Let-7b-5p靶向ETFA减轻高磷诱导的血管平滑肌细胞钙化的机制研究

Let-7b-5p targeting ETFA to inhibit high phosphorus-induced vascular smooth muscle cell calcification

目的:探讨Let-7b-5p调控电子转移黄素蛋白A(ETFA)表达对高磷诱导的血管平滑肌细胞(VSMCs)钙化的影响。方法:体外培养VSMCs,分为正常对照组和钙化组。钙化组用含β-甘油磷酸钠(β-GP)的培养基培养14 d诱导VSMCs钙化,通过茜素红染色和碱性磷酸酶(ALP)染色观察VSMCs钙化情况。分别将Let-7b-5p mimic、Let-7b-5p inhibitor、ETFA siRNA及相应对照转染VSMCs,并诱导细胞钙化。用RT-qPCR法检测细胞Let-7b-5p、ETFA及Runx2表达,Western blotting法检测ETFA和Runx2蛋白表达。双荧光素酶实验验证Let-7b-5p与ETFA的靶向关系。结果:与正常对照组比较,钙化组出现明显的紫红色钙盐结节,Let-7b-5p表达显著下调,ETFA和成骨细胞因子Runx2表达明显上调(均P<0.05)。Pearson相关分析显示,Let-7b-5p与ETFA表达呈负相关关系(r=-0.785,P<0.001),ETFA与Runx2表达呈正相关关系(r=0.962,P<0.001)。Let-7b-5p mimic能显著降低钙化细胞Runx2和ETFA的表达及ALP活性,而Let-7b-5p inhibitor则相反。Let-7b-5p可靶向调控ETFA表达。沉默ETFA后,钙化VSMCs中ETFA和Runx2表达显著下降,钙盐结节明显减少。结论:Let-7b-5p可通过靶向抑制ETFA的表达抑制高磷诱导的VSMCs钙化。

Objective:To invastigate the effect of Let-7b-5p on high phosphorus-induced vascular smooth muscle cells(VSMCs)calcification by targeting electron transfer flavoprotein A(ETFA).Methods:VSMCs were cultured in vitro,and divided into normal control group and calcification group.β-glycerophosphate(β-GP)was used to induce calcification of VSMCs for 14 days.The calcification of VSMCs was observed by Alizarin red staining and alkaline phosphatase(ALP)staining.VSMCs were transfected with Let-7b-5p mimic,Let-7b-5p inhibitor,ETFA siRNA and their negative controls,and then cell calcification was induced.The expressions of Let-7b-5p,ETFA,and Runx2 were detected by RT-qPCR.The protein expression levels of ETFA and Runx2 were determined by western blotting.The targeting relationship between Let-7b-5p and ETFA was verified by dual luciferase reporter assay.Results:Compared with the normal control group,there were more calcium nodules in calcification group,and Let-7B-5P expression was significantly down-regulated,ETFA and osteoblast cytokine Runx2 expressions were significantly up-regulated(P<0.05).Pearson correlation analysis showed that Let-7b-5p was negatively correlated with ETFA expression(r=-0.785,P<0.001),and ETFA was positively correlated with Runx2 expression(r=0.962,P<0.001).Let-7b-5p mimic significantly reduced the Runx2 and ETFA expressions and ALP activity of VSMCs,while Let-7B-5P inhibitor had the opposite effects.Let-7b-5p targeted and regulated ETFA expression.After ETFA silencing,the calcium nodules and the expressions of ETFA and Runx2 in calcified VSMCs were significantly decreased(P<0.05).Conclusion:Let-7b-5p can inhibit high phosphorus-induced VSMCs calcification by suppressing ETFA expression.