Suppression of Dicer Increases Sensitivity to Gefitinib in
Human Lung Cancer Cells
Jui-Chieh Chen, PhD1, Yen-Hao Su, MD2,3, Yang-Hao Yu, MD, PhD4,5, Yi-Wen
Chang, PhD6,7, Ching-Feng Chiu, PhD1, Chi-Feng Tseng, MS1, Hsin-An Chen,
MD, PhD2,3, and Jen-Liang Su, PhD1,8,9,10
1
National Institute of Cancer Research, National Health Research Institutes, Zhunan,
Taiwan; 2Graduate Institute of Clinical Medicine, College of Medicine, Taipei
Medical University, Taipei, Taiwan; 3Department of Surgery, Division of General
Surgery, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan;
4
Department of Internal Medicine, Divisions of Pulmonary and Critical Care
Medicine, China Medical University Hospital, Taichung, Taiwan; 5Graduate Institute
of Clinical Medical Science, China Medical University, Taichung, Taiwan; 6Graduate
Institute of Biochemistry and Molecular Biology, National Yang-Ming University,
Taipei, Taiwan; 7Genomics Research Center, Academia Sinica, Taipei, Taiwan;
8
Graduate Institute of Cancer Biology, China Medical University, Taichung, Taiwan;
9
Department of Biotechnology, Asia University, Taichung, Taiwan;
10
Center for
Molecular Medicine, China Medical University Hospital, Taichung, Taiwan
1
Correspondence author:
Jen-Liang Su, PhD; National Institute of Cancer Research, National Health Research
Institutes, No.35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan; e-mail:
jlsu@nhri.org.tw; tel: + 886-37-246-166 (ext. 35142); fax: + 886-37-586-401
The running head:
Dicer regulate gefitinib sensitivity in NSCLC
Disclosure of potential conflict of interest:
No potential conflicts of interest were disclosed.
2
SYNOPSIS
In the present study, we found that Dicer-upregulated miR-30b/c and
miR-221/222 promote gefitinib resistance of NSCLC cells by downregulation of
caspase-3. We speculate that suppression of Dicer might have a potential for future
applications in gefitinib resistance of NSCLCs.
3
ABSTRACT
Background. Accumulating evidence is revealing an important role of microRNA
(miRNA) in tumor progression and chemotherapeutic resistance. Dicer is a
cytoplasmic endoribonuclease Type III crucial for production of mature microRNAs.
The aberrant expression of Dicer has also been reportedly associated with clinical
aggressiveness, prognosis and patient survival in various cancer types. However, the
molecular mechanisms of Dicer in acquired gefitinib resistance are still not clear.
Methods. In this study, we analyzed the protein level of Dicer between
gefitinib-sensitive (PC9) and gefitinib-resistant (PC9/GR) non-small cell lung cancer
(NSCLC) cell lines by Western blot analysis. Silence and overexpression of the Dicer
were respectively performed to investigate the effects on gefitinib sensitivity, as
assessed by MTT assay and sub-G1 assay of flow cytometry. To further explore the
mechanism of chemoresistance, we examined whether Dicer knockdown leading to
modulating specific miRNAs and and its microRNA target genes.
Results. Dicer expression was significantly increased in PC9/GR compared with
PC9 cells. Knockdown of Dicer restores gefitinib sensitivity in resistant cells, and
overexpression of Dicer enhances resistance to gefitinib in sensitive cells. Silencing of
Dicer induces sensitive to gefitinib in NSCLC cells through the down-regulation of
miR-30b/c and miR-221/222 to increase the protein level of caspase-3 resulting in an
4
increase in gefitinib-induced apoptosis.
Conclusions. Dicer contributes to the resistance to gefitinib in lung cancer. These
results indicate that Dicer may be a potential target for diagnosis and therapy of
patients with resistance to gefitinib.
5
INTRODUCTION
Global cancer statistics indicate that lung cancer is the leading cause of cancer
deaths worldwide.1 Approximately 85% of all lung cancers are non-small cell lung
cancers (NSCLCs), which include adenocarcinoma, squamous cell carcinoma and
large cell carcinoma.2 Gefitinib, an EGFR tyrosine kinase inhibitor (TKI), is a
commonly used therapeutic agent for advanced NSCLCs.3,4 Nevertheless, despite a
positive response to initial treatment, many patients eventually develop resistance to
gefitinib after varying periods of time.5 Therefore, novel approaches to the treatment
of NSCLCs must be developed.
Micro-RNAs (miRNAs) are a class of endogenous non-coding RNAs consisting
of approximately 22 nucleotides in length and are capable of repressing gene
expression by binding to their complementary sequence in the 3' untranslated region
(UTR) of mRNA targets.6 MiRNA biogenesis is required for sequential processing by
Drosha, AGO, and Dicer.7 Recently, emerging evidence is revealing an important role
of miRNAs in anticancer drug resistance and miRNA may be a target for the
development of novel therapeutic strategies to overcome drug resistance.8-11
Dicer is cytoplasmic RNase III-type endoribonuclease that processes precursor
microRNAs into mature microRNAs. Deregulated expression of Dicer has been
reportedly associated with drug resistance in different cancer types.12-16 Nevertheless,
6
whether or not Dicer is involved in the gefitinib resistance of NSCLC cells has not yet
been reported.
A previous study indicated that epidermal growth factor receptor (EGFR) and
MET (the receptor for hepatocyte growth factor) could induce gefitinib resistance in
NSCLCs via upregulation of miR-30b, miR-30c, miR-221, and miR-222. In addition,
the downregulation of miR-103 in NSCLCs has also been shown to increase these
microRNAs production to induce resistance to gefitinib treatment.17 A recent report
showed that miR-103 could attenuate miRNA biosynthesis by targeting Dicer.18 In the
present study, we speculate that expressions of Dicer might play important role in the
development of gefitinib resistance in NSCLCs.
7
MATERIALS AND METHODS
Reagents and antibodies
Gefitinib was obtained from AstraZeneca Pharmaceuticals (Wilmington, DE).
FuGENE 6 was purchased from Roche (Basel, CH). Polyethyleneimine (PEI),
polybrene,
RNase
A,
propidium
iodide,
protease
inhibitor
cocktail
and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased
from Sigma-Aldrich (St Louis, MO). The caspase-3 inhibitor Z-DEVD-FMK was
purchased from R&D Systems Inc. (Minneapolis). All cell culture-related reagents
were purchased from Invitrogen (Carlsbad, CA). The sources of primary antibodies
were as follows: AGO2, Dicer and Drosha, were purchased from Abcam Ltd.
(Cambridge, UK). MET was purchased from Santa Cruz Biotechnology (Santa. Cruz,
CA). Cleaved caspase-3, EGFR and pEGFR were from Cell Signaling Technology
(Beverly, MA). Anti-α-tubulin antibody was purchased from Sigma. All secondary
antibodies were purchased from Jackson Immuno Research (West Grove, PA).
Cell culture
Cells were grown in RPMI (PC9/WT, PC9/GR) or DMEM (A549, H1299)
supplemented with 10% (vol/vol) FBS, 2 mM of L-glutamine and 100 units/ml
penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO2. In order to
generate a resistant cell, PC9 cells were initially treated with 0.2 μM gefitinib for 48 h.
8
After exposure to gefitinib, cells were washed and cultured in drug-free medium. The
surviving cells were continuously exposed to increasing dosages and finally to a
concentration of 10 μM. PC9 resistant cells were termed PC9/GR. For drug treatment
studies, gefitinib was diluted in serum free medium and added to cultures to give the
desired final concentrations.
Cytotoxicity assays
MTT assay was used to evaluate relative cell viability. For in vitro studies with a
caspase-3 inhibitor, cells were pre-incubated with 10 μM of Z-DEVD-FMK at 37°C
for 90 min, followed by treatment with 20 μM gefitinib. Absorbance of the dye was
measured at a wavelength of 570 nm with background subtraction at 630 nm.
Western blot
Cells were lysed in RIPA buffer containing protease inhibitor cocktail. Protein
concentration was determined by the BCA assay (Pierce). The equal amount of total
protein was resolved by SDS-PAGE and transferred to PVDF membranes (Millipore,
Bedford, MA). Membranes were blocked with tris-buffered saline/0.1% Tween 20
(TBST) containing 5% skim milk and then incubated overnight at 4°C with the
indicated primary antibodies. Membranes were washed with TBST and incubated for
1 h at room temperature with the appropriate secondary antibodies conjugated to
horseradish peroxidase. Membranes were then washed and bound antibodies were
9
visualized
using
ECL
reagents
(PerkinElmer,
MA)
and
autoradiography.
Semi-quantitative analysis of band intensities was performed by densitometry using
image analysis software ImageJ.
Lentivirus infection and shRNA knockdown
The pLKO.1-puro-based lentiviral vectors: TRCN0000290426 (shDicer#1) and
TRCN0000290489 (shDicer#2), and the pLKO.1-shscramble were purchased from
the National RNAi Core Facility at Academia Sinica, Taipei, Taiwan. Recombinant
lentiviruses were packaged according to the manufacturer's instruction. Cultured cells
were incubated with lentivirus containing 8 μg/ml polybrene for 24 h, replaced fresh
medium and incubated for another 48 h. For stable cell lines, cells were selected by
puromycin.
RNA isolation, reverse transcription and real-time PCR quantification
Total RNA was isolated using Trizol reagent (Invitrogen) and reverse transcribed
into cDNA using M-MLV reverse transcriptase (Invitrogen) according to the
manufacturer's instructions. Real-time PCR was performed using the Roche
LightCycler 480 (Roche). The relative mRNA levels of the target gene were
normalized to the mean of internal GAPDH. The mature miRNA sequences were
obtained from the Sanger Center miRNA Registry and the stem-loop RT primers were
designed according to Chen and Ridzon et al.19 Expression levels of miRNA genes
10
were based on the amount of the target message relative to that of the RNU6B
transcript, used as a control to normalize the initial input of total RNA. The relative
levels of gene expression were represented as ΔCt = Ct gene – Ct reference, and the
fold change of gene expression was calculated by the 2−ΔΔCt Method. All primers
sequences are shown in Supplemental Table 1.
Cloning and expression of Dicer in PC9 cells
The PCR products were digested with BamH I and EcoR I and cloned into
pcDNA6.1 to generate pcDN6.1-Dicer plasmid. Empty pcDNA6.1 plasmid was used
as control. Cells were transiently transfected using FuGENE 6 as suggested by the
manufacturer. Stable clones were selected for blasticidin resistance using standard
protocols.
Gefitinib-induced cell death
Aliquots of 1 × 106 cells were collected and washed once with PBS and then
fixed with 70% ethanol overnight. Fixed cells were then washed twice in PBS and
stained with 1 µg/ml propidium iodide in PBS containing 10 µg/ml RNase A for 1 h.
The DNA content was measured by flow cytometry. For all assays, 10,000 cells were
counted.
11
RESULTS
Characterization of PC9 and PC9/GR cells
To study molecular mechanisms of acquired gefitinib resistance in NSCLCs, we
established a gefitinib-resistant PC9 subline designated PC9/GR. After isolating,
PC9/GR cells were not growth inhibited by exposure to gefitinib at concentrations up
to 7.5 μM (IC50 = 19.64 μM), whereas PC9 cells were very sensitive to gefitinib, with
an IC50 value of 0.009974 μM (Fig. 1a). As shown in Fig. 1b, expression of Dicer,
but not Drosha or AGO2, was higher in PC9/GR cells than PC9 cells. When serially
passed in the absence of gefitinib for 25 passages, PC9/GR cells regained partial
sensitivity to gefitinib corresponded with a decrease in the expression of Dicer (Fig.
1c). In a previous study, EGFR and MET were addressed to induce gefitinib resistance
in lung cancers through regulation of expression of specific miRNAs. Consistent with
the previous study, we observed an increase in expression levels of EGFR and MET in
lysates from PC9/GR cells treated with gefitinib. However, EGFR and MET were not
obviously increased in PC9 cells at the same treatment (Fig. 1d).
Knockdown of Dicer restores gefitinib sensitivity in gefitinib-resistant cells
To clarify whether Dicer expression is associated with sensitivity to gefitinib in
PC9/GR cells, we knocked down Dicer expression by lentivirus-mediated delivery of
Dicer shRNA. Knockdown efficiency of Dicer was determined by q-PCR (Fig. 2a)
12
and Western blot (Fig. 2b). As shown in Fig. 2c, silencing Dicer expression resulted in
elevated sensitivity to gefitinib in a dose-dependent fashion, suggesting that Dicer is a
key regulator of drug resistance. Furthermore, knockdown of Dicer in PC9/GR had
significantly higher proportion of apoptotic cells under treatment with gefitinib
compared with PC9/GR/shScramble cells, as demonstrated by an increased sub-G1
fraction (Fig. 2d). To extend our observations, we introduced specific shRNAs to
silence Dicer in A549 and H1299 cells, which are insensitive to gefitinib in vitro
(IC50 > 9 μM), and the expression of Dicer was further validated by Western blot
analysis (Fig. 3a, d). The results revealed that knockdown of Dicer conferred
increased sensitivity to gefitinib compared with control shRNA (shScramble), as
assessed by MTT assay (Fig. 3b, e). Moreover, gefitinib-induced apoptotic population
was
also
increased
significantly
in
A549/shDicer
cells
compared
with
A549/shScramble cells, as evidenced by an increased sub-G1 fraction (Fig. 3c).
Overexpression of Dicer enhances resistance to gefitinib in gefitinib-sensitive cells
Next, to confirm whether Dicer expression mediates gefitinib sensitivity, we
generated stable PC9 cells that expressed either control vector or Dicer expression
vector. The Dicer expression level was analyzed by q-PCR (Fig. 4a) and Western blot
(Fig. 4b). The results revealed that overexpression of Dicer in PC9 significantly
increased resistance to gefitinib, as assessed by MTT assay (Fig. 4c). HCC827 cells,
13
which are sensitive to gefitinib in vitro, were also transfected with Dicer-expressing
vectors or control vectors, and Dicer expression was determined by Western blot (Fig.
4d). A similar result is obtained in Dicer stably overexpressed HCC827 cells that
render cells more resistant to gefitinib compared with cells transfected with a control
vector (Fig. 4e).
Silencing of Dicer expression by shRNA suppresses miR-30b/c and miR-221/222 in
human NSCLC cells
Previous research had shown that miR-30b/c and miR-221/222 involved in
gefitinib resistance, we investigated whether Dicer could modulate amounts of these
miRNAs in NSCLC cells. Analysis of four miRNAs from Dicer-silenced PC9/GR
cells revealed reduced amounts of mature miRNAs when compared with
PC9/GR/shScramble cells (Fig. 5a). Similar results were also found in Dicer-silenced
A549 cells (Fig. 5b). These results indicated that Dicer is required for maintenance of
the specific miRNAs expression level, which involved in resistance to gefitinib in
lung cancer.
Identification of a potential target of miR-30b/c and miR-221/222 involved in gefitinib
resistance
To identify cellular targets of miR-30b/c and -221/222, we performed two
independent algorithms, including miRanda and TargetScan. We found that
14
miRs-221/222 and -30b/30c are partially complementary to regions in the 3’UTR of
caspase-3 (Fig. 5c). In addition, we observed a marked up-regulation of the protein
level of cleaved caspase-3 in gefitinib-treated PC9/GR/shDicer cells compared with
PC9/GR/shScramble cells (Fig. 5d), suggesting that depletion of Dicer reduces the
expression of miR-30b/c and miR-221/222 resulting in elevated protein levels of
cleaved caspase-3 to induce apoptosis and increase gefitinib sensitivity. To further
confirm that gefitinib-induced activation of caspase-3 is involved in the induction of
apoptotic cell death, the PC9/GR/shDicer cells were pre-treated with the caspase-3
inhibitor (z-DEVD-fmk) and apoptotic cells were analyzed using flow cytometry. As
shown in Fig. 5e, pre-treatment of PC9/GR/shDicer cells with the caspase-3 inhibitor
significantly blocked the gefitinib-induced apoptosis of these cells. Taken together,
these results suggest that Dicer knockdown induces sensitive to gefitinib treatment in
NSCLC cells through the down-regulation of miR-30b/c and miR-221/222, and
elevation of these miRNAs target, caspase-3, leading to an increase in sensitivity to
gefitinib-induced apoptosis.
15
DISCUSSION
In this study, we investigated whether altered Dicer expression affects sensitivity
to gefitinib in human lung cancer cells. Here, our results indicate that miR-30b/c and
miR-221/222 are downregulated after Dicer silencing, which induce apoptosis in
gefitinib-resistant NSCLC cells by increases in the amount and activity of caspase-3.
Although a previous study has demonstrated that the reduced expression of Dicer
in a significant fraction of lung cancers is associated with shorter postoperative
survival,20 it remains unknown whether Dicer is sufficient to make lung cancer cells
resistant to gefitinib and is correlated significantly with clinical response and survival
following gefitinib treatment in patients.
Deregulated expression of Dicer has been reportedly associated with drug
resistance in different cancer types. In human breast carcinoma cells, a study showed
that knockdown of Dicer in MCF-7 cells leads to significant G1 arrest and increased
sensitivity to cisplatin.12 Moreover, Tamoxifen (Tam) is the most prescribed hormonal
agent for the treatment of breast cancer patients with estrogen receptor α
(ERα)-positive, but acquired Tam resistance frequently occur. Dicer overexpression
leads to an increase in the breast cancer resistance protein (BCRP) resulting in Tam
resistance in MCF-7 cells.14 With regard to the roles of Dicer in the relationship
between tumor cells and immune cells, Dicer is responsible for the generation of the
16
mature miR-222 and -339 to suppress ICAM-1 (intercellular cell adhesion molecule)
expression on tumor cells, which promotes resistance of cancer cells to be killed by
cytotoxic T-lymphocytes (CTLs). However, disruption of Dicer leading to
up-regulation of ICAM-1 can enhance the susceptibility of tumor cells to antigen
specific lysis by CTLs.13 A recent study has also shown that decreasing the expression
of Dicer through specific RNAi can sensitize resistant adenocarcinoma cells to
cisplatin.15
Accumulating evidence suggests that Dicer depletion leads to the general
decrease in the amount of mature miRNAs.21,22 It has also been observed that miRNA
expression in normal tissues is globally higher than in tumors. Moreover, poorly
differentiated tumors had lower global levels of miRNA expression compared with
the more-differentiated samples.23 This might potentially account for the generation or
maintenance of cancer stem cells, recently proposed to be responsible for the source
of resistance to gefitinib therapy in lung cancer.24
A study has reported microRNA-128b is a regulator of EGFR in NSCLC cell
lines. The loss of heterozygosity of the microRNA-128b is frequent in tumor samples
and correlated significantly with clinical response and survival after gefitinib
treatment.25 Moreover, Dicer expression was found inversely correlated with
expression levels of mature let-7 in a panel of human cancer cell lines.26 Reduced
17
let-7 significantly over-expressed the expression of Dicer resulting in increased cell
proliferation of oral cancer cells.27 Interestingly, a study has shown that
overexpression of let-7 in lung adenocarcinoma cells inhibited cancer cell growth in
vitro.28
Furthermore, our study has revealed that knockdown of Dicer in cultured
NSCLC cells triggers activation of caspase-3 and leads to increased sensitivity to
gefitinib. Caspase-3 is a cysteine protease that plays a critical role in the process of
apoptotic cell death. Previously, the caspase-3 was also reported to be associated with
chemotherapeutic resistance in cancer. Overexpression of caspase-3 restores
sensitivity for drug-induced apoptosis in cancer cells with acquired drug
resistance.29,30 Conversely, loss of caspase-3 expression may render cancer cells
resistant to chemotherapy.31,32 In NSCLC patients, caspase-3-positive tumors had a
tendency for the most favorable prognosis.33 Moreover, a member of the inhibitor of
apoptosis family of proteins, survivin, has been shown to inhibit caspases and to
prevent caspase mediated cell death. Targeting of survivin has the potential to
overcome EGFR–TKI resistance in NSCLCs.34
In conclusion, we have demonstrated that Dicer is required for maintenance of
the specific miRNAs expression level, which involved in resistance to gefitinib in
lung cancer. These findings will be important for the further development of novel
18
therapeutic strategy and a biomarker to early diagnosis for gefitinib resistance in lung
cancer patients.
19
ACKNOWLEDGMENT
This work was supported by grants from the National Science Council, Taiwan
(NSC
102-2314-B-039-200,
NSC
102-2314-B-038-028-MY3,
NSC
101-2320-B-400-016-MY3; from National Health Research Institutes, Taiwan
(CA-102-PP-41); from China Medical University Hospital (DMR-101-014); and from
China Medical University (CMU99-TC-22, CMU100-S-22).
20
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FIGURE LEGENDS
FIG. 1
Establishment and characterization of the gefitinib-acquired resistant PC9
cells. (a). Serum-starved PC-9 and PC9/GR cells were exposed to various doses of
gefitinib for 24 h and assayed for survival by MTT assay. Data shown are mean ±
SEM of six independent experiments performed in triplicate. ***p < 0.001. (b). The
protein expression levels of Dicer, Drosha and AGO2 in PC9 and PC9/GR cells were
analyzed by Western blot. (c). Associations between dicer expression and gefitinib
sensitivity were investigated in PC9/GR cells cultured without gefitinib for 25
passages. Cells were treated with gefitinib at the indicated doses for 24 h and assayed
for survival by MTT assay. Protein expression of Dicer at different passages was
analyzed by Western blot. The bars represent the means ± SEM of three independent
experiments, performed using different passages of PC9/GR cells in triplicates. **p <
0.01. (d). A western blot showing dose-dependent effects of gefitinib on the
expression of dicer, MET, p-EGFR, and EGFR. α-tubulin was used as a loading
control.
FIG. 2
Silencing of Dicer in PC9/GR cells by shRNA lentivirus resulted in
increased gefitinib sensitivity. Dicer knockdown efficiency was verified by using
quantitative real-time PCR (a) and western blot analysis (b). Bars represent means ±
26
SEM from three independent experiments in triplicates. α-tubulin was used as a
loading control. (c). Cells were exposed to different concentrations of gefitinib for 24
h and subsequently evaluated by MTT assay. Percentage of cell viability is shown
relative to untreated control. Data are representative of at least three independent
experiments performed in triplicate. ***p < 0.001. (d). Cells were treated with or
without 20 μM gefitinib for 24 h. Sub-G1 cell populations were determined by flow
cytometry in PC9/GR. Bars represent means ± SEM from at least three independent
experiments. *p < 0.05.
FIG. 3
Dicer modulating gefitinib sensitivity also exists in other types of non-small
cell lung cancer (NSCLC) cells. (a). Western blot analysis of Dicer expression in
A549 infected with shDicer and shScramble by lentivirus system. (b). A549/shDicer
and A549/shScramble cells were treated with 10 μM of gefitinib for 48 h and then
analyzed for cell viability by a MTT assay. (c). Dicer was required for resistance of
A549 cells to gefitinib-induced apoptosis. Apoptotic cells with sub-G1 DNA content
were determined by flow cytometry. Bars represent means ± SEM from three
independent experiments in triplicates. *p < 0.05. (d). Western blot analysis of Dicer
expression in H1299 infected with shDicer and shScramble by lentivirus system. (e).
H1299/shDicer and H1299/shScramble cells were treated with 10 μM gefitinib for 48
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h. Sub-G1 cell populations were determined by flow cytometry. Bars represent means
± SEM from at least three independent experiments. *p < 0.05.
FIG. 4
Overexpression of dicer induces gefitinib resistance in PC9 and HCC827
cells. Dicer-expressing vector or control vector was transfected into PC9 cells,
followed by analysis of Dicer expression using real-time PCR (a) and Western blot
(b). Bars represent means ± SEM from three independent experiments in triplicates.
(c). PC9/pcDNA and PC9/Dicer cells were exposed to 5 μM of gefitinib for 24 h and
assayed for survival by MTT assay. Data are representative of at least three
independent experiments performed in triplicate. ***p < 0.001. (d). Western blot
analysis of Dicer in HCC827 cells transfected with Dicer-expressing vector or control.
(e). HCC827/pcDNA and HCC827/Dicer cells were treated with 5 μM of gefitinib for
24 h and measured the function of Dicer in gefitinib-suppressed viability by MTT
assay. Data are representative of at least three independent experiments performed in
triplicate. *p < 0.05.
FIG. 5
Knockdown of Dicer in NSCLC cells decreases expression of miR-30b/c
and miR-221/222 leading to up-regulation of caspase-3 and increased gefitinib
sensitivity. Knockdown of Dicer in PC9/GR cells (a) and A549 cells (b) decreases
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expression of miR-30b/c and miR-221/222. All reactions were normalized to
wild-type cells. Error bars indicate the SEM of three independent experiments. *p <
0.05, **p < 0.01. (c). The miR-30b/c and miR-221/222 are partially complementary to
regions in the caspase 3 mRNAs 3’UTR. (d). PC9/GR/shDicer and PC9/GR/Scramble
cells were untreated or treated with gefitinib and analyzed the cleavaged caspase-3 by
Western blot analysis. (e). The effect of the caspase 3 inhibitor on viability of
Dicer-silenced PC9/GR cells after treated with gefitinib. The cell viability was
measured by MTT assay. Data are representative of at least three independent
experiments performed in triplicate. *p < 0.05.
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