MiR-31 inhibits migration and invasion by targeting SATB2 in triple negative breast cancer

MiR-31 inhibits migration and invasion by targeting SATB2 in triple negative breast cancer

Gene 594 (2016) 47–58 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene Research paper MiR-31 inhibits...

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Gene 594 (2016) 47–58

Contents lists available at ScienceDirect

Gene journal homepage: www.elsevier.com/locate/gene

Research paper

MiR-31 inhibits migration and invasion by targeting SATB2 in triple negative breast cancer Long-ji Luo a,b,1, Fan Yang a,b,1, Jia-ji Ding a,b, Da-li Yan d, Dan-dan Wang b,c, Su-jin Yang b,d, Li Ding b,e, Jian Li b,c, Dan Chen f, Rong Ma f, Jian-zhong Wu f, Jin-hai Tang b,g,⁎ a

Xuzhou Medical University, Xuzhou 221004, China Department of General Surgery, Nanjing Medical University Affiliated Cancer Hospital Cancer Institute of Jiangsu Province, Baiziting 42, Nanjing 210009, China The First Clinical School of Nanjing Medical University, Nanjing 210009, China d The Forth Clinical School of Nanjing Medical University, Nanjing 210009, China e China Pharmaceutical University, Nanjing 21009, China f Research Center of Clinical Oncology, Nanjing Medical University Affiliated Cancer Hospital Cancer Institute of Jiangsu Province, Baiziting 42, Nanjing 210009, China g Department of General Surgery, the First Affiliated Hospital with Nanjing Medical University, Nanjing 210029, China b c

a r t i c l e

i n f o

Article history: Received 1 August 2016 Received in revised form 28 August 2016 Accepted 31 August 2016 Available online 01 September 2016 Keywords: Breast cancer Triple negative breast cancer miR-31 SATB2 Metastasis 5-AZA-CdR

a b s t r a c t Metastasis is the leading cause of death among breast cancer (BCa) patients and triple negative breast cancer (TNBC) as one of BCa subtypes exhibits the worst survival rate due to its highly aggressive and metastatic behavior. A growing body of research has shown that the dynamic expression of microRNAs (miRNAs) was intimately associated with tumor invasion and metastasis. Recent studies have demonstrated miR-31 as a metastasissuppressor in breast cancer, but it is still known little about the mechanism of it suppresses metastasis. The special AT-rich sequence-binding protein-2 (SATB2) has been reported to involve in tumor proliferation and invasion, but its function and relationship with miR-31 in breast cancer is still unknown. Here we found that the expression of miR-31 was downregulated in TNBC tissue and cell line. MiR-31 expression was increased after MDA-MB-231 cell was treated by 5-aza-2′-deoxycytidine (5-AZA-CdR), enhance the expression of miR-31 significantly inhibited MDA-MB-231 cell migration and invasion, downregulation of miR-31 expression could promoted MCF-7 cell migration and invasion. The expression of SATB2 was negatively correlated with miR-31 and was upregulated in MCF-7 and MDA-MB-231. Silencing SATB2 expression significantly inhibited MCF-7 and MDAMB-231 cell proliferation, migration and invasion. Luciferase reporter assays indicated SATB2 is a direct target of miR-31. Taken together, these results suggest miR-31 inhibited TNBC cells migration and invasion through suppressing SATB2 expression. © 2016 Published by Elsevier B.V.

1. Introduction Breast cancer (BCa) is the most common cancer for women and a leading cause of cancer mortality worldwide (Jemal et al., 2009). Triple negative breast cancer (TNBC) as one of BCa subtypes is defined as lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epithelial growth factor receptor-2 (HER-2). TNBC as the representative of the most aggressive BCa subtype, this kind of BCa patients Abbreviations: breast cancer, BCa; triple negative breast cancer, TNBC; the special ATrich sequence-binding protein-2, SATB2; microRNAs, miRNAs; 5-aza-2′-deoxycytidine, 5AZA-CdR; human epithelial growth factor receptor-2, HER-2; 3′untranslated region, 3′ UTR. ⁎ Corresponding author at: Department of General Surgery, Nanjing Medical University Affiliated Cancer Hospital Cancer Institute of Jiangsu Province, Baiziting 42, Nanjing 210009, China. E-mail address: [email protected] (J. Tang). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.gene.2016.08.057 0378-1119/© 2016 Published by Elsevier B.V.

who lose the chance of molecular targeted therapy and hormone therapy, even when treated with adjuvant therapy, there were about 1/3 of this patients who recurrence within 3 years (Berrada et al., 2010), moreover due to its highly metastatic behavior which lead to the worst prognosis in BCa (Carey et al., 2010; Anders and Carey, 2009; Foulkes et al., 2010). However, the molecular mechanism of TNBC metastasis is still poorly understood (Weigelt et al., 2005). Hence, understanding the mechanisms underlying metastasis in TNBC will be key to discover new therapeutic targets. MicroRNAs (miRNAs), a series of small noncoding RNAs molecules of 18–25 nucleotides that regulate gene expressions at the posttranscriptional/translational level. MiRNAs repress translation or induce degradation of their target mRNAs through binding to the 3'untranslated region (3′UTR) (He and Hannon, 2004; Bartel, 2009; Pritchard et al., 2012). Aberrant miRNAs expression play crucial roles in tumorigenesis and tumor progression by regulating their target genes involved in cell apoptosis, proliferation, differentiation, invasion and metastasis

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(Brennecke et al., 2003; Xu et al., 2003; Kawasaki and Taira, 2003; Reinhart et al., 2000; Esquela-Kerscher and Slack, 2006). Increasing evidences supported that miR-31 tightly associated with tumorigenesis and metastasis (Laurila and Kallioniemi, 2013). Dysregulation of mir31 was reported in several malignancies, including bladder (Wszolek et al., 2011), prostate (Lin et al., 2013), colon (Xu et al., 2013). Emerging evidences have implied mir-31 to be a tumor suppressor in lung adenocarcinoma by targeting the RNA binding protein Human antigen R (HuR) (Xu et al., 2016). Valastyan et al. has shown that low miR-31 expression correlates with high risk of metastases in BCa patients, miR-31 as a metastasis-suppressor in BCa and ectopic expression of miR-31 in highly metastatic human breast cancer cell lines impaired their mobility, invasiveness and ability to metastasize (Valastyan et al., 2009). MiR31 also inhibited BCa cells invasion by suppressing guanine nucleotidebinding protein subunit alpha-13(GNA13) expression (Rasheed et al., 2015). A nuclear matrix sequence specific binding protein, Special AT-rich sequence-binding protein 2 (SATB2), which located in the q32-q33 region of human chromosome 2, was involved in gene transcription and chromatin remodeling (Dobreva et al., 2006; FitzPatrick et al., 2003; Britanova et al., 2005). Previous studies have shown that SATB2 played important roles in the central nervous system development, craniofacial patterning and osteoblast differentiation (Dobreva et al., 2006; Britanova et al., 2005; Britanova et al., 2006; Gyorgy et al., 2008). Recently, a growing body of evidences demonstrated that abnormal expression of SATB2 was associated with the occurrence and development in several tumors. SATB2 regulated the expression of genes involved in actin cytoskeleton dynamics to promote osteosarcoma invasion and migration. (Seong et al., 2015). SATB2 as a tumor-suppressor in colorectal cancer (CRC) and gastric cancer (GC) via ERK5 inactivation (Wu et al., 2016; Mansour et al., 2015). In addition, the expression of SATB2 in BCa tissues was significantly higher than in normal tissues and higher SATB2 expression showed a significant association with BCa grade and poorer overall survival (OS) (Patani et al., 2009), however , the function roles of SATB2 in BCa remained poorly understood. In the present study, we measured the expression levels of miR-31 in three BCa cell lines, BCa tissues and adjacent non-tumor tissues, and investigated the clinicopathological and prognostic values of miR-31 in patients with BCa. We found the expression of miR-31 was significantly reduced in TNBC tissues and MDA-MB-231 cell line, and downregulation of miR-31 levels in patients with BCa was associated with shortened survival. Therefore, we demonstrated that miR-31 as a tumor metastasis-suppressor, overexpression of miR-31 significantly inhibited MDA-MB-231 cells migration and invasion. Furthermore, we identified SATB2 may be an oncogene in BCa cells and as a direct and functional target for miR-31, thus serving as a potential therapeutic target for BCa. 2. Materials and methods 2.1. Clinical sample collection Fresh BCa tissue and paired adjacent non-tumor breast tissues were obtained from patients with a diagnosis of primary BCa and then undergoing surgery at Jiangsu Cancer Hospital (Nanjing, China) from 2013 to 2016 (Table 1). None of the patients received chemotherapy or radiotherapy before the surgery. A total of 77 cases of fresh BCa tissue (mean age 52 years; range 36–75) which contains 18 cases of TNBC tissue (mean age 51 years; range 36–64) were freshly frozen in liquid nitrogen until further use. All of the patients were given written informed consent and the study was approved by the Ethics Committee of the Jiangsu Cancer Hospital. 2.2. Cell lines and cultures The human BCa cell lines MCF-7 (low-invasiveness normal breast cancer cells), MDA-MB-231 (high-invasiveness TNBC cells) and HBL-

Table 1 Clinicopathological associations of miR-31 expression in primary BCa. Variable

Number of cases

miR-31 expression Low %

High %

p Value

Age (years) b50 ≥50

33 44

17 (22.08%) 21 (27.27%)

16 (20.78%) 23 (29.87%)

0.820

ERa Negative Positive

31 46

12 (15.58%) 26 (33.77%)

19 (24.67%) 20 (25.98%)

0.165

PRa Negative Positive

39 38

16 (20.78%) 22 (28.57%)

23 (29.87%) 16 (20.78%)

0.174

Her-2b Negative Positive

49 28

23 (29.87%) 15 (19.48%)

26 (33.77%) 13 (16.88%)

0.803

Tumor size ≤2CM N2CM

31 46

14 (18.18%) 24 (31.17%)

17 (22.08%) 22 (28.57%)

0.644

TNM stagec Stage I/II Stage III/IV

66 11

33 (42.86%) 5 (6.49%)

33 (42.86%) 6 (7.79%)

0.999

Lymph node status No metastasis 42 Metastasis 35

15 (19.48%) 23 (29.87%)

27 (35.07%) 12 (15.58%)

0.012

Bold numbers indicate significance at p b 0.05. a The expression of ER and PR in tumor cells ≥1% as positive value (b1% as negative value). b Her-2 positive/negative was performed according to the ASCO (American Society of Clinical Oncology)/CAP(College of American Pathologists)guidelines for HER2 testing in breast cancer c Cancer staging was performed according to the International Union against Cancer's (UIAC) tumor-node-metastasis (TNM) system.

100 (normal breast epithelial cells) were obtained from the American Type Culture Collection (Rockville, Md). MCF-7 and HBL-100 were cultured in RPMI-1640 (KEYGEN, Nanjing China) supplemented with 10% fetal bovine serum (Gibco, life technologies), MDA-MB-231 was grown in L-15(KEYGEN, Nanjing China) supplemented with 10% fetal bovine serum (Gibco, life technologies), all the cell lines culture at 37 °C in a humidified atmosphere of 95% air and 5% CO2. 2.3. Transient miRNA and siRNA transfection Human hsa-miR-31 mimics, inhibitor, negative control and small inhibitory RNA (si-RNA) against human SATB2, NC-siRNA, were synthesized by RIBOBIO (Guangzhou, China). MCF-7, MDA-MB-231 and HBL100 cells were plated in 6-well plates (4.0 × 105 cell/well), after 24 h of cell adherence, then transfected with miR-31 mimic (at 100 nM final concentration), inhibitor (100 nM), or negative control (100 nM) using Lipo2000 (Invitrogen, Carlsbad, CA). Si-RNA and NC-siRNA transfected cells in the same way and all operations following the manufacturer's instructions. After 24 or 48 h post-transfection, cells were harvested for subsequent experiments. 2.4. Total RNA extraction and qRT–PCR The total RNA of cells and tissues were extracted using TRIzol® Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. The primers were synthesized by RIBOBIO (Guangzhou, China) as follows: miR-31 reverse transcription primer: CTCAACTGGTGTCGTG GAGTCGGCAATTCAGTTGAGAGCTATGC; forwards primer: ACACTCCA GCTGGGTGGCAAGATGCTGGC; reverse primer: TGGTGTCGTGGAGTCG. U6 reverse transcription primer: AACGCTTCACGAATTTGCGT; forwards primer: CTCGCTTCGGCAGCACA; reverse primer: AACGCTTCACGAATTT GCGT. For quantitative real-time PCR, RNA was first reversely

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transcribed into complementary DNA (cDNA) using the TaqMan® Reverse Transcription Kit (Applied Biosystems; Life Technologies), and the cDNA was spotted using TaqMan® Assay Kit (Applied Biosystems; Life Technologies) according to the manufacturer's instructions with a ABI 7300 real-time PCR machine (Applied Biosystems, Carlsbad, CA). The relative mRNA or miRNA expression level were calculated using the ΔΔCt method and normalized with β-actin or U6 expression (fold difference relative to β-actin or U6), respectively. All qRT-PCR were performed in triplicate.

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2.9. Wound healing assay The wound healing assay was performed in order to measure cell migration. We drew three black mark lines each well on the back of 6well tissue culture plates before cell was placed. When cells were grown to confluent monolayer, a line was scratched through cells each well and low-serum media (0.5% FBS) was added. The three intersections of black mark and wound lines was selected for measure. The wound widths was visualized at 0, 24, 48, or 96 h. Each experiment was performed in triplicate.

2.5. Drug treatment 2.10. Western blot MDA-MB-231 cells were plated into 6-well plates at a density of 80 × 104 cells/well. All cells were treated with 2.5 μm/L, 5 μm/L and 10 μm/L 5-AZA-CdR, and different concentrations of cells were cultured for 48, 72 and 96 h, and then cells were harvested to assess miR-31 expression. 2.6. Luciferase reporter assay The SATB2 3′-UTR fragment containing putative binding sites for miR-31 was synthesized and cloned into the PGL3-promoter vector, designated SATB2-3'UTR-WT. The SATB2-3′UTR mutant constructs (SATB2-3′UTR-MUT) was designated by substituting the seed region of the miR-31-binding site. BCa cells (5 × 104/well) were incubated in 24-well plates. MiR-31 mimics and mimics control, PGL3-SATB2-3′ UTR-WT or PGL3-SATB2-3′UTR-MUT vector containing firefly luciferase reporter gene and 3′UTR of SATB2 gene (Promega, Madison, WI) were co-transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 48 h, cells were harvested and luciferase activity was analyzed by using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol and normalized with renilla luciferase.

The total cellular protein was extracted using a modified RIPA buffer (Shanghai, China) supplemented with proteinase inhibitor cocktail (Complete mini, Roche). The equal amounts of protein lysates were separated on SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (PVDF; Millipore, USA) by electroblotting. The membrane was blocked with 5% non-fat milk and then was incubated with primary antibodies: SATB2 (1: 1000, Abcam, USA), at 4 °C overnight. The next day, the membranes were washed and then incubated with anti-rabbit secondary antibody for 1 h at room temperature. The intensity of protein bands was quantified using image J software. 2.11. Expression datasets In order to explore the relationship between the expression level of miR-31 and the prognosis of patients, we analyzed the Cancer Genome Atlas project (TCGA dataset, https://tcga-data.nci.nih.gov/tcga/) dataset. A total of 390 patients with miR-31 expression data were enrolled in the present study. 2.12. Statistical analysis

2.7. Cell proliferation assay Cell Counting Kit-8 assay (CCK-8, Dojindo, Kumamoto Prefecture, Kyushu, Japan) was used to detect cell proliferation. After transfection, cells was seeded into 96-well plates (3 × 103 cells/well) and measured. Mixed 10 μL of CCK-8 with 100 μL of culture medium were inserted to each well and incubated in a humidified atmosphere for 0.5 h at 37 °C. Subsequently, the absorbance was detected at 450 nm using CliniBio128 (ASYS-Hitech, Austria). Each experiment was performed in triplicate. 2.8. Cell migration and invasion assay We used a transwell cell migration and Matrigel invasion assay to measure the migration and invasion ability of MCF-7 and MDA-MB231 cell lines. In both assays, 6.5 mm diameter tissue culture inserts with an 8.0 μm pore size (Transwell; Corning, USA) were placed into 24-well tissue culture plates. For migration assays, 4.0 × 104 MCF-7 cells/well and 3.0 × 104 MDA-MB-231 cells/well were added to the upper chamber lined with a non-coated membrane. For invasion assays, Serum-free RPMI1640 or L-15 medium was mixed with Matrigel (1:8; BD Biosciences, Bedford, MA, USA), chamber inserts were coated with 50 μL of the mixture. After the Matrigel was solidified at 37 °C for 3 h, 6.0 × 104 MCF-7 cells/well and 4.0 × 104 MDA-MB-231 cells/well were added to the upper chamber. In both assays, MCF-7 and MDAMB-231 cell were suspended in 200 μL RPMI 1640 and L-15 serumfree medium, and 500 μL RPMI 1640 and L-15 medium supplemented with 20% FBS was placed into the lower chamber, respectively. After 24 h of incubation at 37 °C with 5% CO2, the invaded cells on the lower surface were fixed in 4% paraformaldehyde, stained with crystal violet and counted with a microscope (×100 magnification). Four random fields were analyzed for each insert and the experiment was performed in triplicate.

Statistical analyses were performed using SPSS 17.0 software (SPSS Inc., USA). The data are presented as the mean ± standard deviation (SD) from at least three independent experiments. Paired Wilcoxon test was performed to compare miR-31 expression between BCa tissue and paired adjacent non-tumor breast tissues. Pearson's χ2 test was used to assess the correlations between miR-31 expression and clinicopathological features. Overall survival curves were estimated using the Kaplan–Meier method, and the probability (P) values were calculated using the log-rank test. Differences between groups were assessed by two-tailed Student's t-test. All of the P values were two-sided, and p b 0.05 was considered statistically significant. 3. Results 3.1. The expression of microRNAs in MDA-MB-231 breast cancer cell line From our laboratory has made a series of microarray on TNBC progression in lymph node metastasis during chemotherapy (data not published) and a previously published article in our laboratory on BCa drug resistance microarray (Chen et al., 2014), we screened out 22 miRNAs which may be associated with TNBC invasion. The expression of 22 candidate miRNAs was assayed by qRT-PCR in MDA-MB-231 cells, we found miR-31 expression was specifically attenuated in MDA-MB-231 (Fig.1). So, we focused on the function of miR-31 in TNBC. 3.2. MiR-31 is downregulated in BCa tissue and cell lines The expression of miR-31 was downregulated in 50 of 77 BCa tissues compared to the adjacent non-tumor tissues (Fig. 2A). As shown in Fig. 2B, the expression of miR-31 in BCa tissues was lower than in adjacent non-tumor tissues (p b 0.001), and miR-31 expression was significantly

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Fig. 1. The expression of microRNAs in MDA-MB-231 TNBC cell line. The expression level of miR-31 was the lowest in the 22 candidate miRNAs.

lower in the 18 pairs TNBC tissues specifically (Fig. 2C, p b 0.01). Moreover, the lower expression of miR-31 was associated with shorter survival in BCa patients (Fig. 2D, p b 0.05). In BCa cell lines, miR-31 was significantly downregulated in low-invasiveness MCF-7 cells and highinvasiveness TNBC MDA-MB-231cells compared to normal breast epithelial cells HBL-100. Specifically, the relative expression of miR-31 in MDA-MB-231 decreased by about 85-fold compared with HBL-100 (Fig. 2E). Then, we assessed the association of miR-31 expression with clinicopathological features of the BCa patients recruited. The results showed that low miR-31 expression were associated with lymph node metastasis (p = 0.012, Table 1).

3.3. 5-AZA-CdR increase miR-31 expression in MDA-MB-231 The expression of miR-31 was remarkably downregulated in MDAMB-231. Augoff et al. reported the loss expression of miR-31 in MDAMB-231 was mainly attributed to hyper-methylation of its promoterassociated CpG islands (Augoff et al., 2012). So, MDA-MB-231 cells were treated with 5-AZA-CdR, a DNA methyltransferase inhibitor, through different concentration and different time. We observed that 5-AZA-CdR significantly increased miR-31 expression in dose-and time-dependent manner (Fig. 3A).

Fig. 2. MiR-31 is downregulated in BCa cell lines and tissues. (A) qRT-PCR analysis of miR-31 expression in 77 pairs BCa tissues and their corresponding no tumor tissues. The relative expression of miR-31 was downregulated in 50 BCa tissues compared to the adjacent non-tumor tissues. (B) The expression of miR-31 in BCa tissues was lower than in adjacent nontumor tissues. (C) The expression of miR-31 in 18 pairs TNBC tissues was significantly lower than in their adjacent non-tumor tissues. (D) Kaplan–Meier curves for overall survival in the patients divided according to median miR-31 expression. Patients with low miR-31 had significantly poorer overall survival. (E) Expression levels of miR-31 in three cell lines (HBL-100, MCF-7, MDA-MB-231). The expression of miR-31 was significantly downregulated in TNBC cell line MDA-MB-231. ***p b 0.001.

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3.4. Upregulation of miR-31 inhibits MDA-MB-231 cell migration and invasion, but not affects proliferation To explore the function of miR-31 in TNBC, MDA-MB-231 was transfected with miR-31 mimics which significantly increasing the expression of miR-31 (Fig. 6B). To analyze the effect of miR-31 on cells motility we performed wound healing assay and Transwell experiments. Forcing the expression of miR-31 significantly inhibited cells migration and invasion compared with control group (Fig. 4A and B). The wound healing assay showed the same results that migration ability of MDA-MB-231 was significantly decreased (Fig. 4C). We have confirmed that 5-AZA-CdR significantly increased miR-31 expression in dose-and time-dependent manner. So, through the Transwell experiment we confirmed again that the migration and invasion ability of MDA-MB231 cells treated with 5-AZA-CdR were significantly decreased (Fig. 3B and C). Interestingly, high expression of miR-31 not affected cells proliferation (date not show). 3.5. Downregulation of miR-31 promotes MCF-7 cell migration and invasion, but not affects proliferation We observed the expression of miR-31 was gradually decreased in three BCa cell lines (Fig. 2E). We suspected that low expression of miR-31 could allow low-invasiveness cells to gain high-invasiveness. To do so, MCF-7 cells were transfected with miR-31 inhibitor with high transfection efficiency (Fig. 6E). So the Transwell assay confirmed that down expression of miR-31 in MCF-7 cells was significantly enhanced cell migration and invasion compared with control cells (Fig. 5A and B). The wound healing assay showed the same results that migration ability of MCF-7 cells loss miR-31 was significantly increased (Fig. 5C). But, the miR-31 did not affected MCF-7 proliferation too. 3.6. Negative correlation of the expression of miR-31 and SATB2 in breast cancer The expression of miR-31 in HBL-100, MCF-7 and MDA-MB-231 was gradually decreased (Fig. 2E), whereas the level of STAB2 was gradually increased (Fig. 6A), indicated that the expression of miR-31 and STAB2 was negatively correlated. In order to further prove the relationship, miR-31 mimics, inhibitor and corresponding negative control were successfully transfected into MCF-7(Fig. 6E) and MDA-MB-231(Fig. 6B) cells. We performed qRT-PCR to determine the expression level of miR-31 and SATB2, and western blot to determine the protein expression of SATB2. We observed that SATB2 was decreased along with the increase of miR-31 both in MCF-7 (Fig. 6F and G) and MDA-MB-231

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(Fig. 6C and d), and vice versa. So, SATB2 is inversely correlated with miR-31 expression and SATB2 may be a target gene of miR-31. 3.7. SATB2 is a direct target gene of miR-31 in breast cancer According to four bioinformatic algorithms (TargetScan, Pictar, microRNA and miRecord), we found that 3′-UTR of STAB2 contained a conserved putative target site for miR-31 (Fig. 7A). We have confirmed miR-31 and SATB2 were inversely expressed in BCa cells. This result indirectly indicated that the expression of SATB2 could be mediated by miR-31, so we regarded SATB2 as a potential target of miR-31. To further confirm whether SATB2 was directly regulated by miR-31 through binding to its 3′UTR, the dual luciferase reporter assay was performed. We constructed luciferase reporter vectors that included wild-type (Wt) and mutant (Mut) miR-31 target sequences of the SATB2-3′UTR. The MCF-7 and MDA-MB-231 cells were transfected with the luciferase reporter gene fused to the Wt or Mut SATB2 3′-UTR with miR-31 mimics and mimics control. The assays showed miR-31 significantly inhibited the luciferase activity of Wt SATB2-3′-UTR reporter gene (p b 0.05) and the luciferase activity with the Mt. SATB2-3′-UTR was not affected by miR-31 in MDA-MB-231 (Fig. 7B). However, the dual luciferase reporter assay result was the same as in the MCF-7 (Fig. 7C). Therefore, the results indicated SATB2 is a direct target gene of miR31 in BCa. 3.8. SATB2 as an oncogene in breast cancer We used siRNA to silence SATB2 expression in MDA-MB-231 (Fig. 8A and B). We found that the invasion and migration ability were significantly inhibited after SATB2 was silenced (Fig. 8C and D). Subsequently, we performed CCK-8 assay to verify the effect of SATB2 on MDA-MB231 cells proliferation. From the cell proliferation curve, it can be seen that the proliferation ability of MDA-MB-231 cells were significantly inhibited after SATB2 was silenced (Fig. 8E). In MCF-7cells we observed the similar results. The siRNA of SATB2 was successfully transfected into MCF-7 cells (Fig. 9A and B) and significantly inhibited cells migration and invasion (Fig. 9C and D). The siRNA also inhibited MCF-7 cells proliferation (Fig. 9E). We have confirmed that miR-31 targeted regulation the expression of SATB2, but neither upregulated nor downregulated the expression of miR-31 could affect BCa cells proliferation, so we suspected that the effect of SATB2 on cell proliferation is not affected by miR-31. In order to prove the hypothesis we co-transfection miR31 mimics and inhibitor with Si-SATB2 in MDA-MB-231 and MCF-7 cells respectively. Interestingly, the two BCa cell lines proliferation were affected not by miR-31 but by SATB2 (Fig. 8F and Fig. 9F).

Fig. 3. 5-AZA-CdR increase miR-31 expression in MDA-MB-231. (A) The MDA-MB-231 cells were treated with 5-AZA-CdR through different concentration and different time. 5-AZA-CdR significantly increased miR-31 expression in dose-and time-dependent manner. (B) The MDA-MB-231 cells who were treated by AZA-CdR could significantly inhibited cell migration ability as determined by cell migration assay. The relative migration cells of each group have been shown in the right, the data are means ± SD. (C) The MDA-MB-231 cells who were treated by AZA-CdR could significantly inhibited cell invasion ability as determined by cell invasion assay. The relative invasive cells of each group have been shown in the right, the data are means ± SD. **p b 0.01,*p b 0.05.

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Fig. 4. Upregulation of miR-31 inhibits MDA-MB-231 cell migration and invasion. (A) The MDA-MB-231 cells were transfected with miR-31 mimics could significantly inhibited cell migration ability as determined by cell migration assay. The relative migration cells of each group have been shown in the right, the data are means ± SD. (B) The MDA-MB-231 cells were transfected with miR-31 mimics could significantly inhibited cell invasion ability as determined by cell invasion assay. The relative invasive cells of each group have been shown in the right, the data are means ± SD. (C) The MDA-MB-231 cells were transfected with miR-31 mimics could significantly inhibited cell migration ability as determined by wound healing assay. The relative open would of each group have been shown in the right, the data are means ± SD. ***p b 0.001,*p b 0.05.

The expression of SATB2 was silenced could significantly inhibit BCa cells proliferation, migration and invasion, and the effect of SATB2 on proliferation was independent of miR-31, so we think SATB2 may be an oncogene in BCa.

(Fig. 10D). From Fig. 10E and F, we could see the migration and invasive ability indeed recovered to some extent when the co-transfection group compared to the si-group. From the above experimental results, we believed that miR-31 suppressed breast cancer cell migration and invasion by targeting SATB2, at least partly.

3.9. MiR-31 inhibits breast cancer cell migration and invasion by targeting SATB2

4. Discussion

In order to further test whether the effect of miR-31 on BCa cells migration and invasion was exerted via SATB2, a rescue experiment was performed. MDA-MB-231 was transfected with miR-31 mimics, siSATB2 and both of them (Fig. 10A). As revealed in Fig.10B and C, the co-transfection group was not significantly inhibited cell migration and invasion compared to mimics-group or Si-group, however, the migration and invasive ability recovered to some extent when the cotransfection group compared to the mimics-group. MCF-7 was transfected with miR-31 inhibitor, si-SATB2 and both of them

Breast cancer is the most common cancer in women worldwide, and metastasis is one of the main death factors in cancer patients (Jemal et al., 2009; Varki et al., 2009; Nguyen et al., 2009). The patients of TNBC, which defined as lack of the expression of ER, PR and HER-2, were unavailable for molecular targeted and hormone therapy, companied with worse prognosis (Carey et al., 2010; Anders and Carey, 2009; Foulkes et al., 2010). MiRNAs, a class of highly conserved small non-coding RNAs, play a crucial role in tumor development and procession, through regulating gene expressions at the post-

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Fig. 5. Downregulation of miR-31 promotes MCF-7 cell migration and invasion. (A) The MCF-7 cells were transfected with miR-31 inhibitor could significantly promoted cell migration ability as determined by cell migration assay. The relative migration cells of each group have been shown in the right, the data are means ± SD. (B) The MCF-7 cells were transfected with miR-31 inhibitor could significantly promoted cell invasion ability as determined by cell invasion assay. The relative invasive cells of each group have been shown in the right, the data are means ± SD. (C) The MCF-7 cells were transfected with miR-31 inhibitor could significantly promoted cell migration ability as determined by wound healing assay. The relative open would of each group have been shown in the right, the data are means ± SD. ***p b 0.001, **p b 0.01.

transcriptional/translational level (Calin and Croce, 2006). Previous studies have identified that abnormal expression of miR-31 was involved in different kinds of cancers (Yu et al., 2016; Zhang et al., 2016; Wang et al., 2016; Yang et al., 2013; Ning et al., 2014). Especially, miR31 as a metastasis-suppressor in BCa (Valastyan et al., 2009). However, the mechanism of miR-31 effects on metastasis is still poorly understand. In this study, we found that miR-31 was downregulated in TNBC tissues and remarkable loss in high-invasiveness MDA-MB-231 TNBC cell line, while downregulation of miR-31 expression correlated with lymph node positive cases and shorter overall survival (OS) of BCa patients. These evidences support that miR-31 as a metastasis related microRNA in BCa. We found the expression of miR-31 was significantly repressed in MDA-MB-231, so some mechanisms may inhibit miR-31 expression. While MDA-MB-231 cells were treated with 5-AZA-CdR, the expression of miR-31 was significantly increased in dose- and time-dependent

manner. Viré et al., for the first time elucidated the mechanism of regulation of miR-31, demonstrated that ETS-1 recruits oncogene EMSY and the histone demethylase KDM5B to the miR-31 promoter, thus repressing its transcription (Viré et al., 2014). Recently studies showed that miR-31 expression was silenced by promoter methylation in MDA-MB-231 (Augoff et al., 2012) and the miR-31 promoter also undergoes aberrant methylation in breast cancer patients (Vrba et al., 2013), increasing evidences also showed that some miRNAs were also epigenetically silenced by the promoter DNA methylation in cancers (Kozaki and Inazawa, 2012). Therefore, the loss of miR-31 expression in MDA-MB-231 is a multi-factor effect. In subsequent experiments, enhance the expression of miR-31 could significantly inhibited MDA-MB-231 cells migration and invasion, moreover, we observed that miR-31 expression decreased with the increase of the invasive ability of BCa cell lines and reduced the expression of miR-31 in MCF-7 can increase the cells invasion ability. This result showed miR-31 as an anti-metastatic microRNA in BCa, but

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Fig. 6. Negative correlation of the expression of miR-31 and SATB2 in breast cancer. (A) The relative expression of miR-31 in HBL-100, MCF-7 and MDA-MB-231 was gradually decreased. (B) The miR-31 mimics can enhance the expression of miR-31 and miR-31 inhibitor can repress the expression of miR-31 in the MDA-MB-231 cells. (C) qRT-PCR analysis of STAB2 mRNA expression in the MDA-MB-231 cells after treatment with miR-31 mimics or inhibitor or corresponding control. The expression of STAB2 was normalized to β-actin. (D) Western blot analysis of STAB2 expression in the MDA-MB-231 cells after treatment with miR-31 mimics or inhibitor or corresponding control. β-actin was also detected as a loading control. (E) The miR-31 mimics can enhance the expression of miR-31 and miR-31 inhibitor can repress the expression of miR-31 in the MCF-7 cells. (F) qRT-PCR analysis of STAB2 mRNA expression in the MCF-71 cells after treatment with miR-31 mimics or inhibitor or corresponding control. The expression of STAB2 was normalized to β-actin. (G) Western blot analysis of STAB2 expression in the MCF-7 cells after treatment with miR-31 mimics or inhibitor or corresponding control. β-actin was also detected as a loading control.***p b 0.001, **p b 0.01.

interestingly, both miR-31 mimics and inhibitor did not effected cell proliferation in BCa cell lines. Valastyan et al. has demonstrated that miR-31 reduced invasion, motility, and anoikis resistance, with less effect on proliferation in vitro (Valastyan et al., 2009). Körner et al. also reported that miR-31 increased the sensitivity of BCa cells apoptosis by targeting protein kinase C epsilon type (PRKCE)-mediated downregulation of the anti-apoptotic factor B-cell lymphoma-2 (BCL2), but not effected cell growth (Koerner et al., 2013). Some studies showed that miR-31 suppressed cell proliferation, invasion and promoted apoptosis in vivo and vitro in GCa and lungadenocarcinoma (Xu et al., 2016; Wang et al., 2016). However, miR-31 as an oncogene in cervical cancer and colon cancer to inhibit proliferation and invasion (Xu et al., 2013; Wang et al., 2014). From the above we can see miR-31 acts as tumor suppressor or oncogene to affect cell proliferation and invasion in different kind tumors, but miR-31 had less effect on BCa proliferation in vitro, and clinicopathological analysis showed that miR-31 expression was

not associated with tumor size whereas was associated with lymph node positive cases, therefore further highlighting the anti-metastatic potential of miR-31 in BCa. MiR-31 inhibited BCa invasion targeting certain genes, such as Ras homolog gene family, member A (RhoA) and GNA13 (Valastyan et al., 2009; Rasheed et al., 2015). To further explore the other mechanisms of tumor invasion and metastasis by miR-31in BCa, we use bioinformatics analysis to identify that SATB2 may be a target gene of miR-31 which related to metastasis in BCa. From luciferase reporter assays we confirmed that SATB2 is the target gene of miR-31, so next we focused on SATB2. SATB2 is a conserved transcription factor of the SATB family, which has a structural homology consisting of CUT, homeobox, and PDZ domains, involved in gene transcription and chromatin remodeling (FitzPatrick et al., 2003; Britanova et al., 2005). Previous studies showed that SATB2 played important roles in the central nervous system development and involved in craniofacial patterning and osteoblast

Fig. 7. SATB2 is a direct target gene of miR-31 in breast cancer. (A) Predicted miR-31 target sequence in the 3′UTR of STAB2 and mutant containing 7altered nucleotides in the 3′UTR of STAB2. (B) The analysis of the relative luciferase activities of STAB2-WT, STAB2-MUT in the MDA-MB-231 cells. (C) The analysis of the relative luciferase activities of STAB2-WT, STAB2MUT in the MCF-7 cells. *p b 0.05, **p b 0.01.

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Fig. 8. SATB2 promotes proliferation, migration and invasion in MDA-MB-231 cells. (A) The siRNA represses the expression of SATB2 in the MDA-MB-231 cells. (B) Western blot analysis of STAB2 expression in the MDA-MB-231 cells after treatment with siRNA or control. β-actin was also detected as a loading control. (C) The MDA-MB-231 cells were transfected with siRNA could significantly inhibited cell migration ability as determined by cell migration assay. The relative migration cells of each group have been shown in the right, the data are means ± SD. (D) The MDA-MB-231 cells were transfected with siRNA could significantly inhibited cell invasion ability as determined by cell invasion assay. The relative invasive cells of each group have been shown in the right, the data are means ± SD. (E) The MDA-MB-231 cells were transfected with siRNA could significantly inhibited cell proliferation as determined by CCK-8 assay. (F) The MDA-MB-231 cells were transfected with miR-31 mimics and Si-SATB2, cell proliferation were affected not by miR-31 but by SATB2. ***p b 0.001, **p b 0.01, *p b 0.05.

differentiation (Dobreva et al., 2006; Britanova et al., 2005; Gyorgy et al., 2008). Currently study showed that the downregulation of SATB2 was related to shortened survival in patients with GCa patients, and overexpression of SATB2 suppressed GCa cell migration and proliferation (Wu et al., 2016). Moreover, knockdown SATB2 gene expression significantly inhibited osteosarcoma cells migration and invasion (Seong et al., 2015), corresponding to that, the downregulation of SATB2 was associated with Furman grade and AJCC (American Joint Committee on Cancer) staging and poor OS (Guo et al., 2015). SATB2, which remarkably

upregulated in BCa tissues, was related to tumor grade and poor OS (Patani et al., 2009), with its function in BCa remains unknown. In this study, we found that SATB2 was upregulated in BCa cell lines and silence its expression significantly inhibited BCa cells proliferation, migration and invasion. Since miR-31 targeted regulation of SATB2 expression, but miR-31 has no effect on the proliferation of BCa cells, SATB2 is independent of miR-31 to affect BCa proliferation. So, we conjectured that SATB2 may be an oncogene in BCa. Aprelikova et al. showed that SATB2 potential indirectly regulated the expression of MMP3 and

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Fig. 9. SATB2 promotes proliferation, migration and invasion in MCF-7 cells. (A) The siRNA represses the expression of SATB2 in the MCF-7 cells. (B) Western blot analysis of STAB2 expression in the MCF-7 cells after treatment with siRNA or control. β-actin was also detected as a loading control. (C) The MCF-7 cells were transfected with siRNA could significantly inhibited cell migration ability as determined by cell migration assay. The relative migration cells of each group have been shown in the right, the data are means ± SD. (D) The MCF-7 cells were transfected with siRNA could significantly inhibited cell invasion ability as determined by cell invasion assay. The relative invasive cells of each group have been shown in the right, the data are means ± SD. (E) The MCF-7 cells were transfected with siRNA could significantly inhibited cell proliferation as determined by CCK-8 assay. (F) The MCF-7 cells were transfected with miR-31 inhibitor and Si-SATB2, cell proliferation were affected not by miR-31 but by SATB2. ***p b 0.001, *p b 0.05.

TIMP3, both of which were referred to tumor metastasis, leading to a more aggressive phenotype (Aprelikova et al., 2010). Moreover, Mansour et al. have disclosed that SATB2 suppressed colorectal cancer (CRC) cells proliferation, migration and invasion by inactivation of MEK5/ERK5 signaling, and it is significant that the CUT domain of SATB2 is essential for the suppressive function in CRC (Mansour et al., 2015). These researches gave us some inspiration that which domain of SATB2 plays a crucial role in the progression of BCa, and whether

SATB2 promotes BCa progression via ERK singling pathway, this will be value for further focus in our next study. Furthermore, SATB2 was regulated by several miRNAs, such as miR211, miR-34 s and miR-33a-5p (Wei et al., 2012; Mi et al., 2016; Jiang et al., 2015). SATB2 rescued the miR-211-mediated inhibition of cell invasion and proliferation and is a functional target gene of miR-211 in hepatocellular carcinoma (HCC) (Jiang et al., 2015). In our study, we verified that SATB2 was a target gene of miR-31 and has an inverse

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Fig. 10. MiR-31 inhibits breast cancer cell migration and invasion by targeting SATB2. (A) Western blot analysis of STAB2 expression in the MDA-MB-231 cells after treatment with miR-31 mimics or siRNA or both of them. β-actin was also detected as a loading control. (B) The silent expression of SATB2 rescued the miR-31-mediated inhibition of cell migration in MDA-MB231 cells. The relative migration cells of each group have been shown in the right, the data are means ± SD. (C) The silent expression of SATB2 rescued the miR-31-mediated inhibition of cell invasion in MDA-MB-231 cells. The relative invasive cells of each group have been shown in the right, the data are means ± SD. (D) Western blot analysis of STAB2 expression in the MCF-7 cells after treatment with miR-31 inhibitor or siRNA or both of them. β-actin was also detected as a loading control. (E) The silent expression of SATB2 rescued the miR-31-mediated promotion of cell migration in MCF-7 cells. The relative migration cells of each group have been shown in the right, the data are means ± SD. (F) The silent expression of SATB2 rescued the miR-31-mediated promotion of cell invasion in MCF-7 cells. The relative invasive cells of each group have been shown in the right, the data are means ± SD. ***p b 0.001, **p b 0.01, *p b 0.05.

correlation with miR-31 in BCa. Rescue experiments indicated that the most important effect exerted by miR-31 on BCa cells invasion and migration, which was partially reversed when co-transfection with SATB2. These results demonstrated that SATB2 was a functional target gene of miR-31 in BCa. It is worth mentioning that each miRNA can regulate dozens or even hundreds of genes and multiple miRNAs may regulate the same gene, which affect the activities of whole pathways and networks (Pritchard et al., 2012; Gunaratne et al., 2010). Indeed, miR-31 regulated biological functions of different cancers by targeting different genes, such as HuR (Xu et al., 2016), BCL2 (Koerner et al., 2013), and members of the E2F family of transcription factors E2F1 and E2F2 (Lin et al., 2013). SATB2, as a transcription factor of the SATB family, was regulated by miR-211 (Jiang et al., 2015), miR-34s (Wei et al., 2012) and miR-33a-5p (Mi et al., 2016) to exert distinct biological effects. Although increased expression of miR-31 inhibited the migration and invasion in MDA-MB231 cells can be partially rescued by silencing SATB2, we cannot exclude the possibility that other target genes may also involve in the suppressive effects of miR-31 or other miRNAs affect the expression of SATB2 in TNBC. In conclusion, our study indicates that miR-31 functions as a metastasis-suppressor miRNA and SATB2 may be an oncogene in BCa. Furthermore, miR-31 suppressed BCa cells migration and invasion which is partly through its regulation of SATB2. This study may provide a new direction for the molecular mechanism of breast cancer metastasis, and miR-31 may be a potential therapeutic target for the treatment of BCa. Conflict of interest The authors declare no conflict of interest. Acknowledgements This project is funded by National High Technology Research and Development Program of China (No. 2014AA020604), the National

Natural Science Foundation of China (No. 81272470), the National Key Clinical Specialist Construction Programs of China (No. 2013[544]), Major Program of Natural Science Foundation of Jiangsu Province (No. BL2014090), Natural Science Foundation of Jiangsu Province (No. BK20151579).

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