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分析

Analysis of the mechanism of miR-302b-3p regulating GALNT3 and FGF23/PI3K/AKT pathways in aortic valve calcification

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 ex-pression level and activity changes of related molecules were detected by cell transfection,alizarin red stai-ning,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 rela-tive 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.