欢迎访问《现代生物医学进展》编辑部！

摘要目的：探究miR-204在肺动脉高压（PAH）模型大鼠中的作用和相关分子机制。方法：采用一次性腹腔注射野百合碱（MCT，60 mg/kg）溶液方法构建PAH模型大鼠。将PAH大鼠分为PAH组、NC组和miR-204组，每组10只；另取15只健康SD大鼠作为对照组。采用HE染色检测大鼠肺和右心室组织病变，免疫组织化学染色法检测肺组织α-SMA蛋白表达水平。采用双荧光素酶报告基因分析miR-204和Runx2关系。将人肺动脉平滑肌细胞（PASMCs）分为对照组、低氧组、mimics NC组、miR-204 mimics组、pcDNA3.1组和Runx2组。采用CCK-8法和EdU法检测PASMCs细胞增殖，流式细胞术检测细胞周期分布，Transwell实验检测PASMCs细胞迁移。qRT-PCR检测肺组织和PASMCs细胞中miR-204和Runx2表达水平。Western blot检测肺组织或PASMCs细胞中Runx2、α-SMA、CDKN4、Cyclin E和Cyclin D1蛋白表达水平。结果：与对照组比较，PAH组大鼠肺组织中miR-204表达水平降低（P<0.05），Runx2和α-SMA蛋白表达水平均升高（P<0.05），大鼠肺动脉血管壁增厚，管腔狭窄，心肌细胞肥大。与NC组比较，miR-204组大鼠肺组织中miR-204表达水平升高（P<0.05），Runx2和α-SMA蛋白表达水平均降低（P<0.05），大鼠肺动脉血管壁增厚和心肌细胞肥大得到显著改善。miR-204与Runx2存在靶向结合关系。与对照组比较，低氧组细胞miR-204表达水平和CDKN4蛋白表达水平以及G0/G1期分布比例均降低（P<0.05），Runx2 mRNA和蛋白以及Cyclin E和Cyclin D1蛋白表达水平均升高（P<0.05），细胞增殖水平和迁移数量均升高（P<0.05）。与mimics NC组比较，miR-204 mimics组细胞miR-204表达水平和CDKN4蛋白表达水平以及G0/G1期分布比例均升高（P<0.05），Runx2 mRNA和蛋白以及Cyclin E和Cyclin D1蛋白表达水平均降低（P<0.05），细胞增殖水平和迁移数量均降低（P<0.05）。与pcDNA3.1组比较，Runx2组细胞miR-204表达水平和CDKN4蛋白表达水平以及G0/G1期分布比例均降低（P<0.05），Runx2 mRNA和蛋白以及Cyclin E和Cyclin D1蛋白表达水平均升高（P<0.05），细胞增殖水平和迁移数量均升高（P<0.05）。结论：miR-204在PAH模型大鼠中低表达，过表达miR-204可以抑制PASMCs细胞的增殖和迁移，促进细胞分裂阻滞在G0/G1期，该作用可能与靶向抑制Runx2蛋白表达有关。

英文摘要:

ABSTRACT Objective: To explore the role of miR-204 in pulmonary arterial hypertension (PAH) model rats and its related molecular mechanism. Methods: PAH model rats were constructed by one-time intraperitoneal injection of monocrotaline (MCT, 60 mg/kg) solution. PAH rats were divided into PAH group, NC group and miR-204 group, with 10 rats in each group. Another 15 healthy SD rats were selected as control group. The lesions of lung and right ventricle tissues were detected by HE staining, and the expression level of α-SMA protein in lung tissues was detected by immunohistochemical staining. The relationship between miR-204 and Runx2 was analyzed using dual luciferase reporter genes. Human pulmonary arterial smooth muscle cells (PASMCs) were divided into control group, hypoxia group, mimics NC group, miR-204 mimics group, pcDNA3.1 group and Runx2 group. The proliferation of PASMCs cell was detected by CCK-8 method and EdU method, the cell cycle distribution was detected by flow cytometry, and the migration of PASMCs cell was detected by Transwell assay. The mRNA expression levels of miR-204 and Runx2 in lung tissue and PASMCs cells were detected by qRT-PCR. The protein expression levels of Runx2, α-SMA, CDKN4, Cyclin E and Cyclin D1 in lung tissue or PASMCs cells were detected by Western blot. Results: Compared with the control group, the expression level of miR-204 in lung tissue of rats in PAH group was decreased (P<0.05), the expression levels of Runx2 and α-SMA protein were increased (P<0.05), the pulmonary artery wall was thickened, lumen was narrow, and cardiomyocyte hypertrophy was observed. Compared with the NC group, the expression level of miR-204 in the lung tissues of rats in miR-204 group was increased (P<0.05), and the expression levels of Runx2 and α-SMA protein were decreased (P<0.05), and the thickening of pulmonary artery wall and cardiomyocyte hypertrophy of rats were significantly improved. miR-204 has a targeted binding relationship with Runx2. Compared with the control group, the expression level of miR-204 and CDKN4 protein, and the distribution ratio of G0/G1 phase were decreased in hypoxia group (P<0.05), and the expression levels of Runx2 mRNA and protein, Cyclin E and Cyclin D1 protein were increased (P<0.05). The cell proliferation level and migration number were increased (P<0.05). Compared with the mimics NC group, the expression level of miR-204 and CDKN4 protein, and the distribution ratio of G0/G1 phase were increased in miR-204 mimics group (P<0.05), and the expression levels of Runx2 mRNA and protein, Cyclin E and Cyclin D1 protein were decreased (P<0.05). The cell proliferation level and migration number were decreased (P<0.05). Compared with the pcDNA3.1 group, the expression level of miR-204 and CDKN4 protein, and the distribution ratio of G0/G1 phase were decreased in Runx2 group (P<0.05), and the expression levels of Runx2 mRNA and protein, Cyclin E and Cyclin D1 protein were increased (P<0.05). The cell proliferation level and migration number were increased (P<0.05). Conclusion: miR-204 is underexpressed in PAH model rats, and overexpression of miR-204 can inhibit the proliferation and migration of PASMCs cells and promote cell division arrest in the G0/G1 phase, which may be related to the targeted inhibition of Runx2 protein expression.

查看全文查看/发表评论下载PDF阅读器

关闭