Abstract: 【Abstract】Objective To investigate the effect of microRNA (miRNA)-30b on vascular smooth muscle cells (VSMCs) calcification through the down-regulation of nuclear factor of activated T- cell c1 (NFATc1). Methods Primary rat VSMCs were cultured in vitro and divided into control group and high phosphorus group. Alkaline phosphatase (ALP) in VSMCs was assayed by ELISA. The calcification of VSMCs was determined by alizarin red staining and calcium content measurement. The extent of miRNA-30b, NFATc1 mRNA and Runx2 mRNA in VSMCs were evaluated by quantitative real-time PCR. NFATc1 and Runx2 in VSMCs were measured by western blot. To verify the effect of miR-30b, VSMCs were treated with a miR-30b analogue, mimic-30b, or a miR-30b inhibitor, inhibitor-30b, and the changes of alizarin red staining, calcium content, NFATc1 and Runx2 expression level and ALP activity in the cells were then observed. Results Compared with the control group, high phosphorus environment induced the calcification of VSMCs (t=-13.324, P<0.001), the increase of NFATc1 mRNA (t=- 4.621, P=0.010) and its protein (t=- 4.684, P= 0.009), the increase of Runx2 mRNA (t=- 4.340, P=0.005) and its protein (t=- 3.860, P=0.018), the higher ALP activity (t= -12.636, P<0.001), but the decrease of miR-30b (t=3.822, P=0.019) in the cells. After transfection of the inhibitor-30b, the VSMCs showed the changes very similar to the high phosphorus-treated VSMCs including the increase of calcification (F=606.554, P< 0.001), the increase of NFATc1 mRNA (F=36.427, P<0.001) and its protein (F=22.512, P=0.002), the increase of Runx2 mRNA (F=44.225, P=0.005) and its protein(F=42.832, P<0.001), as well as the higher ALP activity (F=347.356, P<0.001). After transfection with the mimic-30b, however, the VSMCs revealed the opposite changes including the decrease of calcification (F= 93.341, P<0.001), the decrease of NFATc1 mRNA (F=69.841, P<0.001) and its protein (F=6.090, P=0.036), the decrease of Runx2 mRNA (F=44.780, P=0.005) and its protein (F=841.081, P<0.001), as well as the lower ALP activity (F=197.829, P<0.001). Conclusion miRNA- 30b inhibits the calcification of VSMCs, probably through the down-regulation of NFATc1 expression and thus the down-regulation of Runx2 expression to increase the phenotypic transformation of VSMCs.

Key words: Vascular smooth muscle cells, Vascular calcification, Phenotypic transformation, miR-30b, NFATc1