microRNA regulation of the Alpha-1 antitrypsin gene

Introduction: Alpha-1 antitrypsin (AAT) deficiency is a conformational disorder characterised by chronic inflammatory lung disease and liver disease. microRNAs (miRNA) represent drug targets that function as post-transcriptional negative regulatory molecules of target gene expression. Aims and Methods: To identify miRNAs that target AAT gene expression using in silico analysis, to quantify AAT gene and protein expression in monocytic THP-1, 16HBE14o- bronchial epithelial and HepG2 liver cells using qRT-PCR and ELISA, to quantify specific miRNAs expression in these cells, to modulate AAT expression using premiRs and to validate miRNAs targeting AAT using a AAT-3'UTR luciferase reporter plasmid in HEK293 cells. Results: In silico analysis predicts multiple miRNAs that target the AAT gene such as miR- 940 (with multiple binding sites in the 3'UTR), miR-132 and -212. Different cells expressed different levels of AAT and miRNAs. AAT and miR-940 are reciprocally expressed in the three cell lines, with miR-940:AAT ratio decreasing with increased AAT expression. Over-expression with premiR-940, but not premiR 132 and 212, led to a decrease in AAT mRNA and protein levels. All three miRNAs could inhibit expression of an AAT 3'UTR luciferase reported plasmid in HEK293 cells. A combination of pre-miRs over-expressed in HepG2 only resulted in a decrease of AAT mRNA and protein expression involving premiR-940, however, not significantly more than cells transfected with premiR 940 alone. Conclusion: AAT mRNA is a true target for miR 940, 132 and 212. miR 940 is a more effective inhibitor than miR-132 or miR-212 due to attractive inherent properties compared to the other miRNAs tested.