Biology Reference
In-Depth Information
studies directly link
-tubulin class III protein to the activity of GTPases that con-
tribute to cell proliferation and metastasis. Several studies indicate that increases in
b
b
-tubulin class III may contribute to drug resistance in both cell culture and
in vivo
(
Cabral, 2008
). Furthermore, in ovarian cancer, therapy that includes miR-200c re-
placement can augment the effectiveness of standard chemotherapy with taxanes
(
Cittelly et al., 2012
), perhaps, in part, because miR-200c targets
-tubulin class
III. Thus, the study of micro-RNAs can contribute to our understanding of tubulin
regulation and may point the way to novel combination chemotherapy.
In this chapter, we review methods for measuring micro-RNA levels in cell
lysates and in RISC. The focus is on studying alterations in micro-RNA levels
induced by tubulin-binding drugs and the effects of these alterations on
b
-tubulin
isotypes. We provide examples of data collected using these methods and discuss
implications for drug resistance and novel therapies.
b
5.1
METHODS AND MATERIALS
5.1.1
Measuring micro-RNAs
5.1.1.1
Choice of cell line
Cells and tissues vary in the amounts of specific micro-RNAs (
Liang, Ridzon,
Wong, & Chen, 2007
), and this is reflected in the levels of the primary target mRNAs.
For example, we found more than 1000-fold difference in the miR-200c target ZEB1 in
two breast cancer cell lines: MDA-MB-231 cells and MCF7 cells. These two cell lines
represent very different models for breast cancer. MDA-MB-231 cells lack the HER2,
progesterone and estrogen receptors and are considered to be a model for an aggressive
triple negative basal type breast cancer phenotype. MDA-MB-231 cells express high
levels of the epithelial-to-mesenchymal transition (EMT) protein ZEB1 and low levels
of its regulating micro-RNA, miR-200c; whereas, MCF7 cells have estrogen and pro-
gesterone receptors and express low levels of ZEB1 and relatively high levels of
miR-200c. The basal expression level of the micro-RNA of interest can be determined
by quantitative real-time PCR (qRT-PCR), as described below.
5.1.1.2
RNA extraction and qRT-PCR
In common RNA extraction protocols, micro-RNA constitutes about 1% of total
RNA extracted from cells and tissues. For micro-RNA cDNA preparation, we find
it most efficient to use total RNA rather than attempting to purify micro-RNA. The
protocol for preparation of total RNA using organic solvents (TRIzol) has been pre-
viously described in detail (
Lobert, Hiser, & Correia, 2010
). MiScript II RT kits
(SABiosciences-Qiagen Corporation, Valencia, CA) are used to prepare miRNA
cDNA for PCR experiments. We use the reaction reagents, volumes, and tempera-
tures as recommended by the manufacturer (SABiosciences-Qiagen Corporation,
Valencia, CA). Total RNA-containing miRNA is used in the first step, and the
5X miSCript HiSpec buffer is used to prepare the cDNA. We start with 1-2
m
gof
total RNA, as determined by absorbance at 260 nm using a NanoDrop 1000 UV-Vis
