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miR-381 inhibited breast cancer cells proliferation, epithelial-to-mesenchymal transition and metastasis by targeting CXCR4
Biomedicine & Pharmacotherapy 86 (2017) 426–433
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miR-381 inhibited breast cancer cells proliferation, epithelial-to -mesenchymal transition and metastasis by targeting CXCR4 Yubao Xuea,1, Wenjing Xub,1, Wei Zhaoc, Wei Wangd , Dahong Zhange,* , Ping Wuf,* a
Department of Oncology, Huai’an Hospital, Xuzhou Medical University, Huai’an 223002, China Department of laboratory, Huaiyin Hospital of huai’an city, Huai’an 223300, China Department of laboratory, Nanjing Maternity and Child Health Care Hospital, Nanjing 210000, China d Department of Pathology, Nanjing Maternity and Child Health Care Hospital, Nanjing 210000, China e Department of Oncology, Hua’an First People’s Hospital, Nanjing Medical Unversity, Huai’an 223300, China f Department of Pathology, Huai’an Maternity and Child Health Care Hospital, Huai’an 223002, China b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 September 2016 Received in revised form 24 November 2016 Accepted 12 December 2016
Background: MicroRNAs act as posttranscriptional regulators of gene expression in many biological processes, which played a vital role in regulation cancer cells epithelial-to-mesenchymal transition and metastasis. The deregulation of miR-381 has been identified in breast cancer. However, the role and mechanism of miR-381 in breast cancer have not been completely unexplored. Methods: Total RNA was extracted from the tissues of 27 patients with breast cancer and two breast cancer cell lines, respectively. The expression levels of miR-381 were examined by quantitative real-time PCR. The stable overexpress or silence miR-381 expression cells lines and control cells line were constructed by lentivirus infection. Subsequently, cell proliferation, cell migration, invasion assay and western blot assay were performed to detect the biological functions of miR-381 in vitro. Moreover, a luciferase reporter assay was conducted to confirm target associations. Results: In this study, we validated the lower expression of miR-381 in breast cancer tissues than their adjacent non-neoplastic tissues in 27 breast cancer patients. The result also showed that miR-381 was lowly expressed in breast cancer cell lines MCF-7 and MDA-MB-231 than human epithelial cell line MCF-10A. The miR-381 expression was significantly up-regulated under exogenous miRNA-381 treatment in MCF-7 and MDA-MB-231 cells analyzed by quantitative real-time PCR. The results also indicated that an inverse correlation existed between miR-381 expression level and breast cancer cell proliferation, epithelial-to-mesenchymal transition and metastasis. Furthermore, miR-381 was predicted as a regulatory miRNA of CXCR4 in breast cancer, and the data analysis revealed that there was a negatively relationship between miR-381 and CXCR4 expression in breast cancer tissues from the patients. miR-381 played an important role in breast cancer cells proliferation, epithelial-tomesenchymal transition and metastasis by targeting CXCR4. Conclusions: This present study revealed that miR-381 might be considered as a novel therapeutic target for breast cancer treatment. © 2016 Elsevier Masson SAS. All rights reserved.
Keywords: miR-381 Breast cancer CXCR4 Proliferation EMT Metastasis
1. Introduction Breast cancer is one of the most frequently diagnosed cancers [1], which is a major cause of cancer-related death for females worldwide [2]. Although the advances in diagnosis and appropriately systemic therapy, including surgery, chemotherapy and
* Corresponding authors. E-mail addresses: [email protected] (D. Zhang), [email protected] (P. Wu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biopha.2016.12.051 0753-3322/© 2016 Elsevier Masson SAS. All rights reserved.
radiation, had significantly decreased the mortality rates, local recurrence and distant metastasis resulted in poor prognosis [3]. So it is urgent to investigate the underlying mechanisms of breast cancer metastasis. The microRNAs (miRNAs) as an important gene regulation mechanism in epigenetics have attracted widespread attention. miRNAs are a class of endogenous short (about 19–25 nucleotides) noncoding RNAs, which is widely present in eukaryotic cells and plays important regulatory roles in transcription and translation. miRNAs, as important regulators, are involved in the development of many types of human diseases, especially in cancer. The dysregulated expression of miRNAs plays an indispensable role in
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carcinogenesis and cancer progression [4]. There is an increasing amount of evidence that under- and over-expression of several miRNAs in cancer have a close relationship to metastatic progression [5]. For example, miR-143, as an tumor-suppressor, inhibited proliferation and invasion in hepatoma cells [6]; miR-183-5p, an as tumor-promoter, promoteed cells proliferation and inhibited apoptosis in human breast cancer cells [7]. Taken together, miRNAs have been identified as potential candidates for novel diagnostic biomarkers or therapeutic targets of cancer. Epithelial-mesenchymal transition (EMT) is a phenomenon that epithelial tumor cells acquire mesenchymal features. During the EMT, cells lose cell–cell adhesion and convert to fibroblastoid cells with invasive and migratory abilities [8]. Down-regulation of epithelial markers such as E-cadherin and up-regulation of mesenchymal markers including N-cadherin and vimentin are common features of EMT [9]. These changes are accompanied by increasing secretion of matrix metalloproteinase-2 (MMP-2) [10] and cells motility. EMT is regulated by extracellular ligands [11], transcription factors [12] and miRNAs [13–15], which has been proposed to enhance migration, invasion and dissemination in epithelial tumor cells [16,17]. Therefore, EMT is a promising therapeutic target and the inhibition of EMT can restrain or prevent the invasion and metastasis of cancer cells. Recent findings showed that miRNAs play an important role in regulating the EMT process in breast cancer [18]. Previous researches have showed biological functions of miRNA381 in both cancerous and noncancerous conditions. Tang et al. found miR-381 could increase the proliferation of glioma cells in vitro and in vivo through suppressing the glioma suppressor leucine-rich repeat C4 [19], which was firstly reported about the role of miR-381. Subsequently, miR-381 has been reported in colon cancer [20] and prostate cancer [21], suggesting its tumor suppressive function. Recent studies showed that down-regulated expression of miR-381 was associated with metastasis of breast cancer [18,22]. However, the relationship between miR-381 and pathogenesis and metastasis of breast cancer is still unknown. Therefore, this study was designed to investigate the potential molecular mechanisms of miR-381 mediated anti-tumor effects in breast cancer cells. Two human breast cancer cell lines MDA-MB-231 and MCF-7 were transfected with synthetic miR-381 mimics and miR-381 inhibits to observe its effects on the biological behavior of breast cancer cells and to explore its possible mechanisms of action. 2. Materials and methods 2.1. Tissue samples Human clinical samples were collected from surgical specimens from 27 patients with breast cancer at Affiliated Hospital of Xuzhou Medical University according to a standard protocol, before any therapeutic intervention. The corresponding adjacent non-neoplastic tissues from the macroscopic tumor margin were isolated at the same time and used as controls. All subjects provided informed consent before specimen collection. The study protocol was approved by the ethics committee of the Xuzhou Medical University. 2.2. Cell lines and cells transfection Human breast cancer cells lines MDA-MB-231, MCF-7 and human epithelial cell line MCF-10A were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231 cells were cultured in L-15 medium (Gibco, USA) medium supplied with 10% fetal bovine serum (FBS, Gibco, USA), 100 mg/ml streptomycin, 100 IU/ml penicillin in normal air atmosphere at 37 C. MCF-7 cells were cultured in Dulbecco’s
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Modified Eagle’s Medium (DMEM) (Gibco, USA) supplemented with 10% FBS, 100 mg/ml streptomycin, 100 IU/ml penicillin in 5% CO2 atmosphere at 37 C. MCF-10A was cultured in DMEM/F12 (1: 1) (Hyclone, USA) supplemented with 5% horse serum (Gibco, USA), 10 mg/ml insulin, 20 ng/ml EGF, 100 ng/ml cholera toxin and 0.5 mg/ml hydrocortisone. miR-381 mimics and miR-381 inhibitors, negative control (NC) and CXCR4 small interfering RNAs (siRNAs) were purchased from GenePharma Company (Shanghai, People’s Republic of China). Cells transfection was carried out using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s protocol. 2.3. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from the human breast cancer tissues or cells using TRIzol reagent (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using a Firststrand cDNA Synthesis System (Marligen Bioscience, MD). qPCR analysis was performed using the Fast Start Universal SYBR Green Master (Rox) (Roche Applied Science) on an Eppendorf Realplex2 Mastercycler (Eppendorf, Hamburg, Germany). miRNA abundance was normalized to U6. RNA from three separate cell pellets pretreatment was analyzed. Primer pairs used in this study were: miR-381 were 50 -AGTCTATACAAGGGCAAGC TCTC-30 (forward) and 50 -ATCCATGACAGATCCCTACCG-30 (reverse); U6 were 50 -CTCGC TTCGGCAGCACA-30 (forward) and 50 -AACG-CTTCACGAATTTGCGT-30 (reverse). The real-time PCR reactions were performed in triplicate and included no-template controls. 2.4. Cell proliferation assay Cells were seeded in 96-well plates at 5 103 per well. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technology, Rockville, MD, USA) according to the manufacturer’s instructions. The optical density (OD) at 450 nm was recorded on a Microplate Reader (Bio-Rad, Hercules, CA, USA). 2.5. Cell migration and invasion assay Transwell chambers (Corning Incorporated, Corning, NY, USA) with a pore size of 8 mm was used for migration and invasion assays. Briefly, cells were harvested and resuspended in serum-free medium. For migration assay, 4 104 cells in serum-free medium were directly added into the upper chamber. For invasion assay, the melted Matrigel (BD Biosciences, San Jose, CA, USA) was diluted with medium and 30 mL of which was added to the upper chamber of transwell; the chamber was incubated at 37 C for 4 h to allow Matrigel to solidify; and then 1 105 cells in serum-free medium were added into the upper chamber. Medium containing 20% FBS was added to the lower chamber and served as a chemoattractant. After incubation in an incubator under 37 C and 5% CO2 condition for 24 h, cotton swabs were used to gently wipe off the cells remaining on the membrane surface without passing through. The cells that migrated or invaded to the lower surface of the membrane were fixed with 4% paraformaldehyde, stained in 10% crystal violet and wash with PBS, the cells were counted under high-power fields microscopically, 5 randomly selected fields for each chamber; the cell counts in each visual field was recorded. The cell counts passing through the membrane were used to indirectly reflect tumor cells migration and invasion ability. 2.6. Western blot analysis Total cell lysates were extracted from the cells using RIPA buffer (Solarbio, Beijing, China) followed by repetitive pipetting and lysis on
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the ice for 0.5 h; the contents were collected into centrifuge tubes and centrifuged at 12000 r/min; after loading buffer was added to the supernatant, the samples were treated at 100 C for 5 min to denature the proteins; 10 mL sample was loaded into each well for 12% SDS-PAGE and then the proteins were transferred to a PVDF membrane. The membranes were soaked with 8% milk and washed with PBST three times (10 min/time). The membranes were then incubated with the following primary antibodies: rabbit anti-human b-actin antibody, rabbit anti-human MMP-2 antibody, rabbit antihuman MMP-9 antibody, rabbit anti-human N-cadherin antibody, rabbit anti-human Vimentin antibody, rabbit anti-human Ecadherin antibody. All antibodies were purchased from cell signaling technology (Beverly, MA, USA). After incubation for 2 h at room temperature, the membranes were washed with PBST three times again. Then, they were incubated with their respective secondary antibodies for another 2 h. The relative band density was determined using the Tanon 5200 Multifunctional Imaging System (Beijing, China) with the ECL Western Blotting Substrate Kit (Millipore, Billerica, MA, USA). b-actin was used as an internal control. 2.7. Fluorescent reporter assay For the luciferase reporter assay, wild-type 30 UTRs of CXCR4 containing predicted miR-381 target sites were amplified by polymerase chain reaction (PCR) from MDA-MB-231 and MCF-7 cell genomic DNA, and mutant 30 UTRs were obtained by overlap extension PCR method. MDA-MB-231 and MCF-7 cells were transfected with Lipofectamine 2000 (Thermo Fisher Scientific). The transfection mixtures contained 5 pmol of miR-381 and 100 ng of firefly luciferase reporter plasmid, and pRL-TK (Promega Corporation, Fitchburg, WI, USA) was also transfected as normalization control. 48 h after transfection, luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Firefly luciferase activities were normalized to Renilla luciferase activity. Transfections were done in duplicate and repeated at least 3 times in independent experiments.
results indicated that miR-381 was markedly down-regulated in breast cancer tissues compared with adjacent non-cancerous tissues (Fig. 1A), which suggested that the expression of miR-381 may be associated with breast cancer carcinogenesis. We further analyzed miR-381 expression in two breast cancer cells lines (MDA-MB-231, MCF-7) and breast epithelial cell line (MCF-10A). Low expression of miR-381 was shown in breast cancer cells as compared to that of the normal breast epithelial cells (Fig. 1 B). Taken together, these data provided an evidence that miR-381 might has a key role in breast cancer progression. 3.2. miR-381 reduced breast cancer cells proliferation and EMT To confirm the potential functional role of miR-381 in breast cancer, we transfected MDA-MB-231 and MCF-7 cells with miR-381 mimics or miR-381 inhibitors to overexpress or silence miR-381 expression, and the transfection efficiency was detected by qRT-PCR. As showed in Fig. 2A, after tranfected with miR-381 mimics, miR-381 expression was effectively up-regulated, and miR-381 expression was greatly down-regulated in MCF-7 and MDA-MB-231 cells after tranfected with miR-381 inhibitors. A CCK-8 assay was performed to detect the proliferation of MCF-7 and MDA-MB-231 cells after transfection with miR-381 mimics for 24, 48, and 72 h. As demonstrated by CCK-8 proliferation assays, overexpression of miR-381 significantly decreased cell growth compared with the negative control cells. After transfection with miR-381 inhibitor, the proliferation was significantly increased in MCF-7 and MDA-MB-231 cells (Fig. 2B). Subsequently, we found miR-381 significantly reversed EMT of MCF-7 and MDA-MB-231 cells based on the observation that miR381 increased epithelial marker (E-cadherin) and inhibited mesenchymal markers (N-cadherin and vimentin). After transfection with miR-381 inhibitor, the protein of epithelial marker E-cadherin was significantly down-regulated and the protein of N-cadherin and vimentin was significantly up-regulated (Fig. 2C). 3.3. miR-381 suppressed cells migration and invasion
2.8. Statistical analysis Graph Pad Prism 5.0 statistical software was used to analyze the above experimental data. Measurement data were represented as X S.D; ANOVA and paired t-test was used to analyze the intergroup differences. The statistical significance is indicated with asterisks 3. Results 3.1. miR-381 was down-regulated in breast cancer tissues and cell lines We firstly examined the expression level of miR-381 by qRTPCR in breast cancer tissues and paired non-cancerous tissues. The
We further assessed the effects of miR-381 on cells migration and invasion, which were the key determinants of malignant progression and metastasis. We transfected miR-381 mimics in MCF-7 and MDA-MB-231 cells to study the migration and invasion of breast cancer cells. Compared with the cells transfected with control, the ability to migration in breast cancer cells decreased owing to a stable over-expression of miR-381. Furthermore, we observed the effect of miR-381 on invasiveness capacity of breast cancer cells by matrigel invasion assay. When matrigel invasion assay was performed on breast cancer cells, it was found that the invasiveness was lower for breast cancer cells with stable overexpression of miR-381 compared with control cells. To confirm the effects of miR-381 on modulating the malignant phenotypes of breast cancer cells, we also investigated the change of aggressive
Fig. 1. The expression of miR-381 in breast cancer tissues and cells. (A) miR-381 expression in breast cancer tissues and the normal tissues was examined by qRT-PCR. (B) miR381 expression in breast cancer cell lines was examined by qRT-PCR. **P < 0.01 vs control.
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Fig. 2. miR-381 inhibited breast cancer cells proliferation and EMT. (A) MCF-7 and MDA-MB-231 cells transfected with miR-381 mimics or miR-381 inhibitors both had significantly increased or decreased miR-381 expression compared with the cells transfected with negative control. (B) MCF-7 and MDA-MB-231 cells transfected with miR381 mimics or miR-381 inhibitors both had obvious decreased or increased the proliferation rate compared with the control groups. (C) MCF-7 and MDA-MB-231 cells transfected with miR-381 mimics had dramatic increased E-cadherin expression and decreased in vimentin and N-cadherin expression compared to the cells transfected with control; MCF-7 and MDA-MB-231 cells transfected with miR-381 inhibitors had decreased E-cadherin expression and increased vimentin and N-cadherin expression compared to the controls. *P < 0.05, **P < 0.01.
phenotypes of breast cancer cells after reducing expression of miR-381. miR-381 inhibitor transfection was employed to inhibit miR-381 expression in MCF-7 and MDA-MB-231 cells. After transfection with miR-381 inhibitor, the migration and invasion of bread cancer cells were significantly increased compared with the cells transfected with control (Fig. 3A and B). To investigate cells invasive capacity, proteins related to invasion were studied in MCF-7 and MDA-MB-231 cells. MMP-2 and MMP-9 expression was obviously decreased in both MCF-7 and MDA-MB-231 cells treated with miR-381 mimics at the protein level, as detected by western blot analysis. In comparison, the inhibition of miR-381 enhanced the expression of MMP-2 and MMP-9 (Fig. 3C). These results demonstrated that miR-381 played important roles in regulating cells migration and invasion in breast cancer cells and suggested that the down-regulation of miR-381 might contribute to tumor metastasis in breast cancer carcinogenesis. 3.4. CXCR4 is a direct target of miR-381 To explore the possible mechanisms of regulation by miR-381, miR-381 targets were analyzed by using the TargetScan bioinformatics predictions. Software analysis revealed that Chemokine (CX-C motif) receptor 4 (CXCR4), might be a potential target of miR381 based on putative target sequences of the CXCR4 30 UTR (Fig. 4A). We further performed luciferase assay to determine whether miR-381 could directly target 30 UTR of CXCR4. The target sequence of CXCR4 30 UTR (CXCR4-WT) or mutant sequence (CXCR4-Mut) was cloned into luciferase vector. The results showed that miR-381 significantly decreased the luciferase activity of the CXCR4 30 -UTR but not mutant sequence (Fig. 4B). To clarify their regulatory relationship, we first detected the mRNA levels of CXCR4 in miR-381 mimic-transfected MCF-7 and MDA-MB231cells or miR-381 inhibitor-transfected using qRT-PCR. The results revealed that the over-expression of miR-381 obviously decreased the mRNA level of CXCR4. In comparison, the inhibition
of miR-381 enhanced the expression of CXCR4 (Fig. 4C). These results suggested that CXCR4 is a direct target of miR-381. 3.5. CXCR4 suppressed breast cancer cells proliferation, EMT and metastasis We further examined whether CXCR4 is a substantial target of miR-381 involved in regulating the migration and invasion of breast cancer cells. Specific siRNA targeting CXCR4 (si-CXCR4) was transfected into MCF-7 and MDA-MB-231 cells to suppress CXCR4 expression. The CCK-8 assays showed that suppression of CXCR4 in cells dramatically decreased cells proliferation of human breast cancer cells (Fig. 5A). The cell migration and invasion assays showed that cells migration and invasion were suppressed after transfection with si-CXCR4 (Fig. 5B and C). The results of western blot experiments showed that malignant breast cancer cells biology behavior-related protein MMP-2 and MMP-9 decreased, as well as mesenchymal markers (N-cadherin and vimentin) decreased, and E-cadherin protein expression quantity increased after transfection with si-CXCR4 (Fig. 5D). Taken together, our results demonstrated that CXCR4 was a functionally important target of miR-381 and was involved in miR-381 regulated human breast cancer cells proliferation, EMT, migration and invasion. 3.6. miR-381 was negatively correlated to CXCR4 in breast cancer tissues As the above results suggested CXCR4 is a direct target of miR-381 could inhibit cell proliferation, EMT, migration and invasion. Therefore, we performed qRT-PCR to explore the expression pattern of CXCR4 in breast tumor tissues. The data validated that CXCR4 mRNA level was up-regulated in breast cancer tissues compared with paired non-cancerous tissues (Fig. 6A). We also determined CXCR4 expression in two breast cancer cell lines (MDA-MB-231 and MCF-7) and breast epithelial cell line (MCF-10A), qRT-PCR assay and western blot found the
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Fig. 3. miR-381 inhibited the breast cancer cells metastasis. (A) Cell migration was assayed in breast cancer cells with miR-381 transfection. (B) Cell invasion was assayed in breast cancer cells with miR-381 transfection. (C) MMP-2 and MMP-9 expression was assayed in breast cancer cells with miR-381 transfection. *P < 0.05 vs control, **P < 0.01 vs control.
Fig. 4. CXCR4 is a direct target of miR-381 in breast cancer cells. (A) The prediction of the binding between miR-381 and CXCR4 by TargetScan. (B) Luciferase reporter assays were performed to verify the binding of miR-381 in 30 -UTR of CXCR4. (C) qRT-PCR assay was performed to detect the mRNA level of CXCR4 in MCF-7 and MDA-MB-231 cells treated with miR-381 mimics and miR-381 inhibitors. *P < 0.05 vs control, **P < 0.01 vs control.
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Fig. 5. miR-381 inhibited the breast cancer cells metastasis and EMT. (A) CCK-8 assay showed that the si-CXCR4 group had a lower proliferation rate than the control group in the breast cancer cell. (B) Migration assay shows that the number of migrated cells in the si-CXCR4 group markedly decreased compared with that in the control. (C) Invasion assay shows that the number of invaded cells in the si-CXCR4 group remarkably decreased compared to that in the control. (D) The si-CXCR4 group had obvious decreases in MMP-2, MMP-9, vimentin and N-cadherin expression and a significant increase in E-cadherin expression compared to the control. *P < 0.05, **P < 0.01.
mRNA/protein level of CXCR4 was up-regulated (Fig. 6B and C). Furthermore, we also investigated the relationship between CXCR4 and miR-381, as we can see from Fig. 6D, the expression level of CXCR4 was inversely correlated with the level of miR-381. 4. Discussion Increasing reports have demonstrated that miRNAs are an important regulator of tumor progression and metastasis. In breast
cancer, many miRNAs have been identified to regulate known genes that are involved in the pathology of tumorigenesis and metastasis. In the present study, we found miR-381 was downregulated in breast cancer tissues and cell lines. Previous researches also showed other biological functions of miRNA-381 in both cancer tissues and cell lines. Yan Liang [20] and Xinxin He [23] found that up-regulation of miRNA-381 inhibited cells proliferation and invasion in colon cancer cells. This effect was also observed in renal cancer cells, which miRNA-381 could
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Fig. 6. Relationship of miR-381 and CXCR4 in the breast cancer tissues. (A) Data analysis of CXCR4 mRNA expression in breast cancer tissues with the normal tissues. (B) Data analysis of CXCR4 mRNA expression in breast cancer cells (MCF-7 and MDA- MB-231cells) with breast epithelial cell line (MCF-10A). (C) CXCR4 protein expression in breast cancer cells lines (MCF-7 and MDA- MB-231cells) and breast epithelial cells line (MCF-10A). (D) Data analysis of relationship between miR-381 and CXCR4 expression in breast cancer tissues (r = 0.9298). **P < 0.01.
suppressed the proliferation of cancer cells [24]. Our data revealed that miRNA-381 could inhibit breast cancer cells proliferation, migration and invasion. Numerous experimental studies have demonstrated an increase of MMPs, especially MMP-2 and MMP-9 with cancer progression. Secretion of cytokines by cancer cells exerts an important influence in the control of cancer cell behavior such as invasion and adhesion [25]. MMP-2 and MMP-9 are regarded as indispensable cytokines for breast cancer cells invasion [26]. Our result found that miRNA-381 could inhibit the expression of MMP-2 and MMP-9 of MCF-7 and MDA-MB-231 cells. MCF-7 and MDA-MB-231 cell lines had been reported to represent low and high invasive breast carcinoma with different proliferative potentials, but in our study there were no significant differences in the amplitude of miR-381 effects on cells proliferation, migration and invasion which needed to be investigated in the future. Emerging evidence has showed that many miRNAs play important roles in the EMT of cancer cells: microRNA-26b inhibits EMT in hepatocellular carcinoma by targeting USP9X, and miR-23a regulated TGF-b-induced EMT in lung cancer cells [27,28]. Breast cancer pathogenesis is particularly accompanied by the EMT that is considered the first step in distant metastasis. In the EMT process,
epithelial cells lose their characteristics, gain mesenchymal features, and become motile and invasive [8]. Loss of epithelial marker E-cadherin and gain of mesenchymal markers N-cadherin and vimentin are regarded as the most important molecular markers of EMT [9]. Our results showed that miR-381 reversed the expression level of epithelial marker and mesenchymal markers in MCF-7 and MDA- MB-231cells and inhibited EMT. We next predicted and validated the possible targets of miR381 in breast cancer cells as the impact of specific miRNAs on cancer biology depends on their downstream targets. miRNA-381 exerts its biological functions through the regulation of various target genes, such as LRH-1 [20], Twist1 [23] and HES1 [29]. Herein, we used the TargetScan bioinformatics predictions to predict another genes, and the binding site of CXCR4 30 UTR was obviously located at miR-381 seed region. We also identified that miR-381 can suppress the mRNA expression of CXCR4 via directly target its 30 UTR in breast cancer cell. It has been found that chemokines play important roles in cancer cell motility, angiogenesis and tumor metastasis [30]. In particular, CXCR4, a class-A G protein-coupled receptor (GPCR), was reported as the most common over-expressed chemokine receptor in human cancer [31], such as breast [32], pancreatic [33] and prostate cancer [34].
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Patients with high expression of CXCR4 in tumors had poor cancer survival [35]. CXCR4 has been considered as a biomarker for poor prognosis and cancer metastasis [36,37]. To further confirm that miR-381 can directly target CXCR4, a knockdown plasmid of CXCR4 (si-CXCR4) was used, which showed that si-CXCR4 can inhibit the cells proliferation, migration, invasion and EMT of MCF-7 and MDA-MB-231 cells. Moreover, si-CXCR4 suppressed the expression of MMP-2 and MMP-9 of breast cancer cells. In order to further examine the mechanism underlying the effect of miRNA-381 in breast cancer cells, the expression of CXCR4 in breast cancer tissue samples and cell lines was measured. The results demonstrated that CXCR4 was over-expressed in breast cancer tissue and cell lines (MCF-7 and MDA- MB-231). In conclusion, the data here suggested that inhibition of miR-381 might be a potential approach to repress the progression of breast cancer through restoring CXCR4 levels, which might provide us new insights into a potential therapeutic strategy for blocking metastasis in breast cancer. Conflict of interest The authors have no conflict of interest pertaining to this manuscript. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin, D. Forman, F. Bray, Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012, Int. J. Cancer. 136 (2015) E359–E386. [2] C.E. DeSantis, C.C. Lin, A.B. Mariotto, R.L. Siegel, K.D. Stein, J.L. Kramer, R. Alteri, A.S. Robbins, A. Jemal, Cancer treatment and survivorship statistics, CA Cancer J. Clin. 64 (2014) 252–271. [3] X.F. Tan, F. Xia, Long-term fatigue state in postoperative patients with breast cancer, Chin. J. Cancer Res. 26 (2014) 12. [4] S. Vimalraj, P.J. Miranda, B. Ramyakrishna, N. Selvamurugan, Regulation of breast cancer and bone metastasis by microRNAs, Dis. Markers 35 (2013) 369–387. [5] L. Wang, J. Wang, MicroRNA-mediated breast cancer metastasis: from primary site to distant organs, Oncogene 31 (2012) 2499–2511. [6] X. Liu, J. Gong, B. Xu, miR-143 down-regulates TLR2 expression in hepatoma cells and inhibits hepatoma cell proliferation and invasion, Int. J. Clin. Exp. Pathol. 8 (2015) 12738–12747. [7] Y. Cheng, G.X. Xiang, Y.B. Meng, R.Z. Dong, MiRNA-183-5p promotes cell proliferation and inhibits apoptosis in human breast cancer by targeting the PDCD4, Reprod. Biol. (2016), doi:http://dx.doi.org/10.1016/j. repbio.2016.07.002. [8] K. Polyak, R.A. Weinberg, Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits, Nat. Rev. Cancer 9 (2009) 265–273. [9] J. Jiang, Y.L. Tang, X.H. Liang, EMT: a new vision of hypoxia promoting cancer progression, Cancer Biol. Ther. 11 (2011) 714–723. [10] M.E. Muroski, M.D. Roycik, R.G. Newcomer, P.E. Van den Steen, G. Opdenakker, H.R. Monroe, Z.J. Sahab, Q.X. Sang, Matrix metalloproteinase-9/gelatinase B is a putative therapeutic target of chronic obstructive pulmonary disease and multiple sclerosis, Curr. Pharm. Biotechnol. 9 (2008) 34–36. [11] C. Scheel, T. Onder, A. Karnoub, R.A. Weinberg, Adaptation versus selection: the origins of metastatic behavior, Cancer Res. 67 (2007) 11476–11479. [12] J.P. Thiery, Epithelial-mesenchymal transitions in tumour progression, Nat. Rev. Cancer 2 (2002) 442–454. [13] P.A. Gregory, A.G. Bert, E.L. Paterson, G.J. Goodall, The miR-200 family and miR205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1, Nat. Cell Biol. 10 (2008) 593–601.
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miR-381 inhibited breast cancer cells proliferation, epithelial-to -mesenchymal transition and metastasis by targeting CXCR4 Yubao Xuea,1, Wenjing Xub,1, Wei Zhaoc, Wei Wangd , Dahong Zhange,* , Ping Wuf,* a
Department of Oncology, Huai’an Hospital, Xuzhou Medical University, Huai’an 223002, China Department of laboratory, Huaiyin Hospital of huai’an city, Huai’an 223300, China Department of laboratory, Nanjing Maternity and Child Health Care Hospital, Nanjing 210000, China d Department of Pathology, Nanjing Maternity and Child Health Care Hospital, Nanjing 210000, China e Department of Oncology, Hua’an First People’s Hospital, Nanjing Medical Unversity, Huai’an 223300, China f Department of Pathology, Huai’an Maternity and Child Health Care Hospital, Huai’an 223002, China b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 September 2016 Received in revised form 24 November 2016 Accepted 12 December 2016
Background: MicroRNAs act as posttranscriptional regulators of gene expression in many biological processes, which played a vital role in regulation cancer cells epithelial-to-mesenchymal transition and metastasis. The deregulation of miR-381 has been identified in breast cancer. However, the role and mechanism of miR-381 in breast cancer have not been completely unexplored. Methods: Total RNA was extracted from the tissues of 27 patients with breast cancer and two breast cancer cell lines, respectively. The expression levels of miR-381 were examined by quantitative real-time PCR. The stable overexpress or silence miR-381 expression cells lines and control cells line were constructed by lentivirus infection. Subsequently, cell proliferation, cell migration, invasion assay and western blot assay were performed to detect the biological functions of miR-381 in vitro. Moreover, a luciferase reporter assay was conducted to confirm target associations. Results: In this study, we validated the lower expression of miR-381 in breast cancer tissues than their adjacent non-neoplastic tissues in 27 breast cancer patients. The result also showed that miR-381 was lowly expressed in breast cancer cell lines MCF-7 and MDA-MB-231 than human epithelial cell line MCF-10A. The miR-381 expression was significantly up-regulated under exogenous miRNA-381 treatment in MCF-7 and MDA-MB-231 cells analyzed by quantitative real-time PCR. The results also indicated that an inverse correlation existed between miR-381 expression level and breast cancer cell proliferation, epithelial-to-mesenchymal transition and metastasis. Furthermore, miR-381 was predicted as a regulatory miRNA of CXCR4 in breast cancer, and the data analysis revealed that there was a negatively relationship between miR-381 and CXCR4 expression in breast cancer tissues from the patients. miR-381 played an important role in breast cancer cells proliferation, epithelial-tomesenchymal transition and metastasis by targeting CXCR4. Conclusions: This present study revealed that miR-381 might be considered as a novel therapeutic target for breast cancer treatment. © 2016 Elsevier Masson SAS. All rights reserved.
Keywords: miR-381 Breast cancer CXCR4 Proliferation EMT Metastasis
1. Introduction Breast cancer is one of the most frequently diagnosed cancers [1], which is a major cause of cancer-related death for females worldwide [2]. Although the advances in diagnosis and appropriately systemic therapy, including surgery, chemotherapy and
* Corresponding authors. E-mail addresses: [email protected] (D. Zhang), [email protected] (P. Wu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biopha.2016.12.051 0753-3322/© 2016 Elsevier Masson SAS. All rights reserved.
radiation, had significantly decreased the mortality rates, local recurrence and distant metastasis resulted in poor prognosis [3]. So it is urgent to investigate the underlying mechanisms of breast cancer metastasis. The microRNAs (miRNAs) as an important gene regulation mechanism in epigenetics have attracted widespread attention. miRNAs are a class of endogenous short (about 19–25 nucleotides) noncoding RNAs, which is widely present in eukaryotic cells and plays important regulatory roles in transcription and translation. miRNAs, as important regulators, are involved in the development of many types of human diseases, especially in cancer. The dysregulated expression of miRNAs plays an indispensable role in
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carcinogenesis and cancer progression [4]. There is an increasing amount of evidence that under- and over-expression of several miRNAs in cancer have a close relationship to metastatic progression [5]. For example, miR-143, as an tumor-suppressor, inhibited proliferation and invasion in hepatoma cells [6]; miR-183-5p, an as tumor-promoter, promoteed cells proliferation and inhibited apoptosis in human breast cancer cells [7]. Taken together, miRNAs have been identified as potential candidates for novel diagnostic biomarkers or therapeutic targets of cancer. Epithelial-mesenchymal transition (EMT) is a phenomenon that epithelial tumor cells acquire mesenchymal features. During the EMT, cells lose cell–cell adhesion and convert to fibroblastoid cells with invasive and migratory abilities [8]. Down-regulation of epithelial markers such as E-cadherin and up-regulation of mesenchymal markers including N-cadherin and vimentin are common features of EMT [9]. These changes are accompanied by increasing secretion of matrix metalloproteinase-2 (MMP-2) [10] and cells motility. EMT is regulated by extracellular ligands [11], transcription factors [12] and miRNAs [13–15], which has been proposed to enhance migration, invasion and dissemination in epithelial tumor cells [16,17]. Therefore, EMT is a promising therapeutic target and the inhibition of EMT can restrain or prevent the invasion and metastasis of cancer cells. Recent findings showed that miRNAs play an important role in regulating the EMT process in breast cancer [18]. Previous researches have showed biological functions of miRNA381 in both cancerous and noncancerous conditions. Tang et al. found miR-381 could increase the proliferation of glioma cells in vitro and in vivo through suppressing the glioma suppressor leucine-rich repeat C4 [19], which was firstly reported about the role of miR-381. Subsequently, miR-381 has been reported in colon cancer [20] and prostate cancer [21], suggesting its tumor suppressive function. Recent studies showed that down-regulated expression of miR-381 was associated with metastasis of breast cancer [18,22]. However, the relationship between miR-381 and pathogenesis and metastasis of breast cancer is still unknown. Therefore, this study was designed to investigate the potential molecular mechanisms of miR-381 mediated anti-tumor effects in breast cancer cells. Two human breast cancer cell lines MDA-MB-231 and MCF-7 were transfected with synthetic miR-381 mimics and miR-381 inhibits to observe its effects on the biological behavior of breast cancer cells and to explore its possible mechanisms of action. 2. Materials and methods 2.1. Tissue samples Human clinical samples were collected from surgical specimens from 27 patients with breast cancer at Affiliated Hospital of Xuzhou Medical University according to a standard protocol, before any therapeutic intervention. The corresponding adjacent non-neoplastic tissues from the macroscopic tumor margin were isolated at the same time and used as controls. All subjects provided informed consent before specimen collection. The study protocol was approved by the ethics committee of the Xuzhou Medical University. 2.2. Cell lines and cells transfection Human breast cancer cells lines MDA-MB-231, MCF-7 and human epithelial cell line MCF-10A were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231 cells were cultured in L-15 medium (Gibco, USA) medium supplied with 10% fetal bovine serum (FBS, Gibco, USA), 100 mg/ml streptomycin, 100 IU/ml penicillin in normal air atmosphere at 37 C. MCF-7 cells were cultured in Dulbecco’s
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Modified Eagle’s Medium (DMEM) (Gibco, USA) supplemented with 10% FBS, 100 mg/ml streptomycin, 100 IU/ml penicillin in 5% CO2 atmosphere at 37 C. MCF-10A was cultured in DMEM/F12 (1: 1) (Hyclone, USA) supplemented with 5% horse serum (Gibco, USA), 10 mg/ml insulin, 20 ng/ml EGF, 100 ng/ml cholera toxin and 0.5 mg/ml hydrocortisone. miR-381 mimics and miR-381 inhibitors, negative control (NC) and CXCR4 small interfering RNAs (siRNAs) were purchased from GenePharma Company (Shanghai, People’s Republic of China). Cells transfection was carried out using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s protocol. 2.3. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from the human breast cancer tissues or cells using TRIzol reagent (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using a Firststrand cDNA Synthesis System (Marligen Bioscience, MD). qPCR analysis was performed using the Fast Start Universal SYBR Green Master (Rox) (Roche Applied Science) on an Eppendorf Realplex2 Mastercycler (Eppendorf, Hamburg, Germany). miRNA abundance was normalized to U6. RNA from three separate cell pellets pretreatment was analyzed. Primer pairs used in this study were: miR-381 were 50 -AGTCTATACAAGGGCAAGC TCTC-30 (forward) and 50 -ATCCATGACAGATCCCTACCG-30 (reverse); U6 were 50 -CTCGC TTCGGCAGCACA-30 (forward) and 50 -AACG-CTTCACGAATTTGCGT-30 (reverse). The real-time PCR reactions were performed in triplicate and included no-template controls. 2.4. Cell proliferation assay Cells were seeded in 96-well plates at 5 103 per well. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technology, Rockville, MD, USA) according to the manufacturer’s instructions. The optical density (OD) at 450 nm was recorded on a Microplate Reader (Bio-Rad, Hercules, CA, USA). 2.5. Cell migration and invasion assay Transwell chambers (Corning Incorporated, Corning, NY, USA) with a pore size of 8 mm was used for migration and invasion assays. Briefly, cells were harvested and resuspended in serum-free medium. For migration assay, 4 104 cells in serum-free medium were directly added into the upper chamber. For invasion assay, the melted Matrigel (BD Biosciences, San Jose, CA, USA) was diluted with medium and 30 mL of which was added to the upper chamber of transwell; the chamber was incubated at 37 C for 4 h to allow Matrigel to solidify; and then 1 105 cells in serum-free medium were added into the upper chamber. Medium containing 20% FBS was added to the lower chamber and served as a chemoattractant. After incubation in an incubator under 37 C and 5% CO2 condition for 24 h, cotton swabs were used to gently wipe off the cells remaining on the membrane surface without passing through. The cells that migrated or invaded to the lower surface of the membrane were fixed with 4% paraformaldehyde, stained in 10% crystal violet and wash with PBS, the cells were counted under high-power fields microscopically, 5 randomly selected fields for each chamber; the cell counts in each visual field was recorded. The cell counts passing through the membrane were used to indirectly reflect tumor cells migration and invasion ability. 2.6. Western blot analysis Total cell lysates were extracted from the cells using RIPA buffer (Solarbio, Beijing, China) followed by repetitive pipetting and lysis on
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the ice for 0.5 h; the contents were collected into centrifuge tubes and centrifuged at 12000 r/min; after loading buffer was added to the supernatant, the samples were treated at 100 C for 5 min to denature the proteins; 10 mL sample was loaded into each well for 12% SDS-PAGE and then the proteins were transferred to a PVDF membrane. The membranes were soaked with 8% milk and washed with PBST three times (10 min/time). The membranes were then incubated with the following primary antibodies: rabbit anti-human b-actin antibody, rabbit anti-human MMP-2 antibody, rabbit antihuman MMP-9 antibody, rabbit anti-human N-cadherin antibody, rabbit anti-human Vimentin antibody, rabbit anti-human Ecadherin antibody. All antibodies were purchased from cell signaling technology (Beverly, MA, USA). After incubation for 2 h at room temperature, the membranes were washed with PBST three times again. Then, they were incubated with their respective secondary antibodies for another 2 h. The relative band density was determined using the Tanon 5200 Multifunctional Imaging System (Beijing, China) with the ECL Western Blotting Substrate Kit (Millipore, Billerica, MA, USA). b-actin was used as an internal control. 2.7. Fluorescent reporter assay For the luciferase reporter assay, wild-type 30 UTRs of CXCR4 containing predicted miR-381 target sites were amplified by polymerase chain reaction (PCR) from MDA-MB-231 and MCF-7 cell genomic DNA, and mutant 30 UTRs were obtained by overlap extension PCR method. MDA-MB-231 and MCF-7 cells were transfected with Lipofectamine 2000 (Thermo Fisher Scientific). The transfection mixtures contained 5 pmol of miR-381 and 100 ng of firefly luciferase reporter plasmid, and pRL-TK (Promega Corporation, Fitchburg, WI, USA) was also transfected as normalization control. 48 h after transfection, luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Firefly luciferase activities were normalized to Renilla luciferase activity. Transfections were done in duplicate and repeated at least 3 times in independent experiments.
results indicated that miR-381 was markedly down-regulated in breast cancer tissues compared with adjacent non-cancerous tissues (Fig. 1A), which suggested that the expression of miR-381 may be associated with breast cancer carcinogenesis. We further analyzed miR-381 expression in two breast cancer cells lines (MDA-MB-231, MCF-7) and breast epithelial cell line (MCF-10A). Low expression of miR-381 was shown in breast cancer cells as compared to that of the normal breast epithelial cells (Fig. 1 B). Taken together, these data provided an evidence that miR-381 might has a key role in breast cancer progression. 3.2. miR-381 reduced breast cancer cells proliferation and EMT To confirm the potential functional role of miR-381 in breast cancer, we transfected MDA-MB-231 and MCF-7 cells with miR-381 mimics or miR-381 inhibitors to overexpress or silence miR-381 expression, and the transfection efficiency was detected by qRT-PCR. As showed in Fig. 2A, after tranfected with miR-381 mimics, miR-381 expression was effectively up-regulated, and miR-381 expression was greatly down-regulated in MCF-7 and MDA-MB-231 cells after tranfected with miR-381 inhibitors. A CCK-8 assay was performed to detect the proliferation of MCF-7 and MDA-MB-231 cells after transfection with miR-381 mimics for 24, 48, and 72 h. As demonstrated by CCK-8 proliferation assays, overexpression of miR-381 significantly decreased cell growth compared with the negative control cells. After transfection with miR-381 inhibitor, the proliferation was significantly increased in MCF-7 and MDA-MB-231 cells (Fig. 2B). Subsequently, we found miR-381 significantly reversed EMT of MCF-7 and MDA-MB-231 cells based on the observation that miR381 increased epithelial marker (E-cadherin) and inhibited mesenchymal markers (N-cadherin and vimentin). After transfection with miR-381 inhibitor, the protein of epithelial marker E-cadherin was significantly down-regulated and the protein of N-cadherin and vimentin was significantly up-regulated (Fig. 2C). 3.3. miR-381 suppressed cells migration and invasion
2.8. Statistical analysis Graph Pad Prism 5.0 statistical software was used to analyze the above experimental data. Measurement data were represented as X S.D; ANOVA and paired t-test was used to analyze the intergroup differences. The statistical significance is indicated with asterisks 3. Results 3.1. miR-381 was down-regulated in breast cancer tissues and cell lines We firstly examined the expression level of miR-381 by qRTPCR in breast cancer tissues and paired non-cancerous tissues. The
We further assessed the effects of miR-381 on cells migration and invasion, which were the key determinants of malignant progression and metastasis. We transfected miR-381 mimics in MCF-7 and MDA-MB-231 cells to study the migration and invasion of breast cancer cells. Compared with the cells transfected with control, the ability to migration in breast cancer cells decreased owing to a stable over-expression of miR-381. Furthermore, we observed the effect of miR-381 on invasiveness capacity of breast cancer cells by matrigel invasion assay. When matrigel invasion assay was performed on breast cancer cells, it was found that the invasiveness was lower for breast cancer cells with stable overexpression of miR-381 compared with control cells. To confirm the effects of miR-381 on modulating the malignant phenotypes of breast cancer cells, we also investigated the change of aggressive
Fig. 1. The expression of miR-381 in breast cancer tissues and cells. (A) miR-381 expression in breast cancer tissues and the normal tissues was examined by qRT-PCR. (B) miR381 expression in breast cancer cell lines was examined by qRT-PCR. **P < 0.01 vs control.
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Fig. 2. miR-381 inhibited breast cancer cells proliferation and EMT. (A) MCF-7 and MDA-MB-231 cells transfected with miR-381 mimics or miR-381 inhibitors both had significantly increased or decreased miR-381 expression compared with the cells transfected with negative control. (B) MCF-7 and MDA-MB-231 cells transfected with miR381 mimics or miR-381 inhibitors both had obvious decreased or increased the proliferation rate compared with the control groups. (C) MCF-7 and MDA-MB-231 cells transfected with miR-381 mimics had dramatic increased E-cadherin expression and decreased in vimentin and N-cadherin expression compared to the cells transfected with control; MCF-7 and MDA-MB-231 cells transfected with miR-381 inhibitors had decreased E-cadherin expression and increased vimentin and N-cadherin expression compared to the controls. *P < 0.05, **P < 0.01.
phenotypes of breast cancer cells after reducing expression of miR-381. miR-381 inhibitor transfection was employed to inhibit miR-381 expression in MCF-7 and MDA-MB-231 cells. After transfection with miR-381 inhibitor, the migration and invasion of bread cancer cells were significantly increased compared with the cells transfected with control (Fig. 3A and B). To investigate cells invasive capacity, proteins related to invasion were studied in MCF-7 and MDA-MB-231 cells. MMP-2 and MMP-9 expression was obviously decreased in both MCF-7 and MDA-MB-231 cells treated with miR-381 mimics at the protein level, as detected by western blot analysis. In comparison, the inhibition of miR-381 enhanced the expression of MMP-2 and MMP-9 (Fig. 3C). These results demonstrated that miR-381 played important roles in regulating cells migration and invasion in breast cancer cells and suggested that the down-regulation of miR-381 might contribute to tumor metastasis in breast cancer carcinogenesis. 3.4. CXCR4 is a direct target of miR-381 To explore the possible mechanisms of regulation by miR-381, miR-381 targets were analyzed by using the TargetScan bioinformatics predictions. Software analysis revealed that Chemokine (CX-C motif) receptor 4 (CXCR4), might be a potential target of miR381 based on putative target sequences of the CXCR4 30 UTR (Fig. 4A). We further performed luciferase assay to determine whether miR-381 could directly target 30 UTR of CXCR4. The target sequence of CXCR4 30 UTR (CXCR4-WT) or mutant sequence (CXCR4-Mut) was cloned into luciferase vector. The results showed that miR-381 significantly decreased the luciferase activity of the CXCR4 30 -UTR but not mutant sequence (Fig. 4B). To clarify their regulatory relationship, we first detected the mRNA levels of CXCR4 in miR-381 mimic-transfected MCF-7 and MDA-MB231cells or miR-381 inhibitor-transfected using qRT-PCR. The results revealed that the over-expression of miR-381 obviously decreased the mRNA level of CXCR4. In comparison, the inhibition
of miR-381 enhanced the expression of CXCR4 (Fig. 4C). These results suggested that CXCR4 is a direct target of miR-381. 3.5. CXCR4 suppressed breast cancer cells proliferation, EMT and metastasis We further examined whether CXCR4 is a substantial target of miR-381 involved in regulating the migration and invasion of breast cancer cells. Specific siRNA targeting CXCR4 (si-CXCR4) was transfected into MCF-7 and MDA-MB-231 cells to suppress CXCR4 expression. The CCK-8 assays showed that suppression of CXCR4 in cells dramatically decreased cells proliferation of human breast cancer cells (Fig. 5A). The cell migration and invasion assays showed that cells migration and invasion were suppressed after transfection with si-CXCR4 (Fig. 5B and C). The results of western blot experiments showed that malignant breast cancer cells biology behavior-related protein MMP-2 and MMP-9 decreased, as well as mesenchymal markers (N-cadherin and vimentin) decreased, and E-cadherin protein expression quantity increased after transfection with si-CXCR4 (Fig. 5D). Taken together, our results demonstrated that CXCR4 was a functionally important target of miR-381 and was involved in miR-381 regulated human breast cancer cells proliferation, EMT, migration and invasion. 3.6. miR-381 was negatively correlated to CXCR4 in breast cancer tissues As the above results suggested CXCR4 is a direct target of miR-381 could inhibit cell proliferation, EMT, migration and invasion. Therefore, we performed qRT-PCR to explore the expression pattern of CXCR4 in breast tumor tissues. The data validated that CXCR4 mRNA level was up-regulated in breast cancer tissues compared with paired non-cancerous tissues (Fig. 6A). We also determined CXCR4 expression in two breast cancer cell lines (MDA-MB-231 and MCF-7) and breast epithelial cell line (MCF-10A), qRT-PCR assay and western blot found the
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Fig. 3. miR-381 inhibited the breast cancer cells metastasis. (A) Cell migration was assayed in breast cancer cells with miR-381 transfection. (B) Cell invasion was assayed in breast cancer cells with miR-381 transfection. (C) MMP-2 and MMP-9 expression was assayed in breast cancer cells with miR-381 transfection. *P < 0.05 vs control, **P < 0.01 vs control.
Fig. 4. CXCR4 is a direct target of miR-381 in breast cancer cells. (A) The prediction of the binding between miR-381 and CXCR4 by TargetScan. (B) Luciferase reporter assays were performed to verify the binding of miR-381 in 30 -UTR of CXCR4. (C) qRT-PCR assay was performed to detect the mRNA level of CXCR4 in MCF-7 and MDA-MB-231 cells treated with miR-381 mimics and miR-381 inhibitors. *P < 0.05 vs control, **P < 0.01 vs control.
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Fig. 5. miR-381 inhibited the breast cancer cells metastasis and EMT. (A) CCK-8 assay showed that the si-CXCR4 group had a lower proliferation rate than the control group in the breast cancer cell. (B) Migration assay shows that the number of migrated cells in the si-CXCR4 group markedly decreased compared with that in the control. (C) Invasion assay shows that the number of invaded cells in the si-CXCR4 group remarkably decreased compared to that in the control. (D) The si-CXCR4 group had obvious decreases in MMP-2, MMP-9, vimentin and N-cadherin expression and a significant increase in E-cadherin expression compared to the control. *P < 0.05, **P < 0.01.
mRNA/protein level of CXCR4 was up-regulated (Fig. 6B and C). Furthermore, we also investigated the relationship between CXCR4 and miR-381, as we can see from Fig. 6D, the expression level of CXCR4 was inversely correlated with the level of miR-381. 4. Discussion Increasing reports have demonstrated that miRNAs are an important regulator of tumor progression and metastasis. In breast
cancer, many miRNAs have been identified to regulate known genes that are involved in the pathology of tumorigenesis and metastasis. In the present study, we found miR-381 was downregulated in breast cancer tissues and cell lines. Previous researches also showed other biological functions of miRNA-381 in both cancer tissues and cell lines. Yan Liang [20] and Xinxin He [23] found that up-regulation of miRNA-381 inhibited cells proliferation and invasion in colon cancer cells. This effect was also observed in renal cancer cells, which miRNA-381 could
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Fig. 6. Relationship of miR-381 and CXCR4 in the breast cancer tissues. (A) Data analysis of CXCR4 mRNA expression in breast cancer tissues with the normal tissues. (B) Data analysis of CXCR4 mRNA expression in breast cancer cells (MCF-7 and MDA- MB-231cells) with breast epithelial cell line (MCF-10A). (C) CXCR4 protein expression in breast cancer cells lines (MCF-7 and MDA- MB-231cells) and breast epithelial cells line (MCF-10A). (D) Data analysis of relationship between miR-381 and CXCR4 expression in breast cancer tissues (r = 0.9298). **P < 0.01.
suppressed the proliferation of cancer cells [24]. Our data revealed that miRNA-381 could inhibit breast cancer cells proliferation, migration and invasion. Numerous experimental studies have demonstrated an increase of MMPs, especially MMP-2 and MMP-9 with cancer progression. Secretion of cytokines by cancer cells exerts an important influence in the control of cancer cell behavior such as invasion and adhesion [25]. MMP-2 and MMP-9 are regarded as indispensable cytokines for breast cancer cells invasion [26]. Our result found that miRNA-381 could inhibit the expression of MMP-2 and MMP-9 of MCF-7 and MDA-MB-231 cells. MCF-7 and MDA-MB-231 cell lines had been reported to represent low and high invasive breast carcinoma with different proliferative potentials, but in our study there were no significant differences in the amplitude of miR-381 effects on cells proliferation, migration and invasion which needed to be investigated in the future. Emerging evidence has showed that many miRNAs play important roles in the EMT of cancer cells: microRNA-26b inhibits EMT in hepatocellular carcinoma by targeting USP9X, and miR-23a regulated TGF-b-induced EMT in lung cancer cells [27,28]. Breast cancer pathogenesis is particularly accompanied by the EMT that is considered the first step in distant metastasis. In the EMT process,
epithelial cells lose their characteristics, gain mesenchymal features, and become motile and invasive [8]. Loss of epithelial marker E-cadherin and gain of mesenchymal markers N-cadherin and vimentin are regarded as the most important molecular markers of EMT [9]. Our results showed that miR-381 reversed the expression level of epithelial marker and mesenchymal markers in MCF-7 and MDA- MB-231cells and inhibited EMT. We next predicted and validated the possible targets of miR381 in breast cancer cells as the impact of specific miRNAs on cancer biology depends on their downstream targets. miRNA-381 exerts its biological functions through the regulation of various target genes, such as LRH-1 [20], Twist1 [23] and HES1 [29]. Herein, we used the TargetScan bioinformatics predictions to predict another genes, and the binding site of CXCR4 30 UTR was obviously located at miR-381 seed region. We also identified that miR-381 can suppress the mRNA expression of CXCR4 via directly target its 30 UTR in breast cancer cell. It has been found that chemokines play important roles in cancer cell motility, angiogenesis and tumor metastasis [30]. In particular, CXCR4, a class-A G protein-coupled receptor (GPCR), was reported as the most common over-expressed chemokine receptor in human cancer [31], such as breast [32], pancreatic [33] and prostate cancer [34].
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Patients with high expression of CXCR4 in tumors had poor cancer survival [35]. CXCR4 has been considered as a biomarker for poor prognosis and cancer metastasis [36,37]. To further confirm that miR-381 can directly target CXCR4, a knockdown plasmid of CXCR4 (si-CXCR4) was used, which showed that si-CXCR4 can inhibit the cells proliferation, migration, invasion and EMT of MCF-7 and MDA-MB-231 cells. Moreover, si-CXCR4 suppressed the expression of MMP-2 and MMP-9 of breast cancer cells. In order to further examine the mechanism underlying the effect of miRNA-381 in breast cancer cells, the expression of CXCR4 in breast cancer tissue samples and cell lines was measured. The results demonstrated that CXCR4 was over-expressed in breast cancer tissue and cell lines (MCF-7 and MDA- MB-231). In conclusion, the data here suggested that inhibition of miR-381 might be a potential approach to repress the progression of breast cancer through restoring CXCR4 levels, which might provide us new insights into a potential therapeutic strategy for blocking metastasis in breast cancer. Conflict of interest The authors have no conflict of interest pertaining to this manuscript. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin, D. Forman, F. Bray, Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012, Int. J. Cancer. 136 (2015) E359–E386. [2] C.E. DeSantis, C.C. Lin, A.B. Mariotto, R.L. Siegel, K.D. Stein, J.L. Kramer, R. Alteri, A.S. Robbins, A. Jemal, Cancer treatment and survivorship statistics, CA Cancer J. Clin. 64 (2014) 252–271. [3] X.F. Tan, F. Xia, Long-term fatigue state in postoperative patients with breast cancer, Chin. J. Cancer Res. 26 (2014) 12. [4] S. Vimalraj, P.J. Miranda, B. Ramyakrishna, N. Selvamurugan, Regulation of breast cancer and bone metastasis by microRNAs, Dis. Markers 35 (2013) 369–387. [5] L. Wang, J. Wang, MicroRNA-mediated breast cancer metastasis: from primary site to distant organs, Oncogene 31 (2012) 2499–2511. [6] X. Liu, J. Gong, B. Xu, miR-143 down-regulates TLR2 expression in hepatoma cells and inhibits hepatoma cell proliferation and invasion, Int. J. Clin. Exp. Pathol. 8 (2015) 12738–12747. [7] Y. Cheng, G.X. Xiang, Y.B. Meng, R.Z. Dong, MiRNA-183-5p promotes cell proliferation and inhibits apoptosis in human breast cancer by targeting the PDCD4, Reprod. Biol. (2016), doi:http://dx.doi.org/10.1016/j. repbio.2016.07.002. [8] K. Polyak, R.A. Weinberg, Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits, Nat. Rev. Cancer 9 (2009) 265–273. [9] J. Jiang, Y.L. Tang, X.H. Liang, EMT: a new vision of hypoxia promoting cancer progression, Cancer Biol. Ther. 11 (2011) 714–723. [10] M.E. Muroski, M.D. Roycik, R.G. Newcomer, P.E. Van den Steen, G. Opdenakker, H.R. Monroe, Z.J. Sahab, Q.X. Sang, Matrix metalloproteinase-9/gelatinase B is a putative therapeutic target of chronic obstructive pulmonary disease and multiple sclerosis, Curr. Pharm. Biotechnol. 9 (2008) 34–36. [11] C. Scheel, T. Onder, A. Karnoub, R.A. Weinberg, Adaptation versus selection: the origins of metastatic behavior, Cancer Res. 67 (2007) 11476–11479. [12] J.P. Thiery, Epithelial-mesenchymal transitions in tumour progression, Nat. Rev. Cancer 2 (2002) 442–454. [13] P.A. Gregory, A.G. Bert, E.L. Paterson, G.J. Goodall, The miR-200 family and miR205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1, Nat. Cell Biol. 10 (2008) 593–601.
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