MicroRNA expression in lung tissues of asbestos-exposed mice: Upregulation of miR-21 and downregulation of tumor suppressor genes Pdcd4 and Reck

Our plain-English summary of MicroRNA expression in lung tissues of asbestos-exposed mice: Upregulation of miR-21 and downregulation of tumor suppressor genes Pdcd4 and Reck, by Y. Hiraku, J. Watanabe, A. Kaneko, et al (Journal of Occupational Health, 2021).

The study

• Authors: Y. Hiraku, J. Watanabe, A. Kaneko, T. Ichinose, M. Murata
• Journal: Journal of Occupational Health
• Published: 2021;63(1):e12282
• Cited by: 8 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Objectives: Asbestos causes lung cancer and malignant mesothelioma in humans, but the precise mechanism has not been well understood. MicroRNA (miRNA) is a short non-coding RNA that suppresses gene expression and participates in human diseases including cancer. In this study, we examined the expression levels of miRNA and potential target genes in lung tissues of asbestos-exposed mice by microarray analysis.

Methods: We intratracheally administered asbestos (chrysotile and crocidolite, 0.05 or 0.2 mg/instillation) to 6-week-old ICR male mice four times weekly. We extracted total RNA from lung tissues and performed microarray analysis for miRNA and gene expression. We also carried out real-time polymerase chain reaction (PCR), Western blotting, and immunohistochemistry to confirm the results of microarray analysis.

Results: Microarray analysis revealed that the expression levels of 14 miRNAs were significantly changed by chrysotile and/or crocidolite (>2-fold, P < .05). Especially, miR-21, an oncogenic miRNA, was significantly upregulated by both chrysotile and crocidolite. In database analysis, miR-21 was predicted to target tumor suppressor genes programmed cell death 4 (Pdcd4) and reversion-inducing-cysteine-rich protein with kazal motifs (Reck). Although real-time PCR showed that Pdcd4 was not significantly downregulated by asbestos exposure, Western blotting and immunohistochemistry revealed that PDCD4 expression was reduced especially by chrysotile. Reck was significantly downregulated by chrysotile in real-time PCR and immunohistochemistry.

Conclusions: This is the first study demonstrating that miR-21 was upregulated and corresponding tumor suppressor genes were downregulated in lung tissues of asbestos-exposed animals. These molecular events are considered to be an early response to asbestos exposure and may contribute to pulmonary toxicity and carcinogenesis.

© 2021 The Authors. Journal of Occupational Health published by John Wiley & Sons Australia, Ltd on behalf of The Japan Society for Occupational Health.

In plain English

This was an animal experiment in mice. The authors placed asbestos directly into the windpipes of 6-week-old male ICR mice, using standard reference samples of chrysotile or crocidolite at 0.05 mg (low dose) or 0.2 mg (high dose) each time, four times at weekly intervals. Control mice received the liquid alone. The authors then measured microRNAs (small molecules that turn down the activity of genes) and gene activity in lung tissue. In the microarray analysis, the levels of 14 microRNAs changed significantly with chrysotile and/or crocidolite. A follow-up test found that one of them, miR-21, rose with both fibers. Two tumor suppressor genes that miR-21 is predicted to act on, Pdcd4 and Reck, were lower in exposed lungs on some tests but not all. The lungs showed only mild changes and no tumors. The authors raised the possibility that these molecules take part in asbestos-induced lung cancer development. They noted that this type of exposure has been reported to cause lung tumors in rats and hamsters but not in mice, and they called for longer studies in other species and in human tissue.

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: Animal experiment: subacute intratracheal asbestos exposure in mice, with microarray analysis of microRNA and gene expression, database prediction of target genes, and confirmation by real-time PCR, Western blotting and immunohistochemistry
• Who was studied: Male ICR mice, 6 weeks old, from Japan Clea, studied in Japan. The microarray analyses used 4 mice per group. Other tests are reported as 3 to 5 independent experiments.
• Exposure: UICC standard reference chrysotile B and crocidolite were instilled into the trachea at 0.05 mg (low dose) or 0.2 mg (high dose) per instillation, four times at weekly intervals, for totals of 0.2 mg and 0.8 mg. Controls received vehicle (saline with Tween 80) alone. The microRNA microarray used tissue from the 0.05 mg per instillation groups. Lungs were taken one day after the last dose.
• Main results:
  • In the microarray analysis, the levels of 14 microRNAs changed significantly (more than 2-fold, P less than .05) with chrysotile and/or crocidolite compared with controls. Four of them, including miR-21, changed with both fibers.
  • By real-time PCR, miR-21 levels in lung tissue were significantly increased by both chrysotile and crocidolite (P less than .05).
  • Gene microarray: 921 genes (569 up, 352 down) changed in chrysotile-exposed mice and 473 genes (206 up, 267 down) changed in crocidolite-exposed mice, compared with controls.
  • By real-time PCR, Pdcd4 gene activity (mRNA) was not significantly changed by asbestos exposure, although it tended to be lower at the high chrysotile dose.
  • By real-time PCR, Reck was significantly lower at the high dose of both chrysotile and crocidolite (P less than .05). Across all lung tissues, miR-21 levels were negatively correlated with Reck levels (r = −.609, P less than .01).
  • By Western blotting, chrysotile significantly reduced PDCD4 protein (P less than .01). Crocidolite tended to reduce it, but the change was not statistically significant (P = .078).
  • By immunohistochemistry, the proportion of PDCD4-positive cells was significantly decreased by both chrysotile (P less than .01) and crocidolite (P less than .05).
  • By immunohistochemistry, the proportion of RECK-positive cells was significantly decreased by chrysotile (P less than .05) but not by crocidolite.
  • Microscopy showed that chrysotile and crocidolite caused only mild pathological changes in the lungs. No tumors (neoplastic lesions) were seen.
  • miR-133a was significantly lower with chrysotile (P less than .01). Crocidolite did not cause a significant change.
• The authors' conclusion: The authors stated that this was the first study to show miR-21 increased and its potential target genes PDCD4 and RECK decreased in lung tissues of asbestos-exposed animals. They said this raises the possibility that these molecules take part in asbestos-induced lung carcinogenesis.
• Limitations the authors state:
  • The study used a subacute (short-term) exposure model. The authors could not exclude that the microRNA changes were simply a consequence of fiber exposure.
  • Intratracheal asbestos has been reported to cause lung tumors in rats and hamsters but not in mice, the species used here.
  • The authors said further studies in different animal species with long-term asbestos exposure are needed.
  • The authors said human specimens would need to be studied before these microRNAs and genes could be used as biomarkers of disease risk.

Topics

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

Medical terms in this study, explained in our Lexicon: Tumor Suppressor Gene, Lung Cancer, Malignant Mesothelioma, Chrysotile, Crocidolite, Immunohistochemistry, Asbestos Exposure, Carcinogenesis.

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• Genome-wide profiling reveals novel microRNAs in hand-spinning-specific chrysotile exposure — J. Jia, X. Guo, et al. (2019)
• Characterization of pulmonary responses in mice to asbestos/asbestiform fibers using gene expression profiles — N. Yanamala, E.R. Kisin, et al. (2018)

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