Introduction

Esophageal squamous cell carcinoma is a major cause of mortality and morbidity in China, and the total number of cases is predicted to rise as a result of population growth [1]. The biology of esophageal squamous cell carcinoma is of aggressive local invasion, early metastasis, and multidrug resistance (MDR) to chemotherapy. So far, the pathogenic mechanism has not been fully elucidated, resulting in MDR of esophageal cancer.

MicroRNAs (miRNAs) are a class of 22-nucleotide noncoding RNAs that are evolutionarily conserved and function as negative regulators of gene expression [2]. Single-stranded miRNAs can bind messenger RNAs of potentially hundreds of genes at the 3′ untranslated region with perfect or near-perfect complementarity, resulting in degradation or inhibition of the target messenger RNA, respectively. Emerging evidence has revealed that the small RNAs might play key roles in various biological processes, including cell proliferation, apoptosis, tumorigenesis, and MDR [3, 4]. MiRNAs are aberrantly expressed or mutated in human cancer, indicating that they might function as a novel class of MDR-related genes [5]. However, up-to-date miRNAs involved in MDR of esophageal cancer have not been reported yet. Better understanding of changes in miRNA expression during MDR of esophageal cancer might lead to possible improvements in the treatment for esophageal squamous cell carcinoma.

In this report, we showed that miR-27a might mediate MDR of esophageal cancer cells through regulation of MDR1 and apoptosis.

Materials and Methods

Cell Culture

Human esophageal squamous cell lines, ECA109 and TE-13, were routinely maintained in DMEM medium (GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 100 U/ml of penicillin sodium, and 100 μg/ml of streptomycin sulfate, at 37°C in humidified air containing 5% carbon dioxide air atmosphere. Throughout the experiment, the cells were used in logarithmic phase of growth.

MiRNA Transfection

Cells in exponential phase of growth were plated in 60-mm plates at 1 × 106 cells/plate and cultured for 16 h, and then transfected with the antagomirs of miR-27a or control RNA (Lafayette, CO) as described previously [6].

Northern Blot

Total RNAs from cells or tissues were extracted with TriZol Reagent (Invitrogen Life Technologies, Gaithersburg, MD) following the manufacturer’s instruction. RNA isolated was subjected to electrophoresis and Northern blot as described previously [7]. Briefly, 15 μg of total RNA were separated on 15% denaturing polyacrylamide gels, electrotransferred to GeneScreen Plus membranes (PerkinElmer, Waltham, MA), and hybridized using UltraHyb-Oligo buffer. Oligonucleotides complementary to mature miR-27a were end-labeled with T4 Kinase (Invitrogen Corp, Carlsbad, CA) and used as probes. Hybridization was performed according to the instructions of the manufacturer and the membranes were exposed to a storage phosphor screen and imaged using a Typhoon 9410 Variable Mode Imager. Prehybridization, hybridization, and washing were then done.

Quantitative Real-Time Polymerase Chain Reaction

Total RNAs from cells or tissues were extracted with TriZol Reagent (Invitrogen Life Technologies, Gaithersburg, MD) following the manufacturer’s instruction. First strand cDNA synthesis and amplification were performed using OmniscriptRTKit (QIAGEN Valencia, CA). The primers for miR-27a were obtained through Applied Biosystems or Eurogentec North America, Inc. Primers were designed as: MDR1, forward: 5′-CCCATCATTGCAATAGCAGG-3′, reverse: 5′-TGTTCAAACTTCTGC TCCTGA-3′; MRP, forward: 5′-TCATGGTGCCCGTCAATG-3′, reverse: 5′-CGATTGTCTTTGCTCTTCATGTG-3′; Bcl-2, forward: 5′-CATGCTGGGGCC GTACAG-3′, reverse: 5′-GAACCGGCACCTGCACAC-3′; Bax, forward: 5′-ATCCA GGATCGAGCAGGGCG-3′, reverse: 5′-GGTTCTGATCAGTTCCGGCA-3′; Bcl-xL, forward: 5′-GGAGGCAGGCGACGAGTTTGAA-3′, reverse: 5′-AAGGGGGTGGG AGGGTAGAGTGG-3′; Bak, forward: 5′-TCGGATCCAAATGGCTTCGGGGCA AGGCCC-3′, reverse: 5′-GAATTCCTTGGGAGTCATGATTTGAAGA-3′. The quantitative PCR amplifications were performed on the Stratagene 3005P Real-Time PCR system. Comparative real-time PCR was performed in triplicate, including no-template controls. Relative expression was calculated using the comparative Ct method.

In Vitro Drug Sensitivity Assay

Vincristine (VCR), adriamycin (ADR), cisplatin (CDDP), and 5-fludrouracil (5-flu) were all freshly prepared before each experiment. Drug sensitivity was evaluated using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay as described previously [8].

Intracellular ADR Concentration Analysis

Fluorescence intensity of intracellular ADR was determined by flow cytometry. Briefly, cells were seeded into six-well plates (1 × 106 cells/well) and cultured overnight. After addition of ADR to the final concentration of 5 μg/ml, cells continued to be cultured for 1 h. Cells were then harvested (for detection of ADR accumulation) or, alternatively, cultured in drug-free RPMI1640 for another 1 h followed by harvesting (for detection of ADR retention). Then cells were washed with PBS and the mean fluorescence intensity of intracellular ADR was detected using flow cytometry (FCM). The experiment was independently performed three times.

Annexin V Staining

Cells were washed twice with cold PBS and resuspended in 100 μl of binding buffer at a concentration of 1 × 106 cells/ml. Annexin V binds to those cells that express phosphatidylserine on the outer layer of the cell membrane, and propidium iodide stains the cellular DNA of those cells with a compromised cell membrane [9].

Luciferase Assay

The pGL3-MDR1 vector (promoter of MDR1, −136 to +10) and the control vector were established previously in our laboratory [8]. Cells were plated in 12-well plates at 1 × 105 per well. After growth for 16–20 h, 0.4 μg of reporter gene constructs was transfected using LipofectAMINE (Invitrogen) reagent according to the manufacturer’s protocol. This transfection was done concurrently with the transfection of the antagomirs of miR-27a as described above. Cells co-transfected with scrambled antago-miR-NC served as controls. After transfection for 5 h, the transfection mix was replaced with complete medium and incubated for 19 h. Cells were then lysed with 100 μl of 1× reporter lysis buffer, and 30 μl of cell extract were used for luciferase assays.

Western Blot

Cellular proteins were extracted and separated on SDS–PAGE gels, and Western-blot analyses were performed according to standard procedures [10]. Western blotting of β-actin on the same membrane was used as a loading control. Bands were quantified using Quantity One4.0 software (Bio-Rad). The following antibodies were used: anti-P-gp, anti-MRP, anti-Bcl2, and anti-Bax polyclonal antibodies (Santa Cruz Corp); anti-Bcl-xL (San Diego, CA); anti-Bak (PharMingen).

Statistical Analysis

All the data were presented as the mean ± SD, and were analyzed using Prism 5.0 software (GraphPad). The significance of differences from the control values was determined with Student’s t-test or the χ2 test. p < 0.05 was considered statistically significant.

Results

Down-Regulation of miR-27a Might Reverse Drug Resistance of Esophageal Cancer Cells

ECA109 and TE-13 cells were transfected with either the antagomirs of miR-27a or control RNA (Fig. 1a), and the viability of cells was measured using the MTT assay. As is shown in Table 1, the IC50 values of miR-27a antagomir cells for VCR, ADR, 5-flu, and CDDP were significantly decreased compared to the control cells. Taken together, miR-27a affected not only the sensitivity of cells to P-gp-related drugs VCR and ADR but also to P-gp-non-related drugs 5-flu and CDDP.

Fig. 1
figure 1

Effect of miR-27a on ADR intracellular accumulation and releasing of esophageal cancer cells. a Relative level of miR-27a in ECA109 and TE-13 cells after transfection. Total cell RNA extracted from each group was examined by Northern blotting. b, d ADR was added to cells in log phase to a final concentration of 5 μg/ml. Fluorescence intensity analysis of intracellular ADR in esophageal cancer cells; c, e ADR releasing index of esophageal cancer cells. Releasing index = (accumulation value–retention value)/accumulation value. * p < 0.05 versus control cells

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Table 1 IC50 values (μg/ml) of anti-cancer drugs for esophageal cancer cells
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Since MDR of cancer was mainly due to alterations of drug influx and efflux, ADR intracellular accumulation and releasing were explored. As is shown in Fig. 1b, d, increased accumulation of ADR of miR-27a antagomir cells was observed as compared with that of controls (p < 0.01). Consistent with this, miR-27a antagomir cells showed decreased releasing index (Fig. 1c, e).

As the blockade of apoptosis was another important mechanism of MDR, we investigated the capacity of miR-27a antagomir cells to undergo ADR-induced apoptosis by Annexin V staining. Down-regulation of miR-27a could promote ADR-induced apoptosis and the apoptotic rate of miR-27a antagomir cells was significantly higher than that of control cells (Fig. 2).

Fig. 2
figure 2

The effects of antagomirs of miR-27a on the induction of apoptosis in response to ADR. ECA109 (a) and TE-13 (b) cells were incubated with 1.5 μg/ml of ADR for 36 h. Annexin V/propidium iodide binding analyses of cells were presented. Results were representative of three independent experiments. * p < 0.05 versus control cells

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MiR-27a Might Target MDR1

To evaluate whether MDR1 was a genuine target of miR-27a, we analyzed the expression correlation of miR-27a and MDR1 in miR-27a antagomir and control cells by real-time PCR (Fig. 3) and Western blot (Fig. 4). The results showed that down-regulation of miR-27a significantly decreased expression of MDR1, but did not alter the expression of MRP. To elucidate the regulatory effects of miR-27a on the promoter activity of MDR1, luciferase reporter assays were performed. As is shown in Fig. 5, co-transfection of the MDR1 reporter gene with increasing amounts of antagomirs of miR-27a resulted in an essentially linear decrease in MDR1 promoter activity, suggesting that miR-27a might target MDR1.

Fig. 3
figure 3

Effects of antagomirs of miR-27a on mRNA expression of MDR1, MRP, Bcl-2, Bax, Bcl-xL, and Bak in ECA109 (a) and TE-13 (b) cells. Cells were transfected with 1 nM of antagomirs of miR-27a or a control RNA. Forty-eight hours later, total RNAs were extracted from the treated cells and quantitative real-time PCR analysis was performed. The mRNA level of the samples treated with a control RNA was arbitrarily set at 1, and the six genes’ mRNA levels of the esophageal cancer cells were normalized to the control. Results were the mean ± SD of triplicate determinations from one of three identical experiments. * p < 0.05 versus control

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Fig. 4
figure 4

Western-blot analysis of P-gp, MRP, Bcl-2, Bax, Bcl-xL, and Bak in esophageal cancer cells. β-actin was used as an internal control. The blot was visualized by ECL system. Results were representative of three independent experiments

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Fig. 5
figure 5

Luciferase reporter assay to determine the regulatory effect of antagomirs of miR-27a on MDR-1 promoter activity. Transcriptional activity of the MDR1 promoter fused to the luciferase gene in the pGL3Basic vector was assayed by cotransfection of this reporter gene (0.2 μg/well) with increasing amounts of antagomirs of miR-27a (0.2, 0.5, and 1 nM) in Eca109 cells. Cells co-transfected with scrambled antago-miR-NC served as controls. Results were presented as means ± SD (n = 3)

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Effect of mir-27a on Proteins Regulating Apoptosis

To gain insight into the molecular mechanisms involved in miR-27a-mediated apoptosis, the expressions of Bcl-2, Bax, Bcl-xL, and Bak were assessed in the cells. As shown in Figs. 3, 4, the expression of Bcl-2 was decreased and the expression of Bax was increased in response to down-regulation of mir-27a. However, relatively equal levels of Bcl-xL and Bak were detected in all ECA109 and TE-13 derived cell lines. These data strongly suggested that down-regulation of mir-27a might confer drug-induced apoptosis by enhancing the Bcl-2/Bax ratio in esophageal cancer cells.

Discussion

MiRNAs were critical regulators of transcriptional and post-transcriptional gene silencing, which were involved in multiple developmental processes in many organisms. To our knowledge, we have firstly identified miRNA, miR-27a, which might significantly mediate MDR of esophageal cancer.

We hypothesized that MiR-27a was related to MDR of esophageal cancer as follows: firstly, MiR-27a was widely expressed in cancer cells and might function as an oncogene, while a number of investigations had shown that transcription oncogenes, such as c-Myc and cyclin D1, were involved in the MDR regulation of esophageal cancer [11]. Secondly, miR-27a might exhibit oncogenic activity through regulating cell survival and angiogenesis, which was the pathway regulating MDR [12–14]. Lastly, miR-27a might suppress the cdc2/cyclin B inhibitor and thereby facilitate cancer cell proliferation by arresting cells at G2-M [11], while cell cycle regulation is involved in the signal network controlling MDR. Taken together, it could be assumed that after drug treatment, miR-27a would be able to regulate cell cycle progression and angiogenesis, and delicately coordinate with some other gene regulators to ensure the establishment of a drug-defense network.

To obtain a better model in which cells of the same origin can be compared, we transfected ECA109 and TE-13 cells with the antagomirs of miR-27a or control RNA and the effective transfectants were selected. MTT assay revealed that miR-27a antagomir cells showed increased sensitivity to both P-gp-related and P-gp-non-related drugs. ADR was used as a probe to evaluate drug accumulation and retention in cancer cells [15]. As the results showed, miR-27a antagomir cells showed increased ADR accumulation and retention and decreased ADR releasing index. The results indicated that miR-27a had a direct or indirect function of pumping the drug out of the cells.

It should be noted that VCR and ADR were the common substrates for P-gp and MRP. To clarify the association of P-gp and MRP with miR-27a related MDR, we investigated the effects of miR-27a on expression of them. The results showed that miR-27a might mediate the expression of P-gp, which functioned as an ATP-dependent drug-efflux pump. Each case of P-gp-related MDR was related to an increased human MDR1 mRNA level that could be linked either to gene amplification and/or increased gene transcription. It was believed that alterations in MDR1 promoter were important for P-gp function. In this report, the MDR1 gene was further analyzed by reporter gene assay. The results of luciferase reporter assay, real-time PCR, and Western blot suggested that miR-27a might be a regulator of the MDR1 gene. We assumed that the role of miR-27a might depend not only upon the promoter sequences but also upon the association of miR-27a with different cofactors.

However, how miR-27a antagomir cells showed increased sensitivity to 5-flu and CDDP couldn’t be explained by regulation of P-gp, which suggested that other mechanisms may exist. Apoptosis was a common pathway that finally mediated the killing functions of anticancer drugs, which was an important cause of MDR. Thus, we further tested whether the apoptosis-related molecules were involved in miR-27a-related MDR. MiR-27a antagomir cells displayed a higher proportion of apoptosing cells after ADR treatment as compared to control cells. The Bcl-2 family, including Bcl-2, Bcl-xL, Bax and Bak, is a rapidly expanding family of proteins involved in apoptosis and responses of tumor cells to chemotherapy [16]. These proteins were believed to modulate apoptosis by forming homodimers or heterodimers with other Bcl-2 family members [17]. A wide variety of human cancers with poor clinical response to chemotherapy exhibited high levels of Bcl-2 expression. It had been implied that Bcl-2 family expression provided resistance to a wide variety of cell death stimuli, including classical chemotherapeutic drugs and radiation [18]. The changed levels of Bcl-2 and Bax caused by miR-27a might contribute to miR-27a-related MDR of esophageal cancer cells.

In conclusion, miR-27a could possibly mediate drug resistance, at least in part through regulation of MDR1 and apoptosis. Further analysis of the mechanism of biological actions of miR-27a in MDR might help to further understand the mechanisms of MDR in esophageal cancer and generate a new approach to reverse MDR.