微小RNA-297a通过调控成纤维细胞生长因子23表达在血管钙化中的作用及其机制

MicoroRNA-297a regulates vascular calcification by targeting fibroblast growth factor 23

目的通过前期维生素D3结合尼古丁法构建稳定的血管钙化动物模型，观察血管钙化过程中的微小NRA（miRNAs，miR）表达谱变化，并通过生物信息学预测分析miRNA靶基因，探讨miRNA通过调节其靶基因的表达在血管钙化中的作用机制。方法7周龄雄性SD大鼠30只，随机分为两组，每组15只，采用维生素D3（300 kIU/kg）肌肉注射和尼古丁（5 mg/kg）灌胃建立大鼠血管钙化动物模型。对照组给予等体积的生理盐水处理。诱导4周后处死大鼠并分离主动脉组织。提取组织RNA，纯化获得miRNAs，采用Affemitrix miRNA芯片检测血管钙化模型组和对照组主动脉组织中miRNA表达谱变化（设2倍改变为阈值），筛选差异表达的miRNAs。通过生物信息学手段预测miRNA的靶基因。实时定量聚合酶链反应（Real-time PCR）及Western blot杂交分析血管钙化过程中相关靶基因及信号通路中分子表达量的变化。结果通过前期构建稳定的血管钙化动物模型，选择变化倍数较大的mmu-miR-297a作为研究重点；成纤维细胞生长因子23（FGF23）作为mmu-miR-297a的靶基因，在调节血管生长过程中发挥重要的作用；Real-time PCR检测结果表明，在模型组中，mmu-miR-297a的表达量明显下调，Real-time PCR也发现FGF23在模型组大鼠主动脉组织中出现上调，而Klotho则明显下调（P＝0.000）；Western blot分析蛋白水平FGF23及Klotho的表达与mRNA水平结果一致，即FGF23在模型组中上调，Klotho则明显下调。结论通过构建血管钙化动物模型，分离血管组织，利用miRNAs芯片，筛选血管钙化过程中差异表达的miRNAs，发现mmu-miR-297a在血管钙化模型中下调，减少对其靶基因FGF23的抑制，促进了血钙、血磷的异常积累。

ObjectiveVascular calcification is one of the major characteristics in patients with various types of chronic inflammatory disorders. miRNAs have been shown to be involved in many normal biological functions as well as diseases, however, their role in vascular calcification has not received much attention.MethodsIn the current study, we built vascular calcification rat model using vitamin D3 plus nicotine and analyzed miRNA expression profile by miRNA chip assay. Potential target of one selected miRNA with sharpest variation in expression was predicted by both PicTar and TargetScan. The impact of the selected miRNA on the expression of the potential target on both mRNA and protein levels was measured by real-time quantitative polymerase chain reaction （Real-time PCR） and Western blotting, respectively.ResultsThe mmu-miR-297a was selected as the focus of the study on the early construction of stable angiocalcified animal models; As the target gene of mmu-miR-297a, fibroblast growth factor 23 （FGF23） plays an important role in regulating vascular growth; The results of Real-time PCR showed that the expression of mmu-miR-297a was significantly reduced in the model group; Real time PCR also found that FGF23 was up-regulated in the aortic tissue of the model group, while Klotho was significantly reduced （P=0.000）; The expression of Western blotting analysis protein level FGF23 and Klotho was consistent with the mRNA level results, FGF23 was up-regulated in the model group, and Klotho was significantly reduced.ConclusionOur results indicated that FGF23 was target of miR-297a and decreased miR-297a in vascular calcification lead to the increase of FGF23, which together with Klotho might enhance vascular calcification. The findings of this study could provide valuable information for the understanding of mechanisms underlying miR-dependent vascular calcification as well as potential treatment target for the disease.