Abstract

   miRNAs may play effective roles in breast cancer so modulating their
   expression levels could have therapeutic benefits. Recent studies have
   found the combination of miRNA-based therapeutics with conventional
   drugs as promising. This study aimed to find drug-responsive miRNAs,
   and explore their anticancer activities in HER2+ breast cancer cells
   and regulatory role in the trastuzumab response. qRT-PCR-array analysis
   was performed with effective concentrations of tamoxifen and
   trastuzumab treated BT-474, SK-BR-3 and MCF-7 cells. Motility and
   invasion analyses were performed with wound healing and xCELLigence
   impedance-based assays respectively. Viability of cells following mimic
   transfection and drug treatment was assessed by WST-1 assay. Western
   blot analysis was used to assess miR-770-5p regulation of proteins and
   their phosphorylated forms. The clinical relevance of miR-770-5p was
   examined by TCGA data analysis. The qRT-PCR-array results indicated
   that miR-770-5p was responsive in a drug and cell line independent
   manner. Overexpression of miR-770-5p inhibited the motility and cell
   invasion through regulation of AKT and ERK proteins. Additionally,
   miR-770-5p potentiated the effectiveness of trastuzumab. Thus,
   regulating the expression level of miR-770-5p in combination with
   trastuzumab treatment may simultaneously inhibit the downstream
   elements of PI3K and MAPK signalling, thereby blocking the
   proliferation, motility and invasion capacities of HER2+ breast cancer
   cells.

Introduction

   Breast cancer is the most common malignancy in women, constituting
   approximately 30% of all cancer types [[28]1]. Breast cancer is a
   heterogeneous disease with complex clinical behavior and responses to
   therapeutic intervention [[29]2,[30]3]. It is classified based on gene
   expression profiling, including HER2 positive (HER2+), luminal A or B,
   basal-like and presence of hormone receptors [[31]4]. Approximately 70%
   of human breast cancers are estrogen receptor alpha positive (ER+), so
   anti-estrogen therapy is an effective treatment [[32]5]. Tamoxifen
   citrate (TAM), which competes with the estrogen that binds to the
   estrogen receptor (ER), was the first selective estrogen receptor
   modulator (SERM) to be developed [[33]6]. Tamoxifen has been used
   clinically for over 30 years as a partial agonist of ER to reduce the
   risk of recurrence and contralateral neoplasia in breast cancer
   treatment. However, the development of resistance to this drug is
   inevitable because of molecular crosstalk mechanisms in the tumor cells
   [[34]7,[35]8]. Additionally, HER2+ tumors, which constitute 25% of
   breast cancers, are also known to show resistance to tamoxifen and
   standard chemotherapeutic approaches [[36]8–[37]10]. Trastuzumab
   (Herceptin) is a FDA-approved recombinant humanized monoclonal antibody
   developed against the extracellular domain of the HER2 protein, which
   is currently used as a therapy for HER2-overexpressing breast cancer
   patients [[38]11–[39]14]. Elucidation of the molecular mechanism of
   trastuzumab treatment is therefore important as it may contribute to
   determining the resistance mechanisms of tumor cells to this drug.

   MicroRNAs (miRNA), which are 20–25 nucleotides long, non-coding RNAs,
   are endogenous RNA molecules that are evolutionarily conserved and
   repress gene expression post-transcriptionally. These regulatory
   molecules play important roles in various cellular processes, such as
   differentation, cell growth and apoptosis. Since these processes are
   generally dysregulated in cancer, the relationship between miRNAs and
   cancer is quite important and solid [[40]15]. miRNAs are deregulated in
   breast cancer and various types of other human cancers [[41]11,[42]15].
   Since miRNAs may play effective roles in disease progression, they
   represent potential therapeutic targets for cancer as well. Modulating
   miRNA expression levels could provide effective diseases therapies
   [[43]16,[44]17].

   miRNAs play regulatory roles in breast cancer progression and have the
   potential to reverse resistance to drugs like tamoxifen
   [[45]18–[46]20]. A few studies have investigated the relationship
   between drugs and miRNAs. One recently showed that miR-210 levels in
   plasma might be associated with trastuzumab resistance in patients
   [[47]13]. Others found an effect of trastuzumab on the expression of
   miRNAs. However, these studies only focused on the oncogenic and tumor
   suppresor functions of individual miRNAs in trastuzumab sensitive or
   resistant cell lines [[48]14–[49]19] failing to explain the complexity
   of miRNA-mediated drug mechanisms.

   In this study, we determined the expression profiles of miRNAs in
   tamoxifen and trastuzumab-sensitive breast cancer cell lines by
   qRT-PCR-array analysis to explain the common molecular mechanisms of
   these two drugs. Among the differentially expressed miRNAs, only one
   common miRNA, miR-770-5p, was responsive in a drug and cell line
   independent manner. Bioinformatics analysis, together with the
   experimental results, indicated that HER2 signaling was one of the
   targets of miR-770-5p. We showed that overexpression of miR-770-5p
   potentiated the effect of trastuzumab, especially in BT-474 cells. When
   miR-770-5p was overexpreesed in the presence of trastuzumab, there was
   downregulation in the total or phoshorylated levels of AKT and ERK.
   This downregulation of the major regulator proteins of PI3K and MAPK
   signaling may explain the potentiation mechanism of miR-770-5p in HER2+
   cells.

Compliance with ethical standards

   Ethical approval: This article does not contain any studies with human
   participants or animals performed by any of the authors.

Materials and methods

Cell lines and culture

   Three human breast cancer cell lines (BT-474, SK-BR-3, MCF-7) were
   obtained from the American Type Culture Collection and maintained in
   DMEM or McCoy medium supplemented with 10% FCS. All cell lines were
   cultured in humidified air supplemented with 5% CO[2] at 37°C. The
   molecular characteristics of these cell lines are summarized in
   [50]Table 1.

Table 1. Molecular classification of human breast cancer cell lines.

   Cell lines Source Subtype   Immunoprofile
   BT-474            Luminal B ER^+, PR^+, HER2^+
   SK-BR-3           HER2      ER^-, PR^-, HER2^+
   MCF-7             Luminal A ER^+, PR^+, HER2^-
   [51]Open in a new tab

Tamoxifen or trastuzumab treatment and cell proliferation analysis

   Tamoxifen (Cat. No: 54965-24-1), purchased from Tocris (Minneapolis,
   MN, USA), was dissolved in ethanol as a 100 μmol/ml stock solution.
   Trastuzumab (Herceptin) was obtained from Roche (Basel, Switzerland).

   For the drug sensitivity test, two experimental designs were applied.
   First, MCF-7 and BT-474 cells were seeded at densities of 3×10^3 and
   6×10^3 cells/ml respectively and cultured in 8 different concentrations
   of tamoxifen (100, 80, 40, 20, 10, 5, 2, 1 μmol/ml) for 3 days. Control
   cells were treated with the same concentrations of ethanol in the
   culture medium. For trastuzumab treatment, SK-BR-3 and BT-474 cells
   were plated in 96-well microtiter plates at a concentration of
   6×10^3/well, and cultured at 37°C in a humidified atmosphere with 5%
   CO[2] overnight before treatments. The cells were incubated with
   decreasing trastuzumab concentrations (300, 60, 30, 6, 2, 0.5 and 0.1
   μg/ml) for 3 days. Control groups received PBS at a concentration equal
   to that in the drug-treated cells.

   Cell proliferation was measured using WST1 assay. Briefly, the cells
   were plated in 96-well plates at 8× 10^3 per well in a final volume of
   100 μl, before exposure to trastuzumab or tamoxifen at the
   aforementioned concentrations for 3 days. 10 ul of WST-1 reagent was
   added to each well on day 3. Incubation of the cells at 37°C for 3 h
   was followed by measuring absorbance at 480 nm on a Wallac Victor
   Counter (Perkin Ekmer, USA). The IC[50] value, which represents the
   drug concentration required for 50% growth inhibition, was calculated
   with Graphpad Prism version 6.04 software (California, CA, USA).

miRNA expression analysis by quantitative real-time PCR array

   For miRNA profiling, a SYBR green-based miScript miRNA PCR Array
   (MIHS-3216ZG) was used (Qiagen). Endogenous controls, normalization
   controls, miRNA reverse transcription and positive controls were also
   tested for each array. After treatment of the cells with the effective
   concentrations of the drugs, total RNA was isolated using QIAzol
   reagent (Qiagen) according to manufacturer’s instructions, reverse
   transcribed to cDNA and used to measure miRNA expression. The plates
   were run on a Roche Light Cycler 480 instrument to analyze the
   expression of miRNAs using the obtained Ct values. The specificity of
   the miRNA assays was confirmed from the melting curves of the PCR
   products.

Validation of real-time PCR array

   Mature miR-770-5p levels were quantified with Qiagen miRNA assays.
   Quantitative PCR was performed using SYBR mix (Qiagen) on a Roche Light
   Cycler 480. U6 snRNA (MS00033740) was used as the internal control
   (Qiagen). Relative expression levels were calculated using the 2^-ΔCT
   method while fold changes were calculated using the equation 2^-ΔΔCT.

Transfection with miRNA modulator

   4x10^5 cells were seeded in six-well plates and transfected with the
   effective concentration, 25 nM, of either the miRNA mimic (MSY0003948)
   or the negative scrambled control (scr, SI03650318) using HiPerFect
   Transfection Reagent (301705) (Qiagen, Germany). After an incubation
   period of 48 h or 72 h, cells were harvested using a cell scraper in
   ice-cold PBS. The RNA and protein were isolated for further qPCR and
   western blot analysis respectively.

   Viability of breast cancer cells after transfection of miR-770-5p miRNA
   mimic was measured by the same WST1 protocol.

   The synergistic effect of trastuzumab with miR-770-5p was also
   analyzed. For this purpose, cells were treated with miR-770-5p mimic or
   scrambled control RNA with varying doses of the drug for 48 h. After
   incubation, the same WST1 protocol was applied to the cells.

Wound healing assay

   For the wound healing assay, cells (5 × 10^5/well) were seeded into
   six-well plates and cultured under standard conditions. When the cells
   reached confluency, they were transfected with miR-770-5p mimic or the
   scrambled control. A wound was performed with a 20 uL tip on confluent
   BT-474, SK-BR-3 and MCF-7 cells (t[0]). We observed and photographed
   the cells with a microscope at 0, 24, 48 and 72 hours after
   transfection. Gap widths at t[0] and t[final] were measured and the
   average gap width ratio of miR-770-5p transfected cells was normalized
   to that of control cells using Tscracth [[52]21].

Invasion assay

   Cell invasion analysis was performed with xCELLigence real-time cell
   analyser. At 24h post-transfection with miRNA mimic, 4x10^4 BT-474 and
   SK-BR-3 cells in 200 uL of serum-free medium were seeded into the upper
   chamber of the ACEA Biosciences Inc. CIM-plate wells (Cat. No:
   2801038), fitted with a microporous membrane of matrigel separating the
   upper and lower chamber. The lower chamber was filled with culture
   medium supplemented with 10% FBS as a chemoattractant. Cell invasion
   was monitored for 24 hours with xCELLigence real-time cell analyser,
   using CIM-plate and measuring impedance-based signals.

Western blot analysis

   Cells in culture were lysed using Complete Lysis-M kit (Roche). The
   protein concentrations of the lysates were quantified using Coomassie
   Plus Protein Assay Reagent (Thermo Scientific). 10 ug of protein for
   each sample were loaded on to 8% SDS-PAGE gel. Separated proteins were
   transferred to a PVDF membrane (L-08008-001, Advansta) in wet transfer
   buffer. Membranes were blocked with 5% milk in TBST (0.5%) for 1 hour
   at room temperature before incubation at +4°C overnight with the
   following antibodies: HER2 (1:1000, ab8054, Abcam), total AKT (1:1000,
   sc8312, SantaCruz), phospho-AKT (Ser 473, 1:1000, sc-7985-R,
   SantaCruz), total ERK2 (1:1000, sc-154, SantaCruz), phosho-ERK 1/2 (Thr
   202 / Tyr 204, 1:1000, sc-81492, SantaCruz) and beta-actin (1:1000,
   634801, Biolegend) in 3% milk powder-TBST. After incubation with
   HRP-conjugated secondary antibodies, the protein bands were detected
   using WesternBright Sirius Kit (K-12043-D20, Advansta).

Target prediction and pathway analysis

   Predicted miRNA targets were retrieved from the mirWalk2.0 target
   prediction tool, which collects data from 12 different programs
   (mirWalk, miRDB, PITA, MicroT4, miRMap, RNA22, miRanda, miRNAMap,
   RNAhybrid, miRBridge, PICTAR2, TargetScan) [[53]22]. To assess the
   functional enrichment of the gene list, we used Webgestalt (WEB-based
   GEne SeT AnaLysis Toolkit) [[54]23].

Statistical analysis

   All the experiments were performed with a minimum of two biological and
   two technical replicates each. Student’s t-test was performed to test
   the differences, which were considered to be statistically significant
   at a p-value of less than 0.05 between two samples. The nonlinear
   regression (curve fit) method was used to analyze dose-response data
   for mimic transfection with drug only or drug plus miR-770-5p.

Results

Identification of tamoxifen or trastuzumab-responsive microRNAs

   The putative roles of miRNAs in tamoxifen or trastuzumab responses were
   investigated by miRNA qRT arrays to search for differentially-expressed
   miRNAs between three different breast cancer cell lines.

   The miRNA qRT array results showed that, for trastuzumab treatment, 53
   miRNAs were differentially expressed in BT-474 cells (3 upregulated, 50
   downregulated) while there were 101 DE miRNAs for SK-BR-3 cells (6
   upregulated, 95 downregulated) ([55]S1 Table). Those miRNAs that showed
   more than 1.5-fold difference in expression level at p<0.05 were
   selected as DE miRNAs. When the DE miRNA lists for the two cell lines
   were intersected, 64 miRNAs were found to be commonly responsive to
   trastuzumab, of which 62 downregulated and 2 upregulated ([56]Fig 1A).
   The array analysis results indicated that 65 and 68 miRNAs were found
   to be tamoxifen-responsive in MCF-7 and BT-474 cells respectively
   ([57]S1 Table). Among the responsive miRNAs, 19 were upregulated in
   MCF-7 cells while 47 were downregulated. For BT-474 cells, out of 68 DE
   miRNAs, 2 were upregulated and 66 were downregulated. DE miRNAs in
   tamoxifen-treated MCF-7 and BT-474 cells were compared with VENNY and
   17 common downregulated miRNAs and 1 common upregulated miRNAs were
   detected ([58]Fig 1A). When all the DE expressed lists were
   intersected, miR-770-5p was significantly responsive in a cell and
   drug-independent manner with consistent upregulation ([59]Fig 1B).

Fig 1. Trastuzumab and Tamoxifen responsive miRNAs were identified by qRT-PCR
Array.

   [60]Fig 1
   [61]Open in a new tab

   (a) HER2+ and ER+ BT-474 cells were treated with both tamoxifen and
   trastuzumab while ER+ MCF-7 cells and HER2+ SK-BR-3 cells were only
   tretated with tamoxifen and trastuzumab respectively. There was only
   one common responsive miRNA among upregulated miRNAs; 11 miRNAs were
   commonly downregulated in all cell lines. The common responsive miRNAs
   are listed in b.

Targets of miR-770-5p participate in cancer-related pathways

   Having determined that miR-770-5p was a common drug target in each cell
   line, we wanted to further characterize its potential molecular
   function. Through the use of the target prediction software mirWalk2.0
   [[62]20], we identified the predicted targets of miR-770-5p. Pathway
   enrichment anlysis results showed that the target genes enriched
   significantly in pathways related to cancer progression, such as ErbB,
   Insulin and MAPK signaling pathways ([63]Table 2).

Table 2. Pathway enrichment analysis results conducted with the targets of
miR-770-5p.

   KEGG Pathway Name         Number of Genes Statistics
   ErbB Signaling Pathway    47              P = 3.67e-18
   Calcium signaling pathway 71              P = 8.01e-18
   Insulin signaling pathway 64              P = 6.69e-20
   Focal adhesion            91              P = 2.84e-27
   MAPK signaling pathway    109             P = 2.27e-27
   [64]Open in a new tab

   Additionally, we assessed miR-770-5p expression level in breast tumor
   tissues in TCGA breast cancer BRCA (n = 1247) cohort using XenaBrowser
   [[65]24]. miR-770-5p expression was significantly downregulated in
   tumor samples compared to normal samples (f = 10.44, p< 0.00005621)
   ([66]Fig 2), which led us to upregulate the expression of miR-770-5p in
   tumor cells as a strategy to explore its molecular function in cancer
   cells.

Fig 2. Lower expression of miR-770-5p is associated with tumor samples.

   [67]Fig 2
   [68]Open in a new tab

   Expression level of miR-770-5p was downregulated in tumor samples
   compared to normal samples (One-way ANOVA; p<0.00005621, f = 10.44).

Motility and invasion are regulated by miR-770-5p

   To determine the effect of miR-770-5p overexpression on cell motility,
   wound-healing assays were performed after mimic transfection. The wound
   closure in the scratched was quantitated over 72 hours. miR-770-5p
   overexpression decreased the cells’ motility capacity compared to
   scrambled control-transfected cells. Wound closure was 100% in both
   BT-474 and SK-BR-3 cells when transfected with negative control.
   However, it reduced to 20% in both cell lines in a miR-770-5p
   overexpression-dependent manner ([69]Fig 3A). Additionally, monitoring
   with xCELLigence-based invasion assay revealed that miR-770-5p
   overexpression decreased the cells’ invasion kinetics ([70]Fig 3B).
   Assessment of the proliferative activity of the mimic-transfected cells
   showed that miR-770-5p did not affect cell proliferation by itself
   ([71]S1 Fig).

Fig 3. miR-770-5p regulates motility and invasion in BT-474 and SK-BR-3
cells.

   [72]Fig 3
   [73]Open in a new tab

   (a) The rate of motility was assessed by wound-healing assay. Wound
   closure was observed in scrambled control-transfected cells in a
   time-dependent manner while miR-770-5p mimic transfected cells lost
   their motility (n = 2, *p<0.005). (b) Cell invasion analysis was
   performed by xCELLigence real-time cell analyzer measuring
   impedance-based signals. Cell invasion capacity decreased in both of
   the cells transfected with miR-770-5p mimic compared to scrambled
   control-transfected cells (n = 2, **p<0.0001).

Upregulation of miR-770-5p changes cellular response to trastuzumab in HER2
(+) breast cancer cells

   The pathway enrichment analysis results suggested that miR-770-5p might
   function as a mediator of the HER2 signaling pathway ([74]Table 2).
   Furthermore, according to the mirWalk results, HER2 was predicted to be
   regulated by miR-770-5p. Supporting this prediction, we demonstrated
   that upregulation of miR-770-5p slightly decreased the expression level
   of HER2 protein in BT-474 cells ([75]Fig 4A). To clarify whether miRNA
   upregulation in cells by mimic transfection increases the
   growth-inhibitory effect of trastuzumab, the sensitivity of HER2
   overexpressing cells (BT474 and SKBR3) to trastuzumab treatment was
   analyzed by WST1 assay after miR-770-5p mimic transfection. This showed
   that the effect of trastuzumab increased with upregulation of miRNA in
   the cells by decreasing the percentage of living cells from 60% to 40%
   in BT-474 cells and from 60% to 50% in SK-BR-3 cells ([76]Fig 4B). In
   addition, HER2 protein levels decreased significantly in both cell
   lines when the cells were treated with a combination of trastuzumab and
   miR-770-5p. That is, miR-770-5p potentiated the effect of trastuzumab
   in HER2+ cells ([77]Fig 4C).

Fig 4. miR-770-5p overexpression downregulated HER2 and increased the effect
of trastuzumab.

   [78]Fig 4
   [79]Open in a new tab

   (a) The protein expression level of HER2 was slightly diminished in
   both cell lines following miR-770-5p transfection n = 3, p<0.05). (b)
   Combinational treatment of cells with trastuzumab and miR-770-5p
   decreased cell viability in BT-474 and SK-BR-3 cells (n = 3, *p<0.001,
   **p<0.05); HER2 expression also reduced significantly, especially in
   BT-474 cells (c) (**p<0.05).

miR-770-5p regulates cancer cell behavior by targeting several genes involved
in the HER2 signaling pathway

   From the bioinformatics analysis, HER2 was predicted to be the target
   of miR-770-5p and our protein results supported the regulative effect
   of miR-770-5p on HER2. To explore the role of miR-770-5p on the HER2
   signaling pathway, downstream elements were assesed in
   miR-770-5p-overexpressing BT-474 and SK-BR-3 cells. The total or
   phoshorylation states of AKT and ERK, which are major regulator
   proteins of PI3K and MAPK signaling respectively, were examined.

   The protein analysis results showed that total ERK expression level was
   downregulated in both cell lines through miR-770-5p overexpression
   alone compared to control cells, while total AKT level was only
   diminished significantly in SK-BR-3 cells ([80]Fig 5; t-test, p<0.05).
   Hence, the expression levels of total and phosphorylated forms of AKT
   and ERK proteins were analysed in BT-474 cells to determine the
   response to trastuzumab combined with miR-770-5p. In the presence of
   trastuzumab, total protein levels of both AKT and ERK were
   downregulated while p-AKT and p-ERK levels also decreased significantly
   ([81]Fig 6; t-test, p<0.05).

Fig 5. miR-770-5p targeted total ERK in BT-474 and SK-BR-3 cells.

   [82]Fig 5
   [83]Open in a new tab

   Protein expression of total AKT and ERK were detected in BT-474 and
   SK-BR-3 cells transfected with miR-770-5p mimics and scrambled control
   (scr). ERK expression decreased in BT-474 cells after mimic
   transfection (n = 4, *p<0.01). Both the ERK expression and the AKT
   expression, decreased significantly only in SK-BR-3 cells (n = 2,
   **p<0.05).

Fig 6. Trastuzumab treatment together with miR-770-5p modulates AKT and ERK
expression level.

   [84]Fig 6
   [85]Open in a new tab

   miR-770-5p mimic was transfected to BT-474 cells and; total and
   phoshorylated AKT and ERK protein levels were analyzed by Western blot
   72 h after transfection. Combination of trastuzumab with miR-770-5p
   mostly affected pERK and pAKT levels in BT-474 cells (n = 3, *p<0.05).

Discussion

   In this study, we identified a common miRNA response in cells
   expressing ER and HER2 to two different drugs, tamoxifen and
   trastuzumab. According to the qRT-PCR-array results, miR-770-5p was
   significantly responsive, independent of cell and drug type, with
   consistent overexpression. miR-770-5p has been linked to various
   cancers, such as gastric cardia adenocarcinoma [[86]25], ovarian cancer
   [[87]26], non-small cell lung cancer [[88]27] and hepatocellular
   carcinoma [[89]28]. It has also been shown to be downregulated in
   breast cancer. A recent study reported that miR-770-5p is downregulated
   in chemo-resistant triple negative breast cancer (TNBC) tissues while
   its ectopic expression antagonized resistance and metastases by
   targeting STMN1 [[90]29]. One of the deregulated miRNAs may be a
   potential biomarker to predict clinical outcomes in TNBCs recieving
   neoadjuvant chemotherapy [[91]30]. However, the role of miR-770-5p in
   ER+ and HER2+ breast cancer cells, and its response to tamoxifen and
   trastuzumab treatment has not previously been determined. Furthermore,
   our TCGA data analysis, which indicates its significant downregulation
   in breast tumors compared to normal breast tissues, supports its
   possible clinical importance and led us to conduct further functional
   analysis.

   In the pathway enrichment analysis, targets of miR-770-5p were
   significantly enriched in focal adhesion, MAPK and ErbB signaling
   pathways, which are closely related with motility and invasion
   [[92]31]. In parellel with this bioinformatics analysis, the motility
   and invasion capacity of both HER2+ cells decreased with the
   overexpression of miR-770-5p. This decrease in motility and invasion
   may be down to ERK downregulation, which is one of the putative targets
   of miR-770-5p [[93]32,[94]33].

   Since HER2 has been identified as one of the targets of miR-770-5p, and
   pathway enrichment analysis conducted with putative targets of
   miR-770-5p indicated that ErbB signaling is one of the regulated
   pathways, we focused on this and a downregulation of HER2 protein was
   observed, especially in miR-770-5p-restored BT-474 cells. To follow the
   downstream signal through PI3K and MAPK signaling [[95]34], we examined
   the expression levels of two major regulator proteins, AKT and ERK.
   Although total ERK expression was downregulated in both cell lines,
   total AKT downregulation was only observed in SK-BR-3 cells. This can
   be explained by variable mutation profiles as well as the different
   receptor status of two cell lines. c.333G>C (p.K111N) mutation in
   PIK3CA gene in BT-474 cells is well defined, which could explain the
   stable expression level of AKT in miR-770-5p transfected cells
   [[96]35]. Finally, this stability could be due to the crosstalk
   mechanism between HER2 and growth factor receptors [[97]10,[98]36]. It
   was previously shown that complete response rates to trastuzumab-based
   treatment are lower in patients with the highest ER expression levels
   in HER2-positive breast cancers, like ER and HER-positive BT-474 cells,
   compared to ER-negative and HER2-positive SK-BR-3 cells [[99]37]. In
   support of this, when cells were transfected with miR-770-5p mimic in
   the presence of trastuzumab, we observed downregulation in total AKT
   and ERK expression as well as their phosphorylated forms compared to
   scrambled control-transfected cells in both cell lines. Multiple levels
   of crosstalk are present between the PI3K/Akt and MAPK signaling
   pathways, which can compensate for each other [[100]38,[101]39]. Hence,
   miR-770-5p may block this crosstalk in the presence of trastuzumab,
   thereby strengthening the antiproliferative effect of trastuzumab.

   Our qRT-PCR-array data show that miRNA expression levels in tumor cells
   can be potent in therapeutic response, and several preclinical studies
   have assessed the combination of miRNA-based therapeutics with
   chemotherapy [[102]40]. Baldasarri et al. demonstrated that a
   combination treatment with miRNAs in vitro, especially miR-126, miR-9,
   miR-181a and miR-326, magnify the activity of specific breast cancer
   drugs [[103]41]. In another study, restoring miR-375 sensitized cells
   to tamoxifen. This indicates it to be a potential target for
   treatment-resistant breast cancer [[104]19]. miR-218 targeted BRCA1 to
   sensitize breast cancer cells against cisplatin [[105]42] and
   miR-542-3p silencing restored trastuzumab resistance via PI3K-AKT
   pathway regulation in breast cancer cells [[106]43]. These data
   obtained from preclinical studies provide insights for using
   miRNA-based therapies to improve the effectiveness and potentiate the
   anticancer activity of drugs by regulating proliferation, motility or
   invasion [[107]44]. One strategy to improve treatment effectiveness in
   HER2-overexpressing cancers, particularly those that develop resistance
   to HER2-targeted therapies, could be combined inhibition of PI3K and
   MEK. In conclusion, we showed that miR-770-5p downregulated AKT and ERK
   through HER2 signaling and potentiated the activity of trastuzumab in
   BT-474 cells. Here, we could suggest that mediating miR-770-5p in
   combination with trastuzumab treatment could inhibit two important
   pathways simultaneously, thereby blocking the proliferation, motility
   and invasion capacities of HER2+ breast cancer cells ([108]S2 Fig).

   A combination of miR-770-5p with thyrosine kinase inhibitors could also
   make breast cancer treatments more effective. However, preclinical
   animal models are needed to confirm the effectiveness of these combined
   therapies in vivo.

Supporting information

   S1 Table. Differentially expressed miRNA lists.

   (XLSX)
   [109]Click here for additional data file.^ (26.2KB, xlsx)
   S1 Fig. Viability of the cells after miR-770-5p transfection.

   Although the viability of the cells decreased significantly in
   miR-770-5p mimic-transfected BT-474 and SK-BR-3 cells compared to
   scrambled control-transfected cells, the total viability of the cells
   diminished only 20% and 10% for BT-474 and SK-BR-3 cells respectively
   (n = 2, *p<0.02).

   (TIF)
   [110]Click here for additional data file.^ (746.4KB, tif)
   S2 Fig. Proposed action mechanism of miR-770-5p in HER2 signaling.

   miR-770-5p is shown to regulate HER2 signaling by targeting HER2, AKT
   and ERK. Introducing miR-770-5p may reduce the expression of HER2 and
   in the presence of trastuzumab it may downregulate AKT and ERK that
   potentiate the activity of trastuzumab.

   (TIF)
   [111]Click here for additional data file.^ (88.5KB, TIF)
   S3 Fig. Western blot images.

   Original uncropped blots of Figs [112]4, [113]5 and [114]6.

   (PDF)
   [115]Click here for additional data file.^ (207.1KB, pdf)

Acknowledgments