www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 30), pp: 49110-49122 Research Paper Transforming growth factor-β1 promotes breast cancer metastasis by downregulating miR-196a-3p expression Yan Chen1,2,*, Shai Huang2,*, Bo Wu3,*, Jiankai Fang2, Minsheng Zhu2, Li Sun4, Lifeng Zhang1, Yongsheng Zhang5, Maomin Sun4, Lingling Guo2 and Shouli Wang2,6,7 1Department of Surgery, The First Affiliated Hospital of Soochow University, Suzhou 215006, China 2Department of Pathology, School of Biology ﹠ Basic Medical Sciences, Soochow University, Suzhou 215123, China 3Department of Surgery, The People’s Hospital of Sihong County, Sihong 223900, Jiangsu Province, China 4Laboratory Animal Research Center, Soochow University School of Medicine, Suzhou 215123, China 5Department of Pathology, The Second Affiliated Hospital of Soochow University, Suzhou 215004, China 6Laboratory of Molecular Pathology, Soochow University ﹠ Sihong County People’s Hospital, Suzhou 215123, China 7Suzhou Key Laboratory of Tumor Microenvironment and Pathology, Soochow University, Suzhou 215006, China *These authors have contributed equally to this work Correspondence to: Shouli Wang, email: [email protected] Lingling Guo, email: [email protected] Keywords: microRNA, miR-196a-3p, transforming growth factor-ß1, breast cancer Received: December 12, 2016 Accepted: February 23, 2017 Published: March 17, 2017 Copyright: Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Transforming growth factor-β1 is considered a key contributor to the progression of breast cancer. MicroRNAs are important factors in the development and progression of many malignancies. In the present study, upon studies of breast cancer cell lines and tissues, we showed that microRNA -196a-3p is decreased by transforming growth factor-β1 in breast cancer cells and associated with breast cancer progression. We identified neuropilin-2 as a target gene of microRNA -196a-3p and showed that it is regulated by transforming growth factor-β1. Moreover, transforming growth factor- β1-mediated inhibition of microRNA -196a-3p and activation of neuropilin-2were required for transforming growth factor-β1-induced migration and invasion of breast cancer cells. In addition, neuropilin-2 expression was suppressed in breast tumors, particularly in triple-negative breast cancers. Collectively, our findings strongly indicate that microRNA -196a-3p is a predictive biomarker of breast cancer metastasis and patient survival and a potential therapeutic target in metastatic breast cancer. INTRODUCTION Recent results indicate an association between TGF-β1 signaling and microRNAs (miRNAs, miR), as revealed Despite considerable progress in the early diagnosis by our previous studies in rhabdomyosarcoma [8, 9] and and surgical therapy of breast cancers, breast cancer is the those of others about breast cancers [4, 10-13], providing second leading cause of death in the United States [1], and new insight into the nature of cancer. the first cause of cancer-related death in women younger MiRNAs, which are noncoding RNAs that than 45 years followed by lung cancer in China [2]. regulate target gene expression post-transcriptionally, Transforming growth factor-β1 (TGF-β1) is considered show unbalanced expression in cancers [14], and there key contributor to the progression of breast cancer [3, is increasing evidence of the efficacy of miRNA-based 4]; TGF-β1 signaling features a growth inhibitory effect therapies [15]. Considering that TGF-β1 promotes the at an early stage of breast cancer cells, but aggressive progression of breast cancer at advanced stages [3, oncogenic activity at the advanced malignant state [5-7]. 4], we assumed that miRNA expression inhibited by www.impactjournals.com/oncotarget 49110 Oncotarget TGF-β1 must have antitumor potential, as previously miR-196a-3p is a potential therapeutic approach for the demonstrated for miR-450b-5p [8] and miR-411-5p [9]. treatment of breast cancer. Using TGF-β1 knockdown colorectal cancer cells (HT- 29, SW620 and LoVo) and comprehensive locked RESULTS nucleic acid microarray analyses, five significantly raised miRNAs were selected (Tracking Number: miR-196a-3p expression is reduced in breast GSE53338, unpublished data), and their expression cancer in correlation with metastatic potential was examined in breast cancer. We found that TGF-β1 suppressed miR-196a-3p in colorectal cancer and in In a previous study, we identified five TGF-β1 breast cancer and exerted an antitumor effect on breast inhibited miRNAs in colorectal cancer by microarray cancer. analysis (GSE53338), including miR-4313, miR-3691- In the present study, we showed that ectopic 3p, miR-196a-3p, miR-324-3p, and miR-4723-3p. To overexpression of miR-196a-3p reversed TGF-β- investigate whether these miRNAs are correlated with induced breast cancer cell migration and invasion. We the malignant behavior of breast cancer, we examined also identified the protein neuropilin 2 (NRP2) as a the expression of these miRNAs in two different breast downstream target of miR-196a-3p, and showed that cancer cell lines (the noninvasive luminal cell line MCF- blocking NRP2 expression potently antagonized TGF-β 7 and the highly invasive cell line MDA-MB-231) by promigratory effects in invasive breast cancer cells. reverse transcription-polymerase chain reaction(RT- Collectively, our results demonstrate that miR-196a-3p PCR). We found that only miR-196a-3p was markedly and its downstream target NRP2 are involved in TGF- decreased in MDA-MB-231 cells (Figure 1A). To further β-induced cell migration and invasion in invasive breast verify the relationship between miR-196a-3p expression cancer cells, suggesting that restoring the expression of and the malignant behavior of breast cancer, three Figure 1: miR-196a-3p is reduced in breast cancer cell lines and clinical specimens. (A) The expression of TGF-β1-inhibited miRNAs was evaluated by qRT-PCR in breast cancer cell lines with different metastatic potentials. The results are means of three independent experiments ± S.D. (**p< 0.01). (B) qRT-PCR analysis of the effect of TGF-β1 on miR-196a-3p expression in breast cancer cell lines with different metastatic potentials stimulated with or without 5 ng/ml TGF-β 1for 24 h. The results are means of three independent experiments ± S.D. (**p< 0.01,***p< 0.001). (C) qRT-PCR analysis of the mRNA expression of miR-196a-3p in 13 human breast cancer tissues and 11 normal breast samples. (D) miR-196a-3p expression in 12 different kinds of breast cancers (3 luminal A, 3 luminal B, 3 ERBB2 overexpressing, and 3 basal-like cases). The results are means of three independent experiments ± S.D. (*p<0.05,**p< 0.01). www.impactjournals.com/oncotarget 49111 Oncotarget Table 1: Relationship between miR-196a-3p expression and clinicalpathologic variables of breast cancer patients miR-196a-3p expression Parameters Number of cases P value Low (52) High (60) Age(years) <50 49 18 31 0.07 ≥50 63 34 29 Tumor size(cm) <2 63 25 38 0.105 ≥2 49 27 22 TNM stage I+II 70 24 46 0.001* III 42 28 14 Differentiation grade G1+G2 84 34 50 0.029* G3 28 18 10 Lymph node metastasis Yes 56 33 23 0.008* No 56 19 37 ER status Negative 58 32 26 0.054 Positive 54 20 34 PR status Negative 57 30 27 0.18 Positive 55 22 33 HER2 status Negative 57 31 26 0.086 Positive 55 21 34 *P < 0.05 was considered significant. noninvasive breast cancer cell lines (MCF-7, T-47D, these results suggest that miR-196a-3p plays an important and SK-BR-3) and two highly invasive breast cancer role downstream of TGF-β-signal transduction in breast cell lines (BT-549 and MDA-MB-231) were analyzed. cancer. We found that miR-196a-3p was inhibited by TGF-β1 in all tested breast cancer cell lines, and the expression Decreased expression of miR-196a-3p correlates of miR-196a-3p was higher in noninvasive breast cancer with poor clinical outcomes in breast cancer cell lines than in the two highly invasive breast cancer cell lines (Figure 1B). The expression of miR-196a-3p was examined using We then detected miR-196a-3p expression levels real-time qRT-PCR in 112 breast cancer tissue samples in 13 primary tumors and 11 non-cancerous specimens. and the association of miR-196a-3p expression levels As shown in Figure 1C, miR-196a-3p was declined by with clinicopathological features was analyzed. As shown approximately two-fold in tumors compared with non- in Table 1, low miR-196a-3p expression levels were cancerous specimens. Moreover, miR-196a-3p expression closely correlated with lymph node metastasis (p=0.008), was lower in basal like breast cancer than in the other TNM stage (p=0.001), and pathological differentiation subtypes of breast cancer (Figure 1D). Taken together, (p=0.029). However, no significant correlations between www.impactjournals.com/oncotarget 49112 Oncotarget miR-196a-3p expression and age, tumor size, estrogen in Figure 3B, TGF-β induced MDA-MB-231 cell migration receptor (ER) status, progesterone receptor (PR) status and and invasion in the mock and control groups, whereas the human epidermal growth factor receptor 2 (HER2) status miR-196a-3p mimic-treated groups showed no significant were found. Our results indicate that miR-196a-3p may difference in the metastatic ability of MDA-MB-231 play a critical part in the carcinogenesis and progression cells with or without TGF-β1 stimulation. In addition,the of breast cancer. overexpression miR-196a-3p did not affect the proliferative To further investigate the significance of miR- properties of breast cancer cells as determined by the MTT 196a-3p in terms of clinical prognosis, the Kaplan–Meier assay (Supplementary Figure 1).