Abstract:

Objective：To study the change of microRNA during the early stage of high phosphorus induced vascular smooth muscle cell (VSMC) calcification and its related mechanism.Methods:The in vitro calcification model was created through stimulating VSMC cell line A7r5 with high Pi (2.6 mmol/L) for 7 d. The calcification was validated through ocresolphthalein complexone colorimetry to detect the cellular calcium content, real-time PCR to measure the calcification-related gene expression and alizarin red staining to observe the formation of calcium nodules. Based on the cell calcification model, microRNA microarray array was applied to screen the profiles of microRNA expression in VSMC following high Pi stimulation for different periods (0, 3 and 12 h). The array data were analyzed by TAM tool to explore the activated signaling pathway.Results: The calcium content of A7r5 cells induced by high Pi was increased 9.6 times high as cells without Pi treatment (P<0.05). VSMC contractile phenotype genes (SM-α actin, SM22) were down-regulated (P<0.05), while calcification-related genes (BMP2, MSX2, Runx2) were up-regulated (P<0.05) in VSMC stimulated by high Pi. The calcium nodules were obviously formed in cells after 7 d high Pi treatment. In microarray experiment, 680 individual microRNAs were detected in high Pi-treated VSMCs at different time points (0, 3 and 12 h). Among these genes, miR-183, miR-664 and miR-9* were increased whereas miR-542-5P, let-7f and miR-29a were decreased in time-dependent manners. Twenty-six kinds of signaling pathways, including cell apoptosis, differentiation and proliferation, were significantly activated. All these activated pathways were associated with calcification. Conclusion:This study implies that microRNA changed in high Pi-induced VSMCs may involve in the process of calcification.

Key words: Vascular calcification, Muscle, smooth, vascular, microRNA, Signal transduction