Discussion Forum For Health Professionals
RNA vs RNA (A new approach to inactivate miRNA)
Although microRNAs (miRNAs) are known to be a crucial gene regulatory mechanism for numerous biological processes— including development—their aberrant expression has been shown to be associated with diseases such as cancer. To better define the precise cellular function of specific miRNAs, it is necessary to experimentally inactivate them in cells of interest.
One such inactivation approach relies on antisense oligonucleotides directed against miRNAs. While this can be effective in repressing miRNA function, a constant supply of the oligonucleotide is necessary for continued knockdown of steadily expressed miRNAs. This limitation can be overcome by using an enzyme to specifically cleave and inactivate miRNAs.
In a recent paper in Molecular BioSystems, Suryawanshi et al. describe their approach to miRNA inactivation through the use of ribozymes. The authors generated a sequence-specific hammerhead ribozyme targeting the human miR-21, which is overexpressed in glioblastomas and other cancers, and represses target genes involved in apoptosis and cell migration.
The engineered ribozyme was first tested in vitro on a 32P-labeled pre-miR-21 substrate, generating two cleavage products of the expected sizes. To examine its effectiveness in cell culture, the ribozyme was lipofected into MCF-7 cells, which endogenously express high levels of miR-21. As measured by stem-loop real-time PCR following 2 days of treatment, the levels of miR-21 in treated cells were decreased by approximately 50%, which correlated with an increase in the protein levels of one of the main targets of miR-21, programmed cell death protein 4.
Treated MCF-7 cells also showed decreased cell viability, consistent with previous studies showing inhibition of tumor cell growth upon inhibition of miR-21. When the ribozyme was modified with 2′-O-methyl nucleotides, the effects in all of these assays were significantly increased. Future work should allow specific spatiotemporal inhibition of miRNAs using vector-based systems for endogenous expression of the ribozymes.
Suryawanshi et al. Modulation of microRNA function by synthetic ribozymes. Mol. Biosyst.
Posted by: aftabac Posts: 271 :: 28-09-2010 :: | Reply to this Message