Zhang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:14 DOI 10.1186/s13046-018-0681-y RESEARCH Open Access miR-3928v is induced by HBx via NF-κB/ EGR1 and contributes to hepatocellular carcinoma malignancy by down-regulating VDAC3 Qiaoge Zhang1†, Ge Song1†, Lili Yao1, Yankun Liu1,2, Min Liu1, Shengping Li3 and Hua Tang1*† Abstract Background: Hepatitis B virus (HBV) plays a critical role in the tumorigenic behavior of human hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) have been reported to participate in HCC development via the regulation of their target genes. However, HBV-modulated miRNAs involved in tumorigenesis remain to be identified. Here, we found that a novel highly expressed miRNA, TLRC-m0008_3p (miR-3928v), may be an important factor that promotes the malignancy of HBV-related HCC. Methods: Solexa sequencing was applied to profile miRNAs, and RT-qPCR was used to identify and quantitate miRNAs. We studied miR-3928v function in HCC cell lines by MTT, colony formation, migration/invasion, and vascular mimicry (VM) assays in vitro and by a xenograft tumor model in vivo. Finally, we predicted and verified the target gene of miR-3928v by a reporter assay, studied the function of this target gene, and cloned the promoter of miR-3928v and the transcription factor for use in dual-luciferase reporter assays and EMSAs. Results: A variant of miR-3928 (miR-3928v) was identified and found to be highly expressed in HBV (+) HCC tissues. Voltage-dependent anion channel 3 (VDAC3) was validated as a target of miR-3928v and found to mediate the effects of miR-3928v in promoting HCC growth and migration/invasion. Furthermore, HBx protein increased early growth response 1 (EGR1) expression and facilitated its translocation into the nucleus to enhance miR-3928v promoter activity in an NF-κB signaling-dependent manner. Conclusions: miR-3928v is induced by HBx through the NF-κB/EGR1 signaling pathway and down-regulates the tumor suppressor gene VDAC3 to accelerate the progression of HCC. Keywords: miRNA, miR-3928v, HBx, EGR1, VDAC3, Hepatocellular carcinoma Background for the high rate of HCC-related mortality. Therefore, it is HCC is the fifth most common malignant tumor and the important to understand how HBV-related proteins are second leading cause of cancer-related death worldwide involved in the development of HCC. Recent research has [1]. HBV infection is the greatest risk factor for HCC [2]. demonstrated that the hepatitis B virus X (HBx) protein, a Although enormous progress has been made in preventing key HBV regulatory molecule, plays pivotal roles in the ini- HBV infection, diagnosis in the late stages of liver cancer tiation and development of HCC [3, 4]. HBx has been and the highly metastatic nature of HCC are responsible reported to regulate signaling pathways and affect cell cycle progression, apoptosis, DNA repair, and protein degrad- * Correspondence: [email protected]; [email protected] ation [5–7]. Although evidence has indicated that HBx can †Equal contributors contribute to HCC by regulating the expression of several 1 Tianjin Life Science Research Center and Department of Pathogen Biology, miRNAs, the mechanism by which HBx induces HCC Collaborative Innovation Center of Tianjin for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, No. 22 Qi-Xiang-Tai Road, remains unclear. Tianjin 300070, 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. Zhang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:14 Page 2 of 12 miRNAs are a class of conserved small RNAs that can registered by another laboratory while we investigated bind to the 3′UTR of mRNA to modulate the expression the function of miR-3928v in HCC. miR-3928v is in- of target genes [8], which are involved in physiological duced by the HBV-related protein HBx and functions as and pathological processes such as cell proliferation, dif- an oncogene by promoting cell proliferation, migration/ ferentiation, apoptosis, angiogenesis and metastasis [9]. invasion and vascular mimicry (VM). Furthermore, The dysregulation of miRNAs plays an important role in VDAC3 was confirmed to be a direct target of miR- tumorigenesis in humans, as miRNAs function as tumor 3928v and to mediate the role of miR-3928v in HCC suppressors and oncogenes [8, 10]. With regard to HBV- cells. Importantly, we revealed that HBx induces miR- related HCC, previous reports have demonstrated that 3928v expression in an NF-κB/EGR1-dependent manner. HBx is involved in HCC progression via miRNA regula- Collectively, these data indicate that HBx induces miR- tion. For example, HBx induces miR-21 expression, sig- 3928v expression through the NF-κB/EGR1 signaling nificantly promoting cell proliferation in HCC [11]. In pathway, resulting in the down-regulation of the tumor addition, a previous study from our laboratory showed suppressor gene VDAC3 and the progression of HCC. that miR-1269b is induced by HBx in an NF-κB- dependent manner, thereby up-regulating CDC40 to Methods promote HCC cell proliferation and migration [12]. We Tissue and serum specimens and cell lines therefore wondered whether HBx regulates additional Twenty pairs of human hepatic carcinoma and normal miRNAs involved in the pathogenesis of HCC. adjacent tissues were obtained from the Cancer Center EGR1 belongs to the early growth response (EGR) of Sun Yat-sen University. Sixty serum samples from pa- family of transcription factors that includes four mem- tients with HCC, 30 serum samples from healthy people bers: EGR1, EGR2, EGR3, and EGR4. All four proteins and 10 pairs of slides of human hepatoma tissues and contain DNA-binding zinc finger domains and tend to adjacent non-tumor tissues for immunohistochemistry bind GC-rich elements in the promoter regions of target analysis were obtained from TangShan People’s Hospital. genes [13]. EGR1 reportedly has a paradoxical function Written informed consent was obtained from each pa- in HCC. For instance, EGR1 acts as a tumor suppressor tient included in the study, and the samples were used by promoting the transcription of TGF-β, p53 and PTEN in accordance with the standards of the center’s ethics [14]. Conversely, EGR1 acts as an oncogene, promoting committee. The HCC cell lines are described in HCC cell migration [15, 16] and enhancing the drug re- Additional file 1. sistance of HCC cells, likely through autophagy [17]. However, the interaction between HBx and EGR1 in Plasmid construction HCC cells remains to be identified. The miR-3928v expression vector (pri-miR-3928v), Voltage-dependent anion channels (VDACs) belong to VDAC3 expression vector (pcDNA3-Flag-VDAC3), shR- the mitochondrial porin family, are located in the mito- VDAC3 vector, dual-luciferase reporter plasmids and chondrial outer membrane and serve as gatekeepers pGL3-luciferase-miR-3928v promoter were constructed. controlling the entry and exit of mitochondrial metabo- The details of these constructs are provided in Additional lites [18]. In mammals, the VDAC family has three file 1. The sequences of the primers used to generate the members: VDAC1, VDAC2 and VDAC3 [19]. Recent re- above plasmids are listed in Additional file 2.A2′-O- ports have demonstrated that VDACs can recruit the methyl-modified antisense oligonucleotide of miR- ubiquitin ligase Parkin to defective mitochondria to pro- 3928v (ASO-miR-3928v) was commercially synthesized mote mitochondrial autophagy [20]. VDACs also partici- by Genpharm (Shanghai, China). pate in various cellular processes, including reactive oxygen species (ROS) signaling and apoptosis mediated RNA extraction and real-time PCR by the release of cytochrome c or by interactions with RNA was extracted from serum and tissues using a miR- Bcl-2 family members [21, 22]. Thus, VDACs play a key cute miRNA Isolation Kit (Tiangen Biotech, China) role in mitochondrial dysfunction and pathophysio- according to the manufacturer’s instructions. The rela- logical processes. Although VDAC3 was found to inter- tive mRNA level was examined by RT-qPCR as detailed act with HBx [23, 24], miRNAs that regulate VDAC3 in in Additional file 1. The primer sequences are provided HCC cells have not been identified. in Additional file 2. In this study, we aimed to identify new miRNAs that may be involved in HCC oncogenic activity by deep se- Dual-luciferase reporter assays quencing. We identified a novel miRNA that is signifi- Huh7, HepG2 and HepG2.2.15 cells were seeded in 48- cantly highly expressed in HCC tissues. This miRNA, well plates and cultured for approximately 20 h after co- miR-3928v, was identified using Solexa sequencing, and transfection with different plasmids. The procedures are it differs by one base from miR-3928, which was described in detail in Additional file 1. Zhang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:14 Page 3 of 12 MTT, colony formation, migration and invasion assays