Abstract:

Objective To investigate the effect of bone morphogenetic protein 2 (BMP2) small interfering RNA (siRNA) targeted suppression on osteogenic differentiation and calcification of human umbilical arterial smooth muscle cells (HUASMC) induced by interleukin-6 (IL-6). Methods Primary HUASMC cultured in vitro were served as study objective. RNA interference (RNAi) experiment platform was established, and transfection condition was optimized with FAMnegative siRNA. Primary HUASMC were transfected with four candidate siRNA of BMP2 gene, respectively, the expression of BMP2 mRNA was detected by Real-Time PCR, and active suppression siRNA was screened. The thawed primary HUASMC were divided into siRNA transfection group (transfection with active suppression siRNA) and mocking transfection group (control group, without transfection with siRNA), and both group were stimulated with recombinant human IL-6 (10 ng/mL). The expression of BMP2, bone specific alkaline phosphatase (BAP), osteocalcin (OC) and osteopontin (OPN) mRNA was detected by Real-Time PCR after simulation for 12 and 48 h. The thawed primary HUASMC were divided into siRNA transfection group, control group and blank group (without rhIL-6 stimulation, similar to control group for the other treatment). rhIL-6 (50 ng/mL) was added to siRNA group and control group for stimulation. The calcium deposition in cell layers was measured by o-cresolphthalein complexone method before stimulation, 2 d and 4 d after stimulation with rhIL-6, and the results were corrected by total protein content. Results The suitable RNAi experiment platform was successfully established. Twenty hours after stimulation with 10 ng/mL rhIL-6, the expression of BMP2 and BAP mRNA of HUASMC in siRNA transfection group was significantly lower than that in control group (P<0.05). Forty-eight hours after stimulation, the expression of OC and OPN mRNA in siRNA transfection group was significantly lower than that in control group (P<0.05). Before stimulation with 50 ng/mL rhIL-6, there was no significant difference in calcium deposition in HUASMC cell layers among siRNA transfection group, control group and blank group (P>0.05). For siRNA transfection group, calcium deposition in cell layers after stimulation with 50 ng/mL rhIL-6 for 2 d and 4 d was significantly lower than that in control group (P<0.05), and calcium deposition in cell layers after stimulation for 2 d was significantly higher than that in blank group (P<0.05). Conclusion Primary HUASMC cultured in vitro can lead to siRNA targeted suppression of BMP2 gene, and can significantly inhibit the osteogenic differentiation and calcification induced by IL-6.

Key words: bone morphogenetic protein 2, RNA interference, osteoblastic differentiation, calcification, human umbilical artery smooth muscle cell