Regulatory role of miR-18a to CCN2 by TGF-β1 signaling pathway in pulmonary injury induced by nano-SiO2
2018
Yang, Hong | Li, Wenchao | Zhang, Yingjian | Li, Mingyue | Gao, Ying | Lao, Canshan | Shi, Bing
This research is designed to investigate the regulatory effect of miR-18a to the target gene connective tissue growth factor (CTGF, or CCN2), by participating in TGF-β1 signaling pathway and explore the pathogenic mechanism of miR-18a in pulmonary injury induced by nano-SiO₂ based on our early study. miR-18a and expression of TGF-β1 in Chinese hamster lung (CHL) fibroblasts cells stimulated by supernatants of NR8383 cells exposed to 40 μg/ml nano-SiO₂ for 24 h demonstrated 1.58 ± 0.22-fold and 1096.00 ± 2.60 pg/ml increase compared with blank control group analyzed by real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Expression increase of miR-18a and reduction of CCN2 mRNA expression levels and protein gray value ratio detected by Western blotting in CHL cells transfect miR-18a mimics for 48 h. The reverse of CHL cell transfection miR-18a inhibit is also true. The result of miR-18a and CCN2 binding sites tested by luciferase reporter gene assay shows that the report relative fluorescence value of miR-18a mimics wild type on CCN2 is 0.50 ± 0.02 with the control of mimics NC and mutant relative fluorescence report value 0.86 ± 0.04 (P < 0.05). Expression levels of miR-18a, CCN2 mRNA, and protein gray value ratio decreased in CHL cells treated by TGF-β1, respectively, and vice versa treated by TGF-β1corepressor. The results suggest that CCN2 is the target gene regulated by miR-18a and miR-18a participates in TGF-β1 signaling pathway by regulating the expression of CCN2 negatively through CCN2 3’UTR site, and thus may be involved in the development process of pulmonary injury.
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