MCF-7, T47D, BT474, MDA-MB-231 and BT549 breast cancer cell lines as well as MCF-10A breast epithelial cell line were obtained from the American Type Culture Collection (ATCC; Manassas, VI, USA). Cells were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific), 100 U/ml penicillin sodium and 100 mg/ml streptomycin sulfate at 37°C in a humidified incubator with 5% CO₂.

miR-30a mimics, anti-miR-30a and negative control were obtained from Guangzho Ruibo Bio Co., Ltd. (Guangzhou, China). The expression plasmid for pCMV6-ROR1 was purchased from OriGene Technologies Inc. (Rockville, MD, USA). Cell transfections were conducted using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific) according to the manufacturer's protocols.

Recombinant lentiviruses containing miR-30a or scrambled sequences were obtained from GeneCopeia Inc. (Guangzhou, China). For selection of breast cancer stable cell lines, miR-30a-expressing retroviruses were transduced into MDA-MB-231 and BT549 cells in the presence of Polybrene (6 µg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Cells were selected with 2 µg/ml puromycin for 14 days.

All tumor tissues and adjacent normal tissues were collected from breast cancer patients who underwent complete resection at the Sixth Affiliated Hospital of Guangzhou Medical University (Qingyuan, China). Follow-up information was obtained from review of the medical records of the patients. The present study was approved by the Ethics Committee of Southern Medical University Authority.

A cell invasion assay was conducted using 24-well Boyden chambers with 8 µm inserts with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). Cells (1×10⁵) were placed on the inserts in the upper chambers with 200 µl serum-free RPMI-1640 medium at 37°C. A volume of 600 µl of RPMI-1640 containing 10% FBS was placed in the lower chamber. After 48 h, the non-invading cells and matrix were gently removed using cotton swabs. The invasive cells that crossed the inserts to the lower surface, were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet solution (Sigma-Aldrich, St. Louis, MO, USA) and 5 microscopic fields (at a magnification of ×200) were used to count and photograph the cells.

Cells (5×10⁵) were seeded in 6-well plates to grow into a monolayer and cultured until 80–90% confluence at 37°C. After starvation in serum-free medium for 24 h, a linear wound was created using a P200 sterile pipette and washed with PBS. Images were captured using an Olympus IX70 microscope microscope (a magnification of ×200; Olympus Corp., Tokyo, Japan) and the distance migrated was observed at time-points 0, 24 and 48 h.

Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Pierce, Rockford, IL, USA) containing mM phenylmethylsulfonyl fluoride (PMSF) and quantified using the BCA method (Pierce). Then proteins were centrifuged at 12,000 × g at 4°C for 15 min to remove insoluble fragments. The proteins (30 µg) were separated by a 10% SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (PVDF; Millipore, Billerica, MA, USA). The membrane was blocked with 5%-skim milk powder in TBST for 1 h. Primary antibodies against vimentin (cat. no. 5741), N-cadherin (cat. no. 13116), E-cadherin (cat. no. 3195), ZO-1 (cat. no. 13663) and Snail (cat. no. 3879; all diluted at 1:1,000 and were from Cell Signaling Technology, Danvers, MA, USA) were used. ROR1 (cat. no. ab135669; Abcam, Cambridge, MA, USA) and β-actin (cat. no. sc-1615; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were diluted at 1:1,000 and then incubated with the membranes overnight at 4°C. After incubation with HPR-conjugated secondary antibodies (anti-rabbit-IgG; 1:5,000; cat. no. 7074; Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature, the blots were visualized with ECL reagent (Millipore).

Cells grown on coverslips were fixed in 4% paraformaldehyde for 10 min at room temperature, and then blocked with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) for 30 min. The cells were incubated with E-cadherin, N-cadherin or vimentin antibodies overnight at 4°C, followed by incubation with CFTM555 goat anti-rabbit IgG (H+L) (1:1,000; cat. no. 4413; Cell Signaling Technology) for 1 h at room temperature. Cell nuclei were counterstained with DAPI for 10 min, and then analyzed using an Olympus LX70 fluorescence microscope (Olympus Corp.).

Total cellular RNA was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific). For mRNA detection, ROR1 and GAPDH mRNA expression was analyzed using SYBR-Green qRT-PCR according to the manufacturer's instructions (Applied Biosystems; Themo Fisher Scientific). The following primers were used: GAPDH forward, 5′-GACTCATGACCACAGTCCATGC-3′ and reverse, 5′-AGAGGCAGGGATGATGTTCTG-3′; ROR1 forward, 5′-TGCCAGCCCAGTGAGTAATCT-3′ and reverse, 5′-GCCAATGAAACCAGCAATCTG-3′.

For miRNA detection, the reverse-transcribed cDNA was synthesized with the All-in-One™ miRNA First-Strand cDNA Synthesis kit (GeneCopoeia, Rockville, MD, USA). miR-30a expression was determined with the All-in-One™ miRNA qRT-PCR Detection kit (GeneCopoeia) and U6 snRNA was used as the internal control.

The PmiRGLO Vector was used to construct the wild-type and mutated type of ROR1 3′-UTR. While the wild-type 3′-UTR of ROR1 is complementary to the seed region of miR-30a (UGUUUAC), the mutated type 3′-UTR is poorly complementary to the seed region of miR-30a. miR-30a mimics or miR-30a NC (50 nmol/l) and wild-type or mutated type of ROR1 3′-UTR vectors (500 ng) were co-transfected into 293T cells or TNBC cells (ATCC) using Lipotectamine™ 2000 (Invitrogen; Themo Fisher Scientific) following the manufacturer's instructions. The luciferase activities were quantified by Dual-Luciferase reporter assay (Promega, Madison, WI, USA). Relative luciferase activities were calculated by firefly luciferase activities/Renilla luciferase activities.

BT549/miR-30a or BT549/miR-SCR cells (1×10⁶) were subcutaneously injected into 4- to 6-week-old BALB/c nude mice (N=6 per group). Tumors were assessed every 4 days and tumor volumes were calculated using the formula: Volume=length × (width/²)2. The mice were sacrificed after 32 days of implantation and the tumors were excised and weighed. To assay the effect of miR-30a on tumor metastasis, BT549/miR-30a or BT549/miR-SCR cells (1×10⁶) were injected into the tail vein of nude mice (N=6 per group). After 45 days, necropsies were performed. The number of micrometastases in the lungs per tissue section in individual mice were determined from morphological observation of H&E-stained sections. All animal studies were approved by the Animal Experimentation Ethics Committee of Southern Medical University.

The prognostic value of miR-30a was analyzed by a Web-based Kaplan-Meier plotter (http://www.kmplot.com/), which is a meta-analysis tool of gene expression and survival data of 5,143 breast cancer patients (2015 version) using multiple microarray data.

Statistical analyses were conducted using the SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Comparisons between the groups were analyzed using the t-test and the Chi-square test (χ²). The differences were considered to be statistically significant when P<0.05.

We first performed qRT-PCR analyses to determine the expression levels of miR-30a in normal mammary cell lines MCF-10A and a panel of breast cancer cells, including MCF-7, T47D, BT474, MDA-MB-231 and BT549. We revealed that miR-30a was expressed at lower levels in breast cancer cells when compared with the MCF-10A cells (Fig. 1A). Notably, the lowest expression of miR-30a was associated with the highly migratory/invasive TNBC cells (Fig. 1A). Next, we detected miR-30a in 69 primary tumor samples for which estrogen receptor and HER2 expression were available and their matched adjacent normal tissues, and we found that the expression level of miR-30a was significantly lower in tumor tissues compared with the matched adjacent tissues (Fig. 1B). Moreover, the expression level of miR-30a was significantly decreased in TNBC compared with non-TNBC (Fig. 1C). We further investigated possible correlations between the expression of miR-30a and clinicopathological factors. As shown in Table I, there was no association observed between miR-30a and the age of patients (P=0.267). However, we found that patients with low expression of miR-30a demonstrated high histological grade and more lymph node metastasis (P=0.032 and P<0.01, respectively). Notably, we found that decreased miR-30a expression was significantly correlated with triple-negative breast cancer (P<0.01). Furthermore, the association between the expression of miR-30a and the prognosis of breast cancer patients was analyzed using the online Kaplan-Meier survival analysis of the expression data from 579 breast cancer patients (http://www.kmplot.com/). The results indicated that patients with low miR-30a expression had a shorter overall survival time compared to the patients with high miR-30a expression (P<0.05, log-rank test) (Fig. 1D). Collectively, these data demonstrated that miR-30a is downregulated in TNBC cells and tissues, and correlated with TNBC metastasis.

To determine whether miR-30a can affect TNBC cell migration and invasion, we established stable miR-30a-precursor expressing and negative control (miR-SCR) cell lines by lentiviral transfections (Fig. 2A). Wound healing assay indicated that the overexpression of miR-30a in MDA-MB-231 and BT549 cells significantly suppressed cell migration, compared to the control group (Fig. 2B). Moreover, Transwell assays demonstrated that ectopic expression of miR-30a significantly impaired invasion of TNBC cells (Fig. 2C).

Epithelial-mesenchymal transition (EMT) has been revealed to be crucial in promoting cancer progression and metastasis (13). Thus, we investigated whether miR-30a inhibited EMT of TNBC cells by investigating the expression levels of EMT-related markers. Western blot analysis indicated that ectopic expression of miR-30a significantly increased the expression of epithelial marker E-cadherin, but reduced the expression of mesenchymal markers vimentin and N-cadherin in MAD-MB-231 and BT549 cells (Fig. 3A). Moreover, immunofluorescence analysis further confirmed that the expression of E-cadherin was increased, whereas the expression of vimentin and N-cadherin was decreased in miR-30a-expressing BT549 cells (Fig. 3B).

We further assessed the effects of miR-30a overexpression on tumor growth and metastasis in vivo. BT549/miR-30a cells that stably expressed miR-30a or BT549/miR-SCR control cells were implanted into the right flanks of nude mice by subcutaneous injections (N=6 animals/group). We determined that the overexpression of miR-30a resulted in a significant decrease in tumor volumes compared to the control group (Fig. 4A). The weights and size of excised tumors from the miR-30a-overexpressing group were significantly decreased than those from the control group (Fig. 4B and C). We further investigated whether miR-30a suppressed TNBC metastasis in vivo. BT549/miR-30a cells or BT549/miR-SCR cells were injected into the lateral tail vein of nude mice. The results indicated that lung tumor nodules were significantly reduced in the mice injected with the BT549/miR-30a cells compared to the BT549/miR-SCR cells (Fig. 4D and E). These results demonstrated that miR-30a suppressed TNBC growth and metastasis in vivo.

We further investigated the molecular mechanism by which miR-30a inhibits the invasion and metastasis of TNBC cells. According to the prediction by TargetScan database, the 3′-UTRs of receptor tyrosine kinase-like orphan receptor 1 (ROR1) mRNA contain putative miR-30a binding sites (Fig. 5A). To determine whether miR-30a regulates ROR1 by binding to the corresponding 3′-UTRs, we cloned the 3′-UTR from ROR1 into the pmirGLO luciferase reporter vector. We also generated the mutant ROR1-3′-UTR vector, in which the sequence for miR-30a binding on the 3′-UTR was mutated (Fig. 5A). The luciferase assay revealed that transfection of miR-30a mimics significantly decreased the ROR1-3′-UTR wild-type but not the ROR1-3′-UTR mutant luciferase levels in 293T cells (Fig. 5B). In contrast, transfection of anti-miR-30a increased the ROR1-3′-UTR wild-type luciferase activity (Fig. 5B). Similar results were found in TNBC cells. Overexpression of miR-30a significantly inhibited ROR1-3′-UTR wild-type luciferase activity (Fig. 5C). Furthermore, we found that overexpression of miR-30a significantly decreased the levels of ROR1 mRNA in MDA-MB-231 and BT549 cells (Fig. 5D). There results were confirmed by western blot analysis (Fig. 5E). Collectively, these results indicated that miR-30a inhibited the expression of ROR1 by directly targeting the 3′-UTR of ROR1.

To elucidate whether the migration and invasion suppressive effects of miR-30a were mediated by inhibition of ROR1 in TNBC cells, gain-of-function studies were performed. We found that co-transfection with ROR1-expressing plasmids and miR-30a indicated a significant decrease in the expression of E-cadherin but an increase in the expression of N-cadherin and vimentin compared to the miR-30a-transfected cells (Fig. 6A). Using Matrigel invasion assays, we found that overexpression of ROR1 partially restored cell invasion suppression by miR-30a (Fig. 6B and C). Collectively, these data revealed that miR-30a suppressed TNBC migration and invasion by targeting ROR1.