N⁶-methyladenosine-dependent primary microRNA-126 processing activated PI3K-AKT-mTOR pathway drove the development of pulmonary fibrosis induced by nanoscale carbon black particles in rats

Abstract

The pulmonary fibrosis could be caused by long-term inhalation of carbon black (CB) particles. Studies on the mechanisms of pulmonary fibrosis induced by CB are required to develop the stratagem of prevention and treatment on fibrosis. The RNA-binding protein DiGeorge syndrome critical region gene 8 (DGCR8)-dependent pri-miRNAs processing is regulated by N⁶-methyladenosine (m⁶A) modification, which targets the downstream signal pathway. However, its role in pulmonary fibrosis has not been known clearly. In the present study, rats inhaled CB at dose of 0, 5 or 30 mg/m³ for 28 days, 6 h/day, respectively. The rats inhaled CB at dose of 0 or 30 mg/m³ for 14 days, 28 days and 90 days, respectively. In vitro experiments, the normal human bronchial epithelial cell line (16HBE) was treated with CB (0, 50, 100 and 200 μg/mL) for 24 h. In vitro and vivo study, the levels of fibrosis indicators including α-SMA, vimentin, collagen-I and hydroxyproline in CB treatment groups statistically increased in dose- or time- dependent manners compared with the control. After CB treatment, PI3K-AKT-mTOR pathway was activated and regulated by miRNA-126. We found that both of m⁶A modifications of pri-miRNA-126 and its binding with DGCR8 were decreased after CB treatment, which resulted in the reduction of mature miRNA-126 accompanied by accumulation of unprocessed pri-miRNA-126. This work demonstrated that m⁶A modification of pri-miRNA-126 and its binding with DGCR8 decreases blocked miRNA-126 maturation, and then activated the PI3K/AKT/mTOR pathway, which drove the fibro genesis in the lung after CB exposure.

Keywords:

• Carbon black (CB)
• pulmonary fibrosis
• N6-Methyladenosine (m6A)
• miRNA-126
• PI3K/AKT/mTOR signaling pathway

Acknowledgements

Skilled technical assistance from Xiangyu Zhang, Tianxu Liu, Shaoguang Sun, Chunfang Zhao, LeiLei, Haiya Zhang and Heran Xie is greatly appreciated.

Disclaimers

All views expressed in the submitted article are author's own position and not an official position of the institution or funder.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This research was supported by National Natural Science Foundation of China (No. 81573190, 91643108).