Get 20M+ Full-Text Papers For Less Than $1.50/day. Subscribe now for You or Your Team.

Learn More →

miR-200b as a prognostic factor in breast cancer targets multiple members of RAB family

miR-200b as a prognostic factor in breast cancer targets multiple members of RAB family Background: miR-200b has been reported to be a tumor suppressor and a promising therapeutic target in cancer. miR-200b has been associated with epithelial-mesenchymal transition and chemo-resistance in cancer. The aim of this study is to investigate the expression of miR-200b, its prognostic roles and its potential targets in breast cancer. Methods: qRT-PCR was used to detect miR-200b expression in breast cancer tissues and cell lines. In situ hybridization of miR-200b on tissue microarray including 134 breast cancer samples was used to evaluate its prognostic role. Novel targets of miR-200b in breast cancer were predicted and confirmed by luciferase reporter assay and western bloting. Immunohistochemical staining was used for protein detection. The biological effects of miR-200b in breast cancer cells were further confirmed by ectopic expression of its mimics followed by MTT assay and invasion test. Results: miR-200b was downregulated in breast cancer tissues and cell lines and its low-expression correlated with poor outcome in breast cancer patients. Members of RAB family, RAB21, RAB23, RAB18 and RAB3B were predicted to be the targets of miR-200b. The luciferase reporter assay was performed to certificate this prediction. The expressions of RAB21, RAB23, RAB18 and RAB3B were suppressed by transfection of miR-200b in breast cancer cells. Over-expression of miR-200b or knock-down of RAB21, RAB23, RAB18 and RAB3B inhibited breast cancer cell proliferation and invasion in vitro. Conclusions: Our study provides evidence that miR-200b is a prognostic factor in breast cancer targeting multiple members of RAB family. MiR-200b could be a potential therapeutic target in breast cancer. Keywords: miR-200b, RAB family, Breast cancer, Prognosis Background like type (ER-, PR-, HER2-, cytokeratin 5/6+, and/or epi- Breast cancer is the first ranked female malignancy dermal growth factor receptor (EGFR)+). Breast cancer of worldwide, with about 200,000 new incidence and 40,000 HER2 over-expressing or basal-like type would predict deaths per year in US [1]. Generally breast cancers could more recurrence, distant metastasis, and therapy resist- be classified according to the TNM staging system [2] and ance [4]. The comprehensive therapy and prognosis for their molecular groups [3], which include luminal A type breast cancer would depend on both the TNM stage and (estrogen receptor (ER) + and/or progesterone receptor molecular subtype. Although breast cancers with early (PR) +, human epidermal growth factor receptor-2 stage show excellent outcome after therapy, recurrent and (HER2)-), luminal B type (ER + and/or PR+, HER2+), metastatic breast cancer patients remain big problems for HER2 over-expressing type (ER-, PR-, and HER2+), basal- cure [5]. MicroRNAs, also termed miRNAs, are a class of en- * Correspondence: xiexm@sysucc.org.cn dogenous, non-protein coding single-stranded RNA mole- Equal contributors cules with a length of 21–23 nucleotides, which plays a Department of Breast Oncology, Sun Yat-Sen University Cancer Center, 651 crucial role in the post-transcriptional regulation of gene East Dongfeng Road, Guangzhou, Guangdong 510060, People’s Republic of China expression [6]. miRNAs are highly conserved and specific, State Key Laboratory of Oncology in South China, Sun Yat-Sen University and regulate gene expression by binding to the 3′ untrans- Cancer Center, Collaborative innovation center for cancer medicine, 651 East lated region (UTR) of target messengerRNA (mRNAs) Dongfeng Road, Guangzhou, Guangdong 510060, People’s Republic of China Full list of author information is available at the end of the article © 2014 Ye et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ye et al. Journal of Translational Medicine 2014, 12:17 Page 2 of 10 http://www.translational-medicine.com/content/12/1/17 and inhibiting translation or inducing degradation of line 184A, MCF-10A, and several breast cancer cell lines mRNAs. miRNAs has been proved to play vital roles in were also included. Total RNAs were extracted from cancer management, acting as either oncogenes or tumor tissues/cells with TRIzol reagent (Invitrogen). For miR- suppressors [7]. 200b, reverse transcription and qRT-PCR reactions were The miR-200 family (including miR-200a, miR-200b, performed by means of a SYBR-green-containing PCR miR-200c, miR-141 and miR-429) are transcribed from kit (GenePharma, Shanghai, China). U6 snRNA was used two chromosomal locations: miR-200b-200a-429 cluster as an endogenous control for miRNA detection. The at chromosomal location 1p36 and miR-200c-141 cluster expression of miR-200b was quantified by measuring at chromosomal location 12p13 [8]. Recently, the miR- cycle threshold (Ct) values and normalized using the ΔΔCt 200 family has been associated with carcinogenesis and 2- method relative to U6 snRNA. cancer therapy [9]. Dysregulation of the miR-200 family was reported in several malignancies, including ovarian, Patients and specimens for tissue microarray endometrial, lung and gastric cancer [10-12]. The miR- A total of 134 female breast cancer patients who were 200 family has been supposed to be tumor suppressors diagnosed by histo-pathology in Sun Yat-Sen University regulating epithelial-mesenchymal transition (EMT) Cancer Center from October 2001 to September 2006 [13]. miR-200 directly targets EMT-inducing transcrip- were obtained. Specimens were formalin-fixed and em- tional factors ZEB1 and ZEB2, which repress E-cadherin bedded in paraffin by standard methodology after obtained expression. miR-200 could also suppress β-catenin/Wnt during surgery and were stored in the Department of signaling pathway by targeting β-catenin mRNA [14], Specimen and Resource in Sun Yat-Sen University Cancer thus highlighting its roles in cancer invasion and metas- Center. IHC of ER, PR, and HER-2 status were performed tasis. Moreover, mi-200 has been reported to affect the in the Pathology Department of Sun Yat-Sen University chemotherapy and endocrine thrapy resistance in breast Cancer Center. All the patients included in present study cancer [15,16]. Among the miR-200 family, miR-200b is did not receive any chemotherapy and radiation therapy thought to be the fundamental regulator in EMT and before, and their complete clinico-pathological data, inclu- cancer chemo-sensitivity [17]. However, the expression ding age, histological type, lymph nodes status, tumor size, of miR-200b and its prognostic role in breast cancer stage, local relapse, distant metastatic relapse, ER status, remain unclear. PR status and HER-2 status, were available and reviewed. In this study, we investigated the expression of miR- Histological type, reclassified according to the WHO 200b and its prognostic roles in breast cancer patients, classification and stage of tumor, was based on the TNM predicted and further identified multiple members of staging system (American Joint Committee on Cancer RAB family as new targets of miR-200b in breast cancer. classification). Follow-up was updated by review of records and telephone calls. The date of death and the date of Materials and methods relapse were used to calculate estimate overall survival Cell lines and culture (OS) and disease-free survival (DFS). Human mammary epithelial (HME) cell line 184A, This study was approved by the Ethics Committee of MCF-10A, human breast cancer cell lines MDA-MB- SunYat-Sen University Cancer Center Health Authority. 231, MDA-MB-435, MCF-7, T47D, BT-474, BT-483, and The collection and use of tissues followed the proce- mouse breast cancer cell line 4 T1 were obtained from the dures that are in accordance with the ethical standards American Type Culture Collection (Manassas, VA, USA) as formulated in the Helsinki Declaration. and were passaged in our laboratory for less than six months after resuscitation of frozen aliquots. The breast Tissue microarray (TMA) construction cancer cells were cultured in Dulbecco’s modified Eagle’s Representative part of the breast cancer specimens used medium (DMEM, Invitrogen, CA, USA) supplemented for creating tissue microarray were selected by two experi- with 10% fetal bovine serum (FBS, GIBCO, Cappinas, enced pathologists, using hematoxylin and eosin–stained Brazil), in a humidified incubator at 37°C containing 5% sections which were formalin-fixed and embedded in CO2. All cell lines were re-authenticated by short tandem paraffin as mentioned above. TMA block was con- repeat DNA profiling every 6 months after used. structed with MiniCore Control Station (ALPHELYS SARL, France) and designed by TMA Designer tissue Quantitative real-time polymerase chain reaction analysis array design software (ALPHELYS SARL, France). We (qRT-PCR) used 1.0-mm core tissue biopsies and took tissues Expression level of miR-200b was detected in both from paraffin-embedded tissue blocks to two new recipi- breast cancer tissues and cell lines. 40 pairs of tumor ent blocks (one contained 51 samples, and the other 83 tissues and para-carcinoma tissues from breast cancer samples), and one core per case was arrayed. The recipient patients were obtained. Human mammary epithelial cell blocks were cut and placed on slides. Ye et al. Journal of Translational Medicine 2014, 12:17 Page 3 of 10 http://www.translational-medicine.com/content/12/1/17 LNA probes for miR-200b finally HRP-Streptavidin for 15 min. After DAB staining, miR-200b miRCURYTM LNA custom detection probe the results were graded for intensity (0-negative, 1-weak, (Exiqon, Vedbaek, Denmark) was used for ISH. The 5′- 2-moderate, and 3-strong) and percentage of positive cells 3′ sequences (enhanced with LNA) were TCATCATT (0, 1 (1–24%), 2 (25–49%), 3 (50–74%), and 4 (75–100%)) ACCAGGCAGTATTA with a digoxigenin (DIG) label at with discrepancies resolved by consensus. The grades were both the 5′ and 3′ ends. multiplied to determine a score. The scores of tumors were defined as the following rule: negative (score = 0–3) In situ hybridization of miR-200b and scoring system and positive (score > =4). After deparaffinized, the slides were mounted onto flow through slide chambers and placed in a Tecan Construction of luc-UTR vectors Freedom Evo automated hybridization instrument (Tecan, The full-length RAB21, RAB23, RAB18, RAB3B, RAB37, Männedorf, Switzerland) in which the following steps RAB8B, RAB7A, RAP1B, RAP2C 3′-UTR was cloned were performed: proteinase-K treatment 15 μg/ml at 37°C into the EcoRIand HindIII sites of the pMIR-REPORT for 8 min, pre-hybridization in Exiqon hybridization buffer luciferase vector (Ambion, Austin, TX, U.S.) using PCR (Exiqon, Vedbæk, Denmark) at 62°C for 15 min, hybri- generated fragment. A Luc-mut vector of RAB21, RAB23, dization with 40 nM miR-200b probe, stringent washes RAB18, and RAB3B in which the first seven nucleotides with 5 × SSC, 1 × SSC and 0.2 × SSC buffers at 62°C complementary to the miR-200b seed-region were mu- over 33 min, DIG blocking reagent (Roche, Mannheim, tated by site-directed mutagenesis (Stratagene) served as a Germany) in maleic acid buffer containing 2% sheep mutant control. serum at 30°C for 15 min, Streptavidin-HRP -conjugated anti-digoxigenin (diluted 1:500 in blocking reagent, Roche) Luciferase assay at 30°C for 30 min. After washing, the slides were incu- Luc-wt, Luc-mut, and Luc-ctrl were co-transfected bated in DAB at 25°C for 3-5 min. The slides were then within vitro-produced miR-200b into MDA-MB-231 cells. dismantled in water, dehydrated in alcohol solutions The pMIR-REPORT β-galactosidase control vector was and mounted with eukitt mounting medium (VWR, transfected and served as a control. Luciferase activity Herlev, Denmark). was measured in cell lysates 48 h after transfection The intensities of miR-200b staining was scored by using a dual-light luminescent reporter gene assay kit 0–4, according to the standards of 0–1 (no staining), (Applied Biosystems). Results were normalized against 1–2 (weak staining), 2–3(medium staining)and 3–4 β-galactosidase activity. (strong staining). The percentages of miR-200b cells of individual samples were analyzed. Those expression Western blot scores equaled to the intensities * the percentages, and the Cell protein lysates, cytosol protein or nuclear protein was maximum was 4 and the minimum was 0. Individual separated in 10% SDS-polyacrylamide gels, electrophoret- samples were evaluated by at least two pathologists in a ically transferred to polyvinylidene difluoride membranes blinded manner, and those expression scores of greater (Millipore), then detected with mouse monoclonal anti- than 2 were defined high expression, otherwise the expres- body for RAB21 (sc-81917), rabbit polyclonal antibody for sion scores were low. RAB23 (sc-130248) and RAB3B (sc-305), goat polyclonal antibody for RAB18 (polyclonal antibody for RAB) (Santa Immunohistochemical (IHC) staining and scoring system Cruz Biotechnology), mouse monoclonal antibody for β- The Labeled StreptAvidin Biotin Method was used for actin (Abcam) and commercial ECL kit (Pierce). The IHC in our study. After deparaffinizing and rehydrating, intensity of protein fragments was quantified using the slides were treated with 90% methanol/3% H O 2 2 Chemical DocTM XRS + (Bio-Rad). solution for 15 min at room temperature to block endo- genous peroxidase. Then, the slides were soaked in sodium citrate buffer (10 mM Sodium citrate, 0.05% RNA silencing for RAB21, RAB23, RAB18, and RAB3B Tween 20, pH 6.0) under 96°C for 5 min for antigen The sense sequences of siRNA oligonucleotides targeting retrieval. After blocking by BSA, the following antibodies the RAB18, RAB21, RAB23, and RAB3B transcripts, were used: mouse monoclonal antibody for RAB21 (sc- respectively, were as follows: si- RAB18: 5′-UUCUGG 81917), rabbit polyclonal antibody for RAB23 (sc-130248) UUGUAACUUCACGGCT-3′; si- RAB21: 5′-UUAAUA and RAB3B (sc-305), goat polyclonal antibody for RAB18 GGUUGGAUGGCGGTT-3′; si- RAB23: 5′-CUUCAC (polyclonal antibody for RAB) (Santa Cruz Biotechnology). TACUGCUUCGAGTT-3′; and si- RAB3B: 5′-AUAA We added antibodies to the slides for overnight storage at CUUGGAGGGACUGCCTT-3′ (Invitrogen). Scrambled 4°C and then incubated the slides at room temperature siRNA was used as a negative control. Cells were plated in with biotinylated secondary antibody for 20 min, and culture dishes or in 24-well plates for 24 h, and transfected Ye et al. Journal of Translational Medicine 2014, 12:17 Page 4 of 10 http://www.translational-medicine.com/content/12/1/17 Figure 1 miR-200b was down-regulated in breast cancer tissues and cell lines. (A) The expression of miR-200b was detected by qRT-PCR in 40 pairs of normal mammary tissues and breast cancer samples. (B) The expression of miR-200b was detected by qRT-PCR in breast cancer cell lines. with siRNA using Lipofectamine 2000. After 48 h, the 200b mimics were seeded at a density of 5,000 cells per cells were harvested for use in other assays. well in 96-well plates and incubated at 37°C for 24 h. Cells were then incubated an additional 72 h, and the MTT MTT assay assay was performed according to the manufacturer’sin- Cell viability was examined by the 3-(4, 5-dimethyl- structions (Molecular Probes, Eugene, OR). Absorbance thiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) values were determined at 570 nm on a Spectra Max 250 assay. Cells transfected with either scramble or miR- spectrophotometer (Molecular Devices, Sunnyvale, CA). Figure 2 miR-200b as a prognostic factor in breast cancer patients. (A) The ISH staining of miR-200b in breast cancer tissue microarray: ×200. Two miR-200b high samples and two miR-200b low samples were shown as representatives. (B) qRT-PCR results for miR-200b detection in three miR-200b high ISH staining samples and three miR-200b low samples. (C) Survival curves of OS and DFS according to miR-200b expression. Low miR-200b expression correlated with worse outcome. Ye et al. Journal of Translational Medicine 2014, 12:17 Page 5 of 10 http://www.translational-medicine.com/content/12/1/17 Cell invasion and migration assays The relationships between expression of miR-200b and The cell invasion assay was conducted as described pre- clinical parameters in breast cancer patients viously [18]. Briefly, cells were seeded onto the basement We further evaluated the prognostic role of expression membrane matrix present in the insert of a 24-well cul- of miR-200b in breast cancer patients by in situ hybri- ture plate (EC matrix, Chemicon, Temecula, CA). Fetal dization (ISH) staining on tissue microarray. MiR-200b bovine serum was added to the lower chamber as a chemoattractant. After an additional 48 hours, the non- Table 1 Clinico-pathological variables and the expression of miR-200b in total breast cancer patients invading cells and EC matrix were gently removed with a cotton swab. Invasive cells located on the lower side of miR-200b low miR-200b high Total P value (n = 78) (n = 56) Characteristics the chamber were stained with crystal violet, counted (n = 134) No. % No. % and imaged. Age (years) 0.330 <50 82 46 56.1 36 43.9 Statistical analysis >50 52 32 61.5 20 38.5 Data are presented as mean ± SD from at least three Menopause 0.524 separate experiments. Multiple group comparisons were performed using ANOVA with a post hoc test for Yes 63 37 58.7 26 41.3 subsequent individual group comparisons. The distinct No 71 41 57.7 30 42.3 expression of miR-200b between tumor tissues and para- LN infiltrated 0.148 carcinoma tissues was examined by independent samples Yes 80 50 62.5 30 37.5 T-test. The relationships between miR-200b expression No 54 28 51.9 26 48.1 and clinico-pathological parameters were examined by Tumor size (cm) 0.080 chi-square test. Overall survival (OS) or disease-free sur- vival (DFS) curves were calculated by the Kaplan-Meier = < 2 38 18 47.3 20 52.7 method and the log-rank test was used to determine >2 96 60 62.5 36 37.5 the difference in OS or DFS rates between two groups. TNM stage 0.040* Results were considered statistically significant when I-II 78 40 51.3 38 48.7 P ≤ 0.05 was obtained. All the statistical analyses were III- IV 56 38 67.9 18 32.1 performed using SPSS13.0 for Windows (SPSS Inc., Local relapse 0.083 Chicago, IL, USA). Yes 8 7 87.5 1 12.5 No 126 71 56.3 55 43.7 Results Distant The expression of miR-200b in breast cancer tissues and 0.003* metastasis cell lines Yes 34 27 79.4 7 20.6 To evaluate the expression of miR-200b in breast cancer tissues, qRT-PCR was used to detect the expression level No 100 51 61.0 49 49.0 in 40 pairs of tumor tissues and para-carcinoma (normal) ER status 0.155 tissues from breast cancer patients. The results showed Positive 51 33 64.7 18 35.3 that expression of miR-200b in breast cancer tissues was Negative 83 45 54.2 38 45.8 significantly lower than in normal tissues (Figure 1A). In PR status 0.073 comparison with the normal tissues, miR-200b was down- Positive 54 36 66.7 18 33.3 regulated in 77.5% (31/40) of the tumor samples. We further detected expression of miR-200b in cell lines Negative 80 42 52.5 38 47.5 (Figure 1B), including human mammary epithelial (HME) HER-2 status 0.213 cell lines (184A, MCF-10A), human breast cancer cell Positive 22 15 68.2 7 31.8 lines (MDA-MB-231, MDA-MB-435, MCF-7, T47D, Negative 112 63 56.3 49 43.8 BT-474, BT-483), and mouse breast cancer cell line TNBC status 0.472 4 T1. Compared with 184A and MCF-10A, miR-200b TNBC 51 29 56.9 22 43.1 was down-regulated in MCF-7, MDA-MB-435, T47D, BT-483, MDA-MB-231, and 4 T1, especially in the latter Non-TNBC 83 49 59.0 34 41.0 two cell lines. These results revealed that reduced miR- *means statistically significant (p < 0.05). % means percentage within the row. 200b expression was a frequent event in human breast TNBC, triple-negative breast cancer, means ER(−), PR(−), HER-2(−). High cancer tissues and could be involved in breast cancer expression of miR-200b was seen in 41.8% of total patients and correlated carcinogenesis. with early TNM stage (p = 0.040) and fewer metastasis (p = 0.003). Ye et al. Journal of Translational Medicine 2014, 12:17 Page 6 of 10 http://www.translational-medicine.com/content/12/1/17 ISH staining in breast cancer tissues was located in miR-200b targeted multiple members of RAB family the cytoplasm. Two miR-200b high samples and two To determine the function of miR-200b in breast cancer, miR-200b low samples were shown as representatives we used online softwares TargetScan to search for (Figure 2A). To confirm the miR-200b ISH staining potential miR-200b target genes. We found that mem- results, we extracted total RNAs form three miR-200b bers of RAB family are among these candidate target high samples and three miR-200b low samples for genes. A miR-200b-binding site was found in the 3′- qRT-PCR (Figure 2B). The PCR results showed that UTR of RAB21, RAB23, RAB18, RAB3B, RAB37, RAB8B, the ISH staining could reflect the relative level of miR- RAB7A, RAP1B, RAP2C mRNA with perfect base pairing 200b expression in the samples. The clinico-pathologic (Figure 3A). To verify whether these genes were direct characteristics and miR-200b expression of the breast targets of miR-200b, we subcloned the full-length 3′-UTR cancer patients involved in our study are shown in Table 1. of RAB21, RAB23, RAB18, RAB3B, RAB37, RAB8B, In all 134 breast cancer patients, high expression of RAB7A, RAP1B, RAP2C into the luciferase reporter miR-200b was seen in 41.8% of total patients. High vector. However, addition of in vitro-produced miR-200b miR-200b expression correlated with early TNM stage only suppressed the luciferase activity of the 3′-UTR of (p = 0.040) and fewer metastasis (p = 0.003). Further- RAB21, RAB23, RAB18 and RAB3B upon co-transfection more, markedly reduced overall survival (p = 0.0030) of the luciferase vector (wild-type, mutant, or negative con- and disease-free survival (p = 0.0000) were observed in the trol) with the in vitro-produced microRNAs (miR-200b breast cancer patients who had low miR-200b expression mimics or scramble control) into MDA-MB-231 cells compared with the patients who exhibited high expres- (Figure 3B). This inhibition was abolished when the seed sion levels (Figure 2C). In our study, the estimate sequences of the miR-200b target sequences were mutated overall survival and disease-free survival for miR-200b in the Luc-mut vector (Figure 3B). To directly assess the high patients were 109.3 months and 101.2 months, while effects of miR-200b on the expression of RAB21, RAB23, the two for miR-200b low patients were 78.3 months and RAB18 and RAB3B, we transfected miR-200b mimics into 66.2 months respectively. These results indicated that MDA-MB-231 and 4 T1 cells and found that overex- miR-200b could be a prognostic factor in breast cancer pression of miR-200b reduced mRNA and protein level of patients. RAB21, RAB23, RAB18 and RAB3B (Figure 3C). Figure 3 RAB21, RAB23, RAB18 and RAB3B is a direct target of miR-200b. (A) miR-200b binds to the 3′UTRs of RAB21, RAB23, RAB18, RAB3B, RAB37, RAB8B, RAB7A, RAP1B and RAP2C. Predicted binding between miR-200b and the seeds match in the nine predicted genes’ 3′UTRs. (B) Luciferase reporter assays 48 h after transfection with indicated pMIR-Report plasmids and a renilla transfection control plasmid, co-transfected with miR-200b mimics, or relevant scramble controls. Luciferase activities were significantly inhibited on co-transfection of miR-200b mimics and pMIR-Report plasmids of RAB21, RAB23, RAB18 and RAB3B. Mutation in the corresponding binding sites abolished this inhibition. Data shown were the mean ± s.d. of six replicates and were representative of three independent experiments. *P < 0.05 (C) miR-200b regulated the expression of RAB21, RAB23, RAB18 and RAB3B. Western blot analyzed their expression 48 h after transfection with miR-200b mimics or scramble control in MDA-MB-231 and 4 T1 cells. Ye et al. Journal of Translational Medicine 2014, 12:17 Page 7 of 10 http://www.translational-medicine.com/content/12/1/17 The association between expression of miR-200b and regulation of RAB21, RAB23, RAB18 and RAB3B pro- RAB21, RAB23, RAB18 and RAB3B in breast cancer tissues teins by miR-200b in breast cancer tissues. To evaluate the associations between expression of miR- 200b and RAB21, RAB23, RAB18 and RAB3B in breast Over-expression of miR-200b or knock-down of RAB21, cancer tissues, we further detected expression of miR- RAB23, RAB18 and RAB3B inhibited breast cancer cell 200b by ISH staining and expression of RAB21, RAB23, proliferation and invasion RAB18 and RAB3B protein by immunohistochemistry To assess the biological effects of over-expressing (IHC) staining in 10 pairs of tumor tissues and para- miR-200b in breast cancer cells, ectopic expression of carcinoma tissues from breast cancer patients, who had miR-200b mimics were transfected into MDA-MB-231 developed distant metastasis after operation. As shown and 4 T1 cells. Transfection of miR-200b mimics in in Figure 4, compared with normal breast tissue, miR- MDA-MB-231 and 4 T1 cells markedly attenuated cell 200b expression was down-regulated in tumor tissue, proliferation, compared with scramble (Figure 5A). More- while the contrary situations were found for RAB21, over, ectopic expression of miR-200b mimics in MDA- RAB23, RAB18 and RAB3B IHC staining. In summary, MB-231 and 4 T1 cells markedly attenuated cell invasion miR-200b low ISH staining was seen in 90% (9/10) compared with control cells (Figure 5B, C). To identify tumor samples, while positive rates for RAB21, RAB23, the biological effects of RAB21, RAB23, RAB18 and RAB18 and RAB3B IHC staining were 80%, 80%, 90%, RAB3B, specific siRNAs for each were also synthesized. 100% respectively. These results further confirmed the Transfection of siRNAs for RAB21, RAB23, RAB18 and Figure 4 IHC staining of RAB21, RAB23, RAB18 and RAB3B in human breast cancer tissues. 10 pairs of tumor tissues and normal tissues from breast cancer patients, who had developed distant metastasis after operation, were detected by ISH or IHC. Compared with normal tissue, miR-200b expression was down-regulated in tumor tissue, while the contrary situations were found for RAB21, RAB23, RAB18 and RAB3B. Ye et al. Journal of Translational Medicine 2014, 12:17 Page 8 of 10 http://www.translational-medicine.com/content/12/1/17 Figure 5 Over-expression of miR-200b or knock-down of RAB21, RAB23, RAB18 and RAB3B inhibits breast cancer cell proliferation and invasion. (A) Transfection of miR-200b mimics or knock-down of RAB21, RAB23, RAB18 and RAB3B in MDA-MB-231 and 4 T1 cells markedly attenuated cell proliferation: MTT assay at 24 h, 48 h and 72 h after transfection. (B, C) Transfection of miR-200b mimics or knock-down of RAB21, RAB23, RAB18 and RAB3B in MDA-MB-231 and 4 T1 cells inhibited cell invasion: Data shown in Figure B were the mean ± s.d. of three replicates and were representative of three independent experiments. *P < 0.05. The representing images were shown in Figure C. RAB3B also inhibits breast cancer cell proliferation and and histone modifications [23,24]. Moreover, miR-200b invasion (Figure 5A, B, and C). These results indicated is thought to be related to cell differentiation by target- that the biological effects of miR-200b in breast cancer ing GATA-4 [25]. Loss of miR-200b contributes to the cells may attribute to regulation of RAB21, RAB23, breast cancer stem cell status maintaining [26]. Besides, RAB18 and RAB3B, and thus miR-200b could be a thera- miR-200b has also been associated with cancer chemo- peutic target in breast cancer. sensitivity by modulating PTEN, PTPN12 and thus their downstream oncogenes like src and ras [27,28]. Discussion In this study, we investigated the expression of miR- The miR-200 family has been reported to be a funda- 200b and its prognostic role in breast cancer. We mental regulator of EMT, thus highlighting their roles in showed that expression of miR-200b was significantly cancer progression. As a founding member in miR-200 lower in breast cancer tissues than normal tissues. Similar family, miR-200b attracts much focus both in carcino- results were found in breast cancer cell lines. In addition, genesis and cancer therapy in recent years [17]. Down- we analyzed the prognostic role of the expression of miR- regulation of miR-200b has been observed in renal cell 200b in breast cancer patients. We found that low expres- carcinoma [19]. miR-200b as tumor suppressor regulat- sion of miR-200b correlated with advanced clinical stage ing EMT has bee reported in several malignancies, such and more distant metastasis in breast cancer. Since as prostate cancer [20], colon cancer [21], non-small cell miR-200b was reported as a regulator of EMT, which lung cancer [22], and so on. The dysregulation of miR- was thought to be the initiation of metastasis, the re- 200b in cancer could be transcriptional inhibition or sults were reasonable. Further we showed that the patients epigenetic modifications, such like DNA methylation with low miR-200b expression correlated with worse Ye et al. Journal of Translational Medicine 2014, 12:17 Page 9 of 10 http://www.translational-medicine.com/content/12/1/17 outcome, indicating miR-200b as a tumor suppressor Authors’ contributions FY and HT designed the experiments, interpreted the data, and wrote the in breast cancer. manuscript. FY, HT and QL carried out experiments. FY, HT, QL, XX, MW, XL, We predicted the targets of miR-200b in breast BC collected the human samples and clinical data. All authors read and cancers. Besides previously reported ZEB1/ZEB2, we approved the final manuscript. found multiple members of RAB family were also poten- tial targets of miR-200b. We then performed the lucif- Acknowledgments We thank the patients who participated in this study, and Dr. Jingping Yun, erase report assay to identify RAB21, RAB23, RAB18 Mayan Huang, and Xingjuan Yu for assistance on tissue microarray and RAB3B as novel direct targets of miR-200b. The construction and ISH analysis. This work was supported by funds from regulations of RAB21, RAB23, RAB18 and RAB3B by National Natural Science Foundation of China (81272514, 31100935, and 81302318), Key Program of National Natural Science Foundation of China miR-200b were further confirmed in breast cancer (31030061), and the China Postdoctoral Science Foundation (2012 M520075). cell lines. RAB family proteins belong to the large Ras superfamily Author details Department of Breast Oncology, Sun Yat-Sen University Cancer Center, 651 of small GTPases [29]. There are more than 60 members East Dongfeng Road, Guangzhou, Guangdong 510060, People’s Republic of in RAB family in humans, which are specifically local- China. State Key Laboratory of Oncology in South China, Sun Yat-Sen ized to subcellular membrane compartments, regulating University Cancer Center, Collaborative innovation center for cancer medicine, 651 East Dongfeng Road, Guangzhou, Guangdong 510060, intracellular membrane transport. By switching between People’s Republic of China. The Center for Skull Base Surgery and inactive cytosolic GDP-bound forms and active mem- Neurooncology, Changsha, Hunan, People’s Republic of China. brane-associated GTP-bound forms, RAB proteins could Received: 4 September 2013 Accepted: 27 December 2013 control endocytosis, protein secretion, recycling and Published: 21 January 2014 degradation, thus acting as key regulators of intracellular trafficking [30]. Recent studies have provided emerging References evidences for involvement of RAB proteins in tumour 1. Siegel R, Naishadham D, Jemal A: Cancer statistics, 2013. CA Cancer J Clin progression. More and more members of RAB family have 2013, 63:11–30. been reported to be dysregulated in cancers, either as on- 2. Singletary SE, Allred C, Ashley P, Bassett LW, Berry D, Bland KI, Borgen PI, Clark G, Edge SB, Hayes DF: Revision of the American joint committee on cogenes or tumor suppressors [31]. Several RAB proteins cancer staging system for breast cancer. J Clin Oncol 2002, 20:3628–3636. have been linked with tumor migration, invasion and drug 3. Carey LA, Perou CM, Livasy CA, Dressler LG, Cowan D, Conway K, Karaca G, resistance. Among them, up-regulation of Rab3B is re- Troester MA, Tse CK, Edmiston S: Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA: the journal of the ported in prostate cancer, promoting cancer cell survival American Medical Association 2006, 295:2492–2502. [32]. Rab21 is associated with the control of integrin traf- 4. Carey LA, Dees EC, Sawyer L, Gatti L, Moore DT, Collichio F, Ollila DW, ficking, thus modulating adhesion and motility in breast Sartor CI, Graham ML, Perou CM: The triple negative paradox: primary tumor chemosensitivity of breast cancer subtypes. Clin Cancer Res 2007, cancer cells [33]. Another RAB family member, RAB23 13:2329–2334. has been identified as an antagonist of Sonic Hedge- 5. Carlson RW, Allred DC, Anderson BO, Burstein HJ, Edge SB, Farrar WB, Forero hog signaling pathway which is deregulated in many A, Giordano SH, Goldstein LJ, Gradishar WJ: Metastatic Breast Cancer, Version 1.2012 Featured Updates to the NCCN Guidelines. J Natl Compr cancers [34], and found to over-expressed in gastric Canc Netw 2012, 10:821–829. and liver cancer [35]. 6. Liu H: MicroRNAs in breast cancer initiation and progression. Cell Mol Life In our study, we identified RAB21, RAB23, RAB18 Sci 2012, 69:3587–3599. 7. Calin GA, Croce CM: MicroRNA signatures in human cancers. Nat Rev and RAB3B as novel direct targets of miR-200b in Cancer 2006, 6:857–866. breast cancers. Over-expression of miR-200b or knock- 8. Korpal M, Kang Y: The emerging role of miR-200 family of microRNAs in down of the four RAB proteins significantly inhibited epithelial-mesenchymal transition and cancer metastasis. RNA biology 2008, 5:115–119. breast cancer cell proliferation and invasion. However, 9. Mongroo PS, Rustgi AK: The role of the miR-200 family in epithelial- whether the tumor suppressing effects of miR-200b in mesenchymal transition. Cancer Biol Ther 2010, 10:219–222. breast cancer depends dominatingly or partially on its 10. Du Y, Xu Y, Ding L, Yao H, Yu H, Zhou T, Si J: Down-regulation of miR-141 in gastric cancer and its involvement in cell growth. J Gastroenterol 2009, regulation of RAB proteins needs more investigation. 44:556–561. 11. Cochrane DR, Howe EN, Spoelstra NS, Richer JK: Loss of miR-200c: a marker of aggressiveness and chemoresistance in female reproductive cancers. Conclusion J Oncol 2010, 2010:821717. In summary, our study demonstrated that miR-200b 12. Ceppi P, Mudduluru G, Kumarswamy R, Rapa I, Scagliotti GV, Papotti M, Allgayer H: Loss of miR-200c expression induces an aggressive, invasive, could be a tumor suppressor and a potential biomarker and chemoresistant phenotype in non-small cell lung cancer. Mol Cancer in breast cancer patients. Members of RAB family, Res 2010, 8:1207–1216. RAB21, RAB23, RAB18 and RAB3B were novel targets 13. Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, Vadas MA, Khew-Goodall Y, Goodall GJ: The miR-200 family and miR-205 regulate regulated by miR-200b in breast cancer, which could be epithelial to mesenchymal transition by targeting ZEB1 and SIP1. of promising therapeutic significance. Nat Cell Biol 2008, 10:593–601. 14. Shimono Y, Zabala M, Cho RW, Lobo N, Dalerba P, Qian D, Diehn M, Liu H, Competing interests Panula SP, Chiao E: Downregulation of miRNA-200c links breast cancer No potential conflicts of interest were disclosed. stem cells with normal stem cells. Cell 2009, 138:592–603. Ye et al. Journal of Translational Medicine 2014, 12:17 Page 10 of 10 http://www.translational-medicine.com/content/12/1/17 15. Leskela S, Leandro-Garcia LJ, Mendiola M, Barriuso J, Inglada-Perez L, Munoz 34. Eggenschwiler JT, Espinoza E, Anderson KV: Rab23 is an essential negative I, Martinez-Delgado B, Redondo A, de Santiago J, Robledo M, et al: The regulator of the mouse Sonic hedgehog signalling pathway. Nature 2001, miR-200 family controls beta-tubulin III expression and is associated with 412:194–198. paclitaxel-based treatment response and progression-free survival in 35. Hou Q, Wu YH, Grabsch H, Zhu Y, Leong SH, Ganesan K, Cross D, Tan LK, ovarian cancer patients. Endocr Relat Cancer 2011, 18:85–95. Tao J, Gopalakrishnan V: Integrative genomics identifies RAB23 as an 16. Xia W, Li J, Chen L, Huang B, Li S, Yang G, Ding H, Wang F, Liu N, Zhao Q, et al: invasion mediator gene in diffuse-type gastric cancer. Cancer Res 2008, MicroRNA-200b regulates cyclin D1 expression and promotes S-phase entry 68:4623–4630. by targeting RND3 in HeLa cells. Mol Cell Biochem 2010, 344:261–266. 17. Feng B, Wang R, Chen L-B: Review of miR-200b and cancer chemosensitivity. doi:10.1186/1479-5876-12-17 Biomed Pharmacother 2012, 66:397–402. Cite this article as: Ye et al.: miR-200b as a prognostic factor in breast 18. Tang H, Wang Z, Liu X, Liu Q, Xu G, Li G, Wu M: LRRC4 inhibits glioma cell cancer targets multiple members of RAB family. Journal of Translational Medicine 2014 12:17. growth and invasion through a miR-185-dependent pathway. Curr Cancer Drug Targets 2012, 12:1032–1042. 19. Yoshino H, Enokida H, Itesako T, Tatarano S, Kinoshita T, Fuse M, Kojima S, Nakagawa M, Seki N: Epithelial-mesenchymal transition-related microRNA-200s regulate molecular targets and pathways in renal cell carcinoma. J Hum Genet 2013, 58:508–516. 20. He M, Liu Y, Deng X, Qi S, Sun X, Liu G, Zhao M: Down-regulation of miR-200b-3p by low p73 contributes to the androgen-independence of prostate cancer cells. Prostate 2013, 73:1048–1056. 21. Cai ZG, Zhang SM, Zhang H, Zhou YY, Wu HB, Xu XP: Aberrant expression of microRNAs involved in epithelial-mesenchymal transition of HT-29 cell line. Cell Biol Int 2013, 37:669–674. 22. Pacurari M, Addison JB, Bondalapati N, Wan YW, Luo D, Qian Y, Castranova V, Ivanov AV, Guo NL: The microRNA-200 family targets multiple non-small cell lung cancer prognostic markers in H1299 cells and BEAS-2B cells. Int J Oncol 2013, 43:548–560. 23. Castilla MA, Diaz-Martin J, Sarrio D, Romero-Perez L, Lopez-Garcia MA, Vieites B, Biscuola M, Ramiro-Fuentes S, Isacke CM, Palacios J: MicroRNA-200 family modulation in distinct breast cancer phenotypes. PLoS One 2012, 7:e47709. 24. Davalos V, Moutinho C, Villanueva A, Boque R, Silva P, Carneiro F, Esteller M: Dynamic epigenetic regulation of the microRNA-200 family mediates epithelial and mesenchymal transitions in human tumorigenesis. Oncogene 2011, 31:2062–2074. 25. Yao C-X, Wei Q-X, Zhang Y-Y, Wang W-P, Xue L-X, Yang F, Zhang S-F, Xiong C-J, Li W-Y, Wei Z-R: miR-200b targets GATA-4 during cell growth and differentiation. RNA Biol 2013, 10:0–1. 26. Lim Y-Y, Wright JA, Attema JL, Gregory PA, Bert AG, Smith E, Thomas D, Lopez AF, Drew PA, Khew-Goodall Y: Epigenetic modulation of the miR-200 family is associated with transition to a breast cancer stem-cell-like state. J Cell Sci 2013, 126:2256–2266. 27. Meng F, Henson R, Lang M, Wehbe H, Maheshwari S, Mendell JT, Jiang J, Schmittgen TD, Patel T: Involvement of human micro-RNA in growth and response to chemotherapy in human cholangiocarcinoma cell lines. Gastroenterology 2006, 130:2113–2129. 28. Rossi L, Bonmassar E, Faraoni I: Modification of miR gene expression pattern in human colon cancer cells following exposure to 5-fluorouracil in vitro. Pharmacol Res 2007, 56:248–253. 29. Jordens I, Marsman M, Kuijl C, Neefjes J: Rab proteins, connecting transport and vesicle fusion. Traffic 2005, 6:1070–1077. 30. Kelly E, Horgan C, Goud B, McCaffrey M: The Rab family of proteins: 25 years on. Biochem Soc Trans 2012, 40:1337–1347. 31. Recchi C, Seabra MC: Rab GTPases and their interacting proteins in health and disease: novel functions for Rab GTPases in multiple aspects of tumour progression. Biochem Soc Trans 2012, 40:1398. Submit your next manuscript to BioMed Central 32. Tan PY, Chang CW, Chng KR, Wansa KDSA, Sung W-K, Cheung E: Integration and take full advantage of: of regulatory networks by NKX3-1 promotes androgen-dependent prostate cancer survival. Mol Cell Biol 2012, 32:399–414. 33. Pellinen T, Arjonen A, Vuoriluoto K, Kallio K, Fransen JAM, Ivaska J: Small • Convenient online submission GTPase Rab21 regulates cell adhesion and controls endosomal traffic of • Thorough peer review beta1-integrins. J Cell Biol 2006, 173:767–780. • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Translational Medicine Springer Journals

miR-200b as a prognostic factor in breast cancer targets multiple members of RAB family

Loading next page...
 
/lp/springer-journals/mir-200b-as-a-prognostic-factor-in-breast-cancer-targets-multiple-t3ixgAvik8

References (70)

Publisher
Springer Journals
Copyright
Copyright © 2014 by Ye et al.; licensee BioMed Central Ltd.
Subject
Biomedicine; Biomedicine general; Medicine/Public Health, general
eISSN
1479-5876
DOI
10.1186/1479-5876-12-17
pmid
24447584
Publisher site
See Article on Publisher Site

Abstract

Background: miR-200b has been reported to be a tumor suppressor and a promising therapeutic target in cancer. miR-200b has been associated with epithelial-mesenchymal transition and chemo-resistance in cancer. The aim of this study is to investigate the expression of miR-200b, its prognostic roles and its potential targets in breast cancer. Methods: qRT-PCR was used to detect miR-200b expression in breast cancer tissues and cell lines. In situ hybridization of miR-200b on tissue microarray including 134 breast cancer samples was used to evaluate its prognostic role. Novel targets of miR-200b in breast cancer were predicted and confirmed by luciferase reporter assay and western bloting. Immunohistochemical staining was used for protein detection. The biological effects of miR-200b in breast cancer cells were further confirmed by ectopic expression of its mimics followed by MTT assay and invasion test. Results: miR-200b was downregulated in breast cancer tissues and cell lines and its low-expression correlated with poor outcome in breast cancer patients. Members of RAB family, RAB21, RAB23, RAB18 and RAB3B were predicted to be the targets of miR-200b. The luciferase reporter assay was performed to certificate this prediction. The expressions of RAB21, RAB23, RAB18 and RAB3B were suppressed by transfection of miR-200b in breast cancer cells. Over-expression of miR-200b or knock-down of RAB21, RAB23, RAB18 and RAB3B inhibited breast cancer cell proliferation and invasion in vitro. Conclusions: Our study provides evidence that miR-200b is a prognostic factor in breast cancer targeting multiple members of RAB family. MiR-200b could be a potential therapeutic target in breast cancer. Keywords: miR-200b, RAB family, Breast cancer, Prognosis Background like type (ER-, PR-, HER2-, cytokeratin 5/6+, and/or epi- Breast cancer is the first ranked female malignancy dermal growth factor receptor (EGFR)+). Breast cancer of worldwide, with about 200,000 new incidence and 40,000 HER2 over-expressing or basal-like type would predict deaths per year in US [1]. Generally breast cancers could more recurrence, distant metastasis, and therapy resist- be classified according to the TNM staging system [2] and ance [4]. The comprehensive therapy and prognosis for their molecular groups [3], which include luminal A type breast cancer would depend on both the TNM stage and (estrogen receptor (ER) + and/or progesterone receptor molecular subtype. Although breast cancers with early (PR) +, human epidermal growth factor receptor-2 stage show excellent outcome after therapy, recurrent and (HER2)-), luminal B type (ER + and/or PR+, HER2+), metastatic breast cancer patients remain big problems for HER2 over-expressing type (ER-, PR-, and HER2+), basal- cure [5]. MicroRNAs, also termed miRNAs, are a class of en- * Correspondence: xiexm@sysucc.org.cn dogenous, non-protein coding single-stranded RNA mole- Equal contributors cules with a length of 21–23 nucleotides, which plays a Department of Breast Oncology, Sun Yat-Sen University Cancer Center, 651 crucial role in the post-transcriptional regulation of gene East Dongfeng Road, Guangzhou, Guangdong 510060, People’s Republic of China expression [6]. miRNAs are highly conserved and specific, State Key Laboratory of Oncology in South China, Sun Yat-Sen University and regulate gene expression by binding to the 3′ untrans- Cancer Center, Collaborative innovation center for cancer medicine, 651 East lated region (UTR) of target messengerRNA (mRNAs) Dongfeng Road, Guangzhou, Guangdong 510060, People’s Republic of China Full list of author information is available at the end of the article © 2014 Ye et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ye et al. Journal of Translational Medicine 2014, 12:17 Page 2 of 10 http://www.translational-medicine.com/content/12/1/17 and inhibiting translation or inducing degradation of line 184A, MCF-10A, and several breast cancer cell lines mRNAs. miRNAs has been proved to play vital roles in were also included. Total RNAs were extracted from cancer management, acting as either oncogenes or tumor tissues/cells with TRIzol reagent (Invitrogen). For miR- suppressors [7]. 200b, reverse transcription and qRT-PCR reactions were The miR-200 family (including miR-200a, miR-200b, performed by means of a SYBR-green-containing PCR miR-200c, miR-141 and miR-429) are transcribed from kit (GenePharma, Shanghai, China). U6 snRNA was used two chromosomal locations: miR-200b-200a-429 cluster as an endogenous control for miRNA detection. The at chromosomal location 1p36 and miR-200c-141 cluster expression of miR-200b was quantified by measuring at chromosomal location 12p13 [8]. Recently, the miR- cycle threshold (Ct) values and normalized using the ΔΔCt 200 family has been associated with carcinogenesis and 2- method relative to U6 snRNA. cancer therapy [9]. Dysregulation of the miR-200 family was reported in several malignancies, including ovarian, Patients and specimens for tissue microarray endometrial, lung and gastric cancer [10-12]. The miR- A total of 134 female breast cancer patients who were 200 family has been supposed to be tumor suppressors diagnosed by histo-pathology in Sun Yat-Sen University regulating epithelial-mesenchymal transition (EMT) Cancer Center from October 2001 to September 2006 [13]. miR-200 directly targets EMT-inducing transcrip- were obtained. Specimens were formalin-fixed and em- tional factors ZEB1 and ZEB2, which repress E-cadherin bedded in paraffin by standard methodology after obtained expression. miR-200 could also suppress β-catenin/Wnt during surgery and were stored in the Department of signaling pathway by targeting β-catenin mRNA [14], Specimen and Resource in Sun Yat-Sen University Cancer thus highlighting its roles in cancer invasion and metas- Center. IHC of ER, PR, and HER-2 status were performed tasis. Moreover, mi-200 has been reported to affect the in the Pathology Department of Sun Yat-Sen University chemotherapy and endocrine thrapy resistance in breast Cancer Center. All the patients included in present study cancer [15,16]. Among the miR-200 family, miR-200b is did not receive any chemotherapy and radiation therapy thought to be the fundamental regulator in EMT and before, and their complete clinico-pathological data, inclu- cancer chemo-sensitivity [17]. However, the expression ding age, histological type, lymph nodes status, tumor size, of miR-200b and its prognostic role in breast cancer stage, local relapse, distant metastatic relapse, ER status, remain unclear. PR status and HER-2 status, were available and reviewed. In this study, we investigated the expression of miR- Histological type, reclassified according to the WHO 200b and its prognostic roles in breast cancer patients, classification and stage of tumor, was based on the TNM predicted and further identified multiple members of staging system (American Joint Committee on Cancer RAB family as new targets of miR-200b in breast cancer. classification). Follow-up was updated by review of records and telephone calls. The date of death and the date of Materials and methods relapse were used to calculate estimate overall survival Cell lines and culture (OS) and disease-free survival (DFS). Human mammary epithelial (HME) cell line 184A, This study was approved by the Ethics Committee of MCF-10A, human breast cancer cell lines MDA-MB- SunYat-Sen University Cancer Center Health Authority. 231, MDA-MB-435, MCF-7, T47D, BT-474, BT-483, and The collection and use of tissues followed the proce- mouse breast cancer cell line 4 T1 were obtained from the dures that are in accordance with the ethical standards American Type Culture Collection (Manassas, VA, USA) as formulated in the Helsinki Declaration. and were passaged in our laboratory for less than six months after resuscitation of frozen aliquots. The breast Tissue microarray (TMA) construction cancer cells were cultured in Dulbecco’s modified Eagle’s Representative part of the breast cancer specimens used medium (DMEM, Invitrogen, CA, USA) supplemented for creating tissue microarray were selected by two experi- with 10% fetal bovine serum (FBS, GIBCO, Cappinas, enced pathologists, using hematoxylin and eosin–stained Brazil), in a humidified incubator at 37°C containing 5% sections which were formalin-fixed and embedded in CO2. All cell lines were re-authenticated by short tandem paraffin as mentioned above. TMA block was con- repeat DNA profiling every 6 months after used. structed with MiniCore Control Station (ALPHELYS SARL, France) and designed by TMA Designer tissue Quantitative real-time polymerase chain reaction analysis array design software (ALPHELYS SARL, France). We (qRT-PCR) used 1.0-mm core tissue biopsies and took tissues Expression level of miR-200b was detected in both from paraffin-embedded tissue blocks to two new recipi- breast cancer tissues and cell lines. 40 pairs of tumor ent blocks (one contained 51 samples, and the other 83 tissues and para-carcinoma tissues from breast cancer samples), and one core per case was arrayed. The recipient patients were obtained. Human mammary epithelial cell blocks were cut and placed on slides. Ye et al. Journal of Translational Medicine 2014, 12:17 Page 3 of 10 http://www.translational-medicine.com/content/12/1/17 LNA probes for miR-200b finally HRP-Streptavidin for 15 min. After DAB staining, miR-200b miRCURYTM LNA custom detection probe the results were graded for intensity (0-negative, 1-weak, (Exiqon, Vedbaek, Denmark) was used for ISH. The 5′- 2-moderate, and 3-strong) and percentage of positive cells 3′ sequences (enhanced with LNA) were TCATCATT (0, 1 (1–24%), 2 (25–49%), 3 (50–74%), and 4 (75–100%)) ACCAGGCAGTATTA with a digoxigenin (DIG) label at with discrepancies resolved by consensus. The grades were both the 5′ and 3′ ends. multiplied to determine a score. The scores of tumors were defined as the following rule: negative (score = 0–3) In situ hybridization of miR-200b and scoring system and positive (score > =4). After deparaffinized, the slides were mounted onto flow through slide chambers and placed in a Tecan Construction of luc-UTR vectors Freedom Evo automated hybridization instrument (Tecan, The full-length RAB21, RAB23, RAB18, RAB3B, RAB37, Männedorf, Switzerland) in which the following steps RAB8B, RAB7A, RAP1B, RAP2C 3′-UTR was cloned were performed: proteinase-K treatment 15 μg/ml at 37°C into the EcoRIand HindIII sites of the pMIR-REPORT for 8 min, pre-hybridization in Exiqon hybridization buffer luciferase vector (Ambion, Austin, TX, U.S.) using PCR (Exiqon, Vedbæk, Denmark) at 62°C for 15 min, hybri- generated fragment. A Luc-mut vector of RAB21, RAB23, dization with 40 nM miR-200b probe, stringent washes RAB18, and RAB3B in which the first seven nucleotides with 5 × SSC, 1 × SSC and 0.2 × SSC buffers at 62°C complementary to the miR-200b seed-region were mu- over 33 min, DIG blocking reagent (Roche, Mannheim, tated by site-directed mutagenesis (Stratagene) served as a Germany) in maleic acid buffer containing 2% sheep mutant control. serum at 30°C for 15 min, Streptavidin-HRP -conjugated anti-digoxigenin (diluted 1:500 in blocking reagent, Roche) Luciferase assay at 30°C for 30 min. After washing, the slides were incu- Luc-wt, Luc-mut, and Luc-ctrl were co-transfected bated in DAB at 25°C for 3-5 min. The slides were then within vitro-produced miR-200b into MDA-MB-231 cells. dismantled in water, dehydrated in alcohol solutions The pMIR-REPORT β-galactosidase control vector was and mounted with eukitt mounting medium (VWR, transfected and served as a control. Luciferase activity Herlev, Denmark). was measured in cell lysates 48 h after transfection The intensities of miR-200b staining was scored by using a dual-light luminescent reporter gene assay kit 0–4, according to the standards of 0–1 (no staining), (Applied Biosystems). Results were normalized against 1–2 (weak staining), 2–3(medium staining)and 3–4 β-galactosidase activity. (strong staining). The percentages of miR-200b cells of individual samples were analyzed. Those expression Western blot scores equaled to the intensities * the percentages, and the Cell protein lysates, cytosol protein or nuclear protein was maximum was 4 and the minimum was 0. Individual separated in 10% SDS-polyacrylamide gels, electrophoret- samples were evaluated by at least two pathologists in a ically transferred to polyvinylidene difluoride membranes blinded manner, and those expression scores of greater (Millipore), then detected with mouse monoclonal anti- than 2 were defined high expression, otherwise the expres- body for RAB21 (sc-81917), rabbit polyclonal antibody for sion scores were low. RAB23 (sc-130248) and RAB3B (sc-305), goat polyclonal antibody for RAB18 (polyclonal antibody for RAB) (Santa Immunohistochemical (IHC) staining and scoring system Cruz Biotechnology), mouse monoclonal antibody for β- The Labeled StreptAvidin Biotin Method was used for actin (Abcam) and commercial ECL kit (Pierce). The IHC in our study. After deparaffinizing and rehydrating, intensity of protein fragments was quantified using the slides were treated with 90% methanol/3% H O 2 2 Chemical DocTM XRS + (Bio-Rad). solution for 15 min at room temperature to block endo- genous peroxidase. Then, the slides were soaked in sodium citrate buffer (10 mM Sodium citrate, 0.05% RNA silencing for RAB21, RAB23, RAB18, and RAB3B Tween 20, pH 6.0) under 96°C for 5 min for antigen The sense sequences of siRNA oligonucleotides targeting retrieval. After blocking by BSA, the following antibodies the RAB18, RAB21, RAB23, and RAB3B transcripts, were used: mouse monoclonal antibody for RAB21 (sc- respectively, were as follows: si- RAB18: 5′-UUCUGG 81917), rabbit polyclonal antibody for RAB23 (sc-130248) UUGUAACUUCACGGCT-3′; si- RAB21: 5′-UUAAUA and RAB3B (sc-305), goat polyclonal antibody for RAB18 GGUUGGAUGGCGGTT-3′; si- RAB23: 5′-CUUCAC (polyclonal antibody for RAB) (Santa Cruz Biotechnology). TACUGCUUCGAGTT-3′; and si- RAB3B: 5′-AUAA We added antibodies to the slides for overnight storage at CUUGGAGGGACUGCCTT-3′ (Invitrogen). Scrambled 4°C and then incubated the slides at room temperature siRNA was used as a negative control. Cells were plated in with biotinylated secondary antibody for 20 min, and culture dishes or in 24-well plates for 24 h, and transfected Ye et al. Journal of Translational Medicine 2014, 12:17 Page 4 of 10 http://www.translational-medicine.com/content/12/1/17 Figure 1 miR-200b was down-regulated in breast cancer tissues and cell lines. (A) The expression of miR-200b was detected by qRT-PCR in 40 pairs of normal mammary tissues and breast cancer samples. (B) The expression of miR-200b was detected by qRT-PCR in breast cancer cell lines. with siRNA using Lipofectamine 2000. After 48 h, the 200b mimics were seeded at a density of 5,000 cells per cells were harvested for use in other assays. well in 96-well plates and incubated at 37°C for 24 h. Cells were then incubated an additional 72 h, and the MTT MTT assay assay was performed according to the manufacturer’sin- Cell viability was examined by the 3-(4, 5-dimethyl- structions (Molecular Probes, Eugene, OR). Absorbance thiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) values were determined at 570 nm on a Spectra Max 250 assay. Cells transfected with either scramble or miR- spectrophotometer (Molecular Devices, Sunnyvale, CA). Figure 2 miR-200b as a prognostic factor in breast cancer patients. (A) The ISH staining of miR-200b in breast cancer tissue microarray: ×200. Two miR-200b high samples and two miR-200b low samples were shown as representatives. (B) qRT-PCR results for miR-200b detection in three miR-200b high ISH staining samples and three miR-200b low samples. (C) Survival curves of OS and DFS according to miR-200b expression. Low miR-200b expression correlated with worse outcome. Ye et al. Journal of Translational Medicine 2014, 12:17 Page 5 of 10 http://www.translational-medicine.com/content/12/1/17 Cell invasion and migration assays The relationships between expression of miR-200b and The cell invasion assay was conducted as described pre- clinical parameters in breast cancer patients viously [18]. Briefly, cells were seeded onto the basement We further evaluated the prognostic role of expression membrane matrix present in the insert of a 24-well cul- of miR-200b in breast cancer patients by in situ hybri- ture plate (EC matrix, Chemicon, Temecula, CA). Fetal dization (ISH) staining on tissue microarray. MiR-200b bovine serum was added to the lower chamber as a chemoattractant. After an additional 48 hours, the non- Table 1 Clinico-pathological variables and the expression of miR-200b in total breast cancer patients invading cells and EC matrix were gently removed with a cotton swab. Invasive cells located on the lower side of miR-200b low miR-200b high Total P value (n = 78) (n = 56) Characteristics the chamber were stained with crystal violet, counted (n = 134) No. % No. % and imaged. Age (years) 0.330 <50 82 46 56.1 36 43.9 Statistical analysis >50 52 32 61.5 20 38.5 Data are presented as mean ± SD from at least three Menopause 0.524 separate experiments. Multiple group comparisons were performed using ANOVA with a post hoc test for Yes 63 37 58.7 26 41.3 subsequent individual group comparisons. The distinct No 71 41 57.7 30 42.3 expression of miR-200b between tumor tissues and para- LN infiltrated 0.148 carcinoma tissues was examined by independent samples Yes 80 50 62.5 30 37.5 T-test. The relationships between miR-200b expression No 54 28 51.9 26 48.1 and clinico-pathological parameters were examined by Tumor size (cm) 0.080 chi-square test. Overall survival (OS) or disease-free sur- vival (DFS) curves were calculated by the Kaplan-Meier = < 2 38 18 47.3 20 52.7 method and the log-rank test was used to determine >2 96 60 62.5 36 37.5 the difference in OS or DFS rates between two groups. TNM stage 0.040* Results were considered statistically significant when I-II 78 40 51.3 38 48.7 P ≤ 0.05 was obtained. All the statistical analyses were III- IV 56 38 67.9 18 32.1 performed using SPSS13.0 for Windows (SPSS Inc., Local relapse 0.083 Chicago, IL, USA). Yes 8 7 87.5 1 12.5 No 126 71 56.3 55 43.7 Results Distant The expression of miR-200b in breast cancer tissues and 0.003* metastasis cell lines Yes 34 27 79.4 7 20.6 To evaluate the expression of miR-200b in breast cancer tissues, qRT-PCR was used to detect the expression level No 100 51 61.0 49 49.0 in 40 pairs of tumor tissues and para-carcinoma (normal) ER status 0.155 tissues from breast cancer patients. The results showed Positive 51 33 64.7 18 35.3 that expression of miR-200b in breast cancer tissues was Negative 83 45 54.2 38 45.8 significantly lower than in normal tissues (Figure 1A). In PR status 0.073 comparison with the normal tissues, miR-200b was down- Positive 54 36 66.7 18 33.3 regulated in 77.5% (31/40) of the tumor samples. We further detected expression of miR-200b in cell lines Negative 80 42 52.5 38 47.5 (Figure 1B), including human mammary epithelial (HME) HER-2 status 0.213 cell lines (184A, MCF-10A), human breast cancer cell Positive 22 15 68.2 7 31.8 lines (MDA-MB-231, MDA-MB-435, MCF-7, T47D, Negative 112 63 56.3 49 43.8 BT-474, BT-483), and mouse breast cancer cell line TNBC status 0.472 4 T1. Compared with 184A and MCF-10A, miR-200b TNBC 51 29 56.9 22 43.1 was down-regulated in MCF-7, MDA-MB-435, T47D, BT-483, MDA-MB-231, and 4 T1, especially in the latter Non-TNBC 83 49 59.0 34 41.0 two cell lines. These results revealed that reduced miR- *means statistically significant (p < 0.05). % means percentage within the row. 200b expression was a frequent event in human breast TNBC, triple-negative breast cancer, means ER(−), PR(−), HER-2(−). High cancer tissues and could be involved in breast cancer expression of miR-200b was seen in 41.8% of total patients and correlated carcinogenesis. with early TNM stage (p = 0.040) and fewer metastasis (p = 0.003). Ye et al. Journal of Translational Medicine 2014, 12:17 Page 6 of 10 http://www.translational-medicine.com/content/12/1/17 ISH staining in breast cancer tissues was located in miR-200b targeted multiple members of RAB family the cytoplasm. Two miR-200b high samples and two To determine the function of miR-200b in breast cancer, miR-200b low samples were shown as representatives we used online softwares TargetScan to search for (Figure 2A). To confirm the miR-200b ISH staining potential miR-200b target genes. We found that mem- results, we extracted total RNAs form three miR-200b bers of RAB family are among these candidate target high samples and three miR-200b low samples for genes. A miR-200b-binding site was found in the 3′- qRT-PCR (Figure 2B). The PCR results showed that UTR of RAB21, RAB23, RAB18, RAB3B, RAB37, RAB8B, the ISH staining could reflect the relative level of miR- RAB7A, RAP1B, RAP2C mRNA with perfect base pairing 200b expression in the samples. The clinico-pathologic (Figure 3A). To verify whether these genes were direct characteristics and miR-200b expression of the breast targets of miR-200b, we subcloned the full-length 3′-UTR cancer patients involved in our study are shown in Table 1. of RAB21, RAB23, RAB18, RAB3B, RAB37, RAB8B, In all 134 breast cancer patients, high expression of RAB7A, RAP1B, RAP2C into the luciferase reporter miR-200b was seen in 41.8% of total patients. High vector. However, addition of in vitro-produced miR-200b miR-200b expression correlated with early TNM stage only suppressed the luciferase activity of the 3′-UTR of (p = 0.040) and fewer metastasis (p = 0.003). Further- RAB21, RAB23, RAB18 and RAB3B upon co-transfection more, markedly reduced overall survival (p = 0.0030) of the luciferase vector (wild-type, mutant, or negative con- and disease-free survival (p = 0.0000) were observed in the trol) with the in vitro-produced microRNAs (miR-200b breast cancer patients who had low miR-200b expression mimics or scramble control) into MDA-MB-231 cells compared with the patients who exhibited high expres- (Figure 3B). This inhibition was abolished when the seed sion levels (Figure 2C). In our study, the estimate sequences of the miR-200b target sequences were mutated overall survival and disease-free survival for miR-200b in the Luc-mut vector (Figure 3B). To directly assess the high patients were 109.3 months and 101.2 months, while effects of miR-200b on the expression of RAB21, RAB23, the two for miR-200b low patients were 78.3 months and RAB18 and RAB3B, we transfected miR-200b mimics into 66.2 months respectively. These results indicated that MDA-MB-231 and 4 T1 cells and found that overex- miR-200b could be a prognostic factor in breast cancer pression of miR-200b reduced mRNA and protein level of patients. RAB21, RAB23, RAB18 and RAB3B (Figure 3C). Figure 3 RAB21, RAB23, RAB18 and RAB3B is a direct target of miR-200b. (A) miR-200b binds to the 3′UTRs of RAB21, RAB23, RAB18, RAB3B, RAB37, RAB8B, RAB7A, RAP1B and RAP2C. Predicted binding between miR-200b and the seeds match in the nine predicted genes’ 3′UTRs. (B) Luciferase reporter assays 48 h after transfection with indicated pMIR-Report plasmids and a renilla transfection control plasmid, co-transfected with miR-200b mimics, or relevant scramble controls. Luciferase activities were significantly inhibited on co-transfection of miR-200b mimics and pMIR-Report plasmids of RAB21, RAB23, RAB18 and RAB3B. Mutation in the corresponding binding sites abolished this inhibition. Data shown were the mean ± s.d. of six replicates and were representative of three independent experiments. *P < 0.05 (C) miR-200b regulated the expression of RAB21, RAB23, RAB18 and RAB3B. Western blot analyzed their expression 48 h after transfection with miR-200b mimics or scramble control in MDA-MB-231 and 4 T1 cells. Ye et al. Journal of Translational Medicine 2014, 12:17 Page 7 of 10 http://www.translational-medicine.com/content/12/1/17 The association between expression of miR-200b and regulation of RAB21, RAB23, RAB18 and RAB3B pro- RAB21, RAB23, RAB18 and RAB3B in breast cancer tissues teins by miR-200b in breast cancer tissues. To evaluate the associations between expression of miR- 200b and RAB21, RAB23, RAB18 and RAB3B in breast Over-expression of miR-200b or knock-down of RAB21, cancer tissues, we further detected expression of miR- RAB23, RAB18 and RAB3B inhibited breast cancer cell 200b by ISH staining and expression of RAB21, RAB23, proliferation and invasion RAB18 and RAB3B protein by immunohistochemistry To assess the biological effects of over-expressing (IHC) staining in 10 pairs of tumor tissues and para- miR-200b in breast cancer cells, ectopic expression of carcinoma tissues from breast cancer patients, who had miR-200b mimics were transfected into MDA-MB-231 developed distant metastasis after operation. As shown and 4 T1 cells. Transfection of miR-200b mimics in in Figure 4, compared with normal breast tissue, miR- MDA-MB-231 and 4 T1 cells markedly attenuated cell 200b expression was down-regulated in tumor tissue, proliferation, compared with scramble (Figure 5A). More- while the contrary situations were found for RAB21, over, ectopic expression of miR-200b mimics in MDA- RAB23, RAB18 and RAB3B IHC staining. In summary, MB-231 and 4 T1 cells markedly attenuated cell invasion miR-200b low ISH staining was seen in 90% (9/10) compared with control cells (Figure 5B, C). To identify tumor samples, while positive rates for RAB21, RAB23, the biological effects of RAB21, RAB23, RAB18 and RAB18 and RAB3B IHC staining were 80%, 80%, 90%, RAB3B, specific siRNAs for each were also synthesized. 100% respectively. These results further confirmed the Transfection of siRNAs for RAB21, RAB23, RAB18 and Figure 4 IHC staining of RAB21, RAB23, RAB18 and RAB3B in human breast cancer tissues. 10 pairs of tumor tissues and normal tissues from breast cancer patients, who had developed distant metastasis after operation, were detected by ISH or IHC. Compared with normal tissue, miR-200b expression was down-regulated in tumor tissue, while the contrary situations were found for RAB21, RAB23, RAB18 and RAB3B. Ye et al. Journal of Translational Medicine 2014, 12:17 Page 8 of 10 http://www.translational-medicine.com/content/12/1/17 Figure 5 Over-expression of miR-200b or knock-down of RAB21, RAB23, RAB18 and RAB3B inhibits breast cancer cell proliferation and invasion. (A) Transfection of miR-200b mimics or knock-down of RAB21, RAB23, RAB18 and RAB3B in MDA-MB-231 and 4 T1 cells markedly attenuated cell proliferation: MTT assay at 24 h, 48 h and 72 h after transfection. (B, C) Transfection of miR-200b mimics or knock-down of RAB21, RAB23, RAB18 and RAB3B in MDA-MB-231 and 4 T1 cells inhibited cell invasion: Data shown in Figure B were the mean ± s.d. of three replicates and were representative of three independent experiments. *P < 0.05. The representing images were shown in Figure C. RAB3B also inhibits breast cancer cell proliferation and and histone modifications [23,24]. Moreover, miR-200b invasion (Figure 5A, B, and C). These results indicated is thought to be related to cell differentiation by target- that the biological effects of miR-200b in breast cancer ing GATA-4 [25]. Loss of miR-200b contributes to the cells may attribute to regulation of RAB21, RAB23, breast cancer stem cell status maintaining [26]. Besides, RAB18 and RAB3B, and thus miR-200b could be a thera- miR-200b has also been associated with cancer chemo- peutic target in breast cancer. sensitivity by modulating PTEN, PTPN12 and thus their downstream oncogenes like src and ras [27,28]. Discussion In this study, we investigated the expression of miR- The miR-200 family has been reported to be a funda- 200b and its prognostic role in breast cancer. We mental regulator of EMT, thus highlighting their roles in showed that expression of miR-200b was significantly cancer progression. As a founding member in miR-200 lower in breast cancer tissues than normal tissues. Similar family, miR-200b attracts much focus both in carcino- results were found in breast cancer cell lines. In addition, genesis and cancer therapy in recent years [17]. Down- we analyzed the prognostic role of the expression of miR- regulation of miR-200b has been observed in renal cell 200b in breast cancer patients. We found that low expres- carcinoma [19]. miR-200b as tumor suppressor regulat- sion of miR-200b correlated with advanced clinical stage ing EMT has bee reported in several malignancies, such and more distant metastasis in breast cancer. Since as prostate cancer [20], colon cancer [21], non-small cell miR-200b was reported as a regulator of EMT, which lung cancer [22], and so on. The dysregulation of miR- was thought to be the initiation of metastasis, the re- 200b in cancer could be transcriptional inhibition or sults were reasonable. Further we showed that the patients epigenetic modifications, such like DNA methylation with low miR-200b expression correlated with worse Ye et al. Journal of Translational Medicine 2014, 12:17 Page 9 of 10 http://www.translational-medicine.com/content/12/1/17 outcome, indicating miR-200b as a tumor suppressor Authors’ contributions FY and HT designed the experiments, interpreted the data, and wrote the in breast cancer. manuscript. FY, HT and QL carried out experiments. FY, HT, QL, XX, MW, XL, We predicted the targets of miR-200b in breast BC collected the human samples and clinical data. All authors read and cancers. Besides previously reported ZEB1/ZEB2, we approved the final manuscript. found multiple members of RAB family were also poten- tial targets of miR-200b. We then performed the lucif- Acknowledgments We thank the patients who participated in this study, and Dr. Jingping Yun, erase report assay to identify RAB21, RAB23, RAB18 Mayan Huang, and Xingjuan Yu for assistance on tissue microarray and RAB3B as novel direct targets of miR-200b. The construction and ISH analysis. This work was supported by funds from regulations of RAB21, RAB23, RAB18 and RAB3B by National Natural Science Foundation of China (81272514, 31100935, and 81302318), Key Program of National Natural Science Foundation of China miR-200b were further confirmed in breast cancer (31030061), and the China Postdoctoral Science Foundation (2012 M520075). cell lines. RAB family proteins belong to the large Ras superfamily Author details Department of Breast Oncology, Sun Yat-Sen University Cancer Center, 651 of small GTPases [29]. There are more than 60 members East Dongfeng Road, Guangzhou, Guangdong 510060, People’s Republic of in RAB family in humans, which are specifically local- China. State Key Laboratory of Oncology in South China, Sun Yat-Sen ized to subcellular membrane compartments, regulating University Cancer Center, Collaborative innovation center for cancer medicine, 651 East Dongfeng Road, Guangzhou, Guangdong 510060, intracellular membrane transport. By switching between People’s Republic of China. The Center for Skull Base Surgery and inactive cytosolic GDP-bound forms and active mem- Neurooncology, Changsha, Hunan, People’s Republic of China. brane-associated GTP-bound forms, RAB proteins could Received: 4 September 2013 Accepted: 27 December 2013 control endocytosis, protein secretion, recycling and Published: 21 January 2014 degradation, thus acting as key regulators of intracellular trafficking [30]. Recent studies have provided emerging References evidences for involvement of RAB proteins in tumour 1. Siegel R, Naishadham D, Jemal A: Cancer statistics, 2013. CA Cancer J Clin progression. More and more members of RAB family have 2013, 63:11–30. been reported to be dysregulated in cancers, either as on- 2. Singletary SE, Allred C, Ashley P, Bassett LW, Berry D, Bland KI, Borgen PI, Clark G, Edge SB, Hayes DF: Revision of the American joint committee on cogenes or tumor suppressors [31]. Several RAB proteins cancer staging system for breast cancer. J Clin Oncol 2002, 20:3628–3636. have been linked with tumor migration, invasion and drug 3. Carey LA, Perou CM, Livasy CA, Dressler LG, Cowan D, Conway K, Karaca G, resistance. Among them, up-regulation of Rab3B is re- Troester MA, Tse CK, Edmiston S: Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA: the journal of the ported in prostate cancer, promoting cancer cell survival American Medical Association 2006, 295:2492–2502. [32]. Rab21 is associated with the control of integrin traf- 4. Carey LA, Dees EC, Sawyer L, Gatti L, Moore DT, Collichio F, Ollila DW, ficking, thus modulating adhesion and motility in breast Sartor CI, Graham ML, Perou CM: The triple negative paradox: primary tumor chemosensitivity of breast cancer subtypes. Clin Cancer Res 2007, cancer cells [33]. Another RAB family member, RAB23 13:2329–2334. has been identified as an antagonist of Sonic Hedge- 5. Carlson RW, Allred DC, Anderson BO, Burstein HJ, Edge SB, Farrar WB, Forero hog signaling pathway which is deregulated in many A, Giordano SH, Goldstein LJ, Gradishar WJ: Metastatic Breast Cancer, Version 1.2012 Featured Updates to the NCCN Guidelines. J Natl Compr cancers [34], and found to over-expressed in gastric Canc Netw 2012, 10:821–829. and liver cancer [35]. 6. Liu H: MicroRNAs in breast cancer initiation and progression. Cell Mol Life In our study, we identified RAB21, RAB23, RAB18 Sci 2012, 69:3587–3599. 7. Calin GA, Croce CM: MicroRNA signatures in human cancers. Nat Rev and RAB3B as novel direct targets of miR-200b in Cancer 2006, 6:857–866. breast cancers. Over-expression of miR-200b or knock- 8. Korpal M, Kang Y: The emerging role of miR-200 family of microRNAs in down of the four RAB proteins significantly inhibited epithelial-mesenchymal transition and cancer metastasis. RNA biology 2008, 5:115–119. breast cancer cell proliferation and invasion. However, 9. Mongroo PS, Rustgi AK: The role of the miR-200 family in epithelial- whether the tumor suppressing effects of miR-200b in mesenchymal transition. Cancer Biol Ther 2010, 10:219–222. breast cancer depends dominatingly or partially on its 10. Du Y, Xu Y, Ding L, Yao H, Yu H, Zhou T, Si J: Down-regulation of miR-141 in gastric cancer and its involvement in cell growth. J Gastroenterol 2009, regulation of RAB proteins needs more investigation. 44:556–561. 11. Cochrane DR, Howe EN, Spoelstra NS, Richer JK: Loss of miR-200c: a marker of aggressiveness and chemoresistance in female reproductive cancers. Conclusion J Oncol 2010, 2010:821717. In summary, our study demonstrated that miR-200b 12. Ceppi P, Mudduluru G, Kumarswamy R, Rapa I, Scagliotti GV, Papotti M, Allgayer H: Loss of miR-200c expression induces an aggressive, invasive, could be a tumor suppressor and a potential biomarker and chemoresistant phenotype in non-small cell lung cancer. Mol Cancer in breast cancer patients. Members of RAB family, Res 2010, 8:1207–1216. RAB21, RAB23, RAB18 and RAB3B were novel targets 13. Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, Vadas MA, Khew-Goodall Y, Goodall GJ: The miR-200 family and miR-205 regulate regulated by miR-200b in breast cancer, which could be epithelial to mesenchymal transition by targeting ZEB1 and SIP1. of promising therapeutic significance. Nat Cell Biol 2008, 10:593–601. 14. Shimono Y, Zabala M, Cho RW, Lobo N, Dalerba P, Qian D, Diehn M, Liu H, Competing interests Panula SP, Chiao E: Downregulation of miRNA-200c links breast cancer No potential conflicts of interest were disclosed. stem cells with normal stem cells. Cell 2009, 138:592–603. Ye et al. Journal of Translational Medicine 2014, 12:17 Page 10 of 10 http://www.translational-medicine.com/content/12/1/17 15. Leskela S, Leandro-Garcia LJ, Mendiola M, Barriuso J, Inglada-Perez L, Munoz 34. Eggenschwiler JT, Espinoza E, Anderson KV: Rab23 is an essential negative I, Martinez-Delgado B, Redondo A, de Santiago J, Robledo M, et al: The regulator of the mouse Sonic hedgehog signalling pathway. Nature 2001, miR-200 family controls beta-tubulin III expression and is associated with 412:194–198. paclitaxel-based treatment response and progression-free survival in 35. Hou Q, Wu YH, Grabsch H, Zhu Y, Leong SH, Ganesan K, Cross D, Tan LK, ovarian cancer patients. Endocr Relat Cancer 2011, 18:85–95. Tao J, Gopalakrishnan V: Integrative genomics identifies RAB23 as an 16. Xia W, Li J, Chen L, Huang B, Li S, Yang G, Ding H, Wang F, Liu N, Zhao Q, et al: invasion mediator gene in diffuse-type gastric cancer. Cancer Res 2008, MicroRNA-200b regulates cyclin D1 expression and promotes S-phase entry 68:4623–4630. by targeting RND3 in HeLa cells. Mol Cell Biochem 2010, 344:261–266. 17. Feng B, Wang R, Chen L-B: Review of miR-200b and cancer chemosensitivity. doi:10.1186/1479-5876-12-17 Biomed Pharmacother 2012, 66:397–402. Cite this article as: Ye et al.: miR-200b as a prognostic factor in breast 18. Tang H, Wang Z, Liu X, Liu Q, Xu G, Li G, Wu M: LRRC4 inhibits glioma cell cancer targets multiple members of RAB family. Journal of Translational Medicine 2014 12:17. growth and invasion through a miR-185-dependent pathway. Curr Cancer Drug Targets 2012, 12:1032–1042. 19. Yoshino H, Enokida H, Itesako T, Tatarano S, Kinoshita T, Fuse M, Kojima S, Nakagawa M, Seki N: Epithelial-mesenchymal transition-related microRNA-200s regulate molecular targets and pathways in renal cell carcinoma. J Hum Genet 2013, 58:508–516. 20. He M, Liu Y, Deng X, Qi S, Sun X, Liu G, Zhao M: Down-regulation of miR-200b-3p by low p73 contributes to the androgen-independence of prostate cancer cells. Prostate 2013, 73:1048–1056. 21. Cai ZG, Zhang SM, Zhang H, Zhou YY, Wu HB, Xu XP: Aberrant expression of microRNAs involved in epithelial-mesenchymal transition of HT-29 cell line. Cell Biol Int 2013, 37:669–674. 22. Pacurari M, Addison JB, Bondalapati N, Wan YW, Luo D, Qian Y, Castranova V, Ivanov AV, Guo NL: The microRNA-200 family targets multiple non-small cell lung cancer prognostic markers in H1299 cells and BEAS-2B cells. Int J Oncol 2013, 43:548–560. 23. Castilla MA, Diaz-Martin J, Sarrio D, Romero-Perez L, Lopez-Garcia MA, Vieites B, Biscuola M, Ramiro-Fuentes S, Isacke CM, Palacios J: MicroRNA-200 family modulation in distinct breast cancer phenotypes. PLoS One 2012, 7:e47709. 24. Davalos V, Moutinho C, Villanueva A, Boque R, Silva P, Carneiro F, Esteller M: Dynamic epigenetic regulation of the microRNA-200 family mediates epithelial and mesenchymal transitions in human tumorigenesis. Oncogene 2011, 31:2062–2074. 25. Yao C-X, Wei Q-X, Zhang Y-Y, Wang W-P, Xue L-X, Yang F, Zhang S-F, Xiong C-J, Li W-Y, Wei Z-R: miR-200b targets GATA-4 during cell growth and differentiation. RNA Biol 2013, 10:0–1. 26. Lim Y-Y, Wright JA, Attema JL, Gregory PA, Bert AG, Smith E, Thomas D, Lopez AF, Drew PA, Khew-Goodall Y: Epigenetic modulation of the miR-200 family is associated with transition to a breast cancer stem-cell-like state. J Cell Sci 2013, 126:2256–2266. 27. Meng F, Henson R, Lang M, Wehbe H, Maheshwari S, Mendell JT, Jiang J, Schmittgen TD, Patel T: Involvement of human micro-RNA in growth and response to chemotherapy in human cholangiocarcinoma cell lines. Gastroenterology 2006, 130:2113–2129. 28. Rossi L, Bonmassar E, Faraoni I: Modification of miR gene expression pattern in human colon cancer cells following exposure to 5-fluorouracil in vitro. Pharmacol Res 2007, 56:248–253. 29. Jordens I, Marsman M, Kuijl C, Neefjes J: Rab proteins, connecting transport and vesicle fusion. Traffic 2005, 6:1070–1077. 30. Kelly E, Horgan C, Goud B, McCaffrey M: The Rab family of proteins: 25 years on. Biochem Soc Trans 2012, 40:1337–1347. 31. Recchi C, Seabra MC: Rab GTPases and their interacting proteins in health and disease: novel functions for Rab GTPases in multiple aspects of tumour progression. Biochem Soc Trans 2012, 40:1398. Submit your next manuscript to BioMed Central 32. Tan PY, Chang CW, Chng KR, Wansa KDSA, Sung W-K, Cheung E: Integration and take full advantage of: of regulatory networks by NKX3-1 promotes androgen-dependent prostate cancer survival. Mol Cell Biol 2012, 32:399–414. 33. Pellinen T, Arjonen A, Vuoriluoto K, Kallio K, Fransen JAM, Ivaska J: Small • Convenient online submission GTPase Rab21 regulates cell adhesion and controls endosomal traffic of • Thorough peer review beta1-integrins. J Cell Biol 2006, 173:767–780. • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

Journal

Journal of Translational MedicineSpringer Journals

Published: Jan 21, 2014

There are no references for this article.