| Objective:Cardiovascular disease(CVD)is the major cause of death in chronic kidney disease(CKD)patients,and vascular calcification(VC)is an important non-traditional risk factor for CVD.It is of important social and economic value to investigate the pathogenesis of VC.Recent studies have shown that VC is an active process regulated by many factors.The calcification of vascular smooth muscle cells(VSMCs)is the basis of VC,and phosphorus could participate in VSMCs calcification through a variety of signaling pathways.micro RNA(miR)is an important regulatory molecule in vivo,which is involved in a series of physiological and pathological processes,and many miRs also participate in the process of VC.In this study,miR-125 b was selected as the research object,to observe its expression level and significance in VSMCs calcification,and to further explore the mechanism of miR-125 b targeting fibroblast growth factor receptor 2(FGFR2)in VSMCs calcification induced by high phosphorus.Methods:VSMCs cell line(A7R5)was selected for cell experiment.The calcification of VSMCs was induced by high-phosphorus medium,and the establishment of the calcification model was verified by the detection of calcium deposition,MTT test,Alizarin S staining and alkaline phosphatase(ALP)activity.q RT-PCR and Western Blot were used to detect the relative expression levels of miR-125 b and FGFR2 m RNA and protein in VSMCs calcification model induced by high phosphorus.miR-125 b mimics,miR-125 b inhibitors,pc DNA3.1-FGFR2,si RNA-FGFR2 and the negative control were transfected into VSMCs through cell transfection,and by up-regulating or down-regulating miR-125 b and FGFR2,the effect of miR-125 b and FGFR2 on VSMCs calcification was verified.The target gene of miR-125 b were forecasted by bioinformatics method,and the relationship between miR-125 b and FGFR2 was tested by dual luciferase reporter gene experiment.Finally,Western Blot was used to detect the effect of FGFR2 on the expression of downstream pathway proteins such as ERK1/2 and Runx2.Results:1.Compared with the normal control group,the calcium deposits and the ALP activity were significantly increased,the cell activity was significantly reduced,and Alizarin S staining showed that there was obvious red dye deposits in VSMCs in the high-phosphorus group(P<0.05).2.In the VSMCs calcification model induced by high phosphorus,the expression level of miR-125 b was significantly lower than that of the normal control group,while the expression levels of FGFR2 m RNA and protein were significantly higher than that of the normal control group(P<0.05).3.The results of cell transfection showed that calcium deposition and ALP activity were significantly reduced after up-regulating of miR-125 b or down-regulating of FGFR2,while calcium deposition and ALP activity were significantly increased after down-regulating of miR-125 b or up-regulating of FGFR2(P<0.05).4.Through online database query and analysis,FGFR2 may be the target gene of miR-125 b.Dual luciferase reporter gene experiment showed that the relative luciferase activety was significantly decreased in 293 T cells co-transfected with miR-125 b mimic and FGFR2 3’-UTR-WT(P<0.05).5.Western Blot showed that compared with the control group,the expression of p ERK1/2 and Runx2 in the FGFR2 overexpression group was significantly increased,while the expression of p ERK1/2 and Runx2 in the FGFR2 knockdown group was significantly decreased(P<0.05).Conclusion:miR-125 b could inhibit the calcification of VSMCs induced by high phosphorus by targeting the expression of FGFR2.The mechanism may be that FGFR2 regulate the ERK1/2-Runx2 pathway. |
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