Background/Aims: To investigate the cellular effects and clinical significance of microRNA-223 (miR-223) in breast cancer by targeting stromal interaction molecule1 (STIM1). Methods: Breast cancer cell lines (T47D, MCF-7, SKB-R3, MDA-MB-231 and MDA-MB-435) and a normal breast epithelial cell line (MCF-10A) were prepared for this study. MiR-223 mimics, anti-miR-223 and pcDNA 3.1-STIM1 were transiently transfected into cancer cells independently or together, and then RT-qPCR was performed to detect the expressions of miR-223 and STIM1 mRNA, dual-luciferase reporter assay was conducted to examine the effects of miR-223 on STIM1, Western blotting was used to measure the expressions of the STIM1 proteins, MTT and Trans-well assays were performed to detect cell proliferation and invasion. Finally, the correlation of miR-223 and STIM1 was investigated by detecting with ISH and IHC in breast cancer specimens or the corresponding adjacent normal tissues. Results: Compared with normal cells and tissues, breast cancer tissues and cells exhibited significantly lower expression of miR-223, but higher expression of STIM1. MiR-223 could inhibit the proliferation and invasiveness of breast cancer cells by negatively regulating the expressions of STIM1. Reimplantation with STIM1 partially rescued the miRNA-223-induced inhibition of breast cancer cells. Clinical data revealed that high expression of STIM1 and miR-223 was respectively detrimental and beneficial factor impacting patient’s disease-free survival (DFS) rather than overall survival (OS). Moreover, Pearson correlation analysis also confirmed that STIM1 was inversely correlated with miR-223. Conclusion: MiR-223 inhibits the proliferation and invasion of breast cancer by targeting STIM1. The miR-223/STIM1 axis could possibly be a potential therapeutic target for treating breast cancer patients.

Breast cancer is the highest incidence of cancer among women in China [1] and United States [2]. In recent years, great achievements have been made in early detection and individual treatment, however, recurrence and metastasis are still the main reasons for the majority of cancer-related death, which accounts for about 30% of these patients dying of metastatic disease within five years [3].

Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca2+ sensor protein which maintains intracellular calcium homeostasis and regulates intracellular calcium concentrations. Ca2+ is a crucial second messenger modulating various cellular processes,thereby STIM1 is also involved in a variety of biological processes including immune activation [4], cardiovascular damage [5], pulmonary disease [6] and so on. Moreover, it is demonstrated that STIM1 is also associated with the development and metastasis of several cancers [7-10] and has emerged as a target for cancer therapeutics [11]. Yang and his colleagues [12] were the first to report that STIM1 mediated breast cancer cells migration and metastasis in 2009. Two subsequent studies also confirmed the role of STIM1 in breast cancer metastasis [13] and proliferation [14]. These studies suggest that STIM1 may be a potential target to block the progression of breast cancer.

MicroRNAs (miRNAs) are endogenous, small non-coding RNAs with less than 25 nucleotides. They usually negatively regulate a large of genes expression by binding to the 3’-untranslated region (3’-UTR) of target mRNAs [15]. Several studies have shown that miRNAs act as a means of anticancer and play an important role in regulating the invasion and migration of cancer [16-18]. Notably, recent studies revealed that a variety of miRNAs, including miR-152 [19], miR-381 [20], miR-211 [21], miR-223 [22] and so on, could inhibit tumor proliferation, invasion and metastasis in breast cancer by targeting various genes. However, the miRNAs targeting STIM1 in breast cancer are still poorly characterized. Hence, understanding which miRNA targeting STIM1to drive tumor proliferation and metastasis remains of great interest.

In this study, we are committed to find out the miRNAs that regulate STIM1 expression and function in breast cancer. Our results demonstrate that miR-223 interacts with the STIM1 3’UTR and negatively regulates endogenous STIM1 expression. Meanwhile, overexpression of miR-223 and downregulation of STIM1could attenuate breast cancer cellular proliferation, invasion and decrease patient’s recurrence. Taken together, our findings uncover a new regulatory mechanism of STIM1 by miR-223 which may be a therapeutic intervention against breast cancer.

Cell culture and transfection

MCF-10A, MCF-7, T-47D, SKB-R3, MDA-MB-231 and MDA-MB-453 cells were purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, (Chinese Academy of Science, Shanghai, China). Those cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and maintained at 37°C in humidified air with 5% CO2. Once the density of the cell growth reached 80%, it would be handed from generation to generation.

For the transfection assay, cells were transfected with miR-223 mimics (artificially synthesized miR-223 mimics) and anti-miR-223 (miR-223 inhibitor), meanwhile their non-targeting sequence were used as control groups (NC group). For STIM1 rescue experiments, the coding domain sequence of human STIM1 mRNA was inserted into pcDNA 3.1 vector. All the RNAs were synthesized from Shanghai Gene Pharma Co., Ltd., Shanghai, China. The whole transfection experiments were mediated with Lipofectamine2000 for 6 hours, after that the cells were replaced with fresh culture medium and kept on culturing for 24, 48 or 72 hours.

Clinical samples

We collected 165 tumor tissue specimens and 60 corresponding adjacent normal breast tissues preserved in the Tumor Tissue Bank of Tianjin Cancer Hospital from the earlier operated BC patients between January 2007 and December 2009. The protocol was approved by the Institutional Review Board of Tianjin Cancer Institute and Hospital. All patients signed a written consent for the use of their specimens and disease information for future investigations according to the ethics committee.

Protein extraction and Western blot analysis

The total protein of cells was extracted by lysing cells with SDS lysis buffer supplemented with a protease inhibitor cocktail (Sigma). 30 µg of protein lysates were loaded onto 10% SDS-PAGE for electrophoresis and then transferred onto a nitrocellulose membrane. The membrane was subsequently incubated at 4°C overnight with rabbit anti-human STIM1 monoclonal antibody (CST) diluted 1: 1000 and GAPDH diluted 1: 5000. After further washes, the membranes were incubated with the goat anti-rabbit peroxidase–conjugated secondary antibodies (Abcam) at room temperature for 2 hours and the blots were developed using ECL (Millipore).

RNA extraction and Real-time PCR

According to the manufacturer’s instructions, total RNA was extracted from each groups of breast cancer cells using Trizol (Invitrogen ‚ USA) and subsequently reverse transcribed to cDNA using a reverse-transcription PCR system (TaKaRa).The expression levels of miR-223 and STIM1 mRNA were analyzed by using SYBR premix real-time PCR Reagent (TaKaRa). The GAPDH gene was used as an internal control. The primer sequences are shown as follows: miR-223(forward: 5′-GCGTGTATTTGACAAGCTGAGTT-3 and reverse: 5′-GTGTCAGTTTGTCAAATACCCCA-3); STIM1(forward: 5’-TGTGTCTCCCTTGTCCATGC-3’ and reverse, 5’-CATCTGAGGTTTGGGGG-3’).

Luciferase assays

The 3′-UTR of human STIM1 mRNA was amplified by PCR and the primers sequence were: STIM1-3′UTR-F: 5′-TGC TCT AGA CAG CTT GTC CTT CCC TGG GT-3′; STIM1–3′UTR-R: 5′-ACG TCT GAG GGG CAG CAC CTC TTA GAC A-3′. The amplified product was inserted into the pmirGLO vector (Promega). MD-MA-231 cells and MCF-7 cells were utilized for the luciferase assays, which were grown in 6-well plates and transfected with 4µg of the pmirGLO-STIM1 3′-UTR reporter vector per well and 100 pmol of microRNA mimics or microRNA inhibitor and their control RNA. the Site-specific mutants of the 3′-UTR reporters for STIM1 were also tested.

MTT assay and trans-well assay

MTT assay was performed to analyze cell proliferation activity by planting cells (3×103 cells/100µl) into 96-well plates and being cultured for varying periods of time. Then, the cells were incubated in the medium containing a final concentration of 0.5mg/ml of 3-(4, 5-dimethylthylthiazol-2yl-)-2, 5-diphenyl tetrazolium bromide (MTT, Sigma, Aldrich, MO, USA) solution at 37°C for 4h. The generated formazan was dissolved in 150µl DMSO. Absorbance was measured at 490 nm using a microplate reader.

Cells (2 ×105 cells) were plated on the top side of polycarbonate trans-well filter coated with Matrigel in the upper chamber and incubated at 37°C for 22h. The cells inside the upper chamber with cotton swabs were then removed. Migratory and invasive cells on the lower membrane surface were fixed, stained with hematoxylin, and counted for 10 random 100× fields per well. Cell counts are expressed as the mean number of cells per field of view.

Immunohistochemistry (ICH) and in situ hybridization (ISH) staining

All paraffin sections were prepared well by the pathology department, and immunostained according to the manufacturer’s instructions. Briefly, 5-µm-thick sections were subjected to antigen retrieval, endogenous peroxidase being neutralized and then incubated with primary STIM1 rat monoclonal antibody (ab57834; Abcam, UK) at 4°C overnight. Subsequently, the sections were incubated with secondary antibodies for 30 minutes at 37°C. The chromogen reaction was developed in 3, 3′-diaminobenzidine tetrahydrochloride for 5 minutes and hematoxylin was finally used as a light nuclear counterstain. Two independent observers (ZLN. and GL.) examined the slides in a blinded manner, and consensus was reached by repeated examination when results were discordant. The final scores were calculated by multiplying the scores of the intensity with the percentage in stained tumor cells as described in our previous article [23]. Two groups were divided: high expression (5-12); low expression (0-4).

Similarly, to evaluate miRNA-223 expression by ISH, the paraffin sections were incubated at 60°C for 2 hours, deparaffinized in xylene, rehydrated with graded alcohol washes, washed three times with diethyl pyrocarbonate -treated PBS, digested with 5 µg/ml proteinase K at 37°C for 30 minutes, washed and then dehydrated in graded alcohol. Slides were hybridized at 55°C for 3 hours with 10 nmol/L locked nucleic acid -modified digoxigenin-labeled probes for miRNA-223 (Boster, Wuhan, China). After stringency washes (5x, 1x, 0.2x SSC), slides were placed in blocking solution for 15 minutes followed by incubation in alkaline phosphatase conjugated anti-DIG Fab fragment solution for 1 hours. Finally, hematoxylin was used as a light nuclear counterstain. For each sample, a score was given for the percentage of positive cells as follows: 1 to 9% (0 points), 10 to 24% of the cells (1 point), 25 to 49% of the cells (2 points), 50 to 100% of the cells (3 points). Another score was given for the intensity of staining cells as follows: none (0 point), weak staining (1 points), intermediate staining (2 points), and strong staining (3 points). The staining index (SI) was calculated by multiplying the staining intensity with the proportion of positive cells and divided the samples into four grades: 0, negative (-); 1-2, low staining (+); 3-5, medium staining (++); and 6-9, high staining (+++). The following criteria were used to quantify the expression levels of miR-223: high expression (++/+++); low expression, other than high expression.

Statistical analysis

Clinicopathological correlations were tested using Fisher’s exact test and the Mann–Whitney U test for categorical and continuous data, respectively. Pearson correlation analysis was performed to evaluate the correlation between variables. Overall survival (OS) time was calculated from the date of the first curative operation to the date (2016-1) of the last follow-up or death from any cause. Survival analyses were performed by the Cox univariate proportional hazards model. For visualization purposes Kaplan–Meier analyses were used for the survival curves test (Mantel-Cox log-rank test). Multivariate Cox proportional hazard regression analysis was adjusted for relevant clinical covariates. All the tests were two-sided and the level of statistical significance was set at P<0.05. Statistical analyses were performed using GraphPad Prism version 5, and SPSS version 23.

MiR-223 inhibits proliferation and migration in breast cancer cells

First, the basal expression level of miR-223 was probed in five breast cancer cells (T47D, MCF-7, SKB-R3, MDA-MB-231 and MDA-MB-435) and a normal breast epithelial cell (MCF-10A) by using qRT-PCR. The results indicated that the levels of miR-223 were significantly lower in breast cancer cell lines than in the normal breast cells. Meanwhile, it was also different in various breast cancer cells, among which hormone receptor (HR)-negative cells (SKB-R3, MDA-MB-231 and MDA-MB-435) was lower than HR-positive cells (MCF-7 and T47D) (Fig. 1A).

Fig. 1.

miR-223 inhibits proliferation and migration in breast cancer cell lines. (A) MiR-223 expression was detected in 5 breast cancer cell lines (MCF-7, T47D, MDA-MB-231, SKB-R3‚ MDA-MB-435) and a normal breast epithelial cell (MCF-10A) using qRT-PCR. (B) Proliferation activity was evaluated with MTT assay in MDA-MB-231 and MCF-7 cells that were respectively transfected with miR-223 mimics and anti-miR-223 for the indicated time points. (C) Migration was evaluated with trans-well assay in MDA-MB-231 and MCF-7 cells that were transiently transfected with miR-223 mimics and anti-miR-223, respectively. (D) Quantitative results of Fig. 1C. The quantitative values are presented as the mean ± SD of 3 independent experiments. *, P<0.05.

Fig. 1.

miR-223 inhibits proliferation and migration in breast cancer cell lines. (A) MiR-223 expression was detected in 5 breast cancer cell lines (MCF-7, T47D, MDA-MB-231, SKB-R3‚ MDA-MB-435) and a normal breast epithelial cell (MCF-10A) using qRT-PCR. (B) Proliferation activity was evaluated with MTT assay in MDA-MB-231 and MCF-7 cells that were respectively transfected with miR-223 mimics and anti-miR-223 for the indicated time points. (C) Migration was evaluated with trans-well assay in MDA-MB-231 and MCF-7 cells that were transiently transfected with miR-223 mimics and anti-miR-223, respectively. (D) Quantitative results of Fig. 1C. The quantitative values are presented as the mean ± SD of 3 independent experiments. *, P<0.05.

Close modal

To further confirm the role of miR-223 in the biological behavior of breast cancer, MCF-7 and MDA-MB-231 cells with high and low levels of miR-223 were transiently transfected with anti-miR-223 and miR-223 mimics respectively. MTT assay and trans-well assay were utilized to compare the ability of proliferation and migration between the two groups of cells. As shown in Fig. 1 B and C, MCF-7 cells transfected with anti-miR-223 showed significantly higher proliferation and migration activity than the control group. Differently, the proliferation and migration activity of MDA-MB-231 cells were obviously attenuated after transfection with the miR-223 mimic compared with control group (Fig. 1B and C). The differences in cell proliferation were notably observed at 48 and 72 hours after transfection with anti-miR-223 in MCF-7 cell and miR-223 mimics in MDA-MB-231 cell. Taken together, these data demonstrated a critical role of miR-223 in regulating the proliferation and migration of breast cancer cells.

MiR-223 down-regulates STIM1 expression by targeting the 3’-UTR of STIM1

To investigate whether STIM1 could be regulated directly by miRNAs, we used several web-based target analysis tools (TargetScan, miRDB, microRNA, PicTar and starBase) to identify miRNAs that potentially target STIM1. As a result, miR-223 was identified as a potential regulator of STIM1 expression (Fig. 2A).

Fig. 2.

miRNA-223 down-regulates STIM1 expression by targeting the 3’-UTR of STIM1. (A) Schematic diagram of the putative miR-223 binding site in human STIM1 3’-UTR and luciferase constructs with wild-type and mutant (STIM1-3’-UTR) miR-497 target sequences. (B). Luciferase activity of cancer cells in each group. Left, miR-223 mimics in MDA-MB-231 cell. Right, anti-miR-223 in MCF-7 cell. (C). The relative mRNA levels of miRNA-233 and STIM1 evaluated by real-time PCR after a period (24h, 48h, 72h) of intervention with miR-223 mimics or anti-miR-223. Above, miR-223 mimics in MDA-MB-231 cell. Below, anti-miR-223 in MCF-7 cell. (D). The expression of STIM1 protein evaluated by western blot after a period (24h, 48h, 72h) of intervention with miR-223 mimics or anti-miR-223. Left, miR-223 mimics in MDA-MB-231 cell. Right, anti-miR-223 in MCF-7 cell. The quantitative values are presented as the mean ± SD of 3 independent experiments. *, P<0.05.

Fig. 2.

miRNA-223 down-regulates STIM1 expression by targeting the 3’-UTR of STIM1. (A) Schematic diagram of the putative miR-223 binding site in human STIM1 3’-UTR and luciferase constructs with wild-type and mutant (STIM1-3’-UTR) miR-497 target sequences. (B). Luciferase activity of cancer cells in each group. Left, miR-223 mimics in MDA-MB-231 cell. Right, anti-miR-223 in MCF-7 cell. (C). The relative mRNA levels of miRNA-233 and STIM1 evaluated by real-time PCR after a period (24h, 48h, 72h) of intervention with miR-223 mimics or anti-miR-223. Above, miR-223 mimics in MDA-MB-231 cell. Below, anti-miR-223 in MCF-7 cell. (D). The expression of STIM1 protein evaluated by western blot after a period (24h, 48h, 72h) of intervention with miR-223 mimics or anti-miR-223. Left, miR-223 mimics in MDA-MB-231 cell. Right, anti-miR-223 in MCF-7 cell. The quantitative values are presented as the mean ± SD of 3 independent experiments. *, P<0.05.

Close modal

To further understand whether STIM1 could be regulated directly by miR-223, the luciferase reporter assay was used to analyze the effect of miR-223 on gene transcription of STIM1. A wild type (WT) or mutant type (MT) 3’-UTR of the STIM1 gene was inserted into the dual luciferase vector pmirGLO (Fig. 2A). Together with the dual luciferase vector, miR-223 mimics or anti-miR-223 were cotransfected into the MDA-MB-231 or MCF-7 cells respectively. The results revealed that the miR-223 mimics significantly reduced the luciferase activity of the wild-type STIM1 but had no effect on the mutant STIM1 reporter gene in MDA-MB-231 cell (Fig. 2B, left). As expected, anti-miR-223 cotransfected with the wild-type reporter gene in MCF-7 cell, the luciferase activity of the reporter gene was elevated, while in the mutant type, the luciferase activity of the reporter gene was equivalent. (Fig. 2B, right). These results revealed that miRNA-223 directly targets STIM1.

Western blot and RT-PCR experiments were further performed to validate the relationship between miR-223 and STIM1. In MDA-MB-231 cells, transfection with the miR-223mimics induced a down-regulation of STIM1 mRNA and protein expression (Fig. 2 C above and Fig. 2 D left). In contrast, anti-miR-223 up-regulated the protein and mRNA level of STIM1 in MCF-7 cells (Fig. 2 C below and Fig. 2 D right). Altogether, these results further confirmed that STIM1 was a direct target gene of miR-223.

MiR-223 suppresses the proliferation and migration of breast cancer cells by targeting STIM1

To further understand whether miR-223 inhibited proliferation and migration by negatively regulating STIM1, miRNA-223 mimic and pcDNA 3.1-STIM1 or null construct were cotransfected into MDA-MB-231 cell. The western blot results showed that over-expression of STIM1 rescued the miRNA-223-induced decline of STIM1 expression compared with the control group (Fig. 3A). Additionally, MTT and trans-well assay results revealed that the ability of cell proliferation and migration were significantly decreased when MDA-MB-231 cells were transfected with miRNA-223 mimic, however, pcDNA 3.1-STIM1 could partially antagonize this proliferation and migration-inhibiting effect of miRNA-223 mimic (Fig. 3B, C, D). These data suggested that miR-223 restrained cell proliferation and migration by targeting STIM1.

Fig. 3.

MiR-223 suppresses the proliferation and migration of breast cancer cells by targeting STIM1. MDA-MB-231 cells were cotransfected with pcDNA 3.1-STIM1/pcDNA 3.1 and miR-223 mimics/NC and then subjected to (A) western blot, (B) MTT assay, (C and D) trans-well assay. The quantitative values are presented as the mean ± SD of 3 independent experiments. *P<0.05.

Fig. 3.

MiR-223 suppresses the proliferation and migration of breast cancer cells by targeting STIM1. MDA-MB-231 cells were cotransfected with pcDNA 3.1-STIM1/pcDNA 3.1 and miR-223 mimics/NC and then subjected to (A) western blot, (B) MTT assay, (C and D) trans-well assay. The quantitative values are presented as the mean ± SD of 3 independent experiments. *P<0.05.

Close modal

Reverse correlation between STIM1 and miR-223 in breast cancer patients

To explore the expression of STIM1 and miR-223 in breast cancer tissues, immunohistochemistry (IHC) and in situ hybridization (ISH) staining were respectively used in 60 breast cancer tissues and the corresponding adjacent normal tissues. Compared with adjacent non-tumor tissues, the increased expression of STIM1 (58.3%, 35/60 vs. 25%, 15/60; P<0.001) and decreased expression of miR-223 (38.3%, 23/60 vs. 65%, 39/60; P=0.003) were found in cancer tissues (Table 1). In addition, the interaction of STIM1 and miR-223 was further explored in 165 breast cancer tissues with detailed clinicopathological information (Table 2). The results showed that STIM1 positively correlated with large tumor size (P=0.001), lymph node metastasis (P=0.005) and ER (P=0.042), while miR-223 was negatively associated with lymph node metastasis (P=0.005) and ER (P=0.042). Furthermore, Kaplan–Meier and Cox regression analysis revealed that high expressions of STIM1 and miR-223 were respectively detrimental and beneficial factor impacting patient’s DFS rather than OS (Fig. 4C and D). Moreover, Pearson correlation analysis also confirmed that STIM1 inversely correlated with miR-223 (Fig. 4E). Collectively, these findings implied that miR-223 negatively regulated STIM1 in breast cancer.

Table 1.

Expression of STIM1 and miR-223 in tumor and adjacent normal tissues

Expression of STIM1 and miR-223 in tumor and adjacent normal tissues
Expression of STIM1 and miR-223 in tumor and adjacent normal tissues
Table 2.

Clinical and pathologic characteristics with STIM1 and miR-223 expression. m Abbreviations: ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor-2;

Clinical and pathologic characteristics with STIM1 and miR-223 expression. m Abbreviations: ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor-2;
Clinical and pathologic characteristics with STIM1 and miR-223 expression. m Abbreviations: ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor-2;
Fig. 4.

STIM1 and miR-223 in breast cancer patients. (A) Immunohistochemistry analysis of STIM1 expression in 165 breast cancer tissues. (a and b) low expression; (b and d) high expression. (a and c) Magnification, × 200; (b and d) magnification, × 400. (B) In situ hybridization (ISH) staining analysis of miR-223 expression in 165 breast cancer tissues. (a and b) low expression; (b and d) high expression. (a and c) Magnification, × 200; (b and d) magnification, × 400. (C) Kaplan–Meier analysis of disease free survival (DFS) of 165 patients with breast cancer according to the expression of STIM1. (D) Kaplan–Meier analysis of disease free survival (DFS) of 165 patients with breast cancer according to the expression of miR-223. (E) Spearman’s correlation analysis of STIM1 and miR-223 in 165 breast cancer patients.

Fig. 4.

STIM1 and miR-223 in breast cancer patients. (A) Immunohistochemistry analysis of STIM1 expression in 165 breast cancer tissues. (a and b) low expression; (b and d) high expression. (a and c) Magnification, × 200; (b and d) magnification, × 400. (B) In situ hybridization (ISH) staining analysis of miR-223 expression in 165 breast cancer tissues. (a and b) low expression; (b and d) high expression. (a and c) Magnification, × 200; (b and d) magnification, × 400. (C) Kaplan–Meier analysis of disease free survival (DFS) of 165 patients with breast cancer according to the expression of STIM1. (D) Kaplan–Meier analysis of disease free survival (DFS) of 165 patients with breast cancer according to the expression of miR-223. (E) Spearman’s correlation analysis of STIM1 and miR-223 in 165 breast cancer patients.

Close modal

In the present study, we demonstrate that STIM1 is a direct target of miR-223 in breast cancer. By directly binding to the 3’-UTR of STIM1, miR-223 inhibits STIM1 expression, suppresses proliferation and migration of breast cancer cells and decreases breast cancer recurrence in patients.

STIM1 and Orai are two key molecules that mediate store-operated Ca2+ entry (SOCE). SOCE is one of the major routes of calcium entry in nonexcitable cells, in which the depletion of intracellular Ca2+ stores inspires activation of the endoplasmic reticulum (ER)-resident Ca2+ sensor protein STIM to gate and open the Orai Ca2+ channels in the plasma membrane [24]. Accumulating evidence indicates that STIM1 and Orai play crucial roles in promoting tumorigenesis and progression in a variety of cancers [25] in addition to regulating normal physiological functions. In 2009, Yang and his colleagues first validated the association between STIM1 and breast cancer cell migration and metastasis [12]. Recently, it was revealed that STIM1 played an important role in TGF- β -induced proliferation [14] and epithelial-mesenchymal transitions (EMT) [13] in breast cancer. Consequently, it is possible that STIM1 is a promising target for breast cancer therapeutics.

Emerging evidence indicated that miRNAs could function as oncogenes or tumor suppressors by regulating hundreds of downstream target genes. A variety of miRNAs, including miR-152 [19], miR-381 [20], miR-211 [21] and miR-203 [26], have been proved to inhibit tumor proliferation, invasion and metastasis in breast cancer by targeting various genes. In our study, miR-223 was identified as a regulator of STIM1 via qRT-PCR, western blot analysis and luciferase reporter assay. Additionally, HAX-1 [27] and Caprin-1 [22] have also been identified as the target genes of miR-223 in breast cancer.

Usually, miR-223 was considered to be a suppressor in multiple cancers, such as cervical cancer [28], bladder cancer [29], prostate cancer [30] and breast cancer [31]. Our results were consistent with these previous findings. Upregulation of miR-223 could inhibit breast cancer cell proliferation and migration, in contrast, downregulation of miR-223 caused decreased abilities of cell proliferation and migration. Besides STIM1, previous study uncovered two other target genes of miR-223, Caprin-1 [22] and HAX-1 [27], which also contributed to breast cancer proliferation, migration, and invasion. Moreover, we showed that miR-223 expression levels were significantly lower in breast cancer tissues than in the adjacent tissues, and that miR-223 was negatively correlated with some poor prognostic factors. A recent study also confirmed that miR-223 induced by radiotherapy could prevent the relapse of breast cancer [32]. However, other researchers found that overexpression of miR-223 was positively correlated with tumor recurrence and metastasis in patients with colorectal cancer [33], hepatocellular cancer [34] and non-small cell lung cancer [35]. Several in vitro studies also verified that miRNA-223 promoted lung cancer, ovarian cancer and prostate cancer cells invasion via targeting EPB41L3 [36] , SOX11 [37] and SEPT6 [38] respectively. These inconsistent results may be due to the different roles of the target genes of miRNA-223 in the tumor.

In addition to regulating tumor growth, miR-223 was also found to be an early diagnostic marker in oral cancer [39, 40], gastric cancer [41] and colorectal cancer [42] detection. Therefore, miR-223 perhaps will be a potential marker for the early diagnosis of breast cancer.

We confirmed that miR-223 could inhibit the biological behavior of breast cancer by suppressing STIM1 expression. We speculate that the miR-223/STIM1 axis is an attractive target for diagnosis and therapy breast cancer.

Funding/Support: This work was supported by a grant from Tianjin Application Foundation and Advanced Technology Research Program (grant numbers:14JCYBJC24900).

Competing interests: All authors declare that they have no conflicts of interest.

1.
Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J: Cancer statistics in China, 2015 CA. Cancer J Clin 2016; 66: 115-132.
2.
Siegel RL, Miller KD, Jemal A: Cancer Statistics, 2017 CA Cancer J Clin 2017; 67: 7-30.
3.
Cardoso F, Costa A, Senkus E, Aapro M, Andre F, Barrios CH, Bergh J, Bhattacharyya G, Biganzoli L, Cardoso MJ, Carey L, Corneliussen-James D, Curigliano G, Dieras V, El Saghir N, Eniu A, Fallowfield L, Fenech D, Francis P, Gelmon K, Gennari A, Harbeck N, Hudis C, Kaufman B, Krop I, Mayer M, Meijer H, Mertz S, Ohno S, Pagani O, Papadopoulos E, Peccatori F, Pernault-Llorca F, Piccart MJ, Pierga JY, Rugo H, Shockney L, Sledge G, Swain S, Thomssen C, Tutt A, Vorobiof D, Xu B, Norton L, Winer E: 3rd ESO-ESMO International Consensus Guidelines for Advanced Breast Cancer (ABC 3). Ann Oncol 2017; 28: 16-33.
4.
Lioudyno MI, Kozak JA, Penna A, Safrina O, Zhang SL, Sen D, Roos J, Stauderman KA, Cahalan MD: Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation. Proc Natl Acad Sci USA 2008; 105: 2011-2016.
5.
Troupes CD, Wallner M, Borghetti G, Zhang C, Mohsin S, von Lewinski D, Berretta RM, Kubo H, Chen X, Soboloff J, Houser S: Role of STIM1 (Stromal Interaction Molecule 1) in Hypertrophy-Related Contractile Dysfunction. Circulation Res 2017; 121: 125-136.
6.
Lu W, Wang J, Peng G, Shimoda LA, Sylvester JT: Knockdown of stromal interaction molecule 1 attenuates store-operated Ca2+ entry and Ca2+ responses to acute hypoxia in pulmonary arterial smooth muscle. Am J Physiol Lung Cell Mol Physiol 2009; 297:L17-25.
7.
Cui X, Song L, Bai Y, Wang Y, Wang B, Wang W: Stromal interaction molecule 1 (STIM1) regulates growth, cell cycle and apoptosis of human tongue squamous carcinoma cells. Biosci Rep 2017; 37: 1-9.
8.
Perrouin Verbe MA, Bruyere F, Rozet F, Vandier C, Fromont G: Expression of store-operated channel components in prostate cancer: the prognostic paradox. Hum Pathol 2016; 49: 77-82.
9.
Wang Y, Wang H, Li L, Li J, Pan T, Zhang D, Yang H: Elevated expression of STIM1 is involved in lung tumorigenesis. Oncotarget 2016; 7: 86584-86593.
10.
Xu JM, Zhou Y, Gao L, Zhou SX, Liu WH, Li XA: Stromal interaction molecule 1 plays an important role in gastric cancer progression. Oncol Rep 2016; 35: 3496-3504.
11.
Vashisht A, Trebak M, Motiani RK: STIM and Orai proteins as novel targets for cancer therapy. A Review in the Theme: Cell and Molecular Processes in Cancer Metastasis. Am J Physiol Cell Physiol 2015; 309:C457-469.
12.
Yang S, Zhang JJ, Huang XY: Orai1 and STIM1 are critical for breast tumor cell migration and metastasis. Cancer Cell 2009; 15: 124-134.
13.
Zhang S, Miao Y, Zheng X, Gong Y, Zhang J, Zou F, Cai C: STIM1 and STIM2 differently regulate endogenous Ca2+ entry and promote TGF-beta-induced EMT in breast cancer cells. Biochem Biophys Res Commun 2017; 488: 74-80.
14.
Cheng H, Wang S, Feng R: STIM1 plays an important role in TGF-beta-induced suppression of breast cancer cell proliferation. Oncotarget 2016; 7: 16866-16878.
15.
Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281-297.
16.
Zhang G, Zhang W, Li B, Stringer-Reasor E, Chu C, Sun L, Bae S, Chen D, Wei S, Jiao K, Yang WH, Cui R, Liu R, Wang L: MicroRNA-200c and microRNA- 141 are regulated by a FOXP3-KAT2B axis and associated with tumor metastasis in breast cancer. Breast Cancer Res 2017; 19: 1-13.
17.
Das R, Gregory PA, Fernandes RC, Denis I, Wang Q, Townley SL, Zhao SG, Hanson AR, Pickering MA, Armstrong HK, Lokman NA, Ebrahimie E, Davicioni E, Jenkins RB, Karnes RJ, Ross AE, Den RB, Klein EA, Chi KN, Ramshaw HS, Williams ED, Zoubeidi A, Goodall GJ, Feng FY, Butler LM, Tilley WD, Selth LA: MicroRNA-194 Promotes Prostate Cancer Metastasis by Inhibiting SOCS2 Cancer Res 2017; 77: 1021-1034.
18.
Cao Q, Liu F, Ji K, Liu N, He Y, Zhang W, Wang L: MicroRNA-381 inhibits the metastasis of gastric cancer by targeting TMEM16A expression. J Exp Clin Cancer Res 2017; 36: 1-16.
19.
Maimaitiming A, Wusiman A, Aimudula A, Tudahong T, Aisimutula D: Downregulation of MicroRNA-152 and Inhibition of Cell Proliferation, Migration, and Invasion in Breast Cancer. Oncology Res 2017; 25: 28653610.
20.
Xue Y, Xu W, Zhao W, Wang W, Zhang D, Wu P: miR-381 inhibited breast cancer cells proliferation, epithelial-to-mesenchymal transition and metastasis by targeting CXCR4 Biomed Pharmacother 2017; 86: 426-433.
21.
Chen LL, Zhang ZJ, Yi ZB, Li JJ: MicroRNA-211-5p suppresses tumour cell proliferation, invasion, migration and metastasis in triple-negative breast cancer by directly targeting SETBP1. Br J Cancer 2017; 117: 78-88.
22.
Gong B, Hu H, Chen J, Cao S, Yu J, Xue J, Chen F, Cai Y, He H, Zhang L: Caprin-1 is a novel microRNA-223 target for regulating the proliferation and invasion of human breast cancer cells. Biomed Pharmacother 2013; 67: 629-636.
23.
Yang Y, Jiang Z, Wang B, Chang L, Liu J, Zhang L, Gu L: Expression of STIM1 is associated with tumor aggressiveness and poor prognosis in breast cancer. Patholo Res Pract 2017; 213: 1043-1047.
24.
Stathopulos PB, Schindl R, Fahrner M, Zheng L, Gasmi-Seabrook GM, Muik M, Romanin C, Ikura M: STIM1/ Orai1 coiled-coil interplay in the regulation of store-operated calcium entry. Nat Commun 2013; 4: 2963: 1-12.
25.
Jardin I, Rosado JA: STIM and calcium channel complexes in cancer. Biochim Biophys Acta 2016; 1863: 1418-1426.
26.
Zhao S, Han J, Zheng L, Yang Z, Zhao L, Lv Y: MicroRNA-203 Regulates Growth and Metastasis of Breast Cancer. Cell Physiol Biochem 2015; 37: 35-42.
27.
Sun X, Li Y, Zheng M, Zuo W, Zheng W: MicroRNA-223 Increases the Sensitivity of Triple-Negative Breast Cancer Stem Cells to TRAIL-Induced Apoptosis by Targeting HAX-1. PLoS One 2016; 11: 2-14.
28.
Tang Y, Wang Y, Chen Q, Qiu N, Zhao Y, You X: MiR-223 inhibited cell metastasis of human cervical cancer by modulating epithelial-mesenchymal transition. Int J Clin Exp Pathol 2015; 8: 11224-11229.
29.
Guo J, Cao R, Yu X, Xiao Z, Chen Z: MicroRNA-223-3p inhibits human bladder cancer cell migration and invasion. Tumour Biol 2017; 39: 1-7.
30.
Kurozumi A, Goto Y, Matsushita R, Fukumoto I, Kato M, Nishikawa R, Sakamoto S, Enokida H, Nakagawa M, Ichikawa T, Seki N: Tumor-suppressive microRNA-223 inhibits cancer cell migration and invasion by targeting ITGA3/ITGB1 signaling in prostate cancer. Cancer Sci 2016; 107: 84-94.
31.
Cao L, Zhang X, Cao F, Wang Y, Shen Y, Yang C, Uzan G, Peng B, Zhang D: Inhibiting inducible miR-223 further reduces viable cells in human cancer cell lines MCF-7 and PC3 treated by celastrol. BMC Cancer 2015; 15: 1-11.
32.
Fabris L, Berton S, Citron F, D’Andrea S, Segatto I, Nicoloso MS, Massarut S, Armenia J, Zafarana G, Rossi S, Ivan C, Perin T, Vaidya JS, Avanzo M, Roncadin M, Schiappacassi M, Bristow RG, Calin G, Baldassarre G, Belletti B: Radiotherapy-induced miR-223 prevents relapse of breast cancer by targeting the EGF pathway. Oncogene 2016; 35: 4914-4926.
33.
Li ZW, Yang YM, Du LT, Dong Z, Wang LL, Zhang X, Zhou XJ, Zheng GX, Qu AL, Wang CX: Overexpression of miR-223 correlates with tumor metastasis and poor prognosis in patients with colorectal cancer. Med Oncol 2014; 31: 25270282.
34.
Han ZB, Zhong L, Teng MJ, Fan JW, Tang HM, Wu JY, Chen HY, Wang ZW, Qiu GQ, Peng ZH: Identification of recurrence-related microRNAs in hepatocellular carcinoma following liver transplantation. Mol Oncol 2012; 6: 445-457.
35.
Sanfiorenzo C, Ilie MI, Belaid A, Barlesi F, Mouroux J, Marquette CH, Brest P, Hofman P: Two panels of plasma microRNAs as non-invasive biomarkers for prediction of recurrence in resectable NSCLC. PLoS One 2013; 8:e54596.
36.
Liang H, Yan X, Pan Y, Wang Y, Wang N, Li L, Liu Y, Chen X, Zhang CY, Gu H, Zen K: MicroRNA-223 delivered by platelet-derived microvesicles promotes lung cancer cell invasion via targeting tumor suppressor EPB41L3. Mol Cancer 2015; 14: 1-13.
37.
Fang G, Liu J, Wang Q, Huang X, Yang R, Pang Y, Yang M: MicroRNA-223-3p Regulates Ovarian Cancer Cell Proliferation and Invasion by Targeting SOX11 Expression. Int J Mol Sci 2017; 18: 2-12.
38.
Wei Y, Yang J, Yi L, Wang Y, Dong Z, Liu Z, Ou-yang S, Wu H, Zhong Z, Yin Z, Zhou K, Gao Y, Yan B, Wang Z: MiR-223-3p targeting SEPT6 promotes the biological behavior of prostate cancer. Sci Rep 2014; 4: 1-8.
39.
Zhou X, Ji G, Chen H, Jin W, Yin C, Zhang G: Clinical role of circulating miR-223 as a novel biomarker in early diagnosis of cancer patients. Int J Clin Exp Med 2015; 8: 16890-16898.
40.
Tachibana H, Sho R, Takeda Y, Zhang X, Yoshida Y, Narimatsu H, Otani K, Ishikawa S, Fukao A, Asao H, Iino M: Circulating miR-223 in Oral Cancer: Its Potential as a Novel Diagnostic Biomarker and Therapeutic Target. PLoS One 2016; 11:e0159693.
41.
Li BS, Zhao YL, Guo G, Li W, Zhu ED, Luo X, Mao XH, Zou QM, Yu PW, Zuo QF, Li N, Tang B, Liu KY, Xiao B: Plasma microRNAs, miR-223, miR-21 and miR-218, as novel potential biomarkers for gastric cancer detection. PLoS One 2012; 7:e41629.
42.
Chang PY, Chen CC, Chang YS, Tsai WS, You JF, Lin GP, Chen TW, Chen JS, Chan EC: MicroRNA-223 and microRNA-92a in stool and plasma samples act as complementary biomarkers to increase colorectal cancer detection. Oncotarget 2016; 7: 10663-10675.

Y. Yang and Z. Jiang contributed equally to this work.

Open Access License / Drug Dosage / Disclaimer
This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND). Usage and distribution for commercial purposes as well as any distribution of modified material requires written permission. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.