RNA interference refers to the silencing or knocking down of a target gene by degrading its corresponding mRNA using small interfering RNAs (siRNA) or short hairpin RNA (shRNA).
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Achieving prolonged gene silencing
Short hairpin sequences are encoded within a DNA vector and then introduced inside cells using plasmid transfection or viral transduction. The short hairpin RNA may either be in the firm of simple stem loop or a microRNA-adapted shRNA.
The stem-loop consists of 19-29 basepairs of double-stranded RNA that is bridged by single-stranded RNA. The simple stem loop and microRNA- adapted shRNA are transcribed in the nucleus. Along with the target sequences, other reporter molecules, such as fluorescent proteins can also be included in the cassettes to track the cells that express shRNA.
Upon entering the cells, shRNA is processed by Drosha, a ribonuclease enzyme. The final product is then exported out of the nucleus, processed by Dicer (an RNAse enzyme) and incorporated into the RNA-induced silencing(RISC) complex. Subsequently, mRNA is cleaved by RISC leading to suppression of expression - thus, prolonged gene silencing can be achieved by short hairpin RNA.
Using shRNA in gene and cancer therapies
Downregulating target genes using short hairpin RNA offers a new strategy for gene therapy. Various in vitro studies have shown the promising use of RNAi to treat cancers. The key features of malignant cells are uncontrolled growth and altered cell death pathways. Cancer cells may also show invasive growth, destroying the surrounding cells and tissues.
These behaviors are brought about by altered gene expression and signaling pathways. Thus, targeting the genes that are critical components of a signaling pathway can form a potent method to target cancer. Presently, studies have targeted genes that are involved in cell death, cell cycle regulation, signal transduction, and other cancer-related genes (such as telomerase, fatty acid synthase, etc).
Understanding the circadian clock
Circadian rhythms are present in physiology, metabolism, behavior and are existent in a wide range of organisms: from cyanobacteria to humans. These clocks provide these organisms with an internal sense of time.
Apart from the brain, the machinery for the circadian clock is also present in several other cells of the body. Recently, several RNAi-based screens have been used to understand the mechanism behind the circadian oscillations. This was done by knocking down both known and predicted genes involved in circadian rhythms and then assessing the effect on oscillations.
Discovering new HIV targets
The acquired immune deficiency syndrome (AIDS) continues to be one of the major human heath setbacks, and although therapies have been devised to prolong the life and health of HIV-infected patients, it still remains one of the biggest health issues in the modern world.
Human immunodeficiency virus (HIV) also works in an interesting manner where it captures the host machinery to promote its replication. Thus, there is a need to further identify all the proteins that are involved in the process of HIV infection to discover more potent therapeutic targets.
One of the ways this is being done is by using the shRNA method to silence specific genes that are known or predicted to be a part of the HIV infection process. After suppressing the genes, the effects on HIV infection and pathway can be analyzed to confirm the role of those genes. Thus, this strategy may bring forth possibilities for newer antiviral therapies.
Apart from these effects, shRNA strategy is also being used to understand and probe other processes, such as cell migration, epithelial to mesenchymal migration (i.e. another cancer indicator), cell migration pathways, as well as other disease related pathways. Apart from in vitro studies, RNAi is also being extended to understand these processes in vivo in animal models.