南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (9): 1469-1475.doi: 10.12122/j.issn.1673-4254.2023.09.03

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长链非编码RNA H19促进血管钙化：基于抑制Bax抑制因子1/视神经萎缩蛋白1通路

陈韦任，杜 辉，沙 媛，周玉杰，梁 静，陈韵岱，马 茜，吴雪萍，钱 赓   

  1. 首都医科大学附属北京安贞医院心内12病房//北京市心肺血管疾病研究所//冠心病精准治疗北京市重点实验室//首都医科大学冠心病临床诊疗与研究中心，北京 100029；中国人民解放军总医院第二医学中心心血管内科//国家老年疾病临床研究中心，北京 100853；中国人民解放军总医院第一医学中心心血管内科，北京 100853；清华大学附属北京清华长庚医院心血管内科，北京 102218
  • 出版日期:2023-09-20 发布日期:2023-09-28

Long noncoding RNA H19 promotes vascular calcification by repressing the Bax inhibitor 1/optic atrophy 1 pathway

CHEN Weiren, DU Hui, SHA Yuan, ZHOU Yujie, LIANG Jing, CHEN Yundai, MA Qian, WU Xueping, QIAN Geng   

  1. Department of Cardiology, Beijing Anzhen Hospital of Capital Medical University, Beijing Institute of Heart, Lung and Blood Vessel Disease, Beijing 100029, China; Department of Cardiology, Second Medical Center & National Clinical Research Center for Geriatric Diseases, Chinese PLA General Hospital, Beijing 100853, China; Department of Cardiology, First Medical Center, Chinese PLA General Hospital, Beijing 100853, China; Department of Cardiology, Beijing Tsinghua Changgung Hospital, School of Clinical Medicine, Tsinghua University, Beijing 102218, China
  • Online:2023-09-20 Published:2023-09-28

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摘要： 目的 探讨长链非编码RNA H19（lncRNA H19）是否通过抑制Bax抑制因子1/视神经萎缩蛋白1（BI-1/OPA1）通路促进钙沉积、细胞骨型分化和细胞凋亡，进而诱导血管钙化。方法 β磷酸甘油和氯化钙药物诱导大鼠血管平滑肌细胞（VSMC）建立细胞钙化模型，将细胞分为5组：对照组（普通培养基培养14 d）、钙化组（钙化培养基培养14 d）、钙化+siH19组（敲低lncRNA H19表达后钙化培养基培养14 d）、钙化+siH19+BI-1-/-组（敲低lncRNA H19和BI-1表达后钙化培养基培养14 d）和钙化+siH19+OPA1-/-组（敲低lncRNA H19和OPA1表达后钙化培养基培养14 d）。另ApoE-/-糖尿病小鼠使用高脂饲料喂养32周建立动物钙化模型。通过茜素红S染色和von Kossa染色检测钙沉积，通过Western blotting测定Runt相关转录因子2（Runx-2）、骨形态发生蛋白2（BMP-2）观察细胞骨型分化，通过TUNEL染色和半胱氨酸天冬氨酸蛋白酶3检测观察细胞凋亡情况。结果 血管钙化后，lncRNA H19表达明显上升，BI-1和OPA1表达明显下降，而siRNA敲低lncRNA H19表达后，BI-1和OPA1蛋白表达明显上升；抑制BI-1后，OPA1再次下降（P<0.001）。 siRNA敲低lncRNA H19表达后，钙化结节消失，钙含量、Runx-2、BMP-2、活化半胱氨酸天冬氨酸蛋白酶3表达和细胞凋亡率均显著降低（P<0.001）。siRNA敲低lncRNA H19基础上再抑制BI-1或OPA1蛋白，钙化结节出现，钙含量、Runx-2、BMP-2、活化半胱氨酸天冬氨酸蛋白酶3表达和细胞凋亡率均显著增加（P<0.001）。结论 LncRNA H19通过抑制BI-1/OPA1蛋白通路诱导血管钙化，其可能机制和促进钙沉积、细胞骨型分化和细胞凋亡有关。

关键词: 长链非编码RNA H19, Bax抑制因子1, 视神经萎缩蛋白1, 血管钙化, 细胞骨型分化

Abstract: Objective To investigate whether long noncoding RNA H19 (lncRNA H19) induces vascular calcification by promoting calcium deposition, osteogenic differentiation and apoptosis via inhibiting the Bax inhibitor 1/optic atrophy 1 (BI-1/OPA1) pathway. Methods β-glycerophosphate and calcium chloride were used to induce calcification in rat vascular smooth muscle cells (VSMCs), and the effects of siH19, alone or in combination with BI-1 or OPA1 knockdown, on calcification of the cells were investigated. Osteogenic differentiation was assessed by measuring Runt-related transcription factor 2 (Runx-2) and bone morphogenetic protein 2 (BMP-2) expression with Western blotting, and cell apoptosis was evaluated by TUNEL staining and Western blotting. An ApoE-/- diabetic mouse model with high-fat feeding for 32 weeks were given an intraperitoneal injection of siH19, and the changes in calcium deposition in the aortic arch were examined using Alizarin red S staining and von Kossa staining. Results In rat VSMCs with calcification, the expression of lncRNA H19 was significantly increased, and the expressions of BI- 1 and OPA1 were significantly decreased. Downregulation of lncRNA H19 significantly increased the expressions of BI-1 and OPA1 proteins in the cells, and BI-1 knockdown further reduced OPA1 expression (P<0.001). The cells treated with siH19 showed total disappearance of the calcified nodules with significantly reduced expressions of Runx-2, BMP-2 and cleaved caspase-3 and a lowered cell apoptosis rate (P<0.001). Calcified nodules were again observed in the cells with lncRNA H19 knockdown combined with BI-1 or OPA1 knockdown, and the expressions of Runx-2, BMP-2, cleaved-caspase-3 and cell apoptosis rate all significantly increased (P<0.001). In the diabetic mouse model with high-fat feeding, siH19 treatment significantly reduced the calcification area and increased mRNA expressions of BI-I and OPA1 in the aortic arch. Conclusion LncRNA H19 promotes vascular calcification possibly by promoting calcium deposition, osteogenic differentiation and cell apoptosis via inhibiting the BI-1/OPA1 pathway.

Key words: long noncoding RNA H19, Bax inhibitor 1, optic atrophy 1, vascular calcification, osteogenic differentiation