Small interfering RNAs (siRNAs) targeting TGF-beta1 mRNA suppress asbestos-induced expression of TGF-beta1 and CTGF in fibroblasts

Our plain-English summary of Small interfering RNAs (siRNAs) targeting TGF-beta1 mRNA suppress asbestos-induced expression of TGF-beta1 and CTGF in fibroblasts, by T.C. Lai, D.A. Pociask, M. Ferris, et al (Journal of Environmental Pathology, Toxicology and Oncology, 2009).

The study

• Authors: T.C. Lai, D.A. Pociask, M. Ferris, H.T. Nguyen, C.A. Miller, A. Brody, D. Sullivan
• Journal: Journal of Environmental Pathology, Toxicology and Oncology
• Published: 2009;28(2):109-19
• Cited by: 7 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Interstitial lung disease (ILD) afflicts millions of people worldwide. ILD can be caused by a number of agents, including inhaled asbestos, and may ultimately result in respiratory failure and death. Currently, there are no effective treatments for ILD. Transforming growth factor-beta1 (TGF-beta1) is thought to play an important role in the development of pulmonary fibrosis, and asbestos has been shown to induce TGF-beta1 expression in a murine model of ILD. To better define the role of TGF-beta1 in ILD, we developed several small interfering RNAs (siRNAs) that target TGF-beta1 mRNA for degradation. To assess the efficacy of each siRNA in reducing asbestos-induced TGF-beta1 expression, Swiss 3T3 fibroblasts were transfected with TGF-beta1 siRNAs and then treated with chrysotile asbestos for 48 h. Two independent siRNAs targeting TGF-beta1 mRNA knocked-down asbestos-induced expression of TGF-beta1 mRNA by 72-89% and protein by 70-84%. Interestingly, siRNA knockdown of TGF-beta1 also reduced asbestos-induced expression of connective tissue growth factor (CTGF). CTGF can be upregulated by TGF-beta1 and appears to play an important role in the development of pulmonary fibrosis. These results suggest that siRNAs could be effective in preventing or possibly arresting the progression of pulmonary fibrosis. Studies are underway in vivo to test this postulate.

In plain English

This was a laboratory study of cells in dishes, not people or animals. The authors tested four small interfering RNAs (siRNAs) that target the messenger RNA (mRNA) for TGF-β1 for breakdown; they describe TGF-β1 as playing an important role in lung scarring (pulmonary fibrosis). They used Swiss 3T3 fibroblasts, a cell line. Chrysotile asbestos raised TGF-β1 in these cells. At 16.6 micrograms per square centimeter of dish surface, TGF-β1 mRNA rose by 3000% by 48 hours. In cells given an siRNA and then 10 micrograms per square centimeter of chrysotile for 48 hours, one siRNA (S1) cut asbestos-induced TGF-β1 mRNA by 89% and protein by 84%. A second (S2) cut them by 72% and 70%. S1 also reduced asbestos-induced mRNA for connective tissue growth factor (CTGF) to about 15% of control levels. A control siRNA not aimed at TGF-β1 also lowered TGF-β1 protein by 41%. The authors concluded that knocking down TGF-β1 may prevent a fibrogenic cascade induced by asbestos. They noted that off-target effects could not be ruled out and that using a cell line instead of living lungs can be criticized.

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 (in vitro) cell culture experiment
• Who was studied: Swiss 3T3 fibroblast cell line obtained from the American Type Culture Collection, studied in triplicate plates per treatment group. Four siRNAs that target mouse TGF-β1 mRNA for degradation were tested.
• Exposure: Chrysotile asbestos supplied by NIEHS, sheared through needles and sonicated, applied at 0, 5, 10 or 16.6 micrograms per square centimeter of dish surface for 6 to 48 hours. The siRNA experiments used 10 micrograms per square centimeter for 48 hours.
• Main results:
  • In cells not exposed to asbestos, each of the four siRNAs reduced TGF-β1 mRNA by at least 50%. S1 reduced it by 80% and S2 by 74%.
  • In fibroblasts given chrysotile asbestos, TGF-β1 mRNA increased by about 1000% at 24 hours with 5 micrograms per square centimeter, and by 3000% by 48 hours with 16.6 micrograms per square centimeter.
  • In asbestos-treated fibroblasts, S1 reduced asbestos-induced TGF-β1 mRNA by 89% and protein by 84%. S2 reduced them by 72% and 70%.
  • The nontargeting control siRNA did not reduce asbestos-induced TGF-β1 mRNA, but TGF-β1 protein levels were reduced by 41%.
  • Asbestos raised CTGF mRNA in the fibroblasts. In asbestos-treated cells given S1, TGF-β1 mRNA was about 10% of controls and CTGF mRNA about 15% of controls.
  • In the experiment without asbestos, none of the siRNAs significantly reduced two unrelated genes (β-actin, GAPDH). S2 did lower β-actin mRNA by about 40% and GAPDH mRNA by 30%, but this was not statistically significant.
• The authors' conclusion: The authors concluded that knocking down TGF-β1 may prevent a fibrogenic (scar-forming) cascade induced by asbestos.
• Limitations the authors state:
  • Without whole-genome profiling, the authors could not rule out all off-target effects of the siRNAs.
  • The authors acknowledged that using a cell line as a stand-in for primary cells and living lungs can be criticized, though they argued it was reasonable.
  • Because TGF-β1 has many normal functions, the authors stated that knocking it down as a therapy would need to be targeted, since effects elsewhere in the body could follow.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Chrysotile.

Medical terms in this study, explained in our Lexicon: Fibroblast, Interstitial Lung Disease, Pulmonary Fibrosis, Chrysotile.

Related studies

• Fe-doped chrysotile nanotubes containing siRNAs to silence SPAG5 to treat bladder cancer — J. Liu, Y. Zhang, et al. (2021)
• mRNA expression patterns in different stages of asbestos-induced carcinogenesis in rats — H. Sandhu, W. Dehnen, et al. (2000)
• Epithelial-mesenchymal transition involved in pulmonary fibrosis induced by multi-walled carbon nanotubes via TGF-beta/Smad signaling pathway — T. Chen, H. Nie, et al. (2014)
• Association of transforming growth factor beta1 gene polymorphisms and asbestos-induced fibrosis and tumors — S. Helmig, A. Belwe, et al. (2009)
• Changes in DNA 8-hydroxyguanine levels, 8-hydroxyguanine repair activity, and hOGG1 and hMTH1 mRNA expression in human lung alveolar epithelial cells induced by crocidolite asbestos — H.N. Kim, Y. Morimoto, et al. (2001)
• Long-lasting production of TGF-beta1 by alveolar macrophages exposed to low doses of asbestos without apoptosis — Y. Nishimura, T. Nishiike-Wada, et al. (2007)

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