Medical Oncology

, Volume 29, Issue 1, pp 384–391

miR-497 modulates multidrug resistance of human cancer cell lines by targeting BCL2

Authors

  • Wei Zhu
    • Department of OncologyFirst Affiliated Hospital of Nanjing Medical University
  • DanXia Zhu
    • Department of HaematologyFirst Affiliated Hospital of Nanjing Medical University
  • Shiqiang Lu
    • Department of Microbiology and ImmunologyNanjing Medical University
  • Tongshan Wang
    • Department of OncologyFirst Affiliated Hospital of Nanjing Medical University
  • Jian Wang
    • Department of OncologyFirst Affiliated Hospital of Nanjing Medical University
  • Binghua Jiang
    • Cancer Center of Nanjing Medical University
  • Yongqian Shu
    • Department of OncologyFirst Affiliated Hospital of Nanjing Medical University
    • Cancer Center of Nanjing Medical University
    • Department of OncologyFirst Affiliated Hospital of Nanjing Medical University
    • Cancer Center of Nanjing Medical University
Original Paper

DOI: 10.1007/s12032-010-9797-4

Cite this article as:
Zhu, W., Zhu, D., Lu, S. et al. Med Oncol (2012) 29: 384. doi:10.1007/s12032-010-9797-4

Abstract

MicroRNAs (miRNAs) are short non-coding RNA molecules, which posttranscriptionally regulate genes expression and play crucial roles in diverse biological processes, such as development, differentiation, apoptosis, and proliferation. Here, we investigated the possible role of miRNAs in the development of multidrug resistance (MDR) in human gastric and lung cancer cell lines. We found that miR-497 was downregulated in both multidrug-resistant human gastric cancer cell line SGC7901/vincristine (VCR) and multidrug-resistant human lung cancer cell line A549/cisplatin (CDDP) and the downregulation of miR-497 was concurrent with the upregulation of BCL2 protein, compared with the parental SGC7901 and A549 cell lines, respectively. In vitro drug sensitivity assay demonstrated that overexpression of miR-497 sensitized SGC7901/VCR and A549/CDDP cells to anticancer drugs, respectively. The luciferase activity of BCL2 3′-untranslated region-based reporter constructed in SGC7901/VCR and A549/CDDP cells suggested that BCL2 was the direct target gene of miR-497. Enforced miR-497 expression reduced BCL2 protein level and sensitized SGC7901/VCR and A549/CDDP cells to VCR-induced and CDDP-induced apoptosis, respectively. Taken together, our findings first suggested that has-miR-497 could play a role in both gastric and lung cancer cell lines at least in part by modulation of apoptosis via targeting BCL2.

Keywords

MiR-497Multidrug resistanceBCL2

Abbreviations

miRNAs

MicroRNAs.

MDR

Multidrug resistance.

VCR

Vincristine.

CDDP

Cisplatin

5-Fu

5-fluorouracil

VP-16

Etoposide

ADR

Adriamycin

Introduction

One of the leading causes of chemotherapy failure is tumor resistance. In many cases, chemotherapies fail because of drug resistance of cancer cells either intrinsic or acquired after an initial round of treatment [1]. Earlier studies have indicated the cytological mechanisms of drug resistance of cancer cells, such as decreased uptake of water-soluble drugs, increased efflux of hydrophobic drugs and various changes in cells that affect the capacity of cytotoxic drugs to kill cells, including alterations in cell cycle, enhanced DNA repair activity, defective apoptosis pathway, altered metabolism of drugs, etc [25]. Recently, people have realized that multiple paths could cause drug resistance phenotype of cancer cells, such as genetic changes including mutations, translocations, deletions, and amplification of genes or promoter regions and epigenetic changes including aberrant DNA methylation, histone modifications, and non-coding RNA expression [6]. Current studies have also implicated that epigenetic mechanisms do not necessarily require a stable heritable genetic alteration and may play a more important role in acquired drug resistance of cancer cells [7, 8].

The microRNAs (miRNAs) are a group of small non-coding RNAs, which are single stranded and consist of 19–25 nucleotides (∼22 nt). The basic mechanism of miRNA action is that miRNA could imperfectly bind to the 3′UTR of target mRNAs, resulting in translational repression or target mRNA cleavage [9]. Recent studies suggested that the acquisition of drug resistance by cancer cells might be modulated via the changes in miRNA levels [1012]. Emerging evidence has shown that knockdown or reexpression of specific miRNAs by synthetic anti-sense oligonucleotides or miRNAs precursors or mimics could modulate drug resistance [13], for instance, miRNA-21 is upregulated in many chemoresistance cancer cells, transfection with a specific anti-miR-21-sensitized cancer cells to undergo apoptosis [1416]; miR-15b, miR-16, and miR-181b are downregulated in multidrug-resistant gastric cancer cell line SGC7901/VCR, overexpression of miR-15b, miR-16, or miR-181b sensitized cells to anti-cancer drugs via targeting antiapoptotic gene BCL2 [17, 18], etc.

In this study, we reported that miR-497 was downregulated in both multidrug-resistant human gastric cancer cell line SGC7901/VCR and multidrug-resistant human lung cancer cell line A549/CDDP, compared with the parental SGC7901 and A549 cell lines, respectively. We demonstrated that miR-497 might play a role in the development of MDR in human gastric and lung cancer cell lines by targeting the antiapoptotic BCL2.

Materials and methods

Cell culture

Human gastric adenocarcinoma cell line SGC7901 (obtained from Academy of Military Medical Science, Beijing, China) and its multidrug-resistant variant SGC7901/VCR (obtained from the State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xian, China), human lung cancer cell line A549 and its multidrug-resistant variant A549/CDDP (both obtained from Biosis Biotechnology Company, Shanghai, China) were all cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (Gibco BRL, Grand Island, NY) in a humidified atmosphere containing 5% CO2 at 37°C. To maintain the MDR phenotype, vincristine (VCR, with final concentration of 1 μg/ml) and cisplatin (CDDP, with final concentration of 4 μg/ml) were added to the culture media for SGC7901/VCR and A549/CDDP cells, respectively.

miRNA microarray analysis

Prior to experimentation, SGC7901/VCR cells were cultured 1 week without vincristine. Total RNA from SGC7901 and SGC7901/VCR cell lines was isolated with Trizol reagent (Invitrogen, Carlsbad, CA), and miRNA fraction was further purified using a mirVanaTM miRNA isolation kit (Ambion, Austin, TX). The isolated miRNAs from the 2 cell lines were then labeled with Hy3 using the miRCURYTM Array Labelling kit (Exiqon, Vedbaek, Denmark) and hybridized, respectively, on a miRCURY TM LNA microRNA Array (v 8.0, Exiqon) as described [17]. Microarray images were acquired using a Genepix 4000B scanner (Axon Instruments, Union City, CA) and processed and analyzed with Genepix Pro 6.0 software (Axon Instruments).

Quantitative real-time PCR analysis for miRNA

The RNA preparation was as described earlier. The concentration and purity of the RNA samples were determined spectroscopically. Expression of mature miRNA was assayed using stem-loop RT followed by real-time PCR analysis [17]. The SYBR and U6 gene were used for detecting the gene amplification and normalizing each sample, respectively. EzOmicsTM miRNA qPCR Detection Primer Set (Catalog No.BK1010) and EzOmicsTM One-Step qPCR Kit (Cat No. BK2100) which were purchased from Biomics Biotechnologies Co., Ltd (Nantong, China) were used for quantitative real-time PCR analysis for miR-497 and U6 snRNA, respectively. The fold change for miRNA from SGC7901/VCR cells and A549/CDDP cells relative to each control SGC7901 and A549 cells was calculated using the 2−ΔΔCt Method [19], where ΔΔCt = ΔCt SGC7901/VCR -ΔCt SGC7901 or ΔΔCt = ΔCt A549/CDDP -ΔCt A549 and ΔCt = Ct miRNA -Ct U6 snRNA. PCR was performed in triplicate.

In vitro drug sensitivity assay

SGC7901/VCR, A549/CDDP, SGC7901, and A549 cells were plated in 6-well plates (6 × 105 cells/well), 100 nM of the miR-497 mimic or 100 nM miRNA mimic control was transfected in SGC7901/VCR and A549/CDDP cells, while 100 nM of the miR-497 inhibitor or 100 nM miRNA inhibitor control was transfected in SGC7901 and A549 cells, using lipofectamine 2000 (Invitrogen, Long Island, NY, USA) according to the manufacturer’s protocol, respectively. The miR-497 mimic, miRNA mimic control, 2′-O-methyl (2′-O-Me) modified miR-497 inhibitor, and miRNA inhibitor control were chemically synthesized by Shanghai GenePharma Company (Shanghai, China). The sequence of each was shown in supplementary data 1.

Twenty-four hours after, transfection cells were seeded into 96-well plates (5 × 103 cells/well) for next step experiment. After cellular adhesion, freshly prepared anticancer drugs including vincristine (VCR), 5-fluorouracil (5-Fu), cisplatin (CDDP), etoposide (VP-16), and adriamycin (ADR) were added with the final concentration being 0.01, 0.1, 1, and 10 times of the human peak plasma concentration for each drug as previously described [17]. The peak serum concentrations of various anticancer drugs are 0.5 μg/ml for VCR, 10 μg/ml for 5-Fu, 2.0 μg/ml for CDDP, 10 μg/ml for VP-16, and 0.4 μg/ml for ADR [20, 21]; 48 h after the addition of drugs, cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The absorbance at 490 nm (A490) of each well was read on a spectrophotometer. The concentration at which each drug produced 50% inhibition of growth (IC50) was estimated by the relative survival curve. Three independent experiments were performed in quadruplicate.

Dual luciferase activity assay

The 3′UTR of human BCL2 cDNA containing the putative target site for the miR-497 (sequence shown in supplementary data 2) was chemically synthesized and inserted at the XbaI site, immediately downstream of the luciferase gene in the pGL3-control vector (Promega, Madison, WI) by Biomics Biotechnologies Co., Ltd (Nantong, China).

Twenty-four hours before transfection, cells were plated at 1.5 × 105 cells/well in 24-well plates. Two hundred nanograms of pGL3-BCL2-3′-UTR plus 80 ng pRL-TK (Promega) was transfected in combination with 60 pmol of the miR-497 mimic or miRNA mimic control using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol as described, respectively [17]. Luciferase activity was measured 24 h after transfection using the Dual Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to renilla luciferase activity for each transfected well. Three independent experiments were performed in triplicate.

Western blot analysis

SGC7901/VCR and A549/CDDP cells were plated in 6-well plates (6 × 105 cells/well), and 72 h after the transfection of miR-497 mimic or miRNA mimic control, cells were harvested and homogenized with lysis buffer. Total protein was separated by denaturing 10% SDS–polyacrylamide gel electrophoresis. Western blot analysis was performed as described [17]. The primary antibodies for BCL2, β-actin, and α-Tubulin were purchased from Cell signaling Technology, Bioworld Technology and Santa Cruz Biotechnology, respectively. Protein levels were normalized to β-actin or α-tubulin. Fold changes were determined.

Apoptosis assay

Cells were plated in 6-well plates (6 × 105 cells/well). Twenty-four hours after the transfection of miRNA mimic as described above, SGC7901/VCR and A549/CDDP cells were treated by VCR and CDDP, with final concentration of 5 and 20 μg/ml, respectively. Forty-eight hours after the treatment of VCR and CDDP, flow cytometry was used to detect apoptosis of the transfected SGC7901/VCR and A549/CDDP cells by determining the relative amount of AnnexinV-FITC-positive-PI-negative cells as previously described [17], respectively.

Statistical analysis

Each experiment was repeated at least 3 times. Numerical data were presented as mean ± SD. The difference between means was analyzed with Student’s t test. All statistical analyses were performed using SPSS11.0 software (Chicago, IL). Differences were considered significant when P < 0.01.

Results

miR-497 is downregulated in both SGC7901/VCR and A549/CDDP cells, compared with SGC7901 and A549 cells, respectively

miRNA microarray analysis of SGC7901 and SGC7901/VCR cells showed that miR-497 was significantly downregulated in SGC7901/VCR cells compared with the parental SGC7901 cells. The decreased fold change for miR-497 was 10.17-fold. The primary microarray data result was shown in the supplementary data 3. Quantitative real-time PCR for miR-497 further verified that miR-497 was significantly downregulated in both SGC7901/VCR and A549/CDDP cells and the decreased fold changes were 2.46 ± 0.4 and 2.94 ± 0.27, compared with each parental cell, respectively (Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs12032-010-9797-4/MediaObjects/12032_2010_9797_Fig1_HTML.gif
Fig. 1

Real-time quantification of miR-497 by stem-loop RT-PCR showed that miR-497 was downregulated in both SGC7901/VCR and A549/CDDP cell lines, compared with SGC7901 and A549 cell lines, respectively. Triplicate assays were performed for each RNA sample, and the relative amount of miR-497 was normalized to U6 snRNA. Data were shown as fold changes of miR-497 levels in SGC7901/VCR and A549/CDDP cell lines relative to SGC7901 and A549 cell lines, respectively, which was set as 1 (mean ± SD). * P < 0.01

miR-497 modulates MDR of SGC7901/VCR and A549/CDDP cell lines

In both SGC7901/VCR and A549/CDDP cells, MTT assay revealed that those transfected with miR-497 mimic exhibited greatly decreased resistance to VCR, CDDP, VP-16, and ADR but not to 5-Fu compared with the miRNA mimic control transfected cells, respectively, (Fig. 2a, b), while in both SGC7901 and A549 cells, those transfected with miR-497 inhibitor exhibited greatly enhanced resistance to VCR, CDDP, VP-16, and ADR but not to 5-Fu compared with the miRNA inhibitor control–transfected cells, respectively (Fig. 2c, d).
https://static-content.springer.com/image/art%3A10.1007%2Fs12032-010-9797-4/MediaObjects/12032_2010_9797_Fig2_HTML.gif
Fig. 2

miR-497 modulates MDR of human gastric and lung cancer cell lines. (a and b). In both SGC7901/VCR and A549/CDDP cells, those transfected with miR-497 mimic exhibited greatly decreased resistance to VCR, CDDP, VP-16, and ADR but not to 5-Fu, respectively. (c and d). In both SGC7901 and A549 cells, those transfected with miR-497 inhibitor exhibited greatly enhanced resistance to VCR, CDDP, VP-16, and ADR but not to 5-Fu, respectively. * P < 0.01

These results suggested that miR-497 might modulate MDR of SGC7901/VCR and A549/CDDP cell lines.

The anti-apoptotic BCL2 is the target gene of miR-497

TargetScanHuman 5.1 (http://www.targetscan.org) predicted that BCL2 was the target gene of the miR-497 conservely between different species (supplementary data 5). To explore whether BCL2 is the target gene of the miR-497, we constructed a luciferase reporter vector with the putative BCL2 3′ UTR target site for the miR-497 downstream of the luciferase gene (pGL3-BCL2-3′-UTR). Luciferase reporter vector together with the miR-497 mimic or the miRNA mimic control was transfected into SGC7901/VCR and A549/CDDP cells, respectively. In both SGC7901/VCR and A549/CDDP cells, a significant decrease in relative luciferase activity was noted when pGL3-BCL2-3′-UTR was cotransfected with the miR-497 mimic but not with the miRNA mimic control, suggesting that BCL2 is the target gene of the miR-497 (Fig. 3).
https://static-content.springer.com/image/art%3A10.1007%2Fs12032-010-9797-4/MediaObjects/12032_2010_9797_Fig3_HTML.gif
Fig. 3

Dual luciferase assay performed in SGC7901/VCR and A549/CDDP cells suggested that BCL2 is the target gene of the miR-497, in both SGC7901/VCR and A549/CDDP cells, a significant decrease in relative luciferase activity was noted when pGL3-BCL2-3′-UTR was cotransfected with the miR-497 mimic but not with the miRNA mimic control. * P < 0.01

miR-497 modulates MDR by repressing BCL2 protein expression

Worth of note, in our study, the decreased expression of miR-497 in SGC7901/VCR and A549/CDDP cells was concurrent with the overexpression of BCL2 protein, compared with the parental SGC7901 and A549 cells, respectively (Fig. 4a). Since the antiapoptotic BCL2 protein is the target of the miR-497, we hypothesized that the miR-497 might modulate multidrug resistance of cancer cells by repressing the BCL2 protein expression.
https://static-content.springer.com/image/art%3A10.1007%2Fs12032-010-9797-4/MediaObjects/12032_2010_9797_Fig4_HTML.gif
Fig. 4

a The antiapoptotic BCL2 protein was overexpressed in SGC7901/VCR and A549/CDDP cells, compared with the parental SGC7901 and A549 cells, respectively. Representative Western blots were attached beside the graphs. b In both A549/CDDP and SGC7901/VCR cells, 72 h after the transfection, Western Blot demonstrated significantly decreased BCL2 protein level in miR-497 mimic transfected cells compared with the miRNA mimic control transfected cells. Representative Western blots were attached beside the graphs. The results shown represent the mean ± SD from 3 independent experiments. * P < 0.01

To ascertain our hypothesis, we transfected the miR-497 mimic and the control miRNA mimic into SGC7901/VCR and A549/CDDP cells to detect the BCL2 expression level changes, respectively. In both SGC7901/VCR and A549/CDDP cells, 72 h after the transfection, Western Blot demonstrated significantly decreased BCL2 protein level in miR-497 mimic transfected cells compared with the miRNA mimic control transfected cells (Fig. 4b).

These results showed that miR-497 might modulate multidrug resistance of cancer cells at least in part by repressing the BCL2 protein expression.

miR-497 sensitizes SGC7901/VCR and A549/CDDP cells to VCR- and CDDP-induced apoptosis, respectively

The development of drug resistance in various cancer cells has been linked to a reduced susceptibility to drug-induced apoptosis, which was shown to be a consequence, at least in some cases, of overexpression of antiapoptotic proteins, such as BCL2, IAPs, and BCL-XL [17, 22, 23]. Since the miR-497 may modulate multidrug resistance of cancer cells at least in part by repressing the BCL2 protein expression, considering the well-characterized role of BCL2 in apoptosis and drug resistance, we suggested a hypothesis that miR-497 might play a role in the development of MDR at least in part by modulation of apoptosis by targeting BCL2. To confirm this hypothesis, we evaluated VCR- and CDDP-induced apoptosis after transfection with SGC7901/VCR and A549/CDDP cells with the miR-497 mimic and the miRNA mimic control, respectively. In both SGC7901/VCR cells and A549/CDDP cells, a marked increase in apoptosis, as assessed by flow cytometry, was observed in the miR-497 mimic transfected cells after VCR and CDDP treatment, compared with the miRNA mimic control transfected cells, respectively [Fig. 5].
https://static-content.springer.com/image/art%3A10.1007%2Fs12032-010-9797-4/MediaObjects/12032_2010_9797_Fig5_HTML.gif
Fig. 5

miR-497 mimic sensitized SGC7901/VCR and A549/CDDP cells to VCR- and CDDP-induced apoptosis, respectively. In SGC7901/VCR cells, apoptosis evaluated by flow cytometry showed a marked increase in apoptosis in miR-497 mimic transfected cells after VCR treatment, compared with the miRNA mimic control transfected cells. In A549/CDDP cells, apoptosis evaluated by flow cytometry showed a marked increase in apoptosis in miR-497 mimic transfected cells after CDDP treatment, compared with the miRNA mimic control transfected cells. Representative flow cytometry report was attached beside the graphs. The results shown represent the mean ± SD from 3 independent experiments. * P < 0.01

Discussion

One major mechanism of drug resistance in cancer cells is the defective apoptosis pathway [24]. Recently, more and more findings have established that miRNAs modulate drug resistance of cancer cells at least in part through this mechanism [1012]. In our study, we found that the antiapoptotic protein BCL2 was upregulated while the miR-497 was downregulated in both SGC7901/VCR and A549/CDDP cells, compared with SGC7901 and A549 cells, respectively. The mechanistic connection of miR-497 dysregulation with the establishment of multidrug resistant in SGC7901/VCR and A549/CDDP cells was evidenced by the correlation between exogenous overexpression of miR-497 and corresponding changes in the protein levels of its target BCL2, which have a documented importance in the development of cancer cells drug resistance.

microRNA cluster miR-195/miR-497 is located on chromosomal band 17p13.1, both of which are members of the miR-15/107 miRNA gene group, based on their sequence AGCAGC starting at either the first nucleotide or the second nucleotide from the 5′ end of the mature (~22 nt, single stranded) miRNA, a motif which referred to as AGC × 2 [24]. Flavin et al. found that miR-195/miR-497 were downregulated from the microRNA cluster site in primary peritoneal carcinoma relative to ovarian serous carcinoma and they suggested that miR-195/miR-497 might have potential roles as tumor suppressor genes in primary peritoneal tumourigenesis [25]. Not only in primary peritoneal carcinoma, miR-195/miR-497 were also found downregulated in a range of other cancers. miR-195 was found downregulated in hepatocellular, bladder, gastric, prostate carcinoma, and chronic lymphocytic leukemia, while miR-497 was found downregulated in prostate carcinoma and human male breast carcinoma [24]. Moreover, Chang et al. found that miR-195/miR-497 were Myc-repressed miRNAs, which have marked anti-tumorigenic activity in a mouse model of B-cell lymphoma, they also suggested that delivery of Myc-repressed miRNAs might represent a therapeutic strategy for cancer [26]. Although the above studies suggested that miR-195/miR-497 may serve as tumor suppressor genes, the study by Bloomston et al. suggested that the function of miR-497 on pancreatic tumourigenesis maybe more conflicting, they found that miR-497 was upregulated in pancreatic cancer compared with chronic pancreatitis while downregulated in chronic pancreatitis compared with the normal pancreas [27], and the study reminded us that the function of miR-497 on tumourigenesis maybe more complicated. In our study, we found that miR-497 was downregulated in human drug-resistant gastric and lung cancer cell lines, compared with their each parental cell lines. The aberrant DNA methylation of the promoter region of miR-195/497 cluster maybe an important cause of the downregulation of miR-497 in drug resistant cells [24]; however, more research needs to elucidate the underlying mechanism. Overexpression of miR-497 could sensitize SGC7901/VCR and A549/CDDP cells to VCR- and CDDP-induced apoptosis, at least in part via targeting the antiapoptotic BCL2. This was in concordance with recent study by Ke-Jie Yina et al. and they found that mmu-miR-497 could regulate neuronal death in mouse brain after transient focal cerebral ischemia via targeting BCL2 [28].

Worth of note, in our study, miR-497 may have the same effect on increasing the sensitivity of both cells to VCR, CDDP, ADR, and VP-16 but not to 5-Fu, a possible explanation for this phenomenon is that 5-FU may induces apoptosis through the Fas/FasL pathway, at least in some cell types, such that factors that affect the mitochondrial release of cytochrome c (e.g., BCL2 overexpression) will have little impact on the cell response to drug-induced apoptosis [17].

In summary, the findings we reported here presented the first evidence that has-miR-497 might be involved in the development of MDR in human gastric and lung cancer cell lines. has-miR-497 could modulate the resistance of gastric and lung cancer cell lines to some anticancer drugs, at least in part, through targeting BCL2 expression. Our study may have implications for cancer chemotherapy whose efficiency is often impeded by the development of MDR. Therapeutic strategies targeting the MDR-related miRNAs, such as hsa-miR-497, may be another promising way to enhance therapeutic effect. However, it should be noted that our data are derived from cell lines which have been removed from their in vivo context and cannot be considered accurate surrogates for clinical tumors. Thus, future studies to assess the roles of hsa-miR-497 in vivo and in clinical context are warranted.

Acknowledgments

The authors are grateful to the fund support by the National Natural Science Foundation of China (Grant number: 30840095) and the Cancer Center of Nanjing Medical University (Grant number: 08ZLKF02).

Supplementary material

12032_2010_9797_MOESM1_ESM.doc (904 kb)
Supplementary material 1 (DOC 903 kb)
12032_2010_9797_MOESM2_ESM.xls (154 kb)
Supplementary material 2 (XLS 154 kb)

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