Introduction
Breast cancer (BC) is by far the most frequent cancer of women (23% of all cancers), with an estimated 1.15 million new cases worldwide in 2002 [
1]. It is still the leading cause of cancer mortality in women [
1]. Despite research and resources dedicated to elucidating the molecular mechanisms of BC, the precise mechanisms of its initiation and progression remain unclear.
MicroRNAs (miRNAs) are small non-coding RNAs (20 to 24 nucleotides) that post-transcriptionally modulate gene expression by negatively regulating the stability or translational efficiency of their target mRNAs [
2]. After the discovery of miRNAs, and findings indicating that they play a role in cancer, the concept of "oncomirs" was proposed [
3]. In particular,
miR-21 [miRBase: MIMAT0000076] has emerged as a key oncomir, since it is the most consistently up-regulated miRNA in a wide range of cancers [
4‐
7].
Functional studies showed that knockdown of
miR-21 in MCF7 cells led to reduced proliferation and tumor growth [
8,
9]. Knockdown of
miR-21 in MDA-MB-231 cells significantly reduced invasion and lung metastasis [
10]. These data clearly implicate
miR-21 as a key molecule in carcinogenesis, but functional studies that demonstrate cause and effect relationships between
miR-21 and target genes are lacking. Given that miRNAs usually target multiple genes post-transcriptionally,
miR-21 is likely to exert its effects by regulating many genes involved in BC.
The inhibition of miRNAs using antisense oligonucleotides (ASOs) is a unique and effective technique for investigating miRNA functions and targets. Peptide nucleic acids (PNAs) are artificial oligonucleotides constructed on a peptide-like backbone. PNAs have a stronger affinity and greater specificity for DNA or RNA than natural nucleic acids, and are resistant to nucleases [
11]. PNA-based ASOs can be used without transfection reagents, and are highly effective and sequence-specific. They provide long-lasting inhibition of miRNAs, and show no cytotoxicity up to 1 μM [
11]. Therefore, we used a PNA
miR-21 inhibitor for
in vivo investigation.
In this study, we explored the role of miR-21 in the malignant progression of human BC by assaying in vitro and in vivo function after miR-21 knockdown. We also searched for miR-21 targets using gene prediction-based and systematic screening approaches. Two potential target genes eukaryotic translation initiation factor 4A2 (EIF4A2) [NCBI: NM001967] and ankyrin repeat domain 46 (ANKRD46) [NCBI: NM198401] were selected for correlation analysis between protein levels and clinicopathological characteristics as well as prognosis using immunohistochemistry (IHC) on cancer tissue microrrays (TMAs).
Materials and methods
Tissue specimens and TMAs construction
In situ hybridization analysis was performed on fresh samples from BC or fibroadenoma (FA) tissues with paired normal adjacent tissues (NATs, > 2 cm from tumor tissues) obtained from Sun Yat-sen University Cancer Center (SYSUCC) (Guangzhou, China) between January and March 2009. For IHC staining of
miR-21 predicted target genes, formalin-fixed paraffin-embedded tissues were obtained from 99 randomly selected BC patients without neoadjuvant therapy at SYSUCC from January 2000 to November 2004, from whom informed consent and agreement, and clinicopathological information was available. A pathologist reviewed slides from all blocks, selecting representative areas of invasive tumor tissue to be cored. Selected cores were analyzed in duplicate using a MiniCore Tissue Arrayer (Alphelys, Passage Paul Langevin, Plaisir, France) with a 1-mm needle. The diagnosis and histological grade of each case were independently confirmed by two pathologists based on World Health Organization classification [
12]. The clinical stage was classified according to the American Joint Committee on Cancer (AJCC) tumor-lymph node-metastasis (TNM) classification system [
13]. The study was approved by the Research Ethics Committee of SYSUCC (Reference number: YP-2009168). The clinicopathological characteristics and follow-up data of the patients are summarized in Table
1.
Table 1
Clinicopathological characteristics and follow-up data for 99 patients with BC
Median age (range) | 48 (30 to 74) (years) |
Histological type* | |
Ductal | 93/99 (94%) |
Lobular | 1/99 (1%) |
Other | 5/99 (5%) |
Histological grade* | |
I | 22/99 (22%) |
II | 58/99 (58%) |
III | 19/99 (20%) |
Lymph node status at time of primary diagnosis |
Metastasis | 57/99 (58%) |
No metastasis | 42/99 (42%) |
AJCC clinical stage** | |
I | 8/99 (8%) |
II | 68/99 (69%) |
III | 23/99 (23%) |
Overall survival (median, range) | 74 (6 to 112) (months) |
Alive without evidence of cancer | 45/99 (68%) |
Alive with cancer | 14/99 (14%) |
Died of cancer | 40/99 (40%) |
Died of other disease | 0/99 (0%) |
Locked nucleic acid (LNA)-based in situhybridization for miRNA
To study the spatial and temporal expression of miRNAs with high sensitivity and resolution, the miRNA chromogenic
in situ hybridization (CISH) and fluorescein
in situ hybridization (FISH) protocol [
14] were optimized (Additional file
1).
Transfection of LNA-antimiR-21 into BC cells
MCF-7 and MDA-MB-231 cells were maintained in Dulbecco's modified Eagle's medium, supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (GIBCO-Invitrogen, Carlsbad, CA, USA). For transfection, the LNA-antimiR-21 or LNA-control (Exiqon A/S, Skelstedet, Vedbaek, Denmark) were delivered at a final concentration of 50 nM using Lipofectamine 2000 reagent (Invitrogen).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and colony formation assay
Growing cells (2 × 103 cells per well) were seeded into 96-well plates. At 24 h after LNA-transfection, cells were stained with 20 μl sterile MTT dye (5 mg/ml; Sigma-Aldrich Corp, St. Louis, MO, USA), followed by 4 h at 37°C. After supernatant removal, 150 μl of dimethyl sulphoxide (Sigma) was added and thoroughly mixed for 15 minutes. Absorbence was measured with a microplate reader (SpectraMax M5; Molecular Devices Corp., Silicon Valley, CA, USA) at 490 nm. For colony formation assays, cells were seeded in six-well plates (0.5 × 103 cells per well) and cultured for two weeks. Colonies were fixed with methanol for 10 minutes and stained with 1% crystal violet (Sigma) for 1 minute. Each cell group was measured in triplicate.
Wound healing assay
Cells cultured in the presence of 50 nM LNA-antimiR-21 or LNA-control for 24 h were allowed to reach confluence before dragging a 1-mL sterile pipette tip (Axygen Scientific, Inc., Union City, CA, USA) through the monolayer. Cells were washed to remove cellular debris and allowed to migrate for 24 h or 48 h. Images were taken at time 0 h, 24 h and 48 h post-wounding using a digital camera system (Leica DFC 480; Leica Microsystems, Bannockburn, IL, USA). The motility of the cells was determined as repaired area percentage [
15]. Each cell group was measured in triplicate.
Validation of tumor growth-promoting activity of miR-21in an animal model
Five- to six-week-old female BALB/c-nude mice (Slaccas Shanghai Laboratory Animal Co., Ltd., Shanghai, China) were used for experimental tumorigenicity assays. To facilitate estrogen-dependent xenograft establishment, each mouse received 17-estradiol (20 mg/kg; Sigma) intraperitoneally once a week. One week after treatment, equivalent amounts of MCF-7 cells, treated with PNA-antimiR-21 or PNA-control (100 nM for 48 h; Panagene, Inc., Yuseong-gu, Daejeon, Korea) without transfection reagents according to the manufacturer's protocol, were injected subcutaneously (10
7 cells/tumor) into the left axilla of nude mice [
16]. Mice were weighed, and tumor width (W) and length (L) were measured every day. Tumor volume was estimated according to the standard formula: V = ∏/6 × L × W
2, as described previously [
17]. Animals were killed nine days after initial growth of the MCF-7 xenografts was detectable, and tumors were extracted. In all experiments, the ethics guidelines for investigations in conscious animals were followed, with approval from the local Ethics Committee for Animal Research.
mRNA array and data mining
MCF-7 and MDA-MB-231 cells were transfected either with LNA-antimiR-21 or with LNA-control at a final concentration of 50 nM. Total RNAs were isolated from MCF-7 cells 48 h post transfection and from MDA-MB-231 cells 36 h post transfection, respectively, using Trizol Reagent (Invitrogen). The mRNA expression profile was performed using human genome oligo array service V1.0 (Catalog Number 400010; CapitalBio, Beijing, China) as described [
18]. Each sample was analyzed once, and the CapitalBio data preprocess, normalization and filtering were as previously described [
18]. Ratios were defined as marginal signal intensity when there was a substantial amount of variation in the signal intensity within the pixels from 800 to 1,500. All the microarray data have been deposited to the Gene Expression Omnibus (GEO) [
19] and are accessible through GEO Series accession number [GEO: GSE20627].
Relative quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
For validation of mRNA array and quantitative analysis of
miR-21 as well as potential target genes, qRT-PCR was used as previously described [
20]. The primers for qRT-PCR are in Additional file
2. The relative expression was calculated using the equation relative quantification (RQ) = 2
-ΔΔ
CT [
21].
Computational prediction of miR-21target genes
Predicted
miR-21 targets were identified using the algorithms of TargetScan 5.1 [
22], miRBase Targets V5 [
23], miRNAMap 2.0 [
24], PicTar [
25] and miRanda 3.0 [
26].
Luciferase reporter assay
The 3' untranslated region (3' UTR) of mRNA sequence of
ANKRD46 containing predicted
miR-21 binding site was amplified by PCR. PCR primers were listed in Additional file
2. After amplification, PCR products were cloned into the pMIR-REPORT (Applied Biosystems, Foster City, CA, USA), resulting in the pMIR-REPORT-3'
ANKRD46. Mutation of
ANKRD46 was introduced in the predicted
miR-21 binding site by a QuikChange site-directed mutagenesis kit (Stratagene, Foster City, CA, USA). Wild-type
EIF4A2 and mutant
EIF4A2 were cloned into pMD19-T Simple Vector by TaKaRa Biotechnology CO., LTD. (Dalian, Liaoning, China) and then were individually subcloned downstream of the luciferase coding sequence in the pMIR-REPORT (Applied Biosystems). All constructs were verified by DNA sequencing.
For reporter assays, wide-type or mutant reporter constructs (15 ng) were cotransfected into 293T cells in twelve-well plates with miR-21 or miR-control (50 nM; GenePharma, Shanghai, China) and Renilla plasmid (5 ng) using lipofectamine 2000 (Invitrogen). Firefly and Renilla luciferase activities were measured by using a Dual Luciferase Assay (Promega, Madison, WI, USA) 24 h after transfection. Firefly luciferase values were normalized to Renilla, and the ratio of firefly/renilla was presented.
Immunoblot analysis
Cells were harvested and lysed in radioimmune precipitation buffer (Upstate, Lake Placid, NY, USA) at the indicted time post-transfection. Antibodies used for immunoblot analysis were against ANKRD46 (1:500 dilution; sc-87548, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), EIF4A2 (1:1000 dilution; ab31218, Abcam, Cambridge, UK) and GAPDH (1:3,000 dilution; sc-32233, Santa Cruz Biotechnology), as a loading control. All bands were detected using a SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA).
Immunohistochemical staining
IHC and scoring of the estrogen receptor (ER), progesterone receptor (PR) and CerbB2 were performed as previously described [
20]. Slides were incubated with primary antibodies against ANKRD46 (1:150 dilution; sc-87548, Santa Cruz Biotechnology); or EIF4A2 (1:700 dilution; ab31218, Abcam). All slides were processed simultaneously in identical conditions per the manufacturer's instructions. Three observers independently determined consensus scoring of EIF4A2 and ANKRD46 immunostaining using a semi-quantitative estimation according to the percentage of positive cells and the intensity of staining as described previously [
27]. With these data, the composite score was obtained by adding the values of the staining intensity and relative abundance [
28]. Samples with scores lower than the median score were grouped as low protein expression [
29].
Statistical analysis
Spearman's rank correlation test was used for correlation analysis between predicted target gene protein levels and endogenous
miR-21 levels measured previously by qRT-PCR [
20]. Pearson's Chi-Square tests were used to compare target gene expression levels to clinicopathological characteristics. Survival curves were estimated by the Kaplan-Meier method and log-rank test. All analysis used SPSS 16.0 for Windows (SPSS Inc, Chicago, IL, USA). All tests were two-tailed, and the significance level was set at
P < 0.05.
Discussion
miR-21 is a key molecule in a wide range of cancers, and identifying its functional role in BC has direct clinical implications. We show here that knockdown of
miR-21 suppresses cell growth and proliferation of MCF-7 cells
in vitro, and suppresses MCF-7 xenograft growth. This result is consistent with the findings of Si
et al. [
9]. Interestingly, our study suggests that LNA-antimiR-21 also suppresses the growth and proliferation of MDA-MB-231
in vitro, in contrast to a recent report that found no effect of LNA-antimiR-21 on the growth of MDA-MB-231
in vitro or
in vivo, although anti-miR-21-treated tumors were slightly smaller than control tumors [
10]. One possibility could be differences in transfection efficiency, or miRNA ASO potency. Our results suggest that, as an oncomir,
miR-21 also affects cell migration.
MCF-7 cells are hormone-sensitive and difficult to culture
in vivo. Therefore, we used 17-estradiol to facilitate MCF-7 cells growth in nude mice, which is a common technique. Recently, estradiol was shown to down-regulate
miR-21 expression in MCF-7 cells [
32], although another study found estradiol-mediated up-regulation of
miR-21 in MCF-7 cells [
33]. In our study, the
miR-21 knockdown effect was reduced from 5.72 log
2-scale reduction before cell injection to 0.96 log
2-scale reduction after mice sacrifice. Based on our results, we propose that estradiol reduced differences in
miR-21 level between MCF/PNA-antimiR-21 and MCF/PNA-control cells, which would explain, in part, why differences in tumor weight between the two groups were not significance (
P = 0.065). Nonetheless, treatment with anti-miR-21 reduced MCF-7 xenograft growth by approximately 68% for up to nine days.
In vivo results suggested that the PNA-based
miR-21 inhibitor had a subtle yet reproducible inhibitory effect on tumor growth. MCF-7 xenograft tumor sections demonstrated that
miR-21 inhibition induced apoptosis of MCF-7 cells, confirming a previous study [
9]. We also showed that miRNA inhibition can be achieved without transfection or electroporation of human BC cell lines, highlighting the potential of PNA for future therapeutic applications.
ANKRD46, also known as ankyrin repeat small protein (ANK-S), is a 228-amino acid single-pass membrane protein, of unclear function. For the first time, we identify
miR-21 as an important regulator of
ANKRD46 mRNA and protein levels in BC cells. Our data showed that
miR-21 directly interacted with the
ANKRD46 3' UTR and inhibited
ANKRD46 expression, though there was no significant association between
miR-21 and ANKRD46 in resected patient tumors. This discrepancy may be due to three reasons. First, the artificial luciferase reporter assays do not fully recapitulate miRNA regulation
in vivo [
34]; second, the expression of ANKRD46 protein in patient tumors reflected specific time-point feature, which maybe different to the
in vitro subsequent increase of ANKRD46 protein at the time point of observation (the indicated hours after transfection); third, immunohistochemistry (IHC) is conventional a semi-quantitative method with relatively limited sensitivity. IHC may not be sensitive enough to observe the down-regulation of
ANKRD46 by
miR-21. Functional study of
ANKRD46 is required in the future to determine weather
ANKRD46 is a functional target of
miR-21 in BC progression as demonstrated in this study.
EIF4A2, an ATP-dependent RNA helicase, is expressed widely in human tissues [
35]. In this study, we found that
miR-21 and EIF4A2 protein were inversely expressed in resected BC patient tumors. But we did not find
miR-21 binding sites in the
EIF4A2 3' UTR and found no significant increase of EIF4A2 protein upon
miR-21 knockdown in MCF-7 and MDA-MB-231 cells, although
EIF4A2 mRNA increased after anti-miR-21 transfection. Taken together, the data reported here suggest that there maybe unknown indirect interactions between
miR-21 and
EIF4A2 in BC progression. In adult mice, the expression of the two EIF4A isoforms is dependent on cell growth status, with EIF4A1 expressed in all tissues, while EIF4A2 is expressed only in tissues with a low rate of cell proliferation [
36], indicating an anti-proliferative effect for EIF4A2. We for the first time revealed that low EIF4A2 expression correlated with low ERBB2 expression and poor survival of BC patients, suggesting its possible functional role in BC and urging further investigation.
Competing interests
Miss Li Xu Yan and Mrs Yan Zhang are doctoral degree candidates; Miss Qi Nian Wu and Mrs Yang Yang Li are master's degree candidates at SYSUCC. Mr Ding Zhun Liao, Mr Jing Hui Hou and Mrs Jia Fu are technicians at SYSUCC. Dr Mu-Sheng Zeng, Jing Ping Yun, Qiu Liang Wu and Yi Xin Zeng are Professors at SYSUCC. Dr Shao is a Professor and Vice Director at Department of Pathology of SYSUCC. The authors declare that they have not received any reimbursements, fees, funding, or salary, nor hold any stocks or shares in an organization that may in any way gain or lose financially from the publication of this manuscript, either now or in the future. The authors do not hold or are not currently applying for any patents relating to the content of the manuscript. The authors declare that they do not have any other financial or non-financial competing interests.
Authors' contributions
LXY, QNW and YZ carried out the substantial experiment work and drafted the manuscript. JYS designed and financially supported the study. QNW and DZL were responsible for patient samples and tissue array construction. JHH and JF supported the immunohistochemistry. YYL carried out the luciferase reporter assay. JPY, MSZ, QLW and YXZ helped carry out the research design and critically reviewed the final version of the manuscript for submission. All authors read and approved the final manuscript.