新疆医科大学学报

目的 探讨miR-302b-3p调控GALNT3及FGF23/PI3K/AKT通路在主动脉瓣膜钙化的作用机制。方法 进行人心脏瓣膜间质细胞培养与钙化诱导，设置对照组(Control)、钙化模型组(Model)、模型组+miR-302b-3p mimics NC组(miR-302b-3p mimics NC)、模型组+miR-302b-3p mimics组(miR-302b-3p mimics)、模型组+miR-302b-3p inhibitor NC组(miR-302b-3p inhibitor NC)、模型组+miR-302b-3p inhibitor组(miR-302b-3p inhibitor),共6组。通过细胞转染、茜素红染色、碱性磷酸酶(ALP)染色、MTT检测细胞活性、实时荧光定量PCR、Western blot及双荧光素酶报告基因检测实验，检测相关分子的表达水平与活性变化。结果 与Control组比较，Model组中miR-302b-3p、骨桥蛋白(OPN)、骨钙素(OCN)、Runt相关转录因子2(RUNX2)、p-PI3K和p-AKT蛋白的表达水平升高(P均<0.05),细胞增殖活性以及导致ALP和钙结节的能力升高，N-乙酰氨基半乳糖转移酶3(GALNT3)和成纤维细胞生长因子23(FGF23)蛋白的表达水平降低(P均<0.05)。与Model组比较，miR-302b-3p mimics组miR-302b-3p和FGF23蛋白的表达水平升高(P均<0.05),OPN、OCN、RUNX2、GALNT3、p-PI3K和p-AKT蛋白的表达水平降低(P均<0.05),细胞增殖活性以及导致ALP和钙结节的能力降低。miR-302b-3p inhibitor组则呈现相反的变化。双荧光素酶报告基因检测实验结果显示，与miR-302b-3p mimics+wt组相比，miR-302b-3p mimics NC+wt组相对荧光素酶活性升高(P<0.05),miR-302b-3p mimics+mut组与miR-302b-3p mimics NC+mut组相比，相对荧光素酶活性差异无统计学意义(P>0.05)。结论 miR-302b-3p可抑制细胞钙化及增殖，通过负向调控GALNT3的表达水平，调控FGF23/PI3K/AKT通路来影响主动脉瓣膜的钙化过程。

Objective To investigate the mechanism of miR-302b-3p regulating GALNT3 and FGF23/PI3K/AKT pathways in aortic valve calcification. Methods Human cardiac valve interstitial cell culture and calcification induction were performed. 6 groups were set up, including control group, calcification model group, model group+miR-302b-3p mimics NC group(miR-302b-3p mimics NC), model group+miR-302b-3p mimics group(miR-302b-3p mimics), model group+miR-302b-3p inhibitor NC group(miR-302b-3p inhibitor NC) and model group+miR-302b-3p inhibitor group(miR-302b-3p inhibitor).The expression level and activity changes of related molecules were detected by cell transfection, alizarin red staining, alkaline phosphatase staining, MTT assay, real-time fluorescent quantitative PCR, Western blot and dual luciferase reporter assay. Results Compared with control group, the expression levels of miR-302b-3p, OPN, OCN, RUNX2, p-PI3K and p-AKT proteins in model group were increased(all P<0.05), cell proliferation activity and the ability to cause ALP and calcium nodules were increased, and the expression levels of GALNT3 and FGF23 proteins were decreased(all P<0.05). Compared with model group, the expression levels of miR-302b-3p and FGF23 proteins in the miR-302b-3p mimics group were increased(all P<0.05), while the expression levels of OPN, OCN, RUNX2, GALNT3, p-PI3K and p-AKT proteins were decreased(all P<0.05), and the cell proliferation activity and the ability to cause ALP and calcium nodules were decreased. The miR-302b-3p inhibitor group showed the opposite change. The results of dual luciferase reporter assay showed that the relative luciferase activity of miR-302b-3p mimics NC+wt group was higher than that of miR-302b-3p mimics+wt group(P<0.05), and the relative luciferase activity of miR-302b-3p mimics+mut group was not significantly different from that of miR-302b-3p mimics NC+mut group(P>0.05). Conclusion miR-302b-3p can inhibit cell calcification and proliferation, and affect the calcification process of aortic valves by negatively regulating the expression level of GALNT3 and regulating the FGF23/PI3K/AKT pathway.