HBV regulated RhoC expression in HepG2.2.15 cells by enhancing its promoter activity

Journal of Basic Microbiology 2012, 52, 1–8 1

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Research Paper

HBV regulated RhoC expression in HepG2.2.15 cells by enhancing its promoter activity

Yuanyuan Tian*, 1, Yunzhi Liu*, 2, Jialin Qu1, Kai Li1, Dongdong Qin1, Ailong Huang1 and Hua Tang1

1 Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University, Chongqing, China

2 Department of forensic medicine, Chongqing Medical University, Chongqing, China

Many studies showed that RhoC-GTPases are central molecules in oncogenic transformation. The expression of RhoC is significantly increased in hepatocellular carcinoma (HCC). HBV is the major risk factor for HCC. However, it is unknown whether HBV regulating RhoC expression. In this study, we showed the RhoC expression was significantly higher in HepG2.2.15 than that in HepG2 cells. HBV could up-regulate RhoC expression through enhancing the activity of its promoter, and HBs and HBx may involve in this process. After silencing HBs and HBx expressions by using RNA interference, the expression of RhoC in HepG2.2.15 cells could be obviously inhibited. These results would provide useful information for understanding mechanism of HCC induced by HBV infection.

Keywords: Hepatocellular carcinoma (HCC) / HBV / RhoC / Expression

Received: February 02, 2012; accepted: March 14, 2012

DOI 10.1002/jobm.201200063

Introduction*

Hepatocellular carcinoma (HCC) is the fifth most com-mon malignancy and the third leading cause of cancer death worldwide [1–3]. Epidemiologic evidence suggests a number of environmental factors associated with the development of HCC, including viral hepatitis type B (HBV) and C (HCV) infections, dietary aflatoxin, the male gender, and chronic liver disease. Among those, chronic hepatitis B virus (HBV) infection has been iden-tified as the major aetiological factor of HCC [4, 5]. HBV genome encodes four major proteins: the viral polymerase, surface proteins, X protein and core pro-tein. Among these four proteins, the Large Surface proteins (LHBs) have been demonstrated to exert a tu-mor promoter-like function in the development of HCC [6–8]. In addition, HBx are involved in oncogenesis, proliferation, inflammation and immune responses as a transcriptional activator of various cellular genes [9]. Importantly, HBx can mediate activation of Ras-Raf- * Yuanyuan Tian and Yunzhi Liu contributed equally to this work. Correspondence: Hua Tang, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China E-mail: [email protected] Phone: +86-23-68486780

MAPK (Mitogen-activated protein kinase) pathway to hepatocyte transformation [10, 11]. Recently, Colom-bino M et al. found that genes of MAPK pathway, such as BRAF and PIK3CA are somatically mutated in pa-tients of hepatocellular carcinoma [12]. Moreover, Sor-rafenib, a putative multi-targeted kinase inhibitor, is used as a medical treatment of patients with advanced HCC [13, 14]. RhoC (Ras homolog gene family, member C), a small (~22 kDa) signaling G protein, is a member of the Rac subfamily of the Rho family of GTPases. It cycles be-tween inactive GDP-bound and active GTP-bound states and function as molecular switches in signal transduc-tion [15]. Activation of the Rho protein leads to the assembly of the actin-myosin contractile filaments into focal adhesion complexes that lead to cell polarity and facilitate motility. Recently, the RhoC expression has reported to be increased in many human cancers in-cluding breast, melanoma, pancreatic, colon, bladder, hepatocellular, non-small cell lung carcinoma, and primary gastric tumor or cell lines [16–18], and overex-pression of this gene was associated with tumor cell invasion and metastasis. Hakem et al. found that RhoC-deficient mice can still develop tumors but these fail to metastasize, arguing that RhoC is essential for metasta-

2 Y. Tian et al. Journal of Basic Microbiology 2012, 52, 1–8


sis [19]. The latest study found that RhoC can activate formins such as mDia1 and FMNL2 to remodel the cy-toskeleton [20]. Genome-wide analysis of gene expres-sion has revealed that RhoC gene was involved in vascu-lar invasiveness of hepatocellular carcinoma [21]. Wang et al. found that RhoC played a critical role in tumori-genesis, invasion and metastasis of HCC [22]. Taken together, the RhoC expression plays an important role in carcinogenesis of HCC. However, the mechanisms why RhoC was up-regulated in HCC has unknown. Many researchers found that invasion and metastasis are essential hallmarks of malignant cancer cells. Adhesion of tumor cells to host cell layers and subse-quent transcellular migration are important processes in cancer invasion and metastasis, which involves the extracellular matrix (ECM) degradation [23–25]. Because HBx has multifunction in the process of HCC, most efforts in the study of the role of HBV in HCC develop-ment have focused on the HBx involvement in the tumor invasion and metastasis. Recently, several re-ports have demonstrated that HBx is also implicated in the late stages of tumor progression, metastasis and angiogenesis and induces extensive morphological changes and cytoskeleton rearrangements in liver cells [26]. It is known that chronic infection with the HBV is a major risk factor for development of HCC [4], we hy-pothesized that HBV might related to the RhoC expres-sion in HCC. In this study, we demonstrate that HBV increased RhoC expression by activation of the RhoC promoter. Among HBV four proteins, the HBs and HBx had higher effect on RhoC promoter activity. Repres-sion of HBx and HBs expression by siRNA could signifi-cantly reduce RhoC expression. These results indicated that HBs and HBx have a major effect on the up-regulation of the RhoC expressions. Our study would provide a novel mechanism for an understanding of the invasion and metastasis of HBV-related HCC.

Materials and methods

Materials Human hepatoma cell lines HepG2 and HepG2.2.15 were kept by our laboratory. The HBV expression plas-mid pCH-9/3091 constructed by Michael et al., (Heidel-berg University, Germany) was donated from Dr. Lan Lin (Southwest Hospital affiliated of the Third Military Medical University, China). pRL-TK plasmid and dual luciferase assay kit were purchased from Promega. pCMV-Sport6 plasmid was bought from ATCC (Ameri-can Type Culture Collection, America). pCMV-Sport6-

HBx, pCMV-Sport6-HBs, pCMV-Sport6-HBc and pCMV-Sport6-HBp were constructed by our laboratory and their correct expressions were confirmed in HepG2 cells (data not shown). pGL3-Basic, Transfection reagent lipofectamine 2000TM were bought from Invitrogen (America). RhoC monoclonal antibody was purchased from Abcam. Peroxidase-conjugated goat anti-mouse IgG was bought from ZSGB-BIO (China).

Cell culture and transient transfection HepG2 and HepG2.2.15 cells were cultured in MEM medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin at 37 °C in a humidi-fied chamber with 5% CO2. Lipofectamine 2000 reagents were used in all trans-fection experiments. For investigation of HBV regulat-ing the expression of RhoC, HepG2 cells were seeded in 24-wells plate at approx 50% confluence and trans-fected with pCH-9/3091. For Western blot analysis, HepG2.2.15 cells or HepG2 cells were seeded in 6-wells plate at a density of 1 × 106 cells/well, transfected with pCH-9/3091, four HBV expression plasmids and RNAi plasmids, respectively. After 48 h, all transfected cells were collected for extracting proteins. For RhoC pro-moter activity assay, HepG2 cells were seeded in 24-wells plate at a density of 2 × 105 cells/well, trans-fected with pGL3-RhoC-P, pGL3-RhoC-P plus pCH-9/3091, pGL3-RhoC-P plus the expression plasmids of HBs, HBc, HBp, HBx, respectively. Above all, cells were co-transfected with pRL-TK, the control reporter vector individually.

RNA extraction and RT-PCR Total RNA was isolated using Trizol reagent (Invitrogen, America), and cDNA was synthesized from the RNA by M-MLVreverse transcriptase (Promega, America) with random primers. The primer sequences of RhoC and β-actin which was used as an internal quantitative control were as follows: the upstream primers, RhoC: 5′-TATTGCGGACATTGAGGTGG-3′ and β-actin: 5′-GTGGATCAGCAAGCAGGAGT-3′; the downstream primers, RhoC: 5′-TAGCCAAAGGCACTGATCCG-3′ and β-actin: 5′-TGTGTGGACTTGGGAGAGGA-3′. The condi-tion of PCR was as follows: 26 cycles of initial denatur-ing at 95 °C for 5 min, denaturing at 95 °C for 40 s, annealing at 56 °C for 50 s and extending at 72 °C for 1 min and a final extension at 72 °C for 10 min. The bands representing amplified products were analyzed by GELDOC 2000 system. RhoC gene expression was presented by the relative yield of PCR product from target sequence to that from β-actin gene.

Journal of Basic Microbiology 2012, 52, 1–8 HBV regulation RhoC expression 3


Western blot analysis To prepare the proteins for Western blot analysis, HepG2, HepG2.2.15, and transfected cells were lysed in protein lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium dexycholate, 0.1% SDS, and sodium orthovanadate, sodium fluoride, EDTA, leupeptin) and the protein concentrations were deter-mined by using BCA protein concentration determina-tion kit (ZSGB-BIO, China). Each sample hole was added 50 μg of total protein sample. Proteins in a 10% SDS-PAGE were transferred electrophoretically to a PVDF membrane. The membranes were blocked in 5% nonfat dry milk in T-TBS (Tris-HCL-buffered saline supple-mented with 0.5% Tween 20) for 2 h, and incubated with a primary antibody (MoAb anti-RhoC, diluted 1:500; MoAb anti-GAPDH, diluted 1:6000) overnight. GAPDH was used as an internal quantitative control. After washing with T-TBS, the membrane was allowed to react with a suitable secondary antibody conjugated with HRP (diluted 1:10,000). After washed five times with PBS, signals were detected by chemiluminescence ECLTM detection system (Pierce, America).

Construction of plasmids The genomic region flank of the RhoC gene promoter was obtained from Promoter 2.0 Prediction Server database (http://www.cbs.dtu.dk/services/Promoter/). To generate the reporter constructs (pGL3-RhoC-P) driven by RhoC promoter, the full region of the RhoC gene promoter was generated by PCR using the HepG2 cells genomic DNA as the template. The primers sequence of RhoC promoter as follows: Forward: GGAGGTAC-CAAGTCAGACCTAGGAGTGG; Reverse: TTAC-TCGAGTCGGACCTCAGGAGGCCAGAGCCCC. (Underlines were KpnI and XhoI restriction endonuclease sites). The PCR products and pGL3-Basic vectors were digested with KpnI and XhoI. After PCR purification by High PCR product Purification Kit (Roche, Switzerland) and gel extraction by DNA Gel Extraction Kit (QIAGEN, Nether-lands), promoter fragment (around 1 kb) and vector fragment (about 4.8 kb) were ligated by T4 ligase. Re-combinant plasmid was identified by restriction en-zyme digestion and sequencing analysis. It was named pGL3-RhoC-P.

Luciferase assays The activity of RhoC promoter was checked using the Dual-Luciferase Reporter Assay system (Promega, Amer-ica). Growth medium of the pGL3-RhoC-P transfected cells were removed. 1 ml of 1 × passive lysis buffer (PLB)

was added into the cells in 24-wells plate. The plates were shaken gently about 15 min by a shaker. Cells were manually scraped from the culture plates in the presence of 1 × PLB, centrifuged, draw supernatant concluding cellular proteins. 50 μl of Luciferase Assay Reagent II was added into a tube containing 20 μl of supernatant, mixed by pipetting, and placed in a lumi-nometer (Turner TD20/20 luminometer) programmed to perform a 2-sec premeasurement delay, followed by a 10-sec measurement period for each reporter assay. The reaction was then stopped by the addition of 50 μl of Stop & Glo Reagent, which activated the renilla luciferase. The ratio of luminescence from the experi-mental reporter (firefly luminescence) to luminescence from the control vector (Renilla luminescence) was calculated to measure RhoC promoter activity.

Generation of siRNA construct plasmids and RNAi analysis Three putative candidate sequences, the unique 19-bp sequence derived from the mRNA transcript of the HBs or HBx, respectively, targeted for suppression, were designed by Wu et al. [27]. The 60-bp oligonucleo-tides, containing the unique 19-bp sequence in both sense and antisense orientations, were synthesized in the forward and reverse orientation, annealed, and ligated into the pGenesil-1.0 vector (Jing Sai Biological Engineering Technology Co., Ltd. China). The presence of the insert was determined by sequenc-ing. The sequences of six putative candidates were as follows: HBs-siRNA1, 5′GCTCCCGCGTGTCTTGGCC-3′; HBs-siRNA2, 5′-GGTGGACTTCTCTCAATTT-3′; HBs-siRNA3, 5′-GCCAAAATTCGCAGTCCC-3′; HBx-siRNA1, 5′-GCACTTCGCTTCACCTCTG-3′; HBx-siRNA2, 5′-GCAATGTCAACGACCGACC-3′; HBx-siRNA3, 5′-GTTTAAAGACTGGGAGGAG-3′. Their specific inhibi-tion effects (do not inhibit other genes expression of HBV) were checked with RT-PCR analysis (data not shown). For detecting HBs, the primers were: forward 5′-ATGGAGAACATCACATCAGG-3′, reverse 5′-GCAATGTATACCCAGAGACAAAA-3′. For detecting HBx, the primers were: forward 5′-ATGGCTGCTAGGCTGTACTG-3′, reverse 5′-GGCAGAGGTGAAAAAGTTGCA-3′. β-actin was used as an internal quantitative.

Quantification analysis Western blot and RT-PCR results were quantified in densitometry with Quantity One-4.6.2 software (Bio-Rad). The data are the mean value.



Results

HBV increased the expression of RhoC in vitro Two hepatoma cell lines (HepG2 and HepG2.2.15) were chosen to perform our study. After comparison the expressions of RhoC gene between these two cells with RT-PCR and Western blot analysis, we found that RhoC expressions were extremely higher in HepG2.2.15 cells than that in HepG2 cells (Fig. 1A, B). It is known that, HepG2.2.15 cells were derived from HepG2 cells which were transfected with a complex HBV construct. There-fore, we hypothesized that the high RhoC expression in HepG2.2.15 cells may relate to HBV expression. To fur-ther demonstrate the function of HBV in regulation of RhoC expression, PCH-9/3901 (HBV expression plasmid) was transiently transfected into HepG2 cells. After 48 h, the total RNA and proteins were extracted to do RT-PCR and Western blot analysis, respectively. At this time, HBV also increased the expression of RhoC (Fig. 1C, D).

HBV enhanced the RhoC promoter activity To determine how HBV could regulate the expression of RhoC, we generated pGL3-RhoC-P plasmid, which contained promoter region of the RhoC gene and pGL3-

basic luciferase expression vector, to analyze whether HBV could increase the RhoC promoter activity. At first, the activity of pGL3-RhoC-P was confirmed (Table 1). Then pGL3-RhoC-P, pCH9/3091(HBV expres-sion plasmid) and pRL-TK plasmids were cotransfected to HepG2. We found that HBV specifically activated the RhoC promoter activity, up to approx 6-folds (Fig. 2). Therefore, we confirmed that HBV up-regulated the expression of RhoC by modulating its promoter activ-ity.

HBs and HBx played major roles in enhancing RhoC promoter activity among the HBV four major proteins HBV has four open reading frames encoding proteins. To reveal which protein had more obvious influence on the promoter activity, four expression plasmids (pCMV-Sport6-HBs, pCMV-Sport6-HBc, pCMV-Sport6-HBp and pCMV-Sport6-HBx) were constructed, after their expres-sion in HepG2 cells were checked with RT-PCR analysis (data no shown), and these four expression plasmids with pGL3-RhoC-P, pRL-TK vector were cotransfected to HepG2 cells, respectively. After 48h, the RhoC promoter activities were detected. As shown in Fig. 3A, HBs and HBx could significantly enhance the RhoC promoter activities, respectively.

Figure 1. HBV can increase the RhoC expression in vitro. (A) RhoC expressions in HepG2 and HepG2.2.15 cells were analyzed with RT-PCR. β-actin was used as an internal quantitative control. (B) Expressions of RhoC in HepG2 and HepG2.2.15 cells were analyzed with Western blot. GAPDH was used as an internal quantitative control. (C) RT-PCR was performed to analyze the expressions of RhoC in HepG2 cells which influenced by HBV. β-actin was used as an internal quantitative control. pCMV-sport6 was used as control. (D) Western blot analysis was performed to determine RhoC expressions in HepG2 cells which influenced by HBV, GAPDH was used as an internal quantitative control. (E) RT-PCR was performed to analyze the expressions of pCH-9/3091, which could express the major HBV proteins when it was transfected HepG2 cells. β-actin was used as an internal quantitative control. Similar results were obtained from a triplicate experiment.



Table 1. Relative luciferase activity of pGL3-Basic-RhoC-P plasmid.

plasmid pGL3-Basic pGL3-RhoC-P pGL3-DNAJB4-P pRL-TK pCMV-Sport6 relative luciferase activity

NC plasmid + – – + + 0.025 ± 0.007 PC plasmid – – + + – 1.962 ± 0.324 pGL3-RhoC-P plasmid – + – + – 1.425 ± 0.465

The values are means ± SD of three independent experiments (NC plasmid: negative control plasmid, PC plasmid: positive control plasmid) We further performed Western Blot to investigate whether the HBs and HBx can up-regulate the RhoC protein expression. The HepG2 cellular proteins which were transfected with HBs or HBx expression plasmids were extracted for Western Blotting analysis. As shown in Fig. 3B, HBs and HBx could significantly increase the RhoC protein expression (Fig. 3B).

Inhibition of HBs and HBx expression decreased the expression of RhoC To further confirm the critical role of HBx and HBs in the process of up-regulation the RhoC expression, RNA interference expression system was established. RT-PCR analysis was performed to confirm siRNAs specifically knocked down the targeted genes (Fig. 4A). Then, cellu-lar lysates derived from HBs or HBx siRNA transfected HepG2.2.15 cells were analyzed with Western Blot. The

Figure 2. HBV regulated RhoC expression by enhancing its promoter activity. The pGL3-RhoC-P, pRL-TK were co-transfected with or without HBV into HepG2 cells. After 48 h, relative luciferase activities were detected. pGL3-B was used as a negative control. Luciferase activity was normalized with the renilla luciferase activity in cell lysate. The values are means ±SD of three independent experiments.

results showed that expressions of RhoC all decreased after inhibition of HBs and HBx by their siRNAs, respec-tively (Fig. 4B).

Discussion

With more than half a million new cases each year, HCC is the fifth most common tumor worldwide and the third cause of cancer-related deaths [1]. Available information suggests that metastasis is the major cause of death in HCC patients [28]. As is known, HBV is a major cause of HCC throughout the world [4]. Many researchers found that invasion and metastasis are essential hallmarks of malignant cancer cells. Adhesion of tumor cells to host cell layers and subsequent tran-scellular migration are important processes in cancer invasion and metastasis, which involves the extracellu-lar matrix (ECM) degradation [23–25]. Because HBx has multifunction in the process of HCC, most efforts in the study of the role of HBV in HCC development have focused on the HBx involvement in the tumor invasion and metastasis. Recently, several reports have demon-strated that HBx is also implicated in the late stages of tumor progression, metastasis and angiogenesis and induces extensive morphological changes and cy-toskeleton rearrangements in liver cells [26]. The Rho GTPases contribute to a multiple cellular processes, including the cellular cytoskeletal reorgani-zation and motility. Feng et al. had found that some Rho GTPases have oncogenic activity and can promote cancer cell invasion [29]. RhoC is one of the Rho GTPases clearly involved in tumor progression and metastasis. It is involved in cytoskeletal reorganization, especially in the formation of actin stress fibers and focal adhesion contacts [30]. RhoC also regulates intra-cellular membrane trafficking, gene transcription and cell cycle progression [31, 32]. Studies focusing on RhoC confirm that over-expression of RhoC mRNA was asso-ciated with venous invasion, number of tumor nodules, metastatic lesions and poor prognosis in HCC [21, 22]. In addition, suppression of RhoC expression resulted in inhibition of invasion and migration in vitro, and knockdown of the RhoC expression in a HCC metastatic



Figure 3. HBs and HBx increased RhoC expressions. (A) The activities of RhoC promoter obviously increased in presence of HBs and HBx. The pCMV-sport6 group was used as a negative control. Luciferase activity was normalized with the renilla luciferase activity in cell lysate. The values are means ±SD of three independent experiments. (B) HepG2 cells were transfected with pCMV-Sport6-HBs or pCMV-Sport6-HBx. The RhoC expressions were checked with Western blotting. pCMV-sport6 was used as control. GAPDH was used as an internal quantitative control. Similar results were obtained from a triplicate experiment. mouse model resulted in inhibition of metastasis of HCC in vivo [33]. Several recent studies have provided evidence for a role of HBV in tumor invasive and metastasis in differ-ent stages. And a lot of results showed RhoC had been over-expressed in HCC. RhoC played a major role in invasion and metastasis of tumor cells. Therefore, we hypothesized that whether HBV can promote the tu-mor cells invasive and metastasis through up-regulating the RhoC expression. In order to confirm this hypothesis, we performed this study. In this article, we set up a research to study the spe-cific association between the RhoC expression and HBV-related HCC. Our results have shown that HBV could up-regulate the RhoC expression. This result provided new evidence that HBV could promote the metastasis of HCC through up-regulating the RhoC expression. It is known that, in genes expression process, promoter plays an important role. So, we designed the RhoC promoter report plasmid to investigate why HBV could increase the RhoC expression. In our result, HBV could dramatically enhance the RhoC promoter activity. Moreover, HBs and HBx have more influence in this process. Our data indicated that, HBV could up-regulated the RhoC expression, maybe this is one way resulting in HCC metastasis.

Despite our research has indicated that HBV could enhance the RhoC promoter to increase its expression, the mechanism how HBV enhance the promoter is unknown. It is well known, the promoter itself can’t control the gene activity, it must be combined with the transcription factors. Previous results suggested that HBx binds to transcription factors to coactivate tran-scription [34, 35] or to stabilize binding to DNA [36]. Early in the 1996, HBx was found to mediate a pro-longed accumulation of AP-1 DNA binding complexes in Chang cells and in HepG2 cells [37]. In addition, HBx can augment the DNA binding activity of the phos-phorylated form of Sp1 in HepG2 cells [38]. Chung et al. found that HBx could up-regulate AP-1 and NF-κB tran-scriptional activity through activation of ERKs and PI-3K/AKT pathways [39]. However, transcription factors mentioned above are relatively common. Therefore, we presumed that HBs or HBx may combine with tran-scription factors to enhance the promoter activity. The specific mechanism would be the next step of our re-search content. The result described above revealed a new idea of HBV causing HCC metastasis, and might provide a new mentality to therapy the HBV-related HCC.



Figure 4. Inhibition of HBs and HBx expression decreased the RhoC expressions in HepG2.2.15 cells. 3 μg HBs or HBx RNAi was transfected into HepG2.2.15 cells. (A) After 48 h, inhibition effects of HBs and HBx expression were analyzed with RT-PCR. siRNA of HBs and siRNA of HBx had strong interference effect on inhibition of HBs or HBx expressions. β-actin was used as an internal quantitative control. (B) HBs or HBx expression was inhibited by siRNAs in HepG2.2.15 cells, the expressions of RhoC were checked with Western blotting. GAPDH was used as an internal quantitative control. Similar results were obtained from a triplicate experiment.

Acknowledgement

This work was supported by Nature Science Foundation of China (30771924) and Natural Science Foundation Project of CQ CSTC (2010BB5359).

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((Funded by • Nature Science Foundation of China; grant number: 30771924

•Natural Science Foundation Project of CQ CSTC; grant number: 2010BB5359))

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