MiR-211-5p Affects Calcification Of Vascular Smooth Muscle Cells Through Wnt3a/β-catenin Signaling Pathway

Objective:To investigate the effect and mechanism of miR-211-5p on A7r5calcification in rat thoracic artery smooth muscle cells.Methods:1.Construction of A7r5 cell calcification modelA7r5 cell line was subcultured,and calcification was induced by high phosphorus（Na H₂PO₄,2m M）for 12 hours+nano-hydroxyapatite particles（Nano-HAP,200μg/m L）for 14 days.2.Experimental groupingIn the first stage,the A7r5 cells were divided into two groups:the control group without any intervention and the calcification group induced by calcification.The calcification model of A7r5 cells was established successfully,and the expression of miR-211-5P was analyzed.In the second stage,after transfection and drug treatment,the cells were divided into 6groups:control group,calcification group,calcification+miR-211-5P mimic group,calcification+mimic NC group,calcification+miR-211-5P inhibitor group,and calcification+inhibitor NC group.To explore the differences in the level of cell calcification and the expression levels of related proteins（including calcification-related regulatory proteins OPN,Runx2 and Wnt signaling pathway-related proteins Wnt3a,β-catenin and GSK3β）after up-regulating and down-regulating miR-211-5P.The effects of up-regulation and down-regulation of miR-211-5P on cell calcification and Wnt signaling pathway were analyzed.In the third stage,after transfection and drug treatment,the cells were divided into 4 groups:control group,calcification group,calcification+miR-211-5P inhibitor group and calcification+inhibitor+XAV939 group.The effect of Wnt signaling pathway inhibitor on cell calcification after down-regulation of miR-211-5P was explored.To further clarify whether miR-211-5P regulates A7r5 cell calcification through Wnt signaling pathway.3.Alizarin red staining and quantitative analysis were used to determine and analyze the calcium deposition of each group at three stages of cell experiment.4.Alkaline phosphatase（ALP）activity assay was used to detect the ALP activity of each group at three stages of cell experiment.5.Calcium content detection（Micro-plate method of o-cresol phthalein complex ketone）was used to detect the calcium content of cells in each group at three stages of cell experiment.6.Western blot（WB）was used to detect the protein expression levels of Wnt3a,β-catenin,GSK3β,OPN,and Runx2.7.Real-time quantitative PCR（RT-q PCR）was used to detect the expression levels of miR-211-5P in the two groups of cells in the first stage of cell experiment and in the cells transfected with miR-211-5P mimic group and its control group.8.Statistical analysis:SPSS 25.0 software was used for statistical analysis and Graph Pad Prism 8.0 software was used for drawing.The data were expressed asx±s.The t-test was used to compare the data between two groups,and one-way analysis of variance（ANOVA）was used to compare the data between multiple groups,and then multiple comparisons were performed.If P<0.05,indicating that the difference was statistically significant.Results:1.The experimental results of the first stage:the expression level of miR-211-5p was decreased in A7r5 cells after high phosphorus and nano-hydroxyapatite induced calcification.1.1 Alizarin red staining and quantitative analysis:the calcium deposition in the calcification group was significantly higher than that in the control group（P<0.001）.1.2 Alkaline phosphatase activity:the alkaline phosphatase activity of the calcified group was significantly higher than that of the control group（P<0.001）.1.3 Calcium content detection（Micro-plate method of o-cresol phthalein complex ketone）:The calcium content of cells in calcification group was significantly higher than that in control group（P<0.001）.1.4 Western blot（WB）results:The expressions of OPN and Runx2 in calcification group were higher than those in control group（P<0.001）.1.5 Real-time quantitative PCR（RT-q PCR）results:The expression level of miR-211-5p in the calcification group was significantly lower than that in the control group（P<0.001）.Compared with the control group,the calcification of the calcification group was significantly increased,indicating that the calcification model was successfully established.The expression level of miR-211-5p in the calcified group was significantly lower than that in the control group,which was consistent with the results of clinical research.2.The experimental results of the second stage:up-regulation and down-regulation of miR-211-5P affected the expression levels of cell calcification-related regulatory proteins and Wnt/β-catenin signaling pathway related proteins.2.1 Real-time quantitative PCR（RT-q PCR）results:Compared with the cells transfected with miR-211-5P mimic NC group,the expression level of miR-211-5P in the cells transfected with miR-211-5p mimic group was significantly higher（P<0.001）,indicating that the transfection was successful.2.2 Alizarin Red staining and quantitative analysis results:Compared with the control group,the calcium deposition was higher in calcification group,calcification+miR-211-5P mimic group,calcification+mimic NC group,calcification+miR-211-5P inhibitor group and calcification+inhibitor NC group（P<0.001）.Compared with the calcification group,the calcium deposition of the cells in the calcification+miR-211-5P mimic group was lower,while the calcium deposition of the cells in the calcification+miR-211-5P inhibitor group was higher（P<0.001）.2.3 Results of alkaline phosphatase activity:Compared with the control group,alkaline phosphatase activity was higher in calcification group,calcification+miR-211-5P mimic group,calcification+mimic NC group,calcification+miR-211-5P inhibitor group and calcification+inhibitor NC group（P<0.001）.Compared with the calcification group,the ALP activity of the cells in the calcification+miR-211-5P mimic group was lower,while the ALP activity of the cells in the calcification+miR-211-5P inhibitor group was higher（P<0.001）.2.4 Calcium content detection（Micro-plate method of o-cresol phthalein complex ketone）Results:Compared with the control group,the calcium content of cells in calcification group,calcification+miR-211-5P mimic group,calcification+mimic NC group,calcification+miR-211-5P inhibitor group and calcification+inhibitor NC group were higher（P<0.001）.Compared with the calcification group,the calcium content of the cells in the calcification+miR-211-5P mimic group was lower,while the calcium content of the cells in the calcification+miR-211-5P inhibitor group was higher（P<0.001）.2.5 Western blot（WB）results:Compared with the control group,the expression levels of Wnt3a,β-catenin,GSK3β,OPN and Runx2 in calcification group,calcification+miR-211-5P mimic group,calcification+mimic NC group,calcification+miR-211-5P inhibitor group,calcification+inhibitor NC group were higher（P<0.01）.Compared with the calcification group,the expression levels of Wnt3a,β-catenin,GSK3β,OPN and Runx2in the calcification+miR-211-5P mimic group were lower,however,the expression levels of Wnt3a,β-catenin,GSK3β,OPN and Runx2 were higher in calcification+miR-211-5P inhibitor group（P<0.01）.Up-regulation of miR-211-5P inhibited cell calcification and the expression levels of calcification-related regulatory proteins OPN and Runx2as well as Wnt/β-catenin signaling pathway-related proteins Wnt3a,β-catenin and GSK3β.Down-regulation of miR-211-5P increased cell calcification and the expression levels of calcification-related regulatory proteins OPN and Runx2,as well as Wnt/β-catenin signaling pathway-related proteins Wnt3a,β-catenin and GSK3β.3.The experimental results of the third stage:XAV939,an inhibitor of Wnt/β-catenin signaling pathway,reversed the increase in cell calcification caused by down-regulation of miR-211-5P.3.1 Alizarin red staining and quantitative analysis:Compared with the control group,the calcium deposition of cells in calcification group,calcification+miR-211-5P inhibitor group and calcification+inhibitor+XAV939 group were higher（P<0.001）.Compared with the calcification group,the calcium deposition of cells in the calcification+miR-211-5P inhibitor group was higher（P<0.001）.Compared with the calcification+miR-211-5P inhibitor group,the calcium deposition of cells in the calcification+inhibitor+XAV939 group was significantly lower（P<0.001）.3.2 Alkaline phosphatase activity:Compared with the control group,alkaline phosphatase activity was higher in calcification group,calcification+miR-211-5P inhibitor group and calcification+inhibitor+XAV939 group（P<0.001）.Compared with the calcification group,the alkaline phosphatase activity of the cells in the calcification+miR-211-5P inhibitor group was higher（P<0.001）.Compared with the calcification+miR-211-5P inhibitor group,the alkaline phosphatase activity of the calcification+inhibitor+XAV939 group was lower（P<0.001）.3.3 Calcium content detection（Micro-plate method of o-cresol phthalein complex ketone）results:Compared with the control group,the calcium content of cells in calcification group,calcification+miR-211-5P inhibitor group and calcification+inhibitor+XAV939 group were higher（P<0.001）.Compared with the calcification group,the calcium content of cells in the calcification+miR-211-5P inhibitor group was higher（P<0.001）.Compared with calcification+miR-211-5P inhibitor group,the calcium content of cells in calcification+inhibitor+XAV939 group was lower（P<0.001）.3.4 Results of Western blot（WB）:Compared with the control group,the protein expression levels ofβ-catenin,OPN and Runx2 were higher in calcification group,calcification+miR-211-5P inhibitor group and calcification+inhibitor+XAV939 group（P<0.01）.Compared with the calcification group,the expression levels ofβ-catenin,OPN and Runx2 in the calcification+miR-211-5P inhibitor group were higher（P<0.01）.Compared with the calcification+miR-211-5P inhibitor group,the expressions ofβ-catenin,OPN and Runx2 in the calcification+inhibitor+XAV939 group were significantly decreased（P<0.01）.XAV939,an inhibitor of Wnt/β-catenin signaling pathway,reversed the increase in cell calcification caused by down-regulation of miR-211-5P.Conclusions:1.High phosphorus combined with nano-hydroxyapatite particles could induce A7r5 cell calcification.2.MiR-211-5p can inhibit the calcification of A7r5 cells.3.MiR-211-5p could affect cell calcification through Wnt3a/β-catenin signaling pathway in A7r5 cells.