Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is a key mechanism in pulmonary arterial hypertension (PAH). Serotonin (5-hydroxytryptamine, 5-HT) can induce abnormal proliferation of PASMCs. The role of miR-361-3p in serotonin-induced abnormal PASMCs proliferation remains unclear. The miR-361-3p level was analyzed in plasma from PAH patients and normal controls and in human PASMCs (hPASMCs) using RT-PCR. The hPASMCs were transfected with an miR-361-3p mimic and then treated with serotonin. Untransfected hPASMCs were used as the control. Cell proliferation was evaluated using an MTS assay and 5-ethynyl-2′-deoxyuridine (EdU) staining. The cell cycle stages were evaluated using flow cytometry. The association between miR-361-3p and serotonin transporter (SERT) was determined using a luciferase reporter assay and anti-AGO2 RNA immunoprecipitation assay. The protein expression was evaluated via western blotting. The miR-361-3p level was lower in plasma from PAH patients than in plasma from the any of the normal control subjects. The mean pulmonary arterial pressure, pulmonary vascular resistance and pulmonary vascular resistance index were higher in PAH patients whose miR-361-3p level was lower than the median value for patients than in those whose miR-361-3p level was higher than the median. Serotonin treatment reduced miR-361-3p expression in the hPASMCs. MiR-361-3p overexpression suppressed cell proliferation, promoted apoptosis, induced G1 arrest, and decreased the phosphorylation level of ERK1/2 in serotonin-treated hPASMCs. SERT was identified as an miR-361-3p target. Its overexpression alleviated the effect of miR-361-3p overexpression on serotonin-induced hPASMC proliferation and upregulation of phosphorylated ERK1/2. The miR-361-3p level is lower in the plasma of PAH patients. Upregulation of miR-361-3p suppresses serotonin-induced proliferation of hPASMCs by targeting SERT. Our results suggest that miR-361-3p is a potential therapeutic target in PAH.

中文翻译：

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miR-361-3p 的上调通过靶向 SERT 抑制血清素诱导的人肺动脉平滑肌细胞增殖

肺动脉平滑肌细胞（PASMCs）的异常增殖是肺动脉高压（PAH）的一个关键机制。5-羟色胺（5-羟色胺，5-HT）可诱导 PASMC 异常增殖。miR-361-3p 在血清素诱导的异常 PASMCs 增殖中的作用仍不清楚。使用 RT-PCR 分析来自 PAH 患者和正常对照的血浆以及人 PASMC (hPASMC) 中的 miR-361-3p 水平。用 miR-361-3p 模拟物转染 hPASMC，然后用血清素处理。未转染的 hPASMC 用作对照。使用 MTS 测定和 5-乙炔基-2'-脱氧尿苷 (EdU) 染色评估细胞增殖。使用流式细胞术评估细胞周期阶段。使用荧光素酶报告基因测定和抗 AGO2 RNA 免疫沉淀测定确定 miR-361-3p 和血清素转运蛋白 (SERT) 之间的关联。通过蛋白质印迹评估蛋白质表达。PAH患者血浆中的miR-361-3p水平低于任何正常对照受试者的血浆。miR-361-3p水平低于中位值的PAH患者的平均肺动脉压、肺血管阻力和肺血管阻力指数高于miR-361-3p水平高于中位值的患者。血清素处理降低了 hPASMCs 中 miR-361-3p 的表达。MiR-361-3p 过表达抑制细胞增殖，促进细胞凋亡，诱导 G1 期阻滞，并降低血清素处理的 hPASMC 中 ERK1/2 的磷酸化水平。SERT 被鉴定为 miR-361-3p 靶标。其过表达减轻了 miR-361-3p 过表达对血清素诱导的 hPASMC 增殖和磷酸化 ERK1/2 上调的影响。PAH患者血浆中的miR-361-3p水平较低。miR-361-3p 的上调通过靶向 SERT 抑制血清素诱导的 hPASMCs 增殖。我们的结果表明 miR-361-3p 是 PAH 的潜在治疗靶点。