The Potential Role of miR205 and miR205HG in Pulmonary Fibrosis

Abstract Rationale:MicroRNA 205 (miR205) is implicated in the regulation of the epithelial-to-mesenchymal transition (EMT). miR205 and LncRNA miR205 Host Gene (miR205HG) are implicated in fibrogenesis and the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF). Both miR205 and TP63, its upstream regulator, are thought to be exclusively expressed in basal cells. We have previously reported that aberrant basaloid cells, an exclusive cell culture in IPF with EMT phenotype, have higher expression of TP63, but have lower expression of miR-205HG. We sought to characterize the effect of miR205 and miR205HG knockdown in vitro. Methods:miRscope in situ hybridization (ISH) was done on IPF and control tissues to stain for miR205. Induced pluripotent stem cell (iPSC) derived basal cells (iBC) were cultured. Knockdown of miR205 was performed using siRNA, and miR205HG was knocked down using custom LNA antisense gapmers. Multi-level analysis was done using qPCR, nCounter miRNA panel, and bulk RNA sequencing. miR qPCR was done to validate the knockdown of miIR205 mature RNA. qPCR was also done on transcriptional predecessors of mir205 to validate specific binding of siRNAs and on downstream effectors in fibrotic processes and in EMT. Results:ISH revealed miR205 aggregation around fibroblastic foci on IPF tissue [Fig A]. miR205 siRNA knockdown was confirmed by qPCR analysis, and this was specific to miR205, not to its transcriptional predecessors, including miR205HG or pre-miR-205 [Fig B]. While there was no significant change in TP63 when mature miR205 was knocked down in iBCs, as shown by qPCR, This led to over-expression of fibrosis related genes, including MMP7 and GPR87 [Fig 1A]. miR205HG knockdown using Gapmer led to over-expression of TP63, as well as fibrosis related genes including GPR87, COL1A1, CDH2 AND VIM [Fig C]. Conclusion:miR205 and miR205HG seem to play complex, context-dependent roles in regulating IPF. The presence of miR205 positive cells along myofibroblastic foci in IPF tissue highlights the complexity of these pathways in IPF development. Additionally, gapmer-mediated knockdown of MIR205HG shows an abnormal basaloid cell phenotype, characterized by increased expression of the airway basal cell marker, EMT markers, and upregulation of genes which are involved in IPF pathogenesis. Legends: A. miRscope ISH of miR-205 (Red), nuclear stain in blue (DAPI) in IPF tissue B. Gene expression, as analyzed by qPCR, iBC control vs siRNA silencing of mature MiR-205C. Gene expression, as analyzed by qPCR iBC control vs gapmer silencing of miR- 205HG