Knockdown of miR-21 in human breast cancer cell lines inhibits proliferation, in vitro migration and in vivo tumor growth

Introduction MicroRNAs (miRNAs) are a class of small non-coding RNAs (20 to 24 nucleotides) that post-transcriptionally modulate gene expression. A key oncomir in carcinogenesis is miR-21, which is consistently up-regulated in a wide range of cancers. However, few functional studies are available for miR-21, and few targets have been identified. In this study, we explored the role of miR-21 in human breast cancer cells and tissues, and searched for miR-21 targets. Methods We used in vitro and in vivo assays to explore the role of miR-21 in the malignant progression of human breast cancer, using miR-21 knockdown. Using LNA silencing combined to microarray technology and target prediction, we screened for potential targets of miR-21 and validated direct targets by using luciferase reporter assay and Western blot. Two candidate target genes (EIF4A2 and ANKRD46) were selected for analysis of correlation with clinicopathological characteristics and prognosis using immunohistochemistry on cancer tissue microrrays. Results Anti-miR-21 inhibited growth and migration of MCF-7 and MDA-MB-231 cells in vitro, and tumor growth in nude mice. Knockdown of miR-21 significantly increased the expression of ANKRD46 at both mRNA and protein levels. Luciferase assays using a reporter carrying a putative target site in the 3' untranslated region of ANKRD46 revealed that miR-21 directly targeted ANKRD46. miR-21 and EIF4A2 protein were inversely expressed in breast cancers (rs = -0.283, P = 0.005, Spearman's correlation analysis). Conclusions Knockdown of miR-21 in MCF-7 and MDA-MB-231 cells inhibits in vitro and in vivo growth as well as in vitro migration. ANKRD46 is newly identified as a direct target of miR-21 in BC. These results suggest that inhibitory strategies against miR-21 using peptide nucleic acids (PNAs)-antimiR-21 may provide potential therapeutic applications in breast cancer treatment.


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][5][6][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).

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.
Locked nucleic acid (LNA)-based in situ hybridization for miRNA To study the spatial and temporal expression of miR-NAs 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).
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].

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 sitedirected 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.

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.

miR-21 is overexpressed in BC tissues and cell lines
Expression of miR-21 was detected in the cytoplasm in cancerous and luminal epithelial cells, and occasionally in fibroblasts. In BC patients, an increase in miR-21 staining intensity was observed in BC and FA tissues compared with corresponding NATs (Figure 1a). Parallel detection by FISH is shown in Additional file 3. Consistent with the CISH results, quantitative analysis indicated that miR-21 expression was significantly increased by 4.44-to 2.02-fold in BC tissues compared with NATs (P = 0.019, n = 4), and increased in FA tissues by 3.03to 1.89-fold (P = 0.008, n = 4, Figure 1b).

PNA-antimiR-21 inhibits tumor growth in vivo
To address the potential effects of PNA-antimiR-21 in vivo on the growth of BC cells, equal numbers (3 × 10 7 )

Identification of potential miR-21 targets
It is known that animal miRNAs regulate gene expression by inhibiting translation and/or by inducing degradation of target. In our study, most modulated genes in the mRNA differential expression profiles changed by less than two-fold. Since mRNA levels regulated by less than two-fold may still be miRNA targets, we defined differentially expressed genes as no less than 1.3-fold change [31]. Comparative analysis of LNA-antimiR-21  Table 2).
To further screen potential direct targets, we compared the 27 candidate mRNAs with miR-21 targets predicted by TargetScan 5.1, miRBase Targets V.5,  (Table 2). Because miR-21 targets are expected to up-regulated for the LNA-antimiR-21 samples, ANKRD46 and EIF4A2, the two up-regulated genes upon miR-21 knockdown in the two cell lines and predicted by target prediction programs, were selected for further investigation. We further tested whether miR-21 could directly repress the identified mRNA targets through 3' UTR interactions (Figure 5a). Thus, the full-length 3' UTRs of the human genes ANKRD46 and EIF4A2 were cloned into the downstream of the luciferase gene (pMIR-REPORT), respectively. These vectors were then used to assess whether miR-21 could repress luciferase activity in 293T cells. ANKRD46 3' UTR showed a reduction to 54.8% of total luciferase reporter activity, in presence of miR-21, but EIF4A2 3' UTR did not display significant reduction of luciferase levels, compared with the miRcontrol (Figure 5b). These results suggest that miR-21 directly targets ANKRD46 in BC cells.

miR-21 and EIF4A2 proteins are inversely expressed in resected patient tumors in vivo
We examined ANKRD46 [NCBI: NP940683] and EIF4A2 [NCBI: NP001958] protein levels by IHC on TMAs constructed by the BC cases described in Materials and Methods. EIF4A2 was found in the cytoplasm, and ANKRD46 was seen in both the cytoplasm and nucleus (Figure 6a). ANKRD46 low expression was found in 47.5% (staining score < 4; median = 4); EIF4A2 low expression was found in 21.2% (staining score < 7; median = 7) of the 99 BC cases. Next, we investigated the negative regulation of endogenous EIF4A2 and ANKRD46 protein by endogenous miR-21 in vivo. In 99 successfully tested cases out of 113, endogenous miR-21 and EIF4A2 protein levels were inversely expressed in resected patient tumors (r s = -0.283, n = 99, P = 0.005, Spearman's correlation analysis). However, no significant association between miR-21 and ANKRD46 (P = 0.181, Spearman's correlation analysis) was observed. Fold-regulation of mRNAs affected by miR-21 knockdown compared with control-transfected cells (MCF-7 and MDA-MB-231) at indicated time points. Foldchanges for all mRNAs are derived from a single microarray experiment, with two relevant genes confirmed by qRT-PCR ( Figure 4B). Shown are mRNAs changing by at least 1.3-fold in both cell lines. Genes in bold were selected for candidate gene analyses by qRT-PCR.; # marginal signal intensity; NCBI, National Center for Biotechnology Information; GB, GenBank.

Correlation of ANKRD46 and EIF4A2 expression with BC clinicopathological features and prognosis
Using Pearson's Chi-Square test, we found that, in BC tissues, IF4A2 protein correlated with CerbB2 status (P = 0.019), and ANKRD46 protein correlated with ER (P = 0.021) and PR (P = 0.001, Table 3). No significant correlation was observed between EIF4A2, ANKRD46, or other parameters. The five-year overall survival rate of the 99 BC patients was 59.60% (Figure 6b). The fiveyear survival rate in patients with high EIF4A2 protein level was 64.10% (n = 78), significantly higher than those with a low EIF4A2 protein level (42.86%, n = 21; P = 0.044, log-rank test; Figure 6b). The five-year survival rate in patients with high ANKRD46 protein was 53.85% (n = 52), which was not statistically different from those with low ANKRD46 protein (65.96%, n = 47; P = 0.146, log-rank test; Figure 6b).

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-21treated 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 upregulation 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 2scale 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/PNAcontrol 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, Figure 5 ANKRD46 is a direct target of miR-21 in BC cell lines. (a) Predicted alignment of miR-21 with the target site derived from ANKRD46 and EIF4A2 3' UTR, determined with the software miRBase Targets V5 and miRNAMap, respectively. Note the seed matches at the 5' end of miR-21 (grey boxes) and the mutated nucleotides (underlined). (b) Luciferase assays show that miR-21 directly repress ANKRD46 mRNAs through 3' UTR interactions. Part of the 3' UTRs of wild-type ANKRD46 (478-bp length) and EIF4A2 (194-bp length), or the mutations were cloned into pMIR-REPORT vector (Applied Biosystems), downstream of luciferase. These vectors were then cotransfected with synthetic miR-21 (pre-miR-21) or miR-control in 293T cells, and luciferase activity was quantified. The graph shows the percentage of remaining luciferase activity calculated by normalizing the miR-21 expression values on the miR-control values. (c) Western blot to assay ANKRD46 and EIF4A2 after miR-21 knockdown, with GAPDH as equal loading control, followed by densitometric analysis. Cells were treated and harvested as described in Figure 4b. Data in (b) and (c) are mean + SD (n = 3). * P < 0.05. WT, wild-type; Mut, mutant.
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.

Conclusions
We demonstrate that MCF-7 and MDA-MB-231 cells transfected with anti-miR-21 show growth inhibition in vitro and in vivo, as well as cell migration in vitro. In addition, ANKRD46 is newly identified as a direct target of miR-21 in BC. These results suggest that miR-21 inhibitory strategies using PNA-antimiR-21 may have potential for therapeutic applications in BC treatment.

Additional material
Additional file 1: Word document containing the protocol of LNAbased CISH and FISH for miRNA in the present study.  *Two-sided Pearson Chi-Square Test; ** there were no stage IV patients because all the cases selected were surgical patients without neoadjuvant therapy to avoid the effect of the preoperative radiotherapy and/or chemotherapy on miRNAs; EIF4A2, eukaryotic translation initiation factor 4A2 protein; ANKRD46, ankyrin repeat domain 46 protein; ER, estrogen receptor; PR, progesterone receptor; CerbB2, v-erb-b2 erythroblastic leukaemia viral oncogene homolog 2 receptors.