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MicroRNAs—Versatile Regulators of Gene Networks


 

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SALT LAKE CITY—Micro­RNAs (miRNAs) represent a previously unsuspected layer of gene regulation that is probably as complex as transcription, according to Kenneth S. Kosik, MD, who addressed the 133rd Annual Meeting of the American Neurological Association. Whereas transcription works in a combinatorial manner to turn genes on and off, miRNAs function in a complex manner to turn messages on and off by controlling their translation. This class of genes, discovered within the last decade, is of increasing relevance to various disease states, including glioblastoma and synaptic plasticity, said Dr. Kosik, who is Harriman Professor of Neuroscience Research in the Department of Molecular, Cellular, and Developmental Biology and Codirector of the Neuroscience Research Institute at the University of California, Santa Barbara.

miRNA genes are organized in the genome like any other gene, even though their product is a mere 21-nucleotide sequence that does encode a protein. These so-called noncoding transcripts are considerably more common and more abundant than was previously believed. The importance of the miRNAs is underscored by their extraordinary conservation, noted Dr. Kosik. Even 600 million years following the evolutionary diversification of animals, some miRNAs retain the identical ancestral sequence, he said. Although genomes have undergone many changes during this time period, the absence of changes in some miRNAs suggests that they serve a critical and fundamental function for many organisms, which cannot tolerate mutations in these short sequences.

miRNA Biogenesis Pathway
Like a coded gene, miRNA undergoes transcription and splicing but is then diverted to a microprocessor pathway in which it gets cut into a 70-nucleotide stem-loop structure, explained Dr. Kosik. The 70-nucleotide stem-loop structure is then exported from the nucleus into the cytoplasm by Exportin-5 and delivers the double-stranded RNA to the RNA-induced silencing complex (RISC) in the cytoplasm. As prelude to entry into the RISC, the Dicer enzyme cuts the loop from the stem, and the remaining stem is guided toward a target messenger RNA (mRNA). Then, either the target mRNA is degraded or its translation is suppressed by a stable miRNA/mRNA duplex. In this way, miRNAs control the level of specific proteins. Although Watson-Crick binding between the miRNA and the target mRNA does occur, partial complementarity of the two sequences can result in some mismatches. Each of the 500 to 1,000 miRNAs in the human genome may have as many as 300 targets, but they are very challenging to predict because of these mismatches.

miRNAs also probe expression boundaries and are present in cells to maintain their identities. For this reason, the early findings in the area of miRNAs that pertain to medicine have been related to cancer and stem cells. Cells have to cross expression boundaries to acquire new identity states. The loss of “cell identity” that occurs when a cluster of genes in a transcriptome crosses an expression boundary is related to the process by which cancer cells begin to acquire new identity states, said Dr. Kosik. Dysregulation of certain key transcripts regulated by miRNAs causes uncontrollable cell division.

Because miRNAs can affect the levels of some mRNAs, it is possible to find a miRNA signature in a transcriptional profile. To look for this signature, Dr. Kosik and colleagues examined glioma samples for highly correlated fluctuations in the miRNA and mRNA levels of each sample. Specifically, the researchers were looking for “perfect correlations” in the miRNA/mRNA relationships and found a set of 35 outliers that were highly unlikely to occur by chance. Among the findings was a high degree of correlation between miRNA-181c and the tumor suppressor gene dcaf. Despite the lack of a target relationship between these two genes, they changed consistently through every sample, suggesting that a pathway could be built from this informatically inferred relationship.

In another study profiling miRNAs in various tumors, Dr. Kosik and colleagues observed that miR-21 “was massively overexpressed in glioblastomas.” He added, “This has turned out to be the most elevated miRNA in many cancers, including breast, ovarian, and colon. Many miRNAs decline in cancer, and a few go up. miR-21 goes up more than most.” The researchers found that when miR-21 was suppressed, cell numbers declined and the amount of apoptosis increased. In addition, cells that were responsive to doxorubicin became resistant to it. miR-21 was observed to affect apoptotic pathways, directly targeting Apaf-1 as well as several elements in the p53 and transforming growth factor β (TGF-β) pathways.

“We can conclude that miR-21 regulates a functionally related network of genes in the p53 and TGF-β mitochondrial pathways by direct targeting and indirect effects,” Dr. Kosik said. He also noted, “If miRNAs will ever be used therapeutically in gliomas, we face the same problems as the RNA interference field in developing delivery methodologies.”

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