Knockdown and Overexpressionof Mitochondrial ATP/ADP Translocases in Airway Epithelial Cells

Sammanfattning

ADP/ATP translocase — ANT1 and ANT2 — are mitochondrial transporters responsible for ADP/ATP exchange between the mitochondria matrix and cytoplasm. Dysregulation of ANT1 and ANT2 has been implicated in respiratory diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. To study the role of ANT1 and ANT2 in airway epithelial cells, we aimed to establish knockdown and overexpression protocols in A549 cell line - which is a commonly used human lung cancer cell line. siRNA was used for knockdown and adenoviral vectors were used for overexpression. Here, we found that the mRNA expression of ANT2 was about 10-fold higher than that of ANT1 in A549 cell line. The siRNA concentration of 10 nM resulted in efficient knockdown of both ANT1 and ANT2 mRNA 48 hours post transfection without any cellular toxicity observed. Preliminary Western blot data indicated ANT1 knockdown also at the protein level. Overexpression of ANT1 was confirmed at the mRNA level whereas that of ANT2 has not yet been confirmed. In summary, we have successfully established knockdown and overexpression protocols in A549 cells, which could serve as a basis to support further functional studies of ANT1 and ANT2 in primary airway epithelial cells.