Research Communication MiRNA Expression in Breast Cancer Varies with Lymph Node Metastasis and Other Clinicopathologic Features

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1 Research Communication MiRNA Expression in Breast Cancer Varies with Lymph Node Metastasis and Other Clinicopathologic Features Bin Wang 1 Jindong Li 1 Mei Sun 2 Lihua Sun 3 Xingyi Zhang 1 * 1 Department of Thoracic Surgery, Second Hospital of Jilin University, Chang Chun, Jilin , China 2 Department of Pathology, Second Hospital of Jilin University, Chang Chun, Jilin , China 3 Department of Anesthesiology, Second Hospital of Jilin University, Chang Chun, Jilin , China Abstract Breast carcinoma is the most common malignant tumor in females, and lymph node (LN) status is one of the most important prognostic factors in patients with breast cancer. MiRNAs have been shown to have important role in oncogenesis, invasion, and metastasis via epigenetic posttranscriptional gene regulation. However, lymphatic metastasis-related mirnas of breast cancer has not been well documented. The aim of this study was to identify and evaluate mirnas related to breast cancer LN metastasis and to explore the clinical significance of the differentially expressed mirnas in patients with breast cancer. The expression of mir- NAs in patients with primary breast cancer with LN metastasis and that without LN metastases was compared by mirna microarray. We further validated the mirnas (mir-185-5p, mir-339-5p, mir-542-5p, and mir-3923) between LN (n 5 31) and nonlymph node (NLN; n 5 42) group using real-time reverse transcriptase polymerase chain reaction. Furthermore, the relationship between mirna expression and clinical pathological features was analyzed. The mirna microarray initially identified that eight mirnas (mir-206, mir-3923, mir-181a, mir-92a, mir-421, mir-339-5p, mir-3196, and mir-29b) were downregulated in LN metastasis group, whereas five mirnas (mir-542-5p, mir-200a, mir-564, mir-451, mir-30c, mir-200b, mir-191-3p, mir-142-5p, and mir-185-5p) were upregulated in LN group when compared with those in NLN group. In the validation cohort, the expression levels of mir-185-5p and mir p were significantly expressed higher in LN group (P and P , respectively), and the expression levels of mir-339-5p and mir-3923 were significantly expressed lower in LN group (P and P , respectively). Our results indicate the potential role of mir-185-5p, mir-542-5p, mir-339-5p, and mir-3923 in predicting metastasis to the LN and prognosis in breast cancers. VC 2014 IUBMB Life, 66(5): , 2014 Keywords: breast cancer; micrornas; microarray; lymph node metastasis; prognosis VC 2014 International Union of Biochemistry and Molecular Biology Volume 66, Number 5, May 2014, Pages *Address correspondence to: Xingyi Zhang, Department of Thoracic Surgery, Second Hospital of Jilin University, Chang Chun, Jilin , China. binharyida@163.com Received 12 April 2014; Accepted 1 May 2014 DOI /iub.1273 Published online 20 May 2014 in Wiley Online Library (wileyonlinelibrary.com) Introduction Breast cancer is the most common malignant tumor in females, and the incidence of breast cancer is increasing in the developing world (1). Metastasis is one of the basic characteristics of malignant tumors, and lymphatic metastasis is the most common phenomenon for carcinoma, which is closely related to the poor prognosis because effective treatments are still lacking (2). Axillary lymph node (LN) status is one of the most important prognostic indicators in breast cancer, and the detection of nodal metastases is a key factor in recommending adjuvant chemotherapy after surgery (3). Widespread use of mammography has resulted in marked increase in early IUBMB Life 371

2 IUBMB LIFE detection of breast cancer, improvement in therapy, and declining mortality (4). The positive yield of axillary LN dissection also decreases, and LN negative patients do not benefit from axillary dissection but may suffer from its complications (5). Axillary LN dissection is now no longer considered to be the standard treatment in all patients with invasive breast cancer (6). Therefore, it is essential to find predictive factors for axillary LN involvement in early breast cancer. MiRNAs are endogenous, small noncoding single-stranded RNAs of nucleotides in length, found in both plants and animals, which function at post-transcriptional and transcriptional levels as negative regulators of gene expression. The binding of mirnas to complementary sites in the untranslated regions and other regions of protein-coding mrna sequences cause either degradation of the mrna or inhibition of translation (7, 8). It is estimated that up to 60% of genes may be regulated by more than 1,900 human mirnas thus far identified (9, 10). The involvement of mirnas in cancer pathogenesis is now well established, and mirna expression profiles accurately classify tumors at different stages and distinguish among subsets of patients with different molecular pathologies (11 13). There are some reports on the association of the clinicopathogenetic features of breast cancer with mirna expression (12 15). However, lymphatic metastasisrelated mirnas of breast cancer has not been well documented. The aim of this study was to identify and evaluate mirnas related with breast cancer LN metastasis. In this study, we investigated the differences in mirna profiling between primary breast cancers with LN metastasis and that without LN metastasis, and then selected several mirnas and detected the expression of them in 73 breast cancer specimens in an effort to identify the mirnas involved in breast cancer LN metastasis. Furthermore, association between mirna expression and clinicopathologic characteristics as well as Her-2/neu, ER, and PR status in breast carcinoma was determined. Materials and Methods Patient Material A total of 73 invasive ductal breast cancer tissue samples with (i.e., 31) or without (i.e., 42) LN metastasis were obtained from the Department of Oncology, Jilin Medical University, Jilin, China, between December 2011 and March The median age of the patients at surgery was 49 years (ranging from 38 to 65 years), and all patients were female. Both tissue samples used in the experiments were carefully snap-frozen in liquid nitrogen at the time of resection and stored at 280 C until total RNA was extracted. None of the patients of our study received any neoadjuvant chemotherapy or radiotherapy. We obtained written informed consent from each patient, and the Institute Research Medical Ethics Committee of Jilin Medical University granted approval for this study. Of all cases, fresh frozen tissue from 12 patients with breast cancer were used to undertake mirna array (LN metastasis 5 6 cases; LN negative group 5 6 cases), which further validated the mirnas between primary breast cancer with LN metastasis (n 5 31) and nonlymph node (NLN) metastases (n 5 42) group using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and analyzed the relationship between the expression and the clinicopathological characteristics of the specimens. Furthermore, mirna expression was correlated with Her-2/neu, ER, and PR expression, and their expression in breast carcinoma was determined. RNA Isolation Total RNA was extracted and purified using mirvana TM mirna Isolation Kit (Cat. No. AM1560; Ambion, Austin, TX, USA) following the manufacturer s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). MiRNA Expression Array MiRNA microarray was executed by Agilent human mirna array (8*60K, version 16.0; Agilent Technologies, Santa Clara, CA, USA), which contains capture probes targeting 1,205 human mirna sequences registered in the mirbase database (available from: MiRNA molecule in total RNA was labeled by mirna Complete Labeling and Hyb Kit (Cat. No ; Agilent Technologies) following the manufacturer s instructions (labeling section). Each slide was hybridized with 100 ng of Cy3-labeled RNA using mirna Complete Labeling and Hyb Kit (Cat. No ; Agilent Technologies) in hybridization oven (Cat. No. G2545A; Agilent Technologies) at 55 C, 20 rpm, and for 20 h according to the manufacturer s instructions (hybridization section). After hybridization, the slides were washed in staining dishes (Cat. No. 121; Thermo Shandon, Waltham, MA, USA) with Gene Expression Wash Buffer Kit (Cat. No ; Agilent Technologies). The slides were scanned by Agilent Microarray Scanner (Cat. No. G2565BA; Agilent Technologies) and Feature Extraction software 10.7 (Agilent Technologies) with default settings. Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent Technologies). MiRNA Quantitative RT-PCR MiRNAs expression in cancer and noncancer tissues of each patient was detected using SYBR-green real-time RT-PCR on a Light Cycler 480 system according to the manufacturer s instructions. Briefly, 50 ng of total RNA was reverse transcribed using mirna cdna Synthesis Kit (Takara Life Technologies, Japan). The measurement of the expression levels of individual mirnas was performed using mirna sequencespecific primers (Takara Life Technologies). The RT-PCR conditions used were as follows: 95 C for 30 sec, followed by 35 cycles of 95 C for 10 sec and 60 C for 25 sec. MiRNA expression levels were accessed by relative quantification and the fold expression changes determined by 2 2 CT method (16), and U6 RNA was used as endogenous control. 372 Mirna Related to Lymph Node Metastasis

3 for 48 h. Invading cells on the lower surface that passed through the filter were fixed and stained using crystal violet in gluteraldehyde and photographed. Statistical Analysis The delta Ct (DCt) in each sample represents the relative expression amount of mirna: DCt 5 Ct (mirna) 2 Ct (U6). Comparison of mean DCt values between the independent groups was calculated using Mann-Whitney U test. For determining the association between mirnas expression and clinical features, the mirnas expression was represented as 2 2DCT. The Mann-Whitney U test was used to analyze the relationship between the levels of mir-185-5p, mir-339-5p, mir p, and mir-3923 and the clinical features of the patients. A P-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 13.0 software. FIG 1 Hierarchical clustering analysis of micrornas expression data from microarray analysis. Each row indicates a mirna, and each column indicates a sample. The mirna subset tree is on the left, and the sample subset is at the top. Cell Culture Human breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from the American Type Culture Collection. Adherent cells were maintained in Dulbecco s modified Eagle medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 U/mL penicillin, and 100 mg/ml streptomycin in a humidified atmosphere with 5% CO 2 at 37 C. Cell Transfection The following items were purchased from Guangzhou Ribo-Bio (Guangzhou, China): mirna mimics; mirna mimic NC, a negative control for mirna mimic; mirna inhibitor with a sequence complementary to mature mirna; and mirna inhibitor NC, a negative control for mirna inhibitor. Breast cancer cell line MDA-MB-231 ( ) was seeded onto six-well plates and cultured overnight. Cells were then transfected with 20 nm mimics (for mir-339-5p and mir- 3923) or 40 nm inhibitors (for mir-185-5p and mir-542-5p) and the corresponding doses of mir NC using Lipofectamine 2000 according to the manufacturer s instruction. After 48 h, cells were used for further qrt-pcr and transwell analysis. Invasion Assay Invasion of cells was measured using a Matrigel invasion assay (Becton Dickinson, Bedford, MA, USA). Transwell inserts of 8- mm pore size were coated with a final concentration of 1 mg/ml of Matrigel in cold serum-free DMEM. Cells were trypsinized, and cells were added in triplicate wells. The lower chamber of the transwell was filled with 750 ml of culture media containing 0.5% serum as a chemoattractant, along with the treatment of nestin shrna and allowed to incubate at 37 C Results Results of the mirna Array Microarray analysis in breast cancer resulted in the detection of 17 differentially expressed mirnas (LN metastasis vs. NLN metastasis). Figure 1 shows two main gene clusters (clusters I and II) of altogether 17 mirnas. A markedly (more than 1.5-fold) decreased level when compared with NLN group was found in LN group for mir-206, mir-3923, mir-181a, mir-92a, mir-421, mir-339-5p, mir-3196, and mir-29b that belongs to the cluster I. Cluster II grouped upregulated mirnas in LN group; more than 1.5-fold change was detected for mir-542-5p, mir-200a, mir-564, mir-451, mir- 30c, mir-200b, mir-191-3p, mir-142-5p, and mir-185-5p. Real-Time RT-PCR Validation of Selected mirnas Two upregulated (mir-185-5p and mir-542-5p) and two downregulated mirnas (mir-339-5p and mir-3923) with more than 1.5-fold change in their expression over the control were selected for array data validation and for the evaluation of LN metastasis precision by the RT-qPCR. MiR-185-5p and mir-542-5p were significantly expressed higher in LN group (P and P , respectively), while mir-339-5p and mir-3923 were significantly expressed lower in LN group (P and P , respectively; Fig. 1). Correlation Between mirnas Expression and Clinicopathological Parameters To investigate whether the change in the four mirnas expression of breast cancer was associated with any of the available clinical characteristics, we studied the association of mirna expression levels with the clinical and pathological parameters of breast cancer. The correlation analysis of mir-185-5p, mir p, mir-542-5p, and mir-3923 expression with clinicopathologic factors in breast carcinoma is shown in Table 1. The expression of mir-185-5p in patients with stages I and II was , which was higher than, but not significantly different from, that in patients with stages III and IV Wang et al. 373

4 IUBMB LIFE TABLE 1 Association between mirnas and clinicopathological characteristics in patients with breast cancer Clinicopathologic features n mir-185-5p (2 2DCt ) mir-339-5p (2 2DCt ) mir-542-5p (2 2DCt ) mir-3923 (2 2DCt ) Age (years) < P Menopause state Yes No P TNM staging I II III IV P Tumor size (cm) < P ER status Positive Negative P PR status Positive Negative P HER status Positive Negative P All data were calculated using Mann-Whitney U test. Data are presented as mean 6 SD; DCt 5 Ct mirnas 2 Ct U6*; statistically significant (P < 0.05). ( ; Mann-Whitney U test, P ). There was no significant correlation found between mirnas expression and other clinicopathologic characteristics, including tumor size, clinical stage, menopause state, and ER, PR, and HER2 status (Mann-Whitney U test, P > 0.05). MiRNAs Expression in Breast Cancer Cell Lines From the above, we can see that the expression of mir-339-5p, mir-3923, mir-185-5p, and mir-542-5p was correlated with LN metastasis in breast cancers. Thus, we next investigated whether the mirnas can influence the invasion ability of 374 Mirna Related to Lymph Node Metastasis

5 FIG 2 qrt-pcr analyses of mirnas in breast cancer cell lines. MiR-339-5p, mir-3923, mir-542-5p, and mir p expression in breast cancer cell line MDA- MB-231 compared with MCF-7. The expression level of mature mirnas in breast cancer cell lines was normalized to the expression of U6 and presented as the 2 2DDCt value relative to the MCF-7. T-test was used to compare the differential expressions. *P < 0.05 when compared with the control (MCF-7). the breast cancer cells. First, we detected the expression level of the four mirnas in MCF-7 (lower invasion ability) and MDA-MB-231 (higher invasion ability) cells. The results showed that mir-339-5p and mir-3923 were significantly downregulated in MDA-MB-231 cells (P and P , respectively), whereas mir-542-5p was significantly upregulated in MDA-MB-231 cells (P ; Fig. 2). The expression of MiR-185-5p was not significantly different between MCF-7 and MDA-MB-231 cells (Fig. 2). Upregulation of mir-339-5p and mir-3923 Decrease the Invasion of MDA-MB-231 Cells The expression of mir-339-5p, mir-3923, and mir-542-5p was significantly different between MDA-MB-231 and MCF-7 breast cancer cell lines. Next, we explored the effect of mir p, mir-3923, and mir-542-5p on invasion ability of breast cancer cells. As shown in Fig. 4, the invasion ability of cells with overexpression of mir-339-5p and mir-3923 was significantly decreased when compared with control cells (P for mir-339-5p and P for mir-3923, T-test; Fig. 3). However, the invasion ability of cells with inhibited mir-542-5p was not significantly different from control cells (P , T-test; Fig. 3). Discussion Although breast cancer represents a major cause of morbidity and mortality, early detection and the use of aggressive multimodal treatment have successfully resulted in a decrease in the mortality due to this disease (17 19). A common first route of spread for breast carcinoma is through the axillary LNs. The best predictor of survival among patients with breast cancer has long been considered to be axillary LN status. There is evidence that overall survival decreases as the number of positive node increases (3, 20). As breast cancers are being detected and treated at early stages, the incidence of axillary nodal involvement has decreased. Previously, an axillary dissection comprising level I and II nodes was done to provide staging and prognostic information (21). Although axillary dissection reliably assesses the axillary contents and at the same time is associated with less morbidity (22 24), it can be associated with a wide range of complications, including paresthesia due to intercostobrachial nerve injury, wound infection, seroma, drain complications, and lymphedema (25), which develops in 15 20% of patients after breast cancer treatment. Currently, axillary LN biopsy was used to evaluate the presence of node metastases. However, it is associated with complications such as lymphedema, shoulder stiffness, breast edema, seroma formation, upper limb numbness, and brachial plexopathy (26, 27). It is necessary to find new markers to predict LN metastasis so as to guide the optimizing therapy and long-term followup. There has been significant progress in the development of new diagnostic, prognostic, and predictive tools from proteincoding genes in the past decades. However, metastasis of breast cancer has still been a great challenge from biological research to clinical management. Recently, the role of mirnas in mediating cancer metastasis was first demonstrated in 2007 by Zhang et al. (28); however, to the best of our knowledge, few reports had screened the mirnas expression specifically associated with LN metastasis, which was a prognostic factor to breast cancer. With this in mind, we conducted a mirna microarray analysis in a cohort of LN and NLN tumor samples. Following validation of selected mirnas, SYBR-green realtime RT-PCR was undertaken to identify mirnas associated with LN involvement. By microarray analysis, we detected 17 differentially expressed mirnas between LN and NLN groups. Our results showed that mir-206, mir-3923, mir-181a, mir-92a, mir- 421, mir-339-5p, mir-3196, and mir-29b were decreased in LN group, whereas mir-542-5p, mir-200a, mir-564, mir-451, mir-30c, mir-200b, mir-191-3p, mir-142-5p, and mir-185-5p were increased in LN group when compared with NLN group. Then, we chose four of them, mir-185-5p, mir-542-5p, mir p, and mir-3923, for further studying their expression in LN and NLN specimens using real-time RT-PCR, and the results were consistent with the microarray analysis. Some studies demonstrated that lower expression of mir p is highly associated with poor patient survival, Wang et al. 375

6 IUBMB LIFE FIG 3 Downregulation of mir-339-5p and mir-3923 decreased the invasion of MDA-MB-231 cells. Representative images (A) and statistical plots (B) of transwell assays in MDA-MB-231 cells, which were transfected with mirna mimics or inhibitor and negative control (NC; 3200 magnification). The data were described as arithmetic mean 6 SD of three independent experiments. T-test was used to compare the results of transwell between groups (*P < 0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] indicating a potential tumor-suppressive function, and that ectopic overexpression of this mirna decreases the invasive potential of neuroblastoma cell lines in vitro (29, 30). Wu et al. (31) investigated the role of mir-339-5p in the regulation of tumor cell growth, migration, and invasion and target gene expression. Finally, they reported that MiR-339-5p may play an important role in breast cancer progression, suggesting that mir-339-5p should be further evaluated as a biomarker for predicting the survival of patients with breast cancer (31). However, few reports had identified the predictive value of these mirnas for LN metastasis in breast cancer. This study is the first to show the higher expression levels of mir-185-5p and mir-542-5p and the lower expression levels of mir-339-5p and mir-3923 in LN group than NLN group. This observation raises the possibility of using mirna as biomarkers combining with imaging examination to guide surgical decision making. Our finding requires validation in a larger cohort and a long-term follow-up. Furthermore, the mechanisms by which the selected mirnas contribute to the LN metastasis of breast cancer were not investigated. Further work is required to investigate the mechanisms of these mirnas as a diagnostic or therapeutic intervention. Taken together, we reported that a subset of mirnas is differentially expressed in LN group compared with NLN group. The utilization of these mirnas as biomarkers of LN metastases and prognosis in patients with breast cancer has the potential to change current management strategies, where an additional prophylactic lateral neck dissection may be considered appropriate in clinically combining with imaging examination. In this study, the sample number was relatively small, thus we hope that more studies with large samples are needed to investigate this topic. Acknowledgements This study was funded by the China Natural Science Foundation (No ). 376 Mirna Related to Lymph Node Metastasis

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