not been studied yet. Herein, a new iridium(III) complex radical (Ir-TTM) and corresponding ligand radical (ppy-TTM) with a TTM unit have been designed and synthesized, and their radical properties were confirmed by the single crystal structure and EPR spectra. Notably, the ligand radical ppy-TTM shows an efficient red light emission, whereas the iridium complex radical Ir-TTM emits no light, which resulted from the intramolecular quenching effect of the TTM radical unit on the iridium luminescence center. DFT calculations demonstrate that the lowest doublet (D1) excited state of ppy-TTM shows an intramolecular charge transfer character from the 2-phenylpyridine moieties to the TTM unit, whereas the D1 of Ir-TTM exhibits a significant charge transfer character from the iridium luminescence center moieties to the TTM unit, which further explains the luminescence quenching mechanism of the phosphorescent iridium complex radical

Additional file 1: of Kunxian capsules in the treatment of patients with ankylosing spondylitis: a randomized placebo-controlled clinical trial

The CONSORT checklist of the trial. (DOC 82 kb

5′-PDE enzymatic properties.

<p>Effect of (A) pH (the relative activities at various pH levels were measured with several different buffer systems at 60°C), (B) temperature (the relative activities at different temperature were determined at pH 5.0), and (C) metal ions on 5′-PDE activity (determing at 60°C and pH 5.0). (D) <i>V</i>_max and <i>K</i>_M values according to Lineweaver–Burk plots. Each value represents the mean of three independent measurements.</p

Isolation, purification and characterization of 5'-phosphodiesterase from <i>Aspergillus fumigatus</i>

<div><p>5′-Phosphodiesterase (5′-PDE) catalyzes the hydrolysis of ribonucleic acid to obtain a mixture of ribonucleotides, such as 5′-guanosine monophosphate and 5′-adenosine monophosphate. In this study, a 5'-PDE was newly isolated and purified from <i>Aspergillus fumigatus</i>. Following purification, this enzyme exhibited a specific activity of 1036.76 U/mg protein, a molecular weight of 9.5 kDa, and an optimal temperature and pH for enzyme activity of 60°C and 5.0, respectively. However, its activity was partially inhibited by Fe³⁺, Cu²⁺, and Zn²⁺, but slightly improved by the presence of K⁺ and Na⁺. Additionally, chemical-modification experiments were also applied to investigate the structural information of 5'-PDE, in which the residues containing carboxyl and imidazole groups were essential for enzyme activity based on their localization in the 5′-PDE active site. Furthermore, purified 5′-PDE could specifically catalyze the synthesis of ribonucleotides with a <i>V</i>_max 0.71 mmol/mg·min and a <i>K</i>_M of 13.60 mg/mL.</p></div