TGF‐β1‐induced miR‐424 Mediates Pulmonary Myofibroblast Differentiation via Targeting Slit2

다국어 초록 (Multilingual Abstract)

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal interstitial lung disease. Transforming growth factor (TGF)‐β1, a major profibrogenic cytokine, induces differentiation of pulmonary fibroblasts to myofibroblasts that produce high levels of extracellular matrix, leading to loss of lung elasticity and function. Emerging evidence has implicated the importance of microRNAs (miRNAs) in the development of IPF but their regulation and individual pathological roles remain largely unknown. The objective of our study was to define the pro‐fibrotic roles and mechanisms of miRNAs in TGF‐β1‐induced pulmonary fibroblast differentiation.
We used both RNA sequencing and quantitative RT‐PCR strategies to systematically study TGF‐β1‐induced alternations of miRNAs in human lung fibroblasts (HLF). Significantly upregulated miRNAs in HLF were confirmed in lung tissue of IPF patients. Then, we overexpressed the identified miRNAs or their inhibitors to determine their importance in TGF‐β1‐induced HLF differentiation. Western blot and immunofluorescence staining were used to measure the changes in TGF‐β1 signaling and HLF differentiation. Finally, we searched for the putative targets of these miRNAs using online algorithms (Targetscan and mirSVR) and luciferase reporter assays were performed to determine whether the predicted binding site is directly targeted.
We identified miR‐424 as an important TGF‐β1‐induced miRNA in HLF. Quantitative RT‐PCR confirmed that miR‐424 expression was increased by 2.5‐fold in HLF in response to TGF‐β1 and was 2‐fold higher in IPF patients as compared to normal subjects. Overexpression of a miR‐424 inhibitor in HLF reduced TGF‐β1‐induced expression of classic myofibroblast differentiation markers including α‐smooth muscle actin (α‐SMA) and connective tissue growth factor (CTGF), whereas a miR‐424 mimic significantly enhanced TGF‐β1‐induced differentiation. Interestingly, upregulation of miR‐424 was blocked by the Smad3 inhibitor SIS3, suggesting the involvement of the canonical TGF‐β1 signaling pathway. In addition, TGF‐β1‐induced Smad3 phosphorylation in HLF was reduced by the miR‐424 inhibitor. Among the putative targets of miR‐424, Slit2 inhibits TGF‐β1‐induced Smad3 phosphorylation. Luciferase reporter assays showed that miR‐424 represses wild‐type Slit2‐3′UTR reporter activity without effects on a mutant Slit2‐3′UTR reporter, indicating the direct regulation by miR‐424 of the 3′UTR of Slit2 mRNA. Indeed, overexpression of the miR‐424 mimic down‐regulated Slit2 expression in HLF, consistent with the reduced Slit2 expression in IPF patients.
MiR‐424 plays a pro‐fibrotic role in TGF‐β1‐induced HLF differentiation. It functions as a positive feed‐back regulator of the TGF‐β1 signaling pathway via targeting Slit2. Thus, inhibition of miR‐424 may provide a new therapeutic strategy to prevent IPF progression.
Support or Funding Information
Supported by grants from NIH R01HL116849 to YT and AG053553 to KB, Nebraska State LB595 to YT and PWA
This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.