| Breast cancer is the most common malignant tumor in females, and the incidence of breast cancer is increasing in the developing world. 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. 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. Widespread use of mammography has resulted in marked increase in early detection of breast cancer, improvement in therapy, and declining mortality. 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. Axillary LN dissection is now no longer considered to be the standard treatment in all patients with invasive breast cancer. Therefore, it is essential to find predictive factors for axillary LN involvement in early breast cancer.MiRNAs are endogenous, small noncoding single-stranded RNAs of18-25nucleotides 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 the3’-untranslated regions and other regions of protein-coding mRNA sequences cause either degradation of the mRNA or inhibition of translation. It has been estimated that, up to60%of genes may be regulated by more than1,900human miRNAs thus far identified. The involvement of miRNAs in can-cer 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. There are some reports on the association of the clinicopathogenetic features of breast cancer with miRNA expression. 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 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 in73breast can-cer 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.A total of73invasive 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 December2011and March2012. The median age of the patients at surgery was49years (ranging from38to65years), and all patients were female.Both tissue samples used in the experiments were care-fully snap-frozen in liquid nitrogen at the time of resection and stored at280℃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 from12patients with breast cancer were used to undertake miRNA array (LN meta-stasis56cases; LN negative group56cases), which further validated the miRNAs between primary breast cancer with LN metastasis (n=31) and nonlymph node (NLN) metastases (n542) 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.Total RNA was extracted and purified using mirVanaTM 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 Bioanalyzer2100(Agilent Technologies, Santa Clara, CA, USA). MiRNA microarray was executed by Agilent human miRNA microarray (8*60K, version16.0; Agilent Technologies, Santa Clara, CA, USA), which contains capture probes targeting1,205human miRNA sequences registered in the miRBASE database (available from:http:\\www.mirbase.org). MiRNA molecule in total RNA was labeled by miRNA Complete Labeling and Hybration Kit (Cat. No.5190-0456; Agilent Technologies) following the manufacturer’s instructions (labeling section). Each slide was hybridized with100ng of Cy3-labeled RNA using miRNA Com-plete Labeling and Hyb Kit (Cat. No.5190-0456; Agilent Tech-nologies) in hybridization oven (Cat. No. G2545A; Agilent Tech-nologies) at55℃,20rpm, and for20h 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.5188-5327; Agilent Technologies). The slides were scanned by Agilent Microarray Scanner (Cat. No. G2565BA; Agilent Technologies) and Feature Extraction software10.7(Agilent Technologies) with default settings. Raw data were normalized by Quantile algorithm, Gene Spring Software11.0(Agilent Technologies).MiRNAs expression in cancer and non-cancer tissues of each patient was detected using SYBR-green real-time RT-PCR on a Light Cycler480system according to the manufacturer’s instructions. Briefly,50ng 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 sequence-specific primers (Takara Life Technologies). The RT-PCR conditions used were as follows:95℃for30sec, followed by35cycles of95℃for10sec and60℃for25sec. MiRNA expression levels were accessed by relative quantification and the fold expression changes determined by2-△△CT method, and U6RNA was used as endogenous control.Human breast cancer cell lines MCF-7and MDA-MB-231were obtained from the American Type Culture Collection. Adherent cells were maintained in Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA, USA) containing10%fetal bovine serum (HyClone, Logan, UT, USA),100U/mL penicillin, and100mg/mL streptomycin in a humidified atmosphere with5%CO2at37℃.The following items were purchased from Guangzhou RiboBio (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(3×105) was seeded onto six-well plates and cultured overnight. Cells were then transfected with20nM mimics (for miR-339-5p and miR-3923) or40nM inhibitors (for miR-185-5p and miR-542-5p) and the corresponding doses of miR NC using Lipofectamine2000according to the manufacturer’s instruction. After48h, cells were used for further qRT-PCR and transwell analysis.Invasion of cells was measured using a Matrigel invasion assay (Becton Dickinson, Bedford, MA, USA). Transwell inserts of8-mm pore size were coated with a final concentration of1mg/mL of Matrigel in cold serum-free DMEM. Cells were trypsinized, and1×105cells were added in triplicate wells. The lower chamber of the transwell was filled with750mL of culture media containing0.5%serum as a chemoattractant, along with the treatment of nestin shRNA and allowed to incubate at37℃.The delta Ct (DCt) in each sample represents the relative expression amount of miRNA:DCt5Ct (miRNA)2Ct (U6). Comparison of mean DCt values between the independent groups was calculated using Mann-Whitney U test. For deter-mining the association between miRNAs expression and clinical features, the miRNAs expression was represented as2-△△Ct. The Mann-Whitney U test was used to analyze the relationship between the levels of miR-185-5p, miR-339-5p, miR-542-5p, and miR-3923and the clinical features of the patients. A P-value of less than0.05was considered statistically significant. All statistical analyses were performed using SPSS13.0software.Microarray analysis in breast cancer resulted in the detection of17differentially expressed miRNAs (LN metastasis vs. NLN metastasis). Figure1shows two main gene clusters (clusters I and II) of altogether17miRNAs. A markedly (more than1.5-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 than1.5-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.Two upregulated (miR-185-5p and miR-542-5p) and two downregulated miRNAs (miR-339-5p and miR-3923) with more than1.5-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 (P50.002and P50.001, respectively), while miR-339-5p and miR-3923were significantly expressed lower in LN group (P=0.001and P=0.001, respectively).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-339-5p, miR-542-5p, and miR-3923expression with clinicopathologic factors in breast carcinoma is shown in Table1. The expression of miR-185-5p in patients with stages Ⅰ and Ⅱ was0.1360.14, which was higher than, but not significantly different from, that in patients with stages III and IV (0.0860.09; Mann-Whitney U test, P50.051). There was no significant correlation found between miRNAs expression and other clinicopathologic characteristics, including tumor size, clinical stage, menopause state, and ER, PR, and HER2status (Mann-Whitney U test, P>0.05).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 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-3923were significantly downregulated in MDA-MB-231cells (P50.002and P50.001, respectively), whereas miR-542-5p was significantly upregulated in MDA-MB-231cells. The expression of MiR-185-5p was not significantly different between MCF-7and MDA-MB-231cells.The expression of miR-339-5p, miR-3923, and miR-542-5p was significantly different between MDA-MB-231and MCF-7breast cancer cell lines. Next, we explored the effect of miR-339-5p, 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-3923was significantly decreased when compared with control cells (P=0.002for miR-339-5P and P=0.001for miR-3923, T-test). However, the invasion ability of cells with inhibited miR-542-5p was not significantly different from control cells (P=0.742, T-test).Although breast cancer represents a major cause of morbidity and mortality, early detection and the use of aggressive multi-modal treatment have successfully resulted in a decrease in the mortality due to this disease. 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.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. Although axillary dissection reliably assesses the axillary contents and at the same time is associated with less morbidity, it can be associated with a wide range of complications, including paresthesia due to intercostobrachial nerve injury, wound infection, seroma, drain complications, and lymphedema, which develops in15-20%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.It is necessary to find new markers to predict LN metastasis so as to guide the optimizing therapy and long-term follow-up. There has been significant progress in the development of new diagnostic, prognostic, and predictive tools from protein-coding 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 in2007by Zhang et al.; 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 real-time RT-PCR was undertaken to identify miRNAs associated with LN involvement.By microarray analysis, we detected17differentially 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-339-5p, 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-542-5p is highly associated with poor patient survival, indicating a potential tumor-suppressive function, and that ectopic overexpression of this miRNA decreases the invasive potential of neuroblastoma cell lines in vitro. Wu et al. 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 bio-marker for predicting the survival of patients with breast cancer.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-3923in 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 con-sidered 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. |
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