Yang Xue, Qi Fei, Wei Shanchen, Lin Lianjun, Liu Xinmin
Department of Geriatrics, Peking University First Hospital, Beijing, China.
Front Cell Dev Biol. 2021 Mar 12;9:651913. doi: 10.3389/fcell.2021.651913. eCollection 2021.
Recent studies were widely concerned about the role of lncRNAs in hypoxic pulmonary hypertension (HPH). HAS2 was found significantly highly expressed in HPH, but the antisense of HAS2 (HAS2-AS1) has not been explored in HPH, providing a new potential therapeutic target of HPH.
In this study, human fetal lung fibroblast-1 (HFL-1) cells were cultured under hypoxia conditions to stimulate the pathological process of HPH. Transwell and wound-healing assays were used to detect HFL-1 cell migration, and CCK 8 assay was used to detect cell proliferation. The upstream transcription factor of HAS2-AS1 was predicted by JASPAR website, and the binding site between C/EBPβ and HAS2-AS1 was predicted by JASPAR, too. In order to verify the association between C/EBPβ and the HAS2 promoter region, we used chromatin immunoprecipitation (ChIP) and dual luciferase reporter gene detection, western blot to detect the expression of inflammation-related proteins, and qRT-PCR to detect the expression of HAS2-AS1 and HAS2. Idiopathic pulmonary fibrosis (IPF) with HPH patient microarray data was downloaded from the GEO database and analyzed by R software.
Our study showed that HAS2-AS1 and C/EBPβ were highly expressed in hypoxic HFL-1 cells, and the knockdown of HAS2-AS1 expression could inhibit the proliferation, migration, and inflammatory response of HFL-1 cells. C/EBPβ binds to the promoter region of HAS2-AS1 and has a positive regulation effect on the transcription of HAS2-AS1. Furthermore, C/EBPβ can regulate the proliferation, migration, and inflammatory response of HFL-1 cells through HAS2-AS1.
This study suggested that C/EBPβ could upregulate HAS2-AS1 expression and induce HFL-1 cell proliferation, migration, and inflammation response.
近期研究广泛关注长链非编码RNA（lncRNAs）在缺氧性肺动脉高压（HPH）中的作用。已发现HAS2在HPH中显著高表达，但尚未对HAS2的反义链（HAS2-AS1）在HPH中的情况进行研究，这为HPH提供了一个新的潜在治疗靶点。
在本研究中，将人胎儿肺成纤维细胞-1（HFL-1）细胞在缺氧条件下培养以模拟HPH的病理过程。采用Transwell和伤口愈合实验检测HFL-1细胞迁移，采用CCK 8实验检测细胞增殖。通过JASPAR网站预测HAS2-AS1的上游转录因子，同时也通过JASPAR预测C/EBPβ与HAS2-AS1之间的结合位点。为验证C/EBPβ与HAS2启动子区域之间的关联，我们采用染色质免疫沉淀（ChIP）和双荧光素酶报告基因检测，蛋白质免疫印迹法检测炎症相关蛋白的表达，以及qRT-PCR检测HAS2-AS1和HAS2的表达。从GEO数据库下载特发性肺纤维化（IPF）合并HPH患者的微阵列数据并通过R软件进行分析。
我们的研究表明，HAS2-AS1和C/EBPβ在缺氧的HFL-1细胞中高表达，敲低HAS2-AS1表达可抑制HFL-1细胞的增殖、迁移和炎症反应。C/EBPβ与HAS2-AS1的启动子区域结合并对HAS2-AS1的转录具有正调控作用。此外，C/EBPβ可通过HAS2-AS1调节HFL-1细胞的增殖、迁移和炎症反应。
本研究提示，C/EBPβ可上调HAS2-AS1表达并诱导HFL-1细胞增殖、迁移和炎症反应。
