Downregulation of miR-29 contributes to cisplatin resistance of ovarian cancer cells

Authors

  • Pei-Ning Yu,

    1. Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China
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  • Ming-De Yan,

    1. Division of Gastroenterology, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan, Republic of China
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  • Hung-Cheng Lai,

    1. Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China
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  • Rui-Lan Huang,

    1. Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China
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  • Yu-Ching Chou,

    1. School of Public Health, National Defense Medical Center, Taipei, Taiwan, Republic of China
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  • Wen-Chi Lin,

    1. Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China
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  • Li-Tzu Yeh,

    1. Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China
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  • Ya-Wen Lin

    Corresponding author
    1. Department and Graduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, Republic of China
    2. Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China
    • Correspondence to: Ya-Wen Lin, PhD, Department and Graduate Institute of Microbiology and Immunology, National Defense Medical Center, No.161, Section 6, Min-Chuan East Road, Taipei 114, Taiwan, Republic of China, Fax: +886-2-87917654, E-mail: ndmc.yawen@msa.hinet.net or lyw@ndmctsgh.edu.tw

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  • Conflict of interest: Nothing to report

Abstract

Drug resistance is an obstacle to the treatment of ovarian cancer. Using a unique cell model, we have proven previously that a subpopulation of ovarian cancer cells is more resistant to cisplatin than are the original cells. MicroRNAs (miRNAs), small noncoding RNAs, are involved in many biological events in cancer cells. In our study, we explored whether miRNAs are involved in cisplatin resistance of ovarian cancer cells. Cisplatin-resistant cells expressed a lower level of miR-29a/b/c. Manipulation of microRNA-29 (miR-29) expression modulated cisplatin sensitivity of CP70, HeyC2, SKOV3 and A2780 ovarian cancer cells. Knockdown of miR-29a/b/c increased the ability of cells to escape cisplatin-induced cell death partly through upregulation of collagen type I alpha 1 (COL1A1) and increased the activation of extracellular signal-regulated kinase 1/2 and inactivation of glycogen synthase kinase 3 beta. When combined with cisplatin treatment, knockdown of miR-29 decreased the amount of the active form of caspase-9 and caspase-3. Ectopic expression of miR-29 alone or in combination with cisplatin treatment efficaciously reduced the tumorigenicity of CP70 cells in vivo. Our data show that downregulation of miR-29 increases cisplatin resistance in ovarian cancer cells. Taken together, these data suggest that overexpression of miR-29 is a potential sensitizer to cisplatin treatment that may have therapeutic implications.

Abbreviations
AKT

v-akt murine thymoma viral oncogene homolog

COL1A1

collagen type I alpha 1

CP70sps

CP70 side-population spheres

ECM

extracellular matrix

EOC

epithelial ovarian cancer

ERK1/2

extracellular signal-regulated kinase 1/2

GSK3β

glycogen synthase kinase 3 beta

miRNAs

microRNAs

miR-29

microRNA-29

3′-UTR

3′-untranslated region

Epithelial ovarian cancer (EOC) is a common gynecologic malignancy and is one of the ten most common causes of death by cancer in women.[1] The course of treatment for ovarian cancer includes cytoreductive surgery and chemotherapy. Although the combination of surgery and platinum-based therapy can reduce the tumor mass, resistance to platinum-based therapy is still one cause of a poor prognosis.[2, 3] Therefore, more comprehensive studies of ovarian cancer chemoresistance from the molecular to the cellular regulatory levels may help to understand tumor progression further and may lead to improvements in therapy.

MicroRNAs (miRNAs) are a group of small endogenous noncoding RNAs that regulate biological events by interfering with target gene expression through posttranscriptional regulation. Regulation occurs through miRNA binding to the 3′-untranslated region (3′-UTR) of the target gene mRNA and by repressing mRNA translation or inducing mRNA degradation.[4] miRNAs are critical regulators of cell proliferation, invasion, metastasis and drug sensitivity of cancer cells.[5, 6] Iorio et al. demonstrated that miRNAs are aberrantly expressed in ovarian cancer compared to normal ovary tissue.[7] The miRNA expression patterns in drug-resistant ovarian cancer cell lines[8] and in patients who are resistant to platinum-based treatment[9] differ from those of the drug-sensitive group. let-7i and miR-214 have been identified as functional regulators in the cisplatin resistance of ovarian cancer cells.[10, 11] Collectively, these results suggest an association between dysregulation of miRNAs and ovarian oncogenesis.

We have recently reported on a subpopulation of ovarian cancer cells with increased drug resistance. After isolation of the side population from a CP70 ovarian cancer cell line by a combination of Hoechst 33342 dye-exclusion and the spheroid-formation method,[12] we identified sphere-forming cells, which we refer to as “side-population spheroid cells” (CP70sps cells). The side population has been identified as a small group of cells that exhibit a lower intensity of Hoechst 33342 staining and a highly drug-resistant ability in multiple cancers such as lung cancer,[13] neuroblastoma[14] and ovarian cancer.[15, 16] Our recent data showed that the Hoechst dye-exclusion-screened CP70sps cells exhibited greater cisplatin resistance than did the parental CP70 cells and possessed properties of cancer-initiating cells.[12] Because of the critical role of miRNAs in oncogenesis, we planned to investigate whether the difference in miRNA expression between CP70 and CP70sps cells is involved in drug resistance in ovarian cancer cells.

Some studies have demonstrated that expression of the microRNA-29 (miR-29) family is lower in ovarian cancer than in normal ovaries.[7] Other studies have suggested that this family acts as a tumor suppressor in cancer network processes such as apoptosis,[17-19] the cell cycle,[20, 21] epigenetic modification[22] and metastasis[23] in cholangiocarcinoma, acute myeloid leukemia (AML), hepatocellular carcinoma (HCC), mantle cell lymphoma, lung cancer and colorectal cancer. However, the contribution of miR-29 to drug resistance is poorly understood. After analyzing the expression of miRNAs in CP70 and CP70sps cells, we chose miR-29 for further investigation. In our study, we confirmed the differences in the expression of the miR-29 family in different cisplatin sensitivity of ovarian cancer cells, and we explored the effects of miR-29 on cisplatin resistance of ovarian cancer cells in vitro and in vivo.

Material and Methods

Cell lines

The CP70 (cisplatin-resistant cell line), A2780 (cisplatin-sensitive cell line),[24] HeyC2 and SKOV3 (intrinsically cisplatin-resistant cell line, p53 double deletion mutant) ovarian cancer cell lines were used in our study. The cell lines were obtained from Dr. Tim Huang's laboratory (University of Texas Health Science Center, San Antonio, TX) in 2007. The culture condition is described in Supporting Information Materials and Methods.

CP70sps cells were isolated from CP70 cell line according to our recent study.[12] The isolation of CP70sps cells is described in Supporting Information Materials and Methods.

The cell lines used were tested by Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan) for authentication by using the Applied Biosystems AmpFLSTR Identifiler kit (Cat# 4322288; Applied Biosystems, Forest City, CA). Briefly, DNA profiling of 15 short tandem repeat loci was verified and compared manually to the ATCC and European Collection of Cell Cultures (ECACC) database.

RNA isolation, reverse transcription and quantitative polymerase chain reaction

Total RNA isolation from cell lines and tissues was performed using Trizol (Invitrogen, Carlsbad, CA). For evaluation of the mature miR-29 expression, cDNA was synthesized using a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). For analyzing the expression of coding genes, cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen) for further gene expression analysis. Quantitative PCR was performed on an ABI 7500 real-time system (PE Applied Biosystems). The detailed procedure and the sequence of each oligonucleotide are described in Supporting Information Materials and Methods.

Cisplatin sensitivity (cell death), cell viability and cell cycle assay

cis-Diammineplatinum (II) dichloride (Sigma, St. Louis, MO) was dissolved in 0.9% NaCl solution. Drug resistance was studied in ovarian cancer cells treated with different doses of cisplatin for 48 hr. The IC50 (the concentration that inhibited growth by 50%) value was determined by assessing cell viability, which was measured by [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay using Cell Titer 96 AQ One Solution (Promega, Madison, WI) according to the manufacturer's instructions. The detailed procedure is described in Supporting Information Materials and Methods.

To evaluate the effect of miR-29 on cisplatin sensitivity in ovarian cancer cells, cell death was measured using flow cytometry. After drug treatment, the cells were harvested and resuspended in 100 µl of Flow Cytometry Staining Buffer [1× phosphate-buffered saline (PBS) and 2% newborn calf serum] and then stained with propidium iodide (PI) (Sigma) staining solution. The PI fluorescence was measured using the FL-2 channel of the FACSCalibur machine (BD Biosciences, San Jose, CA), and the PI fluorescence-positive cells were defined as dead cells.

PI staining was also used to evaluate the cell cycle. The collected cells were resuspended in 0.5 ml of PBS, fixed with 4.5 ml of 70% ethanol for 4 hr, washed with PBS and stained with PI staining solution [0.1% (wt/vol) Triton X-100 (Sigma), 0.2 mg/ml RNase A (Sigma) and 20 µg/ml PI in PBS]. The FACSCalibur flow cytometer (FL-2 channel) was used to determine the cell cycle distribution.

Transfection

Cells were seeded in 6- or 24-well plates or 6-cm dishes and transfection was performed using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. The miR-29 inhibitors (Dharmacon, Chicago, IL), COL1A1 siRNAs (collagen type I alpha 1; Invitrogen), pcDNA6.2-GW/EmGFP-miR-29 plasmid and luciferase reporter plasmids (pGL3 control, pGL3-COL1A1-3′UTR-wt or pGL3-COL1A1-3′UTR-mu plasmid) were used in our study. The pGL3-COL1A1-3′UTR luciferase reporter constructs contained the 334-bp 3′UTR of COL1A1 (nt 5313–5646 of COL1A1 mRNA). MiR-29 inhibitors are single-stranded RNA oligonucleotides that could bind to the complimentary, mature miRNA strand. The details of reagents and procedures are described in Supporting Information Materials and Methods.

Luciferase reporter gene assay

A luciferase reporter assay was performed using the Dual-Luciferase Reporter Assay System (Promega). The detailed procedure and material are described in Supporting Information Materials and Methods.

Western blot analysis

The cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After gel transference to PVDF (Millipore, Bedford, MA), the antibodies COL1A1 (sc-25974, Santa Cruz Biotechnology, Santa Cruz, CA), p-ERK (#9106), ERK (extracellular signal-regulated kinase; #9102), p-AKT (#9271), AKT (#9272), p-GSK3β (#9336), GSK3β (glycogen synthase kinase 3 beta; #9315), caspase-3 (#9665) (Cell Signaling Technology, Danvers, MA) and casepase-9 (sc-8355, Santa Cruz Biotechnology) were used. Subsequently, suitable secondary antibodies were applied and bands for specific molecules were detected by enhanced chemiluminescence (ECL; Millipore). Each sample was normalized to β-actin (Santa Cruz Biotechnology).

Type I collagen coating

To study the effects of collagen, 4 × 105 cells were seeded in 6-cm dishes coated with 84 μg of human collagen I (ICN Biomedicals, Aurora, OH).[25] After 16 hr, the cells were treated with cisplatin for 48 hr and the percentage of cell death was analyzed by PI staining.

Flow cytometric analysis (evaluation of stemness markers)

Flow cytometric analysis was performed using a FACSCalibur machine. Antibodies to OCT4 (ab19857), NANOG (ab21624), CD44 (ab51037), NESTIN (ab6320) (Abcam, Cambridge, MA) and CD133 (AC133, MACS; Miltenyi Biotech, Auburn, CA) were used to evaluate stemness markers. The staining procedure is described in Supporting Information Materials and Methods.

Analysis of tumorigenicity in vivo

To analyze the effects of miR-29 in vivo, animal experiments were designed as described previously.[26-28] A total of 5 × 106 CP70 cells was injected subcutaneously into both flanks of 4- to 6-week-old female nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. When the tumor had grown to 50 mm3, the mice were given an intraperitoneal injection of a combination of cisplatin (5 mg/kg) and miR-negative control (miR-NC) or pcDNA6.2–GW/EmGFP–miR-29 plasmid (12 µg/tumor). Cisplatin was supplied as a concentrated sterile solution (0.5 mg/ml) in NaCl, citric acid and water (Abic, Netanya, Israel). The miR-NC or pcDNA6.2–GW/EmGFP-miR-29 plasmid was mixed with TurboFect in vivo Transfection Reagent (Fermentas, Glen Burnie, MD) (50 µl total volume) and was injected intratumorally on days 1, 3 and 5. After 15 days, the mice were sacrificed and the tumor weights were recorded. The tumor volume was calculated as 0.5236 × L1 × L22, where L1 is the long axis and L2 is the short axis of the tumor. All animal studies were approved by the Institutional Animal Care and Use Committee at National Defense Medical Center.

Statistical analysis

GraphPad Prism software (version 4.0) was used for statistical analysis. All values are shown as the mean ± SEM. The significance level was defined as p < 0.05. Student's t-test was used to compare the cell viability, cell death, luciferase activity and gene expression.

Results

Expression of miR-29a/b/c differs between cisplatin-resistant ovarian cancer cell lines

Our previous data showed that the cisplatin IC50 is twofold higher in CP70sps cells than in CP70 cells and 20.6-fold higher in CP70sps cells than in A2780 cells, a cisplatin-sensitive ovarian cancer cell line.[12] To explore the relationship between the expression of miRNAs and drug resistance in ovarian cancer cells, we used microarray assays to analyze the expression profiles of miRNAs in CP70 and CP70sps cells. Several miRNAs were upregulated or downregulated in cisplatin-resistant CP70sps cells compared to CP70 cells (Supporting Information Fig. S1a and Table S1). Some upregulated miRNAs, such as miR-141,[29] miR-125b[30] miR-130a,[31] are involved in drug sensitivity of ovarian cancer. Among the downregulated miRNAs (Supporting Information Fig. S1b), let-7, miR-23, miR-29, miR-107 and miR-20 were shown to be downregulated in cisplatin-resistant ovarian cancer cells in another study.[8] Considering the important function of miR-29 in human carcinogenesis, we focused on miR-29a/b/c. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed that the expression of miR-29a/b/c was lower in CP70sps than in CP70 cells, which was consistent with the microarray data (Fig. 1a). These data suggest that the lower expression level of miR-29a/b/c is associated with cisplatin resistance in ovarian cancer cells.

Figure 1.

Modulation of miR-29 affects cisplatin sensitivity in CP70 cells. (a) Quantitative RT-PCR analysis was used to evaluate the expression of miR-29a, miR-29b and miR-29c. (b) After cisplatin treatment for 48 hr, the IC50 dosage of CP70 cells was determined by MTS assay. (c) After cisplatin treatment, the percentages of dead CP70 cells transfected with miR-29a/b/c inhibitors (25 nM) (left panel) or miR-29 plasmid (6 µg) (right panel) were analyzed by flow cytometric analysis. (d) MTS analysis was performed to evaluate the viability of CP70 cells transfected with miR-29a/b/c inhibitors (25 nM) (left panel) or miR-29 plasmid (6 µg) (right panel). (e) Flow cytometric analysis was used to analyze the cell cycle progression of CP70 cells transfected with miR-29a/b/c inhibitors (25 nM) (left panel) or miR-29 plasmid (6 µg) (right panel). U6 was used as the internal control in the quantitative RT-PCR analysis. CT, without inhibitor; SCR, scrambled inhibitor; miR-NC, miRNA-negative control vector. Each value represents the mean ± SEM of triplicate experiments (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005, Student's t-test).

Downregulation of miR-29 promotes resistance to cisplatin-induced cell death in ovarian cancer cells

Before examining the effect of miR-29 reduction on cisplatin resistance of ovarian cancer cells treated with cisplatin for 48 hr, the IC50 cisplatin dosage was determined to be 40 µg/ml (∼133.29 µM) in CP70 cells (Fig. 1b). We then modulated the expression of miR-29 by transfection with miR-29 inhibitors (Supporting Information Fig. S2) and used flow cytometry to confirm a decrease in the percentage of dead CP70 cells after the cells had been treated with cisplatin for 48 hr (Fig. 1c, left). A gain-of-function experiment demonstrated that the ectopic expression of miR-29 sensitized the cells to respond to cisplatin treatment (Fig. 1c, right). We used an MTS assay and cell cycle analysis to confirm that the change in cell death was not caused by the effects of miR-29 on cell viability and cell cycle progression (Figs. 1d and 1e).

Because A2780 cells are a parental cisplatin-sensitive cell line of CP70 cells,[24] the reduction in miR-29-mediated cisplatin resistance was validated by knockdown of miR-29 in A2780 cells (Supporting Information Fig. S3). We also evaluated the expression of miR-29 in HeyC2 and SKOV3 cells to investigate whether the correlation between the reduction in miR-29 expression and cisplatin resistance is a common phenomenon in ovarian cancer cells. More cisplatin-resistant cells expressed a lower level of miR-29 (Figs. 2a and 2b). After knockdown of miR-29 in HeyC2 cells and overexpression of miR-29 in SKOV3 cells (Supporting Information Fig. S4), the cisplatin sensitivity was analyzed as described above. The reduction in miR-29 expression decreased cisplatin-induced cell death in HeyC2 cells, whereas overexpression of miR-29 increased the sensitivity of SKOV3 cells to cisplatin (Fig. 2c). Cell viability and cell cycle progression were not affected in these cells (Fig. 2d and Supporting Information Fig. S3d). Taken together, these results indicate that downregulation of miR-29 confers cisplatin resistance to ovarian cancer cells.

Figure 2.

Modulation of miR-29 affects cisplatin resistance in HeyC2 and SKOV3 cells. (a) MTS analysis was used to compare the cisplatin sensitivity of ovarian cancer cell lines. (b) Quantitative RT-PCR analysis was used to measure the endogenous expression of miR-29a/b/c in ovarian cancer cell lines. (c) After cisplatin treatment for 48 hr, the percentage of dead HeyC2 cells transfected with miR-29a/b/c inhibitors (25 nM) (left panel) and the percentage of dead SKOV3 cells transfected with miR-29 plasmid (6 µg) (right panel) were analyzed by flow cytometric analysis. (d) Flow cytometric analysis was used to analyze the cell cycle progression of HeyC2 cells transfected with miR-29a/b/c inhibitors (25 nM) (left panel) and SKOV3 cells transfected with miR-29 plasmid (6 µg) (right panel). U6 was used as the internal control in the quantitative RT-PCR analysis. CT, without inhibitor; SCR, scrambled inhibitor; miR-NC, miRNA-negative control vector. Each value represents the mean ± SEM of triplicate experiments (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005, Student's t-test).

COL1A1 is a target of miR-29

To identify the genes involved in miR-29-associated cisplatin resistance of ovarian cancer cells, we used the databases PicTar and Target Scan (PicTar: http://pictar.mdc-berlin.de/ and TargetScan: http://targetscan.org/) to predict the candidate targets of miR-29 and then applied quantitative RT-PCR to check the expression of the candidate targets. The expression of COL1A1 was significantly higher in CP70sps cells (with lower miR-29 expression) than in CP70 cells (with higher miR-29 expression) (Fig. 3a). COL1A1 is one of the miR-29a/b/c targets with the highest score and has been identified as a target gene of miR-29 in hepatic stellate cells,[32] systemic sclerosis fibroblasts[33] and cardiac fibroblasts.[34] COL1A1 is a component of type I collagen that is known to be associated with drug resistance.[25, 35] Therefore, COL1A1 might be an important target gene of miR-29 in cisplatin-resistant ovarian cancer cells. The 3′-UTR of COL1A1 contains multiple putative sites and matches the seed sequence of miR-29a/b/c (Fig. 3b). To confirm whether miR-29a/b/c regulates COL1A1 expression by binding to the COL1A1 3′-UTR, we created luciferase reporter constructs containing the 334-bp 3′-UTR of COL1A1 (nt 5313–5646 of COL1A1 mRNA) harboring three wild-type or mutated miR-29-binding sites as reported previously[36] (Fig. 3b). The luciferase activity of the wild-type COL1A1 3′-UTR reporter, but not that of the mutant, was increased in CP70 cells transfected with miR-29 inhibitors (Fig. 3b). Quantitative RT-PCR and Western blot analysis revealed that the expression of COL1A1 increased after knockdown of miR-29 in CP70 cells (Fig. 3c). This inverse correlation between the expression of miR-29 and COL1A1 was also confirmed in A2780, HeyC2 and SKOV3 cells (Supporting Information Fig. S5a). These data indicate that miR-29a/b/c negatively regulates COL1A1 expression through the 3′-UTR of COL1A1 in ovarian cancer cells.

Figure 3.

COL1A1 is a target of miR-29. (a) Quantitative RT-PCR analysis was performed to evaluate the expression of the predicted miR-29a/b/c targets in ovarian cancer cells. (b) Sequence alignment shows the miR-29a/b/c seed sequences, three putative target sites of the COL1A1 3′-UTR and three mutated target sites of the COL1A1 3′-UTR (top panel). Luciferase activity was measured in CP70 cells transfected with luciferase reporters alone or cotransfected with miR-29a/b/c inhibitors (25 nM) and luciferase reporters (bottom panel). (c) Quantitative RT-PCR analysis (top panel) and Western blot analysis (bottom panel) were performed to evaluate the mRNA and protein expression of COL1A1 in CP70 cells transfected with miR-29a/b/c inhibitors (25 and 60 nM). The numbers in the blot represent the ratios between COL1A1 and the internal control. (d) After cisplatin treatment for 24 hr, the percentage of dead cells was analyzed by flow cytometric analysis. CP70 cells were transfected with miR-29 (6 µg) and cultured in type I collagen-coated dishes (top panel) or cotransfected with miR-29s inhibitors (45 nM), and COL1A1 siRNA (140 nM) (bottom panel). GAPDH was used as an internal control in the quantitative RT-PCR analysis. CT, without inhibitor; SCR, scrambled inhibitor; miR-NC, miRNA-negative control vector. Each value represents the mean ± SEM of triplicate experiments (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005, Student's t-test).

Knockdown of COL1A1 sensitized A2780 cells (Supporting Information Fig. S5b, right) and CP70 cells (Supporting Information Fig. S6) to cisplatin treatment. In cancer cells cultured in dishes coated with type I collagen, cisplatin-induced cell death decreased modestly (Fig. 3d, top and Supporting Information Fig. S5b). This finding implies that type I collagen is associated with cisplatin resistance in ovarian cancer cells. Next, we investigated the role of COL1A1 in the regulation by miR-29 of cisplatin sensitivity. In miR-29-overexpressing cells cultured in type I collagen-coated dishes, the effect of miR-29 on cisplatin sensitivity was inhibited (Fig. 3d, top). Under cisplatin treatment, the percentage of dead cells was around 26% in the control (bars 9 and 10), but was reduced to 16% in miR-29 inhibitor-transfected cells (bars 13 and 14). When COL1A1 was knocked down by siRNA in the miR-29 inhibitor-transfected cells, the percentage of dead cells increased to 26% (siCOL1A1#1, bar 15) and 29% (siCOL1A1#2, bar 16). Therefore, we suggest that knockdown of COL1A1 attenuates miR-29 inhibitor-mediated cisplatin resistance (Fig. 3d, bottom). Taken together, these data demonstrate that COL1A1 is a miR-29a/b/c target that contributes to cisplatin resistance in ovarian cancer cells.

Activation of ERK1/2, GSK3β, caspase-9 and caspase-3 is involved in the effects of miR-29 on cisplatin resistance

As noted above, we found that cisplatin resistance induced by downregulation of miR-29 occurs partly through modulation of COL1A1. A previous study has shown that the extracellular matrix (ECM, comprised of type I collagen) can interact with integrin in cells to activate the v-akt murine thymoma viral oncogene homolog (AKT) and ERK cell survival pathways.[37] The activation status of AKT, ERK1/2 and GSK3β is thought to affect cisplatin-induced cell death.[38, 39] Therefore, we wondered whether upregulation of COL1A1, which was induced by reducing miR-29 expression, would alter the activation of these signal pathways in ovarian cancer cells. As shown in Figure 4a, knockdown of miR-29a, −29b and −29c increased the expression of COL1A1, phosphorylation of ERK1/2 (Thr202/Tyr204, active form) and GSK3β (Ser9, inactive form) but not the phosphorylation of AKT (Fig. 4a). Next, we cultured CP70 and A2780 cells in type I collagen-coated dishes and found that type I collagen induced the phosphorylation of ERK1/2 (Thr202/Tyr204) and GSK3β (Ser9) in a dose-dependent manner (Fig. 4b). These data suggest that upregulation of COL1A1, which was induced by knockdown of miR-29, increases the activation of ERK1/2 and inactivation of GSK3β, and that these changes are associated with cell survival in ovarian cancer cells.

Figure 4.

Activation of ERK1/2, GSK3β, caspase-9 and caspase-3 is involved in the effects of miR-29 on cisplatin resistance. (a) Western blot analysis was used to detect the phosphorylation of AKT, ERK1/2 (Thr202/Tyr204) and GSK3β (Ser9) in miR-29a/b/c knockdown cells. (b) Western blot analysis was used to detect the phosphorylation of ERK1/2 (Thr202/Tyr204) and GSK3β (Ser9) in CP70 and A2780 cells cultured in type I collagen-coated dishes. (c) Western blot analysis was used to detect the active form of caspase-9 and caspase-3 in miR-29a/b/c knockdown cells. The numbers in the blot represent the ratios between the analyzed genes and internal control.

Because some articles have shown that cisplatin induces apoptosis by activating caspase-9 and caspase-3,[40] we next examined whether the activation of caspase-9 and cleaved caspase-3 is affected by knockdown of miR-29a, −29b and −29c in ovarian cancer cells treated with cisplatin. The amounts of active form of caspase-9 and cleaved caspase-3 decreased in miR-29-knockdown cells treated with cisplatin for 18 hr (Fig. 4c), suggesting that the inactivation of caspase-9 and caspase-3 may contribute to the decrease in cell death induced by cisplatin in miR-29-knockdown cells. These results suggest that the role of miR-29a/b/c in regulating cisplatin sensitivity in ovarian cancer cells is associated with the activities of ERK1/2, GSK3β, caspase-9 and caspase-3.

Overexpression of miR-29 in CP70 cells increases cisplatin sensitivity and suppresses tumor formation in NOD/SCID mice

The in vitro data confirmed the role of miR-29 in cisplatin resistance in ovarian cancer cell lines. We next explored the effect of miR-29 on cisplatin sensitivity in CP70 cells in vivo using the method for xenograft experiments described before.[26-28] The experimental procedure is shown in Figure 5a. Five million CP70 cells were implanted subcutaneously into both flanks of NOD/SCID mice, and when the tumor had grown to 50 mm3, the mice were treated with cisplatin (5 mg/kg, intraperitoneal injection) and the miR-NC or miR-29 plasmid (12 µg/tumor, intratumoral injection). At the end of the experiment, we confirmed the expression of EmGFP, miR-29, and COL1A1 (Fig. 5b and Supporting Information Fig. S7). The combination of miR-29 and cisplatin reduced tumor formation more effectively in CP70 cells than did the combination of miR-NC and cisplatin (Fig. 5c). Surprisingly, without cisplatin treatment, the tumor formation was suppressed by miR-29 treatment alone (Fig. 5c).

Figure 5.

Overexpression of miR-29 in CP70 cells increases cisplatin sensitivity and suppresses tumor formation in NOD/SCID mice. (a) The diagram illustrates the xenograft experimental protocol. (b) Quantitative RT-PCR analysis was performed to evaluate the expression of miR-29 and COL1A1 in the implanted tumors. (c) Representative photographs show the tumor-implanted mice and tumors injected with miR-29 (29) or empty vector (EV) (left). Tumor weight was measured at the end of the experiment (right). Cisplatin-untreated group, N = 5; cisplatin-treated group, N = 5. U6 and GAPDH were used as internal controls in the quantitative RT-PCR analysis. Values represent the mean ± SEM of triplicate experiments (*p ≤ 0.05, Student's t-test).

Because CP70sps cells express low levels of miR-29 and cancer stem-like cell-related factors,[12] we investigated whether enforced expression of miR-29 would suppress tumor formation by inhibiting the expression of cancer stem-like cell-related factors. First, we checked the expression of the cancer stem-like cell-related factors, including OCT4, NESTIN and NANOG, in the two pairs of implanted tumors (tumors No. 2 and No. 3) that had not been treated with cisplatin. Quantitative RT-PCR showed that the expression of OCT4, NANOG and NESTIN was lower in tumors with ectopic miR-29 expression than in the control tumors (Supporting Information Fig. S8a). We next performed an in vitro loss-of-function experiment in CP70 cells. Flow cytometric analysis showed an increase in the CD44-positive, CD-133-positive, NESTIN-positive and NANOG-positive cell population in miR-29-a/b/c-knockdown cells relative to the scrambled control cells (Supporting Information Fig. S8b). We also evaluated whether overexpression of miR-29 would suppress tumor formation by repressing the cell transformation ability of cancer cells. An in vitro gain-of-function experiment showed that the transformation ability of CP70 cells was inhibited by the overexpression of miR-29 (Supporting Information Fig. S9). These findings indicate that expression of miR-29 is associated with the expression of stemness-related factors and transformation ability of CP70 cells. Taken together, these data suggest that the tumor-suppressive effect of miR-29 reflects its ability to increase cell sensitivity to cisplatin treatment and to decrease the expression of stemness-related factors in ovarian cancer cells.

Discussion

Several studies have used in vitro experiments to examine the role of miRNAs in cisplatin sensitivity of ovarian cancer. These studies have found associations between the expression of miRNAs such as miR-214,[11] miR-376c,[41] miR-141[29] and let-7[10] and the chemotherapy response of ovarian cancer patients. An important finding of our study is that reduction in miR-29 expression increased the cisplatin resistance of ovarian cancer cells partly by upregulating the expression of ECM components (e.g., COL1A1) in vitro and in vivo. We studied the effects of combined cisplatin and miRNA treatment in in vivo experiments to identify whether miR-29 is a potential cisplatin sensitizer. Our study is the first to report on the potential of miR-29 in therapy for ovarian cancer.

We classified miRNAs into groups that were upregulated or downregulated in CP70sps cells compared to CP70 cells, and we studied the function of one of the downregulated miRNAs, the miR-29 family. Other upregulated miRNAs, such as miR-221/222[42] and miR-210,[43] are also important regulators of cancer progression, suggesting that these miRNAs also regulate the function of CP70sps cells. We found similar responses in different members of the miR-29 family—miR-29a, miR-29b and miR-29c—in ovarian cancer cells. Only miR-29b can be imported into the nucleus,[44] implying that the function of miR-29b differs from that of miR-29a and miR-29c. However, the predicted and identified target genes of all miR-29s are nearly the same, and most reports have shown that miR-29s function as a tumor suppressor in various types of cancer. Therefore, we suggest that miR-29a, miR-29b and miR-29c have similar functions in ovarian cancer.

miR-29s regulate a range of target genes, including those for molecules associated with apoptosis, cell cycle, cell adhesion and epigenetic regulation.[17-19, 21, 22, 36] Here, we examined the effects of miR-29 on cisplatin-induced cell death and found that changes in miR-29 expression affected the survival pathway (e.g., phosphorylation of ERK and GSK3β) and apoptosis. We speculate that, in addition to COL1A1, many target genes are involved in a complex regulatory network that contributes to the effects of miR-29 in ovarian cancer.[45] COL1A1 encodes the subunit of type I collagen and is upregulated in progressive ovarian carcinoma.[46] We found that miR-29 knockdown affected the activities of ERK1/2 and GSK3β in cancer cells that exhibited upregulation of COL1A1. Therefore, we suggest that the downregulation of miR-29 in ovarian cancer cells manipulates the surrounding ECM to supply survival signal transduction upon cisplatin treatment. This result is similar to that of a previous study that showed the active role of tumor cells in the induction of the ECM in resisting cisplatin treatment.[25] We also evaluated whether modulation of the miR-29 responses to other chemotherapeutic agents induced cell death. We found that downregulation or overexpression of miR-29 did not affect the sensitivity of ovarian cancer cells to paclitaxel or epirubicin treatment (Supporting Information Fig. S10). The different effects of miR-29 on sensitivity to cisplatin, paclitaxel or epirubicin may reflect the different mechanisms of action of these drugs. Similarly, a previous report demonstrated that miR-200c specifically affects the sensitivity of cancer cells to microtubule-targeting chemotherapeutic agents.[47] Moreover, the expression profile of miRNAs in cisplatin-resistant ovarian cancer cell lines is not consistent with that observed in a paclitaxel-resistant ovarian cancer cell line,[8] implying that different miRNAs specifically regulate the sensitivity of cancer cells to different chemotherapeutic agents.

In vivo experiments showed that only miR-29 treatment efficaciously suppressed the tumorigenicity of CP70 cells. We speculate that there are two possible mechanisms. (i) Overexpression of miR-29 in CP70 xenografts may alter the cross talk between the tumor and surrounding cells. Overexpression of miR-29 may reduce angiogenesis, thus inhibiting tumor growth, as has been demonstrated in HCC.[48] In addition, our data indicated that overexpression of miR-29 in cancer cells may remodel the ECM (COL1A1); moreover, a previous report mentioned that miRNAs are released into the microenvironment,[49] suggesting that these effects affect cancer-associated cells and prevent their support of tumor growth. (ii) Overexpression of miR-29 inhibits the expression of stemness-related markers in CP70 cells (Supporting Information Fig. S8) and the transformation ability of CP70 cells (Supporting Information Fig. S9). We found that CP70sps cells expressed a lower level of miR-29 and had a greater sphere-forming ability than did CP70 cells.[12] Previous study has also shown that repression of miR-29 inhibits cell differentiation, thereby contributing to the promotion of dedifferentiation of breast cancer cells, which leads to enrichment of cancer stem-like cells.[50] These findings imply that miR-29 plays an important role in regulating the characteristics of cancer-initiating cells, which are also associated with tumor formation in vivo. Taken together, our data suggest that miR-29 suppresses the tumorigenicity of CP70 cells as a result of miR-29-induced modulation of the markers of cancer-initiating cells and the tumor microenvironment. The function of miR-29 in ovarian cancer-initiating cells should be investigated thoroughly. To avoid individual variability, we injected the control vector into the tumor on one side and the miR-29 plasmid into the tumor on the other side of the same mouse. However, we cannot exclude the possibility that miRNAs were released into the circulation and affected tumor growth.[49] If so, this problem might be avoided by injecting the miR-29 and vector control into different mice. In preliminary analysis, we evaluated the relationships between the expression of miR-29 and the clinicopathological characteristics of ovarian cancer patients (Supporting Information Table S3). We found that in serous-type ovarian cancer (N = 31), the lower miR-29a level was significantly associated with poor survival (p = 0.014) (Supporting Information Fig. S11a). In addition, the miR-29a level was significantly inversely correlated with COL1A1 expression (Supporting Information Fig. S11b). These results suggest an association between low expression of miR-29a and poor prognosis. However, possibly because of the small sample size (N = 31), the correlations between the expression of miR-29b and miR-29c and survival were not significant (p = 0.051 and p = 0.6649, respectively) (Supporting Information Fig. S11a). We plan to analyze more clinical samples and use an orthotopic ovarian cancer model to confirm the therapeutic potential of miR-29.

In summary, we have demonstrated that the overexpression of miR-29 in ovarian cancer cells increases cisplatin-mediated cytotoxicity partly by modulating the production of COL1A1. Taken together, these findings provide insight into the role of miR-29 in chemotherapy and the potential implications in ovarian cancer therapy.

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