LncRNAH19 Sponges MiR-103 To Promote Calcification Of Vascular Smooth Muscle Cells Through Upregulating Runx2

Objective: Vascular calcification(VC)is a well-documented cardiovascular complication in individuals with Chronic kidney disease(CKD).Furthermore,when VC is present,the risk for cardiovascular mortality and morbidity is significantly increased.The recent accumu lation of data indicates that the vascular smooth muscle cells(VSMCs)can undergo trans-differentiation,which is a key part of VC in CKD patients.Long noncoding RNA(lncRNA)H19 has been reported to play vital role in phenotypic transformation of vascular smooth muscle cells.Whether LncRNA H19 is involved in the regulation of vascular calcification?What is the molecular mechanism? It is unclear.So this study was to investigate the function and molecular mechanism of LncRNA H19 in VSMC calcification.Methods:1.Primary VSMCs were cultured in vitro by explant method and stimulated by high phosphorus;We divided VSMCs into 4 groups: normal group,high phosphorus,high phosphorus+shRNA H19,high phosphorus+ shRNA control.2.After the intervention of 7d,14 d,ALP activity,Alizarin Red staining and quantification of calcium were detected.3.The expression of LncRNA H19,mir-103,mir-675,mRNA Runx2 and mRNA BMP-2 was detected by qRT-PC.4.Western blotting was used to detect the expression of Runx2 and BMP-2 proteins.5.Statistical analysisStatistical analysis was performed using SPSS 17.0 software.The mean comparison between groups was performed by one-way analysis of variance(ANOVA),and the comparison between the two was performed by SNK(Student-Newman-Keuls)test,the results were statistically significant at P < 0.05.Results:1.Calcification of VSMCs induced by high phosphorusAfter the intervention of 7d,14 d,Alizarin Red staining and quantification of calcium were increased(P<0.001).And the results of 14 d were significantly higher than 7d(P<0.001).2.The expression of Runx2 and BMP-2 in VSMCs induced by high phosphorusAfter the intervention of 7d,14 d,ALP activity were increased?qRT-PCR and Western Blot results showed that Runx2 and BMP2 were highly expressed(P<0.05)and the level of Runx2 and BMP2 for 14 d stimulation of high phosphorus is higher than 7d(P<0.05).The expression levels of Runx2 and BMP2 were reduced in cells transfected with shRNA H19 when compared with controls(P< 0.05).3.Expressions of LncRNA H19,mir-103 and mir-675 of VSMCs induced by high phosphorusThe qRT-PCR results show that the level of LncRNA H19/miR-675-5p was significantly upregulated in VSMCs Calcification(P<0.05).As shown in the Figures,the level of LncRNA H19/miR-675-5p for 14 d stimulation of high phosphorus is higher than 7d(P<0.05).miR-103 a levels were significantly reduced after ?-GP treatment as measured by qRT-PCR(P<0.05),and the level of miR-103 a for 14 d stimulation of high phosphorus is lower than 7d(P< 0.05).After knockdown of LncRNA H19,expression of miR-675-5p was significantly reduced(P<0.05)and expression of miR-103 a was significantly upregulated(P<0.05).Conclusion:1.In the calcification process of VSMCs induced by high phosphorus,the expression levels of LncRNA H19,mir-675 were increased,and the expression levels of mir-103 were decresead.2.The LncRNA H19/miR-103/Runx2 signalling pathway may participate in the calcification process of VSMCs induced by high phosphorus.