Biomedical Engineering Reference
In-Depth Information
what they do and how they do it. Over 500 miRRNAs have been found in the human
genome. A recent review in
Nature Reviews
suggests that miRNAs regulate one-third of
human genes [15]. MiRNAs have become recognized as a new class of gene regulators
and, therefore, important for gene modification of cells. MiRNAs are small non-coding
RNAs that modify gene expression by post-transcriptional inhibition of targeted mRNA.
In the nucleus, miRNA is formed from introns and exons as 'primary' or 'pri-miRNA.'
But it is not a messenger RNA - it does not specify or generate a protein. The pri-RNA,
a folded-back structure of 60-70 nucleotides, is processed in the nucleus by the enzymes
Drosha and Pasha. Drosha cuts out the stem-loop structure which is the 'pre-miRNA.' The
pre-miRNA is exported out of the nucleus by exportin and into the cytoplasm where it is
diced up by the enzyme Dicer RNase III, mentioned above in the siRNA process. The same
effect occurs when Dicer cuts the stem-loop into short-length (19-25 nucleotides) inverted
'mature miRNA.' As with siRNA, one strand of the mature miRNA becomes part of the
RISC and targets mRNA by binding to antisense complementary regions and cleaving or
degrading the targeted mRNA. Multiple roles for miRNAs in gene regulation have been
revealed by gene expression analysis polymerase chain reaction (PCR), and by transgenic
mice with knockouts of specific miRNA. Expression arrays are revealing specific miRs in
different tissues and cells from invertebrates to humans. Many miRNAs (miR-1, miR-34,
miR-60, miR-87, miR-124a) are highly conserved between vertebrates and invertebrates
[16], including the small temporal (st)RNAs discovered in
C. elegans
(e.g. let-7 RNA, lin-4)
that are similar to miRNAs in humans. As these stRNAs are critical for cell differentiation
and timing of neural connections, the conservation may indicate functional evolution.
A survey of mouse tissues with northern blotting [16] showed miR-1 is dominant in
the heart (45%). In the liver, miR-122 was 72% of all miRNAs tested and miR-124a was
profound in the mouse brain.
Although the mechanism of miRNA action is principally inhibitory on targeted mRNA,
which is essential for normal growth and differentiation in cell and tissue development,
miRNAs can be involved in cancer. They can be depleted or suppressed, allowing oncogenes
to be overproduced. Kumar
et al
. [17] recently showed that global suppression of miRNAs
in various cancer cell lines increased cancer cell transformation and enhanced tumorogenesis
in mice. To suppress miRNA they targeted Drosha and Dicer with siRNA. Non-cancerous
cells did not become cancerous, but did not grow. This suggests that increasing miRNAs
could be a new gene therapy approach to treating cancer by suppressing oncogenes.
12.1.2 Gene therapy strategies: Delivering genes to cells
12.1.2.1 Non-viral delivery
Non-viral delivery for gene transfer refers to formulations of liposomes, naked DNA and
dendrimers. The main advantage is that they are non-pathogenic and have low toxicity,
they can be easily purified and most significant they can carry a transgene of almost any
size. The disadvantages are low efficiency and transient transgene expression. Naked DNA
injected into heart or muscle cells can be expressed for 2 weeks to several months [18].
12.1.2.2 Electroporation
Electroporation (EP) is used for
in vitro
transfection of DNA into cells. It was first used
by Neumann
et al
. in 1982 [19] to transfer genes into mouse lyoma cells. Later, in 1987,
