Background
Breast cancer is one of the most common types of malignant tumors in women and is the main cause of cancer-related death in women [
1]. Based on GLOBOCAN estimates, there were about 1,671,149 new cases of breast cancer worldwide in 2012, of which 521,907 patients died of breast cancer [
2]. According to the statuses of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2), breast cancer can be classified into four distinct molecular subtypes, including luminal-type, basal type, HER-2 positive, and normal [
3]. Although significant achievements have been made in chemotherapeutic strategies, the acquisition of resistance to adriamycin or other chemotherapeutic drugs is the major clinical obstacle to successful treatment [
4]. Consequently, it is necessary to elucidate the regulatory mechanisms of drug resistance, which will be helpful for designing new and targeted therapeutic strategies that can overcome drug resistance and improve the clinical value of treatments for breast cancer.
MicroRNAs (miRNAs) are highly conserved, endogenous, noncoding RNAs about 22 nucleotides in length. These molecules bind to mRNAs of at the 3′-untranslated region (3′-UTR) with perfect or near-perfect complementarity, resulting in degradation or inhibition of the target gene. The profile of miRNAs was proved to be closely associated with the tumor deterioration and metastasis [
5]. And also the expression of miRNAs could be regulated by the environmental factor such as hypoxia in cancer cells [
6]. Interestingly, one miRNA can modulate hundreds of target genes, and one gene can be repressed by hundreds of miRNAs [
7,
8]. These miRNA-based pathways have been shown to regulate cell proliferation, migration and programmed cell death, including apoptosis and autophagy, in cancer cells [
9‐
12]. In mammals, many studies have shown that miRNAs, such as
miR-
365,
miR-
125b,
miR-
491, and
miR-
133a, play important roles in multidrug resistance (MDR) [
13‐
15]. However, few studies have examined the signaling mechanisms through which miRNAs are involved in the regulation of MDR [
16]. The roles of miRNAs in apoptosis, autophagy, and necroptosis have been examined, with a focus on the impact of these regulatory activities on cancer progression [
17,
18]. Moreover, several miRNAs, including
miR-
27, have been shown to be involved in MDR in cancer [
19], particularly through modulation of apoptosis and autophagy pathways.
miR-
4532 has been shown to be differentially expressed in resistant and sensitive breast cancer cells by next-generation sequencing [
20]. Bioinformatics analysis of
miR-
4532 has shown that hypomethylated in cancer-1 (HIC-1) may be an miRNA target gene involved in the regulation of resistance of cancer cells to chemotherapeutic drugs. The
HIC-
1 gene, located on chromosome 17p13.3, is a tumor-suppressor gene that is frequently silenced or deleted in a variety of human cancers, such as leukemia, liver cancer, pancreatic cancer, and breast cancer [
21‐
24]. HIC-1 is involved in several complex biological functions in the regulation of drug resistance in cancer, including cell survival, cell growth, cell motility, and cell migration [
25]. Many downstream targets of HIC-1 responsible for development, proliferation, and migration, including sirtuin-1, C-X-C chemokine receptor type 7, transcription factor 4, matrix metalloproteinase (MMP) 2, MMP9, and cyclin D1, have been identified [
26‐
30]. However, the mechanisms regulating HIC-1 have not been reported, particularly with regard to how miRNAs regulate HIC-1 in breast cancer cells.
Therefore, in this study, we aimed to elucidate the effects of miR-4532 on the regulation of HIC-1 during acquisition of MDR in breast cancer. Our findings provided important insights into the mechanisms through which miR-4532 regulates HIC-1 expression to affect drug resistance in breast cancer cells.
Materials and methods
Cell lines and cell culture
MCF-7 and MDA-MB-231 human breast cancer cells and 293-T cells were maintained in our laboratory. Adriamycin-resistant MCF-7/ADR and MDA-MB-231/ADR cells were established by induction with gradient concentrations of adriamycin in vitro. The induction method is as follows: using a gradient culture of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 μg/ml adriamycin concentrations, each round screened the surviving cells for the beginning of the next drug resistance concentration, until the cells surviving in 1 μg/ml were MCF-7/ADR and MDA-MB-231/ADR. Cells were cultured in RPMI-1640 (Gibco, USA) supplemented with 10% fetal calf serum (Gibco) and 1% penicillin and streptomycin (Invitrogen, Carlsbad, Ca, USA) at 37 °C in a humidified atmosphere with 5% CO2. To maintain the ADR-resistant phenotype, adriamycin was added to the culture medium at a final concentration of 1 μg/ml, and MCF-7/ADR and MDA-MB-231/ADR cells were cultured for 2 weeks in ADR-free medium prior to their use in experiments.
Human tissue specimens and survival curves
Ten pairs of breast tumor specimens and matched adjacent nontumor tissues were randomly obtained from patients who had undergone mastectomy at the Fourth Affiliated Hospital of Jiangsu University. Informed consent was obtained from all patients, and the study was approved by the Ethics Committee of the Fourth Affiliated Hospital of Jiangsu University and was carried out in strict accordance with the Declaration of Helsinki.
Survival curves were calculated using the Kaplan–Meier method, conducted with the R Bioconductor ‘survival’ package. Kaplan–Meier curves were generated using a database of public microarray datasets (
http://kmplot.com) via website interface 2015.
Small RNAs were extracted from MCF-7/ADR and MCF-7 cells using RISO RNA ISOlation Reagent (Biomics, USA) according to the manufacturer’s instructions, and the samples were placed in dry ice for delivery to Genesky Biotechnologies Inc. (China) for next-generation sequencing analysis of miRNAs. The expression levels of mature miRNAs were then analyzed with a stem-loop kit and qRT-PCR, which was conducted using TaqMan Universal PCR Master Mix, as described by kit instruction. U6 was used as an endogenous control for data normalization, and all reactions were run in triplicate. The miRNA was calculated using the 2
−∆∆Ct method, where
\(\Delta \Delta {\text{Ct}}\, = \,\left( {{\text{Ct}}_{\text{miRNA}} \,{-}\,{\text{Ct}}_{\text{internal reference}} } \right)_{\text{experiment}} \,{-}\,\left( {{\text{Ct}}_{\text{miRNA}} \,{-}\,{\text{Ct}}_{\text{internal reference}} } \right)_{\text{control}}\) [
31].
Target gene prediction and gene ontology (GO) analysis of miRNAs
Cell proliferation assay
Cell Counting Kit 8 (CCK-8) assays were conducted as follows. Briefly, 1000 cells from each group were plated in each well of a 96-well microplate in 150 μl medium with different concentrations of chemotherapeutic drugs. After 48 h of culture, 10 μl of CCK-8 solution was added to the medium, and the cells were incubated for 3 h at 37 °C. The optical density at 570 nm was measured with a microplate spectrophotometer. Three independent experiments were performed, and half-maximal inhibitory concentration (IC50) was derived using the curve fitting method.
Cell invasion assays
The cells were harvested 72 h after transfection with siRNA-HIC-1 or control RNA and were resuspended in medium. The cells were then plated at a density of 2.0 × 106 cells/ml. In total, 0.2 ml cells was added to the upper chamber of transwell chambers (24-well inserts, 8-µm pore size; Millipore, Bedford, MA, USA), and 0.6 ml medium containing 10% fetal bovine serum was added to the lower chamber as a chemoattractant.
Cell apoptosis detection by FCM (flow cytometry)
Apoptotic cells differentiated from viable or necrotic ones were analyzed by combined application of propidium iodide (PI) and annexin V-APC as described [
31]. Samples were washed twice and adjusted at a concentration of 1 × 10
6 cells/ml with ice-cold PBS. A total of 100 μl suspension was added into each Falcon tube, and 10 μl of PI (20 μg/ml) and 10 μl of annexin V-APC were added into the labeled tubes. Cells were incubated at room temperature in the dark for at least 30 min, then PBS binding buffer about 400 μl was added into each tube without washing, and analyzed by the FACSCalibur™ Flow Cytometer (BD Biosciences) as soon as possible (within 30 min).
qRT-PCR analysis of mRNA expression
Total mRNA was extracted from breast tumor tissues, matched adjacent nontumor tissues, and cell lines using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. cDNA was then synthesized using a RevertAid First-Strand cDNA Synthesis Kit (Fermentas, USA). The expression levels of each analyte compared with untreated controls were assessed using the 2
−∆∆Ct method. All experiments were conducted at least in triplicate. The primers used to detect mRNA expression are listed in Additional files
1,
2 and
3.
Small RNA transfection
The miR-4532 mimic (5′-UGUAAACAUCCUACACUCUCAGC-3′) was purchased from a domestic provider in China (Genepharma, Shanghai, China). Cells were plated into 6-well plates at a density of 1 × 105 cells/well. After 24 h, 80 nM miR-4532 mimic and its negative control were transfected into cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. The transfected cells were then harvested for studies after culturing for 48 h. Three independent experiments were performed.
Luciferase reporter assay
For luciferase reporter experiments, the 3′-UTR sequence of HIC-1 was amplified by PCR from human genomic DNA using primers that included XbaI and EcoRI sites. Primers for HIC-1 3′-UTR were as follows: forward, 5′-CTAGTCTAGACTCTGTCGCTGCTGCGCGGCCCTGG-3′ and reverse, 5′-CCGGAATTCTCGCAAGGGCCGGAGGTAGGGCTAG-3′. The PCR products were ligated into the luciferase UTR-report vector (Promega, USA), and mutations within the putative miR-4532 binding sites were introduced using the following primers: luc-HIC1-mut-RP, GGGCCCCTTGTCCCGCGACCCCCGAGCTAAGG and luc-HIC1-mut-FP, CGGGACAAGGGGCCCACGGGGGTGGGATGGGG. Cells were transfected with the UTR-report vector, 20 ng control Renilla luciferase pRL-TK vector (Promega), and 10 nM miR-4532 mimic for the HIC-1-3′-UTR construct using Lipofectamine 2000 reagent. Forty-eight hours after transfection, cells were lysed with a 1× passive lysis buffer, and assays were performed using a Dual-Luciferase Reporter Assay System kit (Promega) according to the manufacturer’s instructions.
Western blot analysis
Total cellular extracts were prepared by homogenization of 3 × 10
6 to 5 × 10
6 cells in radioimmunoprecipitation assay buffer (Beyotime, China). Western blot analysis was performed as described previously [
33]. After separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 15% gels, the gels were immersed in cold transfer buffer (0.025 M Tris, 0.19 M glycine, 20% methanol), and the proteins were transferred to polyvinylidene difluoride membranes. The membranes were then blocked in 3% skim milk powder in PBS Tween-20 (PBST) overnight at 4 °C and immunoblotted with primary antibodies (Cell Signaling Technology, USA) diluted 1:1000 for 1.5 h at room temperature. After washing five times with PBST, the membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG diluted 1:2000 for 1.5 h at room temperature. After washing with PBS five times, the bands were visualized using diaminobenzene or enhanced chemiluminescence (ECL; Thermo, Shanghai, China).
Statistical analysis
Data are shown as means ± standard deviations. Statistical significance between groups was evaluated using Student’s t-tests in SPSS PASW Statistics version 18 Multilingual (SPSS Inc., USA). Results with p value of less than 0.05 were considered statistically significant.
Discussion
Breast cancer is a highly heterogeneous disease with a complex etiology involving genetic and environmental factors. Despite the rapid development of molecular diagnosis and chemotherapy for breast cancer, MDR remains an obstacle to effective treatment [
34]. Previous functional studies have indicated that miRNAs can regulate almost every cellular process, including MDR; however, the roles of miRNAs in MDR remain largely unknown [
35,
36].
In this paper, miR-4532 was found to be expressed in breast cancer cells, and high expression of this miRNA was closely related to drug resistance in breast cancer. Moreover, our analysis showed that miR-4532 regulated HIC-1, a transcriptional repressor involved in the regulation of cell survival, growth control, and DNA damage response. Finally, we also showed that miR-4532 was upregulated in human breast cancers. However, the biological relationship between miR-4532 and its specific target HIC-1 in adriamycin resistance in MCF-7/ADR cells remains unknown; further studies are needed to elucidate this mechanism.
HIC-
1 is a tumor-suppressor gene that is frequently epigenetically silenced or deleted in many human cancers [
37]. Growing evidence has suggested that the protein expression level of HIC-1 is correlated with prognosis in patients with cancer [
38,
39]. For example, HIC-1 mRNA and protein levels were reported to be low or absent in pancreatic ductal adenocarcinoma (PDAC) tissues, and its expression gradually decreased during the progression of PDAC; negative HIC-1 expression predicted advanced pathological stage and poor patient survival [
25]. Moreover, HIC-1 expression was found to be silenced in triple-negative breast cancer [
24]. Although studies have identified HIC-1 as having frequent changes in hypermethylation or loss of heterozygosity in many human cancers [
40,
41], the molecular mechanisms through which HIC-1 inhibits cancer progression remain poorly understood. HIC-1 is a multifunctional, sequence-specific transcriptional repressor that interacts with several major repression and chromatin remodeling complexes [
42,
43]. To date, many studies have showed that the IL-6/Janus kinase/STAT3 signaling pathways are involved in drug resistance, angiogenesis, migration, and other processes in cancers [
44,
45]. IL-6-induced transcriptional factor or cytokines, such as c-Myc and vascular endothelial growth factor, initiate and promote cell growth by triggering proliferation, and MMP2 and MMP9 proteins induce cell migration, which is closely related to metastasis and invasiveness in human cancer [
46,
47]. Consistent with this, we found, for the first time, that HIC-1 negatively regulated the expression of STAT3, which decreased transcriptional activation of STAT3, and this may explain why HIC-1 acted as an independent prognostic predictor of poor survival.
In summary, in this study, we found that miR-4532 targeted HIC-1 to modulate drug resistance and cell migration. HIC-1 was also found to be differentially expressed in breast cancer tissues and to be correlated with prognosis. Our findings are the first to demonstrate the interactions between miR-4532 and its target HIC-1 in the context of chemotherapeutic drug resistance in breast cancer. Accordingly, our results provide new mechanistic insights into the functions of miRNAs as potential therapeutic targets for overcoming MDR in breast cancer.
Authors’ contributions
FF designed and performed experiments, wrote the manuscript. ZXL, WCY, CL, CWP, and XWL gave suggestion on study design. All authors read and approved the final manuscript.