Cross-Talk Between Epigenetic Regulation And Mir-17~92 Cluster Expression In Idiopathic Pulmonary Fibrosis (IPF).

摘要

Interstitial lung disease/idiopathic pulmonary fibrosis (ILD/IPF) is the most progressive form of pulmonary fibrosis disorder and leads to death in patients afflicted. The etiology and pathogenesis of ILD/IPF in poorly understood with no known prevention or cure available.;ILD/IPF is associated with increased expression of certain fibrogenic genes such as CTGF, VEGF, GM-CSF , and TSP-1; however, the precise mechanism responsible for the increased gene and protein expression is not known. Since there are a large number of genes differentially expressed in the lungs of patients with ILD/IPF compared to controls, we speculated that elements like microRNAs (miRNAs) may be involved. miRNAs, also known as miRs, are small regulatory RNAs that alter gene expression by causing degradation, translational repression, or epigenetic regulation resulting in a change in protein expression. Changes in miRNA expression are associated with up to 30% of cancers (Volinia et. al., 2010) and a variety of other diseases (Nana-Sinkam et. al., 2009). Using miRNA profiling, we identified a specific miRNA cluster that was reduced in expression in ILD/IPF lung tissue compared to normal lung tissue and lung tissue from patients with chronic obstructive pulmonary disease (COPD), the miR-17∼92 cluster. The miR-17∼92 polycistron cluster is ∼ 1Kb located within the third intron of the open reading frame13q31.3 (C13orf25) which encodes six microRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1).;This locus is overexpressed in B-cell lymphomas and lung cancer. In ILD/IPF, miR-17∼92 miRNA cluster targets genes, such as metalloproteinases, collagen, and transforming growth factor that are highly expressed.;Our preliminary data indicated decreased expression of the miR-17∼92 miRNA cluster family members in the lungs from patients with ILD/IPF. Notably, miR-19b and miR-20a decreased proportionally to disease severity (and pulmonary function) of ILD/IPF. In ILD/IPF lung tissue, miR-19b and miR-20a expression was reduced and its predicted targets, CTGF, VEGF, TGF-beta, TSP-1 and Ets-1 were up-regulated. Importantly, we noticed that the 5'-region of the promoter is heavily occupied with CpG Islands (>90%) and sought to define if this area was affected by methylation in ILD/IPF, as the mechanism causing downregulation of the cluster expression in ILD/IPF. Our preliminary data suggested that this epigenetic silencing of miR-17∼92 clusters was due to methylation of the promoter, and was increased with the severity of ILD/IPF. Indeed, we found this CpG island methylated in ILD/IPF and by relieving this methylation with 5'-aza-2'-deoxycytidine, enhanced cluster expression and decreased expression of the target genes. In addition, we found that the miR-17∼92 cluster targeted DNA methyltransferases (DNMTs), which we believe are responsible for the methylation events in ILD/IPF. Interestingly, our data showed direct regulation of miR-17, miR-19b , miR-20a and miR-92 on the 3'UTR of DNMT-1 in lung fibroblasts isolated from patients with IPF, indicating a negative feedback mechanism of the cluster to its own promoter though an epigenetic modifier.;In fact, inhibition of DNA methylation by 5'-aza-2'-deoxycytidine in our in vivo murine bleomycin-induced pulmonary fibrosis model recovered miR-17∼92 cluster expression to that of normal lung tissue, rescued the animals' lungs from fibrosis, and decreased fibrotic gene expression in their lungs. Thus, our data indicate that dysregulation of miRNA expression in IPF may contribute to the development and/or progression of the disease. 5'-aza-2'-deoxycytidine treatment, or miR-17∼92 overexpression, could be a therapeutic intervention in ILD/IPF.