Biology Reference
In-Depth Information
to target mRNAs in signaling pathways involved in tumor suppression, as well as
tumorigenesis. Investigating micro-RNA regulation of tubulin isotypes will shed
light on the mechanisms underlying the processes that implicate tubulin isotypes
as biomarkers for aggressive tumors or chemotherapy resistance. The methods dis-
cussed in this chapter include the use of micro-RNA superarrays, next-generation
sequencing, real-time PCR experiments, upregulation of micro-RNAs, and immuno-
precipitation of RNA-induced silencing complex. We will show examples of data
collected using these methods and how these data contribute to understanding pac-
litaxel resistance.
INTRODUCTION AND RATIONALE
Over the past 15 years, micro-RNAs have been studied as mRNA posttranscriptional
silencers (
Bartel, 2004; Esquela-Kerscher &Slack, 2006; Heneghan, Miller, &Kerin,
2010; Yu et al., 2010
). Micro-RNA transcription and processing has been described in
detail (
Bartel, 2004
). Briefly, micro-RNAs are initially transcribed by RNA polymer-
ase II and folded into hairpin secondary structures (pri-miRNA) that are processed in
the nucleus by Drosha and DGCR8 to approximately 75-mers (pre-miRNA). The
double-stranded pre-miRNA is transported from the nucleus by XPO5, and the hair-
pin loop is then cleaved by Dicer1 and further processed in the cytoplasm to a final
micro-RNA of 20-25 nucleotides. The double-stranded micro-RNA interacts with
RNA-induced silencing complex (RISC) composed of Dicer, double-stranded
RNA binding protein TRBP, and Argonaute 2 (
Gregory, Chendrimada, Cooch, &
Shiekhattar, 2005
). One strand of the micro-RNA is released, and the guide strand
remains associated with the catalytic protein, Argonaute 2. The micro-RNA can
now complex with target mRNAs to promote mRNA degradation. Complementarity
of the 5
0
nucleotides at positions 2-8 of micro-RNA (seed sequence) with the 3
0
untranslated region of the target mRNA is essential for the mRNA degradation pro-
cesses. Translational silencing is less well understood.
Hundreds of micro-RNAs have been identified that induce tumorigenesis or sup-
press tumor growth either through degradation of message or by repressing translation
(
Esquela-Kerscher & Slack, 2006; Yu et al., 2010
). For example, one tumor suppressor
micro-RNA, miR-100, in ovarian cancer cells (
Nagaraja et al., 2010
) was implicated in
the regulation of cell proliferation and cell survival through interaction with the PI3/
Akt/mTOR pathway. We recently showed that miR-100 levels are reduced in MCF7
breast cancer cells by paclitaxel treatment and that miR-100 can regulate
b
-tubulin iso-
type classes I, IIA, IIB, and V (
Lobert, Jefferson, & Morris, 2011
).
Recently, much interest has focused on the miR-200 family, in particular
miR-200c. This micro-RNA acts as a tumor suppressor in ovarian cancer cells
and tissues (
Cittelly et al., 2012
). It was shown to regulate
-tubulin isotype class
III (
TUBB3
).
b
-Tubulin class III reduces the stability of microtubules and increases
their dynamic instability (
Kamath, Wilson, Cabral, & Jordan, 2005
). Recent work
suggests that
b
b
-tubulin class III is a “survival factor” (
De Donato et al., 2011
). These
