Valproic acid (VPA) is a commonly used drug to treat epilepsy and bipolar disorders. Known properties of VPA are inhibitions of histone deacetylases and activation of extracellular signal regulated kinases (ERK), which cannot fully explain VPA's clinical features. We found that VPA induces the proteasomal degradation of DICER, a key protein in the generation of micro RNAs. Unexpectedly, the concentration of several micro RNAs increases after VPA treatment, which is caused by the upregulation of their hosting genes prior to DICER degradation. The data suggest that a loss of DICER protein and changes in micro RNA concentration contributes to the clinical properties of VPA. VPA can be used experimentally to down regulate DICER protein levels, which likely reflects a natural regulation of DICER.

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Published on PLOS One, v. 8, issue. 12, e82895.

© 2013 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Figure_S1.pptx (46 kB)
Summary of the Exon-junction array analysis. Kyoto Encyclopedia Genes and Genomes (KEGG) pathway analysis of the genes showing transcriptional changes. The p-value indicates the confidence that the given pathway is changed, calculated with a more stringent version of Fisher’s exact test.

Figure_S2.xls (8465 kB)
Array analysis in Excel format. Excel file summarizing the array analysis.

Figure_S3.pptx (162 kB)
Validation of Array Results by RT-PCR. A. RT-PCR analysis of HEK293 cells that were treated for 0, 6 and 12 hours with 20 mM VPA. B. Statistical evaluation of the changes in three independent experiments. The expression level in untreated HEK293 cells was set to 1. (*: p < 0.05, **: p < 0.01, ***: p < 0.001, n>4).

Figure_S4.pptx (88 kB)
Testing of the DICER antisera. Goats were immunized with the peptide ETSVPGRPGSTKRRQC and final sera were analyzed at the indicated dilutions. Similar to other DICER antisera, a cross reactivity is seen around 60kDa.

Figure_S5.pptx (43 kB)
Quantification of the effect of MG132 and Bafliomycin on VPA mediated DICER degradation. The DICER signal was normalized to beta-actin and the ratio in the water treated control was set to 1; n=3. A representative experiment is shown in Figure 4B.

Figure_S6.pptx (74 kB)
Summary of miRNAs changes after VPA treatment. HEK293 cells were treated with 20 mM VPA and changes in miRNAs monitored by array analysis. The table lists miRNAs with the highest fold-changes and their experimentally validated target genes from the literatures.

Figure_S7.xlsx (150 kB)
Array analysis in Excel format. Summary of miRNAs changes after VPA treatment.

Figure_S8.pptx (61 kB)
Primers used in the Exon-junction array validation by RT-PCR.