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

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

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

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

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