基于小RNA测序和数据库比对探究PRMT1调控的sncRNAs在哮喘中的作用

The Role of PRMT1-regulated sncRNAs in Asthma Based on Small RNA Sequencing and Database Comparison

小非编码RNA(small non-coding RNA,sncRNA)包括microRNA(miRNA)和PIWI蛋白互作RNA(PIWI-interacting RNA,piRNAs),其异常表达参与支气管哮喘(简称哮喘)气道上皮炎症过程。目前的研究多基于哮喘相关sncRNA的异常表达功能研究,少有对sncRNA表达变化原因的探究。我们前期的研究发现,蛋白质精氨酸甲基转移酶1(protein arginine methyltransferase 1,PRMT1)作为哮喘标志性分子,参与上皮细胞炎症的发生和气道下重塑。本文利用过表达人肺上皮细胞BEAS-2B中哮喘标志性基因PRMT1,进行sncRNA深度测序并与哮喘数据库联合分析,将差异表达的sncRNA与5个Gene Expression Omnibus(GEO)测序数据集进行比较,发现23种PRMT1调控的哮喘相关的sncRNA。利用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)验证,筛选出12种miRNA和2种piRNA。将这些sncRNA靶基因与哮喘病人生成的上皮细胞样本mRNA测序结果(GSE18965和GSE43696)进行交叉比对,初步确定哮喘相关基因,并进行Kyoto Encyclopedia of Genes and Genomes(KEGG)预测。分析表明,哮喘相关sncRNA主要靶标是Ras/Rap1-MAPK信号通路,包含53种靶基因。本研究通过小RNA测序(small RNA Seq)技术探寻PRMT1调控的下游sncRNA,比对现有的数据库,识别出PRMT1调节的sncRNA及其目标信号通路Ras/Rap1-MAPK,为哮喘的发病机制研究提供了新的sncRNA分子和信号通路靶点。

Aberrant expression of small non-coding RNAs(sncRNAs), including microRNAs(miRNAs) and PIWI-interacting RNAs(piRNAs), contributes to asthma pathologies and airway epithelial inflammation. The current research is mostly based on the abnormal expression of asthma-related sncRNAs. There is limited evidence to clarify the upstream of sncRNA expression. Our previous studies proved that protein arginine methyltransferase 1(PRMT1), as a hallmark of asthma, is linked to both epithelial inflammation and sub-epithelial remodeling. The aim of this study is to find the PRMT1 regulated sncRNAs which are also related to asthma. Human lung epithelial cell line BEAS-2 B was transfected with either PRMT1 expression plasmid pcDNA3.1-PRMT1 or control plasmid pcDNA3.1+ for 48 hours. Expression profile of small RNA was determined by small RNA deep sequencing. After comparing these sncRNAs with 5 datasets of microarrays(GEO dataset), 23 sncRNAs were related to asthma. Following data validation, 12 microRNAs and 2 PIWI-interacting RNAs target prediction analysis were performed. Through comparing the datasets with the GSE18965 and GSE43696, we focused on 639 overlapped target genes. The signaling pathways analysis of these 639 overlapped genes indicated that Ras/Rap1-mitogen activated protein kinase as a major target for PRMT1 induced sncRNAs. This study implied that the comparison of our database with existing databases can be used to identify PRMT1 regulated asthma related sncRNAs. Ras/Rap1-MAPK pathway may provide novel targets for therapy and diagnostics in asthma.