Hepatitis B Virus X Protein-Induced RORγ Expression to Promote the Migration and Proliferation of Hepatocellular Carcinoma

Aberrant expression of RORγ is implicated in cancer development. A previous study identified that RORγ functions as a tumor promoter to drive hepatocellular carcinoma (HCC) growth. However, its expression and significance in HCC remain unclear. The central finding of this work is that RORγ was overexpressed in HCC due to its dysfunction of promoter methylation, and hepatitis B virus X protein (HBx) can remarkably induce the expression of RORγ in hepatocellular carcinoma through enhancing the transcriptional function. Also, the HBx-induced RORγ could promote the migration and proliferation of hepatoma cells. Hence, these results suggest that RORγ was an important regulator in HCC, and our finding provides new insights into the significance of RORγ in HCC.


Introduction
e retinoic acid receptor-related orphan receptors (RORs) are members of nuclear receptor superfamily, including three subtypes (RORα, RORβ, and RORc), which are a class of ligand-dependent transcriptional factors [1]. Previous studies have revealed that RORs have critical roles in the regulation of physiological and pathological processes, such as development, circadian rhythm, and cellular metabolism, especially in the immune response regulation [2][3][4][5]. Besides, RORct is also a member of the ROR family, which is different from RORc in the N-terminal [6]. RORct is overexpressed in the thymus, and RORc is normally expressed in the kidney, liver, skeletal muscles, adipose tissue, pancreas, and so on [7,8]. e pathogenic mechanism studies have revealed that RORct regulates the differentiation of 17 cells, which is a kind of cells that secrete interleukin 17 (IL-17), a regulator of proinflammatory signaling [9,10]. Summary of the previous reports confirm the role of RORct in the regulation of immune-inflammatory effect. However, the study of RORc in the regulation of signaling pathway remains unclear.
In recent years, the study of RORc in cancer has received more attention. Muscat and colleagues demonstrated RORc expression is downregulated in breast tumor tissues RORc negatively regulates TGFβ/EMT and MaSC pathways, and agonist targeting RORc could inhibit the migration and proliferation of breast cancer cells [11,12]. However, its expression was reported to be highly expressed in nonsmall-cell lung cancer, its expression has a positive correlation with the lymph node metastasis, and the high-expression of RORc showed a poor prognosis [13], suggesting that RORc may perform a different function in different cancers. More interestingly, another study in breast cancer revealed that the high RORc expression represented a low survival rate [14]; therefore, the different functions of RORc were illuminated in breast cancer. And a similar phenomenon was reported in melanoma. Kupper's report showed the growth of melanoma cells was inhibited in the RORcdeficient mice [15]. RORc expression showed inverse correlation with melanoma progression [16]. Accordingly, the explanation of different functions in cancers may be due to the tumor microenvironment, and the study of RORc in cancers is worthy of further research.
Hepatocellular carcinoma (HCC) is a main malignant tumor in the digestive system, contributed to the fifth leading cause of cancer-related death [17]. RORct was reported to be overexpressed in peripheral blood mononuclear cells of HCC patients [18], and the contribution of RORc in HCC still has not been reported. In this work, we firstly examined the expression of RORc in HCC and evaluated the potential mechanism. As hepatitis B virus (HBV) infection is a major risk factor for HCC and hepatitis B virus X protein (HBx) is a major protein in the occurrence and development of HCC [19][20][21], whether RORc is involved in the regulation of HBV-related HCC remains unclear. To formulate the hypothesis, we examined the expression of RORc in the tumor and adjacent tissues and found the hypomethylation of RORc in the liver tumor, and RORc expression was further enhanced in the HCC patients with HBV infection. e most important work is that HBx could induce RORc expression by promoting its transcriptional function. e biological function study demonstrated overexpression of RORc can enhance the migration and growth activity of liver cancer cells. Our finding provides new insights into the role of RORc in HCC.

Clinical Liver Cancer Tissues and Cell Lines.
e liver cancer tissues and corresponding adjacent tissues were collected in the department of hepatobiliary surgery, the Guangxi Zhuang Autonomous Region Peopleʼs Hospital, from the patients who have not undergone any treatment, including chemotherapy and radiotherapy. e tissues were used for the analysis of the RORc expression. e study was approved by the institutional research ethics committee at the Guangxi Zhuang Autonomous Region People's Hospital (GuangXi Science and Technology-2018-30). All patients signed the written consent.

Cell Culture.
e HepG2, SMMC-7721, HEK293T, and Huh7 cell lines were obtained from ATCC (Rockville, MD, USA). HCCLM3 cells were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). ese cell lines were cultured in Dulbecco's modified Eagle medium (DMEM) (GIBCO, USA), supplemented with 10% fetal bovine serum (FBS, GIBCO, USA) with heat inactivation and 100 U/mL penicillin (GIBCO, USA). e cells were cultured in a humidified incubator with 37°C and 5% CO 2 . e passenger of cell lines was no more than 10 times, and liquid nitrogen was used for the storage of cells.

Western Blotting.
e clinical tissues were homogenized with tissue homogenization treatment and centrifuged at 12000 g/min at 4°C for purification of protein. e cells were lysed with RIPA lysis buffer containing the protease inhibitor cocktail (Yeasen, Shanghai, China) on ice for 30 min. e lysis was centrifuged at 4°C for 15 min, and the supernatant was collected and subjected to BCA assay for protein concentration evaluation. A total of 30 μg protein was subjected to SDS-PAGE and transferred to the PVDF membrane. e membrane was incubated with primary antibody overnight and subjected to the second antibody at room temperature for 1 h. e antibodies are as follows: anti-RORc (Abcam, Cambridge, USA, ab78007), anti-GAPDH (Proteintech, USA, 60004-1-Ig), anti-Myc (Santa Cruz, CA, USA, 9E10), anti-HBx (Abcam, Cambridge, USA, ab203540), and anti-β-actin (Proteintech, USA, 23660-1-AP). e enhanced chemiluminescence (ECL) system (Yeasen, Shanghai, China) was used to visualize the protein band. e Quantity One software (Bio-Rad, CA, USA) was used to quantify protein expression.

Bioinformatics Analysis of TCGA Database.
e Cancer Genome Atlas (TCGA) program for liver cancer [22] was assessed in this study. A total of 59 normal liver tissues and 97 liver tumor tissues were compared in its mRNA levels.
e relative expression levels were evaluated by the mediancentered intensity method. e methylation analysis was analyzed with 450k methylation array, and the ratio of the methylation levels between liver tumor and corresponding adjacent normal tissues was calculated. And, the median value of methylation level was subjected to those normal liver tissues due to deficiency. Also, the correlation between the methylation level and gene mRNA expression was examined in liver tumor tissues and adjacent normal tissues.

Oncomine Analysis.
e Guichard Liver microarray data were selected from the Oncomine portal [23]. A total of 86 normal liver tissues and 99 liver tumor tissues were analyzed by a reporter (01-150058071), and the data type was mRNA level. Furthermore, 81 cases of liver cancer patients with hepatitis virus infection information were classified into two groups, 48 patients without HBV infection and 33 patients with HBV infection.

Quantitative Real-Time Reverse Transcription PCR (qRT-PCR).
e cells were washed with cold phosphate buffer solution (PBS) twice and collected with a cell scraper. e cells were lysed with TRIzol RNA extraction reagent (Invitrogen, Gaithersburg, MD) for 10 min at room temperature, and trichloromethane was subsequently added. After vortex oscillation, the reaction solution was stored at 4°C for 10 min and centrifuged at 4°C for 10 min. e supernatant was collected and mixed with isopropanol to precipitate total RNA. With 10 min incubation, the solution was centrifuged for 10 min, and the collected RNA was washed with 75% ethanol. e first-strand cDNA was synthesized with SuperScript reverse transcriptase (Invitrogen, MD, USA). e SYBR green-quantitative polymerase chain reaction (PCR) system (TaKaRa, Shiga, Japan) was used to examine the mRNA levels of indicated genes in the StepOne Real-time PCR system (Applied Biosystems, USA). e relative gene expression was calculated by 2 − ΔΔCT . e primer sequences are as follows: RORc forward, 5′-GTG GGG ACA AGT CGT CTG G-3′, RORc reverse, 5′-AGT GCT GGC ATC GGT TTC G-3′, the amplicon size is 156 bp. GAPDH forward, 5′-AGG TCG GAG TCA ACG GAT TT-3′, GAPDH reverse, 5′-ATC TCG CTC CTG GAA GAT GG-3'. GAPDH is a housekeeping gene to normalize the relative gene expression.

Luciferase Reporter
Assay. e luciferase reporter system of RORc was conducted as follows. e promoter sequence of RORc was obtained for GeneCopoeia (Rockville, MD, USA), a web portal for promoter region clones (http://www. genecopoeia.com) [24]. e detailed RORc promoter sequence in this study contains the core promoter sequence of RORc, which was reported previously [8].
e genomic DNA of HepG2 cells was used as a template to amplify the promoter sequence of RORc. e HepG2 and Huh7 cells were transfected with HBx-overexpressing plasmid or siRNA against HBx (Sigma, Saint Louis, USA), combined with the luciferase reporter plasmid of RORc and Rellina plasmid. After transfection for 24 h, the cells were collected and washed with cold PBS twice. e dual-luciferase reporter system (Promega, USA) was used to examine the luciferase activity. In detail, the collected cells were lysed with a reporter lysis buffer for 30 min on ice. After lysis, the supernatant was transferred into white 96-well microplates, and the firefly luciferase solution was added, followed by the addition of Rellina luciferase substrate. e microplate was then placed into a microplate reader to examine the luciferase reporter activity.

Construction of Stable Overexpressing Cell with RORc and
HBx.
e full sequence of RORc and HBx was cloned into a pCDH-CMV-MCS-EF1-Puro vector. e cDNA of HepG2 cells was used as a template for RORc amplification, and the cDNA of HBx was cloned from a pGFP-HBx plasmid (Addgene, Cat.No.65463). e primer of RORc is as follows: forward, 5′-GCT CTA GAA TGG ACA GGG CCC CAC AGA G-3′, reverse, 5′-CGG AAT TCT CAC TTG GAC AGC CCC ACA GG-3′, and the restriction sites were XbaI and EcoRI. en, the lentivirus plasmid of RORc or HBx combined with the packaging plasmid pCMV-DR8.91 and pCMV-VSV-G was transfected into HEK293T cells. For further incubation with 48 h, the lentiviral particles were collected, and the particles were condensed with lentivirus concentration solution. Also, the quality of particles was evaluated with the viral titer, and the qualified lentiviral particles were stored in an ultra-low temperature freezer. en, the viral particle containing RORc and HBx was applied to incubate the indicated cells, supplemented with polybrene at the concentration of 5 μg/mL. e puromycin selection ( ermo Scientific, Madison, WI, USA) was conducted to screen the positive cells.

Wound-Healing Assay.
e cells were plated in 6-well plates and cultured for 24 h and then subjected to the lentiviral transduction particles packing RORc overexpression and control vector system for 12 h transfection. e scratches were produced across the cell monolayers with yellow tips. And the cells were washed with PBS solution for three times to remove the shedding cells. en, the cells were cultured in fresh culture medium for another 48 h, and the confluence of cells was recorded with PrimoStar microscope (Zeiss, Jena, Germany). e migration index was calculated with the ratio of the scratch area between RORc overexpression and vector cells by Image J software (US National Institutes of Health, Bethesda, MD, USA).

CCK-8 Cell Viability Assay.
e HepG2 cells were seeded in 96-well plates at a density of 5000 cells per well, and the cells were cultured in the indicated culture medium, supplemented with lentiviral particles of RORc overexpression and control vector. e cells were cultured for a different time, and before 3 h of indicated detection time, 10 μL CCK-8 reagent (Yeasen, Shanghai, China) was added into the cells, and the absorbance value was detected by a microplate reader ( ermo Scientific, Madison, WI, USA) at 450 nm.

Colony Formation Assay. HepG2 cells overexpressing
RORc and with vector control cells were seeded in a 3.5 cm dish (500 cells/dish) and cultured for 2 weeks in DMEM (containing 10% FBS). After washing twice gently with PBS, cells fixed with 4% paraformaldehyde and stained with crystal violet. e number of foci containing ≥50 cells was calculated at 40X magnification using an optical microscope (Zeiss, Jena, Germany).

Statistical
Analysis. Data are represented as mean ± standard deviation (SD), which were acquired in at least three independent experiments. e statistical significances of differences were analyzed by using analysis of variance or Student's t-test.

Overexpression of RORc in Liver Cancer Patients.
A previous study revealed that RORc has a different regulation mechanism in different cancers. e study of RORc in liver cancer has not been reported yet. However, RORct as a truncated variant was reported overexpressed in peripheral blood mononuclear cells of HCC patients. en, we examined the RORc expression of liver cancer. As seen in Figure 1(a), compared with 86 cases of normal liver tissues, RORc mRNA levels were significantly increased in the 99 cases of liver tumor tissues. And a similar result was confirmed in the TCGA liver database (Figure 1(b)). To further confirm the overexpression of RORc in the liver tumor tissues, 3 pairs of patients with liver cancer in tumor tissues and corresponding adjacent normal tissues were subjected to western blotting; the protein expression of RORc was also enhanced remarkably (Figures 1(c ese results revealed that RORc was significantly increased in liver tumor at both protein and mRNA levels, suggesting that RORc may play a potential role in the occurrence and development of liver cancer.

Downregulation of RORc Promoter Methylation Activity in Liver
Tumor. In our bioinformatics and western blotting analysis of RORc in liver cancer, we confirmed that RORc was overexpressed in the liver tumor tissues. Next, we tried to explore the overexpression of RORc in liver cancer, as methylation regulation was reported as an important regulatory mechanism for gene expression. en, we hypothesized that methylation regulation may play an important role in the overexpression of RORc in liver tumor tissues. e TCGA analysis of the RORc promoter methylation activity of liver tumor tissues and normal liver tissues revealed that 62% of liver cancer patients express hypomethylation (Figure 2(a)). To accurately evaluate the promoter methylation levels of RORc in tumor and normal liver tissues, we selected 49 cases of patients with complete methylation levels both in tumor and corresponding normal tissues. e result showed that the promoter methylation levels of RORc were notably decreased in the tumor tissues (Figure 2(b)). To directly evaluate the association between methylation levels and gene expression levels of RORc in liver cancer patients, as the potential correlation between the gene expression and methylation level, we analyzed the correlation of RORc promoter methylation levels and its mRNA expression of 190 cases of liver tumor tissues and 50 cases adjacent normal liver tissues. e results showed that both in tumor and normal liver tissues, the promoter methylation levels of RORc were negatively correlated with its mRNA expression (Figures 2(c)-2(d)). Taking this, we RORγ protein levels * * * (d) Figure 1: RORc was overexpressed in liver cancer. (a) e Guichard Liver was assessed from the Oncomine database, and the relative mRNA level was analyzed with the median-centered intensity method, and the adjacent liver tissues were considered as normal liver tissues and taken as the control group. (b) e TCGA database of liver cancer was subjected to analyzing the mRNA levels of RORc, and the adjacent liver tissues were considered as normal liver tissues and taken as the control group. (c) ree patients with liver cancer were subjected in this study, and the tumor tissues and corresponding adjacent normal liver tissues were applied to examine the protein levels of RORc with western blotting. (d) e relative quantitative protein intensity of RORc was evaluated with Quantity One software, and the data are expressed as mean ± standard deviation (SD), n � 3. * * * P < 0.001 was considered statistically significant.
believed that the promoter methylation regulation might be an important reason for the overexpression of RORc in liver tumor tissues.

Liver Cancer Patients with HBV Infection Representing
Higher RORc Expression. RORc was overexpressed in liver tumor. In order to deeply study the expression of RORc in different subtypes of liver cancers, as hepatitis virus infection is a major risk factor of HCC, especially the hepatitis B virus (HBV) infection widely occurs in Asia, we examined the RORc expression in hepatitis virus infection or negative infection in liver cancer patients. Interestingly, we found the expression of RORc was higher in these liver cancer patients with HBV infection than in those without hepatitis virus infection (Figure 3(a)). And, a similar phenomenon was revealed in the analysis of the TCGA database (Figure 3(b)). ese results suggested that hepatitis virus infection might  mediate the RORc expression through regulation of its coactivators in liver cancer.

HBx Increased the Expression of RORc.
Considering the higher expression of RORc in those liver cancer patients with HBV infection, HBV may be involved in the regulation of RORc. And hepatitis B virus X (HBx) protein is the most important factor of HBV-mediated liver cancer progression.
us, we hypothesized that HBx may play an important role in the regulation of RORc. To confirm the hypothesis, HBxoverexpressing plasmid was transfected into the HepG2 cells and examined the expression of RORc. As the results showed, HBx can increase the protein levels of RORc in a dose-dependent manner (Figures 4(a) and 4(b)). Next, we also examined the transcriptional activity of RORc with or without HBx. e RT-PCR result revealed that HBx can enhance the mRNA level of RORc (Figures 4(c) and 4(d)).
ese results suggest that HBx may increase the RORc expression through the promotion of the transcriptional activity.

Promoter Activity of RORc Was Activated by HBx.
As described earlier in this study, HBx can increase the expression of RORc in protein and mRNA levels, suggesting that HBx could regulate RORc through the transcriptional regulation mechanism. To confirm the hypothesis, we conducted the luciferase reporter of RORc promoter. As Figures 5(a) and B showed, HBx could increase the promoter activity of RORc in HepG2 and Huh7 cells in a dose-dependent way. Similarly, the promoter activity of RORc was also enhanced in the stable HBx-overexpressing cells (Figures 5(c) and 5(d)). To further confirm the regulation of HBx on the promoter activity of RORc. e HepG2-HBx and Huh7-HBx cells, which were stably overexpressing HBx, were subjected to HBx siRNA, and the efficiency of knockdown was confirmed at the protein level by western blotting (Figure 5(e)). Furthermore, we observed the luciferase activity of RORc promoter was significantly decreased in a dose-dependent manner (Figures 5(f ) and 5(g)).
us, HBx could activate the promoter activity of RORc. Taken these results, RORc was overexpressed in liver tumors, and HBx could increase RORc expression, we believed that HBx can increase RORc expression through activation of promoter activity.

RORc Promotes the Migration of Liver Cancer Cells.
RORc was overexpressed in liver tumor tissues, and the RORc expression was mediated by the HBx, an important oncogene in the development of liver cancer; these data suggest that RORc may act as a cancer promoter in liver cancer. To further validate the function of RORc, RORc was detected in HepG2, Huh7, SMMC7721, and HCCLM3 cells. And HCCLM3 represented the highest expression of RORc (Figures 6(a) and 6(b)), and as the previous report, among the four cell lines, HCCLM3 was the most malignant grade with higher migration activity [25]. us, whether RORc has a potential role in the migration process of liver cancer cells is unclear. en, HepG2 cells were selected to the next study. e lentiviral particles of overexpressing RORc and control vector were confirmed with RT-PCR (Figure 6(c)), and the effect of RORc on the migration activity of liver cancer cells was evaluated by wound-healing assay. e results showed that overexpression of RORc could significantly promote the migration of HepG2 cells after incubation with lentiviral particles for 24 h. And for another 24 h culture, the migration index was further enhanced (Figures 6(d) and 6(e)).

RORc Enhances the Proliferation Activity of Liver Cancer
Cells. RORc has a potential role in the migration activity of    liver cancer cells, and the effect of RORc on the proliferation activity was examined. As Figure 7(a) revealed, cell viability showed no significant difference after treatment with RORcoverexpressing lentiviral particles for 12 h. After treatment with the above for 24 h, cell viability was enhanced by overexpression of RORc (Figure 7(a)). To confirm the effect of RORc on the regulation of proliferation in liver cancer cells, the colony formation assay was used in this study. e results showed the colony number was increased after overexpression of RORc. us, the cell viability and colony formation assay both represented that RORc accelerated the proliferation activity of liver cancer cells.

Discussion
e current study of RORc in cancers remains still unclear. RORc was reported as an oncogene in non-small-cell lung cancer and melanoma cancer [13,15]. However, RORc was also described as a tumor suppressor gene in breast cancer, suggesting that RORc performs a different function in different organs [11,26]. But the recent study also revealed that even in the same type of cancer, RORc was reported to perform a different function. Considering RORct as an important regulator in the immune regulation network, the immune system was a crucial aspect of the tumor microenvironment. Similarly, RORc is different from RORct at the N-terminal, and RORc is also selectively expressed in the 17 cells and involved in the control of 17 differentiation [27]. ese contribute to the different functions in cancers. Up to date, about the function of RORc in liver cancer was not reported yet. In this study, we intended to study the expression of RORc and its significance in liver cancer cells. e previous study showed that RORct was highly expressed in peripheral blood lymphocyte of liver cancer patients [18], RORc as a similar isoform, the expression and its significance have not been reported, and then we explored the expression of RORc in liver cancer. e bioinformatics analysis and western blotting showed that RORc was highly expressed in the liver tumor tissues compared with the adjacent normal liver tissues (Figure 1). More interestingly, we firstly found that the methylation levels of RORc in liver tumor tissues were downregulated compared with the normal liver tissues (Figures 2(a) and  2(b)). Similar to the current study, the regulation of methylation in the gene expression was an important method [28]. Also, the correlation analysis confirmed that the overexpression of RORc in the liver tissues was due to its hypomethylation levels of the promoter (Figures 2(c) and 2(d)), as numerous studies have revealed that the aberrant DNA methylation was deeply involved in the occurrence and development of some cancers [1,29,30]. In this study, we showed that the aberrant expression of RORc in liver cancer tissues and the methylation regulation contributed to the overexpression of RORc in liver tumor tissues.

HepG2
Huh7 SMMC7721 HCCLM3  (e) e migration index was calculated with the ratio of the scratch area between RORc overexpression and vector cells by Image J software. e data are expressed as mean ± standard deviation (SD), n � 3. * P < 0.05, * * P < 0.01, * * * P < 0.001 were considered statistically significant.
As hepatitis B virus (HBV) infection is a major risk factor of liver cancer [31], hepatitis B virus X protein (HBx) is a major protein for HBV-related liver cancer [32]. And the former study revealed that RORc may be involved in the development of liver disease [33]. en, to evaluate the correlation between HBx and RORc, we test the correlation between both the molecules. e results showed RORc expression was further increased in the HBV-positive liver cancer tissues (Figure 3). And overexpression of HBx in liver cancer cells, the expression of RORc was significantly increased in both protein and mRNA levels ( Figure 4). e transcriptional regulation is an essential manner for gene levels. en, we hypothesized that HBx could promote the transcriptional activity of RORc. And the results also confirmed our hypothesis. Furthermore, to confirm the results, we also knockdown HBx in the HBx-stable expressing cells ( Figure 5). And these results demonstrated that RORc was a novel regulator in HBV-positive liver cancer. However, given that HBx was a coactivator of transcription regulation, HBx could activate some signaling pathways, so it is an important direction to study the regulation of HBx on RORc in further studies. Another important question is that the methylation level of RORc promoter was downregulated in the liver tumor tissues, and the promoter methylation regulation might contribute to the overexpression of RORc in liver tumor tissues, and we also reported HBx mediated the RORc expression. Previous reports show that HBx can regulate some genes via DNA methylation, especially in the methylation of the promoter [34,35]. en, whether HBx could affect the promoter methylation of RORc needs further evaluation.
To confirm the significance of RORc in liver cancer, we evaluated the function of RORc in the regulation of migration and proliferation activity in liver cancer cells. e wound-healing assay revealed that overexpression of RORc could enhance the migration activity of liver cancer cells ( Figure 6). Furthermore, the cell viability and colony assays indicated that RORc has positive regulation in the process of proliferation (Figure 7). ese functional tests initially evidenced that RORc performed as an oncogene in the liver cancer cells.
In conclusion, this study demonstrated that RORc was highly expressed in the liver cancer tissues and the disorder of methylation is an important factor contributing to the overexpression of RORc. More interestingly, we firstly revealed that RORc was close with HBV-related liver cancer and HBx could increase the expression of RORc through the promotion of promoter activity. And in vitro studies also showed RORc was a positive regulator in the migration and proliferation of liver cancer cells, suggesting that RORc was a novel target for liver cancer therapy, especially for the HBV-positive liver cancer.

Data Availability
Data are available from the corresponding author.

Conflicts of Interest
e authors declare that they have no conflicts of interest. (b) HepG2 cells were treated with lentiviral particles overexpressing RORc and control vector for indicated times, and then the colony number was calculated with Image J after crystal violet staining. e data are expressed as mean ± standard deviation (SD), n � 3. * P < 0.05, * * P < 0.01, * * * P < 0.001 were considered statistically significant.