Zhang et al. Journal of Experimental & Clinical 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 . 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 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 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 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 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 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 (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 The tumors were isolated, and the weights were recorded. Following transfection, cells were counted and seeded in All studies were performed according to the American the appropriate wells, and the density, cell viability, col- Association for the Accreditation of Laboratory Animal ony formation rate, migration and invasion were deter- Care Guidelines for the Humane Treatment of Animals mined as described previously [25]. and adhered to national and international standards.

Tube formation assay Statistical analyses To examine the effect of miR-3928v and VDAC3 on VM Two tailed Student’s t-tests and analysis of variance in HCC cells, the cells were transfected with pri-miR- (ANOVA) were used to analyze the significance of differ- 3928v, ASO-miR-3928v, pVDAC3 or shR-VDAC3 and ences between sample means obtained from three inde- the corresponding controls. A 24-well cell culture plate pendent experiments. P ≤ 0.05 indicated statistical was coated with 50 μl of Matrigel (DB Biosciences), significance (*: P < 0.05, **: P < 0.01, ***: P < 0.001). which was allowed to solidify at 37 °C for 1 h. After the Matrigel solidified, 4 × 105 or 5 × 105 cells were seeded on it. After 24 h of culture at 37 °C, tube-like structures Results were observed and photographed using a microscope. HBV induces the expression of the novel miRNA miR- 3928v in HCC tissues and cells Cell cycle and apoptosis analyses via flow cytometry To identify novel miRNAs that may play key roles in Transfected cancer cells were seeded into 6-well plates hepatocarcinogenesis, the miRNA expression profile in and incubated for 24 h in complete medium until the HBV (+) HCC tissues was analyzed by deep sequencing. cells became 60% confluent. The culture medium was We selected TLRC-m0008_3p for further analysis be- then replaced with serum-free culture medium for star- cause of the higher read count and the lowest minimum vation. After 24 h of starvation, the cells were cultured free energy (Additional file 3). Using the PubMed search in complete medium for an additional 24 h. The cells tool blastn, we found that TLRC-m0008_3p may be a were then collected for cell cycle and apoptosis analyses. variant of miR-3928. We therefore named this miRNA Details can be found in Ref. [26]. miR-3928v and further investigated its role in HCC. The predicted secondary structure of the miR-3928v precur- ChIP assays sor and its mature miRNA sequence are shown in Fig. 1a. Chromatin immunoprecipitation (ChIP) assays were per- RT-qPCR was used to examine miR-3928v expression in formed using an EZ-ChIP™ Chromatin Immunoprecipi- hepatoma cell lines. Compared with the immortalized tation Kit (Millipore, Billerica, MA, USA) according to normal liver cell line L02, the HBV (−) cell line HepG2 the manufacturer’s instructions. Huh7 cells were seeded and the HBV (+) cell line HepG2.2.15 showed a signifi- in 10-cm cell culture plates. The cells were lysed, soni- cant up-regulation of miR-3928v (Fig. 1b). Furthermore, cated to shear the DNA and immunoprecipitated with overexpression of pHBV1.3copy (a plasmid containing a anti-EGR1 (Shengyang Wanleibio, China) or control 1.3-unit-length genome of HBV in pUC18 for the ex- antibodies (IgG and GAPDH). qPCR primers were de- pression and replication of HBV [27]) in HBV (−)cell signed using PrimerBLAST (Additional file 2), and lines increased miR-3928v expression (Fig. 1c). To deter- DNA purified from EGR1/DNA crosslinks was ana- mine whether the non-structural HBV protein HBx is lyzed by qPCR. responsible for regulating miR-3928v expression, an ex- pression plasmid harboring HBx was transfected into DNA electrophoretic mobility shift assay HepG2 and Huh7 cells and found to increase miR-3928v Electrophoretic mobility shift assays (EMSAs) were per- expression levels (Fig. 1d). These data indicate that HBV, formed using a LightShift Chemiluminescent EMSA Kit and HBx in particular, induces miR-3928v expression. (Thermo) according to the manufacturer’s protocol. De- In addition, we examined miR-3928v expression levels tails can be found in Ref [12], with some modifications in 20 pairs of liver cancer and adjacent non-tumor tis- as described in Additional file 1. sues. RT-qPCR analysis showed that miR-3928v was highly expressed in HCC tissues (Fig. 1e). Moreover, to Tumor growth in a xenograft mouse model assess the correlation between the miR-3928v level and For the in vivo tumor growth study, 1 × 107 Huh7 cells the clinical progression of HCC, we examined miR- transfected with pri-miR-3928v or control vector were 3928v serum levels in 60 HBV (+) HCC patients and 30 suspended in 110 μLofserum-freeRPMIorDMEMfor healthy controls. miR-3928v serum levels were obviously each mouse. Each female, BALB/c-nu/nu SCID mouse higher in HCC patients than in healthy controls (Fig. 1f), (5–6 weeks old, 6 per group) was injected subcutaneously suggesting that miR-3928v may be involved in HCC in the flank. All mice were killed 8 days after implantation. pathogenesis. Zhang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:14 Page 4 of 12

Fig. 1 HBV induces miR-3928v expression in HCC cells and tissues. a The secondary structure of the miR-3928v precursor and its sequence. b The relative miR-3928v mRNA level in HBV-related HCC cell lines. c The relative miR-3928v mRNA level in HBV (−) cell lines with pHBV1.3copy overexpression. d The effect of the HBx expression plasmid (pHBx) on miR-3928v expression. e The miR-3928v expression level was determined in 20 pairs of tissues from patients with liver cancer. f The miR-3928v mRNA level was determined by RT-qPCR in 60 serum samples from patients with HCC and 30 serum samples from healthy people. (**: P < 0.01) miR-3928v functions as an oncogene in HCC cells transition and increased the proliferation index (PI) To determine the function of miR-3928v in HBV- (Fig. 2d). Taken together, these data indicate that miR- associated HCC, pri-miR-3928v and ASO-miR-3928v 3928v suppresses apoptosis and accelerates cell cycle were used to overexpress or block miR-3928v expression progression to promote HCC cell viability and in HepG2, Huh7 and HepG2.2.15 cells. After the trans- proliferation. fection efficiency was confirmed (Additional file 1: Fig- To further investigate the effect of miR-3928v on mi- ure S1A), MTT assays suggested that miR-3928v gration/invasion and VM in HCC cells, we performed promoted cell viability (Fig. 2a). To address whether the migration/invasion and tube formation assays in HepG2, promotion of cell viability is associated with apoptosis, Huh7 and HepG2.2.15 cells. As shown in Fig. 2e-f, FACS analysis was used to examine cell apoptosis. forced miR-3928v expression promoted migration/inva- Annexin-V staining showed that miR-3928v decreased sion and tube formation, but ASO-mediated inhibition the apoptosis rate of HCC cells (Fig. 2b). Next, colony of miR-3928v repressed these effects. To determine the formation assays showed that miR-3928v promoted col- mechanisms underlying the effects of miR-3928v on cell ony formation (Fig. 2c). FACS analysis was used to mobility and VM, epithelial-mesenchymal transition determine whether cell cycle progression is responsible (EMT) and VM markers were detected by Western blot. for the promotion of cell growth by miR-3928v; the re- As expected, miR-3928v promoted vimentin expression sults showed that miR-3928v accelerated the G1/S and suppressed E-cadherin protein expression (Fig. 2e, Zhang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:14 Page 5 of 12

Fig. 2 (See legend on next page.) Zhang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:14 Page 6 of 12

(See figure on previous page.) Fig. 2 miR-3928v acts as an oncogene. Huh7 and HepG2 cells were transfected with pri-miR-3928v or controls. HepG2.2.15 cells were transfected with ASO-miR-3928v or controls. Twenty-four hours after transfection, cells were seeded into cell culture inserts. a MTT assays were performed to examine cell viability at 48 h or 72 h after transfection. b FACS was used to assess cell apoptosis. c Colony formation assays were used to evaluate cell growth. HepG2 and HepG2.2.15 cells were grown for 14 days, and the colony formation rate was calculated. d FACS was used to analyze changes in cell cycle distribution. e Twenty-four hours after transfection, cells were seeded into cell culture inserts to analyze migration/invasion. The cells were counted in five random fields. Then, the expression levels of EMT-associated markers were examined by Western blot. f The effect of miR-3928v on VM. VM-associated markers were also examined. g A nude mouse tumor xenograft model was utilized to study the effect of miR-3928v on tumor growth in vivo. The miR-3928v level in tumors (left) and the tumor weight (right) are shown underneath the picture. (*: P <0.05, **: P <0.01) right panel), indicating that miR-3928v promotes EMT. (Fig. 3e, right panel). Similarly, RT-qPCR analysis showed Moreover, miR-3928v increased matrix metallopeptidase that VDAC3 mRNA levels in tumor tissue from nude 2 (MMP2) and MMP9 protein expression (Fig. 2f, right mice were almost 45% lower in the pri-miR-3928v group panel), further indicating that miR-3928v promotes VM. than in the control group (Fig. 3f). Taken together, these These results suggest that miR-3928v induces EMT to results indicate that miR-3928v directly targets and down- promote migration/invasion and enhances MMP2 and regulates VDAC3. MMP9 expression to promote VM. To determine the effect of miR-3928v on HCC tumor VDAC3 plays a tumor suppressor role in HCC cells growth, xenograft tumor experiments were performed in To study the role of VDAC3 in HCC malignancy me- mice. The average volume and weight of tumors derived diated by miR-3928v, we performed gain- and loss-of- from Huh7 cells were higher in the miR-3928v group function analyses in HCC cell lines. Cell viability, cell than in the control group (Fig. 2g). In short, miR-3928v growth, migration/invasion and VM, as demonstrated functions as an oncogene to contribute to malignancy in by MTT (Fig. 4a), colony formation (Fig. 4b), migra- vivo and in vitro. tion/invasion (Fig. 4c) and tube formation assays (Fig. 4d), were significantly promoted by shRNA- VDAC3 is a direct target gene of miR-3928v mediated knockdown of VDAC3 in HepG2 and Huh7 To identify the mechanism by which miR-3928v promotes cells and were suppressed by forced VDAC3 expres- the malignancy of HCC cells, bioinformatics analysis sion in HepG2.2.15 cells. Furthermore, FACS analysis (http://www.targetscan.org/vert_71/) was used to predict revealed that VDAC3 increased apoptosis (Add- target genes of miR-3928v. Among over 100 candidate tar- itional file 1: Figure S2) and suppressed the G1/S get genes, we noticed that the 3′UTR of VDAC3 contains transition (Fig. 4e) in HCC cells. EMT assays showed the miR-3928v binding site, and previous reports have that VDAC3 reduced vimentin expression and en- found that HBx can directly interact with VDAC3. There- hanced E-cadherin protein expression, indicating that fore, we chose VDAC3 as a candidate target gene in HCC VDAC3 inhibits EMT (Fig. 4f). Moreover, VDAC3 re- and asked whether HBx modulates miR-3928v to indir- duced MMP2 and MMP9 expression, suggesting that ectly regulate VDAC3 expression. As shown in Fig. 3a,the VDAC3 suppresses VM (Fig. 4g). In conclusion, seed sequence of miR-3928v is complementary to a se- VDAC3 suppressed cell viability, growth and migra- quence within the VDAC3 mRNA 3′UTR. In addition, tion/invasion by inducing apoptosis and inhibiting cell dual-luciferase reporter assays showed that miR-3928v cycle progression and EMT. In addition, VDAC3 re- overexpression in HepG2 cells decreased luciferase re- duced MMP2 and MMP9 expression to suppress VM. porter activity, which was blocked by ASO in HepG2.2.15 These data indicate that VDAC3 may function as a cells, whereas these effects were abolished when the bind- tumor suppressor in HCC cells. ing site in the VDAC3 3′UTR was mutated (Fig. 3a,right panel). Subsequently, RT-qPCR and Western blot analyses Restoration of VDAC3 expression abrogates the showed that miR-3928v decreased VDAC3 mRNA and malignant phenotype of HepG2 cells induced by miR- protein levels in HepG2, Huh7 and HepG2.2.15 cells 3928v overexpression (Fig. 3b). Furthermore, VDAC3 mRNA levels were down- To verify that miR-3928v promotes the malignancy of regulated in these cell lines (Fig. 3c)andHCCtissues HCC cells by directly down-regulating VDAC3, we (Fig. 3d). We also examined VDAC3 expression in tissues performed rescue experiments with a VDAC3 expres- from liver cancer patients and nude mice by immunohis- sion vector (pVDAC3) containing the VDAC3 open tochemistry (IHC) and found that VDAC3 expression was reading frame without the 3′UTR. When HepG2 cells lower in liver cancer tissues than in adjacent normal tis- were co-transfected with pri-miR-3928v or a control sues (Fig. 3e, left panel) and lower in miR-3928v- vector and pVDAC3, the miR-3928v-induced inhib- overexpressing tumor tissues than in control tissues ition of VDAC3 mRNA and protein expression Zhang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:14 Page 7 of 12

Fig. 3 miR-3928v directly targets the 3′UTR of VDAC3 mRNA. a The predicted miR-3928v binding site in the VDAC3 3′UTR and the introduced mutations in the binding site (VDAC3 mut) (left). Dual-luciferase reporter assays were performed to analyze the effect of miR-3928v on the VDAC3 3′UTR (right). # indicates no statistically significant difference. b RT-qPCR (left) and Western blot (right) were performed to examine VDAC3 mRNA and protein expression levels after miR-3928v overexpression or inhibition in HCC cells. c-d The relative VDAC3 mRNA level was determined in HCC cells (c) and tissues (d). e Representative IHC images showing the magnitude of VDAC3 expression in tissues isolated from liver cancer patients (left) and nude mice (right). f VDAC3 mRNA level in tissues isolated from nude mice. (*: P < 0.05, **: P <0.01)

(Fig. 5a) and increases in cell viability, cell cycle, mi- observe its effects in HepG2 cells. As shown in Add- gration/ invasion and VM were abrogated (Fig. 5b-f). itional file 1: Figure S3A-D (left panel), HBV/HBx These results indicate that miR-3928v functions as an promoted HepG2 cell viability, cell growth, migration/ oncogene through VDAC3 and that VDAC3 is a func- invasion and VM. Next, rescue experiments were per- tional target of miR-3928v in HCC cells. formed in HCC cells by co-transfection with the HBx expression plasmid and ASO-miR-3928v. As expected, HBx induces miR-3928v to contribute to the malignancy the ability of HBx to promoted HepG2 cell viability, of HCC cells cell growth, migration/invasion and VM was abolished It has been reported that HBx contributes to HCC by ASO-miR-3928v (Additional file 1:FigureS3A-D, progression [4]. To determine whether HBx-induced right panel). These data suggest that miR-3928v may miR-3928v mediates the oncogenic activities of HCC contribute to the malignancy of HCC cells caused by cells caused by HBV, we first expressed HBx to HBV infection. Zhang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:14 Page 8 of 12

Fig. 4 VDAC3 acts as a tumor suppressor. Huh7 and HepG2 cells were transfected with pshR-VDAC3 or controls. HepG2.2.15 cells were transfected with pVDAC3 or controls. Twenty-four hours after transfection, cells were seeded into cell culture inserts. a MTT assays were performed to evaluate cell viability. b Colony formation assays were used to estimate cell proliferation. c Migration/invasion assays and f Western blot analyses were used to evaluate EMT. d VM and g molecular markers of VM were examined to assess tumor angiogenesis. e FACS assays were used to examine changes in cell cycle distribution. (*: P <0.05)

EGR1 mediates the HBx-induced activation of the miR- presence of a silencer. Therefore, P1307 and P302 were 3928v promoter in an NF-κB signaling-dependent manner used in further studies. To investigate whether the can- To determine the mechanism underlying the up- didate promoter fragments are associated with HBV, regulation of miR-3928v after HBV infection, we ana- P1307 and P302 were transiently transfected into lyzed its location in the . As shown in HepG2.2.15, HepG2 and L02 cells. HBV (+) cells had Fig. 6a, miR-3928v is located on Homo sapiens chromo- the highest promoter activity (Fig. 6b, left), indicating some 22 (NC_000022, chr22: 31,160,061–31,160,083). that HBV may positively regulate the miR-3928v pro- The Promoter 2.0 Prediction Server (http:// moter. The effect of HBV on the miR-3928v promoter www.cbs.dtu.dk/services/Promoter/) was used to predict was further confirmed by transient transfection of the promoter region upstream of miR-3928v. Then, the HBV1.3copy/HBx in Huh7 cells, which resulted in in- miR-3928v promoter was cloned into pGL3-basic to creased luciferase activity (Fig. 6b, right), indicating that generate P1307, with the first base of the miR-3928v HBV/HBx positively regulates miR-3928v promoter ac- precursor being set as + 1 (Fig. 6a, left). Dual-luciferase tivity. Transcription factors are known to be critical reporter assays showed that P1307 increased the relative promoter-activating elements. PromoterScan was used luciferase activity in Huh7 cells (Fig. 6a, right). Further- to predict transcription factors that may bind to P302. more, to map the core promoter of miR-3928v, we con- EGR1 was found to have a putative binding site located structed four additional fragments of P1307 with 5′ at − 194~ − 186 bp in the miR-3928v promoter region terminal deletion mutations, P972, P637, P335 and P302 (Fig. 6c, right). To study the effects of EGR1 on the (Fig. 6a, left). As shown in the right panel of Fig. 6a, miR-3928v promoter, Huh7 cells were co-transfected P972, P637 and P302 displayed high luciferase activity, with P1307 or P302 and an EGR1 expression plasmid with P302 having the highest activity, whereas P335 (pEGR1); EGR1 increased miR-3928v promoter activity showed no luciferase activity, perhaps suggesting the (Fig. 6c, left). When the EGR1 binding site in the miR- Zhang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:14 Page 9 of 12

Fig. 5 VDAC3 is a functional target of miR-3928v. HepG2 cells were co-transfected with pCD3/Flag or pVDAC3 and pri-miR-3928v. RT-qPCR and Western blot analyses (a) and MTT (b), colony formation (c), migration (d), invasion (e) and tube formation assays (f) were performed to analyze whether VDAC3 could functionally rescue the miR-3928v-induced phenotype. (*: P < 0.05, **: P < 0.01, ***: P < 0.001)

3928v promoter was mutated, the luciferase activity de- HBx-induced miR-3928v up-regulation (Fig. 6f), demon- creased (Fig. 6c, right). ChIP and EMSA analyses dem- strating that EGR1 is a critical factor that mediates the onstrated that EGR1 directly binds to the miR-3928v HBx-induced promotion of miR-3928v expression. promoter (Fig. 6d). In addition, RT-qPCR showed that It has been reported that HBx can induce NF-κB sig- EGR1 enhanced miR-3928v expression (Fig. 6e). Further- naling in HCC [28, 29] and that tumor factor more, shRNA-mediated knockdown of EGR1 abolished (TNF)-α,anNF-κB signaling activator, enhances EGR1 Zhang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:14 Page 10 of 12

Fig. 6 HBx up-regulates miR-3928v expression via EGR1. a Schematic diagram of the location of miR-3928v in the human genome; the relative positions of the miR-3928v promoter constructs are shown (left). Luciferase reporter assays were used to examine miR-3928v promoter activity in Huh7 cells (right). b Promoter activity in HBV (+) and HBV (−) cells (left). Then, the promoter activity in Huh7 cells was examined after HBV or HBx overexpression (right). c The effect of EGR1 on promoter activity (left). The EGR1 binding site in the miR-3928v promoter was mutated, and the luciferase activity induced by EGR1 was examined (right). d ChIP analysis (left) and EMSAs (right) revealed that EGR1 directly binds to the miR- 3928v promoter. e The miR-3928v expression level was measured by RT-qPCR after EGR1 overexpression or knockdown. f Blocking EGR1 inhibited HBx-induced miR-3928v promoter activity. g Representative IHC images showing the magnitude of EGR1 expression in tissues isolated from liver cancer patients. h Total and nuclear expression of EGR1 and P65 in Huh7 cells after transfection with HBx was analyzed by Western blot. i An immunofluorescence assay was used to detect the induction of EGR1 expression in the nucleus by HBx in Huh7 cells. j Mechanism by which HBx- induced miR-3928v expression contributes to HCC malignancy. (*: P < 0.05, **: P < 0.01) promotor activity [30, 31]; therefore, we hypothesized ascertain the miRNA expression profile in HBV-positive that HBx may increase EGR1 expression. As shown in HCC tissues. Unexpectedly, we found several novel miR- Fig. 6g, EGR1 expression was higher in liver cancer tis- NAs, among which one highly expressed miRNA, sues than in adjacent non-tumor tissues. Western blot TLRC-m0008_3p (miR-3928v), was interesting. RT- analyses revealed that HBx increased EGR1 expression qPCR results showed that miR-3928v is significantly up- and promoted its nuclear import (Fig. 6h). An immuno- regulated in HBV-positive HCC tissues, patient serum fluorescence assay showed similar results (Fig. 6i). Taken samples and cells. together, our results indicate that HBx may facilitate the Abnormally expressed miRNAs play pivotal roles in activation of miR-3928v expression via EGR1 in an NF- the regulation of tumorigenesis. Therefore, we studied κB signaling-dependent manner. the function of miR-3928v in HCC and found that miR- 3928v suppresses apoptosis and promotes cell cycle pro- Discussion gression to promote cell viability, migration and inva- HBV infection is the major inducer of hepatocarcino- sion. Moreover, the xenograft mouse model confirmed genesis, and defining the functional role of HBV in HCC that miR-3928v functions as an oncogene. Usually, miR- development is extremely important. Abundant evidence NAs are involved in tumorigenesis by binding to the 3′ has revealed the critical role of miRNAs in the regula- UTR of relevant target genes and inhibiting translation tion of tumorigenesis. To determine the mechanism by or inducing mRNA degradation [8]. Therefore, it was which HBV-modulated miRNAs influence the malig- necessary to identify the target gene involved in the nancy of HCC, we first applied deep sequencing to effect of miR-3928v on hepatocarcinogenesis. Zhang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:14 Page 11 of 12

Bioinformatics analysis with TargetScanHuman was used HCC tissues and showed that it functions as an onco- to predict target genes, and this analysis revealed that gene in vivo and in vitro. In addition, HBx induces the VDAC3 3′UTR possesses the miR-3928v binding EGR1 nuclear import in an NF-κB signaling-dependent site. VDAC3 is the least investigated mammalian VDAC manner, thus facilitating miR-3928v expression. Finally, isoform [32]. It has been reported that VDAC3 is tar- miR-3928v down-regulates the tumor suppressor gene geted to the centrosome and transcribed at high levels VDAC3 to promote HCC progression. specifically in the testis [33], and recent reports on VDAC3 are related to mitochondria [33]. However, its Additional files function in HCC is unclear, and little is known about the regulation of VDAC3 expression. Therefore, we ex- Additional file 1: Supplementary methods: Detailed experimental materials amined whether VDAC3 is targeted by miR-3928v and and methods. Figure S1. Plasmid transfection efficiency. Figure S2. VDAC3 explored its function in HCC. Dual-luciferase reporter promotes the apoptosis of HCC cells. Figure S3. HBV/HBx infection enhances the malignancy of HCC cells via miR-3928v. (DOCX 1489 kb) assays showed that miR-3928v binds directly to the Additional file 2: Table S1. Sequences of primers used in this study. VDAC3 3′UTR. Upon further investigation to determine (DOCX 29 kb) the function of VDAC3 in HCC cells, we found that Additional file 3: Table S2. Novel miRNAs identified by Solexa VDAC3 is down-regulated in HCC cells and tissues. In sequencing. (DOCX 30 kb) addition, the VDAC3 expression profile is completely opposite that of miR-3928v. Thus, we conclude that Abbreviations miR-3928v directly targets and negatively regulates EMT: Epithelial-mesenchymal transition; ERG1: Early growth response 1; VDAC3 to contribute to HCC malignancy. HBx: Hepatitis B virus X protein; HCC: Hepatocellular carcinoma; miRNAs: microRNAs; VDAC3: Voltage dependent anion channel 3; HBx is considered the key causative agent of the VM: Vascular mimicry pathogenesis and carcinogenesis of HBV-related liver diseases [3]. For instance, HBx inhibits the proliferation Acknowledgements of NRK-52E cells by inducing apoptosis [34]. HBx also We are grateful to Huijie Gao and Hong Xie for their technical assistance. down-regulates miR-122 in carcinoma [35]. Moreover, Funding Liu et al. verified that the down-regulation of miR-18a This work was supported by the National Natural Science Foundation of by HBx promotes growth factor expression in HCC tis- China (Nos: 81572790; 91629302; 31270818) and the Natural Science sues [36]. Therefore, we next sought to determine the Foundation of Tianjin (12JCZDJC25100). mechanism by which miR-3928v is up-regulated by HBx, Availability of data and materials and we speculated that transcription factors may partici- Please contact the corresponding author for all data requests. pate in the regulatory process. There is abundant evi- dence that HBx can bind directly to transcription factors Authors’ contributions and activate gene transcription, causing dysregulation of HT conceived and designed the experiments; QGZ and GS performed and analyzed most of the experiments; LLY and YKL provided HCC patients cell cycle checkpoint controls, proliferation, apoptosis, serum and performed some experiments; ML contributed to the analyses of and DNA repair [3]. HBx can reportedly increase the data; QGZ and HT wrote the manuscript; SPL provided HCC patients tissue binding of EGR1 to the mPGES-1 promoter [37]. Pro- sample. All authors read and approved the final manuscript. moter Scan predicted that EGR1 is a candidate tran- Ethics approval scription factor involved in miR-3928v regulation. The human samples we used were in accordance with the standards of the Interestingly, EGR1 increased miR-3928v promoter Ethics Commiees of Tianjin Medical University (Ethical Approval No. activity and expression. Moreover, the HBx-mediated in- TMUhMEC2014004). Patients approved to contribute to the liver cancer tissues and reaserch study. crease in miR-3928v promoter activity was abolished by For the in vivo tumor growth study, 6 mice were used. All studies were EGR1 knockdown. Therefore, we propose that HBx en- performed according to the American Association for the Accreditation of hances EGR1 binding to the miR-3928v promoter and Laboratory Animal Care guidelines for humane treatment of animals and adhered to national and international standards. The Ethics Committee of that HBx up-regulates EGR1 to promote miR-3928v ex- Tianjin Medical University agreed to carry out this research (Ethical Approval pression via NF-κB signaling, thereby contributing to the No. TMUaMEC2014004). malignancy of HCC (Fig. 6j). Our present study identi- fied a new HBx-induced pathway involved in tumorigen- Consent for publication esis, and our findings may provide potential biomarkers Not applicable. of HBV-related HCC. Competing interests The authors declare that they have no competing interests. Conclusions In summary, this study provides new insight into the Publisher’sNote mechanism by which HBV contributes to liver carcino- Springer Nature remains neutral with regard to jurisdictional claims in genesis. We found that miR-3928v is highly expressed in published maps and institutional affiliations. Zhang et al. Journal of Experimental & Clinical Cancer Research (2018) 37:14 Page 12 of 12

Author details 20. Sun Y, Vashisht AA, Tchieu J, Wohlschlegel JA, Dreier L. Voltage-dependent 1Tianjin Life Science Research Center and Department of Pathogen Biology, anion channels (VDACs) recruit Parkin to defective mitochondria to Collaborative Innovation Center of Tianjin for Medical Epigenetics, School of promote mitochondrial autophagy. J Biol Chem. 2012;287:40652–60. Basic Medical Sciences, Tianjin Medical University, No. 22 Qi-Xiang-Tai Road, 21. Caterino M, Ruoppolo M, Mandola A, Costanzo M, Orru S, Imperlini E. Tianjin 300070, China. 2The Cancer Institute, Tangshan People’s Hospital, Protein-protein interaction networks as a new perspective to evaluate Tangshan 063001, China. 3Department of Hepatobiliary Oncology, State Key distinct functional roles of voltage-dependent anion channel isoforms. Mol Laboratory of Oncology in Southern China, Cancer Center, Sun Yat-sen BioSyst. 2017;13:2466–76. University, Guangzhou 510060, China. 22. De Pinto V, Guarino F, Guarnera A, Messina A, Reina S, Tomasello FM, Palermo V, Mazzoni C. Characterization of human VDAC isoforms: a peculiar Received: 10 August 2017 Accepted: 15 January 2018 function for VDAC3? Biochim Biophys Acta. 2010;1797:1268–75. 23. Rahmani Z, Huh KW, Lasher R, Siddiqui A. Hepatitis B virus X protein colocalizes to mitochondria with a human voltage-dependent anion channel, HVDAC3, and alters its transmembrane potential. J Virol. 2000;74: References 2840–6. 1. Torre LA, Siegel RL, Ward EM, Jemal A. Global cancer incidence and 24. Waris G, Huh KW, Siddiqui A. Mitochondrially associated hepatitis B virus X mortality rates and trends–an update. Cancer Epidemiol Biomark Prev. 2016; protein constitutively activates transcription factors STAT-3 and NF-kappa B – 25:16–27. via . Mol Cell Biol. 2001;21:7721 30. 2. Iida-Ueno A, Enomoto M, Tamori A, Kawada N. Hepatitis B virus infection 25. Yan Y, Luo YC, Wan HY, Wang J, Zhang PP, Liu M, Li X, Li S, Tang H. and alcohol consumption. World J Gastroenterol. 2017;23:2651–9. MicroRNA-10a is involved in the metastatic process by regulating Eph 3. Fu S, Zhou RR, Li N, Huang Y, Fan XG. Hepatitis B virus X protein in liver tyrosine kinase receptor A4-mediated epithelial-mesenchymal transition and – tumor microenvironment. Tumour Biol. 2016;37:15371–81. adhesion in hepatoma cells. Hepatology. 2013;57:667 77. 4. Zhang XD, Wang Y, Ye LH. Hepatitis B virus X protein accelerates the 26. Ren ZJ, Nong XY, Lv YR, Sun HH, An PP, Wang F, Li X, Liu M, Tang H. Mir- development of hepatoma. Cancer Biol Med. 2014;11:182–90. 509-5p joins the Mdm2/p53 feedback loop and regulates cancer cell 5. Liu H, Yuan Y, Guo H, Mitchelson K, Zhang K, Xie L, Qin W, Lu Y, Wang J, growth. Cell Death Dis. 2014;5:e1387. Guo Y, et al. Hepatitis B virus encoded X protein suppresses apoptosis by 27. Shimizu S, Narita M, Tsujimoto Y. Bcl-2 family proteins regulate the release inhibition of the caspase-independent pathway. J Proteome Res. 2012;11: of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature. – 4803–13. 1999;399:483 7. 6. Cho HK, Kim SY, Kyaw YY, Win AA, Koo SH, Kim HH, Cheong J. HBx induces 28. Liu Y, Lou G, Wu W, Zheng M, Shi Y, Zhao D, Chen Z. Involvement of the the proliferation of hepatocellular carcinoma cells via AP1 over-expressed as NF-kappaB pathway in multidrug resistance induced by HBx in a hepatoma – a result of ER stress. Biochem J. 2015;466:115–21. cell line. J Viral Hepat. 2011;18:e439 46. 7. Saxena N, Kumar V. The HBx oncoprotein of hepatitis B virus deregulates 29. Lim KH, Choi HS, Park YK, Park ES, Shin GC, Kim DH, Ahn SH, Kim KH. HBx- the cell cycle by promoting the intracellular accumulation and re- induced NF-kappaB signaling in liver cells is potentially mediated by the compartmentalization of the cellular deubiquitinase USP37. PLoS One. 2014; ternary complex of HBx with p22-FLIP and NEMO. PLoS One. 2013;8:e57331. 9:e111256. 30. Deckmann K, Rorsch F, Geisslinger G, Grosch S. Dimethylcelecoxib induces 8. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. an inhibitory complex consisting of HDAC1/NF-kappaB(p65)RelA leading to 2009;136:215–33. transcriptional downregulation of mPGES-1 and EGR1. Cell Signal. 2012;24: 460–7. 9. Oliveto S, Mancino M, Manfrini N, Biffo S. Role of microRNAs in translation 31. Zheng C, Ren Z, Wang H, Zhang W, Kalvakolanu DV, Tian Z, Xiao W. E2F1 regulation and cancer. World J Biol Chem. 2017;8:45–56. induces tumor cell survival via nuclear factor-kappaB-dependent induction 10. Song G, Wang R, Guo J, Liu X, Wang F, Qi Y, Wan H, Liu M, Li X, Tang H. of EGR1 transcription in prostate cancer cells. Cancer Res. 2009;69:2324–31. miR-346 and miR-138 competitively regulate hTERT in GRSF1- and AGO2- 32. De Pinto V, Reina S, Gupta A, Messina A, Mahalakshmi R. Role of cysteines in dependent manners, respectively. Sci Rep. 2015;5:15793. mammalian VDAC isoforms’ function. Biochim Biophys Acta. 2016;1857: 11. Yin D, Wang Y, Sai W, Zhang L, Miao Y, Cao L, Zhai X, Feng X, Yang L. HBx- 1219–27. induced miR-21 suppresses cell apoptosis in hepatocellular carcinoma by 33. Reina S, Guarino F, Magri A, De Pinto V. VDAC3 as a potential marker of targeting interleukin-12. Oncol Rep. 2016;36:2305–12. mitochondrial status is involved in cancer and . Front Oncol. 2016; 12. Kong XX, Lv YR, Shao LP, Nong XY, Zhang GL, Zhang Y, Fan HX, Liu M, Li X, 6:264. Tang H. HBx-induced MiR-1269b in NF-kappaB dependent manner 34. He P, Zhang B, Liu D, Bian X, Li D, Wang Y, Sun G, Zhou G. Hepatitis B virus upregulates cell division cycle 40 homolog (CDC40) to promote X protein modulates apoptosis in NRK-52E cells and activates Fas/FasL proliferation and migration in hepatoma cells. J Transl Med. 2016;14:189. through the MLK3-MKK7-JNK3 signaling pathway. Cell Physiol Biochem. 13. Kim HJ, Hong JM, Yoon KA, Kim N, Cho DW, Choi JY, Lee IK, Kim SY. Early 2016;39:1433–43. growth response 2 negatively modulates osteoclast differentiation through 35. Song K, Han C, Zhang J, Lu D, Dash S, Feitelson M, Lim K, Wu T. Epigenetic upregulation of Id helix-loop-helix proteins. Bone. 2012;51:643–50. regulation of MicroRNA-122 by peroxisome proliferator activated receptor- 14. Wang L, Sun H, Wang X, Hou N, Zhao L, Tong D, He K, Yang Y, Song T, gamma and hepatitis b virus X protein in hepatocellular carcinoma cells. Yang J, Huang C. EGR1 mediates miR-203a suppress the hepatocellular Hepatology. 2013;58:1681–92. carcinoma cells progression by targeting HOXD3 through EGFR signaling 36. Liu X, Zhang Y, Wang P, Wang H, Su H, Zhou X, Zhang L. HBX protein- pathway. Oncotarget. 2016;7:45302–16. induced downregulation of microRNA-18a is responsible for upregulation of 15. Tian H, Ge C, Li H, Zhao F, Hou H, Chen T, Jiang G, Xie H, Cui Y, Yao M, Li J. connective tissue growth factor in HBV infection-associated Ribonucleotide reductase M2B inhibits cell migration and spreading by Hepatocarcinoma. Med Sci Monit. 2016;22:2492–500. early growth response protein 1-mediated phosphatase and tensin 37. Liu C, Chen S, Wang X, Chen Y, Tang N. 15d-PGJ(2) decreases PGE(2) homolog/Akt1 pathway in hepatocellular carcinoma. Hepatology. 2014;59: synthesis in HBx-positive liver cells by interfering EGR1 binding to mPGES-1 1459–70. promoter. Biochem Pharmacol. 2014;91:337–47. 16. Grotegut S, von Schweinitz D, Christofori G, Lehembre F. Hepatocyte growth factor induces cell scattering through MAPK/Egr-1-mediated upregulation of snail. EMBO J. 2006;25:3534–45. 17. Peng WX, Xiong EM, Ge L, Wan YY, Zhang CL, Du FY, Xu M, Bhat RA, Jin J, Gong AH. Egr-1 promotes hypoxia-induced autophagy to enhance chemo- resistance of hepatocellular carcinoma cells. Exp Cell Res. 2016;340:62–70. 18. Mazure NM. VDAC in cancer. Biochim Biophys Acta. 2017;1858:665–73. 19. Reina S, Checchetto V, Saletti R, Gupta A, Chaturvedi D, Guardiani C, Guarino F, Scorciapino MA, Magri A, Foti S, et al. VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications. Oncotarget. 2016;7:2249–68.