中国血液净化

中国血液净化 ›› 2021, Vol. 20 ›› Issue (10): 669-673.doi: 10.3969/j.issn.1671-4091.2021.10.006

• 基础研究 • 上一篇    下一篇

微小RNA-30b 通过下调活化T 细胞核因子c1 抑制大鼠血管平滑肌细胞钙化

何雷1,周薇1,白亚玲1,张胜雷1,张东雪1,刘兰1,徐金升1   

  1. 1河北医科大学第四医院肾内科
  • 收稿日期:2021-05-13 修回日期:2021-06-27 出版日期:2021-10-12 发布日期:2021-10-12
  • 通讯作者: 徐金升 xjs5766@126.com E-mail:125657481@qq.com
  • 基金资助:
    河北省临床医学优秀人才培养项目[冀财社(2019)139 号]；河北省医学技术跟踪项目(G2018050)；河北省适宜卫生技术推广项目(P20190009)；河北省重点研发计划项目(20377704D)；河北省医学科学研究重点课题(20150310)

MicroRNA-30b inhibits vascular smooth muscle cell calcification by down-regulating NFATc1

  1. 1Department of Nephrology, The Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, China
  • Received:2021-05-13 Revised:2021-06-27 Online:2021-10-12 Published:2021-10-12

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摘要： 【摘要】目的观察微小RNA(microRNA，miRNA)-30b通过调控活化T细胞核因子c1(nuclear factor of activated T-cell c1, NFATc1)对血管平滑肌细胞(vascular smooth muscle cells, VSMCs)钙化的作用及机制。方法大鼠体外原代培养VSMCs，并分为正常组和高磷组。采用ELISA法测定各组VSMCs碱性磷酸酶(ALP)的活性；采用茜素红染色及钙含量检测各组VSMCs 的钙化；采用实时荧光定量PCR测定各组VSMCs miR-30b及NFATc1和Runx2表达，Western blot检测NFATc1、Runx2的蛋白表达。为进一步验证miR-30b对VSMCs的作用，给予miR-30b的类似物mimic-30b和抑制物inhibitor-30b，检测VSMCs的钙化及NFATc1、Runx2的表达和ALP的活性变化。结果与正常组比较，高磷诱导的VSMCs钙化增加(t=-13.324, P＜0.001)；NFATc1的RNA和蛋白表达量升高(t值分别为为-4.621、-4.684, P值分别为0.010、0.009)；Runx2的RNA和蛋白表达量升高(t值分别为-4.340、-3.860, P值分别为0.005、0.018)；ALP活性升高(t= -12.636, P＜0.001)；miR-30b的表达降低(t=3.822, P=0.019)。转染inhibitor-30b后，inhibitor-30b组较未转染组、inhibitor-NC组的钙含量升高(F=606.554, P＜0.001)；NFATc1 的RNA和蛋白表达量升高(F 值分别为36.427、22.512, P 值分别为＜0.001、0.002)；Runx2 的RNA 和蛋白表达量升高(F 值分别为44.225、42.832，P 值分别为＜0.001、＜0.001)；ALP 活性升高(F=347.356, P＜0.001)。转染mimic-30b后，钙化降低(F=93.341，P＜0.001)；NFATc1 的RNA 和蛋白表达量降低(F值分别为69.841、6.090, P值分别为＜0.001、0.036)；Runx2的RNA和蛋白表达量降低(F值分别为44.780、841.081, P值分别为0.005、＜0.001)；ALP 活性降低(F=197.829，P＜0.001)。结论miRNA-30b 可抑制VSMCs 钙化，可能是通过下调NFATc1，进而下调Runx2表达，从而使VSMCs表型转化减少实现的。

关键词: 血管平滑肌细胞, 血管钙化, 表型转化, miR-30b, T 细胞核因子c1

Abstract: 【Abstract】Objective To investigate the effect of microRNA (miRNA)-30b on vascular smooth muscle cells (VSMCs) calcification through the down-regulation of nuclear factor of activated T- cell c1 (NFATc1). Methods Primary rat VSMCs were cultured in vitro and divided into control group and high phosphorus group. Alkaline phosphatase (ALP) in VSMCs was assayed by ELISA. The calcification of VSMCs was determined by alizarin red staining and calcium content measurement. The extent of miRNA-30b, NFATc1 mRNA and Runx2 mRNA in VSMCs were evaluated by quantitative real-time PCR. NFATc1 and Runx2 in VSMCs were measured by western blot. To verify the effect of miR-30b, VSMCs were treated with a miR-30b analogue, mimic-30b, or a miR-30b inhibitor, inhibitor-30b, and the changes of alizarin red staining, calcium content, NFATc1 and Runx2 expression level and ALP activity in the cells were then observed. Results Compared with the control group, high phosphorus environment induced the calcification of VSMCs (t=-13.324, P<0.001), the increase of NFATc1 mRNA (t=- 4.621, P=0.010) and its protein (t=- 4.684, P= 0.009), the increase of Runx2 mRNA (t=- 4.340, P=0.005) and its protein (t=- 3.860, P=0.018), the higher ALP activity (t= -12.636, P<0.001), but the decrease of miR-30b (t=3.822, P=0.019) in the cells. After transfection of the inhibitor-30b, the VSMCs showed the changes very similar to the high phosphorus-treated VSMCs including the increase of calcification (F=606.554, P< 0.001), the increase of NFATc1 mRNA (F=36.427, P<0.001) and its protein (F=22.512, P=0.002), the increase of Runx2 mRNA (F=44.225, P=0.005) and its protein(F=42.832, P<0.001), as well as the higher ALP activity (F=347.356, P<0.001). After transfection with the mimic-30b, however, the VSMCs revealed the opposite changes including the decrease of calcification (F= 93.341, P<0.001), the decrease of NFATc1 mRNA (F=69.841, P<0.001) and its protein (F=6.090, P=0.036), the decrease of Runx2 mRNA (F=44.780, P=0.005) and its protein (F=841.081, P<0.001), as well as the lower ALP activity (F=197.829, P<0.001). Conclusion miRNA- 30b inhibits the calcification of VSMCs, probably through the down-regulation of NFATc1 expression and thus the down-regulation of Runx2 expression to increase the phenotypic transformation of VSMCs.

Key words: Vascular smooth muscle cells, Vascular calcification, Phenotypic transformation, miR-30b, NFATc1

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