Liu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:64 https://doi.org/10.1186/s13046-018-0734-2 RESEARCH Open Access BRD7 expression and c-Myc activation forms a double-negative feedback loop that controls the cell proliferation and tumor growth of nasopharyngeal carcinoma by targeting oncogenic miR-141 Yukun Liu1,2,3, Ran Zhao2,3, Yanmei Wei2,3, Mengna Li2,3, Heran Wang1,3, Weihong Niu2,3, Yao Zhou2,3, Yuanzheng Qiu4, Songqing Fan5, Yihao Zhan6, Wei Xiong1,2,3, Yanhong Zhou1,2,3, Xiaoling Li1,2,3, Zheng Li1,2,3, Guiyuan Li1,2,3 and Ming Zhou1,2,3* Abstract Background: miR-141 is up-regulated and plays crucial roles in nasopharyngeal carcinoma (NPC). However, the molecular mechanism underlying the dysregulation of miR-141 is still obscure. Methods: Thus, the ChIP-PCR was performed to identify the c-Myc-binding sites in miR-141 and BRD7. qRT-PCR, western blot and immunohistochemistry assays were used to detect the expression of miR-141 and its up/down stream molecules. The rescue experiments on the c-Myc/miR-141 axis were performed in vitro and in vivo. Results: Our results showed that the levels of mature miR-141, pre-miR-141 and pri-miR-141 were downregulated in c-Myc knockdown NPC cells. Meanwhile, c-Myc transactivates the expression of miR-141 by binding its promoter region. Moreover, BRD7 was identified as a co-factor of c-Myc to negatively regulate the activation of c-Myc/miR-141 axis, as well as a direct target of c-Myc. Moreover, restoration of miR-141 in c-Myc knockdown NPC cells notably rescued the effect of c-Myc on cell proliferation and tumor growth, as well as the blocking of PTEN/AKT pathway. Additionally, the expression of c-Myc was positively correlated with that of miR-141andtheclinicalstagesof NPC patients and negatively associated with the expression of BRD7. Our findings demonstrated that BRD7 expression and c-Myc activation forms a negative feedback loop to control the cell proliferation and tumor growth by targeting miR-141. Conclusions: These observations provide new mechanistic insights into the dysregulation of miR-141 expression and a promising therapeutic option for NPC. Keywords: Nasopharyngeal carcinoma, BRD7, C-Myc, Feedback loop, miR-141, AKT pathway * Correspondence: [email protected] 1Hunan Cancer Hospital and The Affiliated Tumor Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan 410013, People’s Republic of China 2Key Laboratory of Carcinogenesis of Ministry of Health and Key Laboratory of Carcinogenesis and Cancer Invasion of Ministry of Education, The Xiangya Hospital, Central South University, Changsha, Hunan 410008, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Liu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:64 Page 2 of 14 Background of the c-Myc/miR-141 axis in NPC in vitro and in vivo. Nasopharyngeal carcinoma (NPC) is a leading form of Additionally, we investigated the clinical significance of head and neck cancer, especially in South China. It has c-Myc and the association of c-Myc expression with high metastatic potential that contributes to a high rate BRD7 and miR-141 in NPC patients. In summary, our of local invasion and early metastasis [1]. Thus, there is findings demonstrated that BRD7 expression and c- a clear need to identify some sensitive biomarkers and Myc activation forms a negative feedback loop that reg- novel therapeutic targets for diagnosis and treatment op- ulates miR-141 transcription and controls cell growth tions of NPC in a premalignant stage. and proliferation in NPC. These observations provide Recently, non-coding RNAs have been demonstrated new mechanistic insights into the dysregulation of miR- to represent most of the human transcriptome and are 141 expression and a promising therapeutic option for frequently dysregulated in disease pathogenesis, includ- NPC in early stages. ing cancer [2]. MicroRNAs (miRNAs) are a class of functional, short non-coding RNAs that are associated Methods with all hallmarks in cancer initiation, progression and Cell lines and tissue samples metastasis [3, 4]. Therefore, considering their tissue- The human NPC cell line 5-8F was kindly provided by the specific profiles and stability, miRNA-based strategies Cancer Center of Sun Yet-Sen University (Guangzhou, are considered as promising options for early detection, China). The HNE1 cell line was purchased from the cell accurate diagnosis and the prediction of responses to center of the Xiangya School of Medicine (Central South treatment in malignancies. miR-141 is a member of the University, Changsha, China). Both cell lines were preserved miR-200 family, and it has been shown to be dysregu- in our laboratory and cultured in RPMI-1640 (Hyclone, Lo- lated in a wide variety of cancers [5, 6]. However, the gan, UT, USA) supplemented with 10% fetal bovine serum mechanisms underlying the dysregulation of miR-141 (FBS) (Gibco-BRL, Invitrogen, Paisley, UK) in a humidified are only beginning to be understood in cancer. incubator with 5% CO at 37 °C. The embryonic kidney cell c-Myc is a transcription factor that generally heterodi- 2 line HEK293 were purchased from the American Type Cul- merizes with other basic-helix–loop–helix (bHLH) pro- ture Collection (ATCC, Manassas, VA, USA) and main- teins at thousands of genomic loci [7], which affects tained in Dulbecco’s modified Eagle’s medium (DMEM) more than 15% of the human transcriptome [8]. Hence, (Hyclone) with 10% FBS (Gibco) at 37 °C in a humidified at- c-Myc plays vital roles in almost every aspect of basic mosphere with 5% CO . cellular processes. Aberrant activation of c-Myc is 2 NPC samples (n = 41) and non-cancerous nasopharyn- known to be a crucial hallmark of numerous cancers [9] geal tissues (n = 27) from healthy donors were collected and often leads to widespread dysregulation of miRNAs at the Second Xiangya Hospital of Central South Univer- [10]. In our previous study, we found that knockdown of sity (Changsha, China). The non-cancerous nasopharyn- c-Myc significantly inhibited cell proliferation and tumor geal tissues were collected from independent patients growth of NPC [11] and downregulated the level of with chronic inflammation of nasopharyngeal mucosa or miR-141 in NPC cells [12]. BRD7, a member of the polyps. The profile of clinicopathological characteristics bromodomain-containing protein family, was identified of the NPC patients is shown in Table 1. All tissue sam- as a critical tumor suppressor in multiple types of can- ples were immediately snap-frozen in liquid nitrogen cers, including NPC and breast cancer [13–15] and also and stored in a freezer at − 80 °C. Tissues for immuno- involved in many physiological processes [16–18]. Grow- histochemistry were fixed in 4% paraformaldehyde and ing evidence has revealed that BRD7, as a ubiquitously then paraffin embedded. Clinicopathological data were expressed nucleic protein [17, 19], is generally involved reviewed, and TNM staging classification was based on in the transcription of specific target genes through a the criteria of the American Joint Committee on Cancer protein-protein interaction (PPI) with other transcrip- (AJCC, 6th edition). tion factors [20–22]. Moreover, BRD7 could negatively regulate the transcription of miR-141 in an indirect manner in NPC [23]. However, the detail mechanism Plasmids, oligonucleotides and transfections underlying BRD7 and c-Myc involvement in the regula- The interfering plasmid (pRNAT-U6.1/shc-Myc) express- tion of miR-141 is still obscure and remains to be fur- ing short hairpin RNA (shRNA) for c-Myc knockdown, ther investigated. the expression plasmid encoding the full-length open In this study, we estimated the expression of BRD7 and reading frame of human c-Myc with 2Flag tags (pIRES2- miR-141 in c-Myc knockdown NPC cells and explored EGFP/2Flag-c-Myc), and the plasmid encoding the full- the mechanism of BRD7 and c-Myc in the transcription of length open reading frame of human BRD7 with 3Flag miR-141. Furthermore, we performed rescue experiments tags (pIRESneo3/3Flag-BRD7) or GFP tag (pEGFP-C2/ to identify the functional role and molecular mechanism BRD7) were constructed by our laboratory. The hsa-miR- Liu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:64 Page 3 of 14 Table 1 Association between the expression of c-Myc, BRD7, miR-141 and NPC clinical pathological features (N = 41) c-Myc BRD7 miR-141 Characteristics (N) H (%) L (%) P H (%) L (%) P H (%) L (%) P Age (year) ≤ 40 (n = 7) 3(42.8) 4(57.2) 0.6786 1(14.2) 6(85.8) 0.4942 4(57.1) 3(42.9) 0.7052 >40(n = 34) 20(58.8) 14(41.2) 9(26.4) 25(73.6) 22(64.7) 12(35.3) Gender Female (n = 9) 6(66.6) 3(33.4) 0.7061 2(22.2) 7(77.8) 0.8639 8(88.8) 1(11.2) 0.0725 Male (n = 32) 17(53.1) 15(46.9) 8(47.1) 24(52.9) 18(56.2) 14(43.8) Histological type DNC (n = 20) 12(60.0) 8(40.0) 0.7557 5(25.0) 15(75.0) 0.9293 14(70.0) 6(30.0) 0.3929 UDNC (n = 21) 11(52.3) 10(47.7) 5(23.8) 16(76.2) 12(57.1) 9(42.9) Clinical stage Stage I (n = 7) 1(14.2) 6(85.8) 0.0140 4(57.1) 3(42.9) 0.0475 2(28.6) 5(71.4) 0.0246 Stage II (n = 19) 10(52.6) 9(47.4) 2(10.5) 17(89.5) 11(57.8) 8(42.2) Stage III-IV (n = 15) 12(80.0) 3(20.0) 4(26.6) 11(73.4) 13(86.6) 2(13.4) Abbreviations: DNC, differentiated non-keratinized nasopharyngeal carcinoma; UDNC, undifferentiated non-keratinized nasopharyngeal carcinoma; H, high expression; L, low expression 141 mimic (miR-141) and negative control (miR-NC) were 141, pre-miR-141, U6, pri-miR-141, c-Myc, BRD7, and purchased from RiBoBio (Guangdong, China).