Background
Hepatocellular carcinoma (HCC) is one of the most malignant tumors in the world. The chronic infection of hepatitis B virus (HBV) is a crucial risk factor in the development of HCC. HBV encoded X protein (HBx) is a key player in the pathogenesis of HBV-associated liver diseases. It is able to transactivate cellular genes associated with processes such as transcription, apoptosis, signaling, and cell growth[
1‐
3]. Our laboratory has reported that HBx plays an important role in the event, such as activating Yes-associated protein (YAP), Lin28A/B and Rab18[
4‐
6]. HBx transgenic (Tg) mice are able to develop hepatitis, steatosis, and dysplasia, culminating in the appearance of HCC in liver[
7‐
9]. However, HBx alone is considered a poor transformer of human and rodent hepatic cells. In support of this, co-transfection with an oncogene, such as H-ras or myc, is necessary for accelerating hepatocarcinogenesis[
10]. As an inducible factor, survivin is abundantly expressed in a hepatoma cell line harboring HBV[
11]. We previously reported that HBx was able to up-regulate survivin in hepatoma cells[
12]. HBx may up-regulate survivin through activation of Wnt/β-catenin signaling[
13‐
15]. Therefore, we supposed that HBx might collaborate with survivin to accelerate hepacarcinogenesis.
Mammalian hepatitis B X-interacting protein (HBXIP) is originally identified as a binding protein of HBx[
16]. Recently, it has been reported that HBXIP serves as a regulator component for the mammalian target of rapamycin (mTOR) Complex 1 activation which regulated cell growth[
17]. We have reported that HBXIP acts as an oncoprotein to promote the development of breast cancer through activating some cellular genes such as S100A4, NF-κB, Interleukin-8 and c-Myc[
18‐
20]. HBXIP up-regulates some membrane-bound complement regulatory proteins through phosphorylated extracellular signal-regulating kinase 1/2 (p-ERK1/2)/NF-κB signaling to accelerate breast tumor growth[
21]. In addition, HBXIP has also been identified as an adaptor for survivin to suppress apoptosis[
22]. Meanwhile, HBx may interfere with the normal function of HBXIP during prometaphase, resulting in genomic instability[
23,
24]. However, the function of HBXIP in the development of HCC mediated by HBx remains unclear.
Accumulating data indicated that aberrant expression of microRNAs (miRNAs) could regulate cancer development including tumorigenesis, metastasis and proliferation by serving as tumor suppressors or oncogenes. MiRNAs have essential roles in the progression of HCC and directly contribute to the development of HCC by targeting a large number of critical protein-coding genes[
25,
26]. Our laboratory has revealed that miR-520b targeting HBXIP and IL-8 inhibits the migration of breast cancer cells[
19], which is down-regulated in breast cancer cells and sensitizes breast cancer cells to complement attack[
27]. Moreover, miR-520b targeting mitogen-activated protein kinase kinase kinase 2 (MEKK2) and cyclinD1 inhibits the proliferation of liver cancer cells[
28]. However, the role of miR-520b in hepatocarcinogenesis mediated by HBx remains ill-defined.
In the present study, we further investigated the role of HBx in the development of HCC. Interestingly, we identify that HBx enhances hepatocarcinogenesis with partner survivin through modulating miR-520b and HBXIP. Our finding provides new insights into the mechanism of hepatocarcinogenesis mediated by HBx.
Discussion
It has been reported that HBx is a key factor in hepatocarcinogenesis[
32]. The co-transfection with an oncogene, such as H-ras or myc, is necessary for accelerating hepatocarcinogenesis[
10]. HBx can promote initiation and progression of hepatocellular carcinoma through cooperating with Kras involved in Ras pathway[
33]. We previously reported that HBx was able to up-regulate survivin in hepatoma cells[
12]. Therefore, we are interested in whether HBx can accelerate hepatocarcinogenesis through cooperating with survivin.
Interestingly, in this study we validated that survivin was up-regulated in the liver tissues from HBx-Tg mice in a time course manner. To demonstrate the significance of survivin up-regulation in HBx-induced hepatocarcinogenesis, we successfully generated an engineered cell line model (termed LO2-X-S) that LO2 cells stable transfected with both HBx and survivin. We previously reported that 3 of 8 mice injected with LO2-X cells stably transfected HBx grew tumors[
29]. Strikingly, in this study we observed that all 8 mice injected with LO2-X-S cells formed tumors. It implies that survivin is an important partner of HBx in tumorigenesis.
Next, to better understand the underlying mechanism of tumorigenesis mediated by HBx with partner survivin, we assessed the miRNA differentiate expression profiles between LO2-X-S cells and LO2-X cells using miRNA microarray assay. Our data showed that miR-520b was down-regulated in LO2-X-S cells, relative to LO2-X cells. We previously reported that HBXIP was a target gene of miR-520b in breast cancer cells[
19]. Therefore, we concerned that miR-520b targeting HBXIP might be involved in the HBx-induced hepatocarcinogenesis. Moreover, we identified that HBx was able to form a complex with survivin and transcription factor Sp1, resulting in the down-regulation of miR-520b through inactivating miR-520b promoter. Consistent with our previous reports[
19,
27,
28], our finding suggests that miR-520b is a novel tumor suppressor gene.
Then, we moved to the investigation of HBXIP in HBx-induced hepatocarcinogenessis. It has been reported that HBXIP functions as a cofactor of survivin in apoptosis suppression[
16,
22]. HBXIP is a critical target of viral HBx for promoting genetic instability through formation of defective spindles and subsequent aberrant chromosome segregation[
23]. HBXIP is a regulator of centrosome duplication, required for bipolar spindle formation in HeLa human carcinoma cells and primary mouse embryonic fibroblast cells[
24]. Our laboratory also shows that HBXIP is a novel oncoprotein in breast cancer[
18,
19]. In this study, we used the model of HBx-Tg mice to evaluate expression of survivin and HBXIP in HBx-induced hepatocarcinogenesis. Strikingly, we observed that both of survivin and HBXIP were markedly up-regulated in HBx-Tg mice, however, the up-regulation of survivin was earlier (at 12 M mice) than that of HBXIP (at 18 M mice). Moreover, the expression of HBXIP stayed lower in the liver at 12 M HBx-Tg mice which didn’t develop HCC. However, the HBXIP was highly expressed in the liver cancer at 18 M HBx-Tg mice that developed HCC. Therefore, it strongly suggests that HBx first up-regulates survivin and then increases the expression of HBXIP through collaborating with survivin. In clinical, we found that 20 peritumor samples, 30 normal liver samples and 10 hepatitis samples were weak staining for HBXIP. But, HBXIP was highly expressed in HCC tissues, positively associating with the expression levels of HBx and survivin. The above data are consistent with the data in HBX-Tg mice.
It has been reported that the global patterns of DNA methylation are altered in many cancers, including HCC[
34‐
36]. To explore the mechanism by HBx and survivin up-regulate HBXIP, we observed the promoter region of HBXIP using epigenetics analysis. Interestingly, we found that there were CpG islands in the promoter region. Thus, we supposed that the methylation regulation might be involved in the up-regulation of HBXIP mediated by HBx and survivin. Accumulating data reported that HBx had an effect on DNA methyltransferase[
37,
38]. However, the role of survivin in methylation regulation has not been reported. Surprisingly, we showed that HBx and survivin resulted in demethylation of HBXIP promoter in the cells. The methylation modification of HBXIP was observed in the cell lines and clinical liver cancer tissues. Therefore, it suggests that HBx up-regulates HBXIP with partner survivin not only through down-regulating miR-520b targeting HBXIP mRNA but also inducing demethylation of HBXIP promoter. The possible mechanism may be related to that the overexpression of both HBx and survivin suppresses the methylase (or methyltransferase), or activate demethylase in the cells. The mechanism needs to be further investigated in detail. Recently, our laboratory has reported that the oncoprotein HBXIP serves as co-activator of transcriptional factors in breast cancer. The nuclear import of oncoprotein HBXIP depends on interacting with c-Fos and phosphorylation of both proteins in breast cancer cells[
39‐
41]. Thus, our finding suggests that HBXIP as a crucial oncoprtein contributes to the HBx-induced hepatocarcinogenesis. In function, we found that miR-520b and HBXIP were responsible for the growth of LO2-X-S cells
in vitro and
in vivo. Therapeutically, all the factors, such as HBx, survivin and HBXIP, may serve as targets in HBV-associated HCC.
Methods
Plasmid constructs
The plasmid pCMV-X was used as a template for subcloning HBx into the pCMV-Tag2B vector (termed Flag-X)[
12]. One 3′-deleted DNA fragment and four 5′-deleted DNA fragments of various sizes were amplified by PCR from the 5′-flanking region of HBXIP. To generate a series of reporter constructs (p-3233/-1673, p-1484/+1, p-804/+1, p-588/+1 and p-168/+1), each fragment was inserted between the Kpn I and Hind III sites in the pGL3-basic vector (Promega, USA). To clone the miR-520b promoter into the pGL3-basic vector, a 1 kb section of the putative promoter region (from −1 to −1000) was amplified by PCR and cloned into the pGL3-basic vector. The transcription factor binding site mutants of the miR-520b promoter were cloned using the Fast Mutagenesis System (TransGen Biotech, China). All primers used are listed in Additional file
2: Table S2.
Cell culture and generation of stable cell lines
The human immortalized liver LO2, HepG2, HepG2.2.15 and NIHT3T cell lines were purchased from Nanjing KeyGEN Biotech Co., Ltd (Nanjing, China). The co-transfection with the plasmids of pCMV-X and pcDNA3-sur[
12] was performed in LO2 cells using Lipofectamine 2000 (Invitrogen, USA) as described[
12]. After 48 hours, the transfected cells were incubated in selection medium containing 500 μg/ml or 800 μg/ml G418 (Genview, USA). The transfection efficiencies were tested by PCR (one primer is from survivin and another is from the vector), western blotting, as indicated. Then, the engineered cell line was termed LO2-X-S. All the engineered cell lines were cultured in RPMI Medium 1640 (GIBCO, USA) containing 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal calf serum (FCS). HepG2 cells, HepG2-X cells[
42] and HepG2.2.15 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, GIBCO) containing 100 U/ml penicillin, 100 μg/ml streptomycin and 10% FCS.
Patient samples
The 22 paired HBV-related HCC tissues and the corresponding nearby noncancerous livers used in this study were obtained from patients who underwent radical resection at Tianjin First Center Hospital (Tianjin, China). Specimens were immediately frozen and stored at −80°C. Clinicopathological information about patients was obtained from patient records and was summarized in Table
1. All patients were diagnosed with primary HCC, and none had received previous radiotherapy or chemotherapy before surgery. Informed consent was obtained from each patient and the study was approved by the Institute Research Ethics Committee at Nankai University.
MiRNA microarray
MiRNA microarrays were performed by CapitalBio (CapitalBio Corp, China) as described in detail on the CapitalBio website (
http://www.capitalbio.com). The miRNAs that showed >2-fold or <0.5 changes in expression were considered to have changed significantly and are shown in (Additional file
2: Table S2).
Total RNA of cells (or liver tissues) were extracted using TRIzol reagent (Invitrogen) and qRT-PCR was performed according to the manufacturer’s instructions. The expression of specific genes or miRNAs was tested by the comparative Ct method using 2
-ΔΔCt[
43]. Primers used are listed in (Additional file
2: Table S2). The HBV DNA in the supernatants of LO2 cells transfected with PCH9/3091 containing a genome length HBV sequence was extracted with a commercial kit (QIAGEN, USA)[
44]. HBV DNA copies were quantified by qRT-PCR according to a diagnostic kit for quantification of HBV DNA (Da An Gene, China).
Western blotting
Western blotting was carried out in the liver of HBx-Tg mice, cell lines and tumor tissues from mice using standard protocols[
29]. The following primary antibodies were used: actin (NeoMarkers, USA), HBXIP,[
45] HBx,[
46] HBs (Zhongshan-Golden Bridge, ZM-0122, China), HBc (Zhongshan-Golden Bridge, ZM-0421, China), Sp1 (McAb, Epitomics, ab77441, USA), survivin (PcAb, Labvision, PA1-16836, USA), Flag (PcAb, Abcam, ab93713, UK) and α-fetoprotein ( PcAb, NeoMarkers, PA5-11480, USA). The data were analyzed by Image-Pro Plus 6.0 software.
RNA interference (RNAi) and miRNA
The plasmid of pSilencer3.0-X encoding HBx mRNA was used in transfection of HepG2.2.15 cells as described above[
12]. The short interfering RNA (siRNA) duplexes targeting survivin, HBXIP[
18,
47] were synthesized by Ribobio Co. Lit. (Guangzhou, China) as previously described. MiR-520b mimics and its inhibitor (anti-miR-520b) for
in vitro transfection, and their respective negative controls were from Ribobio Co. Lit. The sequences of miR-520b mimics and its inhibitor are 5' AAAGUGCUUCCUUUUAGAGGG 3' and 5' CCCUCUAAAAGGAAGCACUUU 3'. The transfected cells were lysed after 48 hours. Western blotting or RT-PCR was used to determine the expression levels of survivin and HBXIP.
Luciferase reporter gene assay
H7402-X, HepG2-X cells or LO2 and various LO2-engineered cell lines were plated in 24-well plates (3× 104 cells/well). The cells were transfected with plasmids using Lipofectamine 2000 (Invitrogen). At 48 hours post-transfection, a standard dual luciferase reporter assay was performed, and the results were normalized using a co-transfected pRL-TK plasmid containing the Renilla luciferase gene (Promega). All experiments were performed at least three times.
Co-immunoprecipitation assay
Immunoprecipitations were performed using anti-flag M2 agarose (Sigma,USA) according to the manufacturer’s instructions. The immunoprecipitated proteins were then identified by western blotting as described above using the anti-HBx antibody, anti-survivin antibody, anti-Sp1 and anti-HBXIP antibody.
Chromatin immunoprecipitation assay (ChIP)
The ChIP assay was performed using the EpiQuikTM Chromatin Immunoprecipitation Kit from Epigentek Group Inc. (Brooklyn, NY). Protein–DNA complexes were immunoprecipitated with HBx, survivin or Sp1, with Anti-RNA polymerase II as a positive control antibody and normal mouse IgG as a negative control antibody. PCR amplification was performed using 1 μl of each DNA sample. Amplification of soluble chromatin prior to immunoprecipitation was used as an input control.
Immunohistochemistry analysis
The normal human liver, hepatitis, liver cirrhosis and hepatocellular carcinoma tissue microarrays were obtained from the Xi'an Aomei Biotechnology Co., Ltd. (Xi'an, China). Immunohistochemical staining of samples was performed as previously reported[
12]. The percentage of cells showing positive nuclear and/or cytoplasmic staining for HBXIP was calculated by reviewing the entire slide. On the basis of the percentage of cells with positive nuclear and/or cytoplasmic staining, staining patterns were classified on a four-grade scale: 0, <10% cells with positive nuclear and/or cytoplasmic staining; 1+, 10–30% positive cells; 2+, 30–50% positive cells; 3+ >50% positive cells. Categorization of immunostaining intensity was performed by two or three independent observers. Light microscopic images were documented using a Nikon Eclipse Ti-U fluorescence microscope (Nikon Corp) with an attached SPOT RT digital camera (Diagnostic Instruments, Inc.).
Gene-specific DNA methylation
Genomic DNA was extracted from the cells (or liver tissues) using a TIANamp Genomic DNA Kit (TIANGEN BIOTECH CO.) according to the manufacturer’s instructions. The methylation statuses of various genes were determined by bisulfite sequencing PCR after sodium bisulfite conversion of the DNA using an EZ DNA Methylation-GoldTM Kit (Zymo Research, USA) according to the manufacturer’s instructions. This method is based on the differential sensitivities of methylated and unmethylated CpG to bisulfite treatment. The modified DNA was then used as a PCR template to amplify either unmethylated or methylated bisulfite-converted complementary sequences of the promoter of interest. The primers were designed using MethPrimer software (
http://www.urogene.org/methprimer/). The primer sequences are given in (Additional file
2: Table S2). PCR products were gel-extracted and subcloned into pMD18-T vectors (TAKARA, China), and five clones from each sample were sequenced for DNA sequencing.
Analysis of cell proliferation
For cell proliferation assays, LO2 or various engineered cells were seeded in 96-well plates (1000 cells/well) for 24 hours before transfection and 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) (Sigma) assays were used to assess cell proliferation every day from the first day until the third day after transfection. The protocol was described previously[
48]. 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay was carried out using the Cell-Light TM EdU imaging detecting kit according to the manufacturer’s instructions (RiboBio, China).
Anchorage-independent colony formation by these cell lines was determined as described previously[
49], with some modifications. For colony formation analysis, LO2 and various engineered cells were seeded in six-well plates. Cells were transfected with RNA interferences targeting HBXIP using Lipofectamine 2000. Cells were grown for 9 to 14 days. Once colonies were visible, they were stained with methylene blue and photographed. All assays were repeated at least three times. The colonies were counted using a dissecting microscope.
Transplantation in nude mice
The tumorigenicity of LO2 or the LO2 engineered cell lines were measured as follows. Cells were harvested by trypsinization, washed twice with sterile phosphate-buffered saline, and resuspended at a concentration of 1 × 107 cells per ml. Aliquots of 0.1-0.2 ml were injected subcutaneously into 4- to 6-week-old Balb/c athymic nude mice. Mice were observed at periodic intervals, and photographs were taken 3–4 weeks after the injection. Tumor volume (V) was monitored by measuring the length (L) and width (W) with calipers and calculated with the formula (L × W2) × 0.5.
Statistical analysis
All values are presented as means ± SD. Each value is the mean of at least three separate experiments in each group. Data were analyzed to compare two groups using a Student’s t test. P values of less than 0.05 were considered to be statistically significant. All statistical analysis was performed using SPSS13.0 software (Chicago, IL).
Acknowledgement
We thank Prof. Xiao Yang from Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing, PR China for providing HBx-Tg mice. This work was supported by grants from the Natural Scientific Foundation of China (No. 81272218, 81000870).
Competing interests
The authors declared that they have no competing interests.
Authors’ contributions
XZ and LY designed the experiments, supervised the project and wrote the manuscripts. WZ and GK designed and performed the experiments, analyzed and interpreted data, and wrote the manuscripts; YG, ZL, TW, and QL performed the experiments, analyzed and interpreted data; QW, NC and HW analyzed and interpreted data. All authors read and approved the final manuscript.