The Second School of Clinical Medicine Southern Medical University Guangzhou Guangdong Province China.
Department of Cardiology 305 Hospital of PLA Beijing China.
J Am Heart Assoc. 2022 Jul 19;11(14):e024147. doi: 10.1161/JAHA.121.024147. Epub 2022 Jul 8.
Background Hypoxia is considered a major leading cause of pulmonary hypertension (PH). In this study, the roles and molecular mechanism of circ_0016070 in PH were studied. Methods and Results The expression of circ_0016070 in serum samples, human pulmonary artery smooth muscle cells and hypoxia/monocrotaline-treated rats was determined by real-time quantitative polymerase chain reaction. Cell viability, migration, and apoptosis were analyzed by Cell Counting Kit-8, wound healing, flow cytometry, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays, respectively. The molecular interactions were validated using RNA immunoprecipitation, chromatin immunoprecipitation, and dual luciferase reporter assays. The levels of phenotype switch-related proteins were evaluated by Western blot and immunohistochemistry. The pathological characteristics were assessed using hematoxylin and eosin staining. circ_0016070 was highly expressed in the serum samples, hypoxia-induced pulmonary artery smooth muscle cells and pulmonary arterial tissues of PH rats. Downregulation of circ_0016070 ameliorated the excessive proliferation, migration, vascular remodeling, and phenotypic transformation but enhanced cell apoptosis in the PH rat model. In addition, micro (miR)-340-5p was verified as a direct target of circ_0016070 and negatively regulated TCF4 (transcription factor 4) expression. TCF4 formed a transcriptional complex with β-catenin to activate TWIST1 (Twist family bHLH transcription factor 1) expression. Functional rescue experiments showed that neither miR-340-5p inhibition nor TWIST1 or TCF4 upregulation significantly impeded the biological roles of circ_0010670 silencing in PH. Conclusions These results uncovered a novel mechanism by which circ_0016070 play as a competing endogenouse RNA of miR-340-5p to aggravate PH progression by promoting TCF4/β-catenin/TWIST1 complex, which may provide potential therapeutic targets for PH.
缺氧被认为是肺动脉高压（PH）的主要原因。在这项研究中，研究了 circ_0016070 在 PH 中的作用和分子机制。
通过实时定量聚合酶链反应测定血清样本、人肺动脉平滑肌细胞和缺氧/单硝酸异山梨酯处理的大鼠中 circ_0016070 的表达。通过细胞计数试剂盒-8、划痕愈合、流式细胞术和 TUNEL（末端脱氧核苷酸转移酶 dUTP 缺口末端标记）分析分别检测细胞活力、迁移和凋亡。使用 RNA 免疫沉淀、染色质免疫沉淀和双荧光素酶报告基因分析验证分子相互作用。通过 Western blot 和免疫组织化学评估表型转换相关蛋白的水平。通过苏木精和伊红染色评估病理特征。
circ_0016070 在 PH 大鼠的血清样本、缺氧诱导的肺动脉平滑肌细胞和肺动脉组织中高表达。下调 circ_0016070 可改善 PH 大鼠模型中过度增殖、迁移、血管重塑和表型转化，但增强细胞凋亡。此外，miR-340-5p 被验证为 circ_0016070 的直接靶标，并负调控 TCF4（转录因子 4）表达。TCF4 与β-catenin 形成转录复合物，激活 TWIST1（Twist 家族 bHLH 转录因子 1）表达。功能恢复实验表明，miR-340-5p 抑制或 TWIST1 或 TCF4 上调均不能显著阻碍 circ_0016070 沉默在 PH 中的生物学作用。
这些结果揭示了 circ_0016070 通过作为 miR-340-5p 的竞争性内源性 RNA 发挥作用的新机制，通过促进 TCF4/β-catenin/TWIST1 复合物加重 PH 进展，这可能为 PH 提供潜在的治疗靶点。
