ed by Western blot analysis. Over-expression of pre-miR-16 down-regulates BCL2 expression compared to the over-expressed negative control. Furthermore CRK levels were reduced by premiR-126 transfection and surprisingly, KRAS was down-regulated not only by pre-let-7d, but also by pre-miR-126 in MIA PaCa-2 cells. As it is well documented that the tumour-suppressor let-7 family regulates KRAS in pancreatic, lung, colon and breast cancers, we concentrated on miR-126 as a novel KRAS targeting miRNA in PDAC. To this end we performed loss of function experiments using specific miRNA inhibitors to further validate this finding. We could demonstrate that in contrast to premiR-126 expression, the down-regulation of miR-126 increases both KRAS and CRK protein levels. Since we could not see any difference in KRAS mRNA levels using either pre-miR-126 or anti-miR-126, this indicates that this miRNA possibly acts on the protein translation step. The data herein demonstrate that the down-regulation of multiple miRNAs in PDAC may contribute to malignant transformation. MiR-126 regulates KRAS protein translation by interacting with a ��seedless��motif in its 39UTR We show that over-expression of miR-126 reduces and conversely its silencing increases KRAS protein levels. In order to evaluate whether miR-126 directly regulates KRAS, we performed a bioinformatic search of potential miR-126 interaction sites in the KRAS mRNA. Using the RNA22 software and the entire KRAS transcript as the input MiRNAs in Benign vs. Malignant Pancreatic Tumors normal non-proliferative cells into benign proliferative cells. Dysregulation of proteins involved in miRNA biogenesis in PDAC, which still need to be characterized, could explain this event. Among the down-regulated miRNAs in our microarray, there are many already described as tumor suppressors through inhibition of known PDAC oncogenes. We show general miR-29 family member 
down-regulation. Amongst their targets are DNMT3A and 3B-methyltransferases, whose levels can increase because of the loss of miR-29, causing CpG island hypermethylation and cancer. We also show down-regulation of let-7 family members , which are already described as negative regulators of KRAS and HMGA2 oncogenes, whose increased activity is very important during PDAC progression. Furthermore, we show down-regulation of both miR-143 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189475 and miR-145, which have recently been described as being transcriptionally down-regulated by the Ras signaling pathway, that in turn directly targets KRAS oncogene in PDAC. This revealed a feed-forward mechanism that potentiates Ras signaling. This was of interest as it is well known that KRAS is one of the main genetic promoters of PDAC and HMGA2 expression levels are associated with the malignant phenotype in pancreatic exocrine tissue, which could in part be explained by the down-regulation of these miRNAs. Interestingly, we could see an 871700-17-3 web up-regulation of KRAS protein, but no change in mRNA levels when comparing normal tissues to PDAC, indicating that the post-transcriptional regulation of KRAS in PDAC may be an essential step. Mutations that result in a constitutively active KRAS are found in.95% of PDAC and are thought to be a crucial initiating event for this disease. Furthermore, PDAC continues to be ��addicted��to KRAS for epithelial differentiation and cell viability, indicating that finding new KRAS regulators is an important step. We show a down-regulation of miR-126 in PDAC, with increased expression of KRAS. As a
