BCL6B expression in hepatocellular carcinoma and its efficacy in the inhibition of liver damage and fibrogenesis.

// Weilin Wang 1, 2, 3, * , Pengfei Huang 1, 3, * , Panyisha Wu 2, 3 , Rong Kong 1 , Jiang Xu 1 , Lufei Zhang 1 , Qifan Yang 1 , Qingsong Xie 1 , Linshi Zhang 2 , Xiaohu Zhou 1 , Linghui Chen 1 , Haiyang Xie 1, 3 , Lin Zhou 1, 3 , Shusen Zheng 1, 2, 3 1 Key Lab of Combined Multi-Organ Transplantation, Ministry of Public Health, Hangzhou 310003, China 2 Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China 3 Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, China * These authors have contributed equally to this work Correspondence to: Shusen Zheng, e-mail: shusenzheng@zju.edu.cn Keywords: BCL6B, hepatocellular carcinoma, liver fibrosis, prognostic biomarker Received: February 2, 2015      Accepted: April 14, 2015      Published: April 27, 2015 ABSTRACT B cell CLL/lymphoma 6 member B (BCL6B) is expressed in many normal tissues but expressed at very low levels in cancer tissues. It was reported that BCL6B inhibits hepatocellular carcinoma (HCC) metastases, but the exact role of BCL6B in HCC remains to be investigated. BCL6B expression was significantly decreased in HCC tissues compared with paired non-cancer tissues. Low BCL6B expression in tumors was correlated with shorter overall survival in patients, and multivariate Cox regression analysis revealed that BCL6B expression was an independent prognostic factor for human HCC patients. Moreover, a positive correlation between BCL6B expression and hepatic cirrhosis was found in an analysis of HCC clinicopathological characteristics. BCL6B expression was increased in rat fibrotic liver samples in response to liver injury. BCL6B transgenic rats were less susceptible to hepatocellular damage, inflammation and fibrosis. In vitro studies demonstrated that BCL6B inhibited the activation of hepatic stellate cells though upregulation of hepatocyte growth factor. In addition, transcriptomic microarray analysis was performed to explore the mechanisms in which BCL6B confers protection from tumorigenesis. In conclusion, BCL6B plays a pivotal role as a prognostic biomarker for HCC, and the restoration of BCL6B may be a novel strategy as an anti-fibrogenic therapy for human HCC.


Western blot analysis
Proteins were extracted from tissues and cell lines. The protein concentration was measured using the BCA protein assay (Thermo Scientific, Waltham, MA, USA). Approximately 50 μg tissue or 30 μg cellular protein lysates were separated by electrophoresis using 12% PAGE gels (Invitrogen) and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking nonspecific binding sites for 2 h with 5% nonfat milk, the membranes were incubated overnight on ice with primary antibodies against BCL6B (ab87228, Abcam, Cambridge, UK) and β-actin (Sigma-Aldrich). After washing the membranes several times in Trisbuffered saline and Tween 20 (TBST) with agitation, detection was performed using the appropriate secondary horseradish peroxidase-linked anti-mouse or anti-rabbit antibody. Membrane-enhanced chemiluminescence was then conducted using a chemiluminescence detection kit (Biological Industries, Beit Haemek, Israel).

Tissue microarray construction and immunohistochemistry
For the construction of TMAs, duplicate 1.0-mm diameter cores of tissue from each sample were punched from paraffin tumor blocks and corresponding non-tumor tissues.
Immunohistochemical analysis was performed using paraffin-embedded tissues. Briefly, serial 4-μm sections were deparaffinized and rehydrated and then subjected to heat-induced epitope retrieval in a microwave oven. Next, the sections were treated with 3% hydrogen peroxide to quench endogenous peroxidase activity, followed by incubation with 5% FBS to block non-specific binding.
After blocking, the sections were incubated overnight at 4°C with primary antibodies against BCL6B (ab87228, Abcam), α-SMA (ab5694, Abcam) or CD3 (ab5690, Abcam). The following day, the sections were incubated with HRP-conjugated secondary antibodies (Invitrogen) and visualized using 3, 3′-diaminobenzidine (Zhongshan Golden Bridge Biotechnology, Beijing, China), followed by counterstaining with hematoxylin. For the negative control, the primary antibody was replaced with phosphate-buffered saline (PBS). The staining scores of the tissue controls on each microarray slide were preevaluated as a quality control for immunostaining.
The optimum cutoff value for the IRS was obtained by receiver operating curve (ROC) analysis, and the area under the curve (AUC) values for the various BCL6B IRS cutoff values for 1-, 3-, 5-and 7-year overall survivals were calculated. The optimum cutoff value for the BCL6B IRS was 4, considering that the predictive value of this cutoff point for death was the best (data not shown). Under these conditions, samples with IRS of 0-4 and 6-12 were classified as low and high BCL6B expression, respectively.

Isolation of liver HSCs
Briefly, cells were isolated from the livers using a two-stage collagenase perfusion technique as described previously(2). Filtered cells were centrifuged at 50 g for 2 min to remove hepatocytes. The remaining nonparenchymal cell (NPC) fraction was collected. For HSC enrichment, the remaining NPC fraction was resuspended in 11.5% OptiPrep (Axis-Shield, Oslo, Norway) and put between a bottom cushion of 15% OptiPrep and a top layer of PBS. After centrifugation at 1500 g for 15 min, we obtained the HSC fraction at the interface between the top and intermediate layer.

Serological analysis
Serum liver enzyme concentrations were quantified using the Advia 1800 analyzer (Siemens Healthcare Diagnostics, Eschborn, Germany) and kits from Bayer HealthCare (Leverkusen, Germany).

Analysis of inflammatory cell recruitment
The number of inflammatory cells in the paraffinembedded liver sections was determined via manual counting of CD3+ cells or hematoxylin-eosin staining. A minimum of five different fields in the evaluated liver sections from three independent wild-type or BCL6B Tg animals were analyzed for CD3+ cells.

Analysis of apoptosis in liver tissues
The livers were isolated, fixed and embedded in paraffin. Apoptosis was determined by counting TdTmediated dUTP nick-end labeling (TUNEL)-positive cells using the ApoAlert ® DNA Fragmentation Assay Kit (Clontech) in accordance with the manufacturer's instructions. A minimum of five different fields in each liver section was used to count the number of signal-positive cells.

Quantification of hepatic cytokine levels
Liver tissue extracts were obtained by homogenization of snap-frozen tissues in Cell Lysis Buffer (Cell Signaling, Danvers, MA, USA) supplemented with 1 mM PMSF and a protease inhibitor cocktail (cOmplete Mini Protease Inhibitor Cocktail Tablets from Roche Diagnostics, Mannheim, Germany) using a homogenizer, followed by sonication using a Sonopuls HD 70 (Bandelin Electronics, Berlin, Germany) and centrifugation. TNF, IL-6 and IL-1 levels in liver tissue homogenates were assessed by sandwich enzyme-linked immunosorbent assay (ELISA) using the rat TNF, IL-6 and IL-1 DuoSet ELISA development kits (R&D Systems Inc., Minneapolis, MN, USA) following the manufacturer's instructions.

siRNA transfection
The double-stranded HGF siRNA duplex and control siRNA duplex were purchased from Invitrogen. For transfection, 60% confluent cells were plated in sixwell plates and incubated overnight; 30 pmol HGF siRNA or control siRNA were transfected into the cells using RNAiMax transfection reagent (Invitrogen) according to the manufacturer's instructions. Cells were collected 48 h after transfection.

Lentiviral infection and stable transfection
Replication-defective lentiviruses encoding the complete BCL6B open reading frame (LV-BCL6B) and a lentivirus vector encoding the green fluorescent protein (LV-Ctrl), as a control, were constructed by Invitrogen. HepG2 and SMMC-7721 cells were seeded at a density of 3 × 10 5 cells/ml in six-well plates for synchronization. After culturing overnight, the cells were treated with lentivirus at a multiplicity of infection (MOI) of 20 pfu per cell in 2% FBS medium with 8 μg/ml Polybrene (Sigma-Aldrich). After infection for 6 h, the medium with the lentiviruses was discarded, and the cells were refreshed with complete growth medium. Infection was efficient, showing almost 90% GFP-positive cells as early as 48 h after infection. Blasticidin S HCl (5 μg/ ml) (Invitrogen) was added to the medium to select for stable transformants; blasticidin-resistant colonies were then expanded. The BCL6B-positive colonies were confirmed by Western blot. The stably transfected cells were used to demonstrate the function of BCL6B in HCC cells.

Cell viability assay
Cell viability assay was performed using the Cell Counting kit-8 (Dojindo Laboratories). The cells were plated in 96-well plates at a density of 3 × 10 3 per well. We added 10 μl of the Cell Counting kit-8 solution to 90-μl growth medium in each well after 24, 48, 72 or 96 h. One hour later, the absorbance at 450 nm was read to determine the cell viability in each well. All experiments were completed in triplicate.

Colony formation assay
For colony formation assays, the LV-CTRL or LV-BCL6B cells were plated (3, 000/dish) in 10-cm dishes. The surviving colonies (> 50 cells) were stained with crystal violet and counted after 10 days of culture. The experiments were performed in triplicate.

Annexin V apoptosis assay
Cells undergoing early and late apoptosis were quantified using a flow cytometer (CYTOMICS FC 500; Beckman Coulter, Miami, FL, USA), following staining with Annexin V-APC and propidium iodide (Sungene Biotech Co., Ltd, Tianjin, China). The assay was repeated three times.

Flow cytometry analysis of cell cycle distribution
Cells used for the cell cycle assays were fixed in 70% ethanol, stored at 4°C for more than 24 h, and stained with DNA PREP (Beckman Coulter) before being subjected to flow cytometry. The experiments were performed in triplicate.

Analysis of cell invasion and migration
Cell invasion and migration assays were performed using 24-well transwell plates (Millipore, Billerica, MA, USA). Filters used for invasion assays were coated with Matrigel (BD Biosciences, San Jose, CA, USA) in the upper compartment before cell seeding. Then, 4 × 10 4 cells were seeded in the filters, and the lower compartment was filled with cell culture medium supplemented with 20% FBS. The invading or migrating cells on the bottom surface were stained with crystal violet and counted after 24 h of culture and then photographed using a digital microscope. The experiments were performed in triplicate, and three fields were counted per filter in each group.

In vivo animal experiments
Male BALB/c nude mice at 4-5 weeks obtained from Shanghai SLAC Laboratory Animal Co. Ltd.
(Shanghai, China) were used in all experiments and kept in laminar flow cabinets under specific pathogen-free conditions. The experiments were performed according to the institutional ethics guidelines. Mice were divided randomly into two groups of 10 mice each. The HCC stably transfected cells were injected subcutaneously into the armpits of mice in a total volume of 100 μl (2 × 10 6 cells in PBS). Approximately 10 days after cell inoculation, the tumor volumes in each mouse were monitored every 2 days by measuring the length and width using a caliper. The volume was calculated as the length × (width) 2 /2. Mice were then sacrificed and the tumors removed and weighed.