Mir‐96 Expression in non‐IPF Fibroblasts Changes their Phenotype from Normal to Fibrotic via FoxO3a Suppression
Authors: Nho Richard 
Published: June 2021

RationaleIdiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease that the precise etiology is unknown. IPF is caused by the hyper‐activation of apoptosis‐resistant (myo)fibroblasts that produce excessive amounts of collagen rich extracellular matrix (ECM). When normal lung fibroblasts interact with a collagen matrix, they undergo apoptosis, which prevents excessive fibro‐proliferation after tissue injury. However, fibroblasts from IPF patients (IPF fibroblasts) elude this negative regulatory property of collagen, promoting lung fibrosis. However, the crucial mechanism that protects IPF fibroblasts from collagen matrix‐driven apoptosis has not been understood.Study objectiveWe found that Forkhead box O3a (FoxO3a) that promotes apoptosis was suppressed as a result of abnormally high non‐coding RNA miR‐ 96 in IPF fibroblasts, and that this deregulation caused them to maintain a highly viable phenotype in response to a collagen rich matrix. Based on this finding, our study objective was to examine whether miR‐96‐dependent FoxO3a alteration can reprogram non‐IPF lung fibroblasts to be fibrotic, thereby subsequently participating in promoting lung fibrosis. For this, we altered miR‐96 levels in non‐ IPF fibroblasts and investigated FoxO3a levels, fibroblast viability and proliferation on an apoptosis‐inducing 3D collagen rich ECM.ResultsForced expression of miR‐96 in non‐IPF fibroblasts by miR‐96 mimic suppressed FoxO3a and its target proteins Bim, p27 and p21, which caused them to acquire apoptosis‐resistant and proliferative properties that were mainly seen in IPF fibroblasts on collagen. Unlike these, expression of miR‐96 in IPF fibroblasts had no significant effects on fibroblast viability and FoxO3a target protein levels potentially due to pre‐existing enhanced miR‐96. In contrast, miR‐ 96 inhibition greatly sensitized IPF fibroblasts to collagen matrix‐mediated apoptosis. To further test the pathological role of miR‐96‐dependent FoxO3a alteration in the development of lung fibrosis, miR‐96 and FoxO3a levels were also examined in C57/BL6 mice (n=8) with lung fibrosis. Real time PCR and Western analysis showed that miR‐96 levels were significantly increased while FoxO3a levels were reduced in fibrotic mouse lungs compared with that of normal mice. In accordance with this finding, miR‐96 levels were also increased while FoxO3a levels were low in lung tissues (n=4) from IPF patients.ConclusionsOur findings suggest that miR‐96 modulation reprograms non‐IPF fibroblast phenotype from normal to fibrotic via FoxO3a alteration, and that the presence of miR‐96 activated fibroblasts plays a role in promoting lung fibrosis via FoxO3a suppression. These results indicate that targeting miR‐96 can be an effective approach to mitigate the development of lung fibrosis.Support or Funding InformationThis abstract is funded by the National Heart, Lung and Blood Institute Grant R01 HL114662 (Richard Nho).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.