miRNA103在高磷诱导的血管平滑肌细胞钙化中的作用

Role of miRNA103 in calcification of vascular smooth muscle cells induced by high phosphorus

目的观察微小RNA103(microRNA103,miR-103)在高磷诱导的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)钙化过程中的作用及机制。方法体外培养大鼠胸主动脉VSMCs,将VSMCs随机分为正常组和高磷(HP)组(给予10 mmol/Lβ-甘油磷酸盐刺激),刺激4 d后,收集细胞。采用ELISA法测定VSMCs中碱性磷酸酶(ALP)的活性变化;采用邻甲酚酞络合酮比色法及茜素红染色检测VSMCs的钙化情况;采用qPCR测定VSMCs中miR-103及成骨转录因子Runx2的表达情况,Western blot检测Runx2蛋白表达情况。为进一步验证miR-103对VSMCs的作用,将细胞分为6组:正常组、NC inhi-bitor组、miR-103 inhibitor组、高磷组、NC mimic+HP组、miR-103 mimic+HP组,测定各组VSMCs钙化情况以及Runx2表达和ALP活性的变化。结果与正常组比较,高磷组大鼠VSMCs钙化增加(P<0.05),Runx2表达及ALP活性显著升高(P<0.05),而miR-103 RNA水平显著降低(P<0.05)。转染miR-103 inhibitor后,与正常组和NC inhibitor组比较,miR-103 inhibitor组VSMCs钙化增加,Runx2表达及ALP活性显著升高(均P<0.05);转染miR-103 mimic后,与高磷组和NC mimic+HP组比较,miR-103 mimic+HP组VSMCs钙化减少,Runx2表达及ALP活性显著降低(均P<0.05)。结论高磷可能是通过抑制miR-103表达,促进VSMCs表型转化,从而诱导VSMCs钙化。

Objective To investigate the effect of microRNA103(miR-103)on calcification in high phosphorus-induced vascular smooth muscle cells(VSMCs)and its mechanism.Methods Primary rat thoracic aorta VSMCs were divided into normal group and high phosphorus(HP)group(10 mmol/Lβ-glycerophosphate).After stimulation for 4 d,the cells were collected.Alizarin red staining and ALP test were used to analyze the level of calcification.Meanwhile,qPCR and Western blot were applied to investigate the mRNA and protein expression levels of miR-103 and osteogenic transcription factor Runx2.To further verify the effect of miR-103 on VSMCs,the VSMCs were divided into six groups:normal group,NC inhibitor group,miR-103 inhibitor group,HP group,NC mimic+HP group and miR-103 mimic+HP group.And then the calcification of VSMCs,the expression of Runx2 and the activity of ALP were measured.Results Compared with normal group,the calcification of VSMCs was increased in HP group(P<0.05).Moreover,Runx2 expression and ALP activity in high phosphorus group were higher than those in normal group(P<0.05),whereas the expression of miR-103 was significantly lower(P<0.05).Compared with normal group and NC inhibitor group,Runx2 expression and ALP activity in VSMCs were significantly increased in miR-103 inhibitor group(P<0.05).Runx2 expression and ALP activity in miR-103 mimic+HP group were significantly lower than those in high phosphorus group and NC mimic+HP group(P<0.05).Conclusion High phosphorus can induce the calcification of VSMCs through inhibiting the expression of miR-103 and promoting the phenotypic transformation of VSMCs.