Mike Wilson, senior scientist and array R&D manager at Asuragen (www.asuragen.com), is investigating the global miRNA expression patterns in pancreatic cancer using Ambion’s (www.ambion.com) mirVana microRNA array system that represents 384 different miRNAs. Asuragen, an Ambion spinoff, currently offers the next-generation microRNA array containing 640 miRNAs applicable to human, mouse, and rat experiments.
“Conventional 2-D hierarchical clustering results are useful for organizing and visualizing the relationships across a collection of samples that represent normal pancreas tissue, samples from patients with chronic pancreatitis, pancreatic carcinoma, and pancreas-derived cell lines,” Wilson says. “For better visualization of comparisons of microRNA expression patterns between two groups, such as the cancerous and normal pancreas, a novel type of visualization method can be particularly informative.”
Wilson’s group identified 20 miRNAs with significantly altered changes in expression when comparing normal pancreas to pancreatic carcinoma. After validation of those results by QRT-PCR, the researchers showed how the expression of a small subset of miRNAs can be used as biomarkers for pancreatic detection. The researchers also investigated the downstream consequences of adding miRNAs to an experimental in vitro cellular system and monitored the effective changes that this had on the mRNA gene-expression profiles.
Asuragen’s validation experiment demonstrated how the system could give relevant results if the changes in gene expression were interpreted in the context of pathway analysis. Finally, pathway analysis revealed that among the top pathways with altered mRNA patterns were those related to cell death, cancer, cellular growth and proliferation, and cell cycle, Wilson adds.
According to Devin Leake, Ph.D., associate director of R&D at Dharmacon(www.dharmacon.com), “Both miRNA mimetics and inhibitors will be essential for defining the role of miRNAs in specific disease states as they simulate gain- and loss-of-function scenarios. The types of disease most likely to be affected are long-term, chronic conditions associated with internally caused disorders, such as cancer, rather than acute diseases with some external cause.”
The miRNAs, which are small, noncoding RNAs, target multiple genes simultaneously. An active or mature miRNA is a short ~22 nucleotide single-stranded RNA that numbers anywhere from tens to thousands of molecules per cell, Dr. Leake explains.
In their active form, these RNAs are incorporated as part of the ribonucleoprotein complex, also known as the RNA-induced silencing complex or RISC complex. The sequence of an miRNA determines, via complementary base pairing, the locations at which its RISC-like complex will bind to the 3´ untranslated regions (3´ UTRs) of target messages.
“Complementarity of as few as seven bases is, in most cases, sufficient to direct this binding,” he continues. “Therefore, one miRNA can find binding sites in the 3´ UTRs of many genes, and as long as there are sufficient copies of active miRNA in a cell, all of those genes could, presumably, be targeted simultaneously. Researchers are asking whether or not all potential binding sites are actually used and whether there is preference of some sites over others in cases where the miRNA is limiting.”
Multiple parameters contribute to improved inhibition including length, composition, and modification.
Dharmacon miRIDIAN™ Inhibitors alter the rate kinetics of the miRNA and inhibitor association. The inhibitors act as an artificial nonhydrolyzable mRNA target that irreversibly binds the miRNA-RISC complex. Once a miRISC complex has bound to an inhibitor, it does not easily dissociate. The inhibitors are assumed to act as competitors for miRISC targeting. Thus, they affect RISC function by preventing the microRNA-programmed RISC complexes from binding to their ordinary targets, he concludes.