A total of 52 BC tissue samples were collected from 52 patients (52 females; 43-68 years old; mean age 55.6±12.5 years) histologically diagnosed with BC between 2015 and 2017 at the Second Affiliated Hospital of Xi’an Medical University (Xi’an, China). In addition, 52 normal tissue samples (52 females; 44-74 years old; mean age 50.2±10.4 years) were obtained from the same hospital. All protocols were approved by the Ethics Committee of the Second Affiliated Hospital of Xi’an Medical University and written informed consent was provided by all participants. The clinical characteristics of the patients with BC are summarized in Table I.

The MCF-7 and MDA-MB-231 BC cells, in addition to the normal Hs578bst cell line, were purchased from the Cell Resource Center of the Shanghai Academy of Sciences (Shanghai, China). The cells were cultured in an atmosphere containing 5% CO₂ at 37°C in DMEM supplemented with 10% FBS (both from Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin and 100 mg/ml streptomycin.

Total RNA was extracted from the BC tissues, MCF-7 cells and MDA-MB-231 cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and was quantified using a photometer. cDNA was synthesized using M-MLV reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. All PCR primers were designed and synthesized by Invitrogen; Thermo Fisher Scientific, Inc. qPCR analysis was performed according to the manufacturer’s protocol. The reaction mixture comprised the following: cDNA (0.5 µl), forward primer (0.5 µl), reverse primer (0.5 µl), 2.5 mM dNTPs (2 µl), 10 U/µl DNA polymerase (0.5 µl), 5X buffer (4 µl) and ddH₂O (12 µl). The thermocycling conditions were as follows: 35 cycles at 95°C for 5 min, 95°C for 15 sec and 56°C for 40 sec. U6 was used as an internal reference. The primers used were as follows: miR-421, forward, 5′-ACA CTC CAG CTG GGA TCA ACA GAC ATT AAT TG-3′ and reverse, 5′-TGG TGT CGT GGA GTC G-3′; U6, forward, 5′-CTC GCT TCG GCA GGA CA-3′ and reverse, 5′-AAC GCT TCA CGA ATT TGC GT-3′; PDCD4, forward, 5′-TCT GGG AAA GGA AGG GGA CTA C-3′ and reverse, 5′-TTC ATA AAC ACA GTT CTC CTG GTC AT-3′; U6 forward, 5′-CTC GCT TCG GCA GCA CA-3′ and reverse, 5′-AAC GCT TCA CGA ATT TGC GT-3′. The results were quantified using the 2^−ΔΔCq method (24).

Tissues slides (1.2×1.2 cm) were incubated with primary PDCD4 antibody (dilution 1:600; cat. no. PA5-20309; Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 30 min and were washed twice with cold PBS. Subsequently, the slides were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) secondary antibody (dilution 1:100; cat. no. 31460; Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min at room temperature. The slides were developed with 3,3′-diaminobenzidine and the nuclei were lightly stained with hematoxylin. The expression of PDCD4 was evaluated by observing at least five fields of the slides under an inverted non-confocal microscope (CKX53 type, 4000K LED light; Olympus Corporation, Tokyo, Japan), with at least 100 cells per field assessed.

miR-421 mimics, miR-421 inhibitors, mock miRNAs and the luciferase reporter plasmid were designed and synthesized by Invitrogen; Thermo Fisher Scientific, Inc. The MCF-7 and MDA-MB-231 cells were transfected with miR-421 mimics, miR-421 inhibitors or mock miRNAs using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol.

The cells were lysed and proteins were quantified using a BCA assay. The proteins (25 µg/lane) were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were incubated with primary antibodies against PDCD4 (dilution 1:600; cat. no. PA5-20309; Invitrogen; Thermo Fisher Scientific, Inc.) and GAPDH (dilution 1:800; cat. no. MA5-15738; Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 30 min. The membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) secondary antibody. The band intensities were evaluated using ImageJ 1.47 software (National Institutes of Health, Bethesda, MD, USA).

A search for putative targets of miR-421 was performed with TargetScan Human7.2 software. Target genes of miR-421 were predicted by TargetScan (http://www.targetscan.org/vert_71/). To determine whether PDCD4 is a direct target gene of miR-421, luciferase reported assays were performed. The luciferase reporter plasmids pGL3-PDCD4 3′UTR wild-type (WT) and pGL3-PDCD4 3′UTR mutant type (Mut) were designed and synthesized by Invitrogen; Thermo Fisher Scientific, Inc. The MCF-7 and MDA-MB-231 cells were seeded in 24-well plates and cultured until they reached 40% confluence. The cells were then transfected with miR-421 mimics, inhibitors or mock miRNA using Lipofectamine^® 2000 according to the manufacturer’s protocol. After 24 h incubation at 37°C, the cells were harvested and luciferase activity was assessed using the Dual-Luciferase Reporter Assay system (Promega Corporation, Madison, WI, USA). Each experiment was performed in triplicate.

Following transfection, the proliferation of the MCF-7, MDA-MB-231 and Hs578bst cells was determined using Cell Counting Kit-8 (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer’s protocol. For each experimental group, six duplicate wells were assessed.

The MCF-7 and MDA-MB-231 cells were cultured and treated as described above, following which cell migration and invasion were assessed using Transwell assays. The filters (Corning Incorporated, Corning, NY, USA) were washed in serum-free DMEM and placed into 24-well plates. The lower Transwell chamber contained DMEM supplemented with 10% FBS. A total of 3×10⁴ BC cells were seeded in the upper chamber with 200 µl DMEM supplemented with 0.1% bovine serum albumin (BD Biosciences, Franklin Lakes, NJ, USA), with a 2 mg/ml Matrigel-coated membrane containing 8-m pores. The Transwell chamber was subsequently incubated at 37°C in an atmosphere containing 5% CO₂ for 24 h. Following incubation, any cells remaining on the upper membrane surface were removed with a cotton swab, whereas cells on the lower surface of the membrane were fixed in 10% formalin at room temperature for 30 min and stained with 0.5% crystal violet. Images were captured of six randomly selected fields of view using an inverted microscope (Nikon Corporation, Otsu, Japan) at x200 magnification. For the migration assay, transfected cells (2×10⁴ cells/chamber) were seeded in the top chamber, as above, without Matrigel. After 24 h, the migrated cells were lysed in glacial acetic acid and the solution was transferred to a 96-well culture plate. Colorimetry was performed at 560 nm to determine the optical density. Each experiment was performed in triplicate.

All results were analyzed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). χ² or Fisher’s exact test was used to compare categorical variables as appropriate. Student’s t-test, Mann-Whitney U test and one-way analysis of variance with Tukey’s post hoc test were used to compare continuous data. P<0.05 was considered to indicate a statistically significant difference.

To investigate the expression of miR-421 in patients with BC with different clinicopathological features, 52 patients with BC were divided into two groups (28 in the miR-421 high expression group and 24 in the low expression group). Patients’ characteristics are summarized in Table I. No significant differences were observed in clinicopathological factors, including estrogen receptor status, progesterone receptor status, human epidermal growth factor receptor-2 status, and Ki-67 status, between the miR-421 high expression group and miR-421 low expression group. However, an older age at diagnosis was associated with a high expression of miR-421 in patients with BC (P<0.001). The pathological stage, histological grade and lymph node status were also closely associated with the expression of miR-421 (P<0.001).

The protein expression of PDCD4 in tissues of patients with BC were analyzed by immunohistochemistry, although only 15 pairs of immunohistochemistry results are presented. PDCD4 protein expression was detected in three sets of five normal and five BC tissues in each set, using immunohistochemistry (Fig. 1A-C), and was decreased substantially in BC tissues compared with normal control tissues.

RT-qPCR analysis was performed to assess the expression of miR-421 in BC tissues, Hs578bst cells, MCF-7 cells and MDA-MB-231 cells (Fig. 2A and B). The expression of miR-421 was significantly increased in the BC tissues and cells compared with the normal controls. By contrast, the protein expression of PDCD4 was markedly downregulated in BC cells compared with the controls (Fig. 2C and D). These results suggested that the over-expression of miR-421 was associated with the downregulation of PDCD4 in BC.

PDCD4 was found as a potential target gene of miR-421. Luciferase reporter assays were performed to further confirm whether PDCD4 is a direct target of miR-421. The 3′-UTR of PDCD4 mRNAs, including putative binding sites of miR-421 together with their corresponding mutated sequences, were cloned into vectors and co-transfected into BC cells with miR-421 mimics, inhibitors or mock miRNA (Fig. 3A). Transfection with miR-421 mimics suppressed the luciferase activity significantly in MCF-7 cells (Fig. 3B) and MDA-MB-231 cells (Fig. 3C); by contrast, miR-421 inhibitors significantly enhanced the luciferase activity in MCF-7 cells (Fig. 3B) and MDA-MB-231 cells (Fig. 3C). The functions of the miR-421 mimics and inhibitors were abrogated when mutated 3′-UTR PDCD4-vector constructs were used, suggesting that miR-421 directly targets the 3′-UTRs of PDCD4.

The results revealed that transfection with miR-421 mimics enhanced the proliferation of Hs578bst (Fig. 4A), MCF-7 (Fig. 4B) and MDA-MB-231 (Fig. 4C) cells; however, transfection with miR-421 inhibitors had the opposite effect. These results suggested that the overexpression of miR-421 accelerates BC cell proliferation in vitro.

The Transwell results revealed that the overexpression of miR-421 significantly promoted the migration of MCF-7 and MDA-MB-231 cells, whereas miR-421 knockdown inhibited cell migration (Fig. 5A). The Matrigel assay revealed that the invasive abilities of the MCF-7 and MDA-MB-231 cells were enhanced following transfection with miR-421 mimics and downregulated by miR-421 inhibitors (Fig. 5B). These results suggested that the expression of miR-421 increases the motility of MCF-7 and MDA-MB-231 BC cells.

The flow cytometry results suggested that the number of apoptotic MCF-7 and MDA-MB-231 cells (Fig. 6A) was significantly increased following transfection with miR-421 inhibitors compared with the control group (Fig. 6B), suggesting that miR-421 affects the apoptosis of MCF-7, MDA-MB-231 cells via regulating PDCD4.

The RT-qPCR and western blot analyses revealed that the expression of miR-421 was increased in MCF-7 and MDA-MB-231 cells transfected with miR-421 mimics, but decreased in cells transfected with miR-421 inhibitors (Fig. 7A). Furthermore, the overexpression of miRNA-421 suppressed the expression of PDCD4 at the mRNA (Fig. 7B) and protein (Fig. 7C and D) levels in MCF-7 and MDA-MB-231 cells. By contrast, miR-421 knockdown upregulated the mRNA and protein levels of PDCD4, suggesting that PDCD4 is directly regulated by miR-421 and may function as an anticancer gene.