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.
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。
Keywords: Bax inhibitor 1; long noncoding RNA H19; optic atrophy 1; osteogenic differentiation; vascular calcification.