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grow cells in 300-cm 2 flasks to 80% confluence before initiating drug treatment. For
these experiments, we use the reagents and procedure of the Magna RIP
kit (Milli-
pore, Billerica, MA), except that
columns
from Miltenyi Biotech Inc. (Auburn, CA) are substituted for the original beads,
and the incubation time of the cell lysate with the Microbeads and the RIPAb
m
MACS
Protein G Microbeads and
m
þ
Ago2 (Argonaute 2) monoclonal antibody (Millipore, Billerica, MA) is 45 min.
The RNA is released from the precipitated complex and purified using the TRIzol
method ( Jedamzik & Eckmann, 2009; Lobert et al., 2010 ). The micro-RNA and
mRNA levels associated with Ago2 are measured using qRT-PCR and normalized
to RNA isolated from cell lysate samples that were treated identically except for the
immunoprecipitation step.
5.2 RESULTS AND DISCUSSION
5.2.1
-Tubulin isotypes and miR-100
We showed that paclitaxel treatment of MCF7 breast cancer cells leads to an increase
in mRNA for specific
b
-tubulin isotypes (classes I, IIA, and V). We hypothesized
that upregulation of b -tubulin isotypes was due to a reduction in micro-RNAs that
regulate
b
-tubulin mRNA. We found that miR-100 is downregulated by paclitaxel
treatment and that transfection of MCF7 cells with miR-100 in the absence and pres-
ence of paclitaxel resulted in decrease in mRNA for
b
b
-tubulin isotype classes I, IIA,
IIB, and V ( Lobert et al., 2011 ; Fig. 5.1 A and B).
Because the increase in specific
-tubulin isotype classes with drug treatment is
associated with reduction in the tumor suppressor miR-100, which is known to be
implicated in cell survival pathways, these data suggest that
b
-tubulin isotypes could
be useful biomarkers for drug resistance. For example, miR-100 downregulates
phosphorylated Akt in ovarian cancer cells reducing cell proliferation ( Nagaraja
et al., 2010 ). A reduction in miR-100 with paclitaxel treatment could increase phos-
phorylated Akt in tumor cells. Our data suggest that paclitaxel resistance and in-
creases in specific
b
-tubulin isotypes (classes I, IIA, and V) could develop as a
result of drug-induced reductions in micro-RNA tumor suppressors such as
miR-100 ( Fig. 5.2 ).
b
5.2.2
-Tubulin isotypes and miR-200c
Triple-negative (basal type) breast cancers that lack estrogen, progesterone, and
HER2/neu receptors are frequently the most difficult to treat. Paclitaxel is commonly
used in chemotherapy protocols for these aggressive tumors ( Anonymous &American
Cancer Society, 2012 ). We studied paclitaxel-induced changes in micro-RNAs in
MDA-MB-231 cells that are frequently used as a model for triple negative breast can-
cers. We found that miR-200c is upregulated about twofold by low dose (40 nM) pac-
litaxel treatment for 24 h ( Fig. 5.3 ). This was associated with a 40-50% reduction in
ZEB1 mRNA, a primary target for miR-200c ( Burk et al., 2008 ). ZEB1 is known to
b
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