MiR-541-5p regulates lung fibrosis by targeting cyclic nucleotide phosphodiesterase 1A

<b>Aim of the Study:</b> Idiopathic pulmonary fibrosis (IPF) is a lethal human disease with short survival time and few treatment options. In this study, we aim to demonstrate that cyclic nucleotide phosphodiesterase 1A (PDE1A), a Ca2+/calmodulin-stimulating PDE family member, plays a critical role in the induction of fibrosis and angiogenesis in the lung. <b>Materials and Methods:</b> To induce pulmonary damage, adult male SD rats were treated with bleomycin in a dose of 6 mg/kg body weight by a single intratracheal instillation. For in vivo silencing of PDE1A in rat lung, a nonspecific control siRNA or PDE1A-specific siRNA was used to treat rat through nasal instillation. Human normal pulmonary fibroblasts MRC-5 and hFL1 and rat lung fibroblasts were used as in vitro model. Immunohistochemistry and immunoflurescence staining were performed to detect PDE1A and α-SMA expression. Reverse transcription-qPCR was performed to detect microRNA and mRNA expression. In vitro wound healing assay was performed to detect pulmonary fibroblasts'mortality ability. <b>Results:</b> In vitro studies showed that PDE1A can stimulate lung fibroblasts to undergo myofibroblastic changes. This led to the identification of miR-541-5p as one of the miRNA candidates associated with bleomycin response. We found that miR-541-5p expression is downregulated in TGF-β-treated lung fibroblasts and the rat pulmonary fibrosis model. Overexpression of miR-541-5p in lung fibroblasts inhibited mortality of human lung fibroblasts. <b>Conclusions:</b> MiR-541-5p is a key effector in lung fibroblastsby by regulating PDE1A expression at protein translation level and its overexpression is protective against bleomycin-induced lung fibrosis.

**研究目的**：特发性肺纤维化（Idiopathic pulmonary fibrosis, IPF）是一种致死性人类疾病，患者生存期短且治疗手段匮乏。本研究旨在证实，环核苷酸磷酸二酯酶1A（cyclic nucleotide phosphodiesterase 1A, PDE1A）——一种钙/钙调蛋白激活的磷酸二酯酶家族成员——在肺纤维化与血管生成的诱导过程中发挥关键作用。
**实验材料与方法**：为构建肺损伤模型，成年雄性SD大鼠接受单次气管内滴注博莱霉素处理，剂量为6mg/kg体重。为在大鼠肺内实现PDE1A的体内沉默，本研究采用非特异性对照小干扰RNA（small interfering RNA, siRNA）或PDE1A特异性小干扰RNA经鼻腔滴注方式对大鼠进行给药。本研究选用人类正常肺成纤维细胞MRC-5、hFL1以及大鼠肺成纤维细胞作为体外实验模型。采用免疫组化与免疫荧光染色检测PDE1A及α-平滑肌肌动蛋白（α-smooth muscle actin, α-SMA）的表达水平；采用逆转录定量聚合酶链反应（reverse transcription-quantitative PCR, RT-qPCR）检测微小RNA（microRNA, miRNA）及信使RNA（messenger RNA, mRNA）的表达水平；采用体外划痕愈合实验检测肺成纤维细胞的存活能力。
**实验结果**：体外实验表明，PDE1A可诱导肺成纤维细胞发生肌成纤维细胞转化。本研究据此筛选出与博莱霉素应答相关的miRNA候选分子miR-541-5p。研究发现，在转化生长因子-β（transforming growth factor-β, TGF-β）处理的肺成纤维细胞及博莱霉素诱导的大鼠肺纤维化模型中，miR-541-5p的表达均出现下调。在肺成纤维细胞中过表达miR-541-5p可抑制人肺成纤维细胞的存活能力。
**研究结论**：miR-541-5p通过在蛋白质翻译水平调控PDE1A的表达，成为肺成纤维细胞中的关键效应分子，其过表达可对博莱霉素诱导的肺纤维化起到保护作用。