miR-125b targets erythropoietin and its receptor and their expression correlates with metastatic potential and ERBB2/HER2 expression

To identify possible targets of human miR-125b, we transfected MCF7 breast cancer cell line with a miR-125b mimic oligonucleotide (Ambion) or a negative control oligonucleotide (#2, Ambion) and we collected total RNA at 24 and 48 hours after transfection. Global gene expression changes were examined using microarray technology (Agilent Whole Human Genome microarray). We applied Sylamer algorithm [28], that searches for enriched sequences in 3′UTR of genes, to the list of genes down-modulated by miR-125b both at 24 and 48 hours after transfection. As expected, miR-125b seed sequence (7- 8-mer) was significantly enriched in down-regulated transcripts (Additional file 1: Figure S1). We found a list of 315 genes (Additional file 2: Table S1) whose 3′UTR contains at least 1 region complementary to the seed sequence of miR-125b (7-mers (C)TCAGGG(A) and 8-mer CTCAGGGA) and whose expression is reduced after transfection, thereby supporting a direct role of the miRNA in causing mRNA down-regulation.

We analyzed the list of genes down-regulated after miR-125b transfection and containing a potential miRNA response element (MRE) with Metacore (GeneGO) software and we found the significantly enriched pathways (Table 1). Among them, there are two pathways concerning Erythopoietin (EPO) signaling and the Metacore network analysis revealed that EPO signaling is deeply influenced by miRNA transfection (Figure 1). Indeed, both EPO and EPzOR (Erythopoietin receptor) are putative targets of miR-125b (Additional file 3: Figure S2) and their downregulation after miR-125b transfection was validated by quantitative RT-PCR (RT-qPCR), both at 24 and 48 hours (Additional file 4: Figure S3).

Transfection experiments were performed in MCF7 and HEK-293 cell lines to validate EPO and EPOR as targets of miR-125b. EPO and EPOR 3′UTRs were cloned downstream Renilla luciferase gene in psiCHECK-2 reporter vector. This vector contains also Firefly luciferase gene that can be used as a reference in Dual-Luciferase Reporter Assay (Promega). These vectors were transfected in two different cell lines together with a vector expressing miR-125b (pIRESneo2- miR125b), while the empty pIRESneo2 vector, pIRES-neo2 vector expressing miR-145 (that does not target EPO/EPOR 3′UTRs) and the mutated version of psiCHECK-2 + 3′UTR plasmids were used as negative controls. The luciferase activity of the constructs containing EPO and EPOR-3′UTR was suppressed of ~30% (p < .05) following ectopic expression of miR-125b (Figure 2) demonstrating that EPO/EPOR expression is under the control of miR-125b.

We validated the opposite modulation of miR-125b, EPO and EPOR by RT-qPCR in a cohort of 42 breast cancer patients and 13 normal samples collected at the University of Ferrara (Figure 3A-C). We found a significant down-regulation of miR-125b and an up-regulation of EPO and EPOR genes (p = 0.0003, 0.03 and 0.002, respectively; unpaired t-test with Welch’s correction). Likewise, a significant inverse correlation between miR-125b and EPO (p = 0.0037) and EPOR (p = 0.03) genes was found (Spearman correlation, r = -039; -0.31, Figure 3D). These observations confirm the role of miR-125b in controlling the up-regulation of Erythropoietin and Erythropoietin receptor in breast cancer.

To evaluate the significance of miR-125b, EPO and EPOR expression in breast cancer, we examined their expression levels in breast cancer patients, grouped according to the following clinical-pathological variables: metastatic recurrence, p53 mutation, grade, stage, lymphnode invasion, proliferative index, estrogen and progesterone receptors positivity, ERBB2 overexpression. We found a significant down-regulation of miR-125b levels in metastatic breast cancers (Figure 4A) compared to not metastatic tumors (p = 0.014, two-tailed Mann Whitney test). A reduced expression of miR-125b can be observed also in locally recurrent breast cancers, although only four samples were available. EPO and EPOR levels were only slightly increased in metastatic cancers without reaching significance (data not shown).

ERBB2 (HER2) gene is a validated target of miR-125b [20]. To confirm the regulation of ERBB2 by miR-125b we examined the miRNA levels in breast cancers strongly positive (positive cells > 50%) intermediately positive (positive cells = 20-50%) and weakly positive or negative (positive cells < 20%) for ERBB2 (Figure 4B). As expected, miR-125b expression is significantly reduced in strongly positive breast cancer (p = 0.04, two-tailed Mann Whitney test). A significant inverse correlation was found between miR-125b expression levels and ERBB2 protein levels as well (Spearman correlation, r = -031; p = 0.04; Figure 4C).

Since an extensive co-expression of ERBB2 and EPOR has been described in breast cancer [29] and ERBB2 protein levels correlates with that of ERBB2 mRNA [30, 31], we evaluated the relationship between ERBB2 and EPOR using gene expression data from 69 breast cancers with low and intermediate levels of ERBB2 (data from proprietary microarray experiments, average or two probes for each gene). Normalized expression data are available in Additional file 5: Table S2. We found a significant positive correlation between EPOR and ERBB2 expression levels (Spearman correlation, r = 0.54; two-tailed p-value < 0.0001; Figure 4D). This result confirms the data obtained from qPCR and IHC data that were previously described.

No significant association was found between miRNA/gene expression levels and grade, stage, lymphnode invasion, proliferative index, estrogen and progesterone receptors positivity.

We hypothesized that ERBB2 and EPOR could act as competing endogenous RNAs (ceRNAs), influencing their respective levels using miR-125b response elements present in their 3′ UTR. Since ERBB2 is frequently amplified or overexpressed in breast cancer [32], we used its 3′UTR as a driver for EPOR levels through miR-125b decoy. We selected two breast cancer cell lines (MDA-MB-453 and MDA-MB-157) characterized by low-intermediate levels of endogenous ERBB2 (Additional file 6: Figure S4), moderate-high EPOR expression [29] and low levels of endogenous miR-125b (if compared to normal breast, data not shown). We transfected both cell lines with pIRES-ERBB2-3UTR plasmid that express ERBB2 3′UTR. We used the empty plasmid as well as not-transfected cells as controls. As represented in Figure 5, we observed a significant increase of EPOR levels (two-tailed unpaired t-test) 24 hours after the transfection in both cell lines, using RT-qPCR assay.

Breast cancer and normal breast tissues were anonymously collected at the University of Ferrara (Italy) and extensively characterized for clinical and bio-pathologic status by two expert pathologists (P.Q. and M.P.). Clinico-pathological information concerning metastasis development, ERBB2/HER2 expression, p53 mutation, grade, stage, lymphnode invasion, proliferative index, estrogen and progesterone receptors positivity was available for all tumor samples and determined as previously described [1]. Total RNA isolation was performed with Trizol (Invitrogen) according to the manufacturer’s instructions.

MDA-MB-453, MDA-MB-157, MCF7 and HEK-293 cell lines were obtained from the ATCC and were cultured with ISCOVE’s Modified Dulbecco’s Medium (Lonza BioWhittaker) with 10% fetal bovine serum (FBS) and gentamicin. For microarray experiments, MCF7 cell line was transfected with 100 nM of miR-125b (Ambion) or Negative Control#2 (Ambion); total RNA was extracted at 24hs and 48 hs after transfection by adding Trizol Reagent (Invitrogen) according to manufacturer’s procedure. The day before transfection cells were seeded in antibiotic-free media. Transfection of miRNAs was carried out using Lipofectamine 2000 (Life Technologies) in accordance with manufacturer’s procedure.

The human EPO and EPOR 3′-UTR target sites were amplified by PCR using the following primers: EPO-3UTR_F: 5′- ATCTCGAGCTCCCTCACCAACATTGCTT-3′; EPO-3UTR_R: 5′- ATGTTTAAACGTCTTCATGGTTCCCACCAC-3′; EPOR-3UTR_F: 5′- ATCTCGAGCCAGCTATGTGGCTTGCTCT-3′; EPOR-3UTR_R: 5′- ATGTTTAAACACTGCAAGGTTGTGGTTTCC-3′ and cloned downstream of the Renilla luciferase gene in psiCHECK-2 vector (Promega). Mutated 3′UTRs, through the deletion of the miR-125b seeds inside EPO and EPOR 3′UTRs, were generated using gBlocks Gene Fragments (IDT). These vectors - psiCHECK2-EPO, psiCHECK2-EPOR and their mutated versions - were used for transfection into MCF7 and HEK-293 cells. Vector expressing miR-125b and miR-145 (used as negative control) were obtained after cloning miR-125b and miR-145 genomic sequences in pIRESneo2 expression vector (Clontech). The level of miR-125b expression in transfected cells was assayed by RT-qPCR (TaqMan MicroRNA Assays, Life Technologies) 24hs and 48 hs after transfection (data not shown). The 3′-UTR of ERBB2 was amplified by PCR from the cDNA of T47D and SK-BR3 cell lines (HER2 positive) and cloned into XbaI and EcoRI sites of pIRESneo2 expression plasmid.

MCF7 and HEK-293 cell lines were seeded in 24-well plates at a cellular concentration of 40,000 and 100,000 cells/well respectively. Cells were transfected with 400 ngs of psiCHECK2-EPO or equimolar amounts of psiCHECK2-EPOR or mutant control vectors together with equimolar amounts of pIRESneo2-miR125b or pIRESneo2-control (miR-145). Transfection was performed using Lipofectamine 2000 and OPTI-MEM I Reduced Serum Medium (GIBCO). Twenty-four hours after transfection Firefly and Renilla luciferase activity were measured using the Dual-Luciferase Reporter Assay (Promega). Each transfection was repeated in triplicate.

MDA-MB-453 (7x10⁵/well) and MDA-MB-157 (2x10⁵/well) were seeded in 6-well plates in antibiotic-free media. The following day, they were transfected with 2.5 μg of pIRESneo2 or pIRES-ERBB2-3UTR using Lipofectamine LTX (Life Technologies) and OPTI-MEM I Reduced Serum Medium according to the manufacturer’s recommendations. 24 hours later, cells were collected using Trizol Reagent for RNA extraction. The primers used for PCR amplification of ERBB2 3′UTR were ERBB2-3UTR_F: 5′-CAGAATTCTGCCAGTGTGAACCAGAAG-3′ ERBB2-3UTR_R: 5′-CATCTAGAGACAAAGTGGGTGTGGAGAA-3′.

Mature miRNAs expression was evaluated by Taqman miRNA assays (Applied Biosystem) specific for miR-125b (miR-125b-5b in miRBase release 19) and RNU6B as reference gene according to the manufacturer’s protocol. Briefly, 5 ng of total RNA was reverse transcribed using the specific looped primer; reverse transcription quantitative PCR was conducted using the standard Taqman miRNA assay protocol on a Biorad-Chromo4 thermal cycler. EPO (assay ID Hs01071097_m1) and EPOR (assay ID Hs00959427_m1) gene expression was assessed by RT-qPCR using Taqman gene expression assays (Applied Biosystem) according to manufacturer’s protocol. 18S was used as reference gene and its expression was assessed using the following primers F: 5′- CTGCCCTATCAACTTTCGATGGTAG-3′; R: 5′- CCGTTTCTCAGGCTCCCTCTC-3′ and KAPA SYBR FAST master mix (Kapa Biosystems). Each sample was analyzed in triplicate. The level of each miRNA/gene was measured using Cq (threshold cycle). The amount of target, normalized on reference gene, was calculated using 2^-ΔCq (Comparative Cq) method. Graphs and statistical analyses were performed using GraphPad Prism 5 software.

Four samples derived from MCF7 transfection with miR-125b at 24 and 48 hrs were used for microarray analysis. Global gene expression was detected using Agilent Whole Human Genome microarray (#G4112F, Agilent Technologies), which represent 41,000 unique human transcripts. About 500 ngs of total RNA were employed in each experiment. RNA labeling and hybridization were performed in accordance to manufacturer’s indications. Agilent scanner and the Feature Extraction software v.10.5 (Agilent Technologies) were used to obtain the microarray raw-data. Raw data have been submitted to ArrayExpress with the following accession number: E-MTAB-1604. Microarray results were analysed using the GeneSpring GX 12 software (Agilent Technologies, Palo Alto, CA). Data transformation was applied to set all negative raw values at 1.0, followed by a normalization on 75^th percentile. A filter on low gene expression was used to keep only the probes expressed in at least one sample. Genes were ordered from the most down-regulated at 24 and 48hrs compared to controls to the most up-regulated and used for sylamer analysis.

Functional Ontology Enrichment analysis and Pathway map visualization was performed using MetaCore pathway analysis by GeneGo (GeneGo Inc.).

Sylamer analysis was performed through the web-interface Sylarray (http://​www.​ebi.​ac.​uk/​enright-srv/​sylarray) to detect miR-125b target sequence in 3′ untranslated regions (3′UTR) from a ranked gene list, sorted from downregulated to upregulated in MCF7 + miR-125b at 24 and 48 hours compared to negative controls, as described in [28]. A significant enrichment and/or depletion of 7–8 nts sequences complementary to the miRNAs seeds in the 3′UTR of ordered mRNAs was calculated using hypergeometric statistic.

Statistical analyses were performed using GraphPad Prism 5 software. Two-tailed Mann Whitney and unpaired t-test, with or without Welch’s correction, were used for statistical comparisons as specified in the text, according to data distribution. Correlations were calculated using log2 transformed data and Spearman r correlation (two-tailed p-value).