Chronic and excessive alcohol misuse results in changes in the expression of selected miRNAs and their mRNA targets in specific regions of the human brain. These expression changes likely underlie the cellular adaptations to long term alcohol misuse. In order to delineate the mechanism by which these expression changes occur, we have measured the expression of six miRNAs including miR-7, miR-153, miR-152, miR-15B, miR-203 and miR-144 in HEK293T, SH SY5Y and 1321 N1 cells following exposure to ethanol. These miRNAs are predicted to target key genes involved in the pathophysiology of alcoholism. Chronic and chronic-intermittent exposure to ethanol, and its removal, resulted in specific changes in miRNA expression in each cell line suggesting that different expression patterns can be elicited with different exposure paradigms and that the mechanism of ethanol’s effects is dependent on cell type. Specifically, chronic exposure to ethanol for five days followed by a five day withdrawal period resulted in up-regulation of several miRNAs in each of these cell lines similar to expression changes identified in post mortem human brain. Thus, this model can be used to elucidate the role of miRNAs in regulating gene expression changes that occur in response to ethanol exposure.
References
[1]
Australian Institute of Health and Welfare (AIHW). Australia’s Health 2008; AIHW: Canberra, Australia, 2008.
Anni, H.; Israel, Y. Proteomics in alcohol research. Alcohol Res. Health 2002, 26, 219–232.
[6]
Liu, J.; Lewohl, J.M.; Dodd, P.R.; Randall, P.K.; Harris, R.A.; Mayfield, R.D. Gene expression profiling of individual cases reveals consistent transcriptional changes in alcoholic human brain. J. Neurochem. 2004, 90, 1050–1058, doi:10.1111/j.1471-4159.2004.02570.x.
[7]
Liu, J.; Lewohl, J.M.; Harris, R.A.; Iyer, V.R.; Dodd, P.R.; Randall, P.K.; Mayfield, R.D. Patterns of gene expression in the frontal cortex discriminate alcoholic from nonalcoholic individuals. Neuropsychopharmacology 2006, 31, 1574–1582, doi:10.1038/sj.npp.1300947.
[8]
Lewohl, J.M.; Wang, L.; Miles, M.F.; Zhang, L.; Dodd, P.R.; Harris, R.A. Gene expression in human alcoholism: Microarray analysis of frontal cortex. Alcohol. Clin. Exp. Res. 2000, 24, 1873–1882, doi:10.1111/j.1530-0277.2000.tb01993.x.
[9]
Mayfield, R.D.; Lewohl, J.M.; Dodd, P.R.; Herlihy, A.; Liu, J.; Harris, R.A. Patterns of gene expression are altered in the frontal and motor cortices of human alcoholics. J. Neurochem. 2002, 81, 802–813, doi:10.1046/j.1471-4159.2002.00860.x.
[10]
Liu, J.; Lewohl, J.M.; Harris, R.A.; Dodd, P.R.; Mayfield, R.D. Altered gene expression profiles in the frontal cortex of cirrhotic alcoholics. Alcohol. Clin. Exp. Res. 2007, 31, 1460–1466, doi:10.1111/j.1530-0277.2007.00444.x.
Pietrzykowski, A.Z.; Friesen, R.M.; Martin, G.E.; Puig, S.I.; Nowak, C.L.; Wynne, P.M.; Siegelmann, H.T.; Treistman, S.N. Posttranscriptional regulation of BK channel splice variant stability by miR-9 underlies neuroadaptation to alcohol. Neuron 2008, 59, 274–287, doi:10.1016/j.neuron.2008.05.032.
[13]
Sathyan, P.; Golden, H.B.; Miranda, R.C. Competing interactions between micro-RNAs determine neural progenitor survival and proliferation after ethanol exposure: Evidence from an ex vivo model of the fetal cerebral cortical neuroepithelium. J. Neurosci. 2007, 27, 8546–8557, doi:10.1523/JNEUROSCI.1269-07.2007.
[14]
Tang, Y.; Banan, A.; Forsyth, C.B.; Fields, J.Z.; Lau, C.K.; Zhang, L.J.; Keshavarzian, A. Effect of alcohol on miR-212 expression in intestinal epithelial cells and its potential role in alcoholic liver disease. Alcohol. Clin. Exp. Res. 2008, 32, 355–364, doi:10.1111/j.1530-0277.2007.00584.x.
[15]
Lewohl, J.M.; Nunez, Y.O.; Dodd, P.R.; Tiwari, G.R.; Harris, R.A.; Mayfield, R.D. Up-regulation of microRNAs in brain of human alcoholics. Alcohol. Clin. Exp. Res. 2011, 35, 1928–1937, doi:10.1111/j.1530-0277.2011.01544.x.
[16]
Kril, J.J.; Harper, C.G. Neuronal counts from four cortical regions of alcoholic brains. Acta Neuropathol. (Berl.) 1989, 79, 200–204.
[17]
Guo, Y.; Chen, Y.; Carreon, S.; Qiang, M. Chronic intermittent ethanol exposure and its removal induce a different miRNA expression pattern in primary cortical neuronal cultures. Alcohol. Clin. Exp. Res. 2012, 36, 1058–1066, doi:10.1111/j.1530-0277.2011.01689.x.
[18]
Lewohl, J.M.; Crane, D.I.; Dodd, P.R. Expression of the alpha 1, alpha 2 and alpha 3 isoforms of the GABAA receptor in human alcoholic brain. Brain Res. 1997, 751, 102–112, doi:10.1016/S0006-8993(96)01396-0.
[19]
Janeczek, P.; Mackay, R.K.; Lea, R.A.; Dodd, P.R.; Lewohl, J.M. Reduced expression of alpha-synuclein in alcoholic brain: Influence of SNCA-Rep1 genotype. Addict. Biol. 2012, doi:10.1111/j.1369-1600.2012.00495.x.
[20]
Ho, A.M.; MacKay, R.K.; Dodd, P.R.; Lewohl, J.M. Association of polymorphisms in RGS4 and expression of RGS transcripts in the brains of human alcoholics. Brain Res. 2010, 1340, 1–9, doi:10.1016/j.brainres.2010.04.049.
[21]
MacKay, R.K.; Colson, N.; Dodd, P.R.; Lewohl, J.M. Differential expression of 14-3-3 isoforms in human alcoholic brain. Alcohol. Clin. Exp. Res. 2011, 35, 1041–1049, doi:10.1111/j.1530-0277.2011.01436.x.
[22]
Cagetti, E.; Liang, J.; Spigelman, I.; Olsen, R.W. Withdrawal from chronic intermittent ethanol treatment changes subunit composition, reduces synaptic function, and decreases behavioral responses to positive allosteric modulators of GABAA receptors. Mol. Pharmacol. 2003, 63, 53–64.
[23]
Olsen, R.W.; Liang, J.; Cagetti, E.; Spigelman, I. Plasticity of GABAA receptors in brains of rats treated with chronic intermittent ethanol. Neurochem. Res. 2005, 30, 1579–1588, doi:10.1007/s11064-005-8836-6.
[24]
Cao, J.-Y.; Qiu, S.; Zhang, J.; Wang, J.-J.; Zhang, X.-M.; Luo, J.-H. Transmembrane region of N-methyl-d-aspartate receptor (NMDAR) subunit is required for receptor subunit assembly. J. Biol. Chem. 2011, 286, 27698–27705.
[25]
Thomas, P.; Smart, T.G. HEK293 cell line: A vehicle for the expression of recombinant proteins. J. Pharmacol. Toxicol. Methods 2005, 51, 187–200, doi:10.1016/j.vascn.2004.08.014.
[26]
Shaw, G.; Morse, S.; Ararat, M.; Graham, F.L. Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells. FASEB J. 2002, 16, 869–871.
[27]
Rani, C.S.; Qiang, M.; Ticku, M.K. Potential role of cAMP response element-binding protein in ethanol-induced N-methyl-d-aspartate receptor 2B subunit gene transcription in fetal mouse cortical cells. Mol. Pharmacol. 2005, 67, 2126–2136, doi:10.1124/mol.104.007872.
Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2？ΔΔCT method. Methods 2001, 25, 402–408, doi:10.1006/meth.2001.1262.
