Long Noncoding RNA FENDRR Inhibits Lung Fibroblast Proliferation via a Reduction of β-Catenin

Our plain-English summary of Long Noncoding RNA FENDRR Inhibits Lung Fibroblast Proliferation via a Reduction of β-Catenin, by L.K. Senavirathna, Y. Liang, C. Huang, et al (International Journal of Molecular Sciences, 2021).

The study

• Authors: L.K. Senavirathna, Y. Liang, C. Huang, X. Yang, G. Bamunuarachchi, D. Xu, Q. Dang, P. Sivasami, K. Vaddadi, M.C. Munteanu, S. Hewawasam, P. Cheresh, D.W. Kamp, L. Liu
• Journal: International Journal of Molecular Sciences
• Published: 2021;22(16)
• Cited by: 17 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and usually lethal lung disease and it has been widely accepted that fibroblast proliferation is one of the key characteristics of IPF. Long noncoding RNAs (lncRNAs) play vital roles in the pathogenesis of many diseases. In this study, we investigated the role of lncRNA FENDRR on fibroblast proliferation. Human lung fibroblasts stably overexpressing FENDRR showed a reduced cell proliferation compared to those expressing the control vector. On the other hand, FENDRR silencing increased fibroblast proliferation. FENDRR bound serine-arginine rich splicing factor 9 (SRSF9) and inhibited the phosphorylation of p70 ribosomal S6 kinase 1 (PS6K), a downstream protein of the mammalian target of rapamycin (mTOR) signaling. Silencing SRSF9 reduced fibroblast proliferation. FENDRR reduced β-catenin protein, but not mRNA levels. The reduction of β-catenin protein levels in lung fibroblasts by gene silencing or chemical inhibitor decreased fibroblast proliferation. Adenovirus-mediated FENDRR transfer to the lungs of mice reduced asbestos-induced fibrotic lesions and collagen deposition. RNA sequencing of lung tissues identified 7 cell proliferation-related genes that were up-regulated by asbestos but reversed by FENDRR. In conclusion, FENDRR inhibits fibroblast proliferation and functions as an anti-fibrotic lncRNA.

In plain English

This laboratory study combined human lung fibroblast experiments with a mouse model of asbestos-induced lung fibrosis to test the effect of the long noncoding RNA FENDRR on fibroblast proliferation. In LL29 fibroblasts (from an IPF patient) stably overexpressing FENDRR (39 ± 11 fold over control), BrdU-measured proliferation fell by 68 ± 3% and cell counts by 42 ± 7%. Silencing FENDRR in normal human pulmonary fibroblasts (79 ± 5% reduction) raised proliferation by 34 ± 8%. RNA immunoprecipitation showed FENDRR bound SRSF9 (2.4 ± 0.30-fold enrichment over IgG), and FENDRR reduced PS6K phosphorylation. FENDRR lowered β-catenin protein by 41 ± 5% without changing its mRNA. Silencing β-catenin or treating with XAV939 reduced proliferation by 26 ± 3% and 54 ± 7%. In C57BL/6J mice given intratracheal crocidolite asbestos (50, 100 or 200 µg), fibrosis was dose-dependent at 3 weeks. In the 100 µg group, the only dose also examined at 2 months, fibrosis had not resolved, and lung Fendrr was reduced. In mice given 200 µg crocidolite, adenoviral FENDRR reduced fibrotic lesions, fibrosis scores and collagen deposition. The authors concluded FENDRR acts as an anti-fibrotic lncRNA.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study: human lung fibroblast cell-culture experiments plus a mouse model of crocidolite asbestos-induced pulmonary fibrosis with adenoviral gene transfer and RNA sequencing
• Who was studied: Two human lung fibroblast types: LL29 cells, from the lungs of an IPF patient, and primary human pulmonary fibroblasts from a healthy subject. Also male and female C57BL/6J mice aged 7-8 or 8 weeks. Group sizes in the adenovirus experiment were VC TiO2 = 15, FENDRR TiO2 = 12, VC Cro = 17 and FENDRR Cro = 15.
• Exposure: Mice received crocidolite asbestos intratracheally at 50, 100 or 200 µg per mouse, with TiO2 (100 µg) as the control; the FENDRR adenovirus experiment used 200 µg. In cell culture, the authors used FENDRR overexpression or silencing, SRSF9 silencing, β-catenin silencing or XAV939, and FGF2.
• Main results:
  • In LL29 fibroblasts overexpressing FENDRR (39 ± 11 fold over control cells), proliferation was inhibited by 68 ± 3% (BrdU assay) and cell numbers were reduced by 42 ± 7%
  • In normal human pulmonary fibroblasts, FENDRR silencing (expression reduced by 79 ± 5%) increased proliferation by 34 ± 8%
  • SRSF9 protein bound endogenous FENDRR in LL29 cells with 2.4 ± 0.30-fold enrichment compared with the IgG control. Silencing SRSF9 (65-68% knockdown by 3 shRNAs) reduced fibroblast proliferation by 39-48%.
  • FENDRR-expressing stable cells had a 41 ± 5% reduction in β-catenin protein compared with control cells, while β-catenin mRNA was unchanged
  • In LL29 cells, adenoviral β-catenin silencing (protein down 49 ± 7%) and XAV939 (protein down 60 ± 6%) reduced fibroblast proliferation by 26 ± 3% and 54 ± 7% respectively
  • In mice, crocidolite produced dose-dependent fibrotic lesions and fibrosis scores at 3 weeks, and fibrosis was not resolved at 2 months. No mice died after asbestos delivery.
  • All three doses (50, 100, 200 µg) were assessed at 3 weeks. Only the 100 µg crocidolite group (n = 9) and TiO2 controls (n = 7) were assessed at 2 months.
  • Lung Fendrr expression in asbestos-treated mice was reduced by 32-48% at 3 weeks and by 51% at 2 months
  • At 2 months, the fibrosis score in the asbestos group differed significantly from controls in male but not in female mice. There was a trend toward an increase in females.
  • The 200 µg crocidolite dose was chosen for further experiments. Adenovirus-mediated FENDRR transfer into mouse lungs reduced asbestos-induced fibrotic lesions, fibrosis scores and collagen deposition, with no apparent male-female differences in fibrosis scores.
  • RNA sequencing of lung tissue found 219 genes differentially expressed in VC-Cro versus VC-TiO2 mice (193 up-regulated, 26 down-regulated). Seventy genes differed between VC-Cro and FENDRR-Cro mice (20 up, 50 down). Seven crocidolite-upregulated genes attenuated by FENDRR were related to cell proliferation.
• The authors' conclusion: The authors concluded that FENDRR acts as an anti-fibrotic lncRNA. In their view, it inhibits fibroblast proliferation by binding SRSF9 and inhibiting mTOR signaling, which reduces β-catenin protein translation.
• Limitations the authors state:
  • The authors stated that competition experiments are still needed to confirm whether the reduced PS6K phosphorylation is due to FENDRR binding SRSF9
  • The authors stated that the mechanism by which asbestos reduces lung FENDRR expression is uncertain
  • The authors noted that further studies are required to explore whether the identified signaling pathways are involved in fibrosis and Fendrr regulation

Topics

Medical terms in this study, explained in our Lexicon: Fibroblast, Idiopathic, Pulmonary Fibrosis, Collagen, Deposition.

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