SMOC2 促进低氧诱导的肺血管平滑肌细胞表型转换

SMOC2 drives hypoxia-induced phenotypic switching in pulmonary vascular smooth muscle cells

摘要

目的：探讨分泌型模块化钙结合蛋白2（secreted modular calcium-binding protein 2，SMOC2）是否参与肺动脉高压（pulmonary hypertension，PH）形成时miR-125a/miR-150介导的肺动脉平滑肌细胞（pulmonary artery smooth muscle cell，PASMC）表型转换。方法：收集PH患者和正常对照者的肺组织及其切片，慢性缺氧诱导小鼠PH模型，SU5416联合慢性缺氧诱导大鼠PH模型，通过Western blot检测PH患者及PH动物模型肺组织SMOC2的表达水平，免疫荧光染色检测SMOC2在肺动脉中细胞定位。转染SMOC2 siRNA、添加SMOC2重组蛋白以及转染miR-125a/miR-150模拟物至人源PASMC，Western blot检测PASMC增殖标志物以及收缩标志物的蛋白表达，利用EdU掺入实验及划痕实验评估PASMC增殖和迁移。双荧光素酶报告基因实验验证SMOC2作为miR-125a/miR-150的靶基因，并检测miR-125a/miR-150对低氧诱导PASMC增殖和迁移的影响。结果：在PH患者、PH模型小鼠和PH大鼠肺组织，以及低氧诱导的PASMC中SMOC2表达显著升高。敲低SMOC2可显著抑制低氧诱导的PASMC增殖与迁移；SMOC2重组蛋白处理则促进PASMC增殖与迁移。双荧光素酶报告基因实验证实SMOC2是miR-125a/miR-150的共同靶基因。过表达miR-125a/miR-150后，PASMC中SMOC2蛋白水平下降。挽救实验表明，SMOC2重组蛋白可部分逆转miR-125a/miR-150过表达对PASMC增殖与迁移的抑制作用。结论：miR-125a/miR-150通过靶向SMOC2调控低氧诱导的PASMC表型转换，为以抑制PASMC表型转换为核心的肺动脉重构治疗提供了潜在新靶点。

Abstract

Objective：To investigate whether secreted modular calcium-binding protein 2（SMOC2）participates in miR-125a/miR-150-mediated phenotypic switching of pulmonary artery smooth muscle cell（PASMC）in pulmonary hypertnesion（PH）. Methods：Human lung tissues and lung sections were collected from pulmonary hypertension（PH）patients and healthy controls. PH animal models were established in mice exposed to chronic hypoxia and in rats treated with SU5416 combined with chronic hypoxia. SMOC2 expression levels in lung tissues from PH patients，mice，and rats were assessed by Western blot，while its cellular localization was determined by immunofluorescence staining. Human PASMC were transfected with SMOC2 siRNA and miRNA mimics，or treated with recombinant SMOC2 protein. PASMC proliferation and migration were evaluated using EdU incorporation and wound healing assays. Dual-luciferase reporter assays was performed to validate SMOC2 as a direct target of miR-125a/miR-150. Results：SMOC2 expression was significantly upregulated in the lung tissues from human and animal PH samples，thus in hypoxic PASMC. Inhibition of SMOC2 attenuated proliferation，migration，and phenotype switching in PASMC，whereas treatment with recombinant SMOC2 protein promoted PASMC proliferation and migration. miR-125a and miR-150 recognized and inhibted SMOC2 mRNA translation in PASMC. miR-125a and miR-150 mimics inhibited hypoxia-induced proliferation and phenotype switch of PASMC. Conclusion：miR-125a/miR-150 regulate hypoxia-induced PASMC phenotypic switching by directly targeting SMOC2，providing a potential therapeutic target for PH treatment.

关键词

肺动脉高压 / 分泌型模块化钙结合蛋白2 / 肺动脉平滑肌细胞 / 表型转换 / miR-125a / miR-150

Key words

pulmonary hypertension / secreted modular calcium-binding protein 2 / pulmonary artery smooth muscle cell / phenotype switching / miRNA-125a / miRNA-150

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基金资助

国家自然科学基金(82500069)

江苏省自然科学基金(BK20161034)