lncITPF对邻近基因ITGBL1及对靶miRNA调控的ceRNA机制在特发性肺纤维化中的作用

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and devastating interstitial lung disease with unclear nature. Until now, IPF remains a disease with incompletely understood molecular mechanism underlying the pathogenesis and few treatment options. So it is very important clinical value to look for its mechanism and target gene for effective prevention and treatment of pulmonary fibrosis. Emerging evidence has purported lncRNA as a new class of players involved in the development and progression of various diseases which was considered as a new therapeutic strategy. In our previous study, we found a novel lncRNA transcript, named lncITPF, was expressed at higher levels in fibrotic tissue from rat when treatment with bleomycin compared with the normal. Interestingly, lncITPF was also upregulated in plasma collected from IPF patients. In the present study, we further identified lncITPF as a novel intronic lncRNA; and demonstrated it promoted the progression of IPF by affecting epithelial-mesenchymal transition and myofibroblasts function. To explore whether the mechanism of lncITPF was related to activating its host gene, integrin β-like 1 (ITGBL1) and acting as K-ras ceRNA to competitive binding miR-615-5p which is benefit for seeking the marker gene/protein in pulmonary fibrosis. Meanwhile, we explored whether lncITPF could be a biomarker for IPF disease and searched the potential drug targeted to lncITPF.

特发性肺纤维化（IPF)是一种病因及机制不清的弥漫性肺间质疾病，研究其发病机制，寻找其生物标志物及药物作用靶点具有重要的临床价值。长链非编码RNA(lncRNA)作为新兴的生物标志物和药物治疗靶点受到高度关注。本项目组在前期研究中，发现了一个尚未报道的大鼠lncRNA，其序列经分析显示在人基因组的13号染色体上有一段高度同源的序列，位于整合素β样1（ITGBL1）基因的内含子区域，我们将其命名为lncITPF，临床研究发现lncITPF在IPF病人中表达显著上调。本项目拟进一步通过临床与基础实验，研究lncITPF与IPF发生发展的相关性、lncITPF对临近基因ITGBL1的调控、及对靶miRNA调控的ceRNA（miR-615-5p/lncITPF/k-ras）机制，明确其能否作为血液中稳定存在的IPF病情进展和/或急性发作的生物标志物；同时为IPF的基因或药物治疗提供作用靶点。

特发性肺纤维化（idiopathic pulmonary fibrosis, IPF）是一种病因不明、慢性进展性纤维化型间质性肺炎。目前IPF疾病面临的突出问题为诊断困难、机制不清、治疗局限和预后不良。近年来，长链非编码RNA（long non-coding RNA，lncRNA）作为新兴的生物标志物和药物治疗靶点受到高度关注。然而，lncRNA在IPF中的作用机制尚不清楚。该项目研究中，我们发现了一个在IPF中上调的新的lncRNA，将其命名为lncITPF。生物信息学和体外翻译实验等证实了lncITPF是一个lncRNA。Northern-blot和cDNA末端快速扩增（rapid amplification of complementary DNA ends，RACE）等实验获得了lncITPF的全长序列。RNA-FISH和核质分离实验表明，lncITPF主要定位于细胞核。RNA测序、染色质免疫沉淀（chromatin immunoprecipitation，ChIP）-qPCR、CRISPR-Cas9和启动子活性分析等实验表明，lncITPF的促纤维化功能依赖于其宿主基因--整合素β样1（integrin β-like 1，ITGBL1），但两者并不共享启动子，也不共转录。荧光素酶报告基因和ChIP-qPCR等实验结果表明，Smad2/3与lncITPF启动子结合，TGFβ1以Smad2/3依赖的通路诱导lncITPF转录。RNA pull-down+蛋白质谱、RNA结合蛋白免疫沉淀(RNA Binding Protein Immunoprecipitation, RIP)等实验表明，lncITPF通过靶向异质性核糖核蛋白L（heterogeneous nuclear ribonucleoprotein L，hnRNP L）调节ITGBL1启动子区H3和H4组蛋白乙酰化促进IPF发生发展。研究结果显示lncITPF还可通过分别吸附miR-615-5p和miR-29b-2-5p调控K-ras和STAT3通路；此外，lncITPF能作为化合物芒柄花素-7-丹酚酸脂和虾青素抗肺纤维化的分子靶点。lncITPF在IPF患者血液中显著高表达且与IPF疾病临床指标密切相关。该项目为IPF提供了潜在的治疗靶点或诊断生物标志物。