Abstract
Hepatitis B virus X protein (HBx) contributes to the development of hepatocellular carcinoma (HCC) through inducing dysregulation of lipogenesis. However, the mechanism by which HBx induces the abnormal lipogenesis is not well known. In this study, we report that the oncogene Rab18, a member of Ras family, enhances the HBx-induced hepatocarcinogenesis through inducing dysregulation of lipogenesis and proliferation. Our data showed that the expression levels of Rab18 were positively associated with those of HBx in clinical HCC tissues. HBx was able to upregulate the expression of Rab18 in p21-HBx transgenic mice and hepatoma cell lines. Next, we identified the mechanism by which HBx upregulated Rab18. The results demonstrated that cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) were able to stimulate Rab18 promoter through activating transcription factor activator protein 1 (AP-1) and cyclic adenosine 3′,5′-monophosphate response element-binding (CREB). In addition, we identified another pathway that HBx activated Rab18. We found that miR-429 was able to directly target the 3′ untranslated region of Rab18, suggesting that Rab18 is one of the target genes of miR-429. Then, we found that HBx was able to downregulate miR-429 in hepatoma cells. The oil red O staining showed that HBx resulted in the dysregulation of lipogenesis through Rab18. Moreover, Rab18 contributed to the HBx-enhanced proliferation of hepatoma cells in vitro and in vivo . HBx enhances hepatocarcinogenesis through leading to the dysregulation of lipogenesis and proliferation of hepatoma cells, involving two pathways such as HBx/COX-2/5-LOX/AP-1/CREB/Rab18 and HBx/miR-429/Rab18.
Introduction
Hepatocellular carcinoma (HCC) is one of the most common and aggressive tumors in the world. The chronic infection of hepatitis B virus (HBV) is a major risk factor ( 1 , 2) . As an oncoprotein encoded by HBV, HBV X protein (HBx) is involved in the pathogenesis of HBV-associated liver diseases ( 3 , 4) . It influences various transcription factors, including p53, nuclear factor-κB, activator protein 1 (AP-1), cyclic adenosine 3′,5′-monophosphate response element-binding (CREB) and TATA binding protein, and is involved in many cell signal transduction pathways, such as the mitogen-activated protein kinase, c-jun N-terminal kinase, phosphatidylinositol 3-kinase and Janus kinase/signal transducers and activators of transcription pathways to regulate a wide variety of genes ( 5–7 ). Moreover, HBx influences many genes regulating cell proliferation, apoptosis, migration, lipogenesis and autophagy ( 5 , 8–10 ). We previously reported that arachidonic acid metabolism, extracellular signal-related kinases, sterol regulatory element-binding protein 1c, fatty acid synthase, cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), Yes-associated protein and long non-coding RNA highly upregulated in liver cancer were involved in the HBx-induced hepatoma ( 7 , 11–13 ). However, the underlying mechanism by which HBx induces hepatocarcinogenesis through inducing dysregulation of lipogenesis is not well documented.
Rab guanosine triphosphatases, which are members of the Ras oncogene superfamily of small guanosine triphosphatases, are regulators of vesicular transport in both exocytic and endocytic pathways in eukaryotic cells ( 14 ). Emerging evidence has revealed the association between dysfunction of the Rab small guanosine triphosphatases and multiple human diseases including cancer ( 14–17 ). However, compared with members of Ras and Rho families, Rab family’s pathophysiological roles in human malignancies are poorly understood. Recent findings have revealed that Rab25 is a determinant of tumor progression and aggressiveness of epithelial cancers, such as colon cancer, ovarian cancer, liver cancer, breast cancer and bladder cancer ( 18 , 19) . Rab5a is involved in the migration of HCC and breast cancer and promotes ovary cancer cell proliferation ( 15 , 20 , 21) . It has been reported that Rab18 is involved in the lipogenesis of 3T3-L1 adipocytes ( 22 ). Moreover, it acts as a novel tumor antigen identified by autologous antibody screening of childhood medulloblastoma complementary DNA libraries ( 23 ). However, little is known concerning whether Rab18 is involved in the lipogenesis of human cancers.
MicroRNAs (miRNAs) are a class of small, non-coding RNAs approximately 19–25 nucleotides that act as post-transcriptional regulators of gene expression through binding to the 3′ untranslated regions (3′UTRs) of the target messenger RNAs (mRNAs) ( 24 ). Extensive evidence suggests that miRNAs may represent a novel class of oncogenes or tumor suppressor genes involved in variety of biological processes, including development, differentiation, cell proliferation, migration, metabolism and lipogenesis ( 24–27 ).
In this study, we report that HBx enhances the hepatocarcinogenesis through upregulating Rab18, resulting in the dysregulation of lipogenesis and proliferation of hepatoma cells. Our finding provides new insights into the mechanism of hepatocarcinogenesis mediated by HBx.
Materials and methods
Cell culture and tissue specimen
The human HCC H7402 and H7402-X (a HBx-transfected H7402 cell line) cells were cultured in RPMI medium 1640 (Gibco, Grand Island, NY) ( 28 , 29) . HepG2, HepG2.2.15 (a HBV-transfected HepG2 cell line), HepG2-X (a HBx-transfected HepG2 cell line) and human embryonic kidney cell line 293T were cultured in Dulbecco’s modified Eagle’s medium (Gibco) ( 13 ). The medium was supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin and 100 U/ml streptomycin. Forty pairs of HBV-related HCC, their adjacent non-tumorous liver tissues and another three cases of HBV-related HCC were collected from patients undergoing resection of HCC in Tianjin First Center Hospital (Tianjin, China). Informed consent was obtained from each patient and the study was approved by the institute research ethics committee at Nankai University.
Immunohistochemistry
The HCC tissue microarrays were obtained from the Xi’an Aomei Biotechnology Co., Ltd (Xi’an, China). These microarrays were composed of 110 HCC tissue samples, which included duplicate core biopsies (1mm in diameter) from fixed, paraffin-embedded tumors. Immunohistochemical staining of samples was performed as previously reported ( 29 ). The primary antibodies used were rabbit anti-Rab18 (Proteintech Group), rabbit anti-Ki67 (Thermo Fisher Scientific, Ely, UK) and rabbit anti-BrdU (Genomapping Technology, Tianjin, China).
Quantitative real-time PCR
Quantitative real-time PCR (qRT–PCR) was explored to detect the expression of Rab18, HBx and miR-429 using Absolute Blue QPCR SYBR green mix according to the manufacturer’s instructions. Double-stranded DNA-specific expression was examined by the comparative Ct method using 2 −ΔΔCt . Primers are listed in Supplementary Table 1 , available at Carcinogenesis Online.
Western blot analysis
The western blot protocol was described previously ( 7 , 30) . Following primary antibodies were used: rabbit anti-Rab18 (Proteintech Group), rabbit anti-HBx (Abcam, Cambridge, UK) and mouse anti-β-actin (Sigma–Aldrich, St Louis, MO).
Treatment of cultured cells
Cells were cultured in 6-well or 24-well plates for 12 h and then were transfected with plasmid or small interfering RNA (siRNA) using lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). pSilencer-X, which produces siRNAs, targeting HBx and pSilencer-control as negative control (NC) were used ( 29 ). The plasmid of pCH-9/3091 contains a genome-length HBV sequence ( 31 ). The plasmids of pCH-9/3091, pCMV-X, pCMV-tag2B, pSilencer-X or pSilencer-control were transfected into cells for qRT–PCR, western blot or luciferase reporter gene assay. Indomethacin (Indo, an inhibitor of COX-2) and NDGA (nordihydroguaiaretic acid, an inhibitor of 5-LOX) were purchased from Sigma–Aldrich. siRNA duplexes and miR-429 mimic were synthesized and purified by RiboBio (Guangzhou, China). The siRNA duplexes sequences are all listed in Supplementary Table 1 , available at Carcinogenesis Online.
Construction of the promoter and 3′UTR of Rab18
The 5′-flanking region (from –792 to +85 Nt) of Rab18 was amplified and inserted into the KpnI/XhoI site, the upstream of the luciferase gene in the pGL3-Basic vector (Promega, Madison, WI), named p977. To construct various lengths of luciferase reporter plasmids of Rab18, the regions (−449/+85, −264/+85, −177/+85 and −30/+85) of Rab18 were inserted into the pGL3-Basic vector, named p534, p349, p262 and p115, respectively. Mutant construct of p534 or p262, which carried a substitution of three nucleotides within the binding sites of AP-1 or CREB, was carried out using overlapping extension PCR. The target site of miR-429 in 3′UTR of Rab18 was inserted into the FseI/XbaI site, the downstream of the luciferase gene in the pGL3-Control vector (Promega). The resulting vectors were sequenced and named pGL3-Rab18 3′UTR wt. Site-directed mutants of the miR-429 target sites in pGL3-Rab18 3′UTR were named pGL3-Rab18 3′UTR mut-1 (1106–1109-deleted), pGL3-Rab18 3′UTR mut-2 (1259–1262-deleted) and pGL3-Rab18 3′UTR mut-1 + 2 (both 1106–1109 and 1259–1262-deleted), respectively. All primers are listed in Supplementary Table 1 , available at Carcinogenesis Online.
Luciferase reporter gene assays
Cells were transferred at 3 × 10 4 cells per well into 24-well dishes. After 12 h, the various promoters or pGL3-Basic was transiently co-transfected with the pRL-TK plasmid (Promega) into cells. After 48 h, cells were collected and lysed in 1× passive lysis buffer. The luciferase activity was determined using the Dual-Luciferase Reporter® Assay System (Promega) according to the manufacturer’s instructions.
Oil red O staining
Cells were seeded in six-well plates and incubated overnight. After cells were transfected with si-control (NC) or si-Rab18 for 3 days, cells were washed twice with phosphate-buffered saline and fixed with 10% formalin. Further processes of oil red O staining were performed according to the manufacturer’s instructions.
Analysis of cell proliferation
For quantitative proliferation assays, HepG2/HepG2-X or H7402/H7402-X cells were seeded onto 96-well plates (1000 cells per well) for 12 h before transfection and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma) assays were explored to evaluate cell proliferation. For the next 3 days, the absorbance value (optical density) at 490nm of each well was measured every day. The protocol was described previously ( 7 ). 5-Ethynyl-2′-deoxyuridine incorporation assay was carried out using the Cell-Light™ 5-ethynyl-2′-deoxyuridine imaging detecting kit according to the manufacturer’s instructions (RiboBio).
Analysis of colony formation
For clonogenicity analysis, 1000 viable transfected cells were placed in six-well plates after 48h and maintained in complete medium for 2 weeks. Colonies were fixed with methanol and stained with methylene blue.
Animal transplantation
Nude mice were housed and treated according to guidelines established by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. We conducted the animal transplantation according to the Declaration of Helsinki. Tumor transplantation in nude mice was performed as described previously ( 11 ). Briefly, cells were pretreated with NC, si-HBx, si-Rab18-1 or si-HBx and Rab18 for 24 h and then were harvested and re-suspended at 2 × 10 7 per ml with sterile normal saline. Groups of 4-week-old female BALB/c athymic nude mice (Experiment Animal Center of Peking, China) (each group, n = 6) were subcutaneously injected at the shoulder with 0.2ml of the cell suspensions. Tumor growth was measured after 5 days from injection and then every 5 days. Tumor volume (V) was monitored by measuring the length (L) and width (W) with calipers and calculated with the formula (L × W 2 ) × 0.5. After 30 days, tumor-bearing mice and controls were killed, and the tumors were excised and measured.
In vivo bromodeoxyuridine labeling
Methods for in vivo bromodeoxyuridine (BrdU) labeling were described previously ( 32 ). Briefly, 24 and 4 h before killing, athymic mice bearing tumors were injected with 350 μl of BrdU (10mg/ml; Sigma) or phosphate-buffered saline as control twice. The tumor tissues were fixed and BrdU incorporation was detected by immunohistochemistry (IHC) using the primary antibody of rabbit anti-BrdU (Genomapping Technology).
Statistical analysis
Each experiment was repeated at least three times. Statistical significance was assessed by comparing mean values (± standard deviation) using a Student’s t- test for independent groups and was assumed for P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***). Association between expression levels of Rab18 and HBx in tumorous tissues was explored using Pearson’s correlation coefficient. Rab18 expression in primary HCC tissues and their corresponding non-tumorous livers were compared using a Wilcoxon signed-rank test.
Results
Rab18 is upregulated by HBx in HCC
The expression of Rab18 is not well known in cancers. To highlight the significance of Rab18 expression in HBV-associated HCC, we examined the expression of Rab18 by IHC staining using tissue microarray containing 110 cases HBV-HCC tissues. Our data showed that the positive rate of Rab18 was 73.64% (81/110) in clinical HBV-HCC tissues ( Figure 1A ). Moreover, we found that the mRNA levels of Rab18 were higher in 40 HBV-infected HCC tissues than their non-cancerous hepatic tissues by qRT–PCR ( P < 0.001, Wilcoxon signed-rank test, Figure 1B ), suggesting that Rab18 plays an important role in the pathogenesis of HCC. Due to the crucial role of HBx in the development of HCC, we examined the association between Rab18 and HBx. Intriguingly, we found that the expression levels of Rab18 were positively correlated with those of HBx in HCC tissues ( r = 0.6952, P < 0.0001, n = 43, Pearson’s correlation, Figure 1C ), suggesting that the upregulation of Rab18 may be associated with HBx in HCC. Moreover, we examined the expression of Rab18 in liver tissues from p21-HBx transgenic mice (C57BL/6) by qRT–PCR and IHC, respectively ( 33 ). Our data showed that the expression of Rab18 was increased in the liver tissues of the 12 month p21-HBx transgenic mice relative to that of wild-type (WT) littermate mice. In addition, the expression of Rab18 was increased in the tumor tissues of the 24 month p21-HBx transgenic mice relative to that in the liver tissues of the 12 month p21-HBx transgenic mice ( Figure 1D and E ). We further evaluated the effect of HBx on Rab18 in HBV-infected HepG2.2.15 cells. Compared with HepG2 cells, the expression levels of Rab18 were increased in the HepG2.2.15 cells ( Figure 2A and B ). Moreover, the expression levels of Rab18 were upregulated in HepG2 cells by transfection of pCH-9/3091 plasmid, which contains a full-length HBV sequence ( Figure 2A and B ), suggesting that HBV is able to upregulate Rab18 in hepatoma cells. Then, we identified that HBx was responsible for the upregulation of Rab18 in HepG2 (or H7402) cells by transient transfection of pCMV-X plasmid, whereas the HBx RNAi resulted in the downregulation of Rab18 in HepG2-X (or H7402-X) cells in a dose-dependent manner ( Figure 2C and D and Supplementary Figure 1 , available at Carcinogenesis Online). Overall, we conclude that HBx is able to upregulate Rab18 in hepatoma cells.
Rab18 is overexpressed in clinical HBV-HCC tissues and p21-HBx transgenic mice. ( A ) IHC staining showed the expression levels of Rab18 in 110 cases of HBV-HCC tissues using tissue microarray. ( B ) The mRNA levels of Rab18 were examined by RT–PCR in 40 pairs of human HBV-HCC tissues and their surrounding non-tumorous tissues ( P < 0.001, Wilcoxon signed-rank test). ( C ) Correlation between mRNA levels of Rab18 and HBx was detected in 43 cases of human HBV-HCC tissues ( P < 0.0001, r = 0.6952, Pearson’s correlation). ( D ) The relative expression levels of Rab18 in 12 month and 24 month p21-HBx transgenic mice (p21-HBx) and WT littermate mice were determined by RT–PCR or western blot, respectively. ( E ) The expression of Rab18 in 12 month and 24 month p21-HBx transgenic mice (p21-HBx) and WT littermate mice was determined by IHC. Statistically significant differences are indicated: * P < 0.05, ** P < 0.01, Student’s t -test. We performed triplicate experiments.
HBx is able to upregulate Rab18 in hepatoma cells. ( A and B ) The relative mRNA and protein levels of Rab18 in HepG2 and HepG2.2.15 (or HepG2 and HepG2 transfected with pCH-9/3091) were detected by RT–PCR and western blot, respectively. ( C and D ) The relative expression levels of Rab18 in pCMV-tag2B (pCMV) or pCMV-X stimulated-HepG2 cells (or in HepG2-X cells transfected with pSilencer-con or pSilencer-X) were detected by RT–PCR and western blot, respectively. Statistically significant differences are indicated: * P < 0.05, ** P < 0.01, Student’s t -test. We performed triplicate experiments.
HBx activates Rab18 promoter through COX-2 and 5-LOX involving AP-1 and CREB
To dissect the molecular mechanism by which HBx upregulated Rab18, we cloned five constructs of different Rab18 promoter region, including −792/+85, −449/+85, −264/+85, −177/+85 and −30/+85. Luciferase reporter gene assays indicated that the region −264/+85 exhibited the maximum luciferase activity ( Figure 3A ), suggesting that the fragment −264/+85 contains the core region of Rab18 promoter. Meanwhile, we found that the regulation sites of HBx covered the regions of −449/−264 and −177/−30 ( Figure 3A ). To further demonstrate the mechanism by which HBx activates Rab18 promoter, we analyzed the transcription factor–binding sites in the regions −449/−264 and −177/−30 of Rab18 promoter using promoter analysis program TF2 SEARCH and Genomatix software suite. The AP-1-binding site in −449/−264 fragment and CREB-binding site in −177/−30 region caught our attention. We found that the promoter activity of −449/+85 (p534) or −177/+85 (p262) was suppressed by si-c-Fos, si-c-Jun or si-CREB, respectively ( Figure 3B and C ). In addition, the enhanced promoter activity of −449/+85 (p534) or −177/+85 (p262) by HBx was restrained when the AP-1- or CREB-binding sites were mutant, respectively ( Figure 3D and E ). It has been reported that COX-2 and 5-LOX are able to activate AP-1 or CREB. Interestingly, we found that the promoter activity of −449/+85 (p534) also could be suppressed by the inhibition of COX-2 and 5-LOX using inhibitor or siRNA ( Figure 3F ). Moreover, the finding was repeatable in 293T cells ( Supplementary Figure 2 , available at Carcinogenesis Online). Thus, our data support the notion that HBx activates the Rab18 promoter through COX-2 and 5-LOX involving the transcription factor AP-1 and CREB.
HBx activates Rab18 promoter through COX-2 and 5-LOX involving AP-1 and CREB. ( A – F ) The activities of Rab18 promoter were measured by luciferase reporter gene assays. (A) HepG2 cells were co-transfected with several Rab18 promoters and pCMV-tag2B (pCMV) or pCMV-X, respectively. (B) HepG2-X cells were co-transfected with Rab18 promoter p534 and NC, si-c-Jun, si-c-Fos or both (si-c-Jun and si-c-Fos), respectively. (C) HepG2-X cells were co-transfected with Rab18 promoter p262 and NC or si-CREB, respectively. (D) HepG2 cells were co-transfected with Rab18 promoter p534 (WT or MUT) and pCMV-tag2B (pCMV) or pCMV-X, respectively. (E) HepG2 cells were co-transfected with Rab18 promoter p262 (WT or MUT) and pCMV-tag2B (pCMV) or pCMV-X, respectively. (F) HepG2-X cells transfected with Rab18 promoter p534 were treated with Indo or NDGA, respectively. HepG2-X cells were co-transfected with Rab18 promoter p534 and NC, si-COX-2 or si-5-LOX, respectively. Statistically significant differences are indicated: * P < 0.05, ** P < 0.01, Student’s t -test. We performed triplicate experiments.
HBx activates Rab18 through downregulating miR-429 directly targeting Rab18 mRNA
Next, we tried to identify another mechanism by which HBx upregulates Rab18. We predicted the miRNAs targeting Rab18 using TargetScan ( http://www.targetscan.org/ ). The results showed that there were two target sites of miR-429 in the 3′UTR of Rab18 ( Figure 4A ). Thus, we cloned the 3′UTR of Rab18 and its mutants, respectively. The luciferase reporter gene assays elucidated that miR-429 could decrease the luciferase activity through targeting the 3′UTR of Rab18, but it failed to work when the two target sites were mutant in HepG2-X cells ( Figure 4B ). Meanwhile, the miR-429 inhibitor upregulated the luciferase activity in HepG2 cells and the upregulation was attenuated when the target sites were mutant ( Figure 4C ). In addition, the mimics of miR-429 were able to downregulate Rab18 in HepG2-X cells. Meanwhile, the miR-429 inhibitor resulted in the upregulation of Rab18 in HepG2 cells ( Figure 4D ). Thus, the data support the notion that Rab18 is one of the targets of miR-429. To reinforce this link, we turned our attention on the effect of HBx on miR-429. Compared with HepG2 and H7402 cells, the expression of miR-429 was decreased in HepG2-X and H7402-X cells, suggesting that HBx is able to downregulate miR-429 in hepatoma cells ( Figure 4E and F ). Together, we conclude that miR-429 is involved in the upregulation of Rab18 mediated by HBx.
HBx activates Rab18 through downregulating miR-429 directly targeting Rab18 mRNA. ( A ) Sequence alignment between miR-429 and the 3′UTR of human Rab18 mRNA. ( B ) Luciferase reporter gene assays showed the effect of miR-429 on the promoter activity of Rab18 3′UTR. Co-transfection was performed using the plasmids such as human Rab18 3′UTR (wt), the miR-429-binding site (1106–1109)-deleted Rab18 3′UTR (mut-1), (1259–1262)-deleted Rab18 3′UTR (mut-2) or both (1106–1109) and (1259–1262)-deleted Rab18 3′UTR (mut-1 + 2) with scrambled or miR-429 in HepG2-X cells. ( C ) HepG2 cells were co-transfected with Rab18 3′UTR wt, mut-1, mut-2 or mut-1 + 2 with anti-miR-NC or anti-miR-429. The promoter activities were measured by luciferase reporter gene assays. ( D ) The protein levels of Rab18 in scrambled or miR-429-stimulated HepG2-X cells (or anti-miR-NC or anti-miR-429-stimulated HepG2 cells) were detected by western blot. ( E ) The expression levels of miR-429 in HepG2/HepG2-X or H7402/H7402-X cells were examined by RT–PCR. Statistically significant differences are indicated: * P < 0.05, ** P < 0.01, Student’s t -test. We performed triplicate experiments.
HBx leads to the dysregulation of lipogenesis and proliferation of hepatoma cells through Rab18
It has been reported that HBx or Rab18 is involved in lipogenesis ( 9 , 22) . In this study, we report that HBx is able to upregulate Rab18. Therefore, we are interested in whether HBx leads to dysregulation of lipogenesis through Rab18. Indeed, the oil red O staining validated that the lipogenesis was decreased by si-Rab18 ( Figure 5A ). Therefore, our data suggest that HBx contributes to dysregulation of lipogenesis through Rab18. To further elucidate the role of Rab18 in regulating proliferation of hepatoma cells, we modulated its expression in HepG2/HepG2-X or H7402/H7402-X cells. As indicated in Figure 5B and D and Supplementary Figure 3 , available at Carcinogenesis Online, the proliferation of hepatoma cells transfected with Rab18 was significantly increased relative to pCMV-transfected cells. Moreover, si-Rab18 suppressed the proliferation of hepatoma cells ( Figure 5C and D and Supplementary Figure 3 , available at Carcinogenesis Online). Colony formation assay indicated that the overexpression of Rab18 resulted in a more than 50% increase in colony numbers of HepG2 cells ( Figure 5E ). In addition, the colony numbers were decreased when the cells were treated with si-Rab18 ( Figure 5E ). To further determine the effect of Rab18 on hepatoma cell proliferation in vivo , the HepG2-X cells were treated with NC, si-Rab18, si-HBx or si-HBx and Rab18, respectively. Then, the pretreated cells were subcutaneously injected into 4-week-old BALB/c athymic nude mice. The tumor weight and volume of the si-Rab18-transfected HepG2-X cells were significantly lower than those of NC-transfected cells ( Figure 6A and B and Supplementary Figure 4 , available at Carcinogenesis Online). Moreover, the overexpression of Rab18 was able to rescue the decreased growth of tumor when HBx was knockdown ( Figure 6A and B and Supplementary Figure 4 , available at Carcinogenesis Online). Then, we evaluated the effect of Rab18 on the proliferation of hepatoma cells by examining the expression of Ki-67, a cell proliferation marker, in the tumor tissues from mice. Compared with NC group, the expression levels of Ki-67 were significantly decreased in si-Rab18-transfected group ( Figure 6C ). Moreover, the induced BrdU incorporation by HBx was partially attenuated by the knockdown of Rab18 in HepG2-X cells ( Figure 6C ). In addition, the reduction of BrdU incorporation mediated by si-HBx could be partially rescued by the overexpression of Rab18 ( Figure 6C ). Thus, we conclude that HBx contributes to the dysregulation of lipogenesis and proliferation of hepatoma cells through Rab18.
HBx leads to the dysregulation of lipogenesis and proliferation of hepatoma cells through Rab18 in vitro . ( A–D ) HepG2 (or HepG2-X) cells were transfected with pCMV-tag2B (pCMV) or pCMV-Rab18 (or pSi-NC, pSi-X, NC, si-Rab18-1, si-Rab18-2), respectively. (A) The effect of Rab18 siRNA on HBx-induced lipogenesis was determined by oil red O staining. (B and C) The effect of Rab18 on the HBx-enhanced cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. (D) The effect of Rab18 on HBx-enhanced cell proliferation was determined by 5-ethynyl-2′-deoxyuridine incorporation assay. ( E ) The effect of Rab18 on HBx-enhanced cell proliferation was determined by colony formation assay. Statistically significant differences are indicated: * P < 0.05, ** P < 0.01, Student’s t -test. We performed triplicate experiments.
HBx enhances proliferation of hepatoma cells through Rab18 in vivo. ( A ) The growth curves of tumors derived from HepG2-X cells transfected with NC, si-HBx, si-Rab18-1 or si-HBx and pCMV-Rab18. ( B ) The image of dissected tumors from nude mice ( n = 6). ( C ) IHC for Ki-67 and BrdU in the tumor tissues from mice. Statistically significant differences are indicated: * P < 0.05, ** P < 0.01, Student’s t -test. We performed triplicate experiments.
Discussion
The dysregulation of lipogenesis plays crucial roles in hepatocarcinogenesis. It has been reported that HBx is implicated in abnormal lipogenesis in HBV-associated HCC ( 9 ). Rab18, as a member of Ras oncogene family, is also involved in the lipogenesis ( 22 ). Therefore, we are interested in whether the oncogene Rab18 is involved in the development of HBV-associated HCC through inducing abnormal lipogenesis.
Rab18 was identified as a new novel tumor antigen in medulloblastoma ( 23 ). However, the expression of Rab18 is less well known in human cancers, such as in HCC. In this study, we first report that Rab18 is highly expressed in HBV-associated HCC tissues. Notably, we observed that the expression levels of HBx were positively correlated to those of Rab18 in clinical HCC tissues. Consistent with the finding, we found that the expressions levels of Rab18 were much higher in the tumorous liver tissues of 24 month p21-HBx transgenic mice. It suggests that HBx may upregulate Rab18 in hepatoma cells. Indeed, as predicted, we showed that HBx was able to upregulate Rab18 in the cells. Thus, we conclude that upregulation of Rab18 mediated by HBx may play an important role in the development of HCC.
To investigate the underlying mechanism of upregulation of Rab18 mediated by HBx, we cloned the promoter and 3′UTR of Rab18. Transcription factor and miRNA-binding sites analysis revealed that AP-1 and CREB or miR-429-binding sites are present in the promoter or 3′UTR of Rab18, respectively. The CREB/bZIP family proteins play an essential role in regulating gene expression and different processes such as lipid metabolism and cell proliferation ( 34 ). CREB has been implicated in hepatocarcinogenesis ( 35 ). AP-1 signaling is also activated in hepatoma cells ( 36 ). HBx as a transactivation factor is involved in the activation of many transcription factors, including AP-1 and CREB ( 37 , 38) . It has been reported that inhibition of COX and 5-LOX is able to decrease the phosphorylated c-Jun and inhibit phosphorylated p38, CREB and AP-1 activity ( 39 ). PGE2, the major metabolite of COX-2, induces the activation of ATF-4 and AP-1 ( 40 ). Moreover, our previous findings showed that HBx enhanced and maintained liver cell proliferation via a positive feedback loop involving COX-2 and 5-LOX ( 7 ). Accordingly, we were interested in whether those factors, such as COX-2, 5-LOX, AP-1 and CREB, are involved in the upregulation of Rab18 mediated by HBx in hepatoma cells. Strikingly, our data showed that HBx was able to activate Rab18 promoter through COX-2 and 5-LOX, involving transcription factor AP-1 and CREB. It has been reported that miR-429 is a p53-upregulated miRNAs in hepatoma cells ( 41 ) and HBx suppresses the expression of p53 ( 42 ). MiR-429 is repressed by the transcription repressors, ZEB1/deltaEF1 ( 43 ), which was upregulated by COX-2 ( 44 ). Therefore, we found another way that HBx upregulates Rab18. Our data showed that Rab18 was one of the target genes of miR-429, while HBx downregulated the expression of miR-429 in hepatoma cells. MiR-429 may be downregulated by HBx involving p53, ZEB1 and COX-2. Taken together, we conclude that HBx uses two pathways to upregulate Rab18 in hepatoma cells, namely, HBx/COX-2/5-LOX/AP-1/CREB/Rab18 and HBx/miR-429/Rab18.
Consistent with the contributions of Rab18 in lipogenesis ( 22 ), we found that Rab18 was involved in the HBx-induced dysregulation of lipogenesis. In addition, Rab18 is linked to the promotion of hepatoma cell proliferation mediated by HBx. Thus, in our system, we show that HBx enhances the dysregulation of lipogenesis and proliferation of hepatoma cells through driving network signalings, including lipid metabolism enzymes, transcriptional factors, miRNAs and oncoproteins.
In summary, we propose a model for the regulation by HBx in hepatocarcinogenesis. HBx enhances the dysregulation of lipogenesis and proliferation of hepatoma cells through upregulating Rab18 using two pathways, such as HBx/COX-2/5-LOX/AP-1/CREB/Rab18 and HBx/miR-429/Rab18 pathways.
Supplementary material
Supplementary Table 1 and Supplementary Data can be found at Supplementary Data
Funding
National Basic Research Program of China (973 Program, No. 2009CB521702); National Natural Science Foundation of China (No. 81071624 and 81272218); Support Program of National Science and Technology of China (No. 2012BAI23B08).
Conflict of Interest Statement: None declared.
Acknowledgements
We thank Dr Xiao Yang (from Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing, China) for kindly providing the HBx transgenic mice.
Abbreviations:
- AP-1
activator protein 1
- BrdU
bromodeoxyuridine
- COX-2
cyclooxygenase-2
- CREB
cyclic adenosine 3′,5′−monophosphate response element-binding
- HBV
hepatitis B virus
- HBx
hepatitis B virus X protein
- HCC
hepatocellular carcinoma
- IHC
immunohistochemistry
- 5-LOX
5-lipoxygenase
- mRNA
messenger RNA
- miRNA
microRNA
- NC
negative control
- qRT–PCR
quantitative real-time PCR
- siRNA
small interfering RNA
- UTR
untranslated regions
- WT
wild-type.
References
