Effects of Bmi1 on proliferation of cranial suture mesenchymal cells in mice

Objective To explore the effects of Bmi1 on proliferation of mouse cranial suture mesenchymal cells. Methods Primary posterior frontal and sagittal suture derived cells were isolated from the 2-5 d old C57BL/6 suckling mice (n=6) of the same brood and cultured. Flow cytometry and multilineage differentiation assay were performed to identify the mesenchymal stem cells (MSCs) characteristics of the 2 kinds of cranial suture-derived cells. The mRNA expression of stem cell related genes, Bmi1, Twist1, Gli1 and Axin2 were detected by real-time quantitative polymerase chain reaction (RT-PCR). Then, the proliferation and downstream protein expression were analyzed after down-regulation of Bmi1 in the sagittal suture derived MSCs by transfecting Bmi1 siRNA. The t test was used to compare the mean between two groups. Statistical significance was set at P<0.05. Results The mouse cranial suture derived cells were successfully cultured in vitro. These cells expressed typical MSCs markers, CD44, CD90, CD73, except for CD34. These cells had osteogenic, adipogenic and chondrogenic differentiation potency. RT-PCR results showed that the mRNA expressions of Bmi1 (0.006 30±0.000 58 vs 0.002 60±0.000 34, t=5.430, P=0.005 6), Twist1(0.000 31±0.000 04 vs 0.000 15±0.000 02, t=3.343, P=0.028 8), Axin2(0.000 33±0.000 03 vs 0.000 17±0.000 05, t=3.067, P=0.037 4) and Gli1 (0.001 10±0.000 13 vs 0.000 60±0.000 33, t=3.956, P=0.016 7) were significantly decreased in the posterior frontal suture MSCs compared with those in sagittal suture derived cells. Among them, Bmi1 has the largest decline. After down-regulation of Bmi1 in sagittal suture MSCs, the protein expression level of Ink4a was significantly up-regulated compared with the control group, and the cell proliferation ability was significantly decreased. Conclusions Inhibition of Bmi1 expression can up-regulate the expression of Ink4a protein and decrease the proliferation ability of suture MSCs, which may lead to craniosynostosis. Key words: Bmi1; Cranial suture; Mesenchymal stem cells; Mouse