Altered microRNA expression in cisplatin-resistant ovarian cancer cells and upregulation of miR-130a associated with MDR1/P-glycoprotein-mediated drug resistance
- Lingyun Yang
- Ningwei Li
- Hongjing Wang
- Xibiao Jia
- Xue Wang
- Juan Luo
Published online on: Friday, May 18, 2012
- Pages: 592-600
- DOI: 10.3892/or.2012.1823
Ovarian cancer is the deadliest cancer of the female reproductive system (1). More than 70% newly diagnosed patients with ovarian cancer have advance stage disease (FIGO stage III or IV) because of no early symptoms and effective biomarker screening (2). Primary cytoreduction followed by platinum-based combined chemotherapy, such as cisplatin coupled with paclitaxel, has become the main therapeutic approach for advanced ovarian cancer (3–6). Cisplatin crosslinks to double stranded DNA and forms DNA adducts, interfering with DNA replication and transcription, which in turn activates cellular apoptosis. However, development of a multidrug resistance phenotype is a major obstacle to successful treatment of ovarian cancer, which leads to 85–90% relapse and only 30% 5-year survival rate (6,7). Several molecular mechanisms of drug resistance have been discovered and characterized including the overexpression of MDR1/P-glycoprotein (P-gp) (8,9), increased anti-apoptotic factors activity (8,10,11), altered expression of β-tubulin subunits (12), degeneration of growth factor receptor (13), and increased DNA repair activity (14,15). Drug efflux induced by increased accumulation of various ATP-binding cassette (ABC) transportors, including ABCB1, also known as P-gp, which is a 170-kDa transmembrane glycoprotein encoded by MDR1 gene on human chromosome 7p21, has been demonstrated to play a significant role in drug resistance (8,9,16).
microRNAs (miRNAs) are a class of short non-coding RNA molecules that bind to the 3′-untranslated region (3′-UTR) of the target mRNAs and act post-transcriptionally as negative regulators of gene expression (17–19). Mature miRNAs, derived from precursor miRNA (pre-miRNA), are incorporated into the RNA-induced silencing complex (RISC), resulting in either cleavage or translational repression of target mRNAs (20,21). It has been reported that miRNA-mediated gene regulation plays critical roles in biological processes including cellular proliferation, differentiation, apoptosis, metabolism and oncogenesis (17,22,23). Recent studies show that miRNAs associated with oncogenesis act as oncogenes or tumor suppressors. For example, miR-155, upregulated in ovarian and lung cancer, acts as an oncogene (24,25), while miR-15a and miR-16a, deregulated in CLL, act as tumor suppressors (26). In addition, further reports show that miRNAs also play a vital role in chemotherapeutic drug resistance (27–29). Analysis of the up- and downregulation and the mechanism of action of miRNAs may be useful to develop novel plans of targeted therapies.
In the current study, we performed miRNAs expression profile analysis between the human ovarian cancer cell line SKOV3 and the cisplatin-resistant cell line SKOV3/CIS followed by qRT-PCT based validation of miR-130a expression. We also examined the role of miR-130a in the development of cisplatin-resistance in ovarian cancer cells, which may provide a potential target for gene therapy.
Materials and methods
The human ovarian cancer cell line SKOV3 and the cisplatin-resistant cell line SKOV3/CIS were cultured in PRMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified incubator with 5% CO2 at 37°C. SKOV3/CIS was alternately fed with medium containing 7.5 μg/ml cisplatin and was regularly tested for maintenance of drug-resistance. Growth and morphology of each cell line were observed and monitored every three days. The cisplatin-resistant cell was maintained in drug-free medium for 1 week before follow-up experiments. Both cell lines were preserved in Gynecological Oncology of Biotherapy Laboratory, Department of Gynecology and Obstetrics, West China Second University Hospital, Sichuan University, Chengdu, Sichuan, China.
miRNA microarray analysis
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and the miRNeasy mini kit (Qiagen, Copenhagen, Denmark) according to the manufacturer’s instructions, which efficiently covered all RNA species including miRNAs. RNA quality and quantity was measured using the NanoDrop spectrophotometer (ND-1000, NanoDrop Technologies) and RNA integrity was determined by gel electrophoresis. The miRCURY™ Power labeling kit (Exiqon, Vedbaek, Denmark) was used for miRNA labeling. Each sample (1 μg) was 3′-end-labeled with Hy3™ fluorescent label using T4 RNA ligase. Then the Hy3™-labeled samples were hybridized on the miRCURY™ LNA Array (v.16.0, Exiqon), which contained more than 1,891 capture probes, covering all human, mouse and rat miRNAs annotated in miRBase 16.0, as well as all viral miRNAs related to these species. In addition, this array contained capture probes for 66 new miRPlus™ human miRNAs which were proprietary miRNAs not found in miRBase. The total 25-μl mixture from Hy3™-labeled samples with 25 μl hybridization buffer were first denatured for 2 min at 95°C, incubated on ice for 2 min and then hybridized to the microarray for 16–20 h at 56°C in a 12-Bay hybridization systems (NimbleGen Systems, Inc., Madison, WI, USA). Following hybridization, the slides were washed, dried and then scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA, USA). Images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Expressed data were normalized using the median normalization. The normalized fluorescence intensity, which was less than the average intensity of the negative controls or 3× SD of the intensity of the negative controls, indicated an unreliable signal value. After normalization, differentially expressed miRNAs were identified through fold-change filtering. Hierarchical clustering was performed using standard correlation as a measure of similarity by the MEV software (v4.6, TIGR). Three microarray chips were applied for each cell line.
Real-time qRT-PCR for miR-130a
Verification of altered miR-130a expression was performed in SKOV3 and SKOV3/CIS by real-time qRT-PCR. Total RNA of each cell line was extracted with TRIzol reagent (Invitrogen) and was assessed by agarose gel electrophoresis and spectrophotometry. Complementary DNA (cDNA) was generated using the AMV first strand cDNA synthesis kit (Invitrogen). Specific miR-130a primers and internal control U6 snRNA primers (Bulge-LoopT™ miRNA qPCR Primers) were designed and synthesized by Guangzhou RiboBio (Guangzhou RiboBio Co., Ltd., Guangzhou, China). Real-time qRT-PCR was performed in a 20-μl reaction volume containing 2 μl cDNA template, 9 μl SYBR-Green I mix (Takara, China), 2 μl forward primers, 2 μl reverse primer and 5 μl RNase-free H2O on the ABI StepOnePlus instrument (Applied Biosystems, USA) using the following protocol: 95°C for 20 sec, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 70°C for 10 sec. Each sample was run in triplicate. The relative expression of miR-130a was analyzed using 2−ΔΔCt method.
The miR-130a-mimic, -inhibitor and miR-negative RNA were designed and chemically synthesized by Guangzhou RiboBio. Twenty-four hours prior to transfection, SKOV3 and SKOV3/CIS cells were seeded into 6-well plates with 2×105 cells/well and cultured in medium without antibiotics. Transfection was performed using Lipofectamine 2000 (Invitrogen) and OPTI-MEM I reduced serum medium (Gibco) according to the manufacturer’s instructions. Green fluorescent protein (GFP) was applied to show the efficiency of transfection system. Cells in each well were transfected with 10 μl miR-130a-mimic or -inhibitor or miR-negative RNA or 2 μl GFP constructed plasmid. The medium was replaced 4–6 h after transfection with new fresh medium.
Real-time qRT-PCR for MDR1 mRNA
Real-time qRT-PCR was applied to detect the expression of MDR1 mRNA after miRNA transfection for 24 h. Total RNA of each cell line was extracted with TRIzol reagent (Invitrogen) and was assessed by agarose gel electrophoresis and spectrophotometry. Reverse transcription was carried out with the AMV first strand cDNA synthesis kit. The β-actin was used as internal control for MDR1 expression normalization. Primers of MDR1 and β-actin were designed using the Primer Premier software (version 5.0). The primer sequences for real-time qRT-PCR were: MDR1 (forward) 5′-ACAAAGTGCTCACCCAGA TTC-3′ (reverse) 5′-CATTTCCTCGCTGATGACACTA-3′; β-actin (forward) 5′-GCTACGAGCTGCCTGACG-3′ (reverse) 5′-TCGTGGATGCCACAGGAC-3′. Real-time qRT-PCR was performed with cycle parameters as 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 1 min. Each sample was run in triplicate. Data analysis was performed using the 2−ΔΔCt method.
Western blot analysis
Cells were collected and washed twice with ice-cold PBS. Total protein was extracted with RIPA added with 1% protease inhibitor phenylmethanesulfonyl fluoride (PMSF) for 15 min on ice, then centrifuged at 12,000 rpm 4°C for 15 min and the supernatants were collected. Protein concentrations were determined by the BCA method using the standard curve. GAPDH expression was used as internal control. Equal amounts of protein for each sample were resolved using 10% SDS-PAGE gels and transferred onto Immun-Blot™ PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA). The bands were blocked with a 5% solution of skim dry milk in TBS containing 0.1% Tween-20 for 2 h and incubated with primary antibodies P-gp (diluted 1:1000; Calbiochem, San Diego, CA, USA) and GAPDH (diluted 1:50000; Kangchen, Shanghai, China) overnight at 4°C. After repeated washing three times with TBS-T, membranes were incubated with horseradish peroxidase (HRP) conjugated anti-mouse secondary antibody (Bio-Rad Laboratories) for 2 h. Blots were detected and developed on X-ray film using the chemiluminescence method.
In vitro drug sensitivity array
SKOV3 and SKOV3/CIS cells were transfected with miR-130a-mimic or -inhibitor or miR-negative RNA for 24 h, and then transferred into 96-well plates with 1.5×104 cells/well for a drug sensitivity array. After cellular adhesion, SKOV3 cells were treated with increasing concentrations of cisplatin (0.3, 0.6, 1.2, 2.4, 4.8 and 9.6 μg/ml) and SKOV3/CIS were exposed to various doses of cisplatin (3, 6, 12, 24, 48 and 96 μg/ml) for 48 h. Following the incubation, 20 μl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, St. Louis, MO, USA) was added to each well and incubated for 4 h. At the end of the incubation, the medium was removed and 150 μl DMSO was added. Absorbance of each well was measured at a wavelength of 570 nm using a microplate reader (Model 680, Bio-Rad Laboratories). The IC50 median inhibitory concentration of cisplatin was calculated through a cell viability curve.
Data and statistical analysis
Data are presented as the means ± standard deviation from at least three replicate experiments. One-way ANOVA was employed to analyze difference of means between groups using SPSS 11.0 software (SPSS Inc., Chicago, IL, USA). Statistical significance was set as P<0.05.
Profiling of differentially expressed miRNAs in SKOV3 and SKOV3/CIS cells
RNA quantity and quality of each cell line were assured and checked by spectrophotometer and gel electrophoresis. As shown in Table I, the results confirmed good quantity and quality of total RNA isolated from each cell line. To identify the critical miRNAs involved in cisplatin resistance, the microarray platform was applied to profile the global expression of mature miRNA. The signal ratio of SKOV3/CIS to SKOV3 wasdetermined, and the normalized fluorescence intensity, which was less than the average intensity of the negative controls or 3× SD of the intensity of the negative controls indicated an unreliable signal value. Differentially expressed miRNAs with statistical significance were selected only when they were altered by at least 2-fold. With these strict criteria, 35 miRNAs exhibited lower expression and 54 miRNAs exhibited higher expression in SKOV3/CIS than those in SKOV3 cells (Tables II and III). Expression of miRNAs in SKOV3/CIS compared to SKOV3 was decreased by 2.02- to 8.23-fold, while increases in expression ranged from 2.02- to 46.74-fold. These results indicated that such differentially expressed miRNAs might play a crucial role in the development of cisplatin resistance in epithelial ovarian cancer. The tumor suppressor PTEN was predicted to be a potential target of miR-130a.
Expression of miR-130a and P-gp in SKOV3 and SKOV3/CIS
We performed real-time qRT-PCR for further validation of miR-130a expression level and western blot analysis for comparison of P-gp expression in SKOV3 and SKOV3/CIS cell lines, respectively. High values of relative threshold cycles indicate the low miRNA quantity, which were normalized by U6 snRNA expression, and GAPDH was used as internal control. As shown in Fig. 1A, the expression levels of miR-130a were on average 24.43-fold higher in SKOV3/CIS cell than in parental SKOV3 cell (P<0.05), which was concordant with the result of miRNA expression profiling. In addition, western blotting showed that the expression level of P-gp was very high in SKOV3/CIS and was absent in SKOV3 cells (Fig. 1B). Overall, these findings suggest that upregulation of miR-130a in SKOV3/CIS was related to MDR1/P-glycoprotein-mediated cisplatin resistance.
Expression of miR-130a and P-gp in SKOV3 and SKOV3/CIS cell lines. (A) The mean fold-change of miR-130a expression was detected by qRT-PCR in SKOV3 and SKOV3/CIS. The relative expression of miR-130a in SKOV3/CIS cells was 24.43-fold higher than that in SKOV3 cells, *P<0.05. Each sample was analyzed in triplicate and normalized to U6 snRNA. Fold-change was figured out by 2−ΔΔCt method. (B) The expression of P-gp, as measured using western blotting, was upregulated in SKOV3/CIS and was absent in SKOV3. GAPDH was used as internal control.
Expression of GFP indicating the high efficiency of the liposome-mediated transfection system
We constructed the plasmid labeled with GFP gene sequences to assess the efficiency of the Lipofectamine 2000-mediated transfection system of ovarian cancer cells. After 48 h of transfection, microphotographs of both cell lines were captured under a fluorescence microscope (Nikon Eclipse 80I, Japan). As presented in Fig. 2, nearly 80% of green fluorescence could be clearly observed and detected in the cytoplasm of each cell line, which indicated that the liposome-mediated transfection system in our experiment, could reach high efficiency.
Expression of GFP in SKOV3 and SKOV3/CIS cell lines (original magnification, ×40). (A) SKOV3 cells were transfected with the plasmid labeled with GFP gene sequences. GFP expression was observed under a fluorescence microscope after 48 h of transfection (A-1, normal light; A-2, fluorescent light). (B) After 48 h of transfection, GFP expression in SKOV3/CIS was observed under a fluorescence microscope (B-1, normal light; B-2, fluorescent light).
Transfection of miR-130a modulating MDR1 and P-gp expression in SKOV3 and SKOV3/CIS
To further investigate whether miR-130a is associated with MDR1/P-gp-mediated drug resistance, we examined the MDR1 mRNA and P-gp expression using qRT-PCR and western blot analysis after transfection. As shown in Fig. 3, the expression of MDR1 mRNA was upregulated in cisplatin sensitive SKOV3 cells transfected with an miR-130a-mimic, while inhibiting miR-130a expression could remarkably decreased MDR1 and P-gp expression in drug resistant SKOV3/CIS cell, compared to miR-negative control (P<0.05). Overall, these results might indicate that upregulation of miR-130a in ovarian cancer cells is likely associated with MDR1/P-glycoprotein-mediated drug resistance.
Expression levels of MDR1 mRNA and P-gp in SKOV3 and SKOV3/CIS cells measured by qRT-PCR and western blotting respectively after transfection for 24 h. (A) Expression levels of MDR1 mRNA in SKOV3 and SKOV3/CIS after transfection with a miR-130a-mimic or -inhibitor or with miR-negative. Compared to the miR negative control, the expression of MDR1 mRNA was upregulated in SKOV3 cell transfected with miR-130a-mimic, while it was remarkably decreased in drug-resistant SKOV3/CIS cells transfected with the miR-130a-inhibitor (*P<0.05, compared to miR-negative control). (B) Expression levels of P-gp in SKOV3/CIS cells after transfection. Transfection with the miR-130a-inhibitor could downregulate P-gp expression in cisplatin-resistant SKOV3/CIS cells (**P<0.05 compared to miR-negative control).
Transfection of miR-130a regulating cisplatin sensitivity in SKOV3 and SKOV3/CIS cells
In vitro drug sensitivity array analysis was carried out to validate the role of miR-130a in cisplatin resistance of ovarian cancer cells. After transfection with a miR-130a-mimic or -inhibitor or with miR-negative, SKOV3 and SKOV3/CIS cells were treated with increasing doses of cisplatin. Cell viability curves are shown in Fig. 4, through which the IC50 of each sample was calculated. We found that cisplatin sensitivity was decreased in SKOV3 cells transfected with the miR-130a-mimic, while downregulating miR-130a expression overcame cisplatin resistance in SKOV3/CIS cells.
Cisplatin sensitivity is regulated by miR-130a in SKOV3 and SKOV3/CIS cells. (A) SKOV3 cells were transfected with miR-negative and miR-130a-mimic for 24 h. After transfection, SKOV3 cells were treated with increasing concentrations of cisplatin (0.3, 0.6, 1.2, 2.4, 4.8 and 9.6 μg/ml) for 48 h. Then the cellular viability was detected using the MTT assay. The IC50 of miR-negative and miR-130a-mimic was 0.935 μg/ml vs. 1.756 μg/ml. (B) SKOV3/CIS cells were transfected with miR-negative and miR-130a-inhibitor for 24 h. After transfection, SKOV3/CIS cells were exposed to increasing doses of cisplatin (3, 6, 12, 24, 48 and 96 μg/ml) for 48 h. Then the cellular viability was detected using the MTT assay. IC50 of miR-negative and miR-130a-inhibitor was 17.402 μg/ml vs. 8.439 μg/ml. Columns and bars represented the means and SE of three independent experiments. (*P<0.05, **P<0.05, compared to the miR-negative control).
Chemotherapy remains an essential component for the treatment of ovarian cancer. Although platinum-based combined therapy has a good initial response, development of multidrug resistance is a major obstacle to successful treatment, which leads to relapse and poor prognosis. Consequently, it is necessary to discover novel targeting specific molecules regulating drug resistance and to develop rational approaches for further therapies. In recent years, accumulating evidence of the role of miRNAs in developing chemoresistance have been reported. Aberrant levels of miRNAs, including up- and downregulation, might modulate the expression of down-stream target proteins, which could be involved in several molecular mechanisms of drug resistance. For instance, Kovalchuk et al(34) demonstrated that downregulation of miR-451 was associated with doxorubicin resistance in MCF-7 breast cancer cells by regulating P-gp expression. Another study reported that miR-15b and miR-16 could regulate multidrug resistance by targeting BCL-2 in human gastric cancer cells (35). Microarray, a high-throughput analysis, can be used to detect a genome-wide miRNA expression profiling and identify the appropriate candidates of aberrant miRNAs (36,37). Therefore, in the present study with the help of miRNA microarray platform, we have comprehensively isolated and analyzed a total of 1,289 human miRNAs between parental SKOV3 and cisplatin-resistant SKOV3/CIS cells, within which we discovered that 35 miRNAs were downregulated and 54 miRNAs were upregulated in the SKOV3/CIS cell line. Interestingly, some of these miRNAs and their downstream targets were previously reported to be involved in drug resistance of malignant cancer, such as miR-221/-222 targeting p27 (Kip1) in tamoxifen-resistant breast cancer (38); miR-27a targeting MDR1/P-gp in paclitaxel-resistant ovarian cancer (16); and miR-31 targeting E2F6 in prostate cancer cells (39). Consequently, these preliminary results, to the best of our knowledge, suggest that these differentially expressed miRNAs and predicted targeted proteins probably play a crucial role in the development of cisplatin resistance in ovarian cancer, which might provide novel insight for future approaches of gene therapies.
We verified the aberrant expression of miR-130a and performed a further investigation for the function of miR-130a in cisplatin resistance. Here, we found that upregulation of miR-130a might be associated with MDR1/P-glycoprotein-mediated drug resistance in SKOV3/CIS cells, while downregulating miR-130a expression might overcome cisplatin resistance. In recent studies, Wang et al(40) have reported that miR-130a expression is increased in non-small cell lung cancer (NSCLC) tissues, which was strongly associated with lymph node metastasis, stage of tumor node metastasis classification and poor prognosis. miR-130a has been reported to be a regulator in antagonizing the inhibitory effects of GAX on endothelial cell proliferation, migration and tube formation, and antagonizing the inhibitory effects of HOXA5 on tube formation in vitro(41). In addition, miR-130a has also been found to be upregulated in MCF-7/ADR breast cancer cells (42). Thereby, we hypothesized that miR-130a acts as an oncogene, which could not only promote angiogenesis, metastasis and growth of cancer cells but also induce multidrug resistance to anticancer chemotherapeutics.
It has been reported that the phosphatidylinositol-3-kinase controlled signal transduction cascade (PI3K/Akt/PTEN/mTOR pathway) and the activity of drug transporters of the ABC superfamily (P-glycoprotein, MRP1, BCRP) were implicated in resistance of tumor cells to anticancer drugs (8,9,43). With the help of TargetScan, the phosphatase and tensin homolog (PTEN) gene was predicted to be a tumor suppressor and a potential target of miR-130a. Furthermore, our results indicate that miR-130a could modulate the expression levels of MDR1 mRNA and P-gp. A recent study showed that at least two mechanisms of drug resistance are interrelated in prostate cancer cells. PTEN and mTOR signaling were shown to be involved in the regulation of MRP1 and BCRP (44). Consequently, we assumed that miR-130a, acting as an intermediate, might regulate cisplatin resistance by activating PI3K/Akt/PTEN/mTOR and ABC superfamily drug transporter pathways in ovarian cancer cells, which means that different mechanisms involved in drug resistance are probably interconnected.
In summary, we identified the differentially expressed miRNAs in SKOV3 and SKOV3/CIS cells, which suggested that these miRNAs and predicted targeted proteins probably play a crucial role in the development of cisplatin resistance in ovarian cancer. Moreover, we found that upregulation of miR-130a might be associated with MDR1/P-gp-mediated drug resistance in SKOV3/CIS cells and played the role of an intermediate in drug-resistance pathways in PI3K/Akt/PTEN/mTOR and ABC superfamily drug transporters in ovarian cancer cells. More efforts are needed to further delineate the biofunctional roles of these miRNAs and relevant signaling pathways, which have significant implications in the development of targeted gene therapy for reversing cisplatin resistance in ovarian cancer.
We are sincerely grateful to Professor Zhao Xia and Manager Yi Tao (Gynecological Oncology of Biotherapy Laboratory, Department of Gynecology and Obstetrics, West China Second University Hospital, Sichuan University) for experimental assistance. This study was supported by the Program for Changjiang Scholars and the Innovative Research Team (PCSIRT) in University (IRT0935) and the local Foundation of Science and Technology Department of Sichuan Province (no. 2009S20161).