MEG3 is increased in idiopathic pulmonary fibrosis and regulates epithelial cell differentiation
Jason J. Gokey, John Snowball, Anusha Sridharan, Joseph P. Speth, Katharine E. Black, Lida P. Hariri, Anne-Karina T. Perl, Yan Xu, Jeffrey A. Whitsett
Jason J. Gokey, John Snowball, Anusha Sridharan, Joseph P. Speth, Katharine E. Black, Lida P. Hariri, Anne-Karina T. Perl, Yan Xu, Jeffrey A. Whitsett
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Research Article Pulmonology

MEG3 is increased in idiopathic pulmonary fibrosis and regulates epithelial cell differentiation

Abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease causing fibrotic remodeling of the peripheral lung, leading to respiratory failure. Peripheral pulmonary epithelial cells lose normal alveolar epithelial gene expression patterns and variably express genes associated with diverse conducting airway epithelial cells, including basal cells. Single-cell RNA sequencing of pulmonary epithelial cells isolated from IPF lung tissue demonstrated altered expression of LncRNAs, including increased MEG3. MEG3 RNA was highly expressed in subsets of the atypical IPF epithelial cells and correlated with conducting airway epithelial gene expression patterns. Expression of MEG3 in human pulmonary epithelial cell lines increased basal cell–associated RNAs, including TP63, KRT14, STAT3, and YAP1, and enhanced cell migration, consistent with a role for MEG3 in regulating basal cell identity. MEG3 reduced expression of TP73, SOX2, and Notch-associated RNAs HES1 and HEY1, in primary human bronchial epithelial cells, demonstrating a role for MEG3 in the inhibition of genes influencing basal cell differentiation into club, ciliated, or goblet cells. MEG3 induced basal cell genes and suppressed genes associated with terminal differentiation of airway cells, supporting a role for MEG3 in regulation of basal progenitor cell functions, which may contribute to tissue remodeling in IPF.

Authors

Jason J. Gokey, John Snowball, Anusha Sridharan, Joseph P. Speth, Katharine E. Black, Lida P. Hariri, Anne-Karina T. Perl, Yan Xu, Jeffrey A. Whitsett

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Figure 5

MEG3-binding sites in the promoters of genes associated with basal cell differentiation.

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MEG3-binding sites in the promoters of genes associated with basal cell ...
ChOP-sequencing data of MEG3-binding sites in BT-549 cells (14) were analyzed using Homer’s Annotate Peak and visualized using integrated genomics viewer to reveal MEG3-binding sites in gene promoters and compared with differentially expressed genes identified by analyzing single-cell RNA sequencing of IPF and control lungs, as described in the Methods. MEG3-binding sites within promoters (arrows) were identified in genes (blue) associated with basal cells, including AXL, ITGB4, KRT15, KRT19, and FOXA2, which are altered in IPF. MEG3-binding sites were detected in the promoters of SOX2, STAT3, and HEY1 and in the TA/ΔN splicing region of TP63 and TP73, as shown as gray peaks with arrows in the gene maps. (A) Expression of basal cell–associated genes in IPF epithelial cell types from single-cell sequencing (5) and location of MEG3-binding sites curated from ChOP data (14). Box-and-whisker plots represent the first and third quartile (box), median (line), mean (+), and minimum and maximum of the data (whiskers). TPM expression values are represented on a log2 scale. (B) Location of MEG3-binding sites in the promoters of STAT3, SOX2, and HEY1 and before the ΔN TP63 and TP73 start sites. Peaks were visualized using Integrative Genomics Viewer software. (C) Western blots of MEG3-transfected HBEC3KT cells were used to assess TP63 splice variants. (D) MEG3 transfection inhibited levels of all ΔNTP63 isoforms and did not alter TAP63 isoforms in HBEC3KT cells. Differences in RNA expressions were determined by ANOVA followed by Holm-Bonferroni post hoc test. *P < 0.01.