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-  2016 

microRNA在高磷诱导血管平滑肌细胞钙化早期的动态变化
Dynamic alteration of microRNA in high phosphorus induced calcification of vascular smooth muscle cell

DOI: 10.3969/j.issn.1671-167X.2016.05.002

Keywords: 血管钙化,, 平滑, 血管, 微RNA, 信号转导
Vascular calcification
, Muscle, smooth, vascular, microRNA, Signal transduction

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Abstract:

目的:观察高磷诱导的血管平滑肌细胞钙化早期microRNA表达变化,分析其可能参与的信号通路途径。方法:采用高磷(无机磷 2.6 mmol/L)刺激大鼠血管平滑肌细胞系A7r5钙化7 d,邻甲酚酞络合酮比色法和考马斯亮蓝法检测细胞内钙含量,RTPCR法检测平滑肌细胞表型和钙化相关基因的表达变化,茜素红染色观察钙结节。microRNA microarray 法检测高磷刺激后0、3、12 h,680种microRNA表达变化。采用TAM软件分析不同时间点间信号激活情况。结果:高磷诱导的A7r5细胞钙盐含量升高9.6倍(P<0.05),平滑肌细胞表型mRNA(SM-α actin,SM22)下调(P<0.05), 钙化相关mRNA(BMP2、MSX2、Runx2)上调(P<0.05),茜素红染色后可见高磷刺激组有明显钙结节。680种microRNA在3个时间点的表达各不相同,只有6种microRNA分别逐级上调或渐渐下调。26种信号通路被明显激活,其中包括细胞凋亡、分化增殖等已知与钙化相关的信号通路。结论:microRNA参与调节血管钙化是一种动态微调的过程,为血管钙化研究带来新的思路。
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.

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