miR-221 and miR-222 target p21 and p27 in airway smooth muscle to elicit hyper proliferation in severe asthmatics

Rationale: Since microRNAs (miRNAs) were first discovered to be produced in human tissues over 10 years ago, they have emerged as important mediators in cellular physiology and pathology. Asthma is characterised by chronic airflow obstruction, chronic airway inflammation and remodeling and the airway smooth muscle (ASM) cells cultured from the biopsies of patients with asthma are of a hyper proliferative phenotype and release greater amounts of the chemokine IL-6. This ASM proliferation has been shown to be increased in response to growth factors such as FCS, and inflammatory mediators such as TGF-β. Ergo, we have examined the role of the miRNAs, miR-221 and miR-222, in both FCS and TGF-β-induced ASM proliferation and IL-6 release. Methods: Human ASM cells were stimulated with FCS ± TGF-β for 8 days. For the determination of chemokine release, supernatants were removed and IL-6 levels determined by DuoSet ELISA (R&D Systems). The degree of cell proliferation was assessed by Cell Proliferation ELISA, BrdU kit (Roche Applied Science) according to the manufacturer's instructions. miRNA and mRNA expression was examined by TaqMan RT-PCR. The function of miR-221 and miR-222 were assessed through transfection with miRNA mimics and inhibitors. Results: Exposure to FCS and TGF-β induced an increase in cellular proliferation, IL-6 release and miRNA-221 and miR-222 expression. This increase was greater in patients with severe asthma than those with non severe asthma. FCS and TGF-β-induced proliferation and IL-6 release were inhibited following transfection with miRNA-222 inhibitors and potentiated in the presence of miRNA-222 mimics, by the targeting of p21 and p27.