Chen W, DU H, Sha Y, Zhou Y, Liang J, Chen Y, Ma Q, Wu X, Qian G
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, Beijing Tsinghua Changgung Hospital, School of Clinical Medicine, Tsinghua University, Beijing 102218, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2023 Sep 20;43(9):1469-1475. doi: 10.12122/j.issn.1673-4254.2023.09.03.
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.
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.
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 (<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 (<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 (<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.
LncRNA H19 promotes vascular calcification possibly by promoting calcium deposition, osteogenic differentiation and cell apoptosis via inhibiting the BI-1/OPA1 pathway.
研究长链非编码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）信号通路促进钙沉积、成骨分化和细胞凋亡，从而诱导血管钙化。
用β-甘油磷酸和氯化钙诱导大鼠血管平滑肌细胞（VSMC）钙化，研究单独使用小干扰RNA H19（siH19）或联合敲低BI-1或OPA1对细胞钙化的影响。通过蛋白质印迹法检测 runt相关转录因子2（Runx-2）和骨形态发生蛋白2（BMP-2）的表达来评估成骨分化，通过TUNEL染色和蛋白质印迹法评估细胞凋亡。对高脂喂养32周的载脂蛋白E糖尿病小鼠模型腹腔注射siH19，用茜素红S染色和冯科萨染色检测主动脉弓钙沉积的变化。
在钙化的大鼠VSMC中，lncRNA H19表达显著增加，BI-1和OPA1表达显著降低。lncRNA H19下调显著增加细胞中BI-1和OPA1蛋白的表达，敲低BI-1进一步降低OPA1表达（<0.001）。用siH19处理的细胞钙化结节完全消失，Runx-2、BMP-2和裂解的半胱天冬酶-3表达显著降低，细胞凋亡率降低（<0.001）。在lncRNA H19敲低联合BI-1或OPA1敲低的细胞中再次观察到钙化结节，Runx-2、BMP-2、裂解的半胱天冬酶-3表达和细胞凋亡率均显著增加（<0.001）。在高脂喂养的糖尿病小鼠模型中，siH19处理显著减少钙化面积，并增加主动脉弓中BI-I和OPA1的mRNA表达。
LncRNA H19可能通过抑制BI-1/OPA1信号通路促进钙沉积、成骨分化和细胞凋亡，从而促进血管钙化。
