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play a significant role in EMT, leading to tumor metastasis (
Burk et al., 2008
). In ad-
dition, miR-200c was shown to target
-tubulin class III in ovarian cancer cells and
tumors (
Cochrane, Spoelstra, Howe, Nordeen, & Richer, 2009; Cittelly et al.,
2012
). We found that
b
-tubulin classes I, III, and IVB are downregulated by 40-nM
paclitaxel treatment in MDA-MB-231 cells and that these tubulin isotypes appear
to be coordinately upregulated with ZEB1 (
Fig. 5.4
A). MiR-200c likely plays a part
in this regulation.
Figure 5.4
Bshowsthat
b
-tubulin classes I and III are significantly
upregulated when ZEB1 is upregulated threefold by transfection into cells. Smaller
increases in
b
-tubulin classes IVB and V also occur with ZEB1 upregulation. We con-
clude that the clinical effectiveness of paclitaxel in triple negative breast cancer may be
in part due to the drug-induced upregulation of the tumor suppressor miR-200c. In ad-
dition, upregulation of
b
-tubulin class III, in tumor tissues
are likely to be associated with an increase in cell survival factors such as ZEB1 and not
a direct effect of
b
-tubulin isotypes, such as
b
-tubulin class III.
Figure 5.5
is a schematic of our findings for
paclitaxel-induced changes in miR-200c in MDA-MB-231 cells.
b
SUMMARY
Over the past 15 years, there has been considerable interest in how noncoding micro-
RNAs contribute to mRNA and protein regulation. More than two decades have
passed since Cleveland and colleagues showed that one or more cellular cofactors
are involved in posttranscriptional regulation of
-tubulins (
Pachter, Yen, &
Cleveland, 1987; Theodorakis & Cleveland, 1992; Yen, Gay, Pachter, &
Cleveland, 1988
); however, little is known about the mechanisms underlying differ-
ential regulation of these proteins. Using drugs as probes to induce changes in
b
-
tubulin isotypes mRNA associated with changes in micro-RNAs offers a window
into the regulation of tubulin genes and the essential cofactors. Furthermore, under-
standing the regulation of
b
-tubulin isotypes by micro-RNAs may clarify oncogenic
and metastatic pathways and the mechanisms underlying drug resistance.
b
gene for normalization. The data are plotted as fold difference relative to control cells treated
with DMSO. The error bar represents the standard deviation from two independent
experiments. (B) Transfection of MCF7 cells with miR-100 in the presence and absence of
400-nM paclitaxel treatment for 24 h. Quantitative RT-PCR data were analyzed using
standard curves for each
g of total
RNA. Experiments were done with triplicate individual cell cultures. A representative
experiment is shown. The error bars represent the standard deviation from duplicate samples
from a single experiment. Panel A:
b
-tubulin isotype, andmRNA copies were normalized to 1
m
b
-tubulin class I; Panel B:
b
-tubulin class IIA; Panel C:
b
-tubulin class IIB; Panel D:
b
-tubulin class III, and Panel E:
b
-tubulin class V. No changes
were found in
-tubulin classes III or IVB, with miR-100 transfection. Student t-test was
used to compare paired samples. The p-values from statistical significance analysis of control
and transfected samples (*) are shown.
b
Figure from
Lobert et al. (2011)
