Effects of cigarette smoke condensate on microrna expression of human bronchial epithelial cells.

Abstract

Lung cancer is the leading cause of cancer death and accounts for 1.2 million deaths annually world-wide and its five year survival is less than 15%. The primary etiology of lung cancer is genetic and epigenetic alterations caused by tobacco smoke. Although smoking-related genetic changes have been well studied, much is not known about the association between lung cancer and epigenetic changes, in particular deregulation of microRNAs. Therefore, our objective in this study was to examine the association between early phenotypic changes and altered microRNA expression in cigarette smoke condensate (CSC)-treated immortalized human bronchial epithelial cells (HBECs). We hypothesized that extended treatment of HBECs with CSC causes morphological and growth alterations and these effects are mediated, at least partly, through deregulation of microRNA expression. The aims of this study were to: 1. Determine CSCinduced early phenotypic alterations in HBECs; 2. Identify microRNAs whose levels are altered in CSC-treated HBECs. HBECs were exposed to least-toxic dose of CSC (5 μg/ml) for 4 months and effects on cell proliferation, morphology, transformation, activation of AKT and ERK, and microRNA expression profile were determined. CSC exposure caused HBECs to become round and elongated and their proliferation is increased by 64%. Western blot analysis also indicated activation of ERK1/2 and AKT. Microarray analysis revealed changes in the expression of 89 microRNAs in CSC-exposed HBECs and 87 of them were down-regulated. Further qRT-PCR analysis revealed altered expression of miR-138, miR-921, miR-293-3p, & IVGN-novel-miR-3526. However only the change in IVGN-novel-miR-3526 was statistically significant (up-regulated by 2.4-fold) (P= 0.03). The expression level of IVGN-novel-miR-3526 in A549 cells found to be 4.6-fold higher iv than untreated HBECs. To our knowledge, this is the first report on the altered level of this microRNA in CSC-treated bronchial cells or lung cancer cells. Overall, this study has provided useful insight on CSC-induced phenotypic alterations and microRNA deregulation in HBECs. Functional assays are required to determine the association between IVGN-novel-miR-3526 deregulation and observed phenotypic changes in CSC-exposed HBECs.