| May
1, 2005 |
| By:
Trisha
M. Wise-Draper, Susanne
I. Wells |
| Pharmaceutical
Discovery |
|
Small inhibitory RNAs (siRNAs) have proven to be valuable tools for
knocking down the expression of specific genes. siRNAs exhibit a high
degree of specificity and have important medical implications, such as
selective oncogene repression in cancer (1). Our research is focused on
studies of human papillomaviruses (HPVs) and their roles in cervical
carcinogenesis. A subset of the so-called high-risk HPVs is
etiologically associated with cervical cancer, with over 97% of such
cancers testing positive for high-risk HPV genomic DNA (2). The viral
oncogenes E6 and E7 are expressed in all cells of the primary and
metastatic tumor, and sustained E6/E7 expression is required for
carcinogenesis. The E6 proteins encoded by the high-risk HPVs are known
to degrade the p53 tumor suppressor, while the E7 proteins inhibit and
degrade the retinoblastoma (Rb) tumor suppressor family proteins.
One frequent characteristic of cervical cancer cells is the
integrated state of HPV genomes together with the disruption of the
viral E2 open-reading frame. The loss of E2 is presumed to be involved
in carcinogenic progression because — at least at sufficiently high
expression levels — it can act as a negative regulator of the viral
E6/E7 promoter in cervical cancer cells. Exogenous reintroduction of E2
into HPV-positive cervical cancer cells such as HeLa results in E2
binding to viral E6/E7 promoter sequences and in efficient repression of
E6/E7 expression. This E2-mediated oncogene repression results in the
reversion of cellular transformation through either cellular senescence
or apoptosis. Though E2 expression is a useful way to inhibit viral
oncogene expression, it has been shown to cause apoptosis in HPV-negative
cancer cells (3). In order to repress E6 and E7 with less associated
toxicity, we chose to use siRNA targeted against the viral oncogenes.
HPV18 E6/E7 is expressed from a bicistronic message, and both oncogenes
can therefore be targeted via a single duplex siRNA.
Efficient RNA interference (RNAi) using duplex siRNA depends upon
high transfection efficiency. Duplex siRNA is not transfected easily
using conventional transfection reagents. In order to quantitate the
percentage of cells that received nucleic acids, we co-transfected a
β-galactosidase (β-gal) marker plasmid. We found that some
transfection reagents worked well for introducing either plasmid DNA
alone or duplex siRNA alone, but that the X-tremeGENE siRNA Transfection
Reagent (Roche Applied Science, Indianapolis, Indiana, USA) worked well
for both plasmid DNA and duplex siRNA. Here, we show that specific
knockdown of the HPV18 E6 and E7 oncogenes using the X-tremeGENE siRNA
Transfection Reagent can efficiently inhibit the growth of HPV-positive
cervical cancer cells.
Materials and Methods One day
prior to transfection, HeLa cells (Howley Laboratory, Boston,
Massachusetts, USA) were plated into 60-mm plates in 4 ml or into 6-well
plates in 2 ml of Dulbecco's Modified Eagle Medium (DMEM) supplemented
with 10% fetal bovine serum (FBS) without antibiotics. Cells were
transfected when they had reached 60-80% confluency.
To quantitate β-gal expression, cells in 6-well plates were
transfected with either the X-tremeGENE siRNA Transfection Reagent or
Reagent A from another supplier. For X-tremeGENE siRNA Transfection
Reagent, either 3 µg β-gal plasmid or 2 µg β-gal plasmid
together with 1 µg of the siRNA (Dharmacon Inc., Lafayette, Colorado,
USA) were diluted in 100 µL of antibiotic-free and serum-free medium.
For each well, a total of 10 µL of X-tremeGENE siRNA Transfection
Reagent was diluted in 100 µL of antibiotic and serum-free medium. The
diluted X-tremeGENE siRNA Transfection Reagent was added drop-wise to
the nucleic acids and allowed to incubate for 15-20 min. at room
temperature. The mixture then was added to the cells drop-wise. On the
next day, the cells were examined microscopically for toxicity and then
fixed and stained for β-gal expression. Blue cells were counted,
and the data are expressed as percent blue cells relative to total cell
number.
For duplex RNA transfection in the absence of plasmid DNA, cells in
60-mm plates were either mock transfected or transfected with 4 µg
siRNA. The transfection procedure was the same as above. Total RNA was
harvested on day three for Northern blot analysis using probes that were
specific for the entire HPV18 E6/E7 or GAPDH open reading frames. Total
protein was isolated after three days for subsequent Western blot
analysis using a p53-specific antibody. To examine cell morphology,
microscopic inspection was performed at three days post-transfection.
Reagent A was used exactly according to the manufacturer's directions.
Table I. HeLa cells were
transfected using the X-tremeGENE siRNA Transfection Reagent or
Reagent A with either β-gal expression plasmid alone or
together with siRNA. Cells were stained for β-gal activity
at 24 h post-transfection and were microscopically examined for
signs of toxicity.
|
Results and Discussion
In order to compare the DNA transfection efficiency of the two reagents
24 h after initial transfection, HeLa cells were stained for β-gal
activity (Table I). Percentages of β-gal positive cells were equal
regardless of the presence or absence of duplex RNA. In both instances,
we did not detect any β-gal positive cells with transfection
reagent A, indicating that plasmid-based transfection was inefficient.
In contrast, use of X-tremeGENE siRNA Transfection Reagent resulted in a
transfection efficiency of 80-90%. We assessed toxicity effects by
microscopic scanning for apoptotic cells or debris and found that there
were only minor effects with both reagents (Table I).
Figure 1. Cells were mock
transfected or transfected with HPV18 E6/E7 siRNA using either
X-tremeGENE siRNA Reagent or Reagent A. a) Expression of HPV18
E6/E7 mRNA was quantitated by Northern blot analysis. b)
Expression of p53 protein was quantitated by Western blot
analysis using equal amounts of total protein, as determined by
Bradford assays. c) Photographs were taken at the same
magnification for an assessment of cellular growth and cell
morphology.
|
Since the β-gal stain indicated
successful co-transfection of only plasmid DNA, we performed functional
assays to determine the siRNA duplex transfection efficiency.
HPV18-positive HeLa cells were either mock transfected or transfected
with HPV18 E6/E7-specific siRNA. Transfected HeLa cells were harvested
for isolation of total RNA and protein on day three, and the extracts
were subjected to Northern blot and Western blot analyses, respectively.
The Northern blot was hybridized to a radioactively-labeled probe
specific for HPV18 E6 and E7 sequences. The blot was stripped and
reprobed for GAPDH as a loading control. A reduction of HPV18 E6/E7 mRNA
levels in the lanes containing the HPV18 E6/E7 siRNA transfected cells
with both reagents (Figure 1a, lanes 2 and 4) over the respective mock-transfected
cells (Figure 1a, lanes 1 and 3) was observed. This reduction was more
significant for the cells transfected with X-tremeGENE siRNA
Transfection Reagent than for those transfected with Reagent A (Figure
1a, lanes 2 and 4).
Equal amounts of total protein were
subjected to Western blot analyses with a p53-specific antibody (Figure
1b). The p53 protein is targeted for degradation by E6, leading to
increased p53 levels if E6 expression is sufficiently reduced by RNAi.
Compared with levels of p53 in the mock controls, increased p53 protein
levels were detected in the HBV18 E6/E7 siRNA transfected HeLa cells
using either reagent (Figure 1b). However, levels of p53 were induced by
Reagent A compared with the X-tremeGENE siRNA Transfection Reagent in
the mock controls (Figure 1b, compare lanes 3 and 1). Minimizing
transfection-associated effects on p53 is important for functional
studies because elevated p53 levels are known to induce either cellular
growth arrest or apoptosis in cultured cells.
Finally, since RNAi directed
against HPV18 E6/E7 has been reported to cause cellular senescence in
HeLa cells (4), we monitored the typical senescence-associated
spindly-cell and large-cell morphology of cells transfected with X-tremeGENE
siRNA Transfection Reagent (Figure 1c). As expected, dramatic cell
growth suppression was observed in HPV18 E6/E7 siRNA transfected HeLa
cells together with the appearance of senescent cells. Similar effects
were not observed in the mock-transfected HeLa cell control. Cells of
the cell line 293 — which does not harbor the HPV oncogenes — were
used as a negative control for this experiment and were unaffected,
suggesting that the RNAi approach was specific.
Conclusion
X-tremeGENE siRNA Transfection Reagent is an advanced product that
allows efficient introduction of both DNA plasmid and duplex RNAs into
cultured cells at minimal toxicity. In the future, this reagent in
conjunction with the HPV18 E6/E7 siRNA will be a valuable tool for the
analysis of molecular pathways downstream from the HPV oncogenes.
References
1. Y. Shi, Trends in Genetics 19, 9-12 (2003).
2. P.M. Howley, Papillomavirinae:
The Viruses and their Replication. B.N. Fields, D.N. Knipe and P.M.
Howley (eds). In: Fields Virology, 3rd ed. (Philadelphia, Lippincott-Raven,
1996).
3. K. Webster et al., J. Biol.
Chem. 275, 87-94 (2000).
4. A.H.S. Hall and K.A. Alexander, J.
Virol. 77, 6066-6069 (2003).
Trisha M. Wise-Draper is a
PhD candidate and Susanne I. Wells is assistant professor of
pediatrics in the Division of Hematology/Oncology at the Children's
Hospital Medical Center in Cincinnati, Ohio, USA. Susanne I. Wells can
be reached at susanne.wells@cchmc.org
.
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