In chronic alcoholism, brain shrinkage and cognitive defects due to neuronal death are well established, though the sequence of molecular events has not been fully explored yet.
We explored the role of microRNAs (miRNAs) in ethanol induced apoptosis of neuronal cells. Ethanol sensitive miRNAs in SH-SY5Y, a human neuroblastoma cell line were identified using real time PCR based Taqman Low Density Arrays (TLDA).
Long-term exposure to ethanol (0.5 percent v/v for 72 hours) produced a maximum increase in expression of miR-497 (474 folds) and miR-302b (322 folds). Similar to SH-SY5Y, long term exposure to ethanol induced miR-497 and miR-302b in IMR-32, another human neuroblastoma cell line.
Using in silico approaches, BCL2 and cyclin D2 (CCND2) were identified as probable target genes of these miRNAs. Co-transfection studies with 3-UTR of these genes and miRNA mimics have demonstrated that BCL2 is direct target of miR-497 and CCND2 is regulated negatively by either miR-302b or miR-497.
Over-expression of either miR-497 or miR-302b reduced expression of their identified target genes and increased caspase-3 mediated apoptosis of SH-SY5Y cells. However, over-expression of only miR-497 increased reactive oxygen species formation, disrupted mitochondrial membrane potential and induced cytochrome C release (mitochondria related events of apoptosis).
Moreover, ethanol induced changes in miRNAs and their target genes were substantially prevented by pre-exposure to GSK-3B inhibitors.
In conclusion, our studies have shown that ethanol induced neuronal apoptosis follows both mitochondria mediated (miR-497 and BCL2 mediated) and non-mitochondria mediated (miR-302b and CCND2 mediated) pathway.
Read Full Text (PDF)
