TTN-AS1 is altered during inhalation therapy in COPD patients and aggravates cigarette smoke extracts-induced lung epithelial cell injury via sponging miR-15b-5p

Abstract

Smoking has been found to contribute to the development of chronic obstructive pulmonary disease (COPD), with incompletely reversible airflow limitation. TTN-AS1 has been confirmed to modulate cell proliferation and apoptosis, but its expression in COPD patients and its function in lung bronchial epithelial cell injury remain elusive. The profiles of TTN-AS1 and miR-15b-5p in COPD patients and cigarette smoke extract (CSE)-induced 16HBE cells (in vitro COPD model) were examined by real-time quantitative PCR (RT-qPCR). We applied TTN-AS1 overexpression plasmids and miR-15b-5p mimics for gain-of-function assay. Cell proliferation was tested by the cell counting kit-8 (CCK-8) assay, while cell apoptosis was evaluated by flow cytometry (FCM), TdT-mediated dUTP nick end labeling (TUNEL) staining, and Western blot (WB), respectively. Meanwhile, the profiles of inflammatory cytokines were testified by enzyme-linked immunosorbent assay (ELISA), and the levels of COX2, iNOS and NF-κB were monitored by WB. Besides, the targeting correlation between TTN-AS1 and miR-15b-5p was predicted by the Starbase database. Moreover, the binding association between the two was further measured by the dual-luciferase reporter assay and RNA immunoprecipitation (RIP) method. Our findings showed that TTN-AS1 expression was dramatically strengthened, while miR-15b-5p profiles were curbed in CSE-treated 16HBE cells and serum of COPD patients (compared with healthy donors). Functionally, overexpressing TTN-AS1 aggravated CSE-induced cell viability inhibition and apoptosis and intensified inflammations in 16HBE. Furthermore, miR-15b-5p up-regulation eased CSE-mediated cell damage and inflammation and reversed the effects of TTN-AS1. Mechanically, TTN-AS1 targeted and repressed miR-15b-5p. In conclusion, TTN-AS1 and miR-15b-5p are promising diagnostic molecules for COPD, and TTN-AS1 aggravates CSE-induced human bronchial epithelial cell apoptosis by regulating miR-15b-5p.